UBE2W Human

What distinguishes UBE2W from other ubiquitin-conjugating enzymes?

UBE2W is the only known E2 enzyme that primarily catalyzes the attachment of ubiquitin to the N-termini of proteins rather than internal lysine residues . This unique activity represents a non-canonical ubiquitination pathway with distinct regulatory consequences. UBE2W shows a clear preference for substrates with intrinsically disordered N-termini, suggesting specialized functions in protein quality control and regulation of disordered proteins . When functioning in cells, UBE2W typically mediates monoubiquitination, which can serve as a priming modification for subsequent extension into polyubiquitin chains by other E2/E3 enzyme pairs .

In which tissues is UBE2W predominantly expressed?

UBE2W is ubiquitously expressed as a single isoform localized to the cytoplasm, but demonstrates tissue-specific expression patterns with notably higher levels in testis and thymus . This expression pattern correlates with phenotypic observations in UBE2W knockout mice, which display disproportionate decreases in the weight of these organs (approximately 50% reduction), suggesting particularly important functional roles in reproductive and immune systems . To study tissue-specific functions, researchers should focus experimental designs on these high-expression tissues while considering the broader systemic effects of UBE2W activity.

What are the known E3 ligase partners of UBE2W?

UBE2W demonstrates functional compatibility with several E3 ubiquitin ligases, including:

• The C-terminus of Hsc-70-interacting protein (CHIP)

• BRCA1-BARD1 complex involved in DNA damage responses

• TRIM5α, an innate immunity factor with anti-retroviral functions

These partnerships determine the specificity and functional outcomes of UBE2W-mediated ubiquitination. When designing experiments to study UBE2W function, researchers should consider which E3 partner is relevant to their biological system of interest, as this dictates substrate selection and downstream signaling consequences.

How does UBE2W recognize and select its substrates?

UBE2W substrate recognition depends primarily on the presence of intrinsically disordered N-terminal regions in target proteins . The enzyme itself contains a partially disordered C-terminus that is critical for recognizing substrates with intrinsically disordered N-termini . Proteomic analyses of UBE2W overexpression systems have identified approximately 73 putative UBE2W substrates, most of which are predicted to have disordered N-termini . This substrate selectivity represents an important quality control mechanism potentially targeting misfolded or unstructured proteins for regulatory modification rather than degradation.

How does N-terminal acetylation affect UBE2W function?

N-terminal acetylation, a common co-translational modification of cellular proteins, directly impacts UBE2W activity by blocking its ability to ubiquitinate the N-terminus. Experimental evidence shows that when the N-terminus is blocked by acetylation, UBE2W can instead conjugate ubiquitin to internal lysine residues through isopeptide bonds . This adaptive function allows UBE2W to maintain its biological activity even when its preferred modification site is unavailable. For example, studies with TRIM5α demonstrated that acetylated TRIM5α was ubiquitinated by UBE2W on internal lysine residues (including K45, K50, K85, K218, K284, and K372), while non-acetylated TRIM5α could receive ubiquitin on its free N-terminus .

What is the impact of UBE2W-mediated ubiquitination on substrate fate?

Contrary to the canonical view of ubiquitination as primarily a degradation signal, N-terminal ubiquitination by UBE2W appears to have distinct regulatory functions:

• Modulation of enzyme activity: N-terminal ubiquitination of deubiquitinases UCHL1 and UCHL5 alters their catalytic activity rather than promoting degradation

• Protein solubility regulation: UBE2W affects the aggregation state of mutant huntingtin protein in Huntington's disease models

• Signaling pathway activation: UBE2W-mediated monoubiquitination of TRIM5α enables subsequent polyubiquitination necessary for retroviral restriction

These diverse outcomes highlight the need for substrate-specific analyses when studying UBE2W function.

What methods are available for detecting N-terminal ubiquitination?

Several complementary approaches have been developed to detect and characterize N-terminal ubiquitination:

Researchers should consider combining multiple approaches for comprehensive characterization of N-terminal ubiquitination events .

How can novel UBE2W substrates be identified?

Identification of endogenous N-terminally ubiquitinated substrates requires sophisticated technical approaches:

• Antibody-based enrichment using monoclonal antibodies that selectively recognize N-terminal diglycine remnants (but not lysine-linked diglycine)

• Comparative proteomics between wild-type and UBE2W-deficient or overexpressing systems

• Mass spectrometry analysis of enriched peptides to map precise modification sites

• Validation using in vitro ubiquitination assays with purified components

• Functional studies to determine the biological consequences of N-terminal ubiquitination

This multi-layered approach has successfully identified functionally relevant substrates including deubiquitinases UCHL1 and UCHL5 .

What are optimal conditions for reconstituting UBE2W activity in vitro?

For successful in vitro reconstitution of UBE2W-mediated N-terminal ubiquitination:

• Required components:

  • Purified recombinant UBE2W protein

  • Ubiquitin

  • E1 enzyme (UBA1)

  • Appropriate E3 ligase partner (e.g., CHIP, BRCA1-BARD1, or TRIM5α)

  • Target substrate protein

• Critical considerations:

  • Substrate N-terminal status (acetylated or free)

  • Buffer composition (typically containing ATP, Mg²⁺)

  • Temperature and incubation time optimization

  • Controls to distinguish specific from non-specific activities

• Analysis methods:

  • SDS-PAGE mobility shift detection

  • Western blotting with ubiquitin-specific antibodies

  • Mass spectrometry to identify specific modification sites

What phenotypes are observed in UBE2W-deficient models?

UBE2W knockout mice exhibit multiple phenotypes that provide insights into the physiological functions of this enzyme:

These diverse phenotypes indicate UBE2W plays important roles in multiple organ systems, though it appears to be non-essential for basic cellular viability .

What role does UBE2W play in neurodegenerative diseases?

UBE2W significantly impacts protein aggregation in Huntington's disease models:

• In cellular models, UBE2W deficiency decreases mutant huntingtin (mHTT) aggregate formation while increasing levels of soluble monomers

• This shift from aggregates to soluble forms correlates with reduced mHTT-induced cytotoxicity

• In HdhQ200 knock-in mice, absence of UBE2W significantly increases levels of soluble monomeric mHTT while reducing insoluble oligomeric species

These findings suggest UBE2W modulates protein aggregation dynamics in neurodegenerative contexts, with potential implications for therapeutic strategies targeting protein homeostasis mechanisms.

How does UBE2W contribute to innate immunity?

UBE2W plays a critical role in TRIM5α-mediated restriction of retroviral infection:

• UBE2W monoubiquitinates TRIM5α, providing a priming modification

• This initial ubiquitination enables subsequent extension into K63-linked polyubiquitin chains by the Ube2N/Ube2V2 complex

• Both UBE2W and Ube2N/Ube2V2 activities are required for efficient restriction of retroviral reverse transcription

• N-terminal acetylation status determines whether ubiquitination occurs at the N-terminus or internal lysine residues

This demonstrates how UBE2W-mediated ubiquitination contributes to innate immune defense mechanisms against retroviruses.

How might UBE2W activity be therapeutically targeted?

Given UBE2W's role in multiple disease-relevant processes, several therapeutic strategies could be explored:

• For neurodegenerative disorders:

  • Inhibitors of UBE2W catalytic activity could potentially reduce protein aggregation

  • Structure-based drug design targeting the UBE2W-substrate interface

• For immune modulation:

  • Enhancers of UBE2W activity might strengthen antiviral responses

  • Targeted delivery of UBE2W modulators to specific immune cell populations

• For reproductive medicine:

  • Restoration of UBE2W function in testicular tissues could address specific fertility issues

Development of these approaches requires deeper understanding of UBE2W structure-function relationships and tissue-specific activities.

What is the relationship between UBE2W and protein disorder?

Proteomic analysis of UBE2W knockout mice revealed a preferential accumulation of disordered proteins in affected tissues . This observation supports a model where UBE2W participates in quality control of intrinsically disordered proteins through several mechanisms:

• Direct recognition of disordered N-termini via UBE2W's own disordered C-terminus

• Modification of disordered proteins to alter their interactions, localization, or degradation

• Potential role in preventing inappropriate aggregation of disordered proteins

Researchers investigating protein disorder should consider UBE2W as an important regulator of this protein class, particularly in contexts of proteostasis and protein quality control.

How do UBE2W-mediated modifications intersect with other ubiquitin pathways?

UBE2W-mediated N-terminal ubiquitination creates a complex regulatory network with other ubiquitin pathway components:

• Hierarchical ubiquitination: UBE2W monoubiquitination serves as a priming modification for subsequent extension by other E2/E3 pairs (e.g., Ube2N/Ube2V2)

• DUB regulation: UBE2W targets deubiquitinases UCHL1 and UCHL5 for N-terminal ubiquitination, modulating their activity and creating potential feedback loops within the ubiquitin system

• Competition with other N-terminal modifications: Acetylation blocks UBE2W-mediated N-terminal ubiquitination, redirecting activity to internal lysines

This integration within the broader ubiquitin network highlights the need for systems-level analyses when studying UBE2W function.

What biochemical features determine UBE2W substrate specificity beyond N-terminal disorder?

While N-terminal disorder is a primary determinant of UBE2W substrate selection, additional factors likely contribute to in vivo specificity:

• Sequence context surrounding the N-terminus

• Co-localization with specific E3 ligase partners

• Accessibility of the N-terminus within protein complexes

• Competition with other N-terminal modifying enzymes

• Tissue-specific expression patterns of UBE2W and potential substrates

Computational analyses combining disorder prediction algorithms like IUPred and DisEMBL with other protein features might help identify common patterns among UBE2W substrates . Experimental validation using site-directed mutagenesis and chimeric protein approaches could further elucidate the determinants of substrate specificity.

What emerging technologies could advance UBE2W research?

Several cutting-edge approaches hold promise for deepening our understanding of UBE2W biology:

• Cryo-electron microscopy to visualize UBE2W-E3-substrate complexes

• Proximity labeling techniques to map UBE2W interactomes in different cellular contexts

• CRISPR-based screening to identify genetic modifiers of UBE2W function

• Single-cell proteomics to uncover cell type-specific UBE2W substrates

• Targeted protein degradation technologies exploiting UBE2W's unique N-terminal activity

These approaches could reveal new insights into UBE2W's biological functions and therapeutic potential.

What remains unknown about UBE2W structure-function relationships?

Despite significant progress, several aspects of UBE2W structure and function remain poorly understood:

• Complete three-dimensional structure of UBE2W, particularly its disordered C-terminus

• Structural basis for recognition of disordered N-termini in substrates

• Conformational changes during the catalytic cycle

• Mechanisms of regulation of UBE2W activity

• Structural determinants of E3 ligase partnership selectivity

Addressing these knowledge gaps would enhance our understanding of this unique enzyme and potentially enable structure-based therapeutic design.

How might UBE2W function be affected by cellular stress conditions?

While UBE2W knockout does not alter cell viability in response to various stressors , specific stress-related functions deserve further investigation:

• Potential role in heat shock response given preference for disordered proteins

• Involvement in oxidative stress response pathways

• Function during endoplasmic reticulum stress and unfolded protein response

• Activity modulation under nutrient deprivation conditions

• Participation in DNA damage response through BRCA1-BARD1 interaction