JMJD7 Human

What is JMJD7 and what are its primary biological functions?

JMJD7 (Jumonji domain-containing protein 7) is a human Fe(II) and 2-oxoglutarate (2OG) dependent oxygenase that belongs to the JmjC family of enzymes . It functions as a bifunctional enzyme with dual catalytic capabilities: (1) as a 2-oxoglutarate-dependent monooxygenase that catalyzes (S)-stereospecific hydroxylation at C-3 of lysine residues in specific substrate proteins, and (2) as an endopeptidase that cleaves histone N-terminal tails . Its primary known substrates are Developmentally Regulated GTP Binding Proteins 1 and 2 (DRG1/2), where it hydroxylates 'Lys-22' of DRG1 and 'Lys-21' of DRG2, promoting their interaction with ribonucleic acids (RNA) . This post-translational modification may play a significant role in protein biosynthesis by modifying the translation machinery .

How is JMJD7 structurally organized and what domains are critical for its function?

JMJD7 possesses an unusual dimeric JmjC fold that has been conserved through evolution from Drosophila melanogaster to humans . The full-length human JMJD7 protein consists of 316 amino acids . As a member of the JmjC family, JMJD7 contains the characteristic JmjC domain responsible for its catalytic activity . This domain coordinates an Fe(II) ion in the active site, which is essential for the enzyme's oxygenase activity . The protein structure facilitates the binding of 2-oxoglutarate as a co-substrate and molecular oxygen, which are necessary for the hydroxylation reaction . The specific molecular architecture enables JMJD7 to recognize and modify lysine residues in a stereospecific manner, while also potentially supporting its reported endopeptidase function .

What cofactors are required for JMJD7 enzymatic activity?

JMJD7 requires specific cofactors for its catalytic activity. As a 2-oxoglutarate (2OG) dependent oxygenase, it uses:

• Fe(II) (ferrous iron) - Essential for the catalytic mechanism as it coordinates with 2OG and molecular oxygen in the active site .

• 2-oxoglutarate (α-ketoglutarate) - Acts as a co-substrate that undergoes oxidative decarboxylation during the hydroxylation reaction .

• Molecular oxygen (O₂) - Required for the oxidation reaction where one oxygen atom is incorporated into the lysyl residue and the other into succinate .

The enzyme's dependence on these cofactors places it within the broader family of non-heme iron oxygenases that catalyze a wide range of oxidative modifications on proteins and nucleic acids . These cofactor requirements are important considerations when designing in vitro assays to study JMJD7 activity, as the enzyme would be inactive without proper supplementation of Fe(II) and 2OG .

What are the recommended methods for expressing and purifying recombinant human JMJD7?

For effective expression and purification of recombinant human JMJD7, researchers should consider the following methodological approach:

• Expression System: Escherichia coli has been successfully used for JMJD7 expression . The bacterial expression system typically employs a pET vector with an N-terminal His-tag for purification purposes.

• Protein Construct: The full-length human JMJD7 (1-316 amino acids) can be expressed with an N-terminal His-tag for purification purposes . The tag sequence typically includes MGSSHHHHHHSSGLVPRGS followed by the JMJD7 sequence .

• Expression Conditions: Optimal expression is generally achieved by inducing protein production at lower temperatures (16-18°C) after the culture reaches mid-log phase to enhance proper protein folding.

• Purification Strategy:

  • Immobilized metal affinity chromatography (IMAC) using Ni-NTA resin to capture the His-tagged protein

  • Size exclusion chromatography to achieve >90% purity as indicated in commercial preparations

  • Anion exchange chromatography may be used as an additional purification step

• Buffer Considerations: Purification buffers should contain components that maintain JMJD7 stability, typically including:

  • HEPES or Tris buffer (pH 7.5-8.0)

  • NaCl (typically 50-300 mM)

  • Glycerol (5-10%)

  • Reducing agent (such as DTT or β-mercaptoethanol)

• Quality Control: The purified protein should be assessed by SDS-PAGE to confirm >90% purity and by mass spectrometry to verify the correct molecular weight and sequence .

How can researchers effectively assay JMJD7 enzymatic activity?

Researchers can effectively assay JMJD7 enzymatic activity using several complementary methods:

• Mass Spectrometry-Based Assays:

  • MALDI-TOF MS or LC-MS/MS can be used to detect hydroxylated peptide products after incubation of JMJD7 with synthetic peptide substrates derived from DRG1 or DRG2 .

  • These methods allow for precise determination of the site of hydroxylation and quantification of product formation.

• Oxygen Consumption Assays:

  • Clark-type oxygen electrode measurements can monitor oxygen consumption during the hydroxylation reaction.

  • This provides real-time kinetic data on enzyme activity.

• 2-Oxoglutarate Decarboxylation Assays:

  • Monitoring the conversion of [1-14C]-labeled 2OG to 14CO2 can be used to quantify enzyme activity.

  • This assay is particularly useful for high-throughput screening of potential inhibitors.

• Fluorescence-Based Assays:

  • Fluorescently labeled peptide substrates can be used to develop FRET-based assays or fluorescence polarization assays.

  • These methods allow for real-time monitoring of substrate modification.

• Coupled Enzyme Assays:

  • The succinate produced during the hydroxylation reaction can be coupled to succinate dehydrogenase activity and monitored spectrophotometrically.

• Reaction Conditions:

  • Standard reaction buffer: 50 mM HEPES pH 7.5, 50-100 mM NaCl

  • Required components: Fe(II) (typically 50-100 μM), 2-oxoglutarate (typically 200-500 μM), and ascorbate (typically 1-2 mM) as a reducing agent

  • Substrate: DRG1/2-derived peptides containing the target lysine residue

  • Temperature: 37°C for physiologically relevant conditions

When studying JMJD7's endopeptidase activity, researchers can use histone peptides or nucleosome substrates with methylated arginine or lysine residues and analyze the cleavage products by gel electrophoresis or mass spectrometry .

What are the key considerations for designing peptide substrates for JMJD7 studies?

When designing peptide substrates for JMJD7 studies, researchers should consider several critical factors:

• Sequence Context: The peptide should contain the target lysine residue (Lys-22 in DRG1 or Lys-21 in DRG2) with sufficient flanking residues to maintain the recognition motif . Typically, peptides of 15-25 amino acids in length are used to ensure proper enzyme recognition.

• Substrate Specificity: JMJD7 has been shown to have a relatively narrow substrate scope compared to other JmjC hydroxylases . Therefore, maintaining the native sequence around the target lysine is crucial for enzyme recognition and activity.

• Lysine Derivatives: When exploring substrate selectivity, various lysine derivatives can be incorporated:

  • (E)-dehydrolysine has been identified as an efficient alternative to lysine for JMJD7-catalyzed C3-hydroxylation

  • γ-Thialysine and γ-azalysine can undergo C3-hydroxylation followed by degradation to formylglycine

  • Methionine and homomethionine residues can replace lysine and undergo S-oxidation catalyzed by JMJD7

• Potential Inhibitory Derivatives: Cysteine/selenocysteine replacements at the lysine position have been shown to efficiently inhibit JMJD7 via cross-linking mechanisms . These can be useful for inhibition studies.

• Peptide Modifications:

  • N-terminal acetylation or biotinylation can be incorporated for detection or immobilization purposes

  • C-terminal amidation may improve peptide stability

  • Fluorescent labels can be introduced for assay development

• Peptide Purity: High purity (>95%) peptides are recommended to ensure reliable and reproducible results in enzymatic assays.

• Solubility Considerations: The hydrophobicity/hydrophilicity balance should be considered to ensure good solubility in aqueous buffers used for enzymatic assays.

How does JMJD7 achieve stereospecific (3S)-hydroxylation of lysine residues?

JMJD7 achieves stereospecific (3S)-hydroxylation of lysine residues through a precisely orchestrated catalytic mechanism that involves:

• Coordination Chemistry: The active site of JMJD7 contains an Fe(II) center that is coordinated by a facial triad of two histidine residues and one aspartate/glutamate residue . This metal center also coordinates the 2-oxoglutarate co-substrate and a water molecule, which is displaced upon substrate binding.

• Substrate Positioning: The lysine residue of the substrate is positioned in the active site such that the C3 position is oriented toward the reactive iron-oxo species that forms during the catalytic cycle . This precise positioning is critical for the stereospecificity of the reaction.

• Reaction Mechanism:

  • The Fe(II) center binds molecular oxygen, leading to the oxidative decarboxylation of 2OG

  • This generates a highly reactive Fe(IV)=O intermediate (ferryl species)

  • The ferryl species abstracts a hydrogen atom from the C3 position of the lysine side chain

  • Subsequent radical rebound results in hydroxylation specifically at the C3 position with (S) stereochemistry

• Structural Constraints: The three-dimensional architecture of JMJD7's active site creates steric constraints that only allow for the formation of the (S) stereoisomer at C3 . The unusual dimeric JmjC fold of JMJD7 likely contributes to this stereoselectivity .

• Evolutionary Conservation: The stereospecific hydroxylation mechanism appears to be conserved from Drosophila melanogaster to humans, suggesting its biological importance .

This precise stereochemical control distinguishes JMJD7 from other hydroxylases and highlights the exquisite specificity of enzyme-catalyzed reactions in biological systems.

What structural features distinguish JMJD7 from other JmjC domain-containing proteins?

JMJD7 possesses several distinctive structural features that set it apart from other JmjC domain-containing proteins:

• Dimeric Architecture: JMJD7 exhibits an unusual dimeric JmjC fold that has been conserved evolutionarily from Drosophila melanogaster to humans . This dimeric organization is uncommon among JmjC domain-containing proteins, which typically function as monomers.

• Bifunctional Catalytic Capacity: Unlike most JmjC proteins that catalyze either hydroxylation or demethylation reactions, JMJD7 uniquely possesses dual catalytic functions as both a hydroxylase and an endopeptidase . This bifunctionality suggests structural elements that can accommodate both reaction types.

• Substrate Binding Pocket: JMJD7 has a relatively narrow substrate scope compared to other JmjC hydroxylases , indicating a more restrictive substrate binding pocket that is highly selective for specific lysine-containing sequences.

• Active Site Configuration: The active site of JMJD7 must be configured to:

  • Allow stereospecific C3-hydroxylation of lysine residues with (S) stereochemistry

  • Accommodate alternative substrates like (E)-dehydrolysine, γ-thialysine, and γ-azalysine

  • Enable S-oxidation of methionine-containing peptides

  • Support peptidase activity on histone tails

• Domain Organization: The full-length human JMJD7 protein (316 amino acids) likely contains additional structural elements beyond the core JmjC domain that contribute to its unique functional properties and substrate recognition.

• Inhibitor Susceptibility: JMJD7's structure enables efficient inhibition via cross-linking with cysteine/selenocysteine-containing peptides , suggesting accessible reactive groups within its structure that can be targeted for inhibition studies.

These distinct structural features make JMJD7 an intriguing subject for structural biology studies and highlight the functional diversity within the JmjC protein family.

What are the emerging therapeutic implications of targeting JMJD7 in human disease?

The emerging therapeutic implications of targeting JMJD7 in human disease are multifaceted, though research in this area is still developing:

• Translational Regulation: JMJD7 hydroxylates DRG1/2 translation factors, promoting their interaction with RNA . This suggests potential roles in regulating protein synthesis and translational fidelity. Targeting JMJD7 could therefore impact diseases characterized by dysregulated translation, including certain cancers and neurodegenerative disorders.

• Epigenetic Modulation: JMJD7's reported endopeptidase activity on histone tails implies a role in epigenetic regulation. As epigenetic dysregulation is implicated in numerous diseases including cancer, developmental disorders, and inflammatory conditions, JMJD7 inhibitors could potentially modulate these pathological processes.

• Inhibitor Design Approaches:

  • The relatively narrow substrate scope of JMJD7 compared to other JmjC hydroxylases suggests that selective inhibitors could be developed with minimal off-target effects

  • DRG1 variants possessing cysteine/selenocysteine instead of lysine efficiently inhibit JMJD7 via cross-linking , providing a template for peptidomimetic inhibitor design

  • Understanding the substrate selectivity profile of JMJD7 enables rational design of selective small-molecule inhibitors

• Structural Conservation: The conservation of JMJD7's unusual dimeric structure from Drosophila to humans suggests fundamental biological importance and potentially conserved disease relevance across species, making it a viable target for therapeutic development.

• Methodological Considerations: Research into JMJD7-targeted therapeutics would benefit from:

  • Development of cell-permeable inhibitors that maintain selectivity for JMJD7

  • Establishment of cellular assays to monitor JMJD7 activity in disease-relevant contexts

  • Investigation of JMJD7 expression patterns in various pathological states

  • Elucidation of the biological consequences of JMJD7 inhibition in vivo

As research on JMJD7 continues to evolve, its potential as a therapeutic target will likely become more clearly defined, particularly in diseases involving translational regulation or epigenetic dysregulation.

How can researchers address the dual functionality of JMJD7 in experimental designs?

Addressing the dual functionality of JMJD7 (hydroxylase and endopeptidase activities) in experimental designs requires careful methodological considerations:

• Activity-Specific Assay Conditions:

  • For hydroxylase activity: Optimize Fe(II), 2-oxoglutarate, and ascorbate concentrations, typically using DRG1/2-derived peptides as substrates

  • For endopeptidase activity: Use histone peptides or nucleosomes with methylated arginine/lysine residues as substrates, without requiring 2OG or Fe(II)

  • Control experiments should be performed with and without cofactors to distinguish between the two activities

• Mutation-Based Approaches:

  • Engineer JMJD7 variants with mutations in residues predicted to be essential for one activity but not the other

  • For example, mutations in the Fe(II)-binding residues would likely abolish hydroxylase activity while potentially preserving endopeptidase function

  • These mutants can be used to dissect the biological relevance of each function independently

• Selective Inhibition:

  • Develop or utilize inhibitors that selectively target one activity

  • 2OG competitive inhibitors would affect hydroxylase activity specifically

  • Protease inhibitors might selectively impair endopeptidase function

• Temporal Separation:

  • Design time-course experiments to determine if the two activities occur sequentially or independently

  • This could reveal whether one activity might regulate the other

• Substrate Engineering:

  • Design substrates that can only undergo one type of modification

  • For hydroxylase activity: Use peptides containing lysine analogs that cannot be cleaved but can be hydroxylated

  • For endopeptidase activity: Design peptides with bonds resistant to hydroxylation but susceptible to proteolytic cleavage

• Cellular Assays:

  • Develop cellular reporter systems that can distinguish between the two activities

  • For example, fluorescent reporters with different readouts for hydroxylation versus proteolytic cleavage

• Mass Spectrometry Analysis:

  • Implement comprehensive mass spectrometry approaches that can simultaneously detect both hydroxylation events and proteolytic cleavage products

  • This allows for monitoring both activities in a single experimental setup

By integrating these approaches, researchers can effectively address the dual functionality of JMJD7 and gain insights into the potential interplay between its hydroxylase and endopeptidase activities.

What strategies can overcome the challenges in developing selective inhibitors for JMJD7?

Developing selective inhibitors for JMJD7 presents several challenges, including distinguishing it from other JmjC domain-containing proteins and addressing its dual functionality. The following strategies can help overcome these challenges:

• Structure-Based Rational Design:

  • Utilize the unusual dimeric JmjC fold of JMJD7 as a structural advantage for designing inhibitors that specifically recognize this dimeric architecture

  • Focus on unique features of the JMJD7 active site that differ from other JmjC enzymes to enhance selectivity

  • Employ computational docking and molecular dynamics simulations to predict binding modes and optimize inhibitor structures

• Peptide-Based Approaches:

  • Design inhibitors based on the observation that DRG1 variants with cysteine/selenocysteine efficiently inhibit JMJD7 via cross-linking

  • Develop peptidomimetics that maintain the key recognition elements of the natural substrates while incorporating reactive groups for covalent modification

  • Create peptide libraries with systematic variations to identify optimized binding sequences

• Focused Substrate Analog Libraries:

  • Leverage the relatively narrow substrate scope of JMJD7 to design substrate analogs that specifically target JMJD7

  • Incorporate γ-thialysine, γ-azalysine, or other lysine derivatives that have shown activity with JMJD7

  • Develop transition state analogs that mimic the geometry of the reaction intermediate

• Bifunctional Inhibitor Design:

  • Create inhibitors capable of simultaneously targeting both the hydroxylase and endopeptidase activities

  • Design molecules that can bind to both active sites in the dimeric JMJD7 structure

  • This approach may enhance selectivity by requiring both binding events for effective inhibition

• Allosteric Inhibition:

  • Target allosteric sites unique to JMJD7 rather than the conserved catalytic site

  • This approach can avoid competition with the high cellular concentrations of 2OG and provide better selectivity

• Fragment-Based Drug Discovery:

  • Screen fragment libraries against JMJD7 to identify novel chemical scaffolds with good ligand efficiency

  • Link or grow promising fragments to develop potent and selective inhibitors

  • This approach may identify binding modes not predicted by substrate-based design

• High-Throughput Screening with Selective Counter-Screening:

  • Develop a screening cascade that first identifies JMJD7 inhibitors, then counter-screens against related JmjC enzymes

  • Implement biochemical assays that specifically detect the unique activities of JMJD7

• Exploiting Co-factor Binding Differences:

  • Design inhibitors that exploit subtle differences in how JMJD7 binds Fe(II) and 2OG compared to other JmjC enzymes

  • Focus on the coordination geometry and electronic properties of the metal center

These strategies, either individually or in combination, can help overcome the challenges in developing selective JMJD7 inhibitors with potential therapeutic applications.

How can contradictory findings about JMJD7 functions be reconciled in experimental work?

Reconciling contradictory findings about JMJD7 functions requires systematic experimental approaches that address potential sources of discrepancy:

• Standardization of Experimental Conditions:

  • Establish consistent protocols for JMJD7 expression, purification, and activity assays

  • Carefully control cofactor concentrations (Fe(II), 2OG) and reaction conditions (pH, temperature, buffer composition)

  • Use full-length JMJD7 (1-316 aa) with defined tags and purification methods

• Cross-validation with Multiple Techniques:

  • Employ orthogonal assay methods to verify findings (e.g., mass spectrometry, NMR, enzymatic assays)

  • Use both in vitro biochemical approaches and cellular/in vivo systems

  • Validate key findings across different experimental platforms and in multiple laboratories

• Comprehensive Substrate Profiling:

  • Systematically test reported substrates (DRG1/2, histones) under identical conditions

  • Include positive and negative controls to validate assay performance

  • Consider context-dependent effects by testing substrates in different sequence contexts or structural states

• Structural Biology Approaches:

  • Determine crystal structures of JMJD7 in complex with different substrates

  • Use structural information to explain mechanistic differences in substrate recognition

  • Investigate how the dimeric structure of JMJD7 might influence its multifunctional nature

• Genetic and Cellular Validation:

  • Perform gene knockout/knockdown studies followed by comprehensive omics analyses

  • Implement rescue experiments with wild-type and mutant JMJD7 variants

  • Use CRISPR-Cas9 to introduce specific mutations that selectively affect one function

• Domain and Mutation Analysis:

  • Create truncated versions and point mutants of JMJD7 to map functional domains

  • Identify residues critical for each reported activity

  • Determine if different functions map to distinct structural regions

• Context-Dependent Regulation:

  • Investigate whether JMJD7 functions are regulated by:

    • Post-translational modifications

    • Protein-protein interactions

    • Cellular localization

    • Metabolic state (oxygen levels, 2OG availability)

• Systematic Review and Meta-analysis:

  • Compile all published data on JMJD7 functions

  • Analyze methodological differences that might explain contradictory results

  • Identify consensus findings and establish confidence levels for different reported functions

• Collaborative Verification:

  • Establish collaborative networks where multiple laboratories test the same hypotheses

  • Share reagents, protocols, and cell lines to minimize technical variability

By implementing these approaches, researchers can systematically address contradictions in the literature and develop a more coherent understanding of JMJD7's multifunctional nature, potentially revealing context-dependent regulation of its diverse activities .

What are the most promising approaches for identifying the complete physiological substrate repertoire of JMJD7?

Identifying the complete physiological substrate repertoire of JMJD7 requires innovative and comprehensive approaches:

• Proteome-Wide Hydroxylation Profiling:

  • Implement mass spectrometry-based proteomics to identify proteins containing 3S-hydroxylysine modifications

  • Use stable isotope labeling (SILAC) combined with JMJD7 knockout/knockdown to quantitatively compare hydroxylation levels

  • Employ enrichment strategies (e.g., antibodies against hydroxylysine or chemical tagging approaches) to enhance detection sensitivity

• Proximity-Based Labeling:

  • Engineer JMJD7 fusion proteins with proximity labeling enzymes (BioID, APEX2, TurboID)

  • Identify proteins that physically interact with JMJD7 in cellular contexts

  • Validate candidate interactors as potential substrates using in vitro assays

• Structural Motif Analysis:

  • Analyze known JMJD7 substrates (DRG1/2) to define consensus recognition motifs

  • Perform bioinformatic screening of the proteome for proteins containing similar sequence motifs

  • Validate predicted substrates experimentally using synthetic peptides and recombinant proteins

• Functional Genomics Approaches:

  • Conduct CRISPR screens to identify genes that genetically interact with JMJD7

  • Perform transcriptomics and proteomics analyses in JMJD7-deficient cells to identify dysregulated pathways

  • Use these pathways to guide the search for direct JMJD7 substrates

• In Vitro Peptide Library Screening:

  • Develop peptide libraries based on the relatively narrow substrate scope of JMJD7

  • Screen these libraries using high-throughput hydroxylation assays

  • Validate hits in cellular contexts

• Activity-Based Protein Profiling:

  • Design activity-based probes that covalently label JMJD7 substrates during the catalytic process

  • Use these probes to capture and identify substrate proteins

• Comparative Evolutionary Analysis:

  • Leverage the conservation of JMJD7 from Drosophila to humans

  • Identify proteins with conserved lysine residues in potential JMJD7 recognition motifs across species

  • Focus validation efforts on evolutionarily conserved candidate substrates

• Integration of Multiple Omics Approaches:

  • Combine proteomics, transcriptomics, and metabolomics data from JMJD7 perturbation experiments

  • Use network analysis to identify central nodes that might represent direct JMJD7 substrates

  • Apply machine learning algorithms to predict high-confidence candidate substrates

• Investigation of Both Enzymatic Activities:

  • Consider both the hydroxylase and endopeptidase activities when searching for substrates

  • Develop assays that can simultaneously detect both types of modifications

These approaches, particularly when used in combination, offer promising strategies for comprehensively mapping the physiological substrate repertoire of JMJD7, which will provide crucial insights into its biological functions and potential therapeutic applications.

How might advanced structural biology techniques reveal new insights into JMJD7 catalytic mechanisms?

Advanced structural biology techniques hold tremendous potential for revealing new insights into JMJD7 catalytic mechanisms:

• Cryo-Electron Microscopy (Cryo-EM):

  • Capture JMJD7 in different conformational states during catalysis

  • Visualize the dimeric architecture of JMJD7 at near-atomic resolution

  • Reveal how substrate binding induces conformational changes that facilitate catalysis

  • Image JMJD7 in complex with larger substrate proteins (DRG1/2) that may be challenging for crystallography

• Time-Resolved X-ray Crystallography:

  • Capture intermediates in the catalytic cycle using techniques like temperature-jump or photo-triggering

  • Visualize the formation and decay of the Fe(IV)=O intermediate during hydroxylation

  • Map the structural changes occurring during substrate binding, catalysis, and product release

• Nuclear Magnetic Resonance (NMR) Spectroscopy:

  • Investigate the dynamics of JMJD7 in solution

  • Monitor chemical shift perturbations upon substrate binding to map interaction interfaces

  • Use relaxation dispersion techniques to detect and characterize transient states during catalysis

  • Employ 19F-NMR with strategically placed fluorine labels to track conformational changes

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

  • Map regions of JMJD7 that undergo conformational changes upon substrate binding

  • Compare dynamics in the presence of different substrates to understand selectivity

  • Investigate how the unusual dimeric structure influences protein dynamics and catalysis

• Single-Molecule Techniques:

  • Use single-molecule FRET to monitor conformational changes during catalysis

  • Apply optical tweezers or atomic force microscopy to investigate mechanical properties

  • Employ single-molecule enzymology to detect potential cooperativity between monomers in the dimeric structure

• Integrative Structural Biology:

  • Combine multiple structural techniques (X-ray crystallography, Cryo-EM, NMR, SAXS)

  • Develop comprehensive structural models that incorporate both static and dynamic information

  • Create computational models of the complete catalytic cycle

• Neutron Crystallography:

  • Visualize hydrogen atom positions to elucidate proton transfer steps during catalysis

  • Distinguish between closely related chemical groups that differ mainly in hydrogen positioning

  • Provide insights into the hydrogen bonding networks critical for substrate recognition

• Advanced Computational Approaches:

  • Apply molecular dynamics simulations to model conformational changes during catalysis

  • Use quantum mechanics/molecular mechanics (QM/MM) calculations to model the energetics of the reaction

  • Employ machine learning approaches to predict binding modes for novel substrates

These advanced structural biology techniques, particularly when applied in combination, have the potential to reveal unprecedented insights into JMJD7's catalytic mechanisms, including how it achieves stereospecific hydroxylation, how the dimeric structure influences function, and how it can catalyze both hydroxylation and proteolytic reactions .

What are the emerging connections between JMJD7 and cellular stress response pathways?

The connections between JMJD7 and cellular stress response pathways represent an emerging area of research with several intriguing directions:

• Oxygen Sensing and Hypoxia Response:

  • As an Fe(II) and 2-oxoglutarate dependent oxygenase, JMJD7 activity is inherently oxygen-dependent

  • Under hypoxic conditions, JMJD7 activity may be compromised, potentially affecting translation via its effects on DRG1/2

  • Investigate whether JMJD7 functions as an oxygen sensor in specific cellular contexts

  • Explore potential cross-talk with hypoxia-inducible factor (HIF) pathways

• Translational Stress Responses:

  • JMJD7 hydroxylates translation factors DRG1/2, affecting their interaction with RNA

  • This suggests JMJD7 may play a role in regulating translation during stress conditions

  • Examine whether JMJD7 activity changes during different types of cellular stress (ER stress, oxidative stress, nutrient deprivation)

  • Investigate if JMJD7-mediated modifications of DRG1/2 influence stress granule formation or composition

• Epigenetic Responses to Stress:

  • JMJD7's reported endopeptidase activity on histone tails implies a potential role in stress-induced epigenetic reprogramming

  • Study whether JMJD7-mediated histone cleavage is regulated by stress conditions

  • Investigate if "tailless nucleosomes" generated by JMJD7 play specific roles in stress-responsive gene expression programs

• Metabolic Stress:

  • As a 2OG-dependent enzyme, JMJD7 activity may be sensitive to fluctuations in cellular metabolism

  • Changes in 2OG levels during metabolic stress could modulate JMJD7 function

  • Explore connections between JMJD7 and pathways sensing energy status (AMPK) or nutrient availability (mTOR)

• Iron Homeostasis:

  • JMJD7 requires Fe(II) for catalytic activity

  • Iron deficiency or perturbation of iron homeostasis could affect JMJD7 function

  • Investigate whether JMJD7 participates in cellular responses to iron limitation or iron overload

• Oxidative Stress:

  • The catalytic mechanism of JMJD7 involves reactive oxygen species as intermediates

  • Explore whether JMJD7 contributes to cellular redox homeostasis

  • Examine if JMJD7 activity is modulated by oxidative stress conditions

• Unfolded Protein Response (UPR):

  • Post-translational modifications by JMJD7 might influence protein folding or quality control

  • Investigate potential connections between JMJD7 and the unfolded protein response pathways

  • Examine if JMJD7 expression or activity is regulated during ER stress

• Experimental Approaches for Investigation:

  • Stress-specific transcriptomics and proteomics in JMJD7-deficient cells

  • Assessment of JMJD7 expression, localization, and activity under different stress conditions

  • Identification of stress-specific JMJD7 interactors and substrates

  • Phenotypic characterization of JMJD7-deficient cells under various stress conditions

These emerging connections between JMJD7 and cellular stress response pathways suggest important roles for this enzyme in cellular adaptation to environmental challenges, potentially opening new therapeutic avenues targeting stress-related pathologies.