Recombinant Human Transmembrane and ubiquitin-like domain-containing protein 2 (TMUB2)

What is the genomic location and structure of the TMUB2 gene?

TMUB2 maps to human chromosome 17, specifically at locus 17q21.31, positioned between ASB16-AS1 (upstream) and ATXN7L3 (downstream). The gene spans approximately 4.99Kb and can be transcribed into three possible mRNA variants . When designing experiments targeting this gene, researchers should account for these variants by using primers that either target conserved regions (for total TMUB2 expression analysis) or isoform-specific regions (for variant-specific studies). Genomic analyses should consider the chromosomal context, as neighboring genes may share regulatory elements or be co-regulated in certain cellular contexts.

What is known about TMUB2 expression patterns across tissues?

TMUB2 demonstrates ubiquitous expression throughout the human body, with expression levels approximately 2.9 times higher than the average human gene . When investigating tissue-specific roles, researchers should establish baseline expression through qPCR or RNA-seq in their specific model systems, as expression levels may vary despite ubiquitous presence. Experimental designs should include appropriate housekeeping genes for normalization, and multiple tissue comparisons should account for this high baseline expression when interpreting fold-changes in experimental conditions.

What is the basic structure of the TMUB2 protein?

The human TMUB2 protein consists of 321 amino acids with a molecular weight of 33.8 kDa and an isoelectric point of 4.73899 . The protein contains three transmembrane regions that anchor it within cellular membranes. For protein isolation experiments, researchers should consider these properties when designing extraction protocols, particularly the need for detergent-based lysis buffers suitable for membrane proteins. The acidic isoelectric point should inform buffer selection for isoelectric focusing or ion exchange chromatography during purification.

How evolutionarily conserved is TMUB2 across species?

TMUB2 demonstrates significant evolutionary conservation, particularly among vertebrates. Sequence identity ranges from 100% in chimpanzees to 47% in zebrafish, with more distant homologs found in invertebrates (23% in ants) and even microorganisms such as algae (41%) and fungi (38%) . This conservation pattern suggests functional importance throughout evolution. When selecting model organisms for TMUB2 studies, researchers should consider this conservation data, as shown in the following comparison table:

For cross-species comparisons, researchers should focus on conserved domains when designing experiments and antibodies .

What are the known paralogs of TMUB2 and their significance?

TMUB1 is the only known paralog of TMUB2, sharing 38% sequence identity and 51% similarity . When investigating TMUB2 function, researchers should consider potential functional redundancy or compensation by TMUB1. Experimental designs should include TMUB1 expression analysis when manipulating TMUB2 levels. For knockout or knockdown studies, researchers should validate antibody specificity to ensure they don't cross-react with TMUB1, and consider double-knockout approaches to account for potential compensatory mechanisms between these paralogous proteins.

What protein-protein interactions have been identified for TMUB2?

TMUB2 has a confirmed interaction with Ubiquitin C (UBC), and proposed interactions with BCL2L13 (BCL2-like 13), SGTA (Small glutamine-rich tetratricopeptide repeat-containing protein), and UBQLN1 (Ubiquilin-1) . For researchers investigating these interactions, co-immunoprecipitation experiments should include appropriate controls to validate specificity. When designing yeast two-hybrid or proximity ligation assays, researchers should consider the transmembrane nature of TMUB2 and select appropriate bait constructs that maintain protein folding and accessibility of interaction domains.

How can researchers effectively investigate the ubiquitin-related functions of TMUB2?

Given its ubiquitin-like domain, researchers studying TMUB2's potential role in ubiquitination pathways should employ deubiquitination assays similar to those used for USP2 studies . Experimental approaches should include:

• In vitro deubiquitination assays using recombinant TMUB2 and polyubiquitinated substrates

• Cellular ubiquitination assays with TMUB2 overexpression or knockdown

• Proteasome inhibition studies to determine if TMUB2 affects protein degradation pathways

These experiments should include appropriate controls, such as catalytically inactive mutants and known deubiquitinating enzymes as positive controls .

What are the key considerations for designing knockout or knockdown studies of TMUB2?

When designing CRISPR-Cas9 knockout or siRNA knockdown experiments for TMUB2, researchers should consider:

• Target specificity: Design guide RNAs or siRNAs that specifically target TMUB2 without affecting TMUB1 or other genes

• Verification methods: Implement multiple verification approaches including genomic sequencing, qPCR, and Western blotting

• Phenotypic assessment: Develop comprehensive assays to detect subtle phenotypes, as ubiquitously expressed proteins often have redundant mechanisms

• Temporal considerations: Implement inducible systems to study acute versus chronic loss of TMUB2 function

Researchers should employ quasi-experimental designs when randomization is not feasible, such as when working with patient-derived samples with differential TMUB2 expression .

How should recombinant TMUB2 protein be produced and purified for functional studies?

For recombinant TMUB2 production, researchers should:

• Select an appropriate expression system: Consider mammalian expression systems for full-length TMUB2 with transmembrane domains, or E. coli for soluble domains (similar to USP2 catalytic domain production)

• Design constructs with affinity tags: Include removable tags positioned to minimize interference with protein function

• Optimize purification protocols: Implement specialized detergent-based extraction methods for membrane proteins

• Validate protein folding: Employ circular dichroism spectroscopy to confirm secondary structure

• Assess functional activity: Develop specific activity assays based on hypothesized TMUB2 functions

Researchers should be prepared to troubleshoot common issues with membrane protein expression, including protein aggregation and low yields.

How can researchers investigate the potential role of TMUB2 in protein trafficking and membrane dynamics?

Given its transmembrane domains, TMUB2 may function in protein trafficking or membrane organization. Researchers should consider:

• Subcellular localization studies using fluorescent protein fusions and co-localization with organelle markers

• Pulse-chase experiments to track protein movement through cellular compartments

• Membrane fractionation assays to determine TMUB2 distribution among different membrane types

• Protein trafficking assays using temperature-sensitive cargo proteins

• Live-cell imaging with photoactivatable or photoconvertible TMUB2 fusions

These approaches should implement appropriate controls and quantification methods to detect subtle changes in localization or trafficking dynamics .

What strategies should be employed to resolve contradictory data in TMUB2 research?

When faced with contradictory results in TMUB2 studies, researchers should:

• Systematically evaluate experimental differences between studies, including cell types, culture conditions, and methodological approaches

• Implement multiple complementary techniques to address the same question

• Consider context-dependency of TMUB2 function across different cellular environments

• Develop collaborations to independently verify key findings

• Employ statistical meta-analysis approaches when sufficient data is available

This systematic approach helps resolve whether contradictions reflect true biological variation or methodological differences.

How can researchers investigate potential tissue-specific functions of TMUB2 despite its ubiquitous expression?

To uncover tissue-specific functions of ubiquitously expressed TMUB2, researchers should:

• Implement tissue-specific conditional knockout models using Cre-lox systems

• Analyze differential interactomes across tissues using proximity labeling approaches

• Examine expression correlations with tissue-specific genes through transcriptomic analysis

• Investigate post-translational modifications that might differ across tissues

• Develop tissue-specific reporter systems to monitor TMUB2 activity in different contexts

These approaches enable detection of subtle tissue-specific functions despite uniform expression levels .

What are the most promising approaches for elucidating the currently unknown function of TMUB2?

To uncover TMUB2's function, researchers should pursue multi-disciplinary approaches including:

• Unbiased interactome analysis using BioID or proximity labeling techniques

• Comparative phenotypic analysis across model organisms with TMUB2 orthologs

• High-throughput genetic interaction screens to identify synthetic lethal or synthetic viable interactions

• Structural biology approaches to determine protein domains and potential binding sites

• Systems biology analysis integrating transcriptomic, proteomic, and metabolomic data from TMUB2-manipulated systems

The combination of these approaches provides complementary insights that can converge on functional hypotheses .