Recombinant Mouse Methionine aminopeptidase 1 (Metap1)

What is Mouse Methionine Aminopeptidase 1 (MetAP1) and what is its primary function?

Methionine aminopeptidase 1 (MetAP1) is an essential enzyme responsible for the co-translational removal of the initiator methionine residue from nascent proteins. This N-terminal methionine processing is a fundamental cellular process conserved from prokaryotes to eukaryotes. In mice, MetAP1 is ubiquitously expressed across tissues and plays critical roles in protein maturation, stability, and cellular functions .

The catalytic mechanism of MetAP1 involves a metal-dependent process where cobalt or manganese ions facilitate the hydrolytic cleavage of the N-terminal methionine. This process is particularly important for proteins where the presence of the initiator methionine would interfere with proper folding, localization, or function .

Methodologically, when studying MetAP1 function, researchers should consider both its direct enzymatic activity (methionine removal) and its downstream effects on protein stability and cellular processes, particularly cell cycle progression where it appears to play a significant role in G2/M phase transition .

How is recombinant mouse MetAP1 typically produced for research applications?

Production of recombinant mouse MetAP1 typically follows established protocols similar to those used for human MetAP1, with specific considerations for the mouse sequence. The recommended methodology involves:

• Expression System Selection: Baculovirus-infected insect cells (such as Spodoptera frugiperda Sf21 cells) provide an effective eukaryotic expression system that ensures proper folding and post-translational modifications .

• Construct Design: The mouse MetAP1 coding sequence (corresponding to the catalytic domain, similar to the His52-Phe386 region in human MetAP1) is typically cloned into an expression vector with an appropriate tag (commonly a C-terminal polyhistidine tag) to facilitate purification .

• Protein Purification: Following expression, the protein undergoes multi-step purification:

  • Initial capture using affinity chromatography (typically Ni-NTA for His-tagged proteins)

  • Further purification by ion-exchange chromatography

  • Final polishing step using size-exclusion chromatography to achieve >95% purity

• Quality Control: Verification of purified recombinant MetAP1 includes:

  • SDS-PAGE analysis for purity assessment

  • Western blotting for identity confirmation

  • Enzymatic activity assay using methionine-containing peptide substrates

  • Mass spectrometry for molecular weight confirmation

When working with recombinant mouse MetAP1, it's essential to verify its enzymatic activity before experimental use, as improper folding or insufficient metal cofactor incorporation can significantly impact function .

What are the optimal storage conditions for maintaining mouse MetAP1 activity?

Maintaining the stability and activity of recombinant mouse MetAP1 requires careful attention to storage conditions. Based on established protocols for similar proteins, the recommended procedures include:

• Short-term Storage (1-2 weeks):

  • Store at 4°C in a buffer containing:

    • 50 mM HEPES or 25 mM Tris (pH 7.5-8.0)

    • 100-150 mM NaCl

    • 10-20% glycerol as a cryoprotectant

    • 0.1 mM CoCl₂ or MnCl₂ (to maintain metalloenzyme activity)

    • Optional: 1 mM DTT to prevent oxidation of cysteine residues

• Long-term Storage:

  • Aliquot the protein solution to minimize freeze-thaw cycles

  • Flash-freeze in liquid nitrogen

  • Store at -80°C in a manual defrost freezer

  • Include glycerol (20-25%) in the storage buffer to prevent ice crystal formation

• Stability Considerations:

  • Avoid repeated freeze-thaw cycles, which can cause significant activity loss

  • When thawing, place on ice and use immediately for optimal activity

  • Consider carrier-free formulations for applications where additives might interfere

Importantly, when preparing the enzyme for activity assays, a brief pre-incubation (5-10 minutes) in activation buffer containing the metal cofactor (typically 0.1 mM CoCl₂) is recommended to ensure maximum enzymatic activity .

How can I accurately measure mouse MetAP1 activity in vitro?

Measuring mouse MetAP1 activity requires careful consideration of assay conditions, substrate selection, and detection methods. The following methodological approach is recommended:

• Assay Buffer Preparation:

  • Activation Buffer: 50 mM HEPES, 0.1 mM CoCl₂, 0.1 M NaCl, pH 7.5

  • Reaction Buffer: 25 mM Tris, pH 8.0

• Substrate Selection:

  • For fluorogenic assays: Met-Gly-Pro-AMC or Met-Pro-pNA

  • For HPLC-based assays: Met-Gly-Met-Met or other synthetic peptides with N-terminal methionine

  • Consider using species-specific substrates when comparing across orthologs

• Standard Assay Protocol:

  • Pre-incubate recombinant MetAP1 (2-4 μg/mL) in activation buffer for 5-10 minutes at room temperature

  • Prepare substrate solution (100-200 μM) in the same buffer

  • Combine equal volumes of enzyme and substrate solutions

  • Incubate reactions for 5-30 minutes at room temperature or 37°C

  • For fluorogenic substrates: Stop reactions by heating at 95-100°C for 5 minutes

  • For two-step assays with Met-Gly-Pro-AMC: Add secondary enzyme (e.g., DPPIV/CD26) to cleave the exposed dipeptide and release the fluorophore

• Data Analysis:

  • Calculate initial reaction velocities from the linear portion of product formation curves

  • Determine kinetic parameters (Km, kcat) using appropriate enzyme kinetic models

  • Include positive and negative controls (heat-inactivated enzyme, known inhibitors)

For inhibitor screening, IC₅₀ determinations require careful concentration-response experiments with at least 8-10 inhibitor concentrations spanning several orders of magnitude .

What role does MetAP1 play in cell cycle regulation and how can this be studied?

MetAP1 has been shown to play a critical role in cell cycle regulation, particularly at the G2/M phase transition. This function appears distinct from its methionine processing activity and represents an important area of research with implications for cancer biology.

• Established Role in Cell Cycle:

  • MetAP1 inhibition or knockdown results in significant G2/M phase delay

  • This effect is distinct from MetAP2 inhibition, which typically causes G1/S arrest

  • The mechanism appears to involve specific protein substrates that regulate mitotic progression

• Methodological Approaches to Study Cell Cycle Effects:

  a) Chemical Inhibition Studies:

  • Treat cells with MetAP1-selective inhibitors (e.g., pyridine-2-carboxylic acid derivatives)

  • Monitor cell cycle distribution using flow cytometry with propidium iodide staining

  • Assess mitotic index using phospho-histone H3 immunostaining

  • Suggested concentrations: 0.5-5 μM of inhibitors like compounds IV-43, IV-71, or IV-62, based on their IC₅₀ values

  b) Genetic Approach:

  • Knockdown MetAP1 using siRNA or shRNA (e.g., construct XH3 has shown ~90% knockdown efficiency)

  • Create inducible knockdown systems to observe temporal effects

  • Perform rescue experiments with wild-type vs. catalytically inactive MetAP1

  c) Mechanistic Studies:

  • Identify MetAP1-dependent proteins involved in G2/M transition using proteomics

  • Examine changes in N-terminal methionine retention in synchronized cell populations

  • Evaluate interactions between MetAP1 and cell cycle regulatory proteins

• Data Collection and Analysis:

When interpreting results, it's important to distinguish between direct MetAP1 enzymatic effects and potential secondary consequences of protein processing disruption. Correlation between MetAP1 inhibition potency (IC₅₀) and G2/M arrest efficacy provides strong evidence for specificity, as demonstrated with compounds like IV-71 that show clear G2/M phase blockade while structurally similar but non-inhibitory compounds (e.g., IV-66B) produce no cell cycle effects .

How do mouse MetAP1 inhibitors compare with human MetAP1 inhibitors in terms of selectivity and potency?

Comparing mouse and human MetAP1 inhibitors is essential for translational research and appropriate experimental design. The current understanding of inhibitor cross-species reactivity offers important insights:

• Structural Basis for Inhibitor Selectivity:

  • Mouse and human MetAP1 share high sequence homology (~90% at amino acid level)

  • The active site architecture is highly conserved across mammalian species

  • X-ray crystallography studies of human MetAP1 with inhibitors like pyridine-2-carboxylic acid derivatives (e.g., compound A602) reveal a metal-dependent binding mechanism requiring a third cobalt ion, which is likely conserved in mouse MetAP1

• Cross-Species Inhibitor Activity:

  • Pyridine-2-carbamic acid core compounds developed for human MetAP1 typically demonstrate similar potency against mouse MetAP1

  • The high conservation of the active site suggests that selectivity profiles (MetAP1 vs. MetAP2) are maintained across species

• Representative Inhibitor Comparison:

*Estimated values based on typical human-mouse enzyme activity correlations; actual values require experimental validation

• Methodological Considerations for Inhibitor Studies:

  • When testing inhibitors across species, perform parallel enzymatic assays under identical conditions

  • For cell-based studies in mouse cell lines, evaluate effective concentrations independently from human cell line data

  • Consider potential differences in cellular uptake, metabolism, and efflux between species

  • For in vivo studies, mouse-specific pharmacokinetic evaluation is essential regardless of in vitro similarity

The inhibition mechanism appears consistent across species, with compounds like IV-43 showing competitive inhibition with respect to substrate binding by increasing Km without affecting Vmax. This mechanism is likely conserved between mouse and human enzymes due to the high structural conservation of the active site .

How does MetAP1 function relate to methionine restriction pathways and metabolic regulation?

The relationship between MetAP1 function and methionine restriction (MR) pathways represents an intriguing intersection between protein processing and metabolic regulation. Research in this area has revealed several important aspects:

• Methionine Restriction and Metabolic Benefits:

  • Methionine restriction extends lifespan and improves several markers of health in rodents

  • MR reduces body weight, fat mass, and improves glucose metabolism and respiration

  • These effects appear to involve multiple pathways including oxidative stress resistance

• MetAP1 in Methionine Metabolism:

  • MetAP1 functions to recycle methionine from newly synthesized proteins

  • Under methionine restriction conditions, efficient methionine recycling becomes more critical

  • The relationship between MetAP1 activity and cellular adaptation to MR remains an active area of investigation

• Experimental Evidence on MetAP-MR Interactions:

  • Studies using knockout models suggest complex relationships between methionine processing enzymes and MR benefits

  • Knockout of methionine sulfoxide reductase A (MsrA), which repairs oxidized methionine, does not prevent metabolic benefits of MR

  • Interestingly, MsrA knockout mice showed even greater response to MR in terms of weight loss and metabolic improvements

• Methodological Approaches to Study MetAP1-MR Interactions:

  a) Mouse Models and Dietary Interventions:

  • Compare wild-type and MetAP1-deficient mice (heterozygous or conditional knockout) under normal and methionine-restricted diets

  • Typical MR diet: 0.15% methionine as proportion of protein

  • Control diet: 0.86% methionine as proportion of protein

  • Monitor physiological parameters: body weight, body composition, food consumption, energy expenditure, glucose metabolism

  b) Cellular Models:

  • Culture cells in methionine-restricted media with or without MetAP1 inhibition/knockdown

  • Measure protein synthesis rates, methionine utilization, and cellular stress responses

  • Analyze changes in protein N-terminal modifications under different methionine availability conditions

  c) Biochemical Analyses:

  • Quantify free and protein-bound methionine levels

  • Measure MetAP1 activity and expression under different methionine concentrations

  • Evaluate oxidative stress markers and antioxidant responses

*Based on extrapolation from MsrA knockout studies, requires experimental validation

Understanding the precise role of MetAP1 in methionine restriction pathways will provide important insights into the fundamental mechanisms of aging, metabolism, and cellular adaptation to nutrient availability.

What controls should be included when designing experiments with MetAP1 inhibitors?

Designing robust experiments with MetAP1 inhibitors requires careful consideration of various controls to ensure specificity, rule out off-target effects, and accurately interpret results. The following comprehensive control strategy is recommended:

• Enzymatic Assay Controls:

  a) Positive and Negative Controls:

  • Positive control: Known MetAP1 inhibitor (e.g., pyridine-2-carboxylic acid derivatives)

  • Negative control: Structurally similar compound without MetAP1 inhibitory activity (e.g., IV-66B)

  • Enzyme-free control: Substrate in buffer to monitor spontaneous hydrolysis

  b) Specificity Controls:

  • Test inhibitors against purified MetAP2 to confirm selectivity

  • Include other metalloproteases to rule out broad-spectrum metalloprotease inhibition

  • Test with different metal cofactors (Co²⁺, Mn²⁺, Zn²⁺) to characterize cofactor-dependent inhibition

• Cell-Based Experiment Controls:

  a) Compound Controls:

  • Dose-response curves with at least 5-6 concentrations spanning IC₅₀

  • Vehicle control (DMSO at equivalent concentration)

  • Structurally related inactive analogs (e.g., IV-66B for pyridine derivatives)

  • Alternative MetAP1 inhibitor with different chemical scaffold

  b) Genetic Controls:

  • MetAP1 overexpression to demonstrate rescue of inhibition phenotype

  • MetAP1 knockdown to recapitulate inhibitor effects

  • MetAP1 catalytic mutants to distinguish enzymatic vs. scaffolding roles

  c) Target Engagement Controls:

  • Measure retention of N-terminal methionine on known MetAP1 substrates

  • Cellular thermal shift assay (CETSA) to confirm direct binding to MetAP1

  • Competition experiments with different inhibitors

• Experimental Timeline Considerations:

  • Perform acute vs. chronic inhibition studies to distinguish immediate vs. adaptive responses

  • Include time-course experiments to capture temporal dynamics of responses

  • For cell cycle studies, include synchronized and asynchronous populations

• Control Table for MetAP1 Inhibitor Experiments:

When analyzing cell cycle effects, the comparison between MetAP1-selective inhibitors (causing G2/M arrest) and MetAP2-selective inhibitors (causing G1 arrest) provides a particularly valuable control to confirm target specificity and distinct biological roles .

How can I design experiments to investigate the role of MetAP1 in specific physiological processes?

Designing experiments to elucidate MetAP1's role in specific physiological processes requires a multi-faceted approach combining genetic, pharmacological, and analytical techniques. Here's a comprehensive experimental design framework:

• Genetic Modulation Approaches:

  a) Loss-of-Function Studies:

  • Conditional knockout models (tissue-specific or inducible) using Cre-loxP system

  • RNA interference using validated siRNA/shRNA constructs

  • CRISPR/Cas9 genome editing to create complete or hypomorphic alleles

  Experimental design considerations:

  • Include heterozygous models to assess dose-dependency of MetAP1 function

  • Use inducible systems to distinguish developmental vs. adult-specific roles

  • When using RNAi, validate knockdown efficiency by both Western blot and enzymatic activity assays

  b) Gain-of-Function Studies:

  • Overexpression of wild-type MetAP1 in relevant cell types

  • Expression of constitutively active MetAP1 variants

  • Rescue experiments in knockdown/knockout backgrounds

  Control considerations:

  • Include catalytically inactive MetAP1 mutants to distinguish enzymatic vs. structural functions

  • Use equivalent expression levels of control proteins to account for non-specific effects of protein overexpression

• Pharmacological Manipulation:

  a) Selective Inhibition:

  • Utilize MetAP1-selective inhibitors (e.g., pyridine-2-carboxylic acid derivatives)

  • Implement dose-response and time-course experiments

  • Compare effects with MetAP2-selective inhibitors to distinguish isoform-specific roles

  Experimental design example for investigating MetAP1 in cell proliferation:

  • Treat cells with inhibitor concentrations spanning 0.1-10× IC₅₀ values

  • Monitor proliferation using multiple methods (cell counting, metabolic assays, BrdU incorporation)

  • Perform cell cycle analysis at multiple timepoints (12, 24, 48 hours)

  • Include washout experiments to assess reversibility of effects

• Functional Readouts Based on Physiological Process:

  a) Cell Cycle and Proliferation:

  • Flow cytometry for cell cycle distribution

  • Immunofluorescence for mitotic markers

  • Time-lapse imaging for division dynamics

  • Colony formation assays for long-term proliferative potential

  b) Metabolism and Stress Response:

  • Metabolic profiling (oxygen consumption, extracellular acidification)

  • Methionine utilization and recycling measurements

  • Oxidative stress markers (ROS levels, antioxidant response)

  c) Protein Processing and Quality Control:

  • Proteomics analysis of N-terminal modifications

  • Protein stability and turnover assays

  • Ubiquitination and proteasomal degradation assessment

• Experimental Design Matrix for MetAP1 Studies:

When designing these experiments, it's crucial to include appropriate controls and to consider the potential compensatory mechanisms that might arise, particularly from MetAP2. The combined use of both genetic and pharmacological approaches provides complementary evidence and helps distinguish specific from non-specific effects .

How can I address inconsistent results in MetAP1 activity assays?

Inconsistent results in MetAP1 activity assays can arise from multiple sources, including enzyme preparation, assay conditions, and detection methods. The following systematic approach will help identify and address common issues:

• Enzyme Quality and Preparation Issues:

  a) Troubleshooting Metal Cofactor Requirements:

  • MetAP1 is a metalloenzyme requiring Co²⁺, Mn²⁺, or other divalent metals for activity

  • Insufficient metal incorporation is a primary cause of low or variable activity

  • Solution: Pre-incubate enzyme with 0.1-0.5 mM CoCl₂ in activation buffer for 10-15 minutes before assay

  • Validation: Test activity with multiple metal concentrations and compare different metal ions (Co²⁺, Mn²⁺, Zn²⁺)

  b) Protein Stability Issues:

  • Freeze-thaw cycles can significantly reduce activity

  • Oxidation of metal-coordinating residues can inactivate the enzyme

  • Solution: Use fresh aliquots for each experiment; include reducing agents (0.5-1 mM DTT) in buffer

  • Validation: Compare activity of fresh preparations vs. stored aliquots

• Assay Condition Optimization:

  a) Buffer Composition Effects:

  • Buffer pH affects MetAP1 activity (optimal range: pH 7.5-8.0)

  • Ionic strength influences enzyme-substrate interactions

  • Solution: Systematically test pH range (7.0-8.5) and salt concentrations (50-200 mM NaCl)

  • Validation: Generate pH-activity and salt-activity profiles to identify optimal conditions

  b) Temperature and Incubation Time Considerations:

  • Reaction rates are temperature-dependent

  • Extended incubation may lead to enzyme inactivation

  • Solution: Maintain consistent temperature (25°C or 37°C); monitor time-course of reaction

  • Validation: Perform multiple time-point measurements to ensure linearity

• Substrate and Detection Issues:

  a) Substrate Quality and Concentration:

  • Peptide substrate degradation over time

  • Suboptimal substrate concentration relative to Km

  • Solution: Use fresh substrate preparations; perform substrate concentration series (0.1-5× Km)

  • Validation: Determine Km for each substrate lot; include internal standard peptides

  b) Detection Method Limitations:

  • Background interference in fluorescence assays

  • Detector sensitivity limitations

  • Solution: Include substrate-only and enzyme-only controls; optimize gain settings

  • Validation: Generate standard curves with product (e.g., AMC for fluorogenic assays)

• Systematic Troubleshooting Table:

• Data Normalization and Statistical Approaches:

  • Include internal reference standards in each assay

  • Use relative activity rather than absolute values when comparing across experiments

  • Apply appropriate statistical methods for outlier identification

  • Consider using robust statistical approaches less sensitive to outliers

By systematically addressing these potential sources of variability, researchers can significantly improve the consistency and reliability of MetAP1 activity assays. For particularly challenging samples or conditions, consider implementing a design of experiments (DOE) approach to efficiently optimize multiple parameters simultaneously .

How does MetAP1 function differ from MetAP2, and what are the implications for targeting either enzyme in research?

MetAP1 and MetAP2 share the fundamental function of N-terminal methionine excision but exhibit important differences in structure, substrate specificity, regulation, and physiological roles. Understanding these differences is critical for designing targeted research approaches:

• Structural and Enzymatic Differences:

  • Both MetAP1 and MetAP2 are metalloenzymes requiring divalent metal ions for activity

  • MetAP2 contains an additional N-terminal domain (~150 amino acids) absent in MetAP1

  • The catalytic domains share approximately 40% sequence identity

  • Crystal structures reveal subtle differences in active site architecture that can be exploited for selective inhibitor design

• Substrate Specificity and Cellular Targets:

  • Both enzymes preferentially process proteins with small side chains at the second position (Ala, Gly, Pro, Ser, Thr, Val)

  • MetAP2 shows higher activity toward substrates with larger residues at the second position

  • Specific protein targets differ between MetAP1 and MetAP2, though complete proteome-wide analyses are still developing

  • The distinct but overlapping substrate preferences suggest partial functional redundancy but also unique roles

• Cell Cycle and Proliferation Effects:

• Research Targeting Implications:

  a) Selective Inhibition Strategies:

  • MetAP1-selective inhibitors (pyridine-2-carboxylic acid derivatives) show >100-fold selectivity over MetAP2

  • Selective inhibition allows dissection of isoform-specific functions

  • For cell cycle studies, the distinct arrest points (G2/M for MetAP1, G1 for MetAP2) provide clear differentiation

  b) Genetic Targeting Considerations:

  • MetAP1 knockout is embryonic lethal in mice, requiring conditional approaches

  • MetAP2 knockout is also embryonic lethal but at different developmental stages

  • Tissue-specific knockouts or inducible systems can circumvent lethality

  • Simultaneous partial inhibition/knockdown of both may reveal synthetic interactions

  c) Experimental Design Recommendations:

  • For cancer research: Compare MetAP1 vs. MetAP2 inhibition across cell lines to identify selective vulnerabilities

  • For cell cycle studies: Use selective inhibitors in combination with synchronization to pinpoint exact phases affected

  • For substrate identification: Perform proteomics after selective inhibition of each isoform

  • For mechanistic studies: Rescue experiments with the alternative isoform can reveal functional overlap

• Therapeutic Relevance:

  • MetAP2 inhibitors (fumagillin derivatives) have been extensively studied as anti-angiogenic agents

  • MetAP1 inhibitors show promising anti-proliferative effects, particularly in leukemia models

  • The different cell cycle arrest points suggest potential synergy in combination therapy

  • MetAP1 may represent an underexplored target for conditions where MetAP2 inhibition has shown limitations

Understanding the distinct roles of MetAP1 and MetAP2 requires careful experimental design with appropriate controls and selective tools. The distinct cell cycle effects (G2/M for MetAP1 vs. G1 for MetAP2) provide a clear phenotypic readout to verify target engagement and specificity in cellular systems .

What are the emerging techniques for studying MetAP1 protein interactions and substrate specificity?

Recent advances in proteomics, structural biology, and cellular imaging have revolutionized the study of MetAP1 interactions and substrate specificity. These cutting-edge approaches provide deeper insights into MetAP1 function:

• Advanced Proteomics Approaches:

  a) N-terminomics for Substrate Identification:

  • Terminal Amine Isotopic Labeling of Substrates (TAILS) to enrich and identify protein N-termini

  • Subtiligase-based N-terminal capture methods

  • Combined Fractional Diagonal Chromatography (COFRADIC) for N-terminal peptide isolation

  Experimental design:

  • Compare N-terminal peptides from control vs. MetAP1-inhibited or depleted samples

  • Quantify N-terminal methionine retention on specific proteins

  • Cross-validate findings with recombinant protein processing assays

  b) Interaction Proteomics:

  • BioID or TurboID proximity labeling to identify proteins in the MetAP1 microenvironment

  • Affinity purification-mass spectrometry with structure-preserving crosslinkers

  • Thermal proteome profiling to identify proteins stabilized by MetAP1 binding

  Methodological considerations:

  • Include appropriate controls (BirA* alone, catalytically inactive MetAP1)

  • Perform comparative studies with MetAP2 to identify unique vs. shared interactors

  • Validate key interactions through reciprocal pulldowns and co-localization studies

• Structural and Biophysical Techniques:

  a) Cryo-EM for Multiprotein Complexes:

  • Visualize MetAP1 in complex with ribosomes and translation machinery

  • Analyze conformational changes upon substrate or inhibitor binding

  • Resolve dynamics of MetAP1 action during co-translational processing

  b) Hydrogen-Deuterium Exchange Mass Spectrometry (HDX-MS):

  • Map conformational changes upon substrate binding

  • Identify allosteric sites affected by inhibitor binding

  • Compare dynamics of mouse vs. human MetAP1 to understand cross-species differences

  c) Advanced NMR Approaches:

  • TROSY-based experiments for studying MetAP1-substrate interactions

  • 19F NMR with fluorinated amino acids to probe substrate binding

  • Real-time NMR to capture transient binding events

• Cellular Imaging and Spatial Localization:

  a) Super-resolution Microscopy:

  • Track MetAP1 localization relative to ribosomes during translation

  • Monitor co-localization with newly synthesized proteins

  • Visualize interactions with cell cycle regulatory machinery

  Experimental setup:

  • STORM or PALM imaging of fluorescently tagged MetAP1

  • Dual-color imaging with ribosomal markers or nascent peptide reporters

  • Live-cell imaging to capture dynamics during cell cycle progression

  b) Bimolecular Fluorescence Complementation (BiFC):

  • Visualize MetAP1 interactions with candidate partners in living cells

  • Monitor spatial and temporal aspects of these interactions

  • Compare interaction patterns between MetAP1 and MetAP2

• Computational and AI-Driven Approaches:

  a) Machine Learning for Substrate Prediction:

  • Train algorithms on known MetAP1 substrates to predict new targets

  • Develop models incorporating N-terminal sequence and structural features

  • Compare predicted substrates across species to identify evolutionarily conserved targets

  b) Molecular Dynamics Simulations:

  • Model MetAP1-substrate interactions at atomic resolution

  • Analyze conformational changes during catalytic cycle

  • Virtual screening for novel inhibitor discovery

• CRISPR-Based Functional Genomics:

  a) Genome-Wide Screens for Synthetic Interactions:

  • CRISPR knockout/activation screens in MetAP1-inhibited backgrounds

  • Identify genes that become essential when MetAP1 function is compromised

  • Discover pathways that compensate for or depend on MetAP1 activity

  b) Base Editing for Precise Manipulation:

  • Introduce specific mutations in MetAP1 substrate sites

  • Modify MetAP1 catalytic residues without disrupting protein structure

  • Engineer cells with altered MetAP1 substrate specificity

These emerging techniques provide complementary approaches to understand MetAP1 function from molecular to cellular levels. Integration of multiple methodologies will be particularly powerful, such as combining proteomics identification of substrates with structural studies of specific MetAP1-substrate complexes and cellular validation through imaging and functional assays .

What is the current understanding of MetAP1 in cancer and its potential as a therapeutic target?

The role of MetAP1 in cancer biology has gained significant attention following discoveries about its function in cell cycle regulation and cellular proliferation. Current research reveals multiple dimensions of MetAP1's relevance in cancer:

• Expression and Prognostic Significance:

  • MetAP1 expression is frequently altered in various cancer types

  • Higher expression often correlates with aggressive phenotypes and poor prognosis

  • The specific pattern varies by cancer type, suggesting context-dependent roles

  • Expression analysis should examine both mRNA and protein levels, as post-transcriptional regulation may be significant

• Functional Roles in Cancer Progression:

  a) Cell Cycle Regulation and Proliferation:

  • MetAP1 inhibition or knockdown induces G2/M arrest in multiple cancer cell types

  • This effect appears mechanistically distinct from MetAP2 inhibition (which causes G1 arrest)

  • The anti-proliferative effect is particularly pronounced in rapidly dividing cells

  b) Apoptosis Induction:

  • MetAP1 inhibitors induce apoptosis in leukemia and other cancer cell lines

  • This appears to be a consequence of sustained G2/M checkpoint activation

  • The apoptotic response varies across cancer types, suggesting biomarker-guided approaches may be needed

  c) Protein Processing and Cancer-Specific Substrates:

  • MetAP1 processes the N-terminal methionine of various proteins involved in cancer progression

  • Cancer cells may have increased dependence on MetAP1 due to elevated protein synthesis rates

  • Identifying cancer-relevant substrates remains an active area of investigation

• Pre-Clinical Evidence as a Therapeutic Target:

  a) Small Molecule Inhibitors:

  • Pyridine-2-carboxylic acid derivatives show selective inhibition of MetAP1 with IC₅₀ values in the low micromolar range

  • These compounds demonstrate anti-proliferative activity against cancer cell lines including:

    • Leukemia cell lines (particularly sensitive)

    • Breast cancer cells (e.g., MCF-7, with IC₅₀ ~2.3 μM for compound IV-43)

    • Cervical cancer cells (HeLa, with IC₅₀ ~2.5 μM for compound IV-43)

  b) Combination Therapy Potential:

  • The G2/M arrest mechanism suggests potential synergy with microtubule-targeting agents

  • Combined inhibition of MetAP1 and MetAP2 might overcome compensatory mechanisms

  • MetAP1 inhibition may sensitize cancer cells to DNA-damaging therapies

• Cancer Type-Specific Considerations:

• Future Directions and Challenges:

  a) Target Validation Strategies:

  • CRISPR-based knockout/knockdown in relevant cancer models

  • Patient-derived xenografts treated with selective inhibitors

  • Correlation of response with molecular features (expression, mutations)

  b) Biomarker Development:

  • Identify predictive biomarkers of response to MetAP1 inhibition

  • Develop pharmacodynamic markers to confirm target engagement

  • Establish molecular signatures of MetAP1 dependency

  c) Inhibitor Optimization Challenges:

  • Improve potency of current scaffolds (sub-micromolar IC₅₀ values)

  • Enhance pharmaceutical properties (solubility, stability, bioavailability)

  • Develop suitable formulations for in vivo delivery

The current evidence supports MetAP1 as a promising cancer target, particularly for hematological malignancies and specific solid tumors. The distinct mechanism of action (G2/M arrest) compared to many existing therapies suggests potential for addressing treatment-resistant cancers and developing novel combination strategies. Further research into cancer-specific dependencies and molecular mechanisms will be essential for translating these findings into clinical applications .

How do post-translational modifications affect MetAP1 activity and regulation?

Post-translational modifications (PTMs) play critical roles in regulating MetAP1 function, cellular localization, and interactions. Understanding these modifications provides insights into the dynamic regulation of this essential enzyme:

• Phosphorylation:

  a) Identified Sites and Kinases:

  • Multiple serine, threonine, and tyrosine phosphorylation sites have been identified in proteomic studies

  • Cell cycle-dependent kinases (CDKs) and mitotic kinases are implicated in regulatory phosphorylation

  • Phosphorylation patterns change during cell cycle progression, particularly at the G2/M transition

  b) Functional Consequences:

  • Phosphorylation can modulate catalytic activity through conformational changes

  • Some phosphorylation events affect protein-protein interactions

  • Cell cycle-regulated phosphorylation may connect MetAP1 activity to mitotic progression

  c) Experimental Approaches:

  • Phospho-specific antibodies to monitor modification status

  • Phosphomimetic (S/T→D/E) and phospho-deficient (S/T→A) mutants to study functional impact

  • In vitro kinase assays to identify direct phosphorylation events

• Redox Regulation:

  a) Metal Center Oxidation:

  • The catalytic metal center (Co²⁺/Mn²⁺) is susceptible to oxidation

  • Oxidative stress can inactivate MetAP1 through metal center disruption

  • This provides a potential mechanism linking cellular redox state to protein processing

  b) Cysteine Oxidation:

  • Conserved cysteine residues can undergo reversible oxidation

  • These modifications may serve as redox sensors, adjusting MetAP1 activity to cellular redox conditions

  c) Methodological Considerations:

  • Maintain reducing conditions during purification and assays

  • Use redox-active compounds to test sensitivity to oxidative inactivation

  • Monitor activity under different redox conditions to assess physiological regulation

• Ubiquitination and Protein Stability:

  a) Degradation Pathways:

  • MetAP1 levels are regulated by ubiquitin-proteasome system

  • Cell cycle-dependent fluctuations in protein levels occur in some cell types

  • Stress conditions can trigger rapid degradation or stabilization

  b) Research Approaches:

  • Proteasome inhibitors to assess turnover rates

  • Ubiquitination site mapping through proteomics

  • Half-life measurements under different cellular conditions

• Other Modifications:

  a) SUMOylation:

  • Potential sites for SUMO modification exist in MetAP1

  • SUMOylation could affect nuclear localization or chromatin association

  • May coordinate with phosphorylation in cell cycle regulation

  b) Acetylation:

  • N-terminal acetylation affects protein stability and interactions

  • Internal lysine acetylation may modulate activity or localization

  c) Methodological Approaches:

  • Site-directed mutagenesis of modification sites

  • Mass spectrometry to identify and quantify modifications

  • Inhibitors of modifying enzymes to assess functional relevance

• Integrated Regulation and Cross-talk:

• Future Research Directions:

  • Map the complete PTM landscape of MetAP1 using advanced proteomics

  • Identify condition-specific modification patterns (cell cycle, stress, differentiation)

  • Determine how PTMs affect substrate specificity

  • Develop tools to monitor real-time changes in modification status

  • Investigate cross-talk between different modifications

Understanding the complex PTM-based regulation of MetAP1 will provide important insights into how this essential enzyme integrates various cellular signals to coordinate protein processing with cell cycle progression, stress responses, and other physiological processes.

How does the role of MetAP1 in aging and longevity intersect with methionine restriction pathways?

The intersection of MetAP1 function with aging, longevity, and methionine restriction represents a fascinating frontier in metabolic research. Current evidence suggests complex relationships between protein methionine processing and longevity pathways:

• Methionine Restriction and Longevity:

  a) Established Benefits of Methionine Restriction (MR):

  • MR extends lifespan in multiple model organisms (yeast, flies, rodents)

  • MR improves several markers of health and metabolism in mammals

  • Specific benefits include reduced adiposity, improved glucose homeostasis, and enhanced insulin sensitivity

  • MR appears to modify oxidative stress resistance and inflammatory pathways

  b) Molecular Mechanisms:

  • Reduced mTOR signaling and enhanced autophagy

  • Altered mitochondrial function and biogenesis

  • Improved cellular stress resistance

  • Modifications in methyl donor availability affecting epigenetic regulation

• MetAP1's Potential Roles in Aging Processes:

  a) Protein Homeostasis (Proteostasis):

  • Proper N-terminal processing is critical for protein stability and function

  • Age-related decline in proteostasis is a hallmark of aging

  • MetAP1 function may become increasingly important under proteotoxic stress conditions

  b) Cell Cycle Regulation:

  • Dysregulated cell cycle control contributes to aging and senescence

  • MetAP1's role in G2/M transition may affect cellular aging processes

  • Senescent cell accumulation is implicated in numerous age-related pathologies

  c) Oxidative Stress Management:

  • MetAP enzymes interact with methionine-based antioxidant systems

  • The relationship with methionine sulfoxide reductases (Msr) suggests involvement in oxidative damage repair

  • MetAP1 may help coordinate protein synthesis with cellular redox status

• Experimental Evidence from MsrA Studies:

  a) Msr Family and Methionine Metabolism:

  • MsrA catalytically reduces oxidized methionine, playing a key role in its redox state

  • MsrA knockout mice (MsrA KO) exhibit altered responses to metabolic interventions

  b) Unexpected Findings with MR:

  • In contrast to expectations, MsrA KO mice showed full benefits of methionine restriction

  • MsrA KO mice actually exhibited more pronounced responses to MR in terms of:

    • Greater weight loss

    • More significant fat mass reduction

    • Potentially enhanced metabolic improvements

  • This suggests complex relationships between methionine processing enzymes and MR benefits

• Integrative Research Approaches:

  a) Genetic Models:

  • Compare MetAP1 conditional knockout or knockdown with MsrA and MsrB models

  • Create double mutant models (e.g., MetAP1+MsrA) to study pathway interactions

  • Use tissue-specific manipulations to identify key sites of MetAP1 function in longevity

  b) Dietary Interventions:

  • Combine MetAP1 modulation with different dietary regimens:

    • Standard diet (0.86% methionine)

    • Methionine restricted diet (0.15% methionine)

    • Caloric restriction

    • Time-restricted feeding

  c) Biomarker and Pathway Analysis:

  • Monitor methionine metabolism markers (homocysteine, SAM, SAH)

  • Assess oxidative stress parameters

  • Analyze protein modification patterns

  • Measure inflammatory cytokines and stress response proteins

• Experimental Design for Aging Studies:

The unexpected findings from MsrA knockout studies under methionine restriction highlight the need for careful investigation of the complex relationships between methionine processing enzymes in aging. Because MsrA KO mice showed enhanced rather than diminished response to MR, it's possible that MetAP1 manipulation might similarly reveal unexpected interactions with dietary interventions and aging pathways .

Understanding these relationships could potentially reveal novel therapeutic targets for age-related diseases and interventions to promote healthy aging.

What computational approaches are being developed to predict and analyze MetAP1 substrates?

The computational prediction and analysis of MetAP1 substrates represent a rapidly evolving area of research, combining bioinformatics, machine learning, and structural biology approaches. These computational tools complement experimental methods and accelerate the discovery of novel MetAP1 substrates and functions:

• Sequence-Based Prediction Algorithms:

  a) N-terminal Sequence Analysis:

  • Classical prediction rules based on the penultimate residue (P1' position)

  • MetAP1 typically processes substrates with small side chains at P1' (Ala, Gly, Pro, Ser, Thr, Val)

  • Machine learning models incorporating wider sequence context (positions P1' to P3')

  • Integration of species-specific sequence preferences from experimental data

  b) Advanced Machine Learning Approaches:

  • Deep learning models trained on proteomics datasets of N-terminal modifications

  • Neural networks that consider sequence composition beyond the immediate N-terminus

  • Ensemble methods combining multiple prediction algorithms to improve accuracy

  • Transfer learning from human to mouse data to enhance cross-species predictions

  c) Implementation Examples:

  • TermiNator3: Predicts N-terminal modifications including methionine excision

  • MetaPred: Machine learning-based predictor of MetAP substrate specificity

  • N-TERMINAL: Deep learning approach for comprehensive N-terminal modification prediction

• Structural and Molecular Dynamics Approaches:

  a) Protein-Protein Docking:

  • Structure-based docking of potential substrates to MetAP1 active site

  • Incorporation of metal ion coordination in docking algorithms

  • Scoring functions optimized for MetAP1-substrate interactions

  b) Molecular Dynamics Simulations:

  • Analyze conformational changes during substrate binding and catalysis

  • Predict binding energy and catalytic efficiency

  • Identify allosteric sites affecting substrate specificity

  • Examine species-specific differences between mouse and human MetAP1

  c) Fragment-Based Virtual Screening:

  • Model N-terminal peptide fragments to identify high-affinity substrates

  • Screen proteome databases for matching N-terminal sequences

  • Prioritize candidates based on structural compatibility and binding energy

• Integrative Computational Approaches:

  a) Multi-omics Data Integration:

  • Combine proteomics, transcriptomics, and metabolomics data to identify MetAP1 substrates

  • Correlate N-terminal modification patterns with MetAP1 expression levels

  • Use network analysis to identify functional clusters of MetAP1 substrates

  b) Systems Biology Models:

  • Mathematical modeling of MetAP1 in the context of protein synthesis and maturation

  • Predict system-wide effects of MetAP1 inhibition or knockout

  • Identify potential compensatory mechanisms and feedback loops

  c) Pathway Enrichment Analysis:

  • Functional annotation of predicted MetAP1 substrates

  • Identification of cellular processes enriched for MetAP1 processing

  • Cross-species conservation analysis of substrate networks

• Computational Tools for Experimental Design:

  a) CRISPR Guide RNA Design:

  • Algorithms to identify optimal targeting sites in MetAP1

  • Tools to predict and minimize off-target effects

  • Design of guides for base editing to create specific mutations

  b) Peptide Substrate Design:

  • Computational design of optimized fluorogenic substrates

  • Prediction of cleavage efficiency and specificity

  • Design of control peptides resistant to MetAP1 processing

  c) Small Molecule Screening Approaches:

  • Virtual screening for novel MetAP1 inhibitors

  • Pharmacophore modeling based on known inhibitors

  • Prediction of selectivity profiles (MetAP1 vs. MetAP2)

• Emerging Computational Frontiers:

The integration of these computational approaches with experimental validation creates a powerful framework for understanding MetAP1 substrate specificity, functional impact, and potential therapeutic applications. As multi-omics datasets continue to expand, these computational methods will become increasingly accurate and biologically relevant for predicting MetAP1 substrates across different species, tissues, and disease states.