COPS7A Human

What is COPS7A and what cellular complex does it belong to?

COPS7A (also known as CSN7A) is a protein subunit of the COP9 signalosome complex encoded by the COPS7A gene in humans. The COP9 signalosome is a highly conserved protein complex involved in various cellular processes including protein degradation via the ubiquitin-proteasome pathway . COPS7A functions as part of this multi-protein complex that regulates cullin-RING ligase (CRL) activity through deneddylation. To verify COPS7A's incorporation into the COP9 signalosome, researchers typically employ co-immunoprecipitation assays using antibodies against other CSN subunits or flag-tagged COPS7A protein followed by western blotting analysis .

What are the known post-translational modifications of COPS7A?

COPS7A undergoes multiple post-translational modifications (PTMs) at specific sites, including:

To investigate these modifications, researchers typically employ mass spectrometry-based approaches coupled with enrichment strategies specific to each PTM type. For phosphorylation studies, titanium dioxide (TiO2) or immobilized metal affinity chromatography (IMAC) enrichment followed by LC-MS/MS analysis is commonly used. For ubiquitination studies, affinity purification with ubiquitin-binding domains followed by MS analysis is the standard approach .

How is COPS7A expression regulated in different human tissues?

COPS7A appears to be expressed across all human tissues and cells investigated according to the Genevestigator expression data library . Expression analysis can be performed using RNA-seq data or protein quantification through western blotting. The Human Protein Atlas provides tissue-specific expression data through antibody-based profiling using immunohistochemistry across 44 normal tissue types . For laboratory verification of expression patterns, researchers typically employ qRT-PCR for mRNA quantification and western blotting with specific anti-COPS7A antibodies for protein detection. When studying expression in specific contexts, such as during cellular differentiation, time-course experiments combined with western blotting are effective methods to track expression changes .

What is the relationship between COPS7A and COPS7B?

COPS7A and COPS7B are paralogous proteins that function as alternative subunits in the COP9 signalosome complex. These variants coexist in human cells, including LiSa-2 preadipocytes, and appear to have both overlapping and distinct functions . While both are necessary for processes such as adipogenesis (as demonstrated by the retardation of adipogenesis when either is downregulated), they exhibit differential expression patterns during adipocyte differentiation. Specifically, endogenous COPS7B expression increases during adipogenesis while COPS7A levels remain constant . To study their distinct roles, researchers can employ variant-specific antibodies for detection and use siRNA-mediated knockdown approaches targeting each variant specifically .

How do COPS7A and COPS7B variants differentially affect adipogenic differentiation?

The COPS7A and COPS7B variants appear to have distinct roles in adipogenic differentiation. Research shows that overexpression of COPS7B accelerates adipogenesis, while overexpression of COPS7A does not produce the same effect. Interestingly, downregulation of either COPS7A or COPS7B leads to retardation of adipogenesis, indicating both are necessary for the process .

To investigate these differential effects, researchers can:

• Establish stable cell lines expressing Flag-tagged COPS7A or COPS7B (using vectors such as pCMV-3Tag-1a)

• Perform transient knockdown using siRNA specific to either COPS7A or COPS7B

• Assess adipogenesis through:

  • Western blot analysis of adipogenic markers (PPAR-γ, CHOP)

  • Oil Red O (ORO) staining to quantify lipid production

  • Microscopy to visualize lipid droplet formation

Experimental models for such studies include human preadipocyte cell lines (like LiSa-2) and mouse embryonic fibroblasts (MEFs) .

What protein interactions are specific to COPS7A versus COPS7B complexes?

COPS7A and COPS7B appear to have different affinities for certain proteins, which may explain their distinct functional roles. Flag-pulldown experiments suggest that CSN7A and CSN7B variants bind differently to proteins such as β-TrCP and USP15 . To comprehensively investigate COPS7A-specific protein interactions, researchers should:

• Perform immunoprecipitation using Flag-tagged COPS7A followed by mass spectrometry to identify binding partners

• Compare interactomes between COPS7A and COPS7B using parallel IP-MS experiments

• Validate key interactions using reciprocal co-immunoprecipitation and western blotting

• Assess functional relevance of specific interactions through domain mapping and mutagenesis studies

• Employ proximity labeling methods (BioID or APEX) to capture transient interactions in living cells

These approaches would help elucidate how COPS7A-specific protein interactions contribute to its unique functions within the COP9 signalosome complex .

How do disease-associated variants of COPS7A affect its function?

Several disease-associated variants of COPS7A have been identified that affect post-translational modification sites:

To investigate the functional impact of these variants, researchers should consider:

• Generating site-specific mutations in COPS7A expression constructs using site-directed mutagenesis

• Expressing wild-type and mutant COPS7A in relevant cell models

• Assessing effects on:

  • COP9 signalosome complex formation and stability using gel filtration or blue native PAGE

  • Deneddylating activity using cullins as substrates

  • Protein-protein interactions compared to wild-type COPS7A

  • Post-translational modification patterns at other sites

  • Cellular phenotypes relevant to the associated cancer type

This systematic approach would provide insights into how these variants contribute to pathogenesis in different cancer types .

What is the role of COPS7A post-translational modifications in regulating CSN complex activity?

COPS7A undergoes numerous post-translational modifications, but their precise roles in regulating CSN complex activity remain largely unexplored. To investigate these relationships, researchers should:

• Generate phospho-mimetic and phospho-deficient mutants of key COPS7A phosphorylation sites (Y137, T219, S232, T240, T249, S255)

• Create lysine-to-arginine mutants to prevent ubiquitination/acetylation at sites K20, K109, K199, K206, K217, K221, K256, K260, K269

• Assess the impact of these mutations on:

  • CSN complex assembly using co-immunoprecipitation followed by western blotting

  • Deneddylating activity of the CSN complex using in vitro and cellular assays

  • Interaction with CRLs and other binding partners

  • Cellular localization using immunofluorescence microscopy

  • Stability and turnover rate of COPS7A using cycloheximide chase assays

Additionally, researchers should investigate the crosstalk between different modifications, as phosphorylation often regulates subsequent ubiquitination or acetylation events. Mass spectrometry approaches could reveal how modifications at one site affect the modification status at other sites .

What methodologies are most effective for studying COPS7A in its native complex?

Studying COPS7A within its native COP9 signalosome complex presents technical challenges. The most effective methodologies include:

• Structural Analysis:

  • Cryo-electron microscopy to visualize COPS7A within the intact CSN complex

  • Hydrogen-deuterium exchange mass spectrometry to map interaction surfaces

• Functional Analysis:

  • CRISPR-Cas9 genome editing to create COPS7A knockout or endogenously tagged cell lines

  • Reconstitution assays with purified components to assess direct contributions to CSN activity

• Interaction Analysis:

  • Proximity labeling techniques (BioID, APEX) to identify neighbors of COPS7A in living cells

  • Crosslinking mass spectrometry to capture transient interactions within the complex

• Expression Analysis:

  • Single-cell RNA sequencing to identify cell types with unique COPS7A expression patterns

  • Fluorescence correlation spectroscopy to measure the stoichiometry of COPS7A in living cells

• Purification Approaches:

  • Flag-pulldowns of tagged COPS7A, which have been shown to be effective alternatives to traditional CSN purification methods from red blood cells

  • Tandem affinity purification to isolate native COPS7A-containing complexes under physiological conditions