UBE2T Human

What is UBE2T and what are its key structural characteristics?

UBE2T, also known as E2 ubiquitin-conjugating enzyme T, FANCT, PIG50, or HSPC150, belongs to the UBE2 superfamily, which plays a fundamental role in the second step of the ubiquitination process. Human UBE2T is a 22.521 kDa protein consisting of 197 amino acids with a basal isoelectric point of 7.78 . The protein structure features:

• A core UBC (ubiquitin-conjugating) folding domain

• A C-terminal extension of approximately 40 residues that is largely unstructured and minimally conserved

• A conserved Lys91 near the catalytic site that undergoes constitutive monoubiquitination in vivo

• Eight lysine sites (Lys28, 48, 91, 136, 141, 156, 182, and 192) that can undergo auto-ubiquitination

• Additional post-translational modification sites including phosphorylation (Thr72, Ser184, and His194) and acetylation (Lys191)

The protein localizes primarily to the nucleus and nucleolus, where it performs its functions in DNA repair pathways .

How does UBE2T function in the ubiquitination pathway?

UBE2T functions as an E2 ubiquitin-conjugating enzyme that catalyzes the transfer of ubiquitin from an E1 enzyme to target proteins. Specifically:

• UBE2T attaches ubiquitin covalently to lysine residues of substrate proteins during the ubiquitination process

• It works in coordination with E3 ubiquitin ligases, particularly FANCL in the Fanconi anemia pathway

• UBE2T specifically mediates the monoubiquitination of FANCD2 on Lys563 following exposure to DNA damaging agents like mitomycin C

• It contributes to the ubiquitination and degradation of BRCA1 in breast cancer cells by interacting with the BRCA1/BARD1 complex

Understanding this pathway has significant implications for developing targeted therapies for conditions where UBE2T is dysregulated.

What role does UBE2T play in the Fanconi anemia DNA repair pathway?

UBE2T serves as the dedicated E2 ubiquitin-conjugating enzyme in the Fanconi anemia (FA) DNA repair pathway, which is crucial for repairing DNA interstrand crosslinks. The mechanism involves:

• Following DNA damage, UBE2T interacts with the RING domain of FANCL (the E3 ligase component of the FA core complex)

• This interaction enables UBE2T to monoubiquitinate FANCD2 on Lys563

• Monoubiquitinated FANCD2 then localizes to DNA damage sites and recruits additional repair factors

• This cascade is essential for efficient DNA interstrand crosslink repair

Disruption of UBE2T function compromises this repair pathway, leading to genomic instability and increased sensitivity to DNA-damaging agents . Methodologically, researchers can assess this function by measuring FANCD2 monoubiquitination levels via Western blotting after exposing cells to DNA-damaging agents like mitomycin C.

How can researchers experimentally disrupt UBE2T to study its function?

Several methodological approaches are used to study UBE2T function through experimental disruption:

• Gene knockout/knockdown approaches:

  • CRISPR-Cas9 gene editing to create UBE2T knockout cell lines

  • siRNA or shRNA to achieve transient or stable knockdown

  • Gene targeting constructs with drug resistance cassettes (e.g., puromycin, blasticidin) for generating targeted integrations

• Point mutations of catalytic residues:

  • Introduction of mutations at key residues like P73K, which has been shown to disrupt UBE2T function

  • Mutation of the catalytic cysteine to abolish ubiquitin conjugation activity

• Allosteric inhibition:

  • Small molecule fragments identified through biophysical screens that bind to allosteric pockets on UBE2T and inhibit its function

• Validation of successful disruption:

  • Southern blot analysis of genomic DNA to confirm targeted integrations

  • Monitoring FANCD2 monoubiquitination levels

  • Assessing cellular sensitivity to DNA-damaging agents as a functional readout

These approaches provide complementary insights into UBE2T function and can be selected based on the specific research question.

How is UBE2T expression altered in different cancer types?

UBE2T expression is frequently dysregulated across multiple cancer types, with significant clinical implications:

Research methods to assess UBE2T expression include:

• Immunohistochemistry of tissue sections

• Analysis of gene expression databases like GEO

• RT-qPCR for mRNA quantification

• Western blotting for protein level assessment

Understanding these expression patterns is crucial for evaluating UBE2T as a potential biomarker or therapeutic target.

What are the mechanisms by which UBE2T promotes cancer progression?

UBE2T contributes to cancer development and progression through multiple mechanisms:

• Impaired DNA repair and genomic instability:

  • Dysregulated UBE2T alters DNA repair efficiency, particularly homologous recombination

  • In multiple myeloma, UBE2T is required for efficient DNA repair by homologous recombination

• Regulation of tumor suppressor proteins:

  • UBE2T mediates the ubiquitination and degradation of BRCA1 in breast cancer cells

  • This degradation can contribute to loss of tumor suppressor function

• Signaling pathway activation:

  • In retinoblastoma, UBE2T promotes proliferation and tumorigenesis via the STAT3 signaling pathway

  • Knockdown of UBE2T reduces cancer cell proliferation

• Cell cycle and apoptosis regulation:

  • UBE2T has been linked to dysregulation in cell cycle progression and apoptosis resistance

  • These effects contribute to the hallmarks of cancer

Research approaches to study these mechanisms include pathway analysis through RNA-sequencing after UBE2T knockdown, protein-protein interaction studies, and cell proliferation/apoptosis assays.

What biophysical and structural biology approaches are used to study UBE2T?

Several sophisticated methodologies are employed to study UBE2T structure and interactions:

• Fragment screening approaches:

  • Differential scanning fluorimetry (DSF) to monitor protein unfolding temperature changes in response to fragment binding

  • Biolayer interferometry (BLI) using biotin-labeled UBE2T attached to streptavidin-coated biosensors

  • One-dimensional 1H NMR spectroscopy for secondary screening

  • Protein-observed NMR spectroscopy for binding site identification

• X-ray crystallography:

  • Co-crystallization with fragments or inhibitors

  • Analysis of structural changes upon binding

  • Identification of druggable pockets

• Protein expression and purification:

  • Expression of truncated forms (e.g., UBE2T 1-154) for structural studies

  • Recombinant expression in E. coli with affinity tags (e.g., N-terminal 6-His tag)

  • Isotopic labeling for NMR studies

• Binding affinity measurements:

  • Isothermal titration calorimetry (ITC) for quantitative binding measurements

  • Surface plasmon resonance for real-time binding kinetics

These approaches provide critical insights into UBE2T structure and function that inform both basic research and drug development efforts.

How can researchers identify and validate UBE2T inhibitors?

Developing and validating UBE2T inhibitors involves a systematic approach:

• Initial screening for binding:

  • Fragment screening using biophysical methods (DSF, BLI, NMR)

  • Virtual screening against known structures

  • High-throughput biochemical assays

• Identification of binding sites:

  • Protein-observed NMR to map chemical shift perturbations

  • X-ray crystallography of protein-inhibitor complexes

  • Mutagenesis studies (e.g., P73K mutation disrupts binding of certain inhibitors)

• Functional validation:

  • In vitro ubiquitination assays to assess inhibition of catalytic activity

  • Monitoring of FANCD2 monoubiquitination in cellular systems

  • Cellular sensitivity to DNA-damaging agents

• Structure-activity relationship studies:

  • Medicinal chemistry optimization of initial hits

  • Analysis of allosteric mechanisms (e.g., the allosteric pocket identified through fragment screening)

A notable example is the discovery of fragments binding to an allosteric pocket on UBE2T that inhibit ubiquitin conjugation in vitro, validating this approach for developing UBE2T inhibitors .

How does UBE2T expression correlate with treatment resistance?

UBE2T expression and activity have significant implications for treatment resistance in cancer:

• Chemotherapy resistance:

  • In multiple myeloma, UBE2T is required for homologous recombination DNA repair

  • Knockdown of UBE2T increases multiple myeloma sensitivity to chemotherapeutic agents

  • This suggests that high UBE2T expression may contribute to chemotherapy resistance through enhanced DNA repair capacity

• Radiotherapy response:

  • UBE2T interacts with multiple proteins involved in response to radiation

  • This suggests a potential role in radioresistance mechanisms

• Methodological assessment:

  • Researchers can measure treatment sensitivity using cell viability assays (e.g., CCK-8 assay) after UBE2T knockdown

  • Flow cytometry can be used to assess cell cycle effects and apoptosis following treatment

  • RNA-sequencing analysis can identify pathways affected by UBE2T modulation

Understanding these relationships provides a rationale for targeting UBE2T as a strategy to enhance treatment efficacy.

What are the current approaches for targeting UBE2T therapeutically?

Several strategies are being developed to target UBE2T for therapeutic purposes:

• Small molecule inhibitors:

  • Allosteric inhibitors that bind to newly identified pockets on UBE2T

  • Fragment-based approaches have identified several compounds that inhibit ubiquitin conjugation in vitro

  • Structure-based drug design informed by crystallography and NMR studies

• Genetic approaches:

  • siRNA or shRNA-based knockdown of UBE2T in tumors

  • This approach has shown promise in reducing tumor growth in animal models

• Combination therapy strategies:

  • Combining UBE2T inhibition with DNA-damaging agents to enhance sensitivity

  • This approach exploits the role of UBE2T in DNA repair pathways

• Biomarker-driven therapy:

  • Assessment of UBE2T expression levels to identify patients likely to benefit from UBE2T-targeted therapy

  • Development of companion diagnostics for patient selection

While UBE2T inhibitor development is still in early stages, the biological rationale and preliminary results suggest significant potential for this therapeutic approach.

What are the key unanswered questions in UBE2T research?

Despite significant progress, several important questions about UBE2T remain unanswered:

• Is UBE2T a typical oncogene, or does it have other functions besides carcinogenesis?

• Does UBE2T have cancer-inhibitory effects in certain contexts?

• Are there mechanisms of action for UBE2T beyond ubiquitin-dependent functions?

• How does UBE2T behave differently across various cancer types?

• What is the full spectrum of UBE2T substrates beyond FANCD2 and BRCA1?

• How do post-translational modifications of UBE2T regulate its function?

Addressing these questions will require integrated approaches combining structural biology, biochemistry, cell biology, and clinical research.

What emerging technologies might advance UBE2T research?

Several cutting-edge technologies hold promise for advancing UBE2T research:

• Proteomics approaches:

  • Ubiquitinome analysis to identify the complete set of UBE2T substrates

  • Proximity labeling methods to map the UBE2T interactome in different cellular contexts

• CRISPR-based screening:

  • Genome-wide CRISPR screens to identify synthetic lethal interactions with UBE2T

  • CRISPR activation/inhibition screens to identify regulators of UBE2T expression

• Single-cell technologies:

  • Single-cell RNA-seq to understand heterogeneity in UBE2T expression within tumors

  • Spatial transcriptomics to map UBE2T expression patterns in tissue contexts

• Advanced structural methods:

  • Cryo-EM studies of UBE2T in complex with E3 ligases and substrates

  • Hydrogen-deuterium exchange mass spectrometry to study conformational dynamics