Recombinant Mouse Uncharacterized protein C19orf55 homolog

What is known about the molecular characteristics of C19orf55/PROSER3?

C19orf55/PROSER3 is a 480 amino acid protein encoded by a gene located on human chromosome 19. It has a molecular weight of approximately 51 kDa . The protein contains a domain of unknown function (DUF4619) and appears to be rich in proline and serine residues as suggested by its alternative name (PROSER3) . The gene corresponding to this protein has the accession number Q2NL68 . Current research indicates it may be expressed in multiple tissues, though specific expression patterns and levels across different cell types require further investigation.

How conserved is C19orf55/PROSER3 across species?

Sequence analysis shows significant conservation across mammalian species, with mouse C19orf55 sharing 72% sequence identity with the human ortholog, while the rat version shares 61% identity . This level of conservation suggests the protein may serve an important biological function that has been maintained throughout mammalian evolution. When designing experiments involving cross-species comparisons or antibody selection, researchers should account for these sequence variations.

What antibodies and reagents are currently available for studying C19orf55/PROSER3?

Several research tools are available for studying this protein:

• Polyclonal antibodies: Rabbit polyclonal antibodies against C19orf55 are commercially available for applications including immunocytochemistry (ICC) and immunohistochemistry (IHC)

• Fluorescent conjugates: Antibodies conjugated with fluorescent tags such as RBITC (rhodamine B isothiocyanate) can be used for immunofluorescence studies with recommended dilutions of 1:50-200

• Recombinant proteins: Though not directly mentioned for C19orf55 in the search results, recombinant protein production approaches similar to those used for other uncharacterized proteins could be applied

What experimental applications are supported by current C19orf55 antibodies?

Current antibodies against C19orf55 have been validated for:

These applications allow researchers to investigate the protein's expression patterns and subcellular localization, which may provide clues to its function. Optimal dilutions should be determined by each researcher based on their specific experimental conditions .

What approaches can be used to determine the function of an uncharacterized protein like C19orf55?

Elucidating the function of uncharacterized proteins requires multiple complementary approaches:

• Bioinformatic analysis: Sequence comparison, domain prediction, and structural modeling can provide initial hypotheses about function. For C19orf55, analysis reveals a DUF4619 domain, which itself is uncharacterized but may provide clues through structural similarities to known domains .

• Protein-protein interaction studies: Methods like proximity-dependent biotin identification (BioID) and co-immunoprecipitation followed by mass spectrometry can reveal interaction partners, as demonstrated with other uncharacterized proteins like FAME . These interacting proteins often share functional pathways.

• Gene expression correlation analysis: Analyzing co-expression patterns across tissues and conditions can suggest functional associations. This approach has been successfully applied to other previously uncharacterized proteins, revealing correlations with genes involved in specific cellular processes .

• Loss-of-function studies: CRISPR/Cas9-mediated knockout or RNAi-mediated knockdown followed by phenotypic analysis can reveal the consequences of protein absence.

• Subcellular localization: Fluorescent tagging and microscopy can determine where the protein functions, providing contextual clues about its role.

How can researchers effectively produce and purify recombinant mouse C19orf55 for structural studies?

Based on approaches used for similar uncharacterized proteins:

What can be inferred about C19orf55 function through comparative genomics approaches?

Comparative genomics has proven valuable for uncharacterized proteins, as demonstrated with FAME (previously 1700011H14Rik/C14orf105/CCDC198) . For C19orf55, researchers could:

• Analyze evolutionary rate: Determine if C19orf55 is evolving rapidly (suggesting adaptation to changing selective pressures) or slowly (suggesting fundamental conserved function). This approach revealed FAME as an exceptionally fast-evolving gene in birds and mammals .

• Identify species-specific variations: Compare sequences across species inhabiting different environments to identify correlations between sequence features and ecological niches.

• Search for functional domains: While C19orf55 contains the uncharacterized DUF4619 domain, further analysis may reveal cryptic functional motifs. For example, FAME analysis revealed a previously unrecognized N-myristoylation site that proved critical for its subcellular localization .

• Examine selection signatures: Analyze patterns of genetic variation to detect signatures of selection, which can indicate functional importance, as observed with Neandertal alleles of FAME flowing into modern humans .

What controls should be included when using antibodies against uncharacterized proteins like C19orf55?

When working with antibodies against poorly characterized proteins, rigorous validation is essential:

• Knockout/knockdown controls: Samples from knockout animals or cells with the target gene knocked down should show no signal, confirming antibody specificity .

• Overexpression controls: Cells overexpressing the tagged protein can serve as positive controls and help determine antibody sensitivity .

• Cross-reactivity testing: Test antibody specificity across multiple species if cross-species studies are planned. For C19orf55, antibodies may recognize human, mouse, and potentially rat and pig proteins, but this requires validation .

• Multiple antibody validation: When possible, use multiple antibodies targeting different epitopes to confirm localization and expression patterns.

• Blocking peptide controls: Pre-incubating antibodies with the immunizing peptide should abolish specific staining.

How can researchers address the challenges of studying proteins with unknown function using structural biology approaches?

Structural biology offers powerful insights into potential functions of uncharacterized proteins:

• Domain-based crystallography: Rather than attempting to crystallize the full-length protein, focus on individual predicted domains, such as the DUF4619 domain in C19orf55 .

• Comparative modeling: Use related proteins with known structures as templates. Even with low sequence identity, structural similarities can provide functional hints.

• Experimental structure determination approaches:

  • X-ray crystallography requires well-diffracting crystals

  • Cryo-EM is increasingly useful for proteins recalcitrant to crystallization

  • NMR spectroscopy works well for smaller domains (<30 kDa)

• Structural analysis of protein-protein interfaces: Similar to the approach used for Protein L , introducing engineered metal ion binding sites (ψ analysis) can reveal important structural interactions during folding or function.

• Functional annotation through structure: Once a structure is obtained, tools like ProFunc, COFACTOR, and COACH can predict binding sites and functional partners based on structural features.

What experimental design considerations are important when studying potential post-translational modifications of C19orf55?

Post-translational modifications (PTMs) often regulate protein function and localization:

• Prediction and verification strategy:

  • Use bioinformatic tools to predict potential PTM sites

  • Verify experimentally using mass spectrometry

  • Confirm functional significance through site-directed mutagenesis

• Potential phosphorylation: As a proline and serine-rich protein (PROSER3), C19orf55 likely contains numerous phosphorylation sites. Similar to the approach used for FAME , create a phosphorylation site map and assess conservation across species.

• Potential myristoylation: If N-terminal glycine is present, N-myristoylation might occur, affecting membrane localization. This can be tested using myristoylation inhibitors (e.g., DDD85646) and by mutating the N-terminal glycine to alanine, as demonstrated with FAME .

• Expression system selection: Ensure the chosen expression system can perform the PTMs of interest. Bacterial systems generally cannot perform complex eukaryotic PTMs.

How can researchers optimize antibody detection of low-abundance proteins like C19orf55?

Detecting low-abundance proteins presents technical challenges:

• Sample preparation optimization:

  • Use enrichment techniques like subcellular fractionation

  • Optimize protein extraction methods for the specific tissue/cell type

  • Consider using proteasome inhibitors if protein degradation is suspected

• Signal amplification methods:

  • Tyramide signal amplification (TSA) can increase sensitivity by 10-100 fold

  • Use highly sensitive detection systems like ECL Prime for western blotting

  • Consider proximity ligation assay (PLA) for detecting protein interactions

• Antibody sensitivity limitations: As noted with FAME research, some antibodies may have sensitivity limitations, making detection in western blot difficult without overexpression .

• Concentration and loading optimization: Determine optimal protein loading amounts and antibody concentrations through systematic titration experiments.

What approaches can resolve contradictory experimental data when characterizing novel proteins?

When studying uncharacterized proteins, contradictory results often emerge:

• Methodological reconciliation: Different methods may reveal different aspects of protein function. For example, ψ analysis with engineered metal ion binding sites revealed more extensive transition state structure in Protein L than standard mutational φ analysis .

• Resolution strategies:

  • Use multiple, complementary techniques to address the same question

  • Consider tissue/cell-specific effects that might explain discrepancies

  • Examine species differences if using models from different organisms

  • Investigate potential isoforms or post-translational modifications

• Non-native conformations: Consider that proteins may adopt non-native conformations during folding or function. The carboxy-terminal hairpin in Protein L's transition state was found to be non-native, explaining discrepancies between experimental and computational approaches .

What emerging technologies might accelerate functional characterization of proteins like C19orf55?

Several cutting-edge approaches show promise for uncharacterized protein research:

• AlphaFold2 and structural prediction: Advanced AI-based structural prediction tools can generate high-confidence structural models even without homologous templates.

• Spatial transcriptomics and proteomics: These technologies provide spatial context for gene and protein expression, potentially revealing functional associations through co-localization.

• Single-cell multi-omics: Integrated analysis of transcriptome, proteome, and metabolome at single-cell resolution can reveal cell-specific functions and interactions.

• CRISPR screening: Genome-wide or focused CRISPR screens can systematically identify genetic interactions and phenotypes associated with C19orf55 disruption.

• Protein engineering approaches: Similar to the engineered metal ion binding sites used to study Protein L , engineered versions of C19orf55 could reveal structural and functional insights.

What are the potential physiological roles of C19orf55 based on its genomic context and evolutionary conservation?

Although C19orf55's function remains unknown, contextual information provides clues:

• Genomic neighborhood: C19orf55 is located on chromosome 19, which has the highest gene density among human chromosomes and houses numerous immunoglobulin superfamily members, potentially suggesting immune-related functions .

• Evolutionary conservation: The 72% sequence identity between human and mouse suggests functional importance . Analysis of conservation patterns across more diverse species could reveal functionally critical regions.

• Expression correlation: Similar to the approach used for FAME , analysis of co-expressed genes could reveal functional associations with specific pathways or cellular processes.

• Disease associations: Chromosome 19 has been linked to various conditions including hypercholesterolemia and insulin-dependent diabetes . Investigation of potential links between C19orf55 variants and disease phenotypes may provide functional insights.