Recombinant Mouse UPF0444 transmembrane protein C12orf23 homolog

What is UPF0444 transmembrane protein C12orf23 homolog in mice?

UPF0444 transmembrane protein C12orf23 homolog (TMEM263) in mice is a plasma membrane protein encoded by the Tmem263 gene. It consists of 115 amino acids with UniProt accession number Q9DAM7 . The "UPF" designation (Uncharacterized Protein Family) reflects its initially unknown function, while "C12orf23 homolog" indicates its homology to a protein originally identified on human chromosome 12. Despite its previous classification as an uncharacterized protein, recent research has revealed its essential role in postnatal growth regulation through the GH/IGF-1 signaling pathway. The protein contains specific transmembrane domains that anchor it within the plasma membrane, positioning it to participate in cellular signaling processes critical for growth regulation .

What is the relationship between C12orf23 homolog and TMEM263?

C12orf23 homolog and TMEM263 refer to the same protein, reflecting the evolution of nomenclature as knowledge about the protein advanced. The original designation "C12orf23" was based purely on its genomic location in humans (Chromosome 12 Open Reading Frame 23), while "TMEM263" is a more functional name indicating its identity as a transmembrane protein. This nomenclature progression is evident in the search results, where the human ortholog is designated as "UPF0444 transmembrane protein C12orf23 (TMEM263)" with UniProt entry name "TM263_HUMAN" and accession number Q8WUH6 . Similarly, research publications have increasingly adopted the TMEM263 nomenclature as the protein's functional significance has become better understood . This dual naming system is common in genomics research as functional characterization follows initial genomic identification.

How is the UPF0444 transmembrane protein C12orf23 homolog stored and handled in laboratory settings?

Proper storage and handling of recombinant mouse UPF0444 transmembrane protein C12orf23 homolog is essential for maintaining its stability and functionality in research applications. The recommended storage conditions include:

• Long-term storage: -20°C or -80°C for extended preservation

• Working aliquots: 4°C for up to one week to minimize freeze-thaw cycles

• Storage buffer: Tris-based buffer with 50% glycerol, optimized for protein stability

Critical handling considerations include avoiding repeated freezing and thawing cycles, which can lead to protein degradation and loss of activity . For ELISA kits detecting this protein, shipping at 4°C is standard, with subsequent storage according to the kit-specific instructions . Laboratory protocol standardization is particularly important, as emphasized in stability information for detection kits: "To minimize performance fluctuations, operation procedures and lab conditions should be strictly controlled" . These careful handling practices ensure experimental reproducibility and reliable research outcomes when working with this protein.

What model organisms are used to study UPF0444 transmembrane protein C12orf23 homolog?

Multiple model organisms have been employed to investigate UPF0444 transmembrane protein C12orf23 homolog (TMEM263), providing complementary insights into its evolutionary conservation and physiological functions:

Additionally, comparative genomic analyses have included TMEM263 homologs from humans (Homo sapiens), chimpanzees (Pan troglodytes), rhesus macaques (Macaca mulatta), cattle (Bos taurus), dogs (Canis lupus), rats (Rattus norvegicus), and western clawed frogs (Xenopus tropicalis) . This multi-species approach enables researchers to identify conserved functional domains and species-specific adaptations, strengthening the understanding of TMEM263's fundamental biological roles. The consistency of growth-related phenotypes across diverse species underscores the protein's evolutionarily conserved function in growth regulation.

What is the role of TMEM263/C12orf23 homolog in the GH/IGF-1 signaling pathway?

TMEM263 plays a crucial regulatory role in the growth hormone (GH)/insulin-like growth factor 1 (IGF-1) signaling axis, with several key mechanisms identified through studies of Tmem263 knockout mice:

• GH Receptor Regulation: Tmem263-null mice exhibit significantly reduced hepatic GH receptor (GHR) expression, compromising the liver's ability to respond to circulating GH .

• JAK2/STAT5 Signaling Modulation: The absence of Tmem263 impairs GH-induced JAK2/STAT5 signaling, a primary intracellular pathway mediating GH's physiological effects .

• IGF-1 Production Control: As a direct consequence of diminished GH signaling, Tmem263-null mice demonstrate markedly low circulating IGF-1 levels. Since IGF-1 is predominantly produced in the liver under GH stimulation and is essential for promoting longitudinal bone growth, this reduction explains the observed growth phenotypes .

• Transcriptional Regulation: The GH signaling deficit substantially alters the expression of GH-regulated genes in Tmem263-null mice. A particularly striking effect is the "feminization" of the male liver transcriptome, resulting in an expression profile resembling wild-type females, hypophysectomized males, or Stat5b-null males .

These findings establish TMEM263 as a causal regulator of postnatal growth through its effects on GH signaling and downstream IGF-1 production. The molecular mechanisms elucidated explain the consistent phenotype of growth impairment observed across species with TMEM263 mutations or deletions.

How does deletion of Tmem263 affect postnatal growth in mice?

Deletion of Tmem263 in mice results in a striking postnatal growth failure phenotype with several distinguishing characteristics:

• Temporally Defined Growth Divergence: Tmem263-null mice exhibit normal body weight during the first two weeks of postnatal life, followed by a dramatic growth deficit emerging by postnatal day 21 (P21) . This timing coincides with the developmental period when the GH/IGF-1 axis becomes increasingly critical for growth regulation, suggesting that Tmem263's function becomes particularly important during this transition.

• Skeletal Development Impairment: Knockout mice demonstrate significant reductions in both bone mass and growth plate length . The growth plate, a cartilaginous region near the ends of long bones, is the primary site of longitudinal bone growth. Its reduced length in Tmem263-null mice directly explains the observed growth restriction.

• Proportional Dwarfism: The growth impairment manifests as "proportional dwarfism," indicating that various body structures are affected relatively uniformly rather than producing disproportionate skeletal abnormalities seen in some other growth disorders.

• Molecular Basis: The growth phenotype directly results from disruption of the GH/IGF-1 axis, with Tmem263-null mice exhibiting:

  • Significantly reduced circulating IGF-1 levels

  • Decreased hepatic GH receptor expression

  • Compromised GH-induced JAK2/STAT5 signaling

This comprehensive characterization establishes Tmem263 as an essential regulator of postnatal growth in mice, with its deletion causing a form of GH insensitivity that manifests as severe postnatal growth restriction.

What are the phenotypic consequences of TMEM263 mutations across different species?

TMEM263 mutations produce remarkably consistent growth-related phenotypes across evolutionarily diverse species, underscoring its conserved role in growth regulation:

The consistency of growth abnormalities across such diverse vertebrate species provides compelling evidence for TMEM263's fundamental and evolutionarily conserved role in growth regulation. The molecular mechanism appears similarly conserved, with disruption of the GH/IGF-1 axis established in mouse models and likely operating in other species as well. This cross-species conservation makes TMEM263 a valuable target for both comparative biology studies and potential therapeutic development for human growth disorders.

What experimental approaches are used to study the function of TMEM263 in vivo?

Researchers have employed a diverse array of experimental approaches to elucidate TMEM263 function in vivo:

• Genetic Engineering and Phenotyping:

  • Generation of Tmem263 knockout mice to assess phenotypic consequences of complete gene deletion

  • Detailed analysis of growth parameters, skeletal development, and molecular alterations in these models

• Genetic Mapping and Candidate Gene Identification:

  • Fine mapping using next-generation sequencing data to identify causative mutations

  • Homozygosity analysis to calculate runs of homozygosity (ROH) and estimate autozygosity

  • Variant filtering based on consequence annotation using tools like VEP (Variant Effect Predictor) and SnpEff

• Mutation Validation Strategies:

  • PCR-RFLP (Polymerase Chain Reaction-Restriction Fragment Length Polymorphism) analysis for genotyping specific mutations

  • Example from chicken studies: Validation of the adw mutation (NM_001006244.1:c.433G>A) using specific primers and Ddel restriction enzyme digestion

• Gene Expression Analysis:

  • RT-qPCR quantification of TMEM263 transcript levels

  • Comparative transcriptomic analysis between wild-type and mutant/knockout animals

  • Assessment of GH-regulated gene expression profiles to characterize downstream effects

• Pathway Analysis:

  • Evaluation of GH-induced JAK2/STAT5 signaling activity

  • Measurement of circulating IGF-1 concentrations

  • Quantification of GH receptor expression levels

• Evolutionary and Comparative Genomics:

  • Phylogenetic analysis of TMEM263 proteins across multiple species

  • Reconstruction of evolutionary relationships using methods such as Neighbor-Joining Trees

• Protein Structure Analysis:

  • Secondary topology prediction of transmembrane domains using TMHMM Server (hidden Markov model-based)

  • Visualization of predicted protein features using specialized tools like Protter 1.0

This multifaceted experimental approach has enabled comprehensive characterization of TMEM263 function from molecular mechanisms to whole-organism phenotypes, establishing its causal role in growth regulation.

How is TMEM263 expression measured in tissue samples?

Researchers employ several complementary techniques to measure TMEM263 expression in tissue samples, each offering distinct advantages:

• Reverse Transcription Quantitative PCR (RT-qPCR):

  • This method quantifies TMEM263 mRNA expression levels with high sensitivity

  • Specific primers are designed for amplification:

    • TMEM263_2F: 5′-GCCACCAGAAGGTTCAATCAAAG-3′

    • TMEM263_2R: 5′-CTGAAGATGCCACCAGTCACA-3′

  • Expression is normalized to housekeeping genes (e.g., 28S rRNA) as internal controls

  • Relative quantification typically employs the ΔΔCt method

  • Technical considerations include running multiple technical replicates (typically three) and including appropriate controls

• Enzyme-Linked Immunosorbent Assay (ELISA):

  • Commercial ELISA kits enable quantification of TMEM263 protein levels:

    • Available for multiple species including human and mouse TMEM263

  • Sample types include tissue homogenates, cell lysates, and biological fluids

  • Detection is typically colorimetric with defined quantification ranges (e.g., 0.156-10 ng/ml for human TMEM263)

  • Critical methodological considerations include sample dilution to within the kit's mid-range for optimal accuracy

• Western Blotting:

  • While not explicitly mentioned in the search results, this technique would complement ELISA by providing information about protein size and potential post-translational modifications

  • Requires validated antibodies against TMEM263

• Immunohistochemistry/Immunofluorescence:

  • These techniques enable visualization of TMEM263 cellular and subcellular localization

  • Particularly valuable for assessing expression patterns within heterogeneous tissues

Each method provides distinct and complementary information: RT-qPCR quantifies transcript abundance, ELISA measures total protein concentration, Western blotting assesses protein size and modifications, and immunostaining reveals spatial distribution patterns. Together, these approaches provide comprehensive characterization of TMEM263 expression in biological samples.

What is the predicted secondary structure and transmembrane topology of TMEM263?

The secondary structure and transmembrane topology of TMEM263 have been computationally predicted using specialized bioinformatic approaches:

While the search results don't provide detailed information about specific secondary structure elements (e.g., precise number and positions of transmembrane helices), TMEM263's classification as a transmembrane protein with plasma membrane localization is consistent with its established role in cellular signaling pathways, particularly the GH/IGF-1 axis.

How do TMEM263 knockout models compare across different species?

The search results provide information primarily on mouse knockout models and chicken mutants, enabling valuable comparative analysis:

Several important comparative observations emerge:

• Consistent Growth Phenotype: Both mouse and chicken models demonstrate dwarfism despite different mutation types, supporting a fundamental conserved role for TMEM263 in growth regulation across vertebrate species.

• Differential Molecular Characterization: The mouse model has undergone more extensive molecular characterization, particularly regarding disruption of the GH/IGF-1 axis, providing mechanistic insights potentially applicable to other species.

• Mutation Type Considerations: The mouse studies employed complete gene deletion, while the chicken model involved a specific nonsense mutation, which could potentially yield different phenotypic nuances depending on whether truncated protein products retain partial functionality.

The consistent growth abnormalities across evolutionarily distant species strongly support TMEM263's fundamental role in growth regulation and suggest that research findings from model organisms likely have direct relevance to human growth disorders involving this gene.

What are the potential research applications for recombinant mouse UPF0444 transmembrane protein C12orf23 homolog?

Recombinant mouse UPF0444 transmembrane protein C12orf23 homolog (TMEM263) offers numerous valuable research applications:

• Antibody Development and Validation:

  • Serving as immunogen for generating and validating antibodies against TMEM263

  • These antibodies enable techniques such as Western blotting, immunohistochemistry, and immunoprecipitation for studying endogenous TMEM263 expression, localization, and interactions

• Protein-Protein Interaction Studies:

  • In vitro binding assays to identify interaction partners

  • Pull-down experiments to validate predicted protein interactions

  • Structure-function relationship studies to map critical binding domains

• Quantitative Assay Standardization:

  • As calibration standards in ELISA and other quantitative assays measuring TMEM263 in biological samples

  • For generating standard curves in absolute quantification methods

• Biochemical Characterization:

  • Investigation of post-translational modifications regulating TMEM263 function

  • Biophysical studies of protein stability and conformational changes

• Structural Biology Approaches:

  • While three-dimensional structure prediction has proven challenging , purified recombinant protein could potentially enable structural studies using X-ray crystallography or cryo-electron microscopy

  • Membrane protein structural biology often requires optimized recombinant protein constructs

• Therapeutic Development:

  • Given TMEM263's established role in growth regulation, recombinant protein could facilitate screens for molecules that modulate its activity

  • Potential development of therapeutic approaches for growth disorders associated with TMEM263 dysfunction

When working with recombinant TMEM263, researchers should consider potential differences from the native protein, as noted in ELISA kit documentation: "Please note that our kits are optimised for detection of native samples, rather than recombinant proteins. We are unable to guarantee detection of recombinant proteins, as they may have different sequences or tertiary structures to the native protein" .

How does TMEM263 contribute to bone mineral density and skeletal development?

TMEM263 plays a crucial role in bone mineral density and skeletal development through several interconnected mechanisms:

• Genetic Association with Bone Mineral Density:

  • Single nucleotide polymorphisms (SNPs) in TMEM263 are associated with bone mineral density variations in human populations

  • This genetic association suggests fundamental involvement in bone mass acquisition or maintenance processes

• Growth Plate Development Regulation:

  • Tmem263-null mice exhibit significantly reduced growth plate length

  • The growth plate is the cartilaginous region near the ends of long bones where longitudinal growth occurs through chondrocyte proliferation and differentiation

  • Reduced growth plate dimensions directly contribute to impaired longitudinal bone growth

• Bone Mass Acquisition:

  • Mice lacking Tmem263 demonstrate pronounced reductions in bone mass

  • This phenotype indicates TMEM263's requirement for normal bone acquisition during development

  • Potentially affects both trabecular and cortical bone compartments, though specific details aren't provided in the search results

• Mechanistic Pathway via GH/IGF-1 Axis:

  • TMEM263's effects on skeletal development appear primarily mediated through its regulation of the GH/IGF-1 axis

  • GH and IGF-1 are established major regulators of skeletal growth and bone mineral density

  • GH stimulates bone growth both directly and indirectly (through IGF-1 induction)

  • IGF-1 promotes chondrocyte proliferation and differentiation in growth plates and stimulates osteoblast activity throughout the skeleton

• Clinical Significance:

  • TMEM263 mutations are associated with severe skeletal dysplasia in at least one human fetus

  • The autosomal dwarf phenotype in chickens with TMEM263 mutations further supports its fundamental role in skeletal development across species

This evidence collectively establishes TMEM263 as a critical regulator of skeletal development through its modulation of GH/IGF-1 signaling. The consistent skeletal phenotypes observed across species highlight TMEM263's potential relevance to human growth disorders and skeletal conditions including growth hormone insensitivity syndromes and potential connections to osteoporosis risk.

What are the best practices for validating mutations in TMEM263 in experimental models?

Rigorous validation of TMEM263 mutations in experimental models requires a systematic approach incorporating multiple complementary methodologies:

• Genetic Mapping and Identification:

  • Employ next-generation sequencing for comprehensive mutation detection

  • Implement a structured filtering pipeline to identify causative variants:

    • Compare variant profiles between affected and unaffected individuals

    • Filter for homozygous variants in recessive conditions

    • Assess predicted functional impact using tools like VEP and SIFT

    • Exclude variants found in unaffected controls or public databases

• Genotype-Phenotype Correlation:

  • Validate the association between specific mutations and phenotypes by genotyping multiple affected and unaffected individuals

  • In the chicken adw study, researchers genotyped both dwarf (adw/adw) and normal-sized (ADW/ADW) chickens to confirm the mutation's association with the phenotype

• Molecular Confirmation Methods:

  • PCR-RFLP Analysis for specific mutations:

    • Primer design targeting the mutation region (e.g., TMEM263_1F: 5′-GTTCAATCAAAGACCACCCG-3′ and TMEM263_1R: 5′-TTGGCTTTAGTCAGACTTGTCCT-3′)

    • Restriction enzyme digestion of PCR products (e.g., Ddel)

    • Fragment analysis by agarose gel electrophoresis with expected pattern differences between mutant and wild-type alleles

• Expression Analysis:

  • Quantify the effect of mutations on gene expression using RT-qPCR

  • Include appropriate controls and multiple technical replicates

  • Normalize to stable reference genes (e.g., 28S rRNA)

• Protein-Level Validation:

  • Assess mutation impact on protein expression, stability, and localization

  • ELISA quantification of protein levels in various biological samples

  • Western blotting to detect truncated or abnormal protein products

• Functional Validation:

  • Generate knockout models or specific mutation knock-in models

  • Assess whether engineered mutations recapitulate phenotypes of naturally occurring mutations

  • The comprehensive phenotyping of mouse Tmem263 knockout models demonstrates this approach

• Cross-Species Validation:

  • Compare phenotypes associated with TMEM263 mutations across different species

  • The consistent growth abnormalities in mice and chickens substantially strengthen the evidence for TMEM263's causal role in growth regulation

These methodologies collectively provide robust validation of TMEM263 mutations and their functional consequences, establishing clear causality rather than mere correlation with observed phenotypes.