Recombinant Human UPF0485 protein C1orf144 (C1orf144)

What is UPF0485 protein C1orf144?

C1orf144 (Chromosome 1 Open Reading Frame 144) is a protein-coding gene located on chromosome 1, which is the largest human chromosome spanning approximately 260 million base pairs and constituting about 8% of the human genome. The protein product has been provisionally designated as UPF0485 protein C1orf144, pending further characterization of its structure and function. The "UPF" prefix indicates an uncharacterized protein family, suggesting that its biological roles remain to be fully elucidated through systematic research .

What expression systems are optimal for producing recombinant C1orf144?

Multiple expression systems can be employed for recombinant C1orf144 production, each with distinct advantages. E. coli and yeast expression systems typically offer superior yields and shorter production timelines, making them cost-effective choices for initial characterization studies. For applications requiring post-translational modifications that may be essential for proper protein folding or activity, insect cells with baculovirus expression systems or mammalian cell expression systems are recommended. These advanced systems can provide the necessary cellular machinery to ensure proper glycosylation, phosphorylation, and other modifications that might be critical for maintaining the native conformation and functionality of C1orf144 .

What is known about the structural characteristics of C1orf144?

The structural characteristics of C1orf144 remain largely undefined, as indicated by its UPF (Uncharacterized Protein Family) designation. The protein belongs to the UPF0485 family, suggesting shared structural motifs with other members of this family. Preliminary structural analyses may involve computational prediction methods, circular dichroism spectroscopy to determine secondary structure content, and ultimately X-ray crystallography or NMR spectroscopy for high-resolution structural determination. Researchers should consider both the native protein structure and potential structural changes that might occur upon interaction with binding partners or activators .

How do C1orf144 activators function at the molecular level?

C1orf144 activators constitute a specialized class of chemical compounds designed to modulate C1orf144 activity. These molecules typically function by interacting with specific regulatory elements within the C1orf144 gene, particularly in promoter or enhancer regions, to enhance transcription and subsequent translation into functional protein products. While the precise mechanisms vary among different activators, they fundamentally act as molecular switches that amplify C1orf144 activity. Some compounds, such as Troglitazone, may indirectly influence C1orf144 expression through activation of nuclear receptors like PPARγ, which regulate gene expression networks. Zinc may also play a role as a cofactor for various transcription factors that could potentially influence C1orf144 expression .

How can researchers design experiments to elucidate the functional role of C1orf144 in cellular processes?

Designing experiments to determine C1orf144 function requires a multi-faceted approach:

• Loss-of-function studies: Implement CRISPR-Cas9 gene editing, RNA interference (RNAi), or antisense oligonucleotides to downregulate or eliminate C1orf144 expression. Following knockdown or knockout, comprehensive transcriptomic and proteomic analyses can identify altered pathways and interacting partners.

• Gain-of-function studies: Overexpress C1orf144 using appropriate vectors and promoters, then evaluate phenotypic changes through cell proliferation assays, metabolic assessments, or functional readouts specific to the cellular context being investigated.

• Subcellular localization: Employ immunofluorescence, cell fractionation, or expression of fluorescently-tagged C1orf144 to determine its intracellular distribution, which can provide crucial insights into potential functions.

• Interactome mapping: Conduct co-immunoprecipitation, yeast two-hybrid assays, or proximity-dependent biotin identification (BioID) to identify proteins that physically interact with C1orf144, establishing a network of potential functional associations .

• Pathway analysis: Similar to investigations with related chromosome 1 proteins (like C1orf74), researchers should examine connections to established signaling pathways, such as MAPK signaling, which has been implicated in the function of other chromosome 1 proteins .

What are the optimal purification strategies for maintaining C1orf144 stability and activity?

Purification of recombinant C1orf144 requires careful consideration of protein stability and activity:

• Affinity chromatography: Design constructs with appropriate affinity tags (His-tag, GST, MBP) positioned to minimize interference with protein folding and function. Consider implementing protease cleavage sites to remove tags post-purification.

• Buffer optimization: Systematically screen buffer compositions to identify conditions that maximize protein stability. Critical parameters include:

  • pH range (typically 6.5-8.0 for most proteins)

  • Salt concentration (150-500 mM NaCl)

  • Reducing agents (DTT, β-mercaptoethanol, TCEP)

  • Stabilizing additives (glycerol, arginine, trehalose)

• Chromatographic sequence: Implement a multi-step purification strategy, potentially including ion exchange, size exclusion, and hydrophobic interaction chromatography after initial affinity purification to achieve high purity.

• Aggregation prevention: Monitor protein aggregation using dynamic light scattering or size exclusion chromatography with multi-angle light scattering (SEC-MALS).

• Activity assays: Develop specific activity assays based on predicted or identified functions to confirm that purified C1orf144 retains its biological activity throughout the purification process .

How might posttranslational modifications affect C1orf144 function, and how can these be preserved in recombinant systems?

Posttranslational modifications (PTMs) potentially critical for C1orf144 function require special consideration:

• PTM prediction and identification: Employ bioinformatic tools to predict potential modification sites, followed by mass spectrometry analysis of natively expressed protein to identify actual PTMs.

• Expression system selection: Based on identified PTMs, select appropriate expression systems:

  • Phosphorylation: Mammalian cells or insect cells

  • Glycosylation: Mammalian cells for complex glycans; yeast for high-mannose structures

  • Acetylation/methylation: Mammalian cells

• Co-expression strategies: Consider co-expressing relevant kinases, phosphatases, or other modifying enzymes to enhance specific PTM acquisition in recombinant systems.

• Site-directed mutagenesis: Create PTM site mutants (e.g., phosphomimetic mutations) to assess the functional significance of specific modifications.

• Preservation protocols: Incorporate phosphatase inhibitors, deacetylase inhibitors, or other PTM-preserving reagents during purification to maintain modification status .

What techniques are most effective for studying C1orf144 gene regulation?

Investigating C1orf144 gene regulation requires a comprehensive approach:

• Promoter analysis: Conduct reporter gene assays using luciferase or fluorescent proteins to identify key regulatory elements in the C1orf144 promoter and enhancer regions.

• Chromatin immunoprecipitation (ChIP): Identify transcription factors binding to the C1orf144 regulatory regions through ChIP-seq or ChIP-qPCR approaches.

• CRISPR activation/interference: Employ CRISPRa or CRISPRi systems to specifically activate or repress transcription from the C1orf144 locus without altering the genetic sequence.

• DNA methylation analysis: Use bisulfite sequencing or methylation-specific PCR to assess the role of DNA methylation in C1orf144 expression regulation.

• Chromosome conformation capture: Implement 3C, 4C, or Hi-C techniques to identify long-range chromatin interactions affecting C1orf144 expression.

• RNA stability assessment: Measure C1orf144 mRNA half-life using actinomycin D chase experiments coupled with qRT-PCR to determine post-transcriptional regulation mechanisms .

How can researchers effectively validate C1orf144 antibodies for experimental applications?

Antibody validation for C1orf144 research requires rigorous testing:

• Specificity validation:

  • Western blot comparing wild-type to knockout/knockdown samples

  • Peptide competition assays to confirm epitope specificity

  • Immunoprecipitation followed by mass spectrometry to confirm target identity

• Application-specific validation:

  • For immunohistochemistry: Tissue microarrays with positive and negative controls

  • For flow cytometry: Comparison with isotype controls and blocking experiments

  • For ChIP: Analysis of known non-target regions as negative controls

• Cross-reactivity assessment: Test against related proteins, particularly other chromosome 1 open reading frame products, to ensure specificity.

• Lot-to-lot consistency: Establish standard operating procedures for validating new antibody lots to ensure experimental reproducibility.

• Reporting standards: Document validation results comprehensively according to the Antibody Validation Guidelines established by the International Working Group for Antibody Validation .

What cell models are most appropriate for investigating C1orf144 function?

Selection of appropriate cell models depends on research objectives:

• Expression profiling: Prior to model selection, analyze C1orf144 expression across tissue types using publicly available RNA-seq datasets to identify physiologically relevant models.

• Primary cells vs. cell lines:

  • Primary cells offer physiological relevance but limited lifespan

  • Immortalized cell lines provide experimental convenience but may have altered signaling networks

• Disease models: If investigating potential disease associations, select models that recapitulate relevant pathological features.

• 3D culture systems: Consider organoids or spheroids for more physiologically relevant microenvironments compared to traditional 2D cultures.

• Genetic background considerations: Utilize isogenic cell lines for precise functional studies, particularly when conducting genetic manipulation experiments.

• Validation across multiple models: Confirm key findings in at least two independent cell models to enhance result reliability .

How should researchers approach the analysis of C1orf144 expression data in normal versus disease states?

Analysis of C1orf144 expression data requires a systematic approach:

• Dataset selection and quality control:

  • Utilize multiple independent datasets (e.g., TCGA, GTEx, GEO)

  • Implement rigorous quality control measures for RNA integrity and sequencing depth

  • Account for batch effects using appropriate statistical methods

• Differential expression analysis:

  • Apply appropriate statistical frameworks (DESeq2, edgeR, limma)

  • Use false discovery rate (FDR) correction for multiple testing

  • Establish biologically meaningful thresholds for fold change interpretation

• Correlation analysis:

  • Identify genes with expression patterns correlated with C1orf144

  • Perform gene set enrichment analysis to identify associated pathways

• Survival analysis:

  • Assess the prognostic significance of C1orf144 expression using Kaplan-Meier plots and Cox regression

  • Consider multivariate models incorporating clinical variables

• Validation with quantitative PCR:

  • Confirm key expression differences using independent sample sets

  • Normalize to multiple reference genes for reliable quantification

This approach mirrors successful analyses performed for other chromosome 1 genes such as C1orf74, which demonstrated significant associations with clinical parameters in cancer contexts .

What are the best practices for determining structure-function relationships for poorly characterized proteins like C1orf144?

Elucidating structure-function relationships for poorly characterized proteins involves:

• Sequence-based predictions:

  • Employ multiple alignment tools to identify conserved domains

  • Use structure prediction algorithms (AlphaFold2, RoseTTAFold) to generate structural models

  • Identify potential functional motifs through specialized databases (Prosite, PFAM)

• Experimental structure determination:

  • Pursue X-ray crystallography for high-resolution static structures

  • Consider NMR spectroscopy for dynamic information and intrinsically disordered regions

  • Implement hydrogen-deuterium exchange mass spectrometry for conformational dynamics

• Functional mapping:

  • Create systematic alanine scanning or domain deletion mutants

  • Assess effects on identified functions through relevant assays

  • Correlate structural features with functional outcomes

• Molecular dynamics simulations:

  • Simulate protein dynamics under physiological conditions

  • Identify potential binding pockets or interaction surfaces

  • Validate computational predictions experimentally

• Integrative approaches:

  • Combine computational predictions with limited experimental data

  • Implement cross-linking mass spectrometry to validate interaction interfaces

  • Use small-angle X-ray scattering (SAXS) to obtain low-resolution structural information in solution .

How can researchers investigate potential interactions between C1orf144 and other proteins or signaling pathways?

Investigating protein interactions and pathway connections requires integrated approaches:

• Affinity purification-mass spectrometry (AP-MS):

  • Express tagged C1orf144 in relevant cell types

  • Perform immunoprecipitation under physiological conditions

  • Identify co-precipitating proteins through mass spectrometry

  • Validate key interactions through reciprocal co-IP or proximity ligation assay

• Yeast two-hybrid and mammalian two-hybrid screening:

  • Identify direct protein-protein interactions

  • Validate positive hits with orthogonal methods

  • Map interaction domains through truncation constructs

• Proximity labeling approaches:

  • Employ BioID or APEX2 fusion proteins to identify proximal proteins

  • Distinguish between stable and transient interactions

  • Map subcellular interaction networks

• Phosphoproteomic analysis:

  • Compare global phosphorylation patterns between control and C1orf144-perturbed systems

  • Identify altered phosphosites to map affected signaling pathways

  • Validate through kinase inhibition studies

• Transcriptomic response profiling:

  • Analyze gene expression changes following C1orf144 manipulation

  • Apply pathway enrichment analysis to identify regulated processes

  • Validate through reporter assays for key pathways

Based on research on related proteins, potential signaling connections may include MAPK pathway components, as demonstrated for other chromosome 1 proteins in cancer contexts .

What are the most promising approaches for therapeutic targeting of C1orf144 in disease contexts?

Therapeutic targeting strategies for C1orf144 should consider:

• Target validation:

  • Establish clear disease association through genetic evidence

  • Demonstrate modifiable phenotypes in disease-relevant models

  • Identify specific molecular mechanisms amenable to intervention

• Small molecule modulators:

  • Screen for compounds that specifically activate or inhibit C1orf144

  • Optimize lead compounds for potency, selectivity, and pharmacokinetic properties

  • Evaluate compounds like Troglitazone that may indirectly modulate C1orf144 through nuclear receptor activation

• RNA-based therapeutics:

  • Design antisense oligonucleotides or siRNAs for specific C1orf144 downregulation

  • Develop mRNA therapeutics for controlled expression in deficiency states

  • Address delivery challenges through lipid nanoparticles or conjugated targeting moieties

• Protein-protein interaction disruptors:

  • Identify critical interaction surfaces through structural studies

  • Design peptide mimetics or small molecules to specifically disrupt pathological interactions

  • Validate target engagement in cellular and animal models

• Gene therapy approaches:

  • Develop viral vectors for C1orf144 delivery in deficiency states

  • Consider gene editing for correction of pathogenic variants

  • Explore controlled expression systems for context-specific activation

Research on related chromosome 1 proteins suggests potential applications in cancer contexts, where altered expression has demonstrated significant associations with clinical outcomes and therapeutic responses .

What considerations are important when developing high-throughput screening assays for C1orf144 modulators?

Developing effective high-throughput screening assays requires:

• Assay design considerations:

  • Select appropriate readouts based on known or predicted functions

  • Establish robust positive and negative controls

  • Optimize signal-to-background ratio and Z' factor (aim for Z' > 0.5)

• Primary screening approaches:

  • Reporter gene assays for transcriptional effects

  • AlphaScreen or FRET assays for protein-protein interactions

  • Phenotypic screens in disease-relevant cell models

• Confirmatory and counter-screening cascades:

  • Implement dose-response confirmations for active compounds

  • Conduct counter-screens to eliminate assay-specific artifacts

  • Assess specificity against related proteins in the same family

• Mechanism of action determination:

  • Thermal shift assays to confirm direct binding

  • Competitive binding assays to identify binding sites

  • Cellular target engagement assays (CETSA, DARTS)

• Data analysis and hit prioritization:

  • Apply appropriate statistical methods for hit identification

  • Prioritize compounds based on potency, selectivity, and structural novelty

  • Implement structure-activity relationship studies for promising scaffolds .