UBE2R2 Human

What is UBE2R2 and what is its role in the ubiquitination pathway?

UBE2R2, also known as CDC34B and UBC3B, is a 238 amino acid member of the Ubiquitin-conjugating (E2) enzyme family with a predicted molecular weight of 27 kDa. It contains an E2 catalytic core domain with an active site cysteine residue essential for forming a thioester bond with ubiquitin . UBE2R2 functions within the ubiquitination cascade by receiving ubiquitin from a Ubiquitin-activating (E1) enzyme, specifically UBE1 (but not UBA6), and then facilitating its transfer to substrate proteins with the help of E3 ligases . This process is crucial for protein degradation signaling and cellular regulation, making UBE2R2 an important player in maintaining cellular homeostasis.

How does the structure of UBE2R2 contribute to its function?

The functional architecture of UBE2R2 includes several critical structural elements that enable its specific activity. The protein contains an acidic loop adjacent to the active site and an extended tail C-terminal to the UBC domain . The acidic loop makes ionic contacts with the donor ubiquitin, specifically between Glu112 in the loop and Arg74 of ubiquitin, which helps stabilize the C-terminal tail of ubiquitin . The C-terminal tail of UBE2R2 wraps around its UBC domain to help bridge the UBC and donor ubiquitin. A cluster of hydrophobic residues in this C-terminal tail positions Tyr190 to act as a pin bridging the UBC and donor ubiquitin . These structural features collectively stabilize the E2~Ub in a closed conformation, which is essential for efficient ubiquitin transfer. Disruption of either the hydrophobic pocket or the Tyr190-UBC interaction significantly inhibits UBE2R2 activity, both in the presence and absence of an E3 ligase .

How is UBE2R2 regulated at the post-translational level?

UBE2R2 undergoes post-translational modification, particularly phosphorylation, which regulates its activity. Casein Kinase 2 phosphorylates UBE2R2 on Ser233, which promotes its interaction with the beta-TrCP ubiquitin ligase (E3) . This interaction facilitates the subsequent ubiquitination and degradation of beta-Catenin, a critical component of the Wnt signaling pathway . This regulatory mechanism illustrates how post-translational modifications of UBE2R2 can influence its substrate specificity and functional interactions within the ubiquitination cascade, ultimately affecting downstream cellular processes.

How does UBE2R2 differ from other E2 enzymes in the ubiquitin pathway?

UBE2R2 belongs to the Ube2r family of E2 enzymes, which distinguishes itself from other E2 enzymes through specific structural and functional features. Unlike some promiscuous E2 enzymes that can both initiate ubiquitination and extend chains, the Ube2r family appears more specialized in ubiquitin chain elongation . The distinguishing features include an acidic loop adjacent to the active site and an extended tail C-terminal to the UBC domain, which are critical for its ubiquitin transfer activity .

While the human genome encodes approximately 40 E2 enzymes, each with a conserved UBC domain, UBE2R2's specificity appears to be determined by these small sequence differences beyond the core UBC domain . In comparison to its paralog UBCH3, with which it shares 78% amino acid sequence identity, UBE2R2's unique structural elements likely contribute to its specific functional niche in the ubiquitination pathway . These molecular distinctions represent important considerations when designing experiments targeting specific E2 enzyme activities or developing selective inhibitors.

What is the significance of UBE2R2 in disease pathogenesis?

Dysregulation of UBE2R2 has been implicated in various pathological conditions, including cancer, neurodegenerative disorders, and inflammatory conditions . As an essential component of the ubiquitination pathway, alterations in UBE2R2 expression or activity can disrupt protein degradation and cellular signaling networks. The protein's role in mediating the ubiquitination and degradation of beta-Catenin through interaction with beta-TrCP ubiquitin ligase suggests its involvement in Wnt signaling regulation , a pathway frequently dysregulated in cancer.

The specific contribution of UBE2R2 to different disease mechanisms remains an active area of research. Studying UBE2R2 levels and activity in pathological contexts can provide valuable insights into disease mechanisms and potentially identify novel therapeutic targets . Researchers should consider examining UBE2R2 expression patterns, activity profiles, and interaction networks in disease models to elucidate its role in pathogenesis and potential value as a biomarker or therapeutic target.

How do the molecular interactions between UBE2R2, E3 ligases, and substrates determine ubiquitination specificity?

The molecular basis for UBE2R2's specificity in ubiquitin transfer involves intricate interactions between the E2 enzyme, E3 ligases, and substrate proteins. The crystal structure of a Ube2r2~Ub conjugate revealed that the acidic loop of Ube2r2 forms an ionic contact with the donor ubiquitin between Glu112 and Arg74 of ubiquitin, which helps stabilize the ubiquitin's C-terminal tail . Additionally, the C-terminal tail of UBE2R2 contains a cluster of hydrophobic residues that position Tyr190 to bridge the UBC domain and donor ubiquitin .

For RING-catalyzed ubiquitylation, E2 enzymes like UBE2R2 determine the exact "language" of the ubiquitin code . The intricate molecular contacts between E3 ligases and E2 enzymes suggest that the balance of substrate chain initiation and chain elongation can be rapidly triggered and quenched . This complex interplay of molecular interactions defines the specificity of the ubiquitination process and represents a critical area for continued investigation. Understanding these molecular determinants could inform structure-based drug design targeting specific ubiquitination events in disease contexts.

What are the optimal approaches for measuring UBE2R2 levels in experimental samples?

For quantitative determination of UBE2R2 levels in experimental samples, sandwich ELISA represents a highly sensitive and specific methodology. The Human UBE2R2 ELISA Kit allows for accurate measurement of UBE2R2 concentrations in serum, plasma, tissue homogenates, and other biological fluids with a sensitivity of <0.094ng/ml and a detection range of 0.156-10ng/ml . This double antibody approach provides reliable quantification with intra-assay coefficients of variation (CV) <8% and inter-assay CV <10% .

Sample preparation protocols vary depending on the starting material:

• For serum: Allow samples to clot for 30 minutes at room temperature, centrifuge for 10 minutes at 1,000×g, collect the serum fraction, and either assay promptly or store at -80°C .

• For plasma: Collect using EDTA or heparin as an anticoagulant, centrifuge at 4°C for 15 mins at 1000×g within 30 mins of collection, and process similarly to serum samples .

• For cell lysates: Solubilize cells in lysis buffer, incubate on ice for 30 minutes, centrifuge at 14,000×g for 5 minutes, and quantify total protein concentration before assay .

• For tissue homogenates: Rinse tissue with PBS, homogenize in PBS with protease inhibitors, subject to freeze-thaw cycles, and centrifuge for 5 mins at 5000×g before collecting the supernatant .

Alternative approaches might include western blotting for semi-quantitative analysis or mass spectrometry-based proteomics for deeper analysis of UBE2R2 post-translational modifications and interaction partners.

What experimental systems are most suitable for studying UBE2R2 function?

Multiple experimental systems can be employed to investigate UBE2R2 function, each with specific advantages depending on the research question:

• In vitro ubiquitination assays: Recombinant human His6-UBE2R2 protein can be used in reconstituted ubiquitination reactions to study the enzyme's catalytic activity, substrate specificity, and chain-building properties . These assays typically include purified E1, E2 (UBE2R2), E3 ligase, ubiquitin, ATP, and substrate proteins.

• Cell-based systems: Human cell lines expressing endogenous or tagged UBE2R2 can be used to study its cellular functions, regulation, and involvement in specific cellular processes. These systems are particularly valuable for investigating UBE2R2's role in degrading specific substrates like beta-Catenin through the beta-TrCP pathway .

• Structural biology approaches: X-ray crystallography, as demonstrated by the crystal structure of a Ube2r2~Ub conjugate , provides valuable insights into the molecular mechanisms of UBE2R2 function. Additional approaches like cryo-EM could further elucidate the structural basis of UBE2R2 interactions with E3 ligases and substrates.

• Genetic models: CRISPR/Cas9-mediated knockout or knockdown models can help establish the physiological relevance of UBE2R2 in specific cellular contexts and disease models.

When selecting an experimental system, researchers should consider the specific aspects of UBE2R2 biology they aim to investigate and choose the approach that best addresses their research question while accounting for technical limitations.

How can researchers effectively manipulate UBE2R2 activity in experimental settings?

Several approaches can be employed to manipulate UBE2R2 activity in experimental settings:

• Genetic manipulation: CRISPR/Cas9 genome editing, siRNA knockdown, or overexpression systems can alter UBE2R2 levels. When designing such experiments, researchers should consider potential compensatory mechanisms from related E2 enzymes, particularly its paralog UBCH3, which shares 78% amino acid sequence identity .

• Site-directed mutagenesis: Based on structural insights, key residues can be mutated to disrupt specific functions. For example, mutations affecting the active site cysteine would abolish thioester formation with ubiquitin, while mutations in the acidic loop (e.g., Glu112) or C-terminal tail (e.g., Tyr190) would disrupt contacts necessary for ubiquitin transfer activity .

• Small molecule inhibitors: Compounds like CC0651 have been shown to bind to Ube2r2~Ub conjugates and provide structural insights into the enzyme's mechanism . Similar inhibitors could be valuable tools for acutely modulating UBE2R2 activity in cellular contexts.

• Manipulation of post-translational modifications: Since UBE2R2 is regulated by phosphorylation at Ser233 by Casein Kinase 2 , manipulating this kinase's activity or introducing phosphomimetic mutations could alter UBE2R2's interactions with E3 ligases like beta-TrCP.

When implementing these approaches, researchers should include appropriate controls and validation experiments to confirm specificity and effectiveness of the manipulation strategy.

How can UBE2R2 research contribute to understanding neurodegenerative disorders?

Dysregulation of the ubiquitin-proteasome system is a common feature in many neurodegenerative disorders, and UBE2R2's role in this system makes it a potential contributor to disease pathogenesis . As a key E2 enzyme involved in protein degradation signaling, alterations in UBE2R2 activity could affect the clearance of pathogenic protein aggregates characteristic of conditions like Alzheimer's disease, Parkinson's disease, and Huntington's disease.

Research approaches to investigate UBE2R2's role in neurodegeneration could include:

• Measuring UBE2R2 levels and activity in post-mortem brain tissue from patients with neurodegenerative disorders compared to age-matched controls.

• Identifying specific neuronal substrates of UBE2R2-mediated ubiquitination that might be relevant to neurodegenerative processes.

• Evaluating the effects of UBE2R2 manipulation in cellular and animal models of neurodegeneration, particularly focusing on protein aggregation and neuronal survival.

• Investigating potential genetic associations between UBE2R2 variants and neurodegenerative disease risk or progression.

Understanding UBE2R2's contribution to neurodegenerative processes could potentially identify new therapeutic strategies focused on modulating specific ubiquitination pathways rather than broadly targeting the proteasome.

What are the current technical challenges in studying UBE2R2-mediated ubiquitination?

Despite significant advances in understanding UBE2R2 function, several technical challenges remain in studying its specific contributions to ubiquitination pathways:

• Specificity in cellular contexts: With approximately 40 E2 enzymes in the human genome , determining which ubiquitination events are specifically mediated by UBE2R2 versus other E2 enzymes remains challenging, particularly for events involving redundant E2 activities.

• Temporal dynamics: The rapid and dynamic nature of ubiquitination events makes capturing the specific role of UBE2R2 in real-time challenging within cellular contexts.

• Substrate identification: Comprehensively identifying physiological substrates of UBE2R2-mediated ubiquitination requires sophisticated proteomics approaches capable of distinguishing direct from indirect effects.

• Structural complexity: While partial structural information exists for UBE2R2 , complete structures of UBE2R2 in complex with different E3 ligases and substrates would provide valuable insights but remain technically challenging to obtain.

Addressing these challenges will require interdisciplinary approaches combining advanced proteomics, structural biology, real-time cellular imaging, and computational modeling to fully elucidate UBE2R2's specific roles within the complex ubiquitination network.