Recombinant Human Transmembrane protein 183A (TMEM183A)

What is TMEM183A and what is its molecular structure?

TMEM183A (Transmembrane protein 183A) is a human protein consisting of 376 amino acids encoded by the TMEM183A gene (previously known as C1orf37) located on chromosome 1 . Structurally, TMEM183A is characterized as a multi-pass membrane protein with predicted transmembrane domains. While the complete three-dimensional structure has not been fully resolved using methods like X-ray crystallography or cryo-EM, sequence analysis suggests it contains multiple hydrophobic regions consistent with membrane-spanning domains .

Methodological approach: To investigate TMEM183A structure, researchers should consider:

• Employing computational prediction tools for transmembrane domains

• Utilizing recombinant expression systems with appropriate detergents for membrane protein purification

• Applying structural biology techniques similar to those used for other membrane proteins, such as the NMR solution structure approach demonstrated for VDAC-1

• Incorporating isotope labeling strategies for high-field triple-resonance TROSY-type experiments to identify secondary structure elements

What post-translational modifications have been identified in TMEM183A?

Multiple post-translational modifications have been identified in human TMEM183A through proteomic analyses :

Methodological approach: Researchers investigating PTMs should:

• Employ phospho-enrichment strategies combined with mass spectrometry

• Utilize site-directed mutagenesis of modified residues to assess functional significance

• Develop modification-specific antibodies for tracking PTM status under various conditions

• Consider the interplay between different modifications, particularly at K57 where both acetylation and sumoylation occur

What is the expression pattern of TMEM183A across human tissues?

TMEM183A appears to be widely expressed across multiple human tissues according to the Human Protein Atlas data . While the search results don't provide comprehensive expression data, researchers should note that TMEM183A has been detected in various tissue types including neural tissues, reproductive organs, and hematopoietic lineages.

Methodological approach: To characterize expression patterns:

• Analyze publicly available RNA-seq datasets from repositories like GTEx

• Employ quantitative PCR with tissue-specific cDNA panels

• Utilize immunohistochemistry with validated antibodies

• Consider single-cell transcriptomics to identify cell-type specific expression patterns within heterogeneous tissues

What are the predicted functional roles of TMEM183A?

TMEM183A is predicted to be involved in regulation of protein stability and may be part of the SCF ubiquitin ligase complex . Its interaction with proteins involved in interferon signaling, apoptosis regulation, and stress response pathways suggests potential roles in immune response and cellular stress adaptation .

Methodological considerations:

• Analyze protein-protein interaction networks using immunoprecipitation followed by mass spectrometry

• Employ functional genomics approaches including CRISPR-Cas9 knockout/knockdown studies

• Assess cellular phenotypes following TMEM183A perturbation in relevant cell types

• Investigate changes in ubiquitination patterns of potential substrate proteins

What experimental approaches are optimal for studying TMEM183A function?

Based on current literature, optimal approaches for TMEM183A functional studies include:

• Genetic knockdown/knockout models:

  • In zebrafish, antisense oligonucleotide (ASO)-based knockdown revealed TMEM183A's potential role in thrombocyte function through gill bleeding assays

  • For mammalian systems, CRISPR-Cas9 or shRNA approaches targeting conserved regions would be appropriate

• Recombinant protein production:

  • Cell-free protein synthesis (CFPS) systems have successfully produced recombinant TMEM183A with Strep-Tag purification

  • The ALiCE® expression system derived from Nicotiana tabacum has demonstrated >70-80% purity for TMEM183A production

• Functional interaction studies:

  • Investigating protein partners identified through STRING database analysis (IFIT3, RNF13, HSF2BP, VRK2)

  • Employing proximity labeling approaches like BioID or APEX to identify membrane-proximal interactors

• Disease-relevant assays:

  • Analysis of cellular phenotypes in patient-derived cells harboring TMEM183A variants

  • Investigation of signaling pathway alterations using phospho-specific antibodies to assess downstream effects

How does TMEM183A interact with other proteins and what signaling pathways might it be involved in?

STRING database analysis reveals several predicted functional partners of TMEM183A with confidence scores :

These interactions suggest TMEM183A may function in:

• Interferon-mediated antiviral response pathways through IFIT3

• ER stress and JNK signaling through RNF13

• Stress response modulation through HSF2BP and VRK2

Methodological approach:

• Perform co-immunoprecipitation studies with tagged TMEM183A

• Use proximity ligation assays to confirm interactions in intact cells

• Employ FRET/BRET approaches for dynamic interaction studies

• Investigate pathway activation states (phospho-JNK, IRF3 translocation) following TMEM183A perturbation

What disease associations have been identified for TMEM183A variants?

Several disease-associated variants have been identified in TMEM183A :

Notably, these variants affect sites of post-translational modification, suggesting potential functional consequences through altered protein regulation.

Methodological considerations:

• Perform case-control studies in cancer cohorts to validate associations

• Create cellular models with variant forms using CRISPR knock-in approaches

• Assess functional consequences of each variant on protein stability, localization, and interaction partners

• Investigate differences in PTM status between wild-type and variant forms

What is known about TMEM183A orthology and evolutionary conservation?

TMEM183A appears to be conserved across multiple species, with orthologs identified in various mammals including elephantulus edwardii (Cape elephant shrew) and microcebus murinus (gray mouse lemur) , suggesting evolutionary conservation of function. The NCBI ortholog database indicates the presence of TMEM183A across different species .

Methodological approach for evolutionary studies:

• Perform multiple sequence alignment of TMEM183A orthologs

• Calculate selection pressure metrics (dN/dS) across protein domains

• Identify highly conserved regions as likely functional domains

• Reconstruct phylogenetic relationships to trace evolutionary history

• Compare expression patterns of orthologs across equivalent tissues in different species

What role might TMEM183A play in thrombocyte function based on zebrafish models?

Research in zebrafish has demonstrated that knockdown of tmem183a results in significantly increased bleeding in gill bleeding assays, suggesting a role in thrombocyte function . This finding provides important insights into potential roles of TMEM183A in hemostasis and platelet function.

Methodological considerations:

• Extend zebrafish findings to mammalian platelet function studies

• Investigate TMEM183A expression during megakaryocyte differentiation

• Assess platelet aggregation, adhesion, and activation markers in TMEM183A-deficient models

• Explore potential interactions with known platelet receptors and signaling proteins

• Consider human platelet transcriptome data to validate expression in human platelets

How can recombinant TMEM183A be effectively produced for structural and functional studies?

Production of recombinant TMEM183A has been achieved using the following methods :

• Expression system selection:

  • The ALiCE® (Almost Living Cell-Free Expression System) derived from Nicotiana tabacum has successfully produced TMEM183A

  • This system maintains the protein production machinery and mitochondria while removing cellular components not required for protein production

• Purification approach:

  • One-step Strep-tag purification has yielded >70-80% purity as determined by SDS-PAGE, Western Blot, and analytical SEC

  • The recombinant protein includes the full sequence (AA 1-376) with a Strep tag

• Quality control metrics:

  • Concentration determination using absorbance at 280nm against a specific reference buffer

  • Expasy's ProtParam tool for absorption coefficient determination

Methodological considerations for optimization:

• Assess different detergents for membrane protein stabilization

• Explore nanodiscs or amphipols for maintaining native-like membrane environment

• Consider codon optimization for expression host

• Evaluate different fusion tags for enhanced solubility and purification

What challenges exist in TMEM183A research and what methodological approaches might overcome them?

Several challenges exist in TMEM183A research:

• Limited structural information:

  • Apply integrative structural biology approaches combining computational prediction, crosslinking mass spectrometry, and low-resolution structural techniques

  • Consider parallel studies of more tractable orthologs from thermophilic organisms

• Functional ambiguity:

  • Employ multi-omics approaches (proteomics, transcriptomics, metabolomics) following TMEM183A perturbation

  • Utilize cell-type specific conditional knockout models to overcome potential developmental effects

• Membrane protein isolation:

  • Optimize detergent selection through stability screening

  • Explore protein stabilization through engineering approaches like thermostabilizing mutations or antibody fragments

• Disease relevance clarification:

  • Implement high-throughput screening of TMEM183A variants in cellular models

  • Develop targeted sequencing panels for TMEM183A in relevant patient cohorts

  • Utilize patient-derived organoids to study TMEM183A function in disease contexts

What are the most promising future research directions for TMEM183A?

Based on current knowledge, the most promising research directions include:

• Structural characterization:

  • Determination of high-resolution structure through cryo-EM or X-ray crystallography

  • Investigation of conformational changes associated with function

• Functional networks:

  • Comprehensive mapping of TMEM183A interactome in different cellular contexts

  • Integration of TMEM183A into known signaling networks

• Disease mechanisms:

  • Detailed investigation of how TMEM183A variants contribute to cancer pathogenesis

  • Exploration of potential roles in other diseases, particularly those involving thrombocyte function

• Therapeutic targeting:

  • Assessment of TMEM183A as a potential diagnostic biomarker

  • Exploration of methods to modulate TMEM183A function in disease contexts