IN THE UNITED STATES PATENT AND TRADEMARK OFFICE Alexandria, Virginia

A UTILITY PATENT APPLICATION for

ARTICLES AND METHODS FOR INHIBITING METHIONINE AMINOPEPTIDASE ACTIVITY by

Eric Skaar, of Nashville, TN;

Caitlin Murdoch, of Nashville, TN; and Andy Weiss, of Nashville, TN.

Assignee: Vanderbilt University

Attorney Docket No. : 11672N/22117WO

RELATED APPLICATIONS

[0001] This is a utility patent application claiming the benefit of priority in U.S. Provisional Application Serial No. 63/328,213 filed April 6, 2022, the entire disclosure of which is incorporated herein by reference.

REFERENCE TO SEQUENCE LISTING SUBMITTED ELECTRONICALLY

[0002] A sequence listing electronically submitted with the present application as an XML file named 11672N_22117W0.xml, created on 04-6-2023 and having a size of 79000 bytes, is incorporated herein by reference in its entirety.

GOVERNMENT INTEREST

[0003] This invention was made with government support under grant numbers P30CA068485 and R01AI150701, awarded by the National Institutes of Health (NIH). The government has certain rights in the invention.

TECHNICAL FIELD

[0004] The present-disclosed subject matter relates to articles and methods for inhibiting methionine aminopeptidase activity. In particular, the presently-disclosed subject matter relates to inhibition of methionine aminopeptidase activity through targeting of ZNG1 or inhibition of metalation of methionine aminopeptidase by ZNG1 for the treatment of diseases and conditions such as obesity and cancer.

BACKGROUND

[0005] Metals are essential micronutrients that are indispensable for cellular processes in all kingdoms of life. Zinc (Zn) is the second most abundant transition metal in humans serving as a structural or enzymatic cofactor for approximately 10% of the proteome (Andreini et al., 2006). Consequently, perturbations in Zn homeostasis are linked to human disease, including growth deficiencies, immune defects, neurological disorders, and cancers (Basu, 2018; Devirgiliis et al., 2007; Fischer Walker and Black, 2004; Prasad, 2013). This is particularly alarming as Zn deficiency is the fifth most important risk factor for mortality in developing countries affecting close to half of the world’s population (Guilbert, 2003). [0006] Members of the ubiquitous G3E family of P-loop GTPases deliver/insert different metal cofactors to client metalloproteins (Haas et al., 2009). This family includes COG0523 proteins, a subgroup found across all branches of life with poorly understood cellular functions (Edmonds et al., 2021; Haas et al., 2009). COG0523 proteins are characterized by a conserved N-terminal GTPase domain and GTP hydrolysis is thought to provide energy for the transfer of metals to client proteins (Jordan et al., 2019). Furthermore, COG0523 proteins have been implicated in cellular Zn homeostasis as the expression of several bacterial and eukaryotic COG0523 members is induced during conditions of Zn starvation (Coneyworth et al., 2012; Haas et al., 2009; Jordan et al., 2019; Mortensen et al., 2014; Ogo et al., 2015). Despite the assignment of COG0523 proteins as putative nucleotide hydrolysis-powered Zn metallochaperones that act under conditions of Zn restriction, conclusive experimental evidence in support of this proposed function has not been provided

[0007] Cellular Zn is present at levels similar to major metabolites like ATP; however, the majority of Zn within the cell is associated with Zn-requiring metalloproteins, Zn-storage proteins, or maintained in vesicular storage (Krezel and Maret, 2017; Sigel et al., 2013; Wellenreuther et al., 2009). This tight regulation of intracellular pools results in extremely low levels of freely available Zn (Ba et al., 2009). During Zn limitation, the metalation of critical metalloproteins is thought to require the hierarchical distribution of Zn to ensure function of these proteins. Akin to other metals like copper (O'Halloran and Culotta, 2000), targeted transfer of Zn to metalloproteins is hypothesized to be mediated by specialized proteins referred to as metallochaperones/metal-insertases (Rosenzweig, 2002), yet no such protein has been identified to date.

[0008] Many proteins require metal co-factors (e.g, zinc) for proper function. One group of zinc-dependent proteins are methionine aminopeptidases (METAPs), which process newly synthesized proteins to ensure their proper function. Due to their importance for de novo protein synthesis and proteostasis, METAPs are uniformly expressed across the human body, and thought to be particularly important for proliferating cells. One hallmark of cancer is accelerated cellular proliferation and increased expression of human METAP2 has been implicated in tumorigenesis. This connection has led to the development of METAP2-inhibitors including TNP-470 as pharmacological interventions to treat various cancers (e.g, prostate). [0009] While generally promising, such treatments are hampered by dose-limiting toxic side effects, therefore restricting their therapeutic potential. Of note, although the role of METAP2 for oncogenesis has been well established, little is known about the influence of METAP 1 on cancer progression. This is particularly important, as METAP 1 could aid in overcoming the effects of METAP2 inhibitors through the redundancy between the METAP proteins. Additionally, METAP enzymes have been linked to other non-communicable diseases including obesity. Known METAP2 inhibitors have been shown to elicit anti-obesogenic activity via inhibition of the sterol regulatory element binding protein (SREB), a factor involved in lipid and cholesterol biosynthesis, thereby placing drugs that target METAP activity as therapies for metabolic syndromes.

[0010] The present inventors have recently identified a novel vertebrate family of proteins that distribute Zn within the cell, which they have named Zn regulated GTPase metalloprotein activator 1 (ZNG1) family. The present disclosure shows that ZNG1 functions as a metallochaperone to deliver zinc to METAP 1, therefore supporting its function. Consequently, targeted inhibition of ZNG1 (e.g., in cancer cells) or interference with ZNG1/METAP1 interaction is believed to decrease METAP 1 activity. Such targeted inhibition/interference shows promise as a treatment for cancer and obesity either alone or in conjunction with known METAP2 inhibitors to circumvent compensatory effects by METAP 1.

SUMMARY

[0011] In accordance with the purposes and benefits described herein, novel methods for treatment of diseases and/or conditions facilitated by cells dependent on metallation are described. In one aspect, a method for treating a disease or condition such as obesity or cancer is described, comprising inhibiting methionine aminopeptidase (METAP) activity. Tn some embodiments, the method includes inhibiting METAP 1 activity. In some embodiments the method includes targeting Zn regulated GTPase metalloprotein activator 1 (ZNG1) family proteins and/or the interaction of METAP and ZNG1 proteins.

[0012] In one aspect of the disclosure, a method of treating a disease or condition characterized by increased activity of a methionine aminopeptidase (METAP) protein is disclosed, comprising interfering with a transfer of Zn to the METAP protein from a Zn- regulated GPTase metalloprotein activator 1 (ZNG1) protein. The disease or condition may be characterized by uncontrolled cellular proliferation or may be a metabolic disease or condition. In embodiments, the disease or condition is obesity or a cancer. The METAP protein may be METAP 1. The method may optionally include administering one or more METAP2 inhibitors.

[0013] In embodiments, the interfering may comprise reducing or preventing the transfer of Zn from the ZNG1 protein to the METAP protein. In other embodiments, the interfering may comprise reducing or inhibiting an activity of the ZNG1 protein. In embodiments, the reducing or preventing the transfer of Zn comprises reducing or preventing an interaction between the ZNG1 protein and the METAP protein. In one possible embodiment, the reducing or preventing an interaction is effected by a peptide comprising the amino acid sequence: MAAVETRVCETDGCSSEAKLQCPTCIKLGIQGSYFCSQECFKGSWATHKLLHKKAKDE K (SEQ ID NO:_70).

[0014] In another aspect of the disclosure, a method of reducing an activity of a methionine aminopeptidase (METAP) protein, comprising interfering with a transfer of Zn to the METAP protein from a Zn-regulated GPTase metalloprotein activator 1 (ZNG1) protein. The METAP protein may be METAP 1.

[0015] In embodiments, the interfering may comprise reducing or preventing the transfer of Zn from the ZNG1 protein to the METAP protein. In other embodiments, the interfering may comprise reducing or inhibiting an activity of the ZNG1 protein. In embodiments, the reducing or preventing the transfer of Zn comprises reducing or preventing an interaction between the ZNG1 protein and the METAP protein. In one possible embodiment, the reducing or preventing an interaction is effected by a peptide comprising the amino acid sequence: MAAVETRVCETDGCSSEAKLQCPTCIKLGIQGSYFCSQECFKGSWATHKLLHKKAKDE K (SEQ ID NO:_70).

BRIEF DESCRIPTION OF THE DRAWINGS

[0016] The novel features of the invention are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are used, and the accompanying drawings of which: [0017] Figure 1. Identification of metalloprotein targets of Zn regulated GTPase metalloprotein activator 1 (ZNG1) in vertebrates. (A) Cladogram and analysis of ZNG1 protein sequence conservation in vertebrates over evolutionary time. Numbers on branches denote nucleotide substitutions per site. (B) Amino acid conservation along the length of vertebrate ZNGls indicates high sequence conservation particularly in the GTPase motifs and at the N-terminus. Lighter shading reflects higher conservation. (C) Yeast-two-hybrid screens using full-length human, mouse, and zebrafish ZNGls identify several unique and shared interacting proteins. (D) Molecular function enrichment analysis of ZNG1 interaction protein PF AM domains. (E) Number and cellular activity of ZNG1 client Zn metalloproteins detected in yeast-two-hybrid screens. (F) ZNG1 interaction domains in Zn metalloproteins shown in E. (G) Conserved ZNG1 interaction domain of METAP1 across species overlaps with a zf-C6H2 domain. Yellow shading indicates minimal conserved region across species.

[0018] Figure 2. ZNG1 interacts with METAP1 via a unique C6H2 Zn finger domain.

(A) Affinity chromatography of mouse (Mm) His-MBP-ZNGl immobilized on amylose resin, with flow-through (FT) and washes (Wl-3) prior to addition of free A-//?? METAP 1, with subsequent FT and W4-6 prior to elution (E) of His-MBP-ZNGl, and detection by SDS PAGE (top 2 panels) and by immunoblot (bottom). (B) DLS analysis of full-length Mm ZNG1 and METAP 1 shows complex formation in vitro. (C) Size exclusion chromatography of full-length Mm ZNG1 and METAP 11-86. (D) Identification of a conserved N-terminal ‘CPELVPF motif in Mm, human (Hs), and zebrafish (Dr) ZNGls and synthetic peptides. (E) Spectra of W45 fluorescence quenching of 3 pM METAP 11.59 upon Mm ZNG1 N-terminus binding. (F) Peptide binding curve from (E) fit to a 1 : 1 binding model. (G) Measured affinities of METAPI1-59 and METAP h.79 binding to Mm, Hs, and Dr ZNG1 N-termini. (H) 'H ¹⁵ N HSQC NMR spectra of MET AP I 1.59 alone (left), in complex with Mm ZNG1 N-terminal peptide (middle), and as N- terminal Mm ZNG1 peptide fusion (right). (I) Chemical shift perturbations in ¹⁵ N METAP 11.59 upon binding to Mm ZNG1 peptide.

[0019] Figure 3. NMR structure of METAP1-ZNG1 complex. the fusion (black) and free domain (red). (B) Structure of the 20 lowest-energy conformers of the full fusion construct, colored by 'H, ^I5 N NOE. (C) Ribbon diagram of the Mm ZNG110-30

METAP h -59 fusion showing Zn coordination. (D) Stereo view of 20 lowest-energy conformers of the A/w ZNG110-30 METAPh-59 fusion, colored by secondary structure, with helices in green and beta strands in purple, and ZNG110-30 in blue. (E) Electrostatic surface map of the METAPh- 59 peptide interface. (F) N-terminal ZNG1 motif among eukaryotic cluster 3 COG0523 proteins.

[0020] Figure 4. ZNG1 enhances METAP1 aminopeptidase activity in vitro. (A) Representative titration of 1.0 pM METAP 1, 1.0 pM quin-2 with Zn. (B) Representative titration of 1.3 pM ZNG1, 1.3 pM quin-2 with Zn. Solid lines show a global fit of independent replicates; dashed lines are simulations of 10-fold greater and weaker affinities. (C) GTPase activity of ZNG1 in the presence or absence of Zn and/or ZmMETAP l . (D) Initial rate of Met release from 750 pM MAHAIHY peptide by 20 nM METAP1 in different metalation states. (E) Activation of 10 pM ZmMETAP I enzymatic activity by 25 pM ZNG1 in the presence or absence of Zn and 500 pM guanine nucleotides. (F) Activation of 10 pM ZnzMETAPl enzymatic activity by 22.5 pM Zn or 25 pM ZnZNGl in the presence or absence of 500 pM guanine nucleotides, all in the presence of 250 pM NTA. METAP 1 reactions in panels E and F were conducted by diluting activation reactions to 20 nM METAP 1 and monitoring cleavage of 750 pM MAHAIHY. (G) Mechanistic model of GTP hydrolysis-coupled Zn transfer from ZNG1 to the METAP 1 active site.

[0021] Figure 5. zngl mutant zebrafish display Zn-dependent sensitivity to Metap inhibition and phenocopy metapl mutant larvae. (A) zngl expression levels following TPEN treatment in WT and zngl" mutant 6 dpf whole zebrafish larvae. (n=4 pools of 30 larvae per genotype / treatment group). (B-D) Measurement of height at anterior anal fin (HAA) (B), standard length (C), and snout to vent length (D) of zngl ^ and WT 6 dpf larvae. Each data point represents an individual larva. (E) Survival of WT and zngl' ' mutant larvae treated with Bengamide B and TPEN (n=3 groups of 5 larvae / genotype / treatment) over 3 days of treatment and terminating at 6 dpf. (F) Brightfield images of 6 dpf metapl mutant larvae treated with 1 pM Bengamide B or vehicle control. Yellow arrows denote abnormal pathologies. Pericardial Edema (PCE), craniofacial defects (CD), yolk sack edema (YSE), and uninflated swim bladder (USB). Scale bars = 1 mm. (G) Quantification of gross pathology score of 6 dpf Bengamide B treated metapl mutant and WT larvae. Pathology scores calculated by taking the sum of each pathology indicated by the yellow arrows in panel F. Each data point represents an individual larva. (H) Brightfield images of 6 dpf zngl mutant larvae treated with 500nM Bengamide B or vehicle control. Scale bars = 1 mm. (I) Quantification of gross pathology score of 6 dpf Bengamide B treated zngl mutant larvae as depicted above. (J) Brightfield images of 6 dpf WT larvae treated with 1 pM Bengamide B or vehicle control. Scale bars = 1mm. (K) Survival of WT and zn /' ' larvae treated with TNP-470 and TPEN (n=3 groups of 5 larvae / genotype / treatment) over 3 days of treatment and terminating at 6 dpf. Data in panels A, E, and K analyzed by two-way ANOVA with Tukey’s multiple comparison test. Data in panels B,C,D, G and I analyzed by Student’s /-test.

[0022] Figure 6. Zngl mutant mice exhibit signatures of mitochondrial dysfunction on a Zn deficient diet. (A) Genotype distribution of WT, Zngl ^+/ ~, and Zngl ’ ’mice. Number of animals per genotype indicated in white text. (B) Percent weight gain of 5-7 week old WT and Zngl mutant mice that were placed on a Zn-deficient diet for 6 weeks (n= 4-5 mice / genotype / sex). (C) Differential protein abundances from WT and Zngl mutant dissected kidneys from 11- 13 week old female mice that were maintained on a low Zn diet for 5 weeks. Proteins that localize to the mitochondria are highlighted in red (n = 5 mice / genotype). (D) IPA analysis of differentially abundant proteins (Zngl' ' /WT with significance of gene enrichment and number of differentially abundant proteins for each pathway depicted, z-scores are denoted within teal bars and indicate the predicted effects on each pathway (positive values: activation; negative values: inhibition, n.a.: no prediction through IPA available due to insufficient evidence in the Knowledge Base). Red bars indicate enriched proteins in Zngl mutant animals and blue bars represent proteins with reduced abundance. Data in panel A analyzed by Chi-squared goodness of fit test. Data in panel B analyzed by two-way ANOVA with Sidak’s multiple comparison test within each sex.

[0023] Figure 7. ZNG1 regulates cellular respiration through stimulation of METAP activity. (A) Expression of Zngl in TKPTS cells treated with TPA. (B) Zngl transcript levels in untreated WT and Zngl mutant TKPTS cells. (C-D) Proliferation of TKPTS cells quantified by CellTrace median fluorescence intensity (MFI) in Zn-deplete conditions (C) and following treatment with METAP2 inhibitor TNP-470 (D) using flow cytometry. (E) Ratio of processed 14-3 -3y protein levels in vehicle or TPA treated Zngl' ' / WT TKPTS cells, (n = 4 / genotype / treatment). (F) Representative TEM images of WT and Zngl mutant TKPTS cells grown in Zn- deplete conditions (scale bars = 500 nm). Red box (top panel) outlines region of higher magnification (bottom panel). (G-H) Non-linear regression analysis of the size distribution of mitochondria (G) and mean signal intensity (H) in WT and Zngl mutant TKPTS cells treated with TPA. (WT n = 112 mitochondria, ZngT n = 101 mitochondria). (I) ATP levels in cell lysates from untreated WT and Zngl mutant TKPTS cells. (J) Mitochondrial membrane potential (AT m) in untreated and T A treated TKPTS cells. (I ) Production of mitochondrial superoxide in TKPTS cells treated with TNP-470 was quantified by flow cytometry. (L-M) Oxygen consumption rates (OCR) by TKPTS cells treated with vehicle or TPA. OCR was normalized by cell number. Data in panels A, B, H, and I analyzed by Student’s /-test. Data in panel E analyzed by Welch’s /-test. Data in panel G analyzed by non-linear regression goodness of fit. Data in panels C, D, J, K, and M analyzed by two-way ANOVA with Tukey’s multiple comparison test.

[00241 Figure 8. Conservation and tissue specific expression of Human ZNGls. (A) Multiple sequence alignment of vertebrate ZNG1 proteins depicted in cladogram of Figure 1 panel A. White shading in the bottom bar indicates high conservation. (B) Expression levels of human ZNG1A, ZNG1B, ZNG1C, ZNG1E, and ZNG1F across different tissue and cell types. Expression data obtained from the human protein atlas (Uhlen et al., 2015). (C) Multiple sequence alignment of human ZNG1 paralogs and consensus sequence.

[0025] Figure 9. Biochemical interaction of ZNG1 and METAP1. (A-C) Affinity chromatography of full-length immobilized zebrafish MBP-Metapl bound amylose (A) or empty control resin (B) with purified zebrafish Zngl passed over (FT, flow through). Columns were serially washed (W). Elution of Metapl (denoted E) resulted in detection of Zngl shown on SDS PAGE (top two panels) and by immunoblot (bottom panel). Immunoblot signal quantified by densitometry (C). (D) Size exclusion chromatography of mouse METAPi-se interaction with full- length ZNG1 visualized by SDS PAGE. (E) Alignment of METAP 1 C2H6 zf domains from zebrafish (Dr), mouse (Mm), human (Hs), and Saccharomyces cerevisiae (Sc) with MYND zf domains from indicated Hs proteins AML-1, BS69, and DEAF-1. (F) Ribbon diagrams of previously determined structures of zf-MYND:peptide complexes (Harter et al., 2016; Liu et al., 2007). (G) Consensus sequence logo of zf-MYND and zf-C6H2 domains. (H) Metal stoichiometries of Mm METAP 11.59 and METAP 11.79 as purified and after loading with Zn as measured by TCP -MS. ‘n.d.‘ (not detected) indicates measured metal concentrations below 0.01 equivalents. (I) Representative spectra of fluorescence quenching of W45 in Mm METAPh.79 by titration with Mm ZNG1 peptide. (J) 'H ¹⁵ N HSQC NMR spectrum o Mm METAP 11.59 in 2:1 complex with Mm ZNG1 N-terminal peptide, showing equal populations in bound and free states. Data in panel C are represented as mean ± SEM.

[0026] Figure 10. NMR structural characterization of ZNG1 and METAP1. (A)

Assigned ^X H ¹⁵ N HSQC NMR spectra o Mm METAP 11.59 alone (left), in 1: 1 complex with ZNG1 N-terminal peptide (middle), and with N-terminally fused Mm ZNG1 N-terminal peptide (right). (B) Chemical shift-based secondary structure predictions for Mm METAP 11.59 alone (top), the 1: 1 complex with Mm ZNG1 N-terminal peptide (middle), and with N-terminally fused Mm ZNG1 N-terminal peptide (bottom). (C) Sequence diagram of fusion construct of Mm ZNG1 N-terminal peptide with Mm METAP 11.59. (D) Overlay of 'H ¹⁵ N HSQC NMR spectra o Mm METAP h.59 in 1 : 1 complex with Mm ZNG1 N-terminal peptide (blue contours) and with N- terminally fused Mm ZNG1 N-terminal peptide (red contours), showing the fusion strategy eliminates peak splitting artifacts. (E) Chemical shift perturbations caused by the fusion construct, relative to the complex. (F) ¹⁵ N Ri relaxation rates for the fusion (black) and free domain (red). (G) ¹⁵ N R2 relaxation rates for the fusion (black) and free domain (red). (H) Surface representation of the fusion structure, colored by ^H^N chemical shift perturbations upon peptide binding. The peptide is shown as blue sticks. P23 is colored black. (I) Ribbon diagram and electrostatic surface representation showing the predicted peptide binding interface of the S. cerevisiae Map Ip zf-C6H2 domain with a Znglp peptide.

[0027] Figure 11. In vitro METAP1 activity assay. (A) Unfolding of METAPI 1.59 as measured by a decrease in W45 fluorescence as a function of EDTA concentration and time. (B) Unfolding of METAPI 1-59 as monitored by ¹⁵ N-HSQC spectra recorded at increasing concentrations of EDTA. METAP 11.59 in the presence of 50 mM EDTA gives spectra indicative of protein aggregation. (C) Metal stoichiometries of Mm METAP 1 as purified, supplemented with excess Zn, or when treated with various metal chelators as measured by ICP-MS. ‘n.d.‘ (not detected) indicates measured metal concentrations below 0.01 equivalents. (D) ZtoMETAP I Zn binding. Representative titration of Zn into 1 pM Zn?METAP I and 0.8 pM mf2. (E) ZNG1 Zn binding. Representative titration of Zn into 7.5 pM ZNG1 and 16 pM mf2. For panels D and E, solid lines represent the global fit from three independent replicates and dashed lines represent simulations of 10-fold greater and weaker affinities. (F) Zn binding affinities of Mm MET API and Mm ZNG1 as measured by competition with quin2 and mf2. (G) ZNG1 GTPase activity as measured by enzyme-coupled assay in Apo and Zn states. (H) Representative time-course analysis of the cleavage of 125 pM MAHAIHY peptide by Z METAP l by monitoring the release of Met. Indicated timepoint was analyzed by LC-ESI-MS (see panel I). (I) Extracted ion chromatograms of product AHAIHY (top) and substrate MAHAIHY (bottom) peptides taken from the indicated 10 min reaction from panel H. (J) Initial rates of MAHAIHY peptide cleavage at varying substrate concentrations by Z METAPl. (K) Michaelis-Menten kinetics of the ZmMETAP l cleavage of the MAHAIHY peptide. (L) Initial rate of cleavage of 750 pM MAHAIHY by 20 nM murine ZmMETAP l or Zn4METAPl in the presence or absence of Apo or Zn-bound murine ZNG1 in the absence of nucleotide. (M) Cleavage of 100 pM MAHAIHY peptide after incubation of 1 pM Dr Zngl and 1 pM Metapl. Data are represented as mean ± SEM. Data in panels M analyzed by one-way ANOVA with Tukey’s multiple comparison test. Significance denoted as: * P < 0.05, ** P < 0.01 , *** P < 0.001 , **** P < 0.0001 .

[0028] Figure 12. Generation of zngl and metapl mutant zebrafish. (A) Schematic of zebrafish zngl locus with gRNA shown by red bar in second exon. Mutant alleles and in silico translations indicated. Arrows indicate screening primer binding sites. (B) Genotype distributions of WT, zngP , and homozygous zngl" 6 dpf larvae. Number of animals indicated in white text. (C) Survival of WT and zngl''' mutant larvae treated with Bengamide B and TPEN (n=3 groups of 5 larvae / genotype / treatment) over the course of 3 days of treatment. (D) Measurement of height at anal fin (HAA) of WT 6 dpf larvae following three days of exposure to 500 nM Bengamide B or vehicle control. Each data point represents an individual larva. (E) Schematic of zebrafish metapl locus with gRNA shown by red bar in first exon. Mutant allele and in silico translations indicated. Arrows indicate screening primer binding sites. (F) Genotype distributions of WT, metap 1 ^/+ , and homozygous metap l' ~ 6 dpf larvae. Number of animals indicated in white text. (G) Percent of WT and metap 1' ' 6 dpf larvae with pericardial edema, uninflated swim bladder, craniofacial abnormalities, or yolk sac edema following treatment with 1 pM Bengamide B or vehicle control for 3 days, (n = 6 - 8 larvae / genotype). (H) Percent of WT and zngl' ' 6 dpf larvae with pericardial edema, uninflated swim bladder, craniofacial abnormalities, or yolk sac edema following treatment with 500 nM Bengamide B of vehicle control for 3 days, (n = 18 - 20 larvae / genotype). (I) Survival of WT and zngl'/' larvae treated with TNP-470 and TPEN (n=3 groups of 5 larvae / genotype / treatment) over the course of 3 days of treatment. Data are represented as mean ± SEM. Data in panels B and F analyzed by Chi-square goodness of fit test. Data in panel D analyzed by Student’s /-test. Significance denoted as: * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.

[0029] Figure 13. Generation of Zngl mutant mice. (A) Schematic of mouse Zngl locus with gRNA shown by red bar in first exon. (B) RT-qPCR of Zngl transcript in WT and Zngl mutant kidneys (n=4 mice / genotype). Normalized to Actb expression. (C) Normalized peak area of ZNG1 peptides in WT and Zngl' ¹ ' tissue (brain) measured using data-independent acquisition mass spectrometry proteomics. (D-E) Breeding patterns of male and female mice resulting from Zngl heterozygous crosses. Number of animals indicated in white text. (F) 5-7 week old male or female mice were placed on a control diet for the course of 6 weeks and percent weight gain measured (n= 5-8 mice / genotype / sex). (G) 5-7 week old female mice were placed on a control or low Zn diet for the course of 6 weeks and tissue Zn levels assessed (n= 6-8 mice / genotype / sex). (H) Summary network of ATPIF1 upstream regulatory connections identified by IPA. Analysis was based on all differentially abundant proteins (P- value < 0.05) from proteomics comparing renal protein content from WT and Zngl mutant mice (Figure 6C). (I) RT-qPCR analysis of transcripts encoding for most differentially abundant proteins in Zngl mutant mice (Figure 6C, Table S3). Normalized to expression of Ppia, Pgam, and 18S genes. For panels A, B and I, protein or RNA was isolated from tissue from 11-13 week old female mice that were maintained on a low Zn diet for 5 weeks. Data are represented as mean ± SEM. Data in panels B, C, and I analyzed by Student’s /-test. Data in panels D and E analyzed by Chi-square goodness of fit test. Data in panel F and G analyzed by two-way ANOVA with Sidak’s post-test. In panel F two-way ANOVA was run for each sex independently. Significance denoted as: * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.

[0030] Figure 14. Generation and validation of Zngl mutant alleles in TKPTS cells. (A)

Sequence alignment of WT and Zngl mutant alleles generated by CRISPR/Cas9. Two independent mutant clones were isolated, clone 1 (A2/A2) and clone 2 (A2/A7) with deletion alleles that result in frameshift mutations. (B) Immunoblot for METAP 1 in WT and Zngl' ¹ ' TKPTS cells. (C) Immunoblot for ZNG1 and Tubulin control in WT, Zngl' ¹ ' , and Zngl' ' + Zngl TKPTS cells. (D) Zngl transcript levels in WT, Zngl clone 1, and Zngl clone 2 TKPTS cells. (E- F) CellTrace fluorescence levels in WT, Zngl clone 1, and Zngl clone 2 TKPTS cells treated with TPA (E). FACS histogram associated with quantification of indicated panel in Figure 7 (F). (G) Proliferation measured by CellTrace fluorescence of untreated and Bengamide B treated WT TKPTS cells. (H-I) CellTrace fluorescence levels in WT, Zngl clone 1, and Zngl clone 2 TKPTS cells treated with TNP-470 (H). Histogram associated with quantification of indicated panel in Figure 7 (I). (J) Proliferation measured by CellTrace fluorescence of untreated and TNP-470 treated WT, Zngl' ¹ ', and Zngl' ' + Zngl TKPTS cells. (K-L) Proteomics was performed on WT and Zngl' ¹ ' TKPTS cells grown in the presence of TPA or Bengamide B. The abundance of iMet-containing and processed N-termini of 14-3-3y (MVDREQLVQK and VDREQLVQK, respectively) in WT and mutant cells upon TPA treatment was determined to assess the % of unprocessed protein (K). Changes to canonical pathways (as determined by IP A) comparing protein signatures from TKPTS cells with impaired Zngl and WT cells upon inhibition of METAP with Bengamide B were determined (L). Depicted are pathways that meet the following criteria for one of the two conditions: significance cutoff of P-value(-loglO) >1.3, |z-score| > 2. Pathways with the highest total score (combined p-value and z-score) are on top, and colorcoding reflects z-score (positive values: activation; negative values: inhibition). Grey boxes denote pathways for which no z-score was determined due to a limited number (<4) proteins affected in a specific pathway. (M) Mitochondrial roundness of WT and Zngl mutant TKPTS cells grown in Zn-deplete conditions as quantified from TEM. (N) TMRM fluorescence in WT, Zngl' ' clone 1, and Zngl' ' clone 2 TKPTS cells. (O) MitoTracker levels WT, Zngl clone 1, and Zngl clone 2 TKPTS cells. (P-Q) MitoSOX levels WT, Zngl' ' clone 1, and Zngl' ' clone 2 TKPTS cells treated with TNP-470. (N) FACS histogram associated with quantification of indicated panel in Figure 7. (R) Mitochondrial superoxide measured by MitoSOX levels in untreated and TNP-470 treated WT, Zngl' ¹ ', and Zngl' ' + Zngl TKPTS cells. (S) Confocal micrographs of WT and Zngl' ' cells illustrating ATPIF1 distribution (magenta), mitochondria (cyan), and DAPI (grey). Scale bar = 20 pm Data are represented as mean ± SEM. Data in panels D and N were analyzed by one-way ANOVA. Data in panels K and M analyzed by Student’s t- test. Data in panels E,G,H,J,O,Q and R were analyzed by two-way ANOVA with Tukey’s multiple comparison test. Significance denoted as: * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.

[0031] Figure 15. Overview of interaction partners of human, murine, and zebrafish ZNG1 as identified by yeast-two-hybrid data. [0032] Figure 16. Structural statistics for NMR solution structure of the fusion of ZNG110-30 with METAPI-59.

[0033] Figure 17. Proteomics hits from low zinc diet.

[0034] Figure 18. Primers.

DETAILED DESCRIPTION

[0035] The details of one or more embodiments of the presently-disclosed subject matter are set forth in this document. Modifications to embodiments described in this document, and other embodiments, will be evident to those of ordinary skill in the art after a study of the information provided in this document. The information provided in this document, and particularly the specific details of the described exemplary embodiments, is provided primarily for clearness of understanding and no unnecessary limitations are to be understood therefrom. In case of conflict, the specification of this document, including definitions, will control.

[0036] The presently-disclosed subject matter relates to articles and methods for inhibiting methionine aminopeptidase (METAP) activity. In some embodiments, the method includes inhibiting METAP 1 activity. In some embodiments, for example, the method includes targeting Zn regulated GTPase metalloprotein activator 1 (ZNG1) family proteins. ZNG1 proteins are metallochaperones that deliver zinc to, or metalate, METAP 1, thereby supporting the function of METAP 1. As such, inhibiting ZNG1 decreases or eliminates delivery of zinc to METAP 1, which consequently decreases or inhibits METAP 1 activity.

[0037] In some embodiments, the method includes targeted inhibition of ZNG1. For example, in some embodiments, the method includes inhibition of ZNG1 in specific cells, such as cancer cells. Any suitable method of targeting ZNG1 may be used, such as, but not limited to, using small molecules, dietary zinc interventions, or interference with the interaction of ZNG1 and METAP.

[0038] In some embodiments, the method includes inhibition of METAP activity by targeted inhibition of the interaction of ZNG1 and METAP to reduce or inhibit metalation of METAP. The targeted inhibition may occur at a specific interaction site between the two proteins.

[0039] Also provided herein are methods of treating a disease involving METAP 1. In some embodiments, the method includes administering one or more inhibitors of ZNG1 to a subject in need thereof. In some embodiments, the one or more inhibitors are targeted. The disease includes any suitable disease involving METAP1, such as, but not limited to, cancer, obesity, diseases involving rapidly proliferating cells, metabolic syndromes, or any other suitable disease. In some embodiments, the method also includes administering one or more METAP2 inhibitors in conjunction with the one or more inhibitors of ZNG1. Without wishing to be bound by theory, it is believed that targeting ZNG1 as a modulator of cellular proliferation and lipid biosynthesis via METAP activity is a novel approach for anti-cancer and anti-obesity therapy. The methods of treating a disease involving METAP 1 may in embodiments comprise any or all of the abovesummarized methods.

[0040] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the disclosure belongs. Any methods and materials similar to or equivalent to those described herein can be used in the practice or testing of the present disclosure, including the methods and materials are described below. While the terms used herein are believed to be well understood by those of ordinary skill in the art, certain definitions are set forth to facilitate explanation of the presently- disclosed subject matter.

[0041] The terms “treatment” or “treating” refer to the medical management of a subject with the intent to cure, ameliorate, reduce, or prevent a disease or condition. As will be recognized by one of ordinary skill in the art, the term “cure” does not refer to the ability to completely eliminate a disease or condition. For example, in some embodiments, a cure can refer to a decrease at a level of 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% decrease. Similarly, as will be recognized by one of ordinary skill in the art, the term “prevent” does not refer to an ability to completely remove any and all symptoms or evidence of a disease or condition.

[0042] Likewise, as will be recognized by one of ordinary skill in the art, the term “inhibiting” or “inhibition” does not refer to the ability to completely inactivate all target biological activity in all cases. Rather, the skilled artisan will understand that the term “inhibiting” refers to decreasing biological activity of a target, such as METAP, such as can occur, for example, when a nucleotide limits the expression of the target gene, when a ligand binding site of the target protein is blocked, or when a non-native complex with the target is formed. Such decrease in biological activity can be determined relative to a control, wherein an inhibitor is not administered and/or placed in contact with the target. For example, in some embodiments, a decrease in activity relative to a control can be about a 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,

11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36,

37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62,

63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88,

89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% decrease. The term “inhibitor” refers to a compound of composition that reduces the expression of and/or decreases the biological activity of a target, such as METAP.

[0043] The terms “subject” or “subject in need thereof’ refer to a target of administration, which optionally displays symptoms related to a particular disease, pathological condition, disorder, or the like. The subject of the herein disclosed methods can be a mammal. Thus, the subject of the herein disclosed methods can be a human, non-human primate, horse, pig, rabbit, dog, sheep, goat, cow, cat, guinea pig, or rodent. The term does not denote a particular age or sex. Thus, adult and newborn subjects, as well as fetuses, whether male or female, are intended to be covered. A patient refers to a subject afflicted with a disease or disorder. The term “patient” includes human and veterinary subjects.

[0044] The term “administering” refers to any method of providing a therapeutic composition to a subject. Such methods are well known to those skilled in the art and include, but are not limited to, oral administration, transdermal administration, administration by inhalation, nasal administration, topical administration, intravaginal administration, ophthalmic administration, intraaural administration, intracerebral administration, rectal administration, and parenteral administration, including injectable means such as intravenous administration, intraarterial administration, intramuscular administration, peritoneal administration, and subcutaneous administration. Administration can be continuous or intermittent. In various aspects, a preparation can be administered therapeutically; that is, administered to treat an existing disease or condition. In further various aspects, a preparation can be administered prophylactically; that is, administered for prevention or amelioration of a disease or condition. [0045] The term “effective amount” refers to an amount that is sufficient to achieve the desired result or to have an effect on an undesired condition. For example, a “therapeutically effective amount” refers to an amount that is sufficient to achieve the desired therapeutic result or to have an effect on undesired symptoms, but is generally insufficient to cause adverse side effects. The specific therapeutically effective dose level for any particular patient will depend upon a variety of factors including the disorder being treated and the severity of the disorder; the specific composition employed; the age, body weight, general health, sex and diet of the patient; the time of administration; the route of administration; the rate of excretion of the specific compound employed; the duration of the treatment; drugs used in combination or coincidental with the specific compound employed and like factors well known in the medical arts. For example, it is well within the skill of the art to start doses of a compound at levels lower than those required to achieve the desired therapeutic effect and to gradually increase the dosage until the desired effect is achieved. If desired, the effective daily dose can be divided into multiple doses for purposes of administration. Consequently, single dose compositions can contain such amounts or submultiples thereof to make up the daily dose. The dosage can be adjusted by the individual physician in the event of any contraindications. Dosage can vary, and can be administered in one or more dose administrations daily, for one or several days.

[0046] All patents, patent applications, published applications and publications, GenBank sequences, databases, websites and other published materials referred to throughout the entire disclosure herein, unless noted otherwise, are incorporated by reference in their entirety.

[0047] Where reference is made to a URL or other such identifier or address, it understood that such identifiers can change and particular information on the internet can come and go, but equivalent information can be found by searching the internet. Reference thereto evidences the availability and public dissemination of such information.

[0048] As used herein, the abbreviations for any protective groups, amino acids and other compounds, are, unless indicated otherwise, in accord with their common usage, recognized abbreviations, or the IUPAC-IUB Commission on Biochemical Nomenclature (see, Biochem. (1972) 1 1 (9): 1726-1732). [0049] Although any methods, devices, and materials similar or equivalent to those described herein can be used in the practice or testing of the presently-disclosed subject matter, representative methods, devices, and materials are described herein.

[0050] In certain instances, nucleotides and polypeptides disclosed herein are included in publicly-available databases, such as GENBANK® and SWISSPROT. Information including sequences and other information related to such nucleotides and polypeptides included in such publicly-available databases are expressly incorporated by reference. Unless otherwise indicated or apparent the references to such publicly-available databases are references to the most recent version of the database as of the fding date of this Application.

[0051] The present application can “comprise” (open ended) or “consist essentially of’ the components of the present invention as well as other ingredients or elements described herein. As used herein, “comprising” is open ended and means the elements recited, or their equivalent in structure or function, plus any other element or elements which are not recited. The terms “having” and “including” are also to be construed as open ended unless the context suggests otherwise.

[0052] Following long-standing patent law convention, the terms “a”, “an”, and “the” refer to “one or more” when used in this application, including the claims. Thus, for example, reference to “a cell” includes a plurality of such cells, and so forth.

[0053] Unless otherwise indicated, all numbers expressing quantities of ingredients, properties such as reaction conditions, and so forth used in the specification and claims are to be understood as being modified in all instances by the term “about”. Accordingly, unless indicated to the contrary, the numerical parameters set forth in this specification and claims are approximations that can vary depending upon the desired properties sought to be obtained by the presently-disclosed subject matter.

[0054] As used herein, the term “about,” when referring to a value or to an amount of mass, weight, time, volume, concentration or percentage is meant to encompass variations of in some embodiments ±20%, in some embodiments ±10%, in some embodiments ±5%, in some embodiments ±1%, in some embodiments ±0.5%, in some embodiments ±0.1%, in some embodiments ±0.01%, and in some embodiments ±0.001% from the specified amount, as such variations are appropriate to perform the disclosed method.

[0055] As used herein, ranges can be expressed as from “about” one particular value, and/or to “about” another particular value. It is also understood that there are a number of values disclosed herein, and that each value is also herein disclosed as “about” that particular value in addition to the value itself. For example, if the value “10” is disclosed, then “about 10” is also disclosed. It is also understood that each unit between two particular units are also disclosed. For example, if 10 and 15 are disclosed, then 11, 12, 13, and 14 are also disclosed.

[0056] As used herein, “optional” or “optionally” means that the subsequently described event or circumstance does or does not occur and that the description includes instances where said event or circumstance occurs and instances where it does not. For example, an optionally variant portion means that the portion is variant or non-variant.

[0057] The presently-disclosed subject matter is further illustrated by the following specific but non-limiting examples. The following examples may include compilations of data that are representative of data gathered at various times during the course of development and experimentation related to the present invention.

EXAMPLES

[0058] All combinations of method or process steps as used herein can be performed in any order, unless otherwise specified or clearly implied to the contrary by the context in which the referenced combination is made.

Experimental model and subject details

Ethics statement

[0059] Mouse and zebrafish studies were approved by the Institutional Animal Care and Use Committees of Vanderbilt University Medical Center (protocol numbers M1900043-00 and Ml 900076-00 respectively) in accordance with the Public Health Service Policy on the Human Care and Use of Laboratory Animals under the Unites States of America National Institutes of Health (NIH) Office of Laboratory Animal Welfare (OLAW).

Zebrafish husbandry [0060] Yeast-two-hybrid data are provided within Figure 15. Proteomics data are provided within Figures 17 and 18. Additionally, all mass spectrometry proteome data were submitted to ProteomeXchange via the PRIDE database under the submission reference number 562287. NMR structure information is provided within Figure 16 and is deposited on PDB (7SEK).

[0061] All zebrafish lines were maintained on a mixed Tubingen (Tii) / AB background on a 14: 10 h light:dark cycle in a recirculating aquaculture system. Embryos were collected from natural matings and maintained in embryo medium (0.03% Instant Ocean Sea Salt in RO water) at a density of < 1 larva / mL at 28°C 14: 10 hour light:dark cycle. At 3 dpf, larvae were split randomly into treatment groups. All larvae used in experiments are of indeterminate sex.

Mouse husbandry and diet

[0062] Experiments were performed using adult age-matched C57BL/6 (Jackson Laboratories) or C57BL/6 ZngT ' (breeding colony) mice. Animals were maintained at the Vanderbilt University Medical Center Animal Facilities and housed in groups of five. For each experiment, ZngT~ mice from multiple litters were included. For routine colony maintenance, mice were fed a standard chow diet (LabDiets; Rodent Chow Diet 5001). For manipulation of organismal Zn levels, mice were fed a defined Zn-free or control diet (Dyets Inc., AIN-93M Purified Rodent Diet with or without Zn supplementation at 29 parts per million). For experimental endpoints, animals were humanely euthanized. All animal experiments were approved and performed in compliance with the Institutional Animal Care and Use Committee (IACUC) of Vanderbilt University.

Cell culture conditions

[0063] Mycoplasma-negative WT parental TKPTS and pooled Zngl CRISPR/Cas9 mutant cells were ordered from Synthego (https://www.synthego.com/). Briefly, cells were propagated in DMEM/F-12 50/50 IX (Coming 10-092-CV) supplemented with 7% heat-inactivated fetal bovine serum (R and D Systems, SI 1150) with insulin (Sigma SLCF5002 - 0.3 mL of lOmg/mL per 500 mL media) under 5% CO2. Cells were passaged and trypsinized using TrypLE express (Thermo Fisher Scientific, 12605010).

Method Details

Yeast- two-hybrid screening and analysis [0064] All yeast-two-hybrid screens were performed by Hybrigenics Services using full- length human ZNG1E, mouse ZNG1, and zebrafish Zngl. Murine Zngl and human ZNG1E sequences were obtained from Genscript (Clone ID OMul2215 and OHu42907 in the pcDNA3.1-C-(k)DYK backbone, respectively). Full length zebrafish Zngl was amplified from 6 days post fertilization (dpf) whole larval cDNA with Pl and P2 (Table S5) and cloned into pCRII TOPO (Invitrogen, K465001) and sequence verified by Sanger sequencing. The sequences described above were used as template for the generation of all subsequent ZNG1 constructs. Yeast-two-hybrid screens to identify interaction partners of ZNG1 proteins from different species were performed on a mixed (A549, H1703, H460) lung tumor cell library (human), an adult kidney library (mouse), or a whole embryo 20 h post fertilization library (zebrafish). Any interacting partners that were scored as experimental artifact were excluded from further analysis.

Recombinant protein cloning, expression, and purification

Cloning

[0065] Full-length murine Zngl was amplified from pcDNA3.1-C-(k)DYK-Zwg/ using primers P3 and P4 (Table S5). PCR products were inserted into the expression vector pLM302 (Center for Structural Biology, Vanderbilt University) containing a 3C protease cleavable N- terminal 6xHis and Maltose Binding Protein (MBP) tag using BrzmHI and EcoRI restriction sites.

[0066] Full-length murine Metapl was amplified from pcDNA3.1-C-(k)DYK-Afetap/ (Gencript, Clone ID OMu05035) using primer pair P5/P6. Vector pLM302 was linearized by PCR (primers P7/P8, Figure 18). Both amplicons were joined using the NEBuilder HiFi DNA Assembly Master Mix (New England Biolabs, E2621S).

[0067] A SUMO-tagged, truncated murine METAP h-86 was prepared by amplifying the N- terminal portion of the murine Metapl construct using primers P9 and P10 (Figure 18). The PCR product was cloned into the expression vector pBG102 carrying a 3C protease cleavable N- terminal 6xHis and SUMO tag (Center for Structural Biology, Vanderbilt University) using the Ba/wHI and EcoRI restriction sites.

[0068] For peptide, metal-binding, and METAP 1 activation studies, murine ZNG1 was amplified using primers Pl 1 and P 12 before being cloned into the Ndel and Bcz/wHI sites in the pHis vector (Sheffield et al., 1999). Mouse Metapl, METAPh.59, and METAPh.79 were cloned into the Ndel and Hindlll sites in the pSUMO vector (Peroutka lii et al., 2011) containing N- terminal 6xHis and SUMO tags using primers P13-P16 (Figure 18) via isothermal assembly (Gibson et al., 2009). The fusion of a 21-residue peptide from murine ZNG1 (ZNGI10-30) onto the N-terminus of the truncated METAPI1.59, used for the NMR structure, was generated by isothermal assembly (Gibson, 2011) of oligonucleotides P17 and P18 (Figure 18) into the TG/i/HI site of the METAP 11.59 construct.

[0069] The full-length zebrafish zngl coding sequence was cloned from 6 dpf whole larval cDNA using primers Pl and P2 into pCRII (Invitrogen) as described above (Figure 18). Full- length zebrafish zngl was subcloned from TOPO pCRII into pLM302 using primer pair P19/P20 (Figure 16) and confirmed via Sanger sequencing. Full-length zebrafish metapl was amplified from 6 dpf whole larval cDNA and cloned into pLM302 using primer pair P21/P22 (Figure 18).

[0070] All constructs were confirmed by Sanger sequencing. Murine 6xHis-SUMO-tagged METAP 11-86 was transformed into A. coli C41(DE3). All other constructs were transformed into E. coli BL21(DE3) for expression.

Recombinant protein expression and purification

[0071] All bacterial expression strains were grown aerobically in Lysogeny Broth (LB) while shaking. Depending on the specific expression vector, media were supplemented with antibiotic to ensure plasmid maintenance, including 50 pg/mL kanamycin, 100 pg/mL ampicillin, or 35 pg/mL chloramphenicol. Bacterial cultures were grown at 37°C to ODeoo = 0.6. Protein expression was initiated with the addition of 0.2-1 mM isopropyl P-D-l- thiogalactopyranoside (IPTG), the temperature was lowered to 18 °C, and the cultures allowed to grow for 14-16 h. Cells were harvested by centrifugation at 4,500x g for 15 min at 4 °C and stored at -20 °C prior to protein isolation.

[0072] For affinity chromatography, size exclusion chromatography, and dynamic light scattering experiments, the protein was isolated as follows. Bacterial pellets were resuspended in 10 mL lysis/wash buffer (20 mM HEPES (pH 7.5), 150 mM NaCl). Lysozyme (Thermo Fisher Scientific, 89833) was added at a final concentration of 1 mg/mL before incubating on ice for 10 min. Cells were lysed by sonication and cellular debris removed by centrifugation (4 °C, 10,000x g, 30 min). The supernatant was loaded on a pre-washed column containing amylose resin (New England Biolabs, E8021). Columns were washed with 10 volumes of lysis/wash buffer before elution of bound protein with lysis/wash buffer containing 10 mM maltose. Protein purity and quantity was assessed by SDS-PAGE and BCA assay (Pierce, 23225 and 23209). To remove the 6His-MBP tag, protein was incubated with HRV 3C protease (Pierce, 88947) for 12-16h (4 °C with agitation). Cleaved tag was removed by loading the mixture on a pre-washed column containing HisPur Cobalt Resin (Thermo Fisher Scientific, 89964). The flow through was collected and protein purity and quantity assessed by SDS-PAGE and BCA assay (Pierce, 23225 and 23209). Purified protein was used immediately or flash frozen and stored at -80 °C.

[0073] For peptide-binding, metal-binding, and METAPl activation assays, ZNG1 and METAP 1 were prepared as follows. Cells expressing untagged ZNG1 were resuspended in 25 mM Tris (pH 8.0), 500 mM NaCl, 2.5 mM EDTA, and 5 mM tris(2- carboxyethyl)phosphine (TCEP). Cells were lysed by sonication for 15 min, and the resulting lysate was centrifuged at 13,000x g for 20 min at 4 °C to remove cellular debris. The resulting supernatant was treated with 0.015% polyethyl enimine on ice to precipitate nucleic acids, which were removed by centrifugation at 13,000x g for 20 min at 4 °C. The supernatant was treated with 40% ammonium sulfate on ice for 30 min to precipitate ZNG1. The mixture was separated by centrifugation at 13,000x g for 20 min at 4 °C, and the resulting pellet containing ZNG1 was resuspended in 25 mM Tris (pH 8.0), 150 mM NaCl, 2.5 mM EDTA, and 5 mM TCEP. This solution was dialyzed into 25 mM Tris (pH 8.0), 50 mM NaCl, 2.5 mM EDTA, and 5 mM TCEP overnight, injected onto a preequilibrated HiTrap Q FF (Cytiva Life Sciences, 17515601), and eluted with a gradient from 50 mM to 1 M NaCl. Fractions of >90% purity were combined for additional purification by size-exclusion chromatography (HiLoad 16/600 Superdex 200 pg, Cytiva Life Sciences 28989335) in 25 mM Tris (pH 8.0), 150 mM NaCl, 2.5 mM EDTA, and 5 mM TCEP. All fractions of >95% purity (by inspection of SDS PAGE gels) were collected and buffer- exchanged 10 ⁶ -fold into chelex-treated (Biorad 141253) 25 mM 4-(2 -hy droxy ethyl)- 1- piperazineethanesulfonic acid (HEPES (pH 7.4)), 150 mM NaCl, and 5 mM TCEP to create the final prep of ZNG1. ZNG1 prepared in this way was devoid of bound metal (<0.01 mol equiv of Zn(II) measured by ICP-MS).

[0074] For SUMO-tagged METAP1 constructs, cells were resuspended in 25 mM Tris (pH 8.0), 500 mM NaCl, and 5 mM TCEP. Cells were lysed by sonication for 15 min, and the resulting lysate was centrifuged at 13,000x g for 20 min at 4 °C to remove cellular debris. The resulting supernatant was injected onto a HisTrap FF (Cytiva Life Sciences, 17525501) column and eluted with a gradient from 0 to 500 mM imidazole. Fractions of >90% purity were combined and treated with SUMO protease for 2 h at room temperature to remove the N- terminal His-SUMO tag and dialyzed into 25 mM Tris (pH 8.0), 500 mM NaCl, and 2 mM TCEP. The cleaved protein product was injected onto a HisTrap FF column and the flow through was collected and concentrated for additional purification by size-exclusion chromatography (HiLoad 16/600 Superdex 200 pg for full-length METAP1 or HiLoad 16/600 Superdex 75 pg for truncated METAP1) in 25 mM Tris (pH 8.0), 150 mM NaCl, 2 mM TCEP. All fractions of >95% purity (by inspection of SDS PAGE gels) were collected and full-length protein was treated with chelator as indicated below. Purified protein was buffer-exchanged 10 ⁶ -fold into chelexed 25 mM HEPES (pH 7.4), 150 mM NaCl, and 2 mM TCEP to create the final prep of MET API, METAPh-59, or MET API 1.79. Notably, we experienced significant decreases in METAP 1 enzymatic activity from frozen protein stocks frozen over extended amounts of time (>1 month) and aliquots that had gone through multiple freeze thaw cycles. Therefore, final preparations of full-length METAP 1 utilized in in vitro experiments in this study were purified, stripped of metal, and aliquoted for -80°C storage all within one day. Moreover, frozen aliquots were thawed no more than twice to ensure maximal METAP 1 enzymatic function. Proteins were analyzed by ICP-MS to identify metal stoichiometries (Figure S2F). To remove excess metal, METAP 1 was treated with either 2 mM EDTA, 10 mM NT A, or 4 molar equivalents N,N,N',N- terakis-(2-pyridylmethyl)ethylenediamine (TPEN) for 1 hour at 4°C and then buffer-exchanged 10 ⁶ -fold into chelexed 25 mM HEPES (pH 7.4), 150 mM NaCl, and 2 mM TCEP. To load Zn(II) into METAPli-59 and METAPI1.79, 50 pM Zn(II) and 1 mM NTA were added to protein solutions for 1 hour at 4 °C and then buffer-exchanged 10 ⁶ -fold into chelexed 25 mM HEPES (pH 7.4), 150 mM NaCl, and 2 mM TCEP. Molar extinction coefficients at 280 nm (35410 M’ ¹ cm’ ¹ for ZNG1, 55350 M’ ¹ cm’ ¹ for METAP1, 6990 M’ ¹ cm’ ¹ for METAP1.59, and 17990 M’ ¹ cm’ ¹ for METAP 11.79) were used to calculate protein concentration.

[0075] Uniformly ¹⁵ N- and ¹⁵ N ¹³ C-labeled METAP I ¹ ’ ⁵⁹ and the ZNGI10.30-METAPI 1.59 fusion were prepared for NMR experiments using the SUMO tagged constructs as described above, except for the following details: The expression strains were grown in M9 minimal media with 1 g/L ¹⁵ N ammonium chloride (Cambridge Isotope Laboratories) as the sole nitrogen source and either 4 g/L unlabeled glucose or 2 g/L ¹³ C-glucose (Cambridge Isotope Laboratories) as the sole carbon source. 10 % ¹³ C-labeled protein was prepared using 1 g ¹⁵ NH4C1, 0.4 g ¹³ Cs - glucose, and 3.6 g unlabeled glucose. The lysis buffer was supplemented with EDTA-free protease inhibitor cocktail (Roche). Nickel-loaded HisTrap FF columns were used to isolate the protein from clarified lysate and after cleavage of the SUMO tag. To ensure that the metal binding sites were homogeneously occupied by Zn(II), the protein was exchanged 10 ³ -fold into Zn-loading buffer (25 mM HEPES, 150 mM NaCl, pH 7.4, treated with chelex and supplemented with 1 mM nitrilotriacetic acid [NTA], 50 M Zn(II), and 2 mM TCEP) using an Amicon ultra centrifugal filter unit (3 kDa cut-off), prior to 10 ³ -fold exchange into NMR buffer (chelex -treated 10 mM sodium phosphate, 150 mM NaCl, pH 7.0, supplemented with 2 mM TCEP). These samples were confirmed by ICP-MS to contain 2.0 equivalents of Zn(II) and less than 0.01 equivalent of Ni(II).

Affinity Chromatography to determine ZNG1-METAP1 interaction

[0076] To probe the interaction of the full-length proteins, His-tagged-MBP-ZNGl (Mouse) was isolated as described above. Protein was loaded onto a previously equilibrated Ni-NTA column (Cytiva Lifesciences, 17-5248-05). The column was washed three times with five column volumes of lysis/wash buffer (10 mM Tris (pH 8) 150 mM NaCl). Following the washes, purified METAP 1 with the tag cleaved was loaded onto the column and washed three additional times. After washing, the complex was eluted using wash buffer containing 500 mM imidazole. Fractions were visualized on a 4-20% Bis-Tris SDS-PAGE gel. Western Blot (methods described below) using a polyclonal METAP1 antibody (PA5-58202, 1:1,000) was performed to confirm identity of METAP 1.

[0077] To test the interaction of zebrafish Zngl and Metapl, His-tagged-MBP-Metapl was isolated as described above, cleaved with HRV 3C protease (Pierce, 88947) passed through a HisPur Cobalt Resin (Thermo Scientific, 89964) column, and dialyzed in lysis/wash buffer overnight at 4 °C. Subsequently, His-tagged-MBP-Zngl was purified as described above with the following exception. Following flow through and washing of the amylose resin (New England Biolabs, E8021), purified Metapl was passed over the column containing immobilized MBP-Zngl or empty resin as control. Columns were subsequently washed with lOOmL of lysis/wash buffer prior to elution and fractions collected. Western blot detection of Zngl (anti- CBWD1, HPA042813) of wash and elution fractions was performed using methods described

15 below. Densitometry of Zngl signal in each fraction was measured using Fiji software (Schindelin et al., 2012).

Size Exclusion Chromatography to determine ZNG1-METAP1 interaction

[0078] The interaction of full-length murine ZNG1 with METAP 11-86 was performed by size exclusion chromatography. Recombinant proteins were purified using methods described above and incubated for 1 h at 4 °C followed by passing over a Superdex 200 increase 10/300 GL column (Cytiva Lifesciences, 28990944). The buffer contained 50 mM Tris (pH 8) and 300 mM NaCl and the experiment was performed with a flow rate of 0.5 mL/min. The resulting fractions were separated and visualized by SDS-PAGE.

[0079] Immunoblot detection ofZNGl and METAPl

[0080] For protein isolation, WT and Zngl mutant TKPTS cells were washed once with warm PBS, trypsinized as described above, and transferred into fresh conical tubes. Samples were centrifuged at 200x g for 10 min. Cells were subsequently washed once with PBS and resuspended in 500pl of cold RIPA buffer (Thermo Fisher Scientific, 89900) supplemented with Halt protease inhibitor (Thermo Fisher Scientific, 78442). Cells were subsequently vortexed for 10 sec and incubated on ice for 20 min prior to centrifugation for 15 min at 15,000x g. Supernatant was transferred into fresh Eppendorf tubes and protein concentration was measured by BCA assay (Thermo Fisher Scientific, 23225). Samples normalized for protein input were run on SDS PAGE gels in IX Tris/Glycine/SDS Buffer (Bio-Rad, 1610732) and transferred using a semi -dry method using Bio-Rad trans blot turbo (25V, 1A, 18 min) onto a nitrocellulose membrane (Li -COR, 926-31092). Transfer efficiency was evaluated using staining with Ponceau S solution (Sigma P7170) following manufacturer’s protocol. Membranes were washed three times in PBS supplemented with 0.2% tween (PBS-T), rocking for 5 min at room temperature. Blocking was performed using Odyssey (PBS) blocking buffer (Li-COR 927-40000) for 1 h at room temperature and subsequently washed three times with PBS-T. Primary antibodies were applied (anti-CBWDl, Sigma, HP A042813, 1 :500 dilution; anti-Tubulin, Cell signaling, 3873S, 1 :1,000 dilution; anti-METAPl, Thermo Fisher Scientific PA5-58202, 1 :1,000) in Odyssey blocking buffer and incubated overnight at 4 °C rocking. Membranes were washed three times with PBS-T for 5 min at room temperature. Secondary antibody solutions (Li-COR), species specific IR800CW or IR680LT, 1:2,000) were applied in Odyssey (PBS) blocking buffer, and membranes were incubated in the dark for 2 h rocking at room temperature. Following secondary antibody staining, membranes were washed three times with PBS-T and visualized using a ChemiDoc MP Imaging System with appropriate channels for detection.

Murine METAP1 methionine aminopeptidase assay

[0081] Methionine aminopeptidase activity was evaluated by quantifying the release of Met from the heptapeptide MAHAIHY (Genscript) as adapted from (Kabir-ud-Din, 2003). Reactions were conducted at 25°C in 25 mM HEPES pH 7.4, 150 mM NaCl, 2 mM TCEP and consisted of 20 nM murine METAP 1 with 0, 1, or 2 molar equivalents of Zn. Reactions were initiated with the addition of varying concentrations of MAHAIHY peptide. 50 pL aliquots were taken every 30 sec from 30-150 sec and the reaction was quenched with the addition of 10 pL glacial acetic acid (Supelco, Sigma-Aldrich). 60 pL of 4% w/v ninhydrin (Sigma- Aldrich) in 100% ACN was added to each quenched reaction and incubated at 37 °C for 1 h to detect free Met. The absorbance at 570 nm was measured and the cleaved Met concentration was determined from a standard curve of 10-500 pM Met. All data are reported as the average of triplicate measurements from each evaluated experimental condition. Midpoint and completed reactions were additionally analyzed by LC-ESI-MS to confirm the generation of the expected AHAIHY cleaved peptide product (Figure 11 I).

Murine ZNG1 activation ofMET Pl assay

[0082] Activation assays were performed in 25 mM HEPES pH 7.4, 150 mM KC1, 2 mM MgC12, 2 mM TCEP. 250 pM NTA was included where indicated. 10 pM Zn2METAPl was incubated with 25 pM apo or ZnZNGl (ZNG1 loaded with 0.9 eq, 22.5 pM, Zn) at 37 °C for 1 h. Reactions were diluted 500-fold in 25 mM HEPES pH 7.4, 150 mM NaCl, 2 mM TCEP to a final concentration of 20 nM METAP 1 and methionine aminopeptidase activity was assayed as described above by initiating the enzymatic reaction with addition of 750 pM MAHAIHY. When using GTP, GDP, or GDPNP, the activation reaction contained 500 pM of the indicated nucleotide. A control activation reaction of Zn-NTA contained 22.5 pM and 250 pM NTA in reaction buffer.

Zebrafish Metapl methionine aminopeptidase activity [0083] A single endpoint assay was used for these experiments. Reaction buffer (20 mM HEPES (pH 7.5), 150 mM NaCl) was chelex treated overnight at 4 °C. 1 pM solutions of zebrafish Metapl and Zngl were made in reaction buffer. To strip Zn from the METAP1 active site, 3 pM of TPEN was incubated with IpM METAP1 at 4 °C for 1 h. To prepare holo-Zngl (Zn bound), Zngl was incubated with 10 pM of ZnCh for 1 h at 4 °C. As a control, Metapl was incubated with reaction buffer alone. Following the 1 h incubation, Zngl and Metapl were washed four times with chel ex-treated reaction buffer using protein concentration columns (Amicon Ultra, UFC500396). After washes, proteins were mixed and incubated at 37 °C for 30 min. Following incubation, 100 pM of the synthetic peptide MAHAIHY (Genscript) was added to reactions and incubated for 90 min at 37 °C. Reactions were stopped by the addition of a final concentration of 2% trifluoroacetic acid and samples were centrifuged at max speed for 5 min. Clear supernatant was transferred into fresh glass vials for HPLC analysis.

[0084] Twenty pL of each sample and standard was analyzed on an Agilent 1260 Infinity II system. Analytes were separated by gradient HPLC on a Supelco Ascends Express Cis column (50 x 2.1 mm, 5 pm) with a Phenomenex SecurityGuard Cis cartridge (3.2 x 8 mm) at a flow rate of 0.4 mL/min using 0.1 % trifluoroacetic acid in water and 0.1% trifluoroacetic acid in acetonitrile as the A and B mobile phases, respectively. The gradient was held at 0% B for 2 min, then ramped to 100% B over the next 18 min. The column was washed at 100% B for 5 min, then equilibrated to 0% B for 5 min. MAHAIHY and AHAIHY were detected using absorbance at 280 nm, and retention times were confirmed using each standard. Methionine aminopeptidase relative activity was determined by calculating the percent of MAHAIHY converted to AHAIHY using the AUC values at 280 nm for each peptide.

Dynamic Light Scattering

[0085] Purified and tag-free murine ZNG1 and METAP 1 were incubated for 1 h at 4 °C before the experiments. The individual proteins and the mixture were filtered through 0.1 pm syringe filters to remove dust and trace residual lipid vesicles. Experiments were performed at 25 °C and data were analyzed using Dynamics 7.5 software (Wyatt Technologies). The DLS software calculates an apparent hydrodynamic radius (Rh) via the Stokes-Einstein equation (Stetefeld et al., 2016). Mag-fura-2 Zn(TT) Competition Assays

[0086] Experiments were performed using an HP8453 UV-vis spectrophotometer or an ISS PCI spectrofluorometer at 25.0 °C. Apo-mf2 (Invitrogen) was monitored by measuring the absorbance at 366 nm or by fluorescence emission at 505 nm upon excitation at 366 nm. Zn-mf2 was monitored by measuring the absorbance at 324 nm or the emission at 505 nm upon excitation at 324 nm. Several [protein]/[mf2] values were used for replicate experiments and analyzed globally to better calculate the stoichiometry. In a typical fluorescence experiment, 1 pM protein and 1 pM mf2 in 2.5 mL were incubated for 10 min in chelexed buffer [25 mM HEPES (pH 7.4), 150 mM NaCl, and 2.5 mM TCEP], and aliquots of Zn(II) were added. The equilibrium time was 2 min between subsequent additions of metal. The peak intensities at 324 and 366 nm from triplicate experiments were globally fit to 1 : 1, 2: 1, 3: 1, and 4: 1 binding models, with theXzn value of mf2 fixed to 5 * 10 ⁸ M ^-1 using Dynafit (Kuzmic, 1996). For ZNG1, the 3:1 binding model provided the best fit of the mf2 data alone, whereas the 4:1 binding model provided the best fit of the METAP1 mf2 data. The selected 4:1 binding model was subsequently used for a simultaneous global fit of triplicate mf2 data sets along with triplicate quin2 datasets for METAP 1.

Quin-2 Zn(II) Competition Assays

[0087] Experiments were performed using an ISS PCI spectrofluorometer at 25.0 °C by monitoring the fluorescence emission of quin2 at 490 nm upon excitation at 353 nm, with quenching of the 490 nm emission maximum as aliquots of Zn(IT) were added. Experiments were performed in triplicate at multiple protein:quin2 ratios. In a typical experiment, 1-2 pM protein and 1-2 pM quin-2 (Sigma 08520) were incubated for 10 min in chelexed buffer [25 mM HEPES (pH 7.4), 150 mM NaCl, and 2.5 mM TCEP] at a total volume of 3 mL, and 0.3 pM aliquots of Zn(II) were added. The equilibrium time was 12 min between subsequent additions of metal. The peak emission intensities at 490 nm from triplicate experiments were globally fit along with triplicate mf2 datasets using Dynafit (Kuzmic, 1996), with the Kz _a value of quin2 fixed to 2 x io ⁿ M ^_1 .

Intrinsic Trp Fluorescence Quenching Assays

[0088] Peptide binding experiments were performed using an ISS PCI spectrofluorometer at 25.0 °C. Intrinsic Trp fluorescence was monitored using = 292 nm and km = 350 nm, with quenching of the 350 nm emission maxima as aliquots of ZNG1 peptide were added. Different [METAP 11-59/1-79] values were used for replicate experiments to better calculate the binding stoichiometry and analyzed globally. In a typical experiment, 5 pM METAP 11.59/1-79 in 2.5 mL were incubated for 10 min in chelexed buffer [25 mM HEPES (pH 7.4), 150 mM NaCl, and 2.5 mM TCEP], and aliquots of ZNG1 peptide were added. The equilibrium time was 2 min between subsequent additions of ZNG1 peptide. The peak emission intensities at 350 nm from triplicate experiments were globally fit to a 1 : 1 binding model using Dynafit (Kuzmic, 1996).

[0089] EDTA titrations of METAP 11-56 were performed using in a Synergy Neo2 plate reader to monitor Trp fluorescence using X _ex = 292 nm and = 350 nm for a series of EDTA (Alfa Aesar A107130B) concentrations ranging from 0.3 pM to 5 mM, for incubation times ranging from 10 min to 6 h at 25 °C. Intensities from triplicate experiments were globally fit to a 2:1 Zn:METAPli-56 binding model using DynaFit to give an upper bound estimate of the affinity (Kuzmic, 1996) with the Kzn value of EDTA fixed to 7.6 x 10 ¹² M ^-1 .

ZNG1 Peptide Synthesis

[0090] The peptides were prepared as C-terminal amides and acetylated at the N-terminus by Fmoc solid-phase synthesis on Chem matrix Rink-Amide resin and purified by reverse-phase HPLC. Peptides were dried and resuspended in water. Calculated molar extinction coefficients at 214 nm were used to determine peptide concentration.

NMR

Assignments

[0091] NMR samples contained 0.5-2 mM uniformly ¹⁵ N- and ¹⁵ N ¹³ C-labeled protein, with 10 % v/v D2O and 0.3 mM 2,2-dimethyl-2-silapentanesulfonic acid (DSS) as an internal reference. An equivalent 2 mM, ¹⁵ N ¹³ C-labeled 100% D2O sample was prepared by lyophilization of a 100% H2O sample, followed by resuspension in D2O (Cambridge Isotope Laboratories). NMR spectra were recorded at 25 °C on a Bruker Avance Neo 600 MHz spectrometer equipped with a cryogenic probe or on a Varian VNMRS 800 MHz spectrometer with a room temperature 5 mm PFG HCN probe in the METACyt Biomolecular NMR Laboratory at Indiana University, Bloomington. Data were collected using Topspin 4.0.9 (Bruker) or VNMRJ 4.2 (Varian), processed using NMRPipe (Delaglio et al., 1995), and analyzed using CARA (http://cara.nmr.ch/doku.php) and NMRF AM-Sparky (Lee et al., 2015), all on NMRbox (Maciejewski et al., 2017).

[0092] Backbone chemical shifts were assigned for each state using the following standard triple-resonance experiments: HNcaCB, CBCAcoNH, and HNCO (Sattler, 1999). Chemical shift assignments were not obtained for the C’ of K59 or C22 in the free or bound states because an HNCO does not include resonances for C’ in terminal residues or in residues preceding proline. An HNcaCO of the fusion construct provided these chemical shifts as well as those of C18 and V22 in the ZNG1 peptide, which also precede proline residues. With one proline in the zf-C6H2 domain and two proline residues in the peptide, 100% of all possible amide 'H, ¹⁵ N atoms are assigned in all three states. 97% of all possible C’ atoms are assigned in the free and bound forms, and 100% in the fusion. 100% of all possible Ca and Cb atoms are assigned in the free domain and fusion, but 98% of all Ca and Cb atoms in the bound form, without assignments for C22 due to chemical exchange broadening. The backbone assignments of METAPh-59 in the absence of peptide are consistent with and expand upon those previously published for human METAPli-83 (Rachineni et al., 2015).

[0093] Aliphatic side-chain assignments were obtained using HBHAcoNH, HcccoNH- TOCSY, hCccoNH-TOCSY, HcCH-TOCSY, hCCH-TOCSY experiments (Sattler, 1999), while hbCBcgcdHD, hbCBcgcdceHE, and H ^ar (CC-TOCSY-CGCBCACO)NH for were used for aromatic side-chains (Lohr et al., 2007; Yamazaki T, 1993). The 3D experiments were collected using non-uniform sampling with Poisson gap scheduling (Hyberts et al., 2010), and reconstructed with istHMS (Hyberts et al., 2012). Sidechain assignments of the peptide-zf-C6H2 fusion construct are essentially complete, with the assignment of the single methionine methyl group confirmed by a 3D ¹³ C-NOESY-HSQC. Stereospecific assignments of methyl groups for all 3 valine residues and all 5 leucine residues in the fusion construct were obtained using a combination of high-resolution W’C-HSQC and constant-time ^^C-HSQC of a 10% Relabeled sample (Hilty et al., 2003). The chemical shifts and protonation states of the Nd and Ne atoms of the two histidine sidechains were assigned using a ^^N-HSQC experiment optimized for ² JNH transfer (Pelton et al., 1993), demonstrating that the Nd atoms are protonated and the Ne atoms coordinate Zn ⁿ A small number of non-amide exchangeable protons were observed in 3D ¹⁵ N-NOESY-HSQC spectra of the fusion construct, and were attributed to the OHI hydroxyl groups of Ser58, Ser65, and Thr68. Chemical shifts were analyzed for indications of secondary structure using TALOSN (Shen and Bax, 2015). All backbone and side chain chemical shifts have been deposited in the BioMagResBank under accession number 51117 (free), 51118 (bound), and 30956 (fusion) (Ulrich et al., 2008).

Dynamics

[0094] NMR relaxation measurements including ¹⁵ N spin relaxation rates, Ri and Ri, and ¹ H- ¹⁵ N heteronuclear NOE (hNOE) values were recorded in an interleaved manner using standard experiments. The relaxation delays used were 0.02, 0.06, 0.10, 0.20, 0.40, 0.60, 0.80, and 1.2 s for Ai and 0.017, 0.034, 0.051, 0.068, 0.085, 0.102, 0.119, 0.136, 0.153, 0.170, 0.204, and 0.238 s for R2. Residue-specific Ri and R2 values were obtained from fits of peak intensities vs. relaxation time to a single exponential decay function, while hNOE ratios were ascertained directly from intensities in experiments recorded with (2 s relaxation delay followed by 3 s saturation) and without saturation (relaxation delay of 5 s). Errors in hNOE values were calculated by propagating the error from the signal to noise. Backbone relaxation rates have been deposited in the BioMagResBank under accession numbers 51117 (free) and 51119 (fusion).

Structure Determination

[0095] Initial structure calculations were performed with CYANA using automatically assigned NOESY peak lists with no further restraints, revealing the expected cross-brace zinc chelation topology (Guntert, 2004). Zn coordination geometry was defined and maintained for subsequent calculations using distance restraints (Zn-Sg = 2.3 A and Zn-Ne2 = 1.95 A). Hydrogen bond restraints were added where supported by NOEs, chemical shift-based secondary structure predictions, and slow rates of hydrogen-deuterium exchange. The structure has been deposited into the Protein Data Bank with accession ID 7SEK.

GTPase activity measurements (malachite green assay)

[0096] Reactions were run in 25 mM HEPES pH 7.4, 150 mM KC1, 2 mM MgCh, 2 mM TCEP. 20 pL reactions of 5 pM ZNG1 in the presence or absence of 5 pM Zn and 5 pM Zn METAPl were initiated with the addition of 500 pM GTP (Sigma 10106399001) and incubated at 37°C for 90 min. GTPase reactions were quenched and inorganic phosphate was detected with the addition of 120 pL of 1 mM malachite green oxalate (Acros Organics), 10 mM ammonium molybdate (Sigma-Aldrich) in 1 M HC1. This reaction was incubated in the dark at ambient temperature for 10 min and then quenched with the addition of 60 pL 35% citric acid. Mixtures were incubated for 10 min and inorganic phosphate concentration was calculated based on the absorbance at 680 nm relative to a standard curve (5-500 pM phosphate, Fluka Analytical).

GTPase activity measurements (coupled assay)

[0097] GTPase activity was measured following a previously described protocol (Ingerman and Nunnari, 2005). Reactions were run in 25 mM HEPES pH 7.4, 150 mM KC1, 2 mM MgCh, 2 mM TCEP in a total volume of 100 pL and included 4 pM ZNG1, 500 pM GTP (Sigma 10106399001), 260 pM NADH (Sigma N8129), 2 mM phosphoenolpyruvate (Sigma 860077), and 1 pL pre-mixed Pyruvate Kinase / Lactate Dehydrogenase (Sigma P0294) at 37 °C. Reactions were monitored in real time by a decrease in absorbance at 340 nm in a Synergy Neo2 plate reader.

Zebrafish mutagenesis

[0098] Targeted deletion of the zngl and metapl genes were performed using CRISPR/Cas9 gene editing targeting the second exon of zngl and first exon of metapl as described elsewhere (Essner, 2016). Briefly, the guide RNA sequences 5’ GACCCACAGCTCAGATCC 3’, targeting zebrafish zngl and 5’ AGGTGGGACACTGGAGCT 3’ targeting metapl were designed using CRISPRscan (Moreno-Mateos et al., 2015) and were synthesized using the oligo-based method (Yin et al., 2015) (P23-P25 Table S5). Cas9 mRNA was generated from Xbal (New England Biolabs) digested pT3TS-nls-zCas9-nls plasmid (Addgene, 46757), and in vitro transcribed using mMESSAGE mMACHINE T3 kit (Thermo Fisher Scientific, AM1348) (Jao et al., 2013). A cocktail consisting of 150 ng/pL of nls-zCas9-nls and 120 ng/pL of gRNA, 0.05% phenol red, 120 mM KC1, and 20 mM HEPES (pH 7.0) was prepared, and approximately 1-2 nL was injected directly into the cell of one cell stage Tubingen zebrafish embryos. Mutagenesis was initially screened using Melt Doctor High Resolution Melting Assay (Thermo Fisher Scientific, 4409535), and identified two independent zngl alleles which were confirmed as deletions by Sanger sequencing of TOPO-cloned PCR products. Subsequent screening of the Al 1 (allele designation vz/2) was performed by PCR (primers P26/P27, Figure 18) and products were resolved on 2% agarose Tris-Borate-EDTA (TBE) gels. Screening of the A5 allele (allele designation vul') was performed by PCR amplification using primers P26 and P27 (Figure 18), followed by purification and Dpnl digest (New England Biolabs). All zngl mutant alleles result in the loss of a single Dpnll restriction site present in the WT sequence. Sanger sequencing identified a 17 base pair deletion in the metapl first exon (allele designation vu3) which was genotyped by PCR using P28/P29 (Table S5) and resolved on 2% agarose Tris-Borate-EDTA (TBE) gels.

Zebrafish pharmacological manipulations

[0099] For all experiments larval zebrafish were bred naturally and collected into embryo media and were maintained at 28 °C on a 14/10 h light/dark cycle for duration of experiments. For survival experiments, zebrafish larvae were split into even densities into 6 or 12 multi -well plates in 5 mL or 1 mL embryo media respectively at 3 dpf. Stock solutions of METAP2 inhibitor TNP-470 (Sigma, T1455) and METAP1/2 inhibitor Bengamide B (Santa cruz, sc- 397521A) were prepared in ACS-grade DMSO (Fisher Scientific, D136) and TPEN (Sigma, P4413) was prepared in ethanol and stored at -20 °C. TNP-470, Bengamide B, and/or TPEN were applied into embryo media as well as a DMSO and/or ethanol vehicle controls at 3 dpf. Zebrafish survival was monitored daily until 6 dpf and all moribund or dead animals were removed. For qPCR experiments, larvae were split at 3 dpf into groups of 20-30 larvae in 20 mL of embryo media supplemented either 20 pM of TPEN or ethanol vehicle control. For imaging experiments, 6 dpf zebrafish larvae treated with 500 nM to 1 pM of Bengamide B or vehicle control were imaged by a Leica Thunder M205FCA stereomicroscope with a Leica DFC9000 GT camera.

Mouse mutagenesis

[00100] Generation of Zngl' ' mice. Zngl' ¹ ' mice were produced using CRISPR-Cas9 genome editing in a C57B1/6J strain (The Jackson Laboratory) through the Vanderbilt Genome Editing Resource (Vanderbilt University). Ribonucleoprotein complexes containing crRNA + tracrRNA (ctRNA) and SpCas9 protein (MilliporeSigma) targeting DNA sequences within Zngl exon 1 and a single stranded DNA donor containing an EcoRT restriction site were delivered by pronuclear injection into 1-cell C57BL/6J embryos. crRNA sequences were crRNA: 5’TGGAGTCACTGGTGTCCTGC crRNA: 5’CCGGGTACTTAGGTAATTAA* and 180 nucleotide ssODN donor sequence: ctagaagagaggccccagcccctacctcgaagattcgtcctcggccaggaggcccgggtg cctgaggagacacggcagcccccctta GAATTC ggacaccagtgactccagaactaaagagctgccgccagtcgcgtttccgcgacaggattc agtcggcgcggggctgatg atgtcacc. FO founder animals were screened for precise Zngl deletions by standard PCR and restriction fragment length polymorphism assays. Deletions were confirmed by Sanger sequencing and verified by a second round of Sanger sequencing in the heterozygous N1 generation. N1 generation Zngl' ' heterozygous mice containing the desired mutation were backcrossed at least three generations into the C57B1/6J strain to allow for segregation of potential off-target editing events prior to familial breeding to produce homozygous offspring. Genotyping of mice from the Zngl mutant breeding colony was performed by Transnetyx using ear punches of individual animals and primer pair P30/P31 (Table S5). Zngl' ' animals will be cryopreserved and are available through the Vanderbilt Cryopreserved Mouse Repository.

Cell line mutagenesis

[00101] Targeted deletion of the first exon of murine Zngl was performed using the guide RNA ‘ATTCCAGTCACAATTGTCAC’. Clonal selection of mutant cells was performed by dilution plating. Individual clonal populations of cells were subsequently screened using primers P32 and P33 (Figure 18) followed by Sanger sequencing of TOPO-cloned PCR products. Two independent mutant clones were identified and harbored a homozygous A2/A2 base pair deletion (clone 1) and a A7/A2 base pair deletion (clone 2).

Mass spectrometry proteomics of murine tissue and cell lines

[00102] Kidneys were isolated from 11-13-week old C57BL/6 or C57BL/6 Zngl' ' mice that were fed a low Zn diet for 5 weeks. Animals were humanely euthanized, kidneys isolated, and flash frozen in liquid nitrogen. For protein isolation, frozen organs were gently thawed on ice and transferred through a 70 pM sterile cell strainers (Thermo Fisher Scientific, 22-363-548) into 5 mL of ice-cold PBS supplemented with Halt protease inhibitor (Thermo Fisher Scientific, 78430). Filters were flushed an additional two times with PBS with protease inhibitor. Cells were pelleted (4 °C, 400x g, 10 min) and pellets resuspended in 500 pL of RIP A buffer with Halt protease inhibitor (Thermo Fisher Scientific, 78430). Following rigorous vortexing, incubation on ice (10 min), and a second round of vortexing, cellular debris were pelleted by centrifugation (4 °C, 10,000x g, 10 min) and supernatant transferred into fresh tubes for further analysis.

[00103] Protein concentrations were determined using the 660 Protein Assay (Pierce,

22660). Equal amounts of protein (200 pg) were processed for LC-MS/MS using s-traps (Protifi) (HaileMariam et al., 2018; Zougman et al., 2014). Briefly, proteins were reduced with dithiothreitol (DTT), alkylated with iodoacetamide (IAA), acidified using phosphoric acid, and combined with s-trap loading buffer (90% MeOH, lOOmM TEAB). Proteins were loaded onto s- traps, washed, and finally digested with Trypsin/Lys-C (1 :100, w:w; enzyme:protein) overnight at 37 °C. Peptides were eluted and dried with a vacuum concentrator. Peptides were resuspended in H2O/1% acetonitrile/0.1% formic acid for LC-MS/MS analysis.

[00104] Peptides were separated using a 75 pm x 50 cm C18 reversed-phase-HPLC column (Thermo Fisher Scientific, 164570) on an Ultimate 3000 UHPLC (Thermo Fisher Scientific) with a 120 min gradient (2-32% ACN with 0.1% formic acid) and analyzed on a hybrid quadrupole-Orbitrap instrument (Q Exactive Plus, Thermo Fisher Scientific). Full MS survey scans were acquired at 70,000 resolution. The top 10 most abundant ions were selected for MS/MS analysis.

[00105] Raw data files were processed in MaxQuant (v 1.6.14.0, www.maxquant.org) and searched against the current UniProt Mus musculus protein sequences database. Search parameters included constant modification of cysteine by carbamidomethylation and the variable modifications, methionine oxidation and protein N-term acetylation. Proteins were identified using the filtering criteria of 1% protein and peptide false discovery rate. Protein intensity values were normalized using the MaxQuant LFQ function (Cox et al., 2014).

[00106] Label free quantitation analysis was performed using Perseus (v 1.6.14.0), software developed for the analysis of -omics data (Tyanova et al., 2016). LFQ Intensity values were Log2 -transformed, and then filtered to include proteins containing at least 60% valid values (reported LFQ intensities) in at least one experimental group. Finally, the missing values in the filtered dataset were replaced using the imputation function in Perseus with default parameters (Tyanova et al., 2016). Statistical analyses were carried out using the filtered and imputed protein groups file. Statistically significant changes in protein abundance are determined using Welch’s t-test P-values and z-scores.

Mass Spectrometry Proteomic Quantification of Zngl Peptides

[00107] Murine brain tissue was homogenized in NP40 buffer (50 mM trizma base, 150 mM sodium chloride, 1 mM EDTA, and 1% nonidet 40) using cycles of sonication. Protein concentration was quantified using a Bicinchoninic Acid assay (Sigma Aldrich, QPBCA). For each sample, 10 pg of protein was suspended in 8 M urea containing 50mM ammonium bicarbonate to a final volume of 90 pL. Proteins were reduced using a final concentration of 20 mM dithiothreitol (DTT, Thermo Scientific A39255) at 37 °C for 45 min, followed by alkylation using 20 mM iodoacetamide (IAA, Thermo Scientific A39271) at room temperature for 15 min. Samples were diluted to reduce the concentration of urea to <1 M using 50 mM ammonium bicarbonate and proteins were digested using 1 pg of LysN (Thermo Scientific 90300) at 50 °C for 2 h. Oasis mixed-mode cation exchange (MCX) solid phase extraction cartridges (Waters Corporation, Milford, MA 186000252) were used to remove lipids and other contaminants from the digested peptides. Peptide samples were dried using a centrifugal evaporator.

[00108] Dried digested samples were reconstituted in HPLC-grade water containing 0.1% formic acid by vortexing and centrifuged briefly to remove any particulates. A total of 250 ng of each sample was desalted and concentrated using Cl 8 EvoTips (EvoSep EV2001). Liquid chromatography mass spectrometry was performed using an EvoSepOne (EvoSep) coupled to an Orbitrap Fusion (ThermoFisher Scientific) equipped with a Nanospray Flex Ion source. Chromatography was performed using a 15 cm long column containing ReproSil-Pur C18 1.9 pm beads by Dr Maisch C18 and a fused silica emitter tip (20 pm inner diameter) (EvoSep EVI 112, EVI 111). Peptides were eluted from the column using an EvoSep 20 samples/day Whisper method. Mass spectrometry was performed on an Orbitrap Fusion (ThermoFisher Scientific) equipped with a Nanospray Flex Ion source. MSI was performed using Orbitrap detection at a resolution of 60,000 with a standard AGC target. Data independent acquisition was performed using quadropole isolation with nonoverlapping windows of 16 m/z from precursor masses 400-1008 with HCD fragmentation (33% collision energy) and Orbitrap detection with a resolution of 30,000. The normalized peak areas for Zngl peptides were calculated using Skyline (MacLean et al., 2010).

Gene Expression Analysis

[00109] For RNA isolation from dissected mouse tissue, mice were humanely euthanized, tissues harvested and immediately flash frozen in liquid nitrogen and stored at -80C. Mouse tissues were transferred into Lysing matrix B tubes (MP, 6911100) containing 700 pl of RLT buffer (Qiagen, 74104) with 1% (v/v) B-mercaptoethanol. Samples were homogenized by bead beating 6 times for 40 s at 6 m/s. Tubes were kept on ice for 1 min in between steps. Samples were centrifuged and homogenate was added to a fresh tube containing 600 l of phenol:chloroform:iAA (Fisher Scientific, BP17521-100) then vortexed for 30 s. Samples were spun at max speed for 15 minutes and the aqueous phase was transferred to a tube containing 700pl of 100% ethanol. Subsequent isolation steps were performed using the Qiagen RNeasy Kit following manufacturer's protocol (Qiagen). gDNA was depleted from final RNA samples by treatment with TURBO DNA-free Kit (Invitrogen). For RNA isolation from zebrafish, pools of 20-30 whole larvae were collected into 1 mL of TRIzol (Thermo Fisher Scientific, 15596026) and stored at -80 °C. For TKPTS cell lines, cells were washed with PBS and collected with 500 pL TRIzol from multi-well plates and stored at -80 °C. Zebrafish larvae and cell lines were homogenized by passing samples 10-15 times through a 27-gauge needle. RNA was isolated following the manufacturer’s protocol with the following modification: a second wash with 70% ethanol (prepared in DEPC-treated H2O) was performed. RNA was treated with DNasel following manufacturer’s instructions (New England Biolabs, M0303L). cDNA from RNA isolated by any of the methods described above was synthesized using the iScript kit (Bio-Rad, 1708891). Quantitative PCR was performed in duplicate-quadruplicate 25 pL reactions using 2X iQ SYBR Green SuperMix (BioRad, 1708882) run on an BioRad CFX96 Real-Time System instrument using gene-specific primers (zebrafish:P34-P39, mice: P40-P43, cell lines: 44-69, Table S5). Data were analyzed with the AACt method (Livak and Schmittgen, 2001) or presented as relative expression values.

Transfection of Zngl mutant cells

[00110] To generate linearized DNA for transfection, pcDNA3. l-C-(k)DYK-Zn 7 _mwr;W e was digested with Seal. Cells were grown to 80-90% confluency in 6-well TC treated plates and transfected with lipof ectamine reagent using manufacturer’s protocol (Lipofectamine 3000, Invitrogen L3000-008). Briefly, 2.5 pg of linear DNA was mixed with P3000 reagent and opti- MEM media (Gibco, 11058-021) to prepare the DNA solution. DNA solution and lipofectamine reagent solutions were mixed at a 1 : 1 ratio and incubated for 15 min at room temperature. DNA- lipid complexes were added to cells dropwise and cells were incubated overnight at 37 °C at 5% CO2. G418 (RPI G64500-10) was added at a concentration of 300 pg/ml in complete DMEM/F12 50/50 complete cell culture media described above. G418 resistant cells were diluted and clonally expanded to identify clonal populations of transfected cells. Expression of full-length ZNG1 was validated using Western blot against ZNGL

Immunofluorescence

[00111] WT and Zngl mutant TKPTS cells were seeded onto sterile coverslips in 6-well tissue culture treated multi-well plates. At 60-80% confluency, cells were washed three times with warm phenol red free DMEM (Thermo Fisher Scientific, 21041025) prior to fixation with 4% paraformaldehyde solution (PF A, Alfa aesar, J61899) for 15 min at 37 °C then washed three times for 5 min with PBS. Cells were subsequently permeabilized by incubation in 0.2% Triton X-100 in PBS for 10 min at room temperature. Following permeabilization, cells were washed three times for 5 min with PBS at room temperature. Cells were blocked using Odyssey (PBS) blocking buffer (LI-COR) for 1 h at room temperature or overnight at 4 °C. Primary antibodies were diluted in Odyssey blocking solution and incubated overnight at 4 °C rocking (anti-OxPhos Complex V inhibitor Clone 5E2, Invitrogen A21355, 1:200). Cells were washed three times with PBS for 5 min. Secondary antibodies (Thermo Fisher Scientific) and Hoechst (Invitrogen, H3570) were added to Odyssey blocking buffer (Hoechst, 1 : 1,000, Alexa -conjugated secondaries 1 :2000) and cells were incubated for 2 h rocking at room temperature. Following secondary staining, cells were washed three times with PBS and mounted onto slides with 20 uL of ProLong Gold antifade reagent (Invitrogen, 2273617). Slides cured in the dark overnight at room temperature prior to imaging. Slides were imaged on the Zeiss LSM 880 using the 63X oil objective (1.40 Plan-APOCHROMAT OIL, WD=0.19mm).

Transmission Electron Microscopy

[00112] WT and Zngl mutant TKPTS cells treated with 3 pM TPA (Sigma, 723134) were fixed in 2.5% glutaraldehyde in 0.1 M cacodylate for 1 hour at room temperature followed by 24 hours at 4C. The cells were sequentially postfixed in 1% tannic acid, followed by 1% OsO4, then enblock stained with 1% uranyl acetate. Samples were dehydrated in a graded ethanol series and gradually infiltrated with a Quetol 651 formulation Spurrs resin using propylene oxide as the transition solvent. The resin was polymerized at 60oC for 48 hours. Thin sections were cut using a Leica UC7 ultramicrotome with a nominal thickness of 70 nm and collected onto 300 mesh copper grids. All samples were post-stained with 2% uranyl acetate and lead citrate. [00113] TEM imaging was performed on a Tecnai T12 operating at 100 keV using an AMT nanosprint CMOS camera. Single images were acquired using the AMT Capture Engine software. Images for quantification were acquired using serialEM to acquire montages of entire gridsquares, using identical lens currents between samples. These montages were reconstructed using the IMOD/etomo software suite. Mitochondria size, shape, and electron density quantification was performed using the FIJI ROI manager to manually segmentation all mitochondrial cross-sections within randomly selected cells until at least 100 mitochondria were measured for each treatment.

Flow cytometry measurement of cell proliferation and mitochondrial superoxide

[00114] TKPTS cells were seeding at 2,000 to 4,000 cells per well of 24-or 12-well tissue culture treated multi-well plates and incubated overnight at 37 °C. Cells were staining with CellTRACE violet (Invitrogen, C34557) as described by manufacturer's protocol. Briefly, CellTRACE solution was prepared in PBS at a 1 : 1,000 dilution, added to the cells, and cells were incubated for 20 min at 37 °C. Cells were then washed two times in cell culture media supplemented with 7% FBS, and replaced with fresh cell culture media supplemented with desired chemical treatments (3 pM TP A, Sigma, 723134; 5 pg/ml TNP470, Sigma, T1455; or 50 nM Bengamide B, Santa cruz, sc-397521A). Stock solutions of drugs were prepared in ACS- grade DMSO (Fisher Scientific, D136). For growth in Zn deplete conditions, TPA was chosen because it exhibits lower cytotoxicity towards cell lines compared to other chelators (e.g. TPEN) (Huang et al., 2013; Lo et al., 2020). After three days of growth, cells were stained with MitoTracker far-red (Invitrogen, M22426; 1:2500 dilution for 20 min at 37 °C) and MitoSOX red (Molecular probes, M36008 : 1,1000 for 20 min at 37 °C) following manufacture’s protocols. Cells were washed with PBS, trypsinized, and transferred to Eppendorf tubes prior to fixation in 4% PFA (Alfa aesar, J61899). Cells were fixed for 20 min at room temperature, centrifuged at 200x g for 10 min and washed with PBS prior to resuspension in 300 pl FACS buffer (2% FBS, 0.02% sodium azide in PBS). All flow cytometry data were collected using a BD LSRII flow cytometer with FACSDIVA software and analyzed using FlowJo (FlowJo LLC). Samples were gated forward scatter height (FSC-H) by forward scatter area (FSC-A) and side scatter height (SSC-H) by side scatter area (SSC-A) to remove doublet populations. The singlet population was gated SSC-A by FSC-A to isolate live cells. The resulting cell population was then assessed for assay-specific fluorescent markers and the median fluorescence intensity (MFI) quantified.

Seahorse XF96 Cell Mitochondrial Stress Test

[00115] 8,000 WT or Zngl mutant vehicle treated TKPTS cells or TKPTS cells serially passaged in 3 pM TPA (Sigma, 723134) were plated in individual wells of seahorse plates in complete DMEM F12 media described above. Cells were resuspended in mitochondria assay media (Seahorse Agilent pH 7.4 media + 1 mM pyruvate + 10 mM glucose + 2 mM glutamine). No TKPTS cells were added to the four wells in the corners of the plate as a background control. For the mitochondrial stress test, the cartridge was sequentially loaded with oligomycin (15pM), FCCP (15 pM), and rotenone/antimycin A (5 pM) (diluted in mitochondria assay media) for injection into the wells. To normalize across wells by cell density, the Seahorse plate was imaged using a BioTek Cytation 5. Oxygen consumption and extracellular acidification rates were collected using a Seahorse XFe96 Analyzer and analyzed using Wave Desktop software (Agilent Technologies).

Measuring cellular ATP production

[00116] Cellular ATP levels were measured from WT and Zngl mutant TKPTS cell lysates following manufacturer’s protocols (Sigma, MAKI 13). Briefly, 20,000 cells/well WT and Zngl mutant cells were seeded in clear-bottom black 96-well plates (Costar, 3603) and incubated for 36 h at 37 °C under 5% CO2. ATP levels were measured using Cell Titer Gio 2.0 (Promega, G9241). To normalize to cell number, cells were incubated with Hoechst (1 : 1,000 in PBS) for 15 min at 37 °C in cell culture incubator then washed with PBS prior to the addition of 100 pL of fresh PBS. Hoechst signal was measured in the BioTek Cytation 5 (ex/em 350/461). Subsequently, 100 pL of Cell Titer Gio reagent was applied directly to the wells and incubated in the dark for 15 min at room temperature. Luminescence was measured in the BioTeck Cytation 5 and expressed as relative light units (RLU).

Tetramethylrhodamine methyl ester (TMRM) assay

[00117] WT and Zngl mutant TKPTS cells untreated or treated with 3 pM TPA (Sigma, 723134) were seeded at a density of 4,000 cells / well in clear-bottom black 96-well plates (Costar, 3603) and incubated overnight at 37° C under 5% CO2. Adherent cells were stained with MitoTracker deep red (Invitrogen, M22426) as described above. TMRM (Invitrogen, T668) staining solution was prepared following manufacturer's instructions. Briefly 100 mM TMRM was prepared in phenol red-free cell growth medium and was added to adherent cells at a quenching concentration of 200 nM as described previously (Monteith et al., 2013). Cells were incubated for 1-2 h at 37 °C at 5% CO2. TMRM and MitoTracker fluorescence was measured using a BioTek Cytation 5 (TMRM- ex/em 548 nm/574 nm and MitoTracker- ex/em 644 nm/665 nm). For each genotype and treatment condition, 4-8 independent wells were analyzed and TMRM signal was normalized to MitoTracker signal.

Determination of metal levels by inductively coupled plasma mass spectrometry (ICP-MS) [00118] Mouse tissue homogenate were acid digested in 400 pL of Optima grade nitric acid (Thermo Fisher Scientific, A467-500) and 100 L of Ultratrace hydrogen peroxide (Sigma, 95321) at 65 °C for 48 h. After digestion, the acid content was diluted to below 10 % with 3.5 mL of UltraPure water (Invitrogen, 10977-023) Elemental quantification on acid-digested samples was performed using an Agilent 7700 inductively coupled plasma mass spectrometer (Agilent) attached to a Teledyne CETAC Technologies ASX-560 autosampler (Teledyne CETAC Technologies). The following settings were fixed for the analysis Cell Entrance = -40 V, Cell Exit = -60 V, Plate Bias = -60 V, OctP Bias = -18 V, and collision cell Helium Flow = 4.5 mL/min. Optimal voltages for Extract 2, Omega Bias, Omega Lens, OctP RF, and Deflect were determined empirically before each sample set was analyzed. Element calibration curves were generated using ARISTAR ICP Standard Mix (VWR, BDH82026-108). Samples were introduced by peristaltic pump with 0.5 mm internal diameter tubing through a MicroMist borosilicate glass nebulizer (Agilent). Samples were initially up taken at 0.5 rps for 30 s followed by 30 s at 0.1 rps to stabilize the signal. Samples were analyzed in Spectrum mode at 0.1 rps collecting three points across each peak and performing three replicates of 100 sweeps for each element analyzed. Sampling probe and tubing were rinsed for 20 s at 0.5 rps with 2% nitric acid between every sample. Data were acquired and analyzed using the Agilent Mass Hunter Workstation Software version A.01.02.

Computational tools

[00119] Sequence analysis, alignments and cladograms were created using CLC Genomics Workbench (version 20.0.1.). Visualization of pairwise alignments was generated using Sequence Demarcation Tool (SDTvl.2) (Muhire et al., 2014). Pathway analysis of proteins with significantly altered abundance (as determined by Welch’s /-test) was performed using the Ingenuity Pathway Analysis tool (IP A, Qiagen). Structural multiple sequence alignments were generated using ESPript (Robert and Gouet, 2014). An iterative HMMsearch was used to identify variations in the ZNG1 N-terminal motif (Eddy, 2009). HMMsearch was also used to identify matches to the refined motif in yeast, human, mouse, and zebrafish genomes. Sequence logo motifs were generated using WebLogo 3.7.4 (Crooks et al., 2004). AlphaFold2 modeling of the yeast Maplp-Znglp interaction was performed using the ColabFold AlphaFold2_advanced jupyter notebook with max_recycles set to 48 (Mirdita et al., 2021).

Statistical Methods

[00120] All experiments were repeated at least two times and statistical analyses were performed with GraphPad Prism v.9 or OriginPro. Data are presented as mean ± SEM unless otherwise noted in figure legend. For comparisons between 2 groups a two tailed student’s t-test or Mann-Whitney test was applied. For comparisons between 3 or more groups, a one-way ANOVA with Tukey’s multiple comparisons test was used. For experiments with 2 independent variables, a two-way ANOVA was performed with Tukey’s or Sidak’s multiple comparisons test. Significance was set as P < 0.05, and denoted as: * P < 0.05, ** P < 0.01, *** < 0.001, **** P < 0.0001. Sample sizes are indicated in the figure legends.

Results

ZNGls are ancient COG0523 proteins with conserved metalloprotein interactions.

[00121] To interrogate the role of vertebrate ZNGls in Zn homeostasis, the evolutionary conservation of ZNG1 proteins was evaluated by aligning ZNG1 amino acid sequences from vertebrates spanning an evolutionary distance of over 400 million years (Howe et al., 2021; Kumar et al., 2017). While many vertebrates, including mice and zebrafish, encode for a single ZNG1 protein, a chromosomal duplication event within the primate lineage led to the expansion of the COG0523 gene family to five intact ZNG1 paralogs (ZNG1 A-F with ZNG1DP being a pseudogene) (Wong et al., 2004). There is over 60% sequence conservation across all analyzed ZNG1 proteins (Figure 1 A and SI A). Among the analyzed ZNG1 members, the known catalytic motifs and metal-binding residues of the GTPase domain are uniformly present and conserved (Figure IB) (Edmonds et al., 2021; Haas et al., 2009). Of note, several regions outside of the GTPase domain are also conserved, including the N-terminus (Figure IB). [00122] Despite broad evolutionary conservation, the in vivo functions of COG0523 proteins, including ZNG1, have yet to be determined in part due to a lack of knowledge of interaction partners. To identify potential ZNG1 client proteins, three independent yeast-two- hybrid screens were performed with full-length ZNGls as bait. To assess species-specificity as well as evolutionarily conservation of interactions, the screens were performed using zebrafish Zngl, mouse ZNG1, and human ZNG1E proteins. ZNG1E was chosen as the representative human protein due to its consistently high expression across different tissue types and high similarity to the consensus sequence of the human ZNG1 proteins (Figure SIB, C) (Uhlen et al., 2015). Of note, species-specific nomenclature referring to genes and proteins from the representative backgrounds will be maintained throughout the manuscript (e.g. human: ZNG1E, ZNG1E; mouse: Zngl, ZNG1; zebrafish: zngl, Zngl). For each screen between 9 and 27 putative ZNG1 interacting partners were identified (Figure 1C, Table SI). Importantly, while each dataset revealed species-specific interaction partners, the Zn-finger protein methionine aminopeptidase 1 (METAP1) was identified in each of the three screens. These findings suggest that ZNG1 interacts with both common and distinct proteins across vertebrate species.

[00123] In addition to identifying interaction partners, the yeast-two-hybrid approach also discerned predicted Selected Interacting Domains (SID) of target proteins. SIDs were analyzed to identify the presence of specific protein domains (defined by PF AM or SMART) that are partially or completely contained within each SID (Table SI). To test if ZNG1 supports distinct cellular processes, an enrichment analysis of identified PF AM domains was performed using the dcGO platform (Fang and Gough, 2013). A high prevalence of domains with functions in protein and chromatin binding as well as factors with regulatory roles were identified (Figure ID). Since ZNG1 expression is associated with conditions of Zn limitation (Ogo et al., 2015), we hypothesized that ZNG1 aids in the mobilization of cellular Zn during limitation and that proteins with known Zn-binding domains would be interaction partners enriched in these data sets. Indeed, 12 Zn metalloproteins were identified across all three screens, including proteins involved in transcription, protein processing, and the cell cycle (Figure IE). For most of these proteins, the predicted site of interaction with ZNG1 harbors a Zn finger (zf) motif, including C2H2 (CyszHisz), C6H2 (CyseHisz, also referred to as MYND-like), or MYND (myeloid, Nervy, and DEAF-1) (Figure IF), consistent with known roles of zf domains in facilitating protein-protein interactions (Brayer et al., 2008; Fedotova et al., 2017; Gross and McGinnis, 1996). Because METAP 1 was the single conserved ZNG1 interaction identified in all yeast-two- hybrid screens, the METAP 1-ZNG1 interaction was selected for additional investigation. To further define the region of METAP1 that interacts with ZNG1, the results from the three screens were compared to predict a minimal METAP 1 -interacting region (Figure 1G). This analysis suggested that an N-terminal segment of 108 residues within METAP 1 containing the zf-C6H2 domain is the site of interaction with ZNG1.

The METAP1 zf-C6H2 interacts with a conserved motif in ZNG1.

[00124] The interaction of full-length recombinant ZNG1 and METAP 1 from mouse and zebrafish was confirmed by affinity chromatography (Figures 2A and 9A-C) and by dynamic light scattering (DLS) (Figure 2B). Masses obtained from DLS for murine ZNG1 and METAP 1 individually, as well as for the equimolar complex, were consistent with theoretical masses (Figure 2B). Size exclusion chromatography was performed using murine ZNG1 and N- terminally truncated METAPb-sg containing the complete zf-C6H2 domain (Figure 9D). The observed shift in elution volume of the 1: 1 mixture, compared to individual proteins, indicates that the N-terminus of METAP 1 is sufficient to mediate the interaction of ZNG1 with METAP 1 (Figures 2C and 9D).

[00125] Determination of a minimal binding site on ZNG1 for the interaction with METAP 1 was informed by the observation that zf-C6H2 domains resemble zf-MYND domains, with four pairs of conserved cysteine and histidine residues as well as conserved aromatic residues (Figure S2E-G). No structure has been determined for a zf-C6H2 domain, but structures of zf-MYND domains in AML1/ETO, DEAF-1, and BS69 reveal a ppa architecture, two Zn ions coordinated in an interdigitated pattern, and a binding interface for peptides containing a PxLxP motif (Figure S2E,G) (Harter et al., 2016; Kateb et al., 2013; Liu et al., 2007). The METAP1 zf- C6H2 domain also binds two Zn ions, even when truncated to METAP 11.59 or METAP 11.79, as measured by inductively coupled plasma-mass spectrometry (ICP-MS) (Figure S2H). Moreover, a conserved CPELVPI sequence at the N-terminus of ZNG1 homologs matches the PxLxP motif that zf-MYND binds (Figures IB and 2D).

[00126] METAP1 binding of ZNG1 peptides was confirmed by monitoring quenching of W45 fluorescence in murine METAP 11.59 and METAP 11.79, upon addition of chemically synthesized N-terminal ZNG1 peptides from human, mouse, and zebrafish (Figures 2D,E and 9 I). These data reveal a 1: 1 binding stoichiometry and a.Ko ~ 0.1 pM (Figure 2F-G), which is > 100-fold tighter than the zf-MYND domains with their cognate peptides (Kateb et al., 2013; Liu et al., 2007). Mouse, human, and zebrafish ZNG1 peptides bound with similar affinities to both METAP 11-59 and METAP 11-79 (Figure 2G), indicating that the N-terminal 59 residues are sufficient for binding. Combined, these findings identify the specific regions of interaction between ZNG1 and METAP 1.

[00127] NMR spectroscopy was used to confirm peptide binding and to explore the interaction in more detail. ^X H- ¹⁵ N HSQC spectra of ¹⁵ N-enriched METAP 11.59 show excellent signal dispersion and uniform intensities indicating a stable, well-folded domain (Figure 2H, left). Addition of a half-molar equivalent of ZNG1 peptide produces a second set of peaks of equal intensity (Figure 9 J), indicating a complex in the slow chemical exchange regime on the NMR time scale; addition of a full molar equivalent of ZNG1 peptide reveals a saturated complex. (Figure 2H, middle panel). These results confirm tighter binding than previously observed for MYND domains (Kateb et al., 2013; Liu et al., 2007), and motivate a more detailed analysis of the peptide binding interface.

Solution structure of the METAP1 zf-C6H2 : ZNG1 peptide complex.

[00128] An NMR chemical shift perturbation assay was used to identify the METAP1 residues most directly involved in peptide binding. Complete backbone chemical shift assignments for both the peptide-free and peptide-bound states of METAPh.59 (Figure S3A) reveals that residues 21-24 are particularly affected by peptide binding, suggesting direct contact with the peptide (Figure 21), while residues 1-18 and 27-32 are relatively unaffected and likely distant from the binding site. Further, chemical shift-based secondary structure predictions reveal that the MET API domain has a ppapa architecture in the free and bound forms (Figure 10B, top, middle), similar to but distinct from the ppa architecture of zf-MYND domains (Figure 9E) (Shen and Bax, 2015). The secondary structure is minimally perturbed by peptide binding, as in zf-MYND domains (Liu et al., 2007).

[00129] Further NMR studies of the bound form of METAPh.59 were not possible due to peak splitting for many residues, a limitation circumvented by studying a fusion construct inspired by the structure determination of the AML1/ETO zf-MYND bound to the SMRT peptide (Liu et al., 2007). The ZNG1 peptide was expressed at the N-terminus of METAP 11.59 (Figure IOC). The ¹⁵ N-labeled fusion reproduces the spectra of the bound form, without the multiplets observed previously (Figures 2H and 10D). Minimal perturbations to chemical shifts, localized to the N-terminus of METAP 1 validate the utility of the fusion construct (Figures 10A right; 10B bottom; 10E). Moreover, backbone dynamics show that the linker region is flexible and that the dynamics of the domain are consistent in the free and fusion forms (Figures 3A,B and 10F,G). The central region of the peptide encompassing the CPELVPI sequence has elevated heteronuclear NOE values indicative of tight binding. Additionally, the fusion construct enables direct observation of the ZNG1 peptide resonances, facilitating structure determination. An initial, low-resolution 3D solution NMR structure of the ZNG110.30 METAP 11.59 fusion relying exclusively on automated NOE assignments with no additional constraints clearly shows the same cross-brace Zn-binding topology as in a zf-MYND domain. Znl is coordinated by C9, Cl 4, C36, and C40 and Zn2 by C22, C25, H48 and H52 (Figure 3C).

[00130] The final solution structure shows the novel zf-MYND-like fold, with an additional oc-helix and p-strand (Figure 3D, Figure 16). This strand pairs with the C-terminal strand to form a 1,3,2 antiparallel p-sheet, in which strand 2 hydrogen bonds with the peptide, consistent with the pattern of ¹ H, ¹⁵ N chemical shift perturbations (Figure 10H). The negatively charged peptide binds a hydrophobic groove in a positively charged face of the domain, surrounded by conserved lysine residues, K19, K27, K42, K49, and K53 (Figures 3E and 9E-F). In the hydrophobic groove, C18’ of ZNG1 contacts the W45 side chain in METAP1, while P19’ contacts F41, the L21’ sidechain inserts into the deep pocket lined by F35 and F41, the V22’ sidechain makes hydrophobic contacts with the conserved P23, and the 124’ sidechain packs against Y34. Sidechains of Q21 and Q38 in METAP1 form hydrogen bonds with the ZNG1 peptide backbone. Collectively, these data reveal the molecular features of the ZNG1 -METAP 1 interaction, showing that the high affinity comes from a combination of electrostatic and hydrophobic interactions, as well as hydrogen bonds, consistent with the conservation of these residues in METAP 1 and in ZNG1.

ZNG1 supports METAP1 function in a Zn-dependent manner.

[00131] Based on our previous work with bacterial COG0523 proteins, we hypothesize that ZNG1 uses GTP hydrolysis to transfer Zn from the high affinity CxCC binding site into the MET API active site, thereby maintaining MET API activity under conditions of low cellular Zn. In addition to the two Zn ions coordinated by the zf-C6H2 domain, METAP 1 binds two metal ions in the active site (Addlagatta et al., 2005). Recombinant murine METAP1 can therefore be loaded with Zn to four molar equivalents (termed Z METAPl). Removal of Zn by extended exposure to EDTA disrupts the zf fold (Figure 11 A,B); however, gentler treatment with chelators including nitrilotriacetic acid (NT A; Xz _n ~10 nM) selectively removes metal from the active site while leaving the zf domain intact, generating ZnzMETAPl (Figure 11C). Mag-fura-2 (mf2) and quin-2 Zn competition experiments with ZmMET AP I show two high affinity (Xzni ~ 8 pM, K7r0 ~ 20 pM) and two low affinity Zn binding sites (Figures 4A and 1 ID). Murine ZNG1 binds three molar equivalents of Zn (Figure 1 IE), with one high affinity site (Kz.ni ~ 5 pM) likely coordinated by the CxCC motif in the core GTPase domain (Figure 4B) (Jordan et al., 2019) and two low affinity sites. Like other COG0523 proteins of the same phylogenetic grouping (Edmonds et al., 2021), GTP hydrolysis is slow and slightly inhibited when Zn is bound solely at the high-affinity site (ZnZNGl) (Figure 4C,1 IF) (Sydor et al., 2013). However, ZnZNGl GTPase activity is stimulated by ZmMETAP l , while ApoZNGl is not (Figure 4C).

[00132] To assess the effect of ZNG1 on METAP 1 activity in vitro, we first determined initial rates of aminopeptidase activity for the various Zn-bound forms of METAP 1 by quantifying released methionine (Met) from the heptapeptide MAHAIHY, a known METAP 1 target (Xiao et al., 2010) (Figure 11H-K). Active site-bound Zn is required for activity as Zn?METAP I exhibits no turnover, while ZmMETAP I partially recovers the activity of fully active Z METAPl (Figure 4D). Incubation of ApoZNGl and ZmMETAP l shows no activation ofZmMETAP l , while addition of ZnZNGl recovers small amounts of Z METAPl activity (Figure 4E). ZnZNGl shows no activation of fully metalated Z METAPl (Figure 1 IL), consistent with a regulatory impact only on undermetalated METAPL We next assessed the nucleotide dependence of ZnZNGl activation of ZmMETAP l by incubation with GTP, GDP, or nonhydrolyzable GTP analog, GDPNP. In the presence of GTP, ZnZNGl nearly fully activates METAP1, and GTP hydrolysis is required for this activation as ZnZNGl in the presence of GDPNP exhibited no activation (Figure 4E). Although GDP shows some ZnZNGl -dependent activation of MET API above that of ZnZNGl alone, it is far less than that of GTP (Figure 4E). This could result from a conformational change in ZNG1 after GTP hydrolysis that may facilitate release of Zn into the METAP1 active site (Jordan et al., 2019). We performed the same activation experiments in the presence of NTA to ensure Zn-dependent activation of ZmMETAP l is due to Zn transfer from ZNG1 directly to METAP1, rather than from Zn in solution. The GTP-bound form of ZnZNGl again exhibits significant activation of ZmMETAP I above that of NTA-bound Zn, while GDPNP has no effect (Figure 4F). Similarly, ZnZNGl in the presence and absence of GDP shows only minor activation of ZroMETAP l activity (Figure 4F). Zebrafish Zngl also stimulates apo-Metapl activity (Figure S4M). Together, these data establish that ZNG1 activates ZnzMETAPl in a Zn- and GTP hydrolysis-dependent manner (Figure 4G).

Zngl promotes in vivo Metap function in zebrafish.

[00133] To study the in vivo role of Zngl, we turned to a zebrafish model. Zebrafish possess a single copy of zngl, which has high sequence similarity to mammalian ZNG1 homologs (Figure 1 A). Based on the reported Zn-dependent repression of ZNG1 (Coneyworth et al., 2012; Ogo et al., 2015), we first sought to assess zngl expression in a zebrafish. To modulate organismal Zn levels, chemical treatment of zebrafish larvae was performed via immersion in system water containing the Zn chelator N,N,N',N'-Tetrakis(2-pyridylmethyl)ethylenediamine (TPEN). Treatment of zebrafish larvae with TPEN resulted in a significant induction of zngl transcript in WT larvae (Figure 5A). To investigate Zngl-Metapl interaction in vivo, zngl mutant zebrafish (hereafter referred to as zngl") were generated using CRISPR/Cas9 gene editing (Figures 5A, 12A). zngl mutant fish survived to adulthood under Zn-replete conditions yet morphometric analysis revealed zngl mutant larvae exhibit moderate developmental delay with reduced height at anterior anal fin (HAA), standard length, and snout to vent length at 6 days post fertilization (dpf) (Figures 5B-D, 12B) (Parichy et al., 2009).

[00134] We hypothesized a role of Zngl in maintaining Metapl function and Zn homeostasis in zebrafish. Zebrafish encode for a single copy of Metapl with high sequence similarity to mammalian METAP1, and two paralogs of Metap2 (Metap2a and Metap2b). Previous studies indicate functional redundancy between METAP 1 and METAP2 suggesting that zebrafish Metap2 paralogs could compensate for Metapl activity in zngl" animals (Frottin et al., 2006; Li and Chang, 1995; Xiao et al., 2010). To investigate the effects of impaired Metapl function on organismal health, Metapl activity in vivo was modulated using pharmacologic inhibition of Metap enzymes in larval zebrafish. WT and zngl' ¹ ' larvae were exposed to the established METAP 1/2 inhibitor Bengamide B in Zn-deplete or replete conditions and survival was monitored over three days of treatment (Emilio Quinoa, 1986; White et al., 2017). zngl mutant larvae displayed increased sensitivity to Bengamide B with 100% lethality when treated with 1 pM of inhibitor as compared to WT controls which remained viable (Figures 5E, 12C). We hypothesized that inhibition of Metap activity in zngl mutant animals would be exacerbated in conditions of low Zn, consistent with in vitro data suggesting that Zngl supports metallation of Metapl under Zn limiting conditions. Indeed, co-treatment of zngl mutant larvae with a sublethal dose of Bengamide B (500 nM) and TPEN resulted in decreased viability as compared to treatment with 500 nM Bengamide B alone (Figures 5E, 12C). Notably, WT animals treated with Bengamide B also displayed significantly reduced HAA (Figure S5D) mirroring the defects observed in zngl ^ larvae, indicating a functional link between Zngl and Metapl in vivo. Combined, these data suggest Metap activity is impaired in zngl mutant larvae, especially in Zn-deplete conditions.

[00135] To further evaluate Zngl modulation of in vivo Metapl activity, metapl mutant zebrafish were generated by CRISPR/Cas9 (hereafter referred to as metap 1") (Figure S5E,F). As expected, pharmacologic inhibition of total Metap activity in metap 1'' larvae resulted in significantly higher morphologic abnormalities as compared to WT controls at 6 dpf (Figure 5F,G). These abnormalities included pericardial edema (PCE), yolk sack edema (YSE), uninflated swim bladder (USB), and craniofacial defects (CD) (Figures 5F, 12G) (Raghunath and Perumal, 2018). Importantly, zngl mutant larvae exhibited similar defects when exposed to the maximum tolerable dose of Bengamide B (Figures 5H-J, 12H) consistent with a role for Zngl in the promotion of Metapl activity in vivo.

[00136] Finally, to test if Metapl can compensate for Metap2-inhibition in WT animals but not in zngl deficient animals (Frottin et al., 2006; Xiao et al., 2010), the METAP2 specific inhibitor TNP-470 was applied by immersion and larval survival was assessed. Similar to what was observed following treatment with Bengamide B, zngl" larvae displayed decreased survival compared to WT animals following TNP-470 exposure which became more pronounced in Zn- deplete conditions (Figures 5K, 121). Cumulatively, these data suggest that Metapl activity is perturbed in zngl mutant larvae and, consequently, the pharmacologic potency of both Metap inhibitors is enhanced in the absence of Zngl, particularly during Zn deprivation.

Zngl' ¹ ' mice are sensitive to dietary Zn starvation and display signs of mitochondrial dysfunction. [00137] To investigate the impact of ZNG1 in a mammalian model, Zngl mutant mice (hereafter referred to as Zngl'^ were generated using CRISPR/Cas9. The resulting deletion spanning the annotated start codon as well as 50 amino acids within exon 1 (including the CPELVPI motif) was subsequently confirmed at the transcriptional and protein level (Figure 13A-C). Zngl'' animals were viable and did not display anomalies in gross morphology. Nevertheless, breeding patterns showed non-mendelian inheritance (Figures 6A and 13D, E), suggesting that ZNG1 functions in early development. We next assessed if ZNG1 effects Zn homeostasis during conditions of dietary Zn deprivation. WT and Zngl' ' mice were administered a low Zn diet for 6 weeks and monitored for weight gain and organ specific Zn levels (Figure 6B). Mice deficient in Zngl displayed decreased weight gain when fed a low Zn diet (Figure 6B) yet no difference in food uptake was observed. Notably, both WT and Zngl' ' animals gained weight at similar rates when fed a control diet (Figure 13F). While a low Zn diet was shown to effectively decrease Zn levels in various organ systems, differences in organ Zn levels between WT and mutant animals were not observed (Figure 13G). These data suggest that loss of Zngl leads to failure to access existing Zn pools, rather than affecting organ-specific total Zn levels.

[00138] Due to the importance of MET API for maintaining cellular proteostasis (Hu et al., 2006; Jonckheere et al., 2018), the impact of Zngl deficiency on the global proteome in Zn- deplete conditions was evaluated from WT and Zngl' ¹ ' animals fed a low Zn diet for 5 weeks. Kidneys were selected for this analysis because ZNG1 proteins are highly expressed in human renal tissue (Figure 8) (Uhlen et al., 2015). Sixty-three differentially abundant proteins were identified in the Zngl mutant compared to the WT control (±2 -fold cutoff, Figure 6C, Figure 17). Analysis of predicted localizations of these proteins using the UniProt database (UniProt, 2021) suggested that a large number localize to the mitochondria, particularly those with decreased abundance (Figure 6C, D). Ingenuity Pathway Analysis (IP A) was performed to predict cellular processes affected by loss of Zngl and revealed that numerous affected proteins play roles in canonical mitochondrial pathways (Figure 6D) (Kramer et al., 2014). Notably, ZPA also revealed that loss of Zngl in Zn-starved animals significantly affects proteins connected to mitochondrial dysfunction, further suggesting negative effects on mitochondrial integrity. While most of the mitochondrial proteins were decreased in abundance in mutant animals (Figure 6C, D), some proteins in this group were significantly elevated in Zngl" mice. One of the proteins with the largest increase in abundance (+7.2-fold) in the mutant animals was the mitochondrial ATPase inhibitory factor 1 (IF1, ATPIF1) (Garcia-Bermudez and Cuezva, 2016). ATPIF1 is considered the master regulator of ATP synthase, and blocks both ATP synthesis and hydrolysis activities (Chen et al, 2014). Subsequent IPA upstream regulator analysis (URA) (Kramer et al., 2014) identified ATPIF as a main regulatory node (E- value of overlap = 2.4E-33) with experimental linkage to a subset of (mitochondrial) proteins identified as being negatively affected by loss of ZNG1 (Figure 13H). To evaluate if changes in protein abundance were a result of altered protein processing, transcriptomic analysis of a subset of the most differentially abundant proteins was performed. These data revealed modest changes in transcript levels, with some changes reaching statistical significance, indicating effects of Zngl deficiency at both the transcript and protein level (Figure 131). Together, these results suggest that ZNG1 is important for maintaining mitochondrial function and organismal homeostasis during Zn starvation.

ZNG1 promotes cellular proliferation and respiration in Zn-deplete conditions.

[00139] Given that Zngl mutant mice displayed signs of impaired Zn homeostasis as well as disruption of mitochondrial function, a cell culture model was generated to further characterize the role of ZNG1. Murine proximal tubular epithelial cells (TKPTS) were selected as a model since mitochondrial dysfunction was detected in murine Zngl' ' renal tissue, and TKPTS cell lines express both ZNG1 and MET API (Figures 7A, B and 14A-C). Zngl mutant TKPTS cells (Figure 14A) were generated using CRISPR/Cas9 and decreased mRNA expression (Figures 7B and 14D) and protein abundance (Figure 14C) of ZNG1 were confirmed. The expression of Zngl in TKPTS cells was dependent on Zn availability (Figure 7A). While Zngl' ' and WT cells grew at similar rates in untreated media, growth in the presence of Zn chelator tris(2-pyridylmethyl)amine (TP A) significantly slowed proliferation of the Zngl mutant cells compared to WT as measured by increased retention of CellTrace dye (Figures 7C and 14E, F). Of note, exposure of WT TKPTS cells to the METAP 1/2 inhibitor Bengamide B similarly reduced cellular proliferation, thereby phenocopying the growth impairment observed in Zngl mutant cells in Zn-deplete conditions (Figure 14G). Because ZNG1 supports proper function of METAP 1 in vitro (Figure 4), we postulated that Zngl -deficient cells would display increased sensitivity to METAP inhibition. Due to the overlapping substrate specificity of METAP 1 and METAP2, TKPTS cells were exposed to the METAP2-specific inhibitor TNP-470. This strategy increases cellular reliance on METAP1 thereby permitting the evaluation of ZNG1 -dependent METAP 1 activity. Indeed, Zngl'' cells exhibited slowed proliferation relative to WT when exposed to TNP-470 (Figures 7D and 14H, I) and complementation with full-length Zngl significantly enhanced proliferation of Zngl mutant cells (Figure 14J). Notably, decreased proliferation and increased sensitivity to METAP-inhibition have been reported in METAP 1- deficient HAP1 cells (Jonckheere et al., 2018), which is consistent with the possibility that defects observed in Zngl' ^! ' cells may in part be mediated by dysregulated METAP 1 activity.

[00140] To further assess a potential impairment of MET API function in the Zngl' ¹ ' background, the abundance and iMet-status of the known METAP-substrate 14-3-3y were probed though proteomic profiling (Towbin et al., 2003). Analysis of WT and Zngl' ' cells grown in Zn-replete and Zn-limiting (TPA-treated conditions) conditions revealed reduced levels of 14- 3-3y in Zngl mutant cells (Figure 7E). We hypothesized that this reduction was a result of impaired METAP 1 -dependent iMet removal, and accordingly detected a trend towards higher iMet retention at the 14-3 -3y N-terminus in TPA treated Zngl' ¹ ' mutant cells as compared to WT (Figure 14K). Taken together, these data are indicative of a reduction of METAP1 activity in Zngl' ¹ ' cells. To further test if the proteomic changes in the Zngl mutant resemble those of METAP-deficient cells, proteomic analysis was performed on WT cells treated with the METAP 1/2 inhibitor Bengamide B and the results compared to the TPA-treated Zngl mutant cells (Figure 17). IPA was performed and significantly affected pathways in both datasets determined. Subsequent comparison of the two analyses showed remarkable overlap of the pathways affected by loss of Zngl and pharmacological inhibition of METAP (Figure 14L). Notably, pathways significantly enriched in both datasets included oxidative phosphorylation, TCA cycle (Figure 14L), and mitochondrial dysfunction (P-value(-loglO) of 3 and 10.5 for Zngl' ¹ ' and Bengamide B-treated, respectively). Collectively, these findings demonstrate that mutation of Zngl phenocopies a METAP-deficient system, and that mitochondrial processes and function are affected by loss of ZNG1.

[00141] The reduced proliferation of Zngl mutant cells (Figure 7C,D), in combination with proteomic signatures of mitochondrial distress in Zngl' ' cell lines and mice (Figures 6C and 14L) are consistent with a restriction in mitochondrial energy production. Since mitochondrial morphology reflects function (Picard et al., 2013), Zn starved WT and Zngl mutant cells were imaged using transmission electron microscopy (TEM). Zngl mutant cells were morphologically distinct (Figure 7F) and displayed a significant change in mitochondrial cross-sectional area (Figure 7G) in the absence of alterations of mitochondrial roundness, suggesting that mitochondria from Zngl' ' cells are swollen (Figure S7M). Additionally, mitochondria of Zngl' ' cells displayed increased electron density (Figure 7H). Similar alterations in mitochondrial morphology have previously been associated with uncoupling of the mitochondrial membrane potential from ATP synthesis (Hackenbrock et al., 1971; Huet et al., 2018; Weissert et al., 2021). Consistent with this, cellular ATP levels quantified from the lysates of Zng '- TKPTS cells were decreased compared to WT (Figure 71). Together, these data suggest that ZNG1 contributes to maintaining mitochondrial metabolic homeostasis, and that a loss of ZNG1 renders cells less capable of sustaining the energy necessary for growth during conditions of stress.

[00142] To assess broad changes in oxidative phosphorylation, mitochondrial membrane potential was quantified using tetramethylrhodamine methyl ester (TMRM) at a quenching concentration where a decrease in fluorescence intensity correlates to an increase in membrane potential (Monteith et al., 2013; Scaduto and Grotyohann, 1999). TMRM fluorescence was decreased in Zngl' ' cells compared to WT cells in conditions of Zn starvation, indicating mitochondria within Zngl mutant cells are hyperpolarized (Figure 7J and 14N), consistent with reduced ATP synthase activity. Mitochondrial superoxide (Ch"’) production, a byproduct of electron leakage during electron shuttling across the inner mitochondrial membrane (Forman and Kennedy, 1974; Loschen et al., 1974), was quantified using the fluorescent dye, MitoSOX. Mitochondrial OZ' levels in Zngl mutant cells were lower compared to WT cells, and this phenotype is exacerbated upon METAP2 inhibition (Figures 7K and S7O-R). These results suggest that electron shuttling is decreased in Zngl mutant cells despite hyperpolarization of the mitochondria.

[00143] To further interrogate the implications of loss of Zngl on oxidative phosphorylation, the oxygen consumption rate (OCR) of WT and Zngl' ¹ ' cells grown in media alone or in the presence of TPA was quantified by extracellular flux analysis. While Zngl' ¹ ' cells exhibited a trend towards decreased basal and maximal respiration compared to WT cells, pretreatment of mutant cells with TPA significantly impaired basal and maximal respiration, ATP production, and proton leak (Figure 7L, M). Notably, Zngl' ' cells showed accumulated ATPIF1 puncta colocalizing with mitochondrial structures (Figure 14S), consistent with proteomic analysis of Zngl mutant animals (Figure 6C, D). These results support a model whereby loss of Zngl negatively effects METAP 1 activity, particularly during Zn starvation. In turn, impaired Metapl -dependent processing of the proteome likely disrupts mitochondrial homeostasis. Specifically, Zngl mutant cells display hyperpolarization of the mitochondrial membrane with decreased oxidative phosphorylation, consistent with ATPIF1 -dependent inhibition of ATP synthase. Taken together, these findings demonstrate that ZNG1 activates METAP 1 activity and ZNG1 plays a critical role in regulating mitochondrial function and energy metabolism during Zn starvation.

Discussion

[00144] Zn is an essential structural and enzymatic cofactor for many proteins in all branches of life. Consequently, vertebrates have developed sophisticated systems for Zn uptake, intracellular distribution into organelles, and efflux to maintain organismal and cellular Zn homeostasis (Eide, 2006). While numerous aspects of Zn transport and trafficking have been well characterized, mechanisms for allocating Zn within the cell and to individual proteins remain largely unexplored. Distribution of this essential nutrient to “high priority” (Eide, 2006) proteins has been attributed to hypothetical Zn metallochaperones. However, functionally validated interactions with target proteins and bona fide Zn metallochaperone function have not been reported. In this study, evolutionarily divergent systems were used to establish a role for ZNG1 in regulating cellular Zn homeostasis, identifying METAP 1 as a conserved client of ZNG1 and providing mechanistic insights into ZNG1 -dependent METAP 1 activation. Specifically, the ZNG1 N-terminus and METAP 1 zf-C6H2 were identified as the primary interacting domains and structurally characterized. Upon interaction, METAP 1 activates the GTPase activity specifically of Zn-bound ZNG1. GTP hydrolysis drives Zn transfer from ZNG1 into the METAP 1 catalytic site either through the energy provided by phosphodiester bond cleavage or a conformational change upon hydrolysis, thereby metalating and activating METAP 1. Importantly, the Zn binding affinities of ZNG1 and MET API are similar, suggesting that Zn transfer may have some element of kinetic control; alternatively, GDP modestly lowers the affinity of ZNG1 for Zn, facilitating capture by METAP 1. In any case, the Zn- and GTP- dependent activation of METAP1 by ZNG1 is consistent with the assignment of ZNG1 as a metallochaperone.

[00145] The ZNG1-METAP1 interactions described in this study are likely to be more broadly conserved across eukaryotes. Consistent with our findings, this interaction was also suggested by a large-scale screen of the human interactome (Huttlin et al., 2015) and identified by a recent study that investigated Zn lp in Saccharomyces cerevisiae (Pasquini et al ). The ZNG1-METAP1 interaction in vertebrates is mediated by a conserved N-terminal CPELVPI motif in ZNG1 and the zf-C6H2 domain of METAP 1. Our solution structure of the interaction domain reveals that the conserved features, including the hydrophobic binding pockets and the electrostatic surface of METAP1 required for ZNG1 binding, may well be shared among other ZNG1 clients or binding partners. Further sequence analysis reveals that approximately half of all eukaryotic ZNG1 homologs (Edmonds et al., 2021) share an N-terminal “[D/E]n\|/PxLVp” motif, in which a series of negatively charged residues [E/D] _n are followed by a non-aromatic hydrophobic residue (rp), and a conserved proline, leucine, and valine (Figure 3F). Our structure is fully consistent with this revised motif, and therefore likely captures key features that describe an evolutionarily conserved ZNG1 -METAP 1 interaction. Indeed, the N-terminus of S. cerevisiae Znglp lacks the exact vertebrate CPELVPI motif, but matches the more general “[D/E] _n \|/PxLVp” pattern. Although the sequence identity of the yeast and mouse Maplp/METAPl N-terminal domains is less than 40% (Figure S2E), AlphaFol d2 modeling of the Znglp-Maplp interaction predicts a nearly identical fold and similar peptide interface (Jumper et al., 2021) (Figure S3I), where positive charges surround a hydrophobic groove and engage negatively charged ZNG1 peptides in both complexes. These findings suggest that our structure of the ZNG1 -METAP 1 interaction domain may be representative of how evolutionarily distant ZNG1 homologs interact with their targets.

[00146] The structure and revised ZNG1 motif also provide insight into what proteins may compete with ZNG1 to perform its metallochaperone function. While many proteins contain a more general PxLxP sequence, very few match the “[D/E] _n \|/PxLVp” pattern identified here.

One notable candidate is in the C-terminus of the p-subunit of the nascent polypeptide-associated complex (NAC), which protrudes from the ribosome exit tunnel (Gamerdinger et al., 2019; Lin et al., 2020). This motif likely explains how the zf-C6H2 domain localizes MET API to the ribosome in the presence of NAC (Nyathi and Pool, 2015; Vetro and Chang, 2002). The high affinity of ZNG1 for METAP 1 may enable it to outcompete NAC under conditions of low cellular Zn.

[00147] Our zebrafish and mouse models of Zngl deficiency link ZNG1 to mitochondrial function, organismal development, and Zn homeostasis. The connection between MET API and mitochondrial integrity is not without precedent. A previous study that utilized transcriptomic and proteomic approaches to characterize the effects of MET API loss in human HAP I cells found changes to mitochondrial transcript and protein levels as a result of METAP I mutation (Jonckheere et al., 2018). Such data offer a potential explanation as to how mutation of Zngl in TKPTS cells results in mitochondrial dysfunction and attenuated cellular respiration, and ultimately explain the detrimental effects observed in Zngl deficient animals. Collectively, these findings highlight the importance of ZNG1 for survival during Zn limitation, likely by ensuring functional METAPl-dependent NME and maintaining animal energy balance during such conditions.

[00148] In conclusion, we established the vertebrate COG0523 ZNG1 protein family as a major feature of the vertebrate adaptive response to severe Zn deprivation. Using biochemical, structural, genetic, and pharmacological approaches across evolutionarily divergent models, including zebrafish and mice, we demonstrate a critical role for ZNG1 proteins in regulating cellular Zn homeostasis. Collectively, these data reveal the existence of a family of Zn metallochaperones and place ZNG1 at the center of a new paradigm for intracellular Zn trafficking. While these data show a clear physical interaction between ZNG1 and the metalloprotein METAP 1, we also provide structural insights into how ZNG1 could interact with other targets. These findings reveal an evolutionary conserved strategy to ensure targeted Zn distribution to a network of high-priority client proteins during conditions of severe Zn starvation.

[00149] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference, including the references set forth in the following list:

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[00258] It will be understood that various details of the presently disclosed subject matter can be changed without departing from the scope of the subject matter disclosed herein. Furthermore, the foregoing description is for the purpose of illustration only, and not for the purpose of limitation. Obvious modifications and variations are possible in light of the above teachings. All such modifications and variations are within the scope of the appended claims when interpreted in accordance with the breadth to which they are fairly, legally and equitably entitled.