UBE2V2 Human

What is UBE2V2 and what is its primary function in human cells?

UBE2V2, also known as the human homologue of yeast MMS2, is a catalytically inactive protein that forms a functional complex with UBE2N (Ubc13). Despite lacking enzymatic activity itself, UBE2V2 is crucial for the ubiquitination pathway as it works with UBE2N to synthesize Lys63-linked ubiquitin chains . These chains can either be unanchored or attached to target proteins, playing significant roles in facilitating responses to various forms of DNA damage . UBE2V2 contains one conserved cysteine residue (C69) that has been identified as a privileged sensor for reactive electrophilic species, establishing it as a bridge between redox signaling and ubiquitin-dependent pathways .

The UBE2N/UBE2V2 complex functions with ubiquitin ligases (E3s), including RNF111 and RNF8, to coordinate protein modification through ubiquitination . This process is fundamental to cellular processes including DNA damage repair, signal transduction, and protein quality control.

What is the molecular structure and key features of human UBE2V2?

Human UBE2V2 is a 17 kDa protein containing a single cysteine residue (C69) . When forming a complex with UBE2N (18 kDa), the functional unit contains distinct domains that facilitate ubiquitin transfer. The specific structural features include:

• A highly conserved cysteine (C69) that is present from humans to yeast

• An N-terminal region that facilitates complex formation with UBE2N

• A ubiquitin-conjugating enzyme variant (UEV) fold that structurally resembles E2 enzymes but lacks catalytic activity

When expressed recombinantly with an N-terminal His-tag, the protein maintains its ability to form functional complexes with UBE2N and participate in ubiquitination reactions .

How is UBE2V2 conserved across species and what does this indicate about its function?

Alignments of UBE2V2 across vertebrate species show remarkable conservation, particularly of the C69 residue, which is preserved from humans to yeast . This high degree of evolutionary conservation suggests that UBE2V2 serves a fundamental cellular function that has been maintained throughout eukaryotic evolution.

The conserved nature of UBE2V2 and its homologues is particularly evident in comparison with related proteins:

• Human UBE2V2 (Mms2) and UBE2V1 (Uev1) share homologous structures

• The analogous cysteine residue (C69 in UBE2V2 and C94 in UBE2V1) is conserved across species

• Saccharomyces cerevisiae and Schizosaccharomyces pombe homologues (Ubc6) maintain similar structural features

This conservation underscores the protein's essential role in cellular processes, particularly in DNA damage response pathways that are fundamental to genomic integrity across eukaryotic organisms.

How does UBE2V2 interact with UBE2N to form a functional complex?

The UBE2V2/UBE2N interaction represents a fascinating example of functional complementation where a catalytically inactive protein (UBE2V2) enhances the activity of an enzymatically active partner (UBE2N). The complex formation occurs through specific protein-protein interfaces that position both proteins for optimal ubiquitin transfer .

When forming a complex with UBE2N, UBE2V2 contributes to:

• Specific recognition of ubiquitin molecules for Lys63-linked chain formation

• Proper orientation of the acceptor ubiquitin for chain extension

• Enhanced interaction with E3 ubiquitin ligases like RNF111 and RNF8

The recombinant human His6-UBE2N/UBE2V2 complex is typically formulated at 0.88 mg/ml (25 μM) in 50 mM HEPES pH 7.5, 200 mM NaCl, 10% Glycerol (v/v), and 2 mM TCEP . This formulation maintains the stability of the complex and preserves its activity for in vitro applications. For experimental ubiquitination reactions, an initial concentration of 0.1-1 μM of the complex is typically recommended .

What is the significance of the C69 residue in UBE2V2 for electrophile sensing?

The C69 residue in UBE2V2 has emerged as a critical sensor for reactive electrophilic species (RES) such as 4-hydroxynonenal (HNE) . This finding, identified through the "G-REX" technique, represents a novel function for UBE2V2 beyond its role in ubiquitination:

• C69 demonstrates kinetic privilege in detecting electrophiles under physiological conditions

• This cysteine is conserved from humans to yeast, suggesting evolutionary importance

• Modification of C69 by electrophiles like HNE leads to allosteric activation of the UBE2N partner enzyme

• This activation promotes enhanced K63-linked ubiquitination and stimulates H2AX-dependent DNA damage response

Notably, the HNE-sensing ability of UBE2V2 (with one cysteine) surpasses that of UBE2V1 (with three cysteines), demonstrating that electrophile sensitivity is not simply correlated with cysteine content but depends on the specific biochemical environment around the reactive residue . The significance of this site was verified through multiple approaches, including:

• G-REX proteome-wide identification

• Targeted T-REX modification in live cells

• LC-MS/MS verification of site-specific modification

This redox-sensing capability positions UBE2V2 as a "Rosetta stone" bridging redox signaling and ubiquitin-dependent pathways in the maintenance of genome integrity .

How can recombinant UBE2N/UBE2V2 complex be optimally used in in vitro ubiquitination assays?

For optimal use of recombinant UBE2N/UBE2V2 complex in ubiquitination assays, researchers should consider several factors that affect reaction efficiency and specificity:

Recommended reaction conditions:

• Initial UBE2N/UBE2V2 complex concentration: 0.1-1 μM

• Buffer system: 50 mM HEPES pH 7.5 with 200 mM NaCl

• Reducing agent: 2 mM TCEP to maintain cysteine residues in reduced state

• Temperature: Typically 30-37°C for human protein interactions

• E1 enzyme: Required for initial ubiquitin activation

• E3 ligase: Appropriate E3 (e.g., RNF111, RNF8) should be included for substrate specificity

• ATP and Mg²⁺: Essential cofactors for the ubiquitination cascade

When working with carrier-free preparations, special attention should be paid to protein stability, as the absence of carrier proteins like BSA can affect shelf-life and activity . For applications where the presence of carrier proteins might interfere, such as certain mass spectrometry analyses or specific enzymatic assays, the carrier-free version is preferred .

Reaction parameters will need optimization for each specific experimental system, particularly when investigating novel substrates or E3 ligase combinations.

What experimental approaches are best for studying UBE2V2 function in cellular contexts?

Several complementary approaches have proven effective for investigating UBE2V2 function in cellular systems:

1. CRISPR/Cas9-mediated knockout cell lines:
The UBE2V2 knockout in HCT 116 colorectal cancer cells represents a valuable tool for studying the consequences of UBE2V2 ablation . These cells allow researchers to:

• Investigate altered protein turnover in cancer cells

• Unveil mechanisms contributing to tumor progression

• Study resistance to therapies in the absence of UBE2V2-dependent ubiquitination

• Test drug efficacy in UBE2V2-deficient backgrounds

2. Site-specific modification approaches:
The G-REX and T-REX platforms enable controlled release of reactive electrophiles in vivo, allowing researchers to:

• Target specific cysteines for modification under physiological conditions

• Identify first-responding innate cysteines that bind electrophiles

• Verify site-specific modifications through mass spectrometry analysis

3. Functional ubiquitination assays:
Monitoring K63-linked ubiquitination in cellular contexts can be achieved through:

• Immunoprecipitation of target proteins followed by ubiquitin chain-specific antibody detection

• Monitoring DNA damage response markers like H2AX phosphorylation

• Analyzing protein complex formation through co-immunoprecipitation studies

These methodologies provide complementary insights into UBE2V2 function, from biochemical mechanisms to cellular consequences of UBE2V2 activity or its absence.

How does UBE2V2 contribute to DNA damage response pathways?

UBE2V2 plays a critical role in DNA damage response (DDR) through its ability to form K63-linked ubiquitin chains in conjunction with UBE2N . The contribution of UBE2V2 to DDR includes:

• Formation of K63-linked ubiquitin chains on histone H2AX and other chromatin-associated proteins

• Recruitment of DNA repair factors to sites of damage

• Stimulation of H2AX-dependent DNA damage response pathways

• Enabling specific signaling cascades required for various DNA repair mechanisms

The UBE2N/UBE2V2 complex works with E3 ligases like RNF8 to facilitate these responses to DNA damage . Additionally, the electrophile-sensing capability of UBE2V2 through its C69 residue represents an intriguing mechanism by which oxidative stress (which often accompanies DNA damage) can trigger enhanced DDR signaling .

This dual functionality - both as a component of the ubiquitination machinery and as a redox sensor - positions UBE2V2 as a central coordinator that integrates different cellular stress response pathways to maintain genome integrity.

What are the implications of UBE2V2 knockout in cancer research?

UBE2V2 knockout cell lines, particularly in cancer models like HCT 116 colorectal cancer cells, have emerged as valuable tools for investigating the role of ubiquitination in cancer biology . The implications of UBE2V2 knockout include:

1. Understanding ubiquitin signaling in cancer:

• Altered protein turnover in the absence of UBE2V2-dependent K63 ubiquitination

• Uncovering mechanisms contributing to tumor progression that rely on UBE2V2 function

• Evaluating resistance to therapies linked to ubiquitin-dependent pathways

2. Drug development applications:

• Screening novel anti-cancer compounds in a UBE2V2-deficient background

• Identifying synthetic lethal interactions with UBE2V2 deficiency

• Developing targeted strategies based on ubiquitination pathway dependencies

3. Mechanistic studies:

• Dissecting the role of ubiquitin-conjugating enzymes in cancer progression

• Evaluation of DNA damage response efficiency in the absence of UBE2V2

• Understanding compensatory mechanisms that may emerge in UBE2V2-deficient cells

These knockout models facilitate significant enhancements in the accuracy of cancer mechanistic studies and therapeutic evaluations, potentially leading to improved outcomes in developing targeted cancer treatments .

How does UBE2V2 bridge redox and ubiquitin signaling pathways?

UBE2V2 serves as a functional bridge between redox and ubiquitin signaling pathways, earning its description as a "Rosetta Stone" linking these two crucial cellular regulatory systems . This integrative function is facilitated by several key mechanisms:

• Electrophile sensing through C69: The conserved C69 residue acts as a privileged sensor for reactive electrophilic species (RES) like 4-hydroxynonenal (HNE), which are products of oxidative stress

• Allosteric activation of UBE2N: When C69 is modified by electrophiles, UBE2V2 undergoes conformational changes that allosterically hyperactivate its binding partner UBE2N, despite UBE2N itself not being directly modified

• Enhanced ubiquitination activity: This activation promotes increased K63-linked ubiquitination of client proteins, particularly those involved in DNA damage response

• Functional specificity: Despite structural similarities between UBE2V1 and UBE2V2, electrophile modification triggers responses specific to each protein, highlighting the precise nature of this signaling mechanism

The linkage between these pathways is particularly significant because it provides a direct mechanistic connection between oxidative stress (which generates electrophilic species) and the ubiquitin-dependent pathways that regulate critical cellular processes like DNA repair. This mechanism enables cells to respond to oxidative damage not only through direct antioxidant defenses but also by enhancing DNA repair capacity through ubiquitin-dependent signaling.

What are the determinants of chemoselectivity in UBE2V2-related ubiquitination?

The chemoselectivity of ubiquitination reactions involving UBE2V2 depends on several factors that collectively determine the specificity and efficiency of ubiquitin transfer:

• Complex formation: The precise structural arrangement of the UBE2N/UBE2V2 complex creates a specific binding interface that determines interaction specificity with both ubiquitin and E3 ligases

• Active site architecture: Although UBE2V2 itself lacks catalytic activity, subtle rearrangements in the active site region of the complex influence preferential activity toward specific substrates

• E3 ligase cooperation: The UBE2N/UBE2V2 complex works with specific E3 ligases like RNF111 and RNF8, which contribute additional layers of substrate specificity

• Lysine specificity: The UBE2N/UBE2V2 complex specifically synthesizes K63-linked ubiquitin chains, distinguishing it from other E2 enzymes that might generate different linkage types

• Redox modification effects: Modification of UBE2V2's C69 residue by electrophiles can alter the activity and potentially the specificity of the complex, providing an additional regulatory mechanism

These determinants collectively ensure that ubiquitination reactions involving UBE2V2 proceed with appropriate specificity, targeting the correct substrates with the proper ubiquitin chain types needed for specific cellular functions, particularly in DNA damage response pathways.