Recombinant Pongo abelii Transmembrane protein 231 (TMEM231)

What experimental models and systems are available for studying TMEM231 function?

Multiple experimental models have been developed to study TMEM231 function:

Mammalian Cell Culture Systems:

• Mouse embryonic fibroblasts (MEFs) from wild-type and TMEM231-deficient mice have been used to assess localization of transition zone proteins

• Human patient-derived fibroblasts carrying TMEM231 mutations provide important disease models

Animal Models:

• Mouse models: Tmem231-knockout mice on C57BL/6 background die at embryonic day E15.5, while those on C57BL/6-CD1 mixed background survive until birth, enabling study of later developmental stages

• Caenorhabditis elegans: Transgenic nematodes expressing GFP-tagged TMEM-231 allow visualization of protein localization in ciliated sensory neurons

Recombinant Protein Systems:

• LAP-tagged (Localization and Affinity Purification) Tmem231 has been used for protein interaction studies through mass spectrometry

• Epitope-tagged versions (Flag-tagged Tmem231, V5-tagged B9d1) enable coimmunoprecipitation experiments to study protein interactions

For optimal results in studying TMEM231 function, researchers should select models appropriate to their specific research questions, with consideration for species-specific differences in protein function and expression patterns.

How does TMEM231 participate in ciliary transition zone assembly and function?

TMEM231 plays a critical role in transition zone (TZ) assembly and function through several interconnected mechanisms:

Assembly of the MKS Complex:
TMEM231 and B9d1 are reciprocally required for their localization to the TZ . Experiments show that in Tmem231-/- mouse embryonic fibroblasts (MEFs), B9d1 fails to localize to the TZ, and conversely, in B9d1-/- MEFs, Tmem231 no longer concentrates at the TZ . Additionally, Tmem231 and B9d1 are both essential for other MKS complex components like Mks1 and Tmem67 (Mks3) to properly localize to the TZ .

Diffusion Barrier Formation:
As part of the TZ, TMEM231 contributes to the formation of a diffusion barrier that regulates protein entry into the cilium. Experimental evidence indicates that TMEM231 mutations disrupt the localization of ciliary membrane proteins including Arl13b and Inpp5e, resulting in ciliopathy phenotypes .

Evolutionary Conservation:
The functional importance of TMEM231 in TZ assembly is underscored by its evolutionary conservation. In C. elegans, TMEM-231 also localizes to the TZ and depends on MKS-2 and MKS-5 for its proper localization . Reciprocally, TMEM-17 and MKS-2 require TMEM-231 for their TZ localization in nematodes .

These findings demonstrate that TMEM231 functions as a critical organizer of the MKS complex at the TZ, with significant implications for understanding ciliopathy pathogenesis.

What methodological approaches are recommended for detecting pathogenic variants in TMEM231?

Detection of pathogenic TMEM231 variants requires a comprehensive approach combining multiple methodologies:

Next-Generation Sequencing Approaches:

• Whole-exome sequencing (WES) has been successfully employed to identify novel TMEM231 variants in patients with ciliopathies

• Targeted gene panels focusing on ciliopathy-associated genes can be more cost-effective for clinical diagnosis

Variant Verification and Classification:

• Sanger sequencing remains essential for confirming variants identified through NGS and for family segregation studies

• Classification of variants should follow ACMG guidelines, with splice site variants often classified as pathogenic when they meet PVS1+PM2+PP4 criteria

Alternative Splicing Analysis:

• cDNA TA cloning analysis is critical for evaluating the impact of splice site variants

• RT-PCR can be employed to assess whether variants affect mRNA expression levels

Functional Characterization:

• Immunofluorescence analysis of patient tissues (such as kidney) can reveal alterations in cilia formation and structure

• Protein modeling using tools like SWISS-MODEL and ProtScale can predict structural changes and hydrophobicity alterations resulting from missense variants

Third-Generation Sequencing Considerations:
Due to limitations of short-read NGS in detecting gene conversions and distinguishing pseudogenes, third-generation long-read sequencing technologies (such as nanopore sequencing) should be considered as complementary approaches for comprehensive genetic diagnosis .

When investigating ciliopathy patients with only one heterozygous TMEM231 variant detected, additional testing for large deletions or gene conversions should be performed, as these may be missed by standard sequencing methods .

Producing functional recombinant TMEM231 for research applications presents significant challenges due to its transmembrane nature. Based on current research approaches, the following methodological framework is recommended:

Expression Systems:

• Mammalian cell expression systems (such as HEK293 or CHO cells) are preferred for maintaining proper post-translational modifications and membrane topology

• Insect cell systems (Sf9, High Five) can provide higher yields while maintaining eukaryotic processing capabilities

• Bacterial systems are generally less suitable due to the transmembrane nature of the protein, though specialized strains designed for membrane protein expression could be considered for fragments

Fusion Tags and Constructs:

• Localization and Affinity Purification (LAP) tags have been successfully used for TMEM231, enabling both visualization and purification

• Epitope tags such as FLAG or V5 tags positioned at either the N- or C-terminus facilitate detection and purification

• When designing constructs, careful consideration of tag placement is essential to avoid disrupting transmembrane domains

Solubilization and Purification:

• Detergent screening is critical for effective solubilization - mild detergents like DDM (n-Dodecyl β-D-maltoside) or LMNG (lauryl maltose neopentyl glycol) are recommended starting points

• Lipid nanodisc reconstitution can provide a more native-like environment for structural and functional studies

• Two-step affinity purification followed by size exclusion chromatography can improve purity while maintaining protein integrity

Quality Control:

• Western blotting to confirm protein integrity and expected molecular weight (approximately 36 kDa)

• Circular dichroism spectroscopy to verify proper secondary structure

• Dynamic light scattering to assess homogeneity and aggregation status

Functional Validation:

• Interaction studies with known binding partners like B9d1, Mks1, and other MKS complex components using co-immunoprecipitation or surface plasmon resonance

• Liposome reconstitution assays to assess membrane insertion and orientation

For the specific case of Pongo abelii TMEM231, researchers should note that while the protein is evolutionarily conserved, species-specific differences may affect experimental outcomes, particularly in interaction studies with proteins from different species.

How does TMEM231 dysfunction contribute to the pathogenesis of ciliopathies, and what are potential therapeutic targets?

TMEM231 dysfunction contributes to ciliopathy pathogenesis through several interconnected pathways, providing multiple potential therapeutic targets:

Molecular Mechanisms of Pathogenesis:

• Disrupted Transition Zone Formation:
  TMEM231 mutations compromise the assembly of the MKS complex at the transition zone (TZ), as evidenced by the failure of B9d1, Mks1, and Tmem67 to localize properly in Tmem231-deficient cells . This structural disruption forms the foundation for downstream pathogenic events.

• Compromised Ciliary Membrane Composition:
  Experimental evidence shows that TMEM231 mutations disrupt the localization of key ciliary membrane proteins including Arl13b and Inpp5e . This alteration in ciliary composition affects multiple signaling pathways.

• Impaired Hedgehog Signaling:
  Tmem231-mutant mouse embryos exhibit abrogated Hedgehog signaling, leading to developmental abnormalities including polydactyly and neural tube defects . The Hedgehog pathway is crucial for embryonic patterning and organogenesis.

• Tissue-Specific Manifestations:
  The widespread expression of TMEM231 in ciliated cells explains the multi-organ nature of associated ciliopathies. Phenotypic manifestations include:

  • Renal cysts due to disrupted ciliary signaling in kidney tubules

  • Hepatic ductal plate malformations affecting bile duct development

  • Neural tube defects and brain malformations due to impaired neuronal migration and patterning

  • Polydactyly resulting from aberrant limb bud signaling

Potential Therapeutic Approaches:

• Gene Therapy and Gene Editing:

  • AAV-mediated gene delivery could potentially restore functional TMEM231 in affected tissues

  • CRISPR-Cas9 approaches might correct specific mutations, particularly in cases with founder mutations

• Targeting Downstream Signaling Pathways:

  • Hedgehog pathway modulators could potentially address developmental abnormalities

  • mTOR inhibitors might ameliorate kidney cyst formation based on research in other ciliopathies

• Protein Replacement Strategies:

  • Cell-penetrating peptide conjugates of key functional domains could potentially rescue some aspects of TMEM231 function

  • Exosome-mediated delivery of functional TMEM231 protein might bypass membrane insertion challenges

• Small Molecule Chaperones:

  • For missense mutations that affect protein folding and stability (like p.Asn90Ile and p.Pro125Ala ), small molecule chaperones could potentially enhance the stability and function of mutant proteins

• RNA-Based Therapeutics:

  • Antisense oligonucleotides could target specific splice site mutations (such as c.583-1G>C ) to restore normal splicing

  • mRNA therapy delivering functional TMEM231 transcripts could provide transient protein expression

Development of these therapeutic approaches requires deeper understanding of tissue-specific TMEM231 functions and careful consideration of intervention timing, as many ciliopathy manifestations arise during embryonic development.