Recombinant Human Transmembrane protein 62 (TMEM62)

What is the basic structure of human TMEM62?

Human TMEM62 is a 643-amino acid transmembrane protein with an N-terminal region that can be tagged (commonly with histidine) for recombinant expression. The full protein sequence includes multiple transmembrane domains and conserved regions that contribute to its function. The amino acid sequence, as identified in recombinant production, is: MAAVLALRVVAGLAAAALVAMLLEHYGLAGQPSPLPRPAPPRRPHPAPGPGDSNIFWGLQISDIHLSRFRDPGRAVDLEKFCSETIDIIQPALVLATGDLTDAKTKEQLGSRQHEVEWQTYQGILKKTRVMEKTKWLDIKGNHDAFNIPSLDSIKNYYRKYSAVRRDGSFHYVHSTPFGNYSFICVDATVNPGPKRPYNFFGILDKKKMEELLLLAKESSRSNHTIWFGHFTTSTILSPSPGIRSIMSSAIAYLCGHLHTLGGLMPVLHTRHFQGTLELEVGDWKDNRRYRIFAFDHDLFSFADLIFGKWPVVLITNPKSLLYSCGEHEPLERLLHSTHIRVLAFSLSSITSVTVKIDGVHLGQAVHVSGPIFVLKWNPRNYSSGTHNIEVIVQDSAGRSKSVHHIFSVQENNHLSFDPLASFILRTDHYIMARVLFVLIVLSQLTILIIFRYRGYPELKEPSGFINLTSFSLHVLSKINIFYYSVLLLTLYTVLGPWFFGEIIDGKFGCCFSFGIFVNGHFLQGSITFIIGILQLAFFNIPLMAYMCWSLLQRCFGHNFRSHLHQRKYLKIMPVHLLMLLLYIWQVYSCYFLYATYGTLAFLFSPLRTWLTLLTPVLIRYVWTLNSTKFGIFMVQLKSHLSS .

How is recombinant TMEM62 typically produced for research applications?

Recombinant human TMEM62 is typically expressed in E. coli expression systems with N-terminal histidine tags to facilitate purification. The production process involves expressing the full-length protein (amino acids 1-643) or specific domains of interest. For optimal research applications, the recombinant protein is typically purified using affinity chromatography and supplied as a lyophilized powder that requires reconstitution before use . Expression in mammalian cell systems is also possible for studies requiring post-translational modifications, though bacterial expression remains common for basic structural and functional studies.

What are the recommended storage and handling conditions for recombinant TMEM62?

Recombinant TMEM62 protein should be stored at -20°C to -80°C immediately upon receipt. For working with the protein, researchers should briefly centrifuge the vial before opening to ensure all material is at the bottom. The lyophilized protein should be reconstituted in deionized sterile water to a concentration of 0.1-1.0 mg/mL. For long-term storage, it is recommended to add glycerol (5-50% final concentration) and aliquot the solution to avoid repeated freeze-thaw cycles, which can compromise protein integrity. Working aliquots can be stored at 4°C for up to one week . The reconstituted protein is typically stored in Tris/PBS-based buffer with 6% trehalose at pH 8.0 to maintain stability .

What experimental methods are most effective for studying TMEM62 expression patterns?

Based on research practices evidenced in the literature, effective methods for studying TMEM62 expression include:

• RNA-Seq Analysis: This has been successfully employed to identify differential expression of TMEM62 between experimental groups, such as in studies comparing long-term versus short-term survival in ovarian cancer patients .

• Quantitative Real-Time PCR: For validation of expression levels identified in broader screening approaches.

• Western Blotting: Using specific antibodies against TMEM62 or its tagged versions to detect protein expression levels.

• Immunohistochemistry: For examining tissue-specific expression patterns, particularly in cancer specimens.

• Single-Cell RNA Sequencing: Especially useful for understanding cell-type-specific expression, as demonstrated in studies examining host responses to viral infection .

For developmental studies, expression profiling can be conducted across different tissues and developmental stages, similar to approaches used in Xenopus models where TMEM62 expression has been documented across embryonic stages and in tissues including brain, ectoderm, intestine, and reproductive organs .

What is the significance of TMEM62 in ovarian cancer research?

TMEM62 has emerged as a potential molecular predictor of long-term survival in high-grade serous ovarian carcinoma (HGSC). In a study analyzing samples from patients with long-term survival (LTS, ≥10 years) versus short-term survival (STS, <3 years), TMEM62 was identified among 11 genes differentially expressed between these groups. The study revealed that TMEM62 expression positively correlates with better patient outcomes .

Functional validation studies using OVCAR5 and OVCAR8 ovarian cancer cell lines demonstrated that overexpression of TMEM62 resulted in decreased cell proliferation, suggesting a potential tumor-suppressive role. This was further confirmed in an in vivo model using OVCAR8-TMEM62-TetON cells injected into female athymic nude mice, where higher expression of TMEM62 was associated with reduced tumor burden .

These findings suggest that TMEM62 may function as a tumor suppressor in ovarian cancer, and strategies to restore or enhance its expression could represent a novel therapeutic approach for HGSC.

How does TMEM62 affect cellular proliferation in experimental cancer models?

In experimental models using ovarian cancer cell lines (OVCAR5 and OVCAR8), researchers have demonstrated that TMEM62 overexpression leads to decreased proliferation rates . The mechanism appears to be linked to longevity-regulating pathways, specifically the KEGG HSA04213 pathway, with which TMEM62 shows positive correlation at the RNA level .

The anti-proliferative effect was confirmed through both in vitro and in vivo experiments. In the mouse model using OVCAR8-TMEM62-TetON, induction of TMEM62 expression resulted in significantly reduced tumor burden compared to controls with low TMEM62 expression . This suggests that TMEM62 may regulate cell cycle progression or promote apoptosis in cancer cells, though the precise molecular mechanisms require further investigation.

How is TMEM62 involved in immune responses?

TMEM62 appears to have connections to immune regulation, particularly in the context of host responses to viral infections. Analysis of tumors from long-term versus short-term ovarian cancer survivors using CIBERSORT revealed higher levels of follicular helper T cells in tumors from long-term survivors, which also corresponded with higher TMEM62 expression . This suggests potential crosstalk between TMEM62 expression and T cell-mediated immune responses.

Additionally, TMEM62 has been identified in studies analyzing the invariant host response to viral pandemics, including research on severe acute respiratory syndromes. Using single-cell RNA sequencing and human lung histology, researchers implicated TMEM62 in the lung epithelial and myeloid cell response to viral infection . This positions TMEM62 as potentially important in the broader context of innate immune responses.

What signaling pathways interact with TMEM62?

TMEM62 shows significant correlation with the longevity-regulating pathway (KEGG HSA04213) at the RNA level . This suggests involvement in cellular processes that regulate lifespan and aging, which may partly explain its tumor-suppressive effects.

The protein has also been identified in analyses of ACE2-related gene networks, particularly in the context of viral infections and host responses . In these studies, TMEM62 emerged as part of gene clusters that reflect host immune responses, specifically in reactive myeloid cells and epithelial tissues.

Further research is needed to fully characterize the direct protein interactions and downstream effectors of TMEM62 signaling, as the current literature provides evidence of correlation but lacks detailed mechanistic explanations of how TMEM62 exerts its effects on cell proliferation and immune regulation.

What experimental approaches are recommended for manipulating TMEM62 expression?

Based on successful approaches documented in the literature:

• Inducible Expression Systems: The OVCAR8-TMEM62-TetON model demonstrates the utility of tetracycline-inducible systems for controlled expression of TMEM62 in both in vitro and in vivo studies .

• Lentiviral/Retroviral Transduction: For stable overexpression of TMEM62 in cell line models.

• CRISPR/Cas9 Gene Editing: For knockout or knockdown studies to assess loss-of-function effects.

• siRNA/shRNA Approaches: For transient knockdown experiments to study immediate effects of TMEM62 reduction.

When designing such experiments, researchers should consider:

• The use of tagged versions (e.g., His-tag, FLAG-tag) to facilitate detection and purification

• Cell-type specific promoters for targeted expression in heterogeneous systems

• Dose-dependent induction systems to study threshold effects

What challenges exist in studying transmembrane properties of TMEM62?

As a transmembrane protein, TMEM62 presents several technical challenges:

• Protein Solubility: The hydrophobic transmembrane domains can cause aggregation during recombinant expression and purification.

• Structural Analysis: Traditional structural biology techniques like X-ray crystallography are challenging for membrane proteins. Alternative approaches such as cryo-EM or NMR may be more suitable.

• Functional Reconstitution: Studies of transport or channel functions require reconstitution in artificial membrane systems or careful design of cellular assays.

• Interaction Studies: Identifying protein-protein interactions is complicated by the membrane environment and may require specialized approaches like proximity labeling or split-reporter systems.

To address these challenges, researchers might consider using truncated versions of TMEM62 focusing on specific domains, employing detergent screens for solubilization, or utilizing cell-based assays that preserve the native membrane environment.

What are promising areas for future TMEM62 research?

Based on current findings, several research directions warrant further investigation:

• Mechanistic Studies: Detailed investigation of how TMEM62 regulates cell proliferation and its exact role in longevity-regulating pathways.

• Clinical Correlations: Expanded studies on TMEM62 expression across larger patient cohorts and additional cancer types beyond ovarian cancer.

• Therapeutic Development: Exploration of approaches to modulate TMEM62 expression or activity as potential cancer therapies.

• Immune Regulation: Further characterization of the relationship between TMEM62 expression and T cell responses, particularly in the tumor microenvironment.

• Viral Response Mechanisms: Deeper investigation into TMEM62's role in host responses to viral infections, particularly respiratory viruses.

• Developmental Biology: Studies of TMEM62 function across different developmental stages, building on expression data from model organisms like Xenopus .

How might TMEM62 research impact personalized medicine approaches?

The correlation between TMEM62 expression and survival outcomes in ovarian cancer suggests potential applications in personalized medicine:

• Prognostic Biomarker: TMEM62 expression levels could be developed as a biomarker to help predict patient outcomes and guide treatment intensity.

• Treatment Stratification: Patients with low TMEM62 expression might benefit from more aggressive treatment approaches or novel therapies targeting pathways regulated by TMEM62.

• Therapeutic Targeting: Strategies to upregulate TMEM62 or mimic its downstream effects could represent personalized therapeutic approaches for patients with low endogenous expression.

• Combination Therapies: Understanding TMEM62's relationship with immune responses could inform immunotherapy combinations, particularly for patients with specific TMEM62 expression patterns.

To advance these applications, larger validation studies correlating TMEM62 expression with treatment responses and long-term outcomes will be essential.