Recombinant Rat Transmembrane protein 183 (Tmem183)

What is Transmembrane Protein 183 (Tmem183) and what are its key structural features?

Transmembrane protein 183 (Tmem183), also known as Tmem183a or MNCb-2755, is a 375 amino acid membrane protein that belongs to the TMEM183 family . The protein contains multiple transmembrane domains that anchor it within cellular membranes. While detailed structural analyses are still emerging, the protein likely adopts a conformation similar to other transmembrane proteins, with both extracellular and intracellular domains that facilitate its biological functions. The gene encoding TMEM183 is located on chromosome 1 in mice, which houses over 1,500 genes including those encoding nuclear receptor coactivators, coatomer complex subunits, synaptotagmins, and olfactory receptors .

Sequence analysis of Tmem183 reveals conserved domains that suggest potential functional roles in cellular signaling or transport processes. Unlike other well-characterized transmembrane proteins such as Tmem43, which has been extensively studied (with a known sequence of 400 amino acids for the rat variant) , Tmem183 represents an emerging area of investigation requiring further characterization.

How does Tmem183 expression vary across different tissues in rats?

TMEM183 expression patterns demonstrate tissue specificity, though comprehensive expression profiling across rat tissues remains incomplete. By analogy to other transmembrane proteins in the same family, Tmem183 likely exhibits differential expression across various tissue types. For example, TMEM182, another member of the transmembrane protein family, shows notable abundance in muscle and adipose tissue .

When designing experiments to analyze Tmem183 expression, researchers should consider:

• Using quantitative RT-PCR to measure transcript levels across different tissues

• Implementing Western blot analysis with validated antibodies for protein expression assessment

• Employing immunohistochemistry for spatial localization within tissue sections

• Comparing expression levels during different developmental stages

A systematic analysis of expression patterns would provide valuable insights into potential tissue-specific functions of Tmem183 in rats.

What are the recommended validation methods for confirming the identity of recombinant Tmem183 protein?

Multiple orthogonal approaches should be employed to validate recombinant Tmem183 protein identity:

Mass spectrometric characterization is particularly valuable for definitive identification, as demonstrated with other recombinant rat transmembrane proteins . This approach can provide detailed information about post-translational modifications and confirm the amino acid sequence, enabling researchers to verify the integrity of the recombinant protein before proceeding with functional studies.

What expression systems are most appropriate for producing functional recombinant rat Tmem183?

The selection of an appropriate expression system is critical for obtaining functional recombinant Tmem183. Based on experimental approaches used for similar transmembrane proteins:

What signaling pathways are potentially modulated by Tmem183 based on structural homology?

While specific information about Tmem183 signaling is limited, insights can be gained from other transmembrane proteins in the same family. By analyzing structural homology and conserved domains, several potential signaling pathways may be implicated:

• Wnt/β-catenin Signaling: Similar transmembrane proteins like TMEM182 interact with integrin-linked kinase (ILK), which increases Ser473 phosphorylation of AKT, promoting GSK-3β phosphorylation at Ser9 . This inhibits β-catenin degradation, enhancing its nuclear accumulation and transcriptional activity.

• Cell Adhesion and Cytoskeletal Organization: Based on the involvement of chromosome 1-localized genes in processes related to cell structure and interaction .

• Membrane Transport: As a transmembrane protein, Tmem183 may participate in ion or small molecule transport across cellular membranes.

Experimental approaches to investigate these pathways include:

• Co-immunoprecipitation to identify protein interaction partners

• Phosphorylation analysis of downstream signaling molecules (western blotting)

• Reporter assays for transcriptional activation

• Cellular localization studies using fluorescently tagged Tmem183

Understanding these pathways is crucial for elucidating Tmem183's biological function and potential role in disease processes.

How can researchers investigate protein-protein interactions involving Tmem183?

Investigating protein-protein interactions for transmembrane proteins requires specialized techniques:

For transmembrane proteins like Tmem183, membrane-based assays such as membrane yeast two-hybrid or split-ubiquitin systems are particularly valuable as they are designed specifically for membrane proteins that may not properly localize to the nucleus in conventional yeast two-hybrid systems.

When designing these experiments, researchers should:

• Consider the topology of Tmem183 to ensure interaction domains are accessible

• Use appropriate controls to validate interactions

• Confirm interactions using multiple independent techniques

• Validate biological relevance through functional assays

What phenotypic changes are associated with Tmem183 dysregulation in cellular and animal models?

Understanding phenotypic changes associated with Tmem183 dysregulation provides insights into its functional significance. While specific data on Tmem183 phenotypes is limited, potential areas of investigation based on transmembrane protein biology include:

• Cellular Morphology: Changes in cell shape, size, or membrane architecture

• Cell Viability and Proliferation: Effects on growth rate, cell cycle progression, or apoptosis

• Differentiation Capacity: Impact on lineage commitment or differentiation potential

• Cellular Localization: Altered distribution of cellular components or organelles

• Tissue Integrity: Changes in tissue architecture or function in animal models

For example, studies of similar proteins like TMEM182 have shown that its overexpression inhibits myocardial differentiation of human induced pluripotent stem cells by maintaining Wnt/β-catenin signaling in an activated state . This suggests that transmembrane proteins can significantly impact cellular differentiation processes.

Experimental approaches should include:

• Loss-of-function (knockdown/knockout) studies

• Gain-of-function (overexpression) studies

• Rescue experiments to confirm specificity of observed phenotypes

• Tissue-specific manipulations in animal models

Detailed phenotypic characterization using multiple parameters will provide a comprehensive understanding of Tmem183's biological roles.

What are the critical considerations for developing specific antibodies against rat Tmem183?

Developing specific antibodies against transmembrane proteins presents unique challenges:

• Epitope Selection:

  • Analyze the Tmem183 sequence to identify hydrophilic, surface-exposed regions

  • Focus on N- or C-terminal regions that typically extend into aqueous environments

  • Avoid transmembrane domains that are poorly immunogenic

  • Consider species conservation if cross-reactivity is desired

• Antigen Preparation Options:

  • Synthetic peptides corresponding to selected epitopes

  • Recombinant protein fragments expressed in E. coli

  • Full-length protein in detergent micelles or nanodiscs

  • DNA immunization encoding Tmem183 fragments

• Validation Strategy:

  • Western blot analysis with positive and negative controls

  • Immunoprecipitation followed by mass spectrometry

  • Immunofluorescence with subcellular localization analysis

  • Testing on tissues from Tmem183 knockout models

  • Peptide competition assays

• Potential Pitfalls:

  • Conformational epitopes may be lost in denatured proteins

  • Cross-reactivity with homologous proteins

  • Low expression levels limiting detection

  • Detergent sensitivity affecting epitope recognition

An effective validation process is critical before using antibodies in research applications, as demonstrated in studies of other transmembrane proteins where antibody specificity enables detailed characterization of protein expression and localization .

What computational approaches can predict Tmem183 structure and function?

Computational methods offer valuable insights into transmembrane protein structure and function:

• Structural Prediction:

  • Transmembrane helix prediction using algorithms like TMHMM, PHDhtm, or MEMSAT

  • Ab initio modeling with specialized tools for membrane proteins (e.g., Rosetta Membrane)

  • Homology modeling based on structurally characterized transmembrane proteins

  • Molecular dynamics simulations to predict stability and conformational changes

• Functional Prediction:

  • Motif identification using tools like PROSITE or ELM

  • Gene ontology annotation based on sequence similarity

  • Protein-protein interaction prediction using tools like STRING

  • Virtual screening for potential ligands or binding partners

• Evolutionary Analysis:

  • Multiple sequence alignment to identify conserved residues

  • Phylogenetic analysis to understand evolutionary relationships

  • Positive selection analysis to identify functionally important residues

• Integrative Approaches:

  • Combined structure-function prediction using machine learning

  • Network analysis to place Tmem183 in biological pathways

  • Systems biology modeling of potential regulatory networks

These computational approaches provide testable hypotheses that can guide experimental design and interpretation of results.

What is the potential involvement of Tmem183 in rat disease models?

While specific information about Tmem183 in disease models is limited, transmembrane proteins on chromosome 1 have been implicated in various conditions:

• Neurological Disorders: Chromosome 1-localized genes have been associated with conditions such as infantile neuroaxonal dystrophy in mouse models . Investigating Tmem183's expression in neuronal tissues and its potential role in neurological function could reveal involvement in related disorders.

• Autoimmune Conditions: Given that chromosome 1 genes have been linked to autoimmune myocarditis , Tmem183 may play a role in immune regulation or inflammation.

• Cancer: Chromosome 1 genes have been implicated in lung carcinomas . Research examining Tmem183 expression changes in tumor versus normal tissues could identify potential oncogenic or tumor-suppressive roles.

• Cardiovascular System: By analogy to TMEM182, which affects myocardial differentiation through Wnt/β-catenin signaling , Tmem183 may have roles in cardiovascular development or function.

Research approaches to investigate disease relevance include:

• Expression analysis in tissues from disease models

• Genetic association studies in rat models of human disease

• Functional studies in relevant cell types

• Therapeutic modulation of Tmem183 expression or function

How can recombinant Tmem183 be utilized to develop screening assays for potential therapeutic compounds?

Recombinant Tmem183 provides a valuable tool for developing screening assays:

• Binding Assays:

  • Surface plasmon resonance (SPR) to detect direct interactions

  • Fluorescence-based thermal shift assays to identify stabilizing compounds

  • AlphaScreen or FRET-based assays for high-throughput screening

• Functional Assays:

  • Activity-based assays if enzymatic function is identified

  • Cell-based reporter systems measuring downstream pathway activation

  • Phenotypic screens in Tmem183-expressing cell lines

• Structural Studies for Rational Design:

  • X-ray crystallography or cryo-EM of purified Tmem183

  • Fragment-based screening approaches

  • Computer-aided drug design based on structural models

• Assay Development Considerations:

  • Protein stability in screening conditions

  • Signal-to-background optimization

  • Assay miniaturization for high-throughput formats

  • Inclusion of appropriate positive and negative controls

When establishing these assays, researchers should ensure that recombinant Tmem183 maintains native conformation and activity, possibly by incorporating it into appropriate membrane mimetics such as nanodiscs or liposomes.

How can researchers resolve issues with Tmem183 solubility and stability during purification?

Maintaining transmembrane protein solubility and stability requires specialized approaches:

• Detergent Screening:

  • Test multiple detergent classes (maltoside, glucoside, fos-choline)

  • Evaluate detergent concentration effects

  • Consider detergent mixtures for improved stability

• Buffer Optimization:

  • Adjust pH to optimize protein stability

  • Test different salt concentrations (typically 150-300 mM)

  • Include stabilizing agents (glycerol, trehalose, specific lipids)

• Additives for Enhanced Stability:

  • Cholesterol or other sterols for membrane protein stability

  • Specific lipids that may be required for function

  • Small molecule stabilizers identified through thermal shift assays

• Alternative Solubilization Approaches:

  • Amphipols for improved stability after initial detergent solubilization

  • Nanodiscs for a more native-like membrane environment

  • Styrene maleic acid lipid particles (SMALPs) for detergent-free extraction

• Purification Modifications:

  • Reduce temperature during all purification steps

  • Minimize exposure time during chromatography

  • Include fresh protease inhibitors throughout the process

Systematic optimization of these parameters will significantly improve purification outcomes for challenging transmembrane proteins like Tmem183.