Recombinant Human BRICHOS domain-containing protein C16orf79 (C16orf79)

What is the molecular structure of human C16orf79/BRICD5 protein?

Human C16orf79 (now known as BRICD5) is a membrane protein that contains one BRICHOS domain . The open reading frame (ORF) size is 687 base pairs, encoding a protein predicted to be an integral component of the cell membrane . The BRICHOS domain is approximately 100 amino acids long and has been found in several proteins associated with dementia, respiratory distress, and cancer. The domain's secondary structure typically consists of alpha helices and beta sheets that contribute to its functional properties in protein processing and quality control.

What are the key functional domains of C16orf79/BRICD5?

The primary functional domain in C16orf79/BRICD5 is the BRICHOS domain . BRICHOS domains are believed to have chaperone activity, potentially playing a role in protein folding and processing. While the complete functional characterization of C16orf79's BRICHOS domain remains under investigation, it likely contributes to the protein's predicted role in regulating cell population proliferation . Research should focus on domain-specific mutagenesis studies to elucidate the precise function of this domain within the context of C16orf79.

What is the genomic location and organization of the C16orf79/BRICD5 gene?

The gene encoding C16orf79/BRICD5 maps to human chromosome 16p13.3 . Chromosome 16 is notable for encoding over 900 genes in approximately 90 million base pairs, constituting nearly 3% of human cellular DNA . The C16orf79 gene is identified with Gene ID 283870 and has a RefSeq number of NM_182563 . For researchers conducting genomic studies, it's important to note that chromosome 16 is associated with various genetic disorders, including giant axonal neuropathy (GAN gene), Rubinstein-Taybi syndrome (CREBBP gene), and Crohn's disease (NOD2 gene) .

What is the expression pattern of C16orf79/BRICD5 across different human tissues?

Expression analysis of C16orf79/BRICD5 should be conducted using a combination of RNA-seq data from tissue panels and immunohistochemistry. While comprehensive expression data is not explicitly provided in the available search results, researchers should utilize public databases such as GTEx, Human Protein Atlas, and ENCODE to analyze the tissue-specific expression patterns. For experimental validation, qRT-PCR with tissue-specific cDNA panels and western blotting with anti-C16orf79 antibodies (such as sc-136580) can be employed to quantify expression levels across different tissues .

How can I effectively silence C16orf79/BRICD5 expression in experimental models?

For effective silencing of C16orf79/BRICD5, researchers can utilize adenovirus-mediated shRNA delivery systems. Ready-to-use adenoviruses expressing shRNA for silencing human BRICD5 are available (e.g., shADV-202770) . These viral vectors feature U6 promoters driving shRNA expression and can include optional reporter genes such as eGFP, CFP, YFP, RFP, or mCherry for tracking transduction efficiency . For experimental design, consider:

• Using appropriate control viruses (e.g., Ad-GFP-U6-shRNA, Ad-U6-Luc-RNAi)

• Optimizing multiplicity of infection (MOI) based on target cell type

• Confirming knockdown efficiency via qRT-PCR and western blotting

• Assessing cellular phenotypes 48-72 hours post-transduction

To enhance specificity and reduce off-target effects, CRISPR-Cas9 genome editing can be employed as an alternative approach for creating stable knockout cell lines.

What are the recommended approaches for studying protein-protein interactions involving C16orf79/BRICD5?

To study protein-protein interactions involving C16orf79/BRICD5, researchers should employ a multi-method approach:

• Co-immunoprecipitation (Co-IP): Using antibodies against C16orf79 (such as sc-136580) to pull down protein complexes, followed by mass spectrometry identification of binding partners.

• Proximity labeling techniques: BioID or APEX2 fusion proteins can be created by fusing the biotin ligase to C16orf79 to identify proximal interacting proteins in living cells.

• Yeast two-hybrid screening: Using the BRICHOS domain or full-length C16orf79 as bait to screen human cDNA libraries.

• Protein complementation assays: Split-luciferase or split-GFP systems to validate specific interactions in mammalian cells.

For membrane proteins like C16orf79, consider using membrane-specific techniques such as membrane yeast two-hybrid or MYTH (membrane yeast two-hybrid) systems that are designed for integral membrane proteins.

How can I assess the functional impact of C16orf79/BRICD5 in cell proliferation experiments?

Given that C16orf79/BRICD5 is predicted to be involved in the regulation of cell population proliferation , comprehensive cell proliferation assays should be conducted:

• Gene Knockdown/Knockout Approaches:

  • Use adenoviral shRNA vectors (shADV-202770) for transient knockdown

  • Generate CRISPR-Cas9 knockout cell lines for long-term studies

• Proliferation Assays:

  • Real-time cell analysis (RTCA) for continuous monitoring

  • BrdU incorporation to measure DNA synthesis

  • Ki-67 immunostaining to identify proliferating cells

  • Colony formation assays for long-term proliferation effects

• Cell Cycle Analysis:

  • Flow cytometry with propidium iodide staining

  • EdU pulse-chase experiments to track cell cycle progression

• Rescue Experiments:

  • Re-express wild-type or mutant C16orf79 in knockout cells to confirm specificity

Compare effects across multiple cell lines representing different tissues to establish tissue-specific functions.

What is the potential role of C16orf79/BRICD5 in cancer and how can synthetic lethality approaches be applied?

The potential role of C16orf79/BRICD5 in cancer should be investigated through synthetic lethality approaches, which identify gene pairs where simultaneous perturbation leads to cell death . Given C16orf79's predicted involvement in cell proliferation regulation , researchers should:

• Analyze public cancer genomics databases:

  • Check C16orf79 expression, mutation, and copy number alterations across different cancer types in TCGA, ICGC, and cBioPortal databases

  • Perform survival analysis correlating C16orf79 expression with patient outcomes

• Employ CRISPR-Cas9 screening:

  • Conduct genome-wide CRISPR screens in cell lines with C16orf79 knockdown/knockout to identify synthetic lethal partners

  • Validate top candidates using individual knockdown experiments

• Drug sensitivity testing:

  • Test whether C16orf79 expression levels correlate with sensitivity to specific cancer therapeutics

  • Screen compound libraries in C16orf79-modulated cells to identify selective vulnerabilities

• In vivo validation:

  • Develop xenograft models with C16orf79-depleted cancer cells to assess tumor growth dynamics

  • Test candidate synthetic lethal drug combinations in these models

This approach could potentially identify novel targeted therapeutic strategies for cancers with altered C16orf79 expression or function.

How can recombinant C16orf79/BRICD5 protein be optimally produced and purified for structural studies?

For optimal production and purification of recombinant C16orf79/BRICD5 protein for structural studies:

• Expression System Selection:

  • For membrane proteins like C16orf79 , consider specialized expression systems:

    • Mammalian expression (HEK293, CHO cells) for proper folding and post-translational modifications

    • Insect cell systems (Sf9, High Five) using baculovirus vectors

    • Cell-free expression systems optimized for membrane proteins

• Construct Design:

  • Include affinity tags (His6, FLAG, or Strep-tag II) for purification

  • Consider fusion partners (MBP, SUMO) to enhance solubility

  • For structural studies, create constructs with flexible regions removed based on disorder prediction algorithms

• Solubilization and Purification Strategy:

  • Screen detergents (DDM, LMNG, GDN) for optimal extraction from membranes

  • Employ two-step affinity chromatography followed by size exclusion chromatography

  • Consider amphipol or nanodisc reconstitution for maintaining native-like environment

• Quality Assessment:

  • Verify protein homogeneity by dynamic light scattering and analytical SEC

  • Confirm structural integrity using circular dichroism spectroscopy

  • Assess thermal stability using differential scanning fluorimetry

• Structural Analysis Options:

  • X-ray crystallography (requires detergent screening for crystal formation)

  • Cryo-EM (particularly suitable for membrane proteins)

  • NMR spectroscopy for dynamic studies of specific domains

Commercial recombinant proteins are available for benchmarking , but custom expression is often necessary for structural biology applications.

What are the key differences between human C16orf79/BRICD5 and its orthologs in model organisms?

Understanding the evolutionary conservation of C16orf79/BRICD5 provides insights into its fundamental biological functions:

*Estimated identity ranges based on typical conservation patterns of membrane proteins

For comparative studies, researchers should:

• Perform multiple sequence alignments focusing on the BRICHOS domain conservation

• Analyze synteny of genomic regions containing BRICD5 across species

• Compare tissue expression patterns between orthologs

• Assess functional complementation by expressing orthologs in human cell knockout models

The availability of recombinant proteins from multiple species facilitates cross-species functional comparisons.

How does the BRICHOS domain in C16orf79/BRICD5 compare functionally to BRICHOS domains in other proteins?

The BRICHOS domain, present in C16orf79/BRICD5 , belongs to a family found in several proteins with diverse functions:

• Comparative BRICHOS Analysis:

  • BRICHOS domains (~100 amino acids) appear in proteins associated with dementia, respiratory distress, and cancer

  • Key BRICHOS-containing proteins include:

    • Surfactant protein C (proSP-C) - involved in pulmonary surfactant function

    • Integral Membrane Protein 2B (ITM2B/BRI2) - linked to familial British and Danish dementias

    • Chondromodulin-I precursor (TNMD) - involved in cartilage development

• Functional Comparison Methodologies:

  • Structural analysis: Compare C16orf79 BRICHOS domain using homology modeling based on available BRICHOS structures

  • Chaperone activity assays: Measure ability to prevent protein aggregation compared to other BRICHOS domains

  • Domain swapping experiments: Replace BRICHOS in C16orf79 with domains from other proteins to assess functional conservation

• Experimental Approaches:

  • Recombinant expression of isolated BRICHOS domains from different proteins

  • Thermal shift assays to compare stability profiles

  • Binding assays to identify domain-specific interaction partners

  • Aggregation prevention assays using amyloidogenic peptides

Understanding the unique properties of C16orf79's BRICHOS domain may reveal its specific cellular functions and potential roles in pathological conditions.

What are the most promising future research directions for C16orf79/BRICD5?

Based on current knowledge, several promising research directions for C16orf79/BRICD5 emerge:

• Comprehensive functional characterization:

  • Define the precise role in cell proliferation regulation through systematic phenotypic analysis of knockout models

  • Identify the subcellular localization and trafficking mechanisms of the protein

• BRICHOS domain-specific research:

  • Determine the chaperone activity of C16orf79's BRICHOS domain compared to other family members

  • Resolve the structure of the domain to understand functional mechanisms

• Disease associations:

  • Investigate potential roles in cancer through synthetic lethality approaches

  • Explore associations with diseases linked to chromosome 16p13.3

• Systems biology approaches:

  • Map the complete interactome of C16orf79/BRICD5

  • Integrate multi-omics data to place C16orf79 in relevant cellular pathways

• Therapeutic potential:

  • Develop small molecule modulators of C16orf79 function

  • Explore biologics targeting extracellular portions of the protein

The availability of research tools including recombinant proteins and silencing vectors facilitates these investigations.

What methodological challenges need to be addressed when working with C16orf79/BRICD5?

Researchers working with C16orf79/BRICD5 face several methodological challenges:

• Membrane protein-specific difficulties:

  • Challenges in expression and purification of full-length protein for structural studies

  • Need for specialized detergents and lipid environments to maintain native conformation

  • Potential toxicity when overexpressed in certain systems

• Functional assessment limitations:

  • Limited knowledge of natural ligands or binding partners

  • Potential redundancy with other BRICHOS-containing proteins

  • Need for tissue-specific models to capture context-dependent functions

• Technical considerations:

  • Developing specific antibodies against different epitopes for diverse applications

  • Establishing appropriate controls for knockdown experiments

  • Creating physiologically relevant overexpression models

• Experimental design recommendations:

  • Use multiple independent shRNA or siRNA sequences when performing knockdown studies

  • Complement genetic approaches with pharmacological tools when available

  • Include rescue experiments to confirm specificity of observed phenotypes

  • Employ both in vitro and in vivo models to validate findings