UBE2S Human

What is UBE2S and what is its primary function in human cells?

UBE2S is an E2 ubiquitin-conjugating enzyme that catalyzes the formation of K11-linked polyubiquitin chains. Its primary function involves protein ubiquitination, a post-translational modification process that regulates protein degradation and cellular signaling pathways. UBE2S specifically promotes K11-linked polyubiquitination, which differs from the classical K48-linked chains that typically mark proteins for proteasomal degradation .

To study UBE2S function, researchers typically employ techniques such as RNA interference (shRNA or siRNA) to knock down UBE2S expression or overexpression systems using lentiviral vectors. For instance, the cDNA sequence of UBE2S can be inserted into pCDH lentivirus vectors and transfected into cells to study gain-of-function effects . Expression changes are then validated using quantitative real-time PCR and Western blot analysis.

How is UBE2S expression regulated in normal versus cancer tissues?

UBE2S expression is significantly upregulated in multiple cancer types compared to normal tissues. In hepatocellular carcinoma (HCC), both mRNA and protein levels of UBE2S are higher in tumor tissues than in adjacent normal tissues . Similarly, in lung cancer, immunohistochemical assays and TCGA database analyses have demonstrated elevated UBE2S expression in cancer tissues compared to normal tissues .

To investigate UBE2S expression levels, researchers commonly employ:

• Immunohistochemistry (IHC) on tissue microarrays

• Western blot analysis of tissue or cell lysates

• Quantitative real-time PCR for mRNA expression

• Analysis of public databases such as TCGA

For instance, a study of lung adenocarcinoma used microarrays containing 30 tumor samples across different stages and 30 adjacent normal samples, which were subjected to immunohistochemistry staining of UBE2S .

What signaling pathways does UBE2S interact with?

UBE2S interacts with multiple signaling pathways that are critical for cancer development and progression:

• Cell cycle regulation: UBE2S interacts with TRIM28 in the nucleus to enhance ubiquitination of p27, facilitating its degradation and promoting cell cycle progression .

• Wnt/β-Catenin pathway: UBE2S modifies β-Catenin at K19 via K11-linked polyubiquitin chain, enhancing β-Catenin stability and promoting its cellular accumulation .

• VHL/HIF-1α and VHL/JAK2/STAT3 pathways: UBE2S interacts directly with VHL to upregulate these pathways, enhancing malignant properties of HCC cells .

• P53, Integrin, and ILK signaling: Microarray analysis of UBE2S knockdown cells revealed regulation of genes involved in these pathways .

To study these interactions, researchers use co-immunoprecipitation assays, pathway-specific reporter assays, and gene expression profiling following UBE2S manipulation.

How does the dimeric state of UBE2S affect its function and regulation?

UBE2S has been discovered to exist in a dimeric state that confers auto-inhibition by blocking a catalytically critical ubiquitin binding site. This dimerization is stimulated by the lysine-rich C-terminal extension of UBE2S, which is also required for the recruitment of this E2 to the APC/C (Anaphase-Promoting Complex/Cyclosome) and is auto-ubiquitinated as substrate abundance becomes limiting .

Researchers investigating UBE2S dimerization have employed:

• Nuclear magnetic resonance (NMR) spectroscopy to detect concentration-dependent changes in molecular tumbling

• Pull-down experiments using differentially tagged UBE2S (His6- and HA-tagged)

• Crystallography to determine protein structure (PDB: 6S98 for wild-type UBC domain and PDB: 6S96 for C118A mutant)

The structural data revealed significant differences in crystal packing between wild-type and mutant UBE2S:

Dimerization-deficient UBE2S mutants showed more rapid turnover in cells and did not promote mitotic slippage during prolonged drug-induced mitotic arrest, suggesting that dimerization attenuates the auto-ubiquitination-induced turnover of UBE2S when the APC/C is not fully active .

What is the significance of UBE2S mutations in cancer development?

Whole-exome sequencing of HCC tissues has identified novel somatic mutations in UBE2S, including p.Gly57Ala and p.Lys63Asn. Functional predictions indicated that these amino acid substitutions are potentially deleterious . While wild-type UBE2S promotes cancer progression, these mutations may further enhance its oncogenic properties.

To study these mutations, researchers can employ:

• Site-directed mutagenesis to generate mutant UBE2S constructs

• Functional assays comparing wild-type and mutant UBE2S effects on:

  • Cell proliferation (MTT, BrdU incorporation)

  • Invasion and migration (Transwell, wound healing)

  • Cell cycle progression (flow cytometry)

  • In vivo tumor growth (xenograft models)

• Structural analyses to determine how mutations affect protein conformation and interaction capabilities

How does UBE2S influence cell cycle regulation in cancer cells?

UBE2S plays a critical role in cell cycle progression, particularly at the G1/S phase transition. Mechanistically, UBE2S interacts with TRIM28 in the nucleus, and together they enhance the ubiquitination of p27 to facilitate its degradation and promote cell cycle progression .

Research methodologies to investigate this include:

• Flow cytometry analysis of cell cycle distribution following UBE2S knockdown or overexpression

• BrdU incorporation assays to measure DNA synthesis and cell proliferation

• Western blot analysis of cell cycle regulators (p27, cyclins, CDKs)

• Co-immunoprecipitation to detect UBE2S interactions with TRIM28 and other cell cycle proteins

• Ubiquitination assays to measure p27 ubiquitination levels

Studies have demonstrated that UBE2S knockdown in HCC and lung cancer cells leads to G1 phase arrest, decreased BrdU incorporation, and increased p27 levels . Conversely, UBE2S overexpression promotes G1/S transition and enhances cell proliferation. These findings establish UBE2S as a key regulator of cell cycle progression in cancer cells.

What are the optimal techniques for studying UBE2S protein-protein interactions?

Several complementary techniques can be employed to study UBE2S protein-protein interactions:

• Co-immunoprecipitation (Co-IP): This is the primary method used to detect interactions between UBE2S and partners like TRIM28, VHL, and β-Catenin. Researchers typically use antibodies against UBE2S or its tagged version (HA, Flag, His) to pull down protein complexes, followed by immunoblotting for potential interacting partners .

• Proximity ligation assay (PLA): This technique provides visualization of protein interactions in situ with high specificity and sensitivity.

• Fluorescence resonance energy transfer (FRET) or bimolecular fluorescence complementation (BiFC): These techniques allow for real-time visualization of protein interactions in living cells.

• Yeast two-hybrid screening: This can be used for unbiased identification of novel UBE2S-interacting proteins.

• Mass spectrometry-based approaches: After immunoprecipitation, mass spectrometry can identify novel interacting partners in an unbiased manner.

For studying UBE2S dimerization specifically, researchers have used pull-down experiments with His6- and HA-tagged UBE2S, along with NMR analyses under reducing conditions to prevent disulfide-induced aggregation .

How can researchers effectively modulate UBE2S expression for functional studies?

To modulate UBE2S expression for functional studies, researchers have employed several approaches:

• RNA interference:

  • Short hairpin RNA (shRNA): Lentiviral vectors expressing shRNA targeting UBE2S can be used for stable knockdown. Studies have used constructs like shUBE2S-1 and shUBE2S-2 to ensure specificity and reproducibility .

  • Small interfering RNA (siRNA): For transient knockdown experiments.

• Overexpression systems:

  • The cDNA sequence of UBE2S can be inserted into lentivirus vectors (e.g., pCDH) and transfected into cells along with packaging vectors (PSPAX2 and PDM2G) .

  • Inducible expression systems can provide temporal control over UBE2S expression.

• CRISPR-Cas9 gene editing:

  • For complete knockout or introducing specific mutations.

• Small molecule inhibitors:

  • Cephalomannine has been identified through high-throughput screening to inhibit UBE2S expression and significantly attenuate HCC progression .

Validation of expression modulation should be performed using:

• Quantitative real-time PCR for mRNA levels

• Western blot for protein levels

• Functional assays to confirm phenotypic changes

What are the recommended experimental models for studying UBE2S in cancer?

Researchers have employed various experimental models to study UBE2S in cancer:

• Cell line models:

  • HCC cell lines (HepG2, Hep3B, Huh7, SMMC-7721)

  • Lung cancer cell lines (A549, H1299)

  • Colorectal cancer cell lines

• Patient-derived samples:

  • Tissue microarrays containing multiple patient samples

  • Fresh frozen tissues for protein and RNA extraction

• Animal models:

  • Xenograft models: Cancer cells with manipulated UBE2S expression are injected subcutaneously or orthotopically into immunodeficient mice to assess tumor growth and metastasis .

  • Patient-derived xenografts (PDX): These maintain tumor heterogeneity and better recapitulate human tumor characteristics.

• 3D culture systems:

  • Spheroid cultures and organoids can bridge the gap between 2D cell cultures and in vivo models.

For analyzing clinical relevance, researchers commonly use:

• Immunohistochemistry on tissue microarrays

• Analysis of public databases (TCGA, GEO)

• Correlation of UBE2S expression with clinicopathological features and patient survival

How does UBE2S expression correlate with clinical outcomes in cancer patients?

High UBE2S expression has been consistently associated with poor clinical outcomes across multiple cancer types:

To establish these correlations, researchers typically:

• Perform IHC on tissue microarrays containing samples from patients with comprehensive clinical data

• Use Kaplan-Meier survival analysis to correlate expression with patient outcomes

• Conduct multivariate Cox regression analysis to determine if UBE2S is an independent prognostic factor

• Analyze public databases (TCGA) to validate findings in larger cohorts

These clinical correlations reinforce the biological significance of UBE2S in cancer progression and highlight its potential as a prognostic biomarker.

What therapeutic strategies target UBE2S in cancer treatment?

Several therapeutic approaches targeting UBE2S are being explored:

• Small molecule inhibitors:

  • Cephalomannine was identified through a luciferase-based high-throughput screen to inhibit UBE2S expression and significantly attenuate HCC progression both in vitro and in vivo .

  • Structure-based drug design targeting the UBE2S active site or dimerization interface represents another approach.

• Combination therapies:

  • Downregulation of UBE2S expression enhanced the sensitivity of HCC cells to sorafenib in vivo and in vitro , suggesting potential synergistic effects with established cancer therapies.

• Gene therapy approaches:

  • Delivery of UBE2S-targeting shRNA or siRNA could potentially suppress tumor growth.

• Targeting UBE2S-regulated pathways:

  • Inhibitors of downstream pathways activated by UBE2S, such as Wnt/β-Catenin, VHL/HIF-1α, or JAK2/STAT3 signaling, may be effective in UBE2S-overexpressing tumors.

For preclinical evaluation of these approaches, researchers should:

• Assess efficacy in multiple cell line and animal models

• Evaluate specificity using rescue experiments

• Determine potential toxicities in normal cells

• Investigate mechanisms of resistance

How can UBE2S be used as a biomarker for cancer diagnosis and prognosis?

UBE2S has significant potential as a biomarker for cancer:

To develop UBE2S as a clinical biomarker, researchers should:

• Validate expression patterns in large, multi-center cohorts

• Develop standardized assays for UBE2S detection

• Determine optimal cutoff values for high versus low expression

• Evaluate UBE2S in combination with other biomarkers for improved accuracy

• Assess UBE2S in liquid biopsies (circulating tumor cells, cell-free DNA) for non-invasive testing

What is the role of UBE2S in cellular differentiation and development?

UBE2S has been implicated in cellular differentiation processes, particularly in embryonic stem (ES) cell differentiation into mesoendoderm lineages. Research has shown that Ube2s-promoted β-Catenin accumulation partially released the dependence on exogenous molecules for the process of ES cell differentiation into mesoendoderm lineages .

Research approaches to investigate this role include:

• In vitro differentiation assays with ES cells

• Gene expression profiling during differentiation

• Lineage tracing in development models

• Knockout or knockdown studies in developmental systems

This emerging area of research suggests that UBE2S may have important functions beyond cancer, potentially playing roles in embryonic development and tissue homeostasis.

How do different ubiquitin chain topologies mediated by UBE2S affect substrate fate?

UBE2S specifically catalyzes the formation of K11-linked polyubiquitin chains, which differs from the classical K48-linked chains typically associated with protein degradation. UBE2S modifies β-Catenin at K19 via K11-linked polyubiquitin chain, resulting in enhanced stability rather than degradation .

To investigate ubiquitin chain topology and its effects:

• Use ubiquitin mutants (with specific lysine residues mutated to arginine)

• Employ mass spectrometry to identify ubiquitination sites and chain types

• Perform in vitro ubiquitination assays with purified components

• Use linkage-specific antibodies to detect specific ubiquitin chain types

Understanding how different ubiquitin chain topologies affect substrate fate could reveal new mechanisms of protein regulation and potentially identify novel therapeutic approaches targeting specific ubiquitination events.