UBE2H Human

What is UBE2H and what is its fundamental role in cellular processes?

UBE2H (Ubiquitin Conjugating Enzyme E2 H) is a member of the E2 ubiquitin-conjugating enzyme family in humans (gene ID: 7328). Also known by synonyms E2-20K, GID3, UBC8, UBCH, and UBCH2, UBE2H plays a critical role in the ubiquitination pathway, which targets abnormal or short-lived proteins for degradation .

The ubiquitination process involves a cascade of three enzyme classes working sequentially: ubiquitin-activating enzymes (E1s), ubiquitin-conjugating enzymes (E2s) like UBE2H, and ubiquitin-protein ligases (E3s) . This enzymatic pathway is essential for maintaining cellular protein homeostasis through the ubiquitin-proteasome system (UPS).

When investigating UBE2H function, researchers typically employ in vitro ubiquitination assays with purified components and cell-based systems where UBE2H expression can be genetically modulated to observe downstream effects on protein degradation pathways.

What is known about the evolutionary conservation of UBE2H across species?

UBE2H demonstrates remarkable evolutionary conservation, indicating its fundamental importance in cellular function. According to available data, the human UBE2H protein sequence is 100% identical to its mouse homolog and shares 98% identity with both frog and zebrafish homologs . This high degree of conservation suggests strong evolutionary pressure to maintain UBE2H structure and function.

For researchers studying UBE2H in model organisms, this conservation provides confidence that findings may be translatable across species. Comparative genomic approaches can effectively identify critical functional domains that have been preserved through evolution, potentially highlighting regions essential for enzymatic activity.

What methodologies are most effective for detecting UBE2H expression in different tissues?

For researchers investigating UBE2H expression patterns, several complementary methodologies are recommended:

• RNA-level detection:

  • RT-qPCR provides a sensitive approach for quantifying UBE2H mRNA levels in tissues and blood samples

  • RNA-sequencing (RNA-seq) offers comprehensive transcriptomic analysis with FPKM (Fragments Per Kilobase of transcript per Million mapped reads) values to normalize expression levels

• Protein-level detection:

  • Western blotting with specific antibodies against UBE2H

  • Immunohistochemistry for tissue localization studies

Based on published findings, UBE2H mRNA is highly expressed in primary cortical neurons . Interestingly, UBE2H mRNA levels were elevated in the blood of Alzheimer's disease models (5xFAD mice) and in peripheral blood mononuclear cells (PBMCs) from severe AD patients, while no significant changes were observed in brain tissues .

How should researchers interpret differences in UBE2H expression between blood and brain tissue?

The discrepancy between UBE2H expression in blood versus brain tissue presents an important methodological consideration. Research has shown that while UBE2H mRNA is significantly increased in blood samples from AD models and patients, it shows no significant changes in brain tissue .

When designing experiments:

• Always collect and analyze both central (brain) and peripheral (blood) tissues when possible

• Consider using paired samples from the same subjects to enable direct comparisons

• Control for confounding factors that might influence tissue-specific expression

• Explore the potential role of extracellular vesicles (EVs), which may concentrate UBE2H from blood to cortex

This tissue-specific expression pattern suggests complex regulatory mechanisms that warrant careful experimental design and interpretation of results.

What evidence supports UBE2H as a biomarker for Alzheimer's disease?

Several lines of evidence support the potential of UBE2H as a biomarker for Alzheimer's disease:

• Differential expression in AD: UBE2H mRNA transcription is significantly increased in blood samples from AD models (5xFAD mice) compared to wild-type controls . This finding is corroborated in human studies, where UBE2H mRNA expression levels are elevated in peripheral blood mononuclear cells (PBMCs) from severe AD patients .

• Specificity among UBE2 family members: Among the UBE2 subfamily genes (including UBE2L6, UBE2B, UBE2C, UBE2O, and UBE2M), only UBE2H mRNA shows significant elevation in AD blood samples, suggesting specificity for the disease process .

• Correlation with disease severity: Data from human patients suggest that UBE2H mRNA expression levels correlate with disease severity, with higher levels observed in severe AD patients compared to mild cases or normal controls .

For researchers investigating UBE2H as a biomarker, RT-qPCR provides a quantitative approach for measuring UBE2H mRNA levels in clinical samples, while RNA-seq offers a more comprehensive assessment that can reveal correlations with other gene expression changes.

How does UBE2H contribute to protein degradation pathways in neurodegenerative conditions?

UBE2H functions within the ubiquitin-proteasome system (UPS), which is often dysregulated in neurodegenerative disorders. Research findings suggest several mechanisms through which UBE2H influences protein degradation in these conditions:

• Regulation of polyubiquitination: Depletion of UBE2H through siRNA knockdown decreases intracellular polyubiquitination, indicating its important role in the ubiquitin-dependent system in the cortex .

• Independence from known AD pathways: Notably, depletion of UBE2H did not affect the levels of Tau or Parkin proteins, suggesting that UBE2H contributes to ubiquitin-dependent pathways distinct from these well-known AD-related proteins .

• Potential role in mitophagy: Previous studies have identified UBE2 subfamily members as co-factors for Parkin-dependent mitophagy, a process important for removing damaged mitochondria that is often impaired in neurodegenerative disorders .

Experimental approaches to study UBE2H in this context should include:

• Knockdown/knockout studies to assess effects on global ubiquitination patterns

• Identification of specific substrate proteins targeted by UBE2H-mediated ubiquitination

• Investigation of interactions between UBE2H and relevant E3 ligases in neuronal cells

What are recommended protocols for studying UBE2H-mediated ubiquitination in vitro?

For researchers investigating UBE2H enzymatic activity and substrate specificity, the following experimental approach is recommended:

• Protein purification and activity assays:

  • Express and purify recombinant UBE2H, preferably with affinity tags for easy purification

  • Conduct in vitro ubiquitination assays with purified E1, UBE2H, candidate E3 ligases, and potential substrates

  • Use Western blotting or mass spectrometry to detect ubiquitinated products

• Analysis of ubiquitin chain types:

  • Employ linkage-specific antibodies to determine the types of ubiquitin chains formed (K48, K63, etc.)

  • Use ubiquitin mutants lacking specific lysine residues to confirm chain types

  • Consider mass spectrometry-based approaches for comprehensive ubiquitin linkage analysis

• Interaction studies:

  • Perform pull-down assays to identify proteins that interact with UBE2H

  • Use surface plasmon resonance or isothermal titration calorimetry to measure binding affinities

  • Conduct structural studies (X-ray crystallography, NMR) to characterize interaction interfaces

These approaches will help elucidate the specific role of UBE2H in the ubiquitination cascade and identify its preferred E3 partners and substrates.

How can researchers effectively modulate UBE2H expression for functional studies?

Several genetic approaches have been successfully employed to modulate UBE2H expression:

• RNA interference (RNAi):

  • siRNA-mediated knockdown has been successfully used to deplete UBE2H, resulting in decreased intracellular polyubiquitination

  • Design multiple siRNAs targeting different regions of UBE2H mRNA

  • Include appropriate controls (scrambled siRNA) and validate knockdown efficiency at both mRNA and protein levels

• CRISPR-Cas9 genome editing:

  • Generate stable knockout cell lines for long-term studies

  • Consider inducible CRISPR systems for temporal control of UBE2H depletion

  • Design guide RNAs targeting conserved regions of the UBE2H gene

• Overexpression systems:

  • Use expression vectors with strong promoters (CMV, EF1α) for consistent expression

  • Consider tagged versions (FLAG, HA, GFP) to facilitate detection and immunoprecipitation

  • Employ inducible expression systems (Tet-On/Off) to control expression levels

When designing these experiments, it's critical to validate the specificity of your approach, as manipulation of UBE2H may affect multiple cellular pathways due to its role in protein homeostasis.

How does UBE2H relate to other ubiquitin-conjugating enzymes in neurodegenerative disease research?

UBE2H is one of approximately 40 E2 enzymes encoded in mammals, each with distinct but sometimes overlapping functions. In the context of neurodegenerative disease research:

• Differential expression patterns:

  • RNA-seq and RT-qPCR analyses have shown that among UBE2 subfamily genes (UBE2H, UBE2L6, UBE2B, UBE2C, UBE2O, and UBE2M), only UBE2H mRNA is significantly increased in blood from AD patients

  • This specificity suggests a unique role for UBE2H in AD pathogenesis compared to other E2 enzymes

• Functional specialization:

  • Each E2 enzyme typically cooperates with specific E3 ligases, allowing for targeted regulation of different cellular processes

  • The UBC domain found in all E2s facilitates activation and binding of E3s, but structural differences contribute to functional specialization

  • Extensions of the amino- or carboxyl-terminus on each E2 confer distinct enzymatic activities

• Experimental considerations:

  • When studying UBE2H, researchers should consider potential compensatory mechanisms involving other E2 enzymes

  • Comparative studies examining multiple E2 enzymes can help identify unique versus redundant functions

  • Family-wide approaches (siRNA libraries targeting all E2s) can reveal functional relationships

Understanding how UBE2H functions within the broader context of E2 enzymes provides important insights into its specific contributions to neurodegenerative disease processes.

What approaches should be used to investigate UBE2H genetic variants in patient populations?

UBE2H has been identified as highly polymorphic, with mutations associated with neurodegenerative conditions including amyotrophic lateral sclerosis (ALS) and autistic disorder . To investigate these genetic variations:

• Genetic screening strategies:

  • Targeted sequencing of UBE2H in patient cohorts with neurodegenerative disorders

  • Integration with genome-wide association studies (GWAS) data

  • Case-control studies comparing variant frequencies between patients and healthy individuals

• Functional characterization of variants:

  • Expression of wild-type and variant UBE2H in cellular models

  • Assessment of effects on ubiquitination activity, protein stability, and subcellular localization

  • Evaluation of interactions with known binding partners

• Genotype-phenotype correlations:

  • Detailed clinical characterization of patients carrying UBE2H variants

  • Analysis of potential modifiers that influence disease presentation

  • Longitudinal follow-up to assess effects on disease progression

• In silico analysis:

  • Structural modeling to predict functional consequences of amino acid substitutions

  • Evolutionary conservation analysis to identify critical residues

  • Prediction of effects on protein-protein interactions

These approaches can help elucidate how specific UBE2H polymorphisms affect protein function and contribute to disease pathogenesis, potentially identifying patient subgroups for targeted therapeutic strategies.

What are the most promising therapeutic approaches targeting UBE2H?

Given the potential role of UBE2H in neurodegenerative disorders, several therapeutic strategies warrant investigation:

• Small molecule modulators:

  • Development of compounds that enhance or inhibit UBE2H activity

  • High-throughput screening assays using recombinant UBE2H

  • Structure-based drug design targeting the UBC domain or protein interaction interfaces

• Gene therapy approaches:

  • Viral vector-mediated delivery of UBE2H in deficiency states

  • Antisense oligonucleotides or siRNAs for specific knockdown in cases of pathological overexpression

  • CRISPR-based approaches for correction of pathogenic variants

• Biomarker-based applications:

  • Utilizing circulating UBE2H mRNA as a biomarker for patient selection in clinical trials

  • Monitoring UBE2H levels as a pharmacodynamic marker for treatment response

  • Development of companion diagnostics for targeted therapies

Researchers pursuing these approaches should consider the tissue-specific regulation of UBE2H and the discrepancy between its expression in blood versus brain tissue, which may affect drug delivery strategies and biomarker utility.

How can researchers reconcile contradictory findings regarding UBE2H expression in different tissues?

The observation that UBE2H mRNA is significantly increased in blood from AD patients but shows no change in brain tissue presents an interesting paradox . To address this discrepancy:

• Investigation of tissue-specific regulation:

  • Analysis of tissue-specific transcription factors controlling UBE2H expression

  • Epigenetic profiling of the UBE2H promoter in different tissues

  • Assessment of post-transcriptional regulation mechanisms (miRNAs, RNA-binding proteins)

• Extracellular vesicle (EV) studies:

  • Isolation and characterization of EVs from blood and cerebrospinal fluid

  • Analysis of UBE2H mRNA content in different EV populations

  • Investigation of EV transport mechanisms between blood and brain

• Translation and post-translational regulation:

  • Comparison of UBE2H protein levels with mRNA expression across tissues

  • Analysis of protein stability and degradation rates in different cell types

  • Investigation of post-translational modifications affecting UBE2H function

• Methodological considerations:

  • Use of multiple detection methods to confirm expression patterns

  • Careful selection of reference genes for normalization in different tissues

  • Consideration of cellular heterogeneity within tissue samples

These approaches can help elucidate the mechanisms underlying tissue-specific UBE2H expression patterns and their relevance to disease pathogenesis.