UBE2J2 Human

What is UBE2J2 and what is its primary function in human cells?

UBE2J2, also known as Ubc6, is a human ubiquitin-conjugating enzyme (E2) that functions primarily in the ubiquitination of proteins at the endoplasmic reticulum (ER). It belongs to the J subfamily of E2s in humans, alongside UBE2J1 . UBE2J2 works as a critical component in the ubiquitin-proteasome system, particularly in ER-associated degradation (ERAD) pathways, where it facilitates the tagging of misfolded or unwanted proteins with ubiquitin for subsequent degradation by the proteasome .

To study UBE2J2's basic function, researchers typically employ recombinant protein expression systems, co-immunoprecipitation assays to identify binding partners, and in vitro ubiquitination assays using purified components. Cell-based assays with UBE2J2 knockdown or overexpression can provide insights into its physiological roles in protein quality control and degradation pathways.

How is UBE2J2 regulated in human cells?

UBE2J2 itself is subject to regulation through proteasomal degradation. Research has demonstrated that UBE2J2 is unstable, and incubation of transfected cells with proteasome inhibitors increases its steady-state protein levels . Interestingly, pharmacological induction of the unfolded protein response (UPR) does not significantly alter ectopic UBE2J2 protein levels, suggesting that UBE2J2 regulation is not primarily controlled by ER stress pathways .

The effect of proteasomal inhibition on UBE2J2 levels is abolished if the enzyme is inactivated or truncated to disrupt its ER-localization, indicating that both its catalytic activity and subcellular localization are important for its regulation . For studying UBE2J2 regulation, pulse-chase experiments combined with immunoprecipitation are effective methods to determine protein half-life, while subcellular fractionation and immunofluorescence microscopy can be used to monitor its localization.

What experimental systems are best suited for studying UBE2J2 function?

Multiple experimental systems have proven effective for studying UBE2J2:

• In vitro systems: Recombinant UBE2J2 expression in bacterial systems, followed by purification for biochemical assays, allows for detailed characterization of its enzymatic properties and substrate specificity .

• Permeabilized cell systems: These have been instrumental in demonstrating UBE2J2's ability to interact with E3 ligases like mK3 and to promote ubiquitination of various substrates .

• Cell culture models: Human cell lines with RNAi-mediated knockdown or CRISPR-Cas9 knockout of UBE2J2 are valuable for studying its cellular functions and identifying substrate proteins .

• Reconstituted ubiquitination assays: Systems containing purified E1, UBE2J2, E3 ligases, and potential substrates allow for precise characterization of UBE2J2's ubiquitination activity and specificity .

The choice of experimental system depends on the specific research question, with permeabilized cells being particularly useful for studying UBE2J2's activity in a near-native environment while maintaining experimental control.

How does UBE2J2 contribute to ERAD and what are its known substrates?

UBE2J2 plays a crucial role in ERAD by facilitating the ubiquitination of misfolded or unwanted proteins at the ER. Studies using viral E3 ligases like murine K3 (mK3) have shown that UBE2J2 can ubiquitinate MHC class I heavy chains (HCs), targeting them for degradation . Inhibition of UBE2J2 by shRNA results in impaired ubiquitination and induced stabilization of these HC substrates, demonstrating UBE2J2's essential role in this process .

Methodologically, identifying UBE2J2 substrates typically involves:

• Proteomics approaches comparing ubiquitination patterns in UBE2J2-depleted versus control cells

• Co-immunoprecipitation followed by mass spectrometry to identify interacting proteins

• In vitro ubiquitination assays with candidate substrates

• Cycloheximide chase experiments to assess protein stability in the presence or absence of UBE2J2

Current evidence suggests UBE2J2's substrate range extends beyond canonical ERAD targets, potentially including proteins with hydroxylated amino acids as ubiquitination sites.

What are the structural features of UBE2J2 that distinguish it from other E2 enzymes?

UBE2J2 possesses several distinct structural features:

• Catalytic cysteine environment: Unlike many E2s, UBE2J2 has a flexible loop near its catalytic cysteine (Cys94), spanning from Leu95 to Thr104, which cannot be traced in the UBE2J2 apo crystal structure (PDB ID: 2F4W) . This region includes two proline residues at positions 102 and 106, making it inherently flexible and potentially adopting an ordered conformation upon forming a thioester bond with ubiquitin.

• Lack of HPN motif: UBE2J2 lacks the conserved HPN motif that is critical for reactivity toward lysine in canonical E2 enzymes, yet it retains a critical aspartic acid (Asp99) that is present in other lysine-specific E2s .

• Critical residues: Key residues for UBE2J2 activity include Asp99, Phe100, His101, and Pro102, which are near the catalytic cysteine and interact with the C-terminal of ubiquitin when bound to the catalytic cysteine .

To study these structural features, researchers use X-ray crystallography, molecular modeling, and site-directed mutagenesis experiments. Mutation of these critical residues to alanine and subsequent functional assays have helped determine their roles in UBE2J2's activity.

How can researchers effectively measure UBE2J2 activity in experimental settings?

Several methodologies can effectively measure UBE2J2 activity:

• Discharge assays: These track the rate at which UBE2J2~Ub thioester discharges ubiquitin onto various nucleophiles over time. For example, studies have shown UBE2J2 actively discharges on both lysine and serine residues with similar rates but shows no discharge on threonine .

• MALDI-TOF mass spectrometry: This technique can be used to analyze UBE2J2-catalyzed ubiquitin adduct formation with different substrates, providing precise molecular weight information for reaction products .

• In vitro ubiquitination assays: Using purified components (E1, UBE2J2, E3 ligases, substrates, and ubiquitin), these assays can be monitored by SDS-PAGE and western blotting to detect ubiquitinated products .

• Permeabilized cell assays: These semi-in vitro systems maintain the cellular architecture while allowing the introduction of exogenous components. They have been valuable for studying UBE2J2's role in ubiquitinating HC substrates in the context of mK3 ligase activity .

A typical experimental setup for measuring UBE2J2 activity would include recombinant UBE2J2, E1 enzyme, ATP, ubiquitin (often tagged for detection), and potential substrates or nucleophiles, with detection methods appropriate to the specific experimental question.

What is the molecular basis for UBE2J2's unique chemoselectivity toward hydroxylated amino acids?

UBE2J2 exhibits extraordinary chemoselectivity, capable of ubiquitinating not only lysine residues but also serine residues and even simple hydroxyl-containing molecules like glycerol and glucose . The molecular basis for this unique property appears to involve specific residues near the catalytic cysteine:

• Critical residues for hydroxyl group reactivity: Mutation studies have revealed that Asp99, Phe100, His101, and Pro102 are crucial for UBE2J2's ability to react with hydroxylated substrates. When mutated to alanine, these variants showed significant reductions in their ability to discharge ubiquitin onto hydroxyl groups .

• Differential effects of mutations: The Asp99Ala mutant showed a 50-70% reduction in reactivity toward lysine, glycerol, and glucose, and completely blocked the formation of Ub-Serine adducts. Mutations of Phe100 and His101 to alanine nearly abolished discharge on hydroxyl groups (serine, glycerol, and glucose) but only partially reduced discharge on lysine .

• Hybrid nature: UBE2J2 functions as a "hybrid" E2-conjugating enzyme, possessing some features of canonical E2s (conserved histidine and proline residues) but lacking the asparagine residue typically considered essential for isopeptide bond catalysis .

Methodologically, researchers use a combination of structural biology techniques (X-ray crystallography, molecular modeling), site-directed mutagenesis, and in vitro discharge assays with various nucleophiles to understand this chemoselectivity. Advanced molecular dynamics simulations can also provide insights into how UBE2J2 accommodates different nucleophiles in its active site.

How does UBE2J2 interact with E3 ligases and how specific are these interactions?

UBE2J2 displays specific interactions with certain E3 ligases, particularly those involved in ERAD pathways. Key insights include:

• Selective E3 interactions: Studies using the viral E3 ligase murine K3 (mK3) have shown that UBE2J2 can effectively interact with this ligase to promote ubiquitination of MHC class I heavy chain substrates, whereas other potential ERAD-associated E2s cannot interact with mK3 .

• Functional specificity: In the context of mK3-mediated ubiquitination, inhibition of UBE2J2 by shRNA resulted in impaired ubiquitination and stabilization of HC substrates, demonstrating that UBE2J2 is the primary E2 supporting mK3 function in vivo .

• E2-E3 pair functionality: The UBE2J2-mK3 pair is capable of conjugating ubiquitin on both lysine and serine residues of substrates, whereas other E2s like Ube2d1 (UBE2D1) can only ubiquitinate lysine residues when working with mK3 .

To study these interactions, researchers employ methods such as:

• Yeast two-hybrid screens to identify interacting E3 ligases

• Co-immunoprecipitation assays to confirm interactions in cellular contexts

• In vitro binding assays with purified components to measure binding affinities

• FRET or BRET assays to monitor interactions in real-time

• Structural studies of E2-E3 complexes using X-ray crystallography or cryo-EM

Understanding these E2-E3 specificities is crucial for delineating the unique functions of UBE2J2 in cellular ubiquitination pathways.

What experimental approaches can distinguish between UBE2J2-mediated ubiquitination of lysine versus hydroxylated amino acids?

Distinguishing between UBE2J2-mediated ubiquitination of lysine versus hydroxylated amino acids requires specialized experimental approaches:

• Mutant substrate analysis: Using substrates where all lysines are mutated to arginine (K→R) and all serines/threonines are mutated to alanine (S/T→A) in various combinations. This approach has been used with HC mutants having only 1S or only 1K on the tail to demonstrate UBE2J2's ability to ubiquitinate either residue type .

• Discharge assays with specific nucleophiles: Time-course experiments measuring UBE2J2~Ub discharge onto specific nucleophiles like acetylated lysine (Ac-K), acetylated serine (Ac-S), or acetylated threonine (Ac-T) can reveal differential reactivity patterns .

• Mass spectrometry analysis: MALDI-TOF MS can precisely identify ubiquitin adducts formed with different nucleophiles, confirming the chemical nature of the linkage .

• Linkage-specific antibodies or chemical treatments: Ubiquitin conjugated via ester bonds (to serine/threonine) can be distinguished from isopeptide bonds (to lysine) through their differential sensitivity to mild alkaline hydrolysis, which cleaves ester but not isopeptide bonds.

• Site-directed mutagenesis of UBE2J2: Mutations like Asp99Ala, Phe100Ala, and His101Ala differentially affect UBE2J2's ability to ubiquitinate lysine versus hydroxylated residues, providing tools to distinguish these activities .

These approaches, often used in combination, allow researchers to dissect the mechanisms and specificities of UBE2J2-mediated ubiquitination of different substrate residues.

How does the evolutionary conservation of UBE2J2 inform our understanding of its function?

Phylogenetic analysis reveals that UBE2J2 belongs to a distinct evolutionary branch within the E2 family:

• Evolutionary conservation: UBE2J2 is part of a well-defined branch that includes yeast and S. pombe Ubc6, and A. thaliana UBC33, while a separate branch includes human UBE2J1 and A. thaliana UBC32 .

• Conserved structural elements: Comparative analysis of UBE2J2 homologs across species reveals conserved regions that likely support its specialized function in noncanonical ubiquitination .

• Functional divergence: The evolutionary separation between the UBE2J1 and UBE2J2 branches suggests functional specialization, with UBE2J2 potentially evolving to handle specific types of ubiquitination reactions.

Methodologically, researchers use multiple sequence alignments (MSAs) with tools like MAFFT and HMMER hmmsearch, followed by phylogenetic tree construction using programs like IQ-TREE . These approaches, combined with structural analysis and functional studies, help identify conserved motifs that are critical for UBE2J2's specialized activities.

The evolutionary conservation of UBE2J2 across species suggests its fundamental importance in cellular processes, potentially extending beyond ERAD to other ubiquitination-dependent pathways that may have evolved with eukaryotic compartmentalization.

What are the most recent techniques for studying UBE2J2 dynamics in living cells?

Recent advances have expanded the toolbox for studying UBE2J2 dynamics in living cells:

• CRISPR-Cas9 genome editing: Creation of endogenously tagged UBE2J2 (e.g., with fluorescent proteins or small epitope tags) allows monitoring of its expression, localization, and interactions without overexpression artifacts.

• Proximity labeling techniques: BioID or TurboID fused to UBE2J2 can identify proximal proteins in living cells, providing insights into its local interaction network at the ER membrane.

• Live-cell fluorescence imaging: FRAP (Fluorescence Recovery After Photobleaching) or photoactivatable fluorescent protein fusions can track UBE2J2 mobility and dynamics at the ER membrane.

• Ubiquitination sensors: Fluorescent reporters that detect ubiquitination events can be combined with UBE2J2 manipulation to monitor its activity in real-time.

• Quantitative proteomics: SILAC or TMT labeling approaches combined with UBE2J2 knockout or inhibition can globally profile its impact on the ubiquitinome.

These techniques, often used in combination, provide unprecedented insights into UBE2J2's spatial and temporal dynamics in cellular contexts, helping to connect its biochemical properties observed in vitro with its physiological functions in living cells.

How can molecular modeling and simulation approaches enhance our understanding of UBE2J2 function?

Molecular modeling and simulation approaches offer powerful insights into UBE2J2 function:

• UBE2J2~Ub complex modeling: Models of UBE2J2 in complex with ubiquitin in both open and closed conformations have been generated by superposing UBE2J2 and ubiquitin structures onto template crystal structures like UbcH5c~Ubiquitin (PDB ID: 3UGB) . These models reveal potential interaction surfaces and conformational changes during the ubiquitination cycle.

• Molecular dynamics simulations: These can analyze the stability of the UBE2J2~Ub thioester linkage, the flexibility of key regions like the catalytic loop, and the behavior of critical residues like His94 .

• Pocket volume calculations: Computational approaches have been used to define and measure pocket volumes in UBE2J2's active site, providing insights into substrate accommodation .

• Dihedral angle analysis: Tools like MDAnalysis can examine the conformational flexibility of key residues, such as the sidechain dihedral angles of His94, which may play important roles in UBE2J2's function .

For researchers interested in applying these approaches, typical methodologies include:

• Using software like Maestro Schrödinger for model preparation and refinement

• Applying the OPLS3e force field for energy minimization

• Employing visualization tools like PyMOL or Chimera X for structural analysis

• Running molecular dynamics simulations with packages like GROMACS or AMBER

These computational approaches complement experimental methods by providing atomic-level insights into the mechanisms of UBE2J2-mediated ubiquitination.

What are the implications of UBE2J2's noncanonical ubiquitination activity for disease processes?

The unique ability of UBE2J2 to ubiquitinate hydroxylated amino acids raises important questions about its involvement in disease processes:

• Protein misfolding diseases: Given UBE2J2's role in ERAD, dysregulation of its activity could potentially contribute to diseases associated with ER stress and protein misfolding, such as neurodegenerative disorders or certain types of diabetes.

• Cancer biology: Alterations in ubiquitination pathways are frequently observed in cancer. UBE2J2's contribution to protein degradation pathways may have implications for tumor suppressor or oncogene regulation.

• Viral pathogenesis: The documented interaction between UBE2J2 and viral E3 ligases like mK3 suggests that viruses may exploit this E2 enzyme for immune evasion, raising questions about UBE2J2's role in viral infections.

• Noncanonical ubiquitination in pathology: The ability to ubiquitinate hydroxylated residues may represent an underexplored mechanism for protein regulation in disease contexts, potentially affecting proteins where conventional lysine ubiquitination is not possible.

Future research methodologies to address these questions might include:

• Patient-derived samples analyzed for UBE2J2 expression or mutations

• Disease models with UBE2J2 manipulation (knockout, knockdown, or overexpression)

• Proteomics approaches to identify disease-specific UBE2J2 substrates

• High-throughput screens for small molecule modulators of UBE2J2 activity

Understanding these disease connections could potentially reveal UBE2J2 as a therapeutic target or biomarker.

What techniques are being developed to selectively inhibit or modulate UBE2J2 activity?

Development of selective UBE2J2 modulators is an emerging area with several promising approaches:

• Small molecule inhibitors: Structure-based drug design targeting UBE2J2's active site or its interface with E3 ligases could yield selective inhibitors. The unique structural features of UBE2J2, particularly its catalytic cysteine environment and the critical residues Asp99, Phe100, His101, and Pro102 , offer potential binding sites for small molecules.

• Peptide-based inhibitors: Peptides mimicking E3 binding regions or substrate recognition motifs could selectively interfere with UBE2J2 function.

• Targeted protein degradation: PROTAC (Proteolysis Targeting Chimera) approaches could be developed to specifically target UBE2J2 for degradation.

• Allosteric modulators: Compounds targeting regions outside the active site could modulate UBE2J2's activity or selectivity without completely inhibiting it.

• Gene editing approaches: CRISPR-based technologies for precise modification of UBE2J2 in disease contexts could provide therapeutic benefits while minimizing off-target effects.

Methodological approaches for developing these modulators include:

• High-throughput screening of chemical libraries

• Fragment-based drug discovery

• Computational docking and virtual screening

• Structure-activity relationship studies

• PROTAC design and optimization

• Cellular assays measuring UBE2J2 activity in the presence of candidate modulators

The development of selective UBE2J2 modulators would not only provide valuable research tools but could also have therapeutic potential for diseases involving dysregulated ERAD or protein quality control.

How does UBE2J2 coordinate with other components of the ubiquitin-proteasome system in complex cellular processes?

Understanding UBE2J2's coordination with other components of the ubiquitin-proteasome system represents a complex frontier in research:

• E2-E2 cooperation: Evidence suggests that UBE2J2 might work sequentially or cooperatively with other E2 enzymes in polyubiquitin chain formation. While UBE2J2 might initiate ubiquitination, other E2s could potentially extend chains or modify chain topology .

• Integration with quality control pathways: How UBE2J2-mediated ubiquitination interfaces with other ER quality control mechanisms, such as those involving chaperones, remains to be fully elucidated.

• Regulation by deubiquitinating enzymes (DUBs): The interplay between UBE2J2-mediated ubiquitination and DUB activity could create dynamic regulation of substrate fate.

• Cooperation with the proteasome: UBE2J2's role in generating degradation signals that are recognized by the proteasome, potentially including non-canonical ubiquitin linkages to hydroxylated residues, represents an important area for investigation.

• Crosstalk with other post-translational modifications: How UBE2J2-mediated ubiquitination interacts with other modifications like phosphorylation or glycosylation could reveal complex regulatory networks.

Advanced methodological approaches to study these interactions include:

• Proteomics profiling of ubiquitination changes in response to UBE2J2 manipulation

• Reconstituted systems with purified components to test sequential or cooperative activity

• Live-cell imaging of fluorescently tagged UBE2J2 and other system components

• Network analysis of genetic or physical interactions between UBE2J2 and other factors

• In vitro assays with defined ubiquitin mutants to study chain topology preferences

Unraveling these complex interactions will provide a more complete understanding of UBE2J2's role in cellular proteostasis and potential therapeutic interventions in related disorders.