C10ORF54 Human

What is C10ORF54 and what are its alternative names in scientific literature?

C10ORF54, or Chromosome 10 Open Reading Frame 54, is a protein belonging to the immunoglobulin superfamily that functions as a transmembrane molecule. In scientific literature, this protein is known by numerous synonyms including V-Domain Ig Suppressor Of T Cell Activation (VISTA), Stress-Induced Secreted Protein-1 (SISP-1), Death Domain1alpha (DD1alpha), B7-H5, GI24, PP2135, VSIR, and PD-1H . When conducting literature searches or designing experiments, researchers should include these alternative designations to ensure comprehensive coverage. The most commonly used designation in recent immunology literature appears to be VISTA, particularly in the context of immune checkpoint research.

What is the structural composition and molecular characteristics of human C10ORF54?

Human C10ORF54 (VISTA) is characterized as a glycosylated polypeptide with the following key features:

The amino acid sequence includes specific regions responsible for its immunomodulatory functions . When conducting experimental work with this protein, researchers should account for the difference between theoretical molecular weight and observed migration pattern on electrophoresis gels due to post-translational modifications.

Where is C10ORF54 expressed in human tissues and what methods best detect its expression?

C10ORF54 (VISTA) exhibits a specific expression pattern across human tissues that researchers should consider when designing experimental approaches. It is expressed on embryonic stem cells (ESCs), bone cells, and various tumor cell surfaces . The protein functions as a transmembrane molecule, suggesting its importance in cell-cell interactions and signaling pathways. Based on its role in immune regulation, researchers should expect expression in tissues with significant immune cell infiltration.

For detection and quantification of C10ORF54 expression, multiple methodological approaches are recommended:

• Immunohistochemistry with validated antibodies provides the most reliable approach for tissue localization studies and spatial distribution analysis.

• Flow cytometry remains the gold standard for analyzing expression on specific cell populations in suspension, allowing for multi-parameter analysis.

• Western blotting can confirm protein size and expression levels, though researchers should anticipate the 28-40 kDa band rather than the theoretical 19.1 kDa size.

• qPCR for mRNA expression analysis allows for sensitive detection but should be complemented with protein-level confirmation.

Each method has distinct advantages and should be selected based on the specific research question being addressed.

How does C10ORF54 interact with embryonic stem cells (ESCs)?

C10ORF54 demonstrates several notable interactions with embryonic stem cells that researchers investigating stem cell biology should consider:

• It supports differentiation of ESCs

• It enhances BMP4-induced signaling in ESCs

• Interestingly, C10ORF54 expression is downregulated following BMP4 exposure

This suggests a complex feedback loop where C10ORF54 enhances BMP4 signaling, but BMP4 ultimately leads to reduced C10ORF54 expression. To effectively study these interactions, researchers should consider time-course experiments measuring C10ORF54 expression levels following BMP4 treatment, knockdown or knockout studies to assess the impact of C10ORF54 depletion on ESC differentiation, and co-immunoprecipitation studies to identify binding partners in the BMP4 signaling pathway.

These approaches will help elucidate the precise mechanisms through which C10ORF54 influences stem cell behavior, which may have implications for both developmental biology and regenerative medicine applications.

What are the recommended storage conditions for C10ORF54 recombinant proteins in laboratory settings?

For optimal stability and activity of C10ORF54 recombinant proteins in research applications, follow these evidence-based storage protocols:

The typical formulation includes protein solution at 0.5mg/ml concentration in Phosphate-Buffered Saline (pH 7.4) with 10% glycerol for stability . Multiple freeze-thaw cycles should be avoided to maintain protein integrity.

Before designing experiments, researchers should validate protein activity after storage using appropriate functional assays to ensure the protein has maintained its biological properties. This step is critical for reproducible results, particularly when studying signaling functions. Activity testing protocols should reflect the specific functions being investigated, whether immunomodulatory or stem cell-related activities.

How does C10ORF54 (VISTA) contribute to immune evasion mechanisms in tumor microenvironments?

C10ORF54 (VISTA) functions as an immune checkpoint molecule that can dampen T cell activity in tumor microenvironments. Current research positions VISTA among a group of inhibitory molecules that tumors may upregulate as an immune evasion mechanism .

The current understanding of VISTA's role includes:

• It acts similarly to other immune checkpoint molecules (BTLA, TIM3, LAG3, IDO1)

• It can contribute to the "non-inflamed" tumor phenotype where T cells are excluded or functionally suppressed

• Its expression may represent an adaptive response to immune pressure, potentially contributing to acquired resistance to immunotherapy

To effectively study VISTA's contribution to immune evasion, researchers should use multi-parameter flow cytometry to analyze VISTA expression alongside other checkpoint molecules, perform spatial analysis of VISTA expression relative to tumor-infiltrating lymphocytes using multiplexed immunohistochemistry, and conduct functional assays measuring T cell activation in the presence of VISTA-expressing tumor cells.

Emerging research suggests that VISTA may be particularly important in contexts where other checkpoint pathways (PD-1/PD-L1, CTLA-4) are being therapeutically targeted, potentially serving as an alternative immune evasion mechanism when primary checkpoint pathways are blocked .

What methodological approaches are most effective for studying C10ORF54's role in immunoediting?

Studying C10ORF54's role in immunoediting requires sophisticated experimental approaches that can track the dynamic interaction between immune cells and tumor cells over time. Based on current research methodologies, investigators should consider:

• Longitudinal sampling approaches:

  • Serial tumor biopsies before, during, and after immunotherapy

  • Analysis of circulating tumor DNA to track genomic evolution under immune pressure

  • Development of patient-derived xenograft models to study evolution under controlled conditions

• Multi-omics analytical frameworks:

  • Integration of transcriptomics, proteomics, and genomics data

  • Analysis of neoantigen presentation alongside C10ORF54 expression

  • Next-generation sequencing and epitope prediction to follow the "immunological history" of tumors

• Advanced in vivo models:

  • Genetically engineered mouse models with inducible C10ORF54 expression

  • Humanized mouse models with reconstituted human immune systems

  • Autochthonous tumor models that more closely recapitulate natural tumor evolution

As noted in the literature, rigorous evidence for immunoediting of individual tumor cells in humans is challenging to obtain with current technologies . Researchers should therefore design studies that can provide indirect evidence through careful analysis of tumor evolution under immune pressure, particularly focusing on the relationship between immune checkpoint molecule expression and neoantigen presentation or recognition by T cells.

How can researchers differentiate between the effects of C10ORF54 and other immune checkpoint molecules in experimental designs?

Distinguishing the specific contributions of C10ORF54 (VISTA) from other immune checkpoint molecules requires carefully designed experiments:

• Sequential and combinatorial blockade experiments:

  • Use blocking antibodies against C10ORF54 alone, other checkpoints alone, and in combination

  • Analyze additive versus synergistic effects to determine pathway independence

  • Conduct time-course experiments to identify primary versus compensatory mechanisms

• Genetic manipulation approaches:

  • Generate cell lines with CRISPR-Cas9 knockout of C10ORF54 while preserving other checkpoint molecules

  • Create matched cell lines with knockouts of different checkpoint molecules

  • Develop inducible overexpression systems to study dosage effects

• Pathway-specific readouts:

  • Identify downstream signaling pathways unique to C10ORF54, such as its potential role in MMP14-mediated MMP2 activation

  • Develop reporter systems specific to C10ORF54 activation

  • Use phospho-flow cytometry to analyze pathway-specific phosphorylation events

When interpreting results, researchers should be aware that compensatory upregulation of alternative checkpoint pathways often occurs when one pathway is blocked, necessitating comprehensive analysis of the immune checkpoint landscape . This complexity underscores the importance of multi-parameter approaches that can capture the dynamic and interconnected nature of immune checkpoint regulation.

What are the current contradictions in research findings regarding C10ORF54's immunomodulatory functions?

Several contradictions and knowledge gaps exist in our understanding of C10ORF54's immunomodulatory functions that researchers should address:

• Dual roles in stem cell biology:

  • C10ORF54 enhances BMP4-induced signaling in ESCs

  • Yet it is downregulated following BMP4 exposure

  • This apparent paradox suggests context-dependent functions that require further investigation

• Relationship to interferons:

  • In the broader immune checkpoint literature, many checkpoint molecules (like PD-L1) are upregulated in response to IFNγ

  • It remains unclear whether C10ORF54 follows similar patterns of regulation or has distinct regulatory mechanisms

• Predictive value for immunotherapy response:

  • Whether C10ORF54 expression levels correlate with response to immune checkpoint blockade therapy is not firmly established

  • Inconsistencies exist regarding whether C10ORF54 expression represents a primary or adaptive resistance mechanism

• Interaction with MMP pathways:

  • UniProt annotation suggests C10ORF54 may stimulate MMP14-mediated MMP2 activation

  • How this function relates to its immunomodulatory roles remains poorly characterized

  • The molecular mechanisms connecting these seemingly disparate functions need clarification

Researchers addressing these contradictions should design experiments that include appropriate controls for contextual factors, measure multiple parameters simultaneously, account for temporal dynamics in expression patterns, and consider heterogeneity within tumor and immune cell populations.

How does C10ORF54 expression correlate with response to immune checkpoint inhibitors in clinical studies?

The correlation between C10ORF54 (VISTA) expression and response to established immune checkpoint inhibitors represents an important frontier in immunotherapy research:

• Potential compensatory role:

  • VISTA may be upregulated in tumors that develop resistance to PD-1/PD-L1 or CTLA-4 blockade

  • This upregulation could represent an adaptive resistance mechanism similar to other inhibitory signaling molecules

• Biomarker development considerations:

  • Pre-treatment VISTA expression may have different implications than post-treatment expression

  • Spatial distribution of VISTA relative to immune infiltrates likely matters more than absolute expression levels

• Methodological approach to correlation studies:

  • Immunohistochemistry scoring systems need standardization

  • Multi-parameter analysis including VISTA alongside other checkpoint molecules provides more meaningful data

  • Longitudinal sampling is essential to distinguish pre-existing from therapy-induced expression

To properly study these correlations, researchers should collect matched pre- and post-treatment samples, especially focusing on cases of acquired resistance to first-generation checkpoint inhibitors, where VISTA may play a particularly important role . The relationship between mutation burden, neoantigen load, and VISTA expression should also be explored, as higher mutation burden has been associated with better response to checkpoint inhibitors in some studies .

What are the optimal protocols for isolating and purifying C10ORF54 for research purposes?

For researchers needing to isolate and purify C10ORF54 protein for experimental use, the following protocol outline is recommended based on published methodologies:

• Expression system selection:

  • Insect cell expression systems (e.g., Sf9 Baculovirus) have proven effective for producing human C10ORF54

  • These systems provide appropriate post-translational modifications, particularly glycosylation

• Construct design considerations:

  • Include a C-terminal His-tag (8 amino acids) for purification purposes

  • Ensure the construct contains the extracellular domain (amino acids 33-194)

  • Consider codon optimization for the expression system

• Purification strategy:

  • Use nickel affinity chromatography for initial capture via the His-tag

  • Follow with size exclusion chromatography to remove aggregates and impurities

  • For highest purity (>95%), consider an additional ion exchange chromatography step

• Quality control metrics:

  • Verify purity using SDS-PAGE (should exceed 95%)

  • Confirm identity using mass spectrometry

  • Test biological activity using appropriate functional assays

The resulting protein should be a single, glycosylated polypeptide chain with a theoretical molecular mass of 19.1 kDa, though it will typically migrate at 28-40 kDa on SDS-PAGE due to glycosylation . This discrepancy should be expected and documented as evidence of proper post-translational modification rather than contamination.

How can researchers effectively measure C10ORF54 activity in different cellular contexts?

Measuring C10ORF54 (VISTA) activity requires appropriate functional assays that reflect its biological roles across different experimental settings:

• T cell suppression assays:

  • Co-culture T cells with VISTA-expressing cells or recombinant VISTA protein

  • Measure T cell proliferation using CFSE dilution or tritiated thymidine incorporation

  • Assess cytokine production (IL-2, IFN-γ) by ELISA or intracellular cytokine staining

  • Evaluate changes in T cell activation markers (CD69, CD25) by flow cytometry

• Signal transduction analysis:

  • Investigate MMP14-mediated MMP2 activation, as suggested by UniProt annotation

  • Use zymography to assess MMP2 activity in the presence and absence of VISTA

  • Conduct phospho-western blots to identify downstream signaling events

• Stem cell differentiation models:

  • Establish ESC differentiation assays with and without VISTA

  • Assess BMP4 signaling pathway activation using reporter constructs

  • Measure differentiation markers appropriate to the lineage being studied

The choice of assay should be guided by the specific aspect of VISTA biology being investigated and the cellular context most relevant to the research question. Researchers should also consider developing more complex 3D culture systems or organoid models that better recapitulate the in vivo environment where C10ORF54 functions.

What experimental models are most appropriate for investigating C10ORF54's role in immunotherapy resistance?

When investigating C10ORF54's potential role in immunotherapy resistance, researchers should select experimental models that recapitulate key aspects of the tumor-immune interaction:

• In vitro models:

  • Co-culture systems pairing tumor cells with tumor-infiltrating lymphocytes

  • 3D organoid cultures that incorporate immune components

  • Ex vivo tumor slice cultures that maintain the original tumor microenvironment

• Mouse models:

  • Syngeneic mouse models with intact immune systems

  • Humanized mouse models with reconstituted human immune components

  • Genetically engineered mouse models that develop autochthonous tumors

• Patient-derived models:

  • Patient-derived xenografts in immunodeficient mice

  • Patient-derived organoids co-cultured with autologous immune cells

  • Ex vivo analysis of tumor specimens from patients receiving immunotherapy

• Experimental design for resistance studies:

  • Include sequential sampling before, during, and after checkpoint blockade

  • Compare "responder" and "non-responder" populations

  • Analyze the evolution of C10ORF54 expression under therapeutic pressure

These approaches allow researchers to investigate whether C10ORF54 contributes to primary or acquired resistance to existing immunotherapies, an important distinction for developing targeted therapeutic strategies. Models that permit longitudinal sampling are particularly valuable for understanding the dynamics of resistance development.

What bioinformatic approaches are recommended for analyzing C10ORF54 expression data across different tissue types?

Analyzing C10ORF54 expression patterns across tissues requires sophisticated bioinformatic approaches:

• Data sources integration:

  • Combine RNA-seq data from sources like GTEx, TCGA, and Human Protein Atlas

  • Incorporate single-cell RNA-seq datasets to resolve cell type-specific expression

  • Leverage brain-specific datasets from Allen Brain Atlas for neurological studies

• Normalization and batch correction:

  • Apply appropriate normalization methods (TPM, FPKM, or DESeq2)

  • Implement batch correction algorithms like ComBat or Harmony

  • Consider tissue-specific normalization factors when comparing across tissues

• Co-expression network analysis:

  • Use WGCNA (Weighted Gene Co-expression Network Analysis) to identify modules

  • Construct protein-protein interaction networks incorporating C10ORF54

  • Identify transcription factors potentially regulating C10ORF54 expression

• Comparative analysis approaches:

  • Develop tissue-specific expression profiles as benchmarks

  • Compare normal versus disease state expression patterns

  • Analyze expression in response to various stimuli or treatments

Researchers should complement computational predictions with experimental validation to confirm the biological relevance of observed expression patterns across different tissues and cell types. The Harmonizome platform contains numerous datasets that can be leveraged for understanding C10ORF54's functional associations across different biological contexts .

How can researchers develop effective assays to study the interaction between C10ORF54 and its binding partners?

Developing assays to study C10ORF54 interactions requires careful consideration of both the protein's structure and its functional context:

• Protein-protein interaction assays:

  • Surface Plasmon Resonance (SPR) for kinetic and affinity measurements

  • Bio-Layer Interferometry (BLI) as an alternative label-free interaction analysis

  • Isothermal Titration Calorimetry (ITC) for thermodynamic parameters

  • Microscale Thermophoresis (MST) for interactions in complex solutions

• Cell-based interaction assays:

  • Flow cytometry-based binding assays using fluorescently-labeled proteins

  • FRET or BRET approaches for detecting interactions in living cells

  • Split reporter systems (luciferase, GFP) for monitoring interactions

  • Proximity Ligation Assay (PLA) for visualizing interactions in fixed samples

• Structural biology methods:

  • X-ray crystallography of C10ORF54 with binding partners

  • Cryo-EM for larger complexes

  • Hydrogen-Deuterium Exchange Mass Spectrometry for mapping interaction surfaces

  • NMR spectroscopy for dynamic interaction studies

The most effective approach will depend on the specific binding partner being studied and the question being addressed. When investigating potential interactions with MMP14 or MMP2, researchers should pay particular attention to the UniProt annotation suggesting C10ORF54 may stimulate MMP14-mediated MMP2 activation . This function suggests a potential role for C10ORF54 in extracellular matrix remodeling, which may connect to its other known functions in immune regulation and stem cell biology.