Recombinant Human Coiled-coil domain-containing protein 91 (CCDC91)

What is CCDC91 and what are its primary cellular functions?

CCDC91 (Coiled-coil domain-containing protein 91) is a protein involved in the regulation of membrane traffic through the trans-Golgi network (TGN) . Also known by aliases HSD8 and p56, this protein is encoded by a gene located on chromosome band 12p11.22 in humans . The primary function of CCDC91 appears to be mediating vesicular transport processes, particularly those involved in protein trafficking from the Golgi apparatus to other cellular compartments.

Recent functional analysis has revealed that CCDC91 plays a crucial role in elastin transport and secretion . When CCDC91 is disrupted, cells exhibit distended Golgi cisternae, cytoplasmic vesicle accumulation, and abnormal presence of lysosomes . Additionally, immunostaining of CCDC91-depleted cells demonstrates tropoelastin accumulation in the Golgi and formation of abnormal extracellular aggregates, suggesting its importance in the extracellular matrix organization pathway .

What experimental models are available for studying CCDC91?

Several experimental models have been developed to investigate CCDC91 function:

• CCDC91 Knockout Cell Lines: HEK293-based CCDC91 knockout cell lines are commercially available for research . These models allow investigation of cellular behaviors in the absence of CCDC91 expression.

• siRNA/shRNA Knockdown Systems: Studies have employed short hairpin RNA (shRNA) sequences designed based on the CCDC91 reference sequence (Gene Bank Accession No. NM_018318.5) cloned into lentiviral vectors to reduce CCDC91 expression in human skin fibroblasts (HSF) .

• CRISPR/Cas9 Knockout Models: Targeted disruption of specific CCDC91 exons has been achieved using CRISPR/Cas9 technology. For example, researchers have designed guide RNAs to target exon 11 of CCDC91 to recapitulate disease-specific splicing mutations .

The efficacy of these models can be verified through several methodological approaches:

• Quantitative real-time PCR (Q-PCR) using SYBR Premix Ex Taq TM II on platforms such as LightCycler 480

• Western blot analysis of protein expression

• Direct DNA sequencing of modified regions to confirm genetic alterations

What is the evidence linking CCDC91 to autism spectrum disorders?

Multiple lines of evidence support the association between CCDC91 and autism spectrum disorders (ASD):

• The SFARI Gene database assigns CCDC91 a score of 2, indicating strong evidence for association with ASD .

• Five out of six research reports document relevance to autism , with multiple identified variants:

• The gene was identified as a novel ASD candidate in Gonzalez-Mantilla et al. (2016) based on the presence of two potentially pathogenic loss-of-function variants in ASD cases .

• Ruzzo et al. (2019) further supported this association by identifying CCDC91 within shared genetic networks impacted by both inherited and de novo genetic risk for autism .

How do CCDC91 mutations affect protein function and cellular processes?

Research has revealed several mechanisms by which CCDC91 mutations disrupt normal cellular processes:

• Golgi Structure Disruption: Immunofluorescence analysis of CCDC91-depleted cells shows significant structural impairment of the Golgi apparatus . This disruption appears as distended Golgi cisternae when visualized using GM130 markers.

• Vesicular Transport Abnormalities: Loss of CCDC91 function results in cytoplasmic vesicle accumulation, suggesting defective trafficking between the Golgi and other cellular compartments .

• Elastin Processing Defects: A splicing mutation (1101 + 1 G > A) causing exon 11 skipping results in a 59-amino-acid-residue loss (L309-Q367del), leading to abnormal tropoelastin accumulation in the Golgi and formation of extracellular aggregates .

• Lysosomal Abnormalities: CCDC91 dysfunction is associated with abnormal lysosome presence, potentially affecting protein degradation pathways .

Interestingly, research indicates that while CCDC91 mutations significantly impact elastin processing, they do not appear to affect fibrillin-1 microfibril assembly or lysyl oxidase activity , suggesting a specific role in elastin transport rather than generalized disruption of extracellular matrix formation.

What methodologies are optimal for investigating CCDC91 gene variants?

Researchers have employed several complementary approaches to characterize CCDC91 variants:

• Genome-wide SNP Genotyping: Employing platforms such as Illumina Human 660W-Quad_v1 BeadChips covering >655,000 SNP and CNV probes .

• Multipoint Parametric Linkage Analysis: Software such as MERLIN (version 1.1.2) has been used to identify susceptibility regions containing CCDC91 .

• Whole-exome Sequencing (WES): Libraries generated with capture of exonic regions using tools like Roche NimbleGen's SeqCap EZ Human Exome Library v3.0 (64 Mb), followed by sequencing on platforms like Illumina Hiseq 2000 .

• Variant Filtering Pipeline:

  • Alignment to human reference genome (hg19) using Burrows-Wheeler analysis

  • Sequential filtering against databases (dbSNP138, 1000 Genomes project, Hapmap 8)

  • Confirmation of identified variants through Sanger sequencing

• RNA Analysis: RT-PCR of RNA extracted from peripheral blood mononuclear cells (PBMCs) using TRIzol Reagent and cDNA synthesis with PrimeScript TMII 1st Strand cDNA Synthesis Kit .

For researchers investigating novel CCDC91 variants, this multifaceted approach allows comprehensive characterization from initial discovery through functional validation.

How can CRISPR/Cas9 technology be optimized for CCDC91 functional studies?

Based on published methodologies, optimal CRISPR/Cas9 approaches for CCDC91 include:

• Guide RNA Design: Design specific guide RNAs targeting critical exons (such as exon 11) using validated CRISPR design tools (http://crispr.mit.edu/)[2].

• Vector Systems: Cloning gRNA sequences into vectors such as pSpCas9-2A-Puro (PX459) using restriction enzymes like BbsI .

• Transfection Protocol:

  • For HEK293T cells, Lipofectamine 2000 has proven effective

  • Following 8-hour transfection, culture in fresh complete DMEM

  • Selection with 3 μg/ml puromycin

• Validation of Knockout Efficiency:

  • Sequencing of cDNA to confirm exon deletion

  • Protein expression verification through Western blotting

  • Immunofluorescence analysis to confirm cellular phenotype

These approaches allow precise modification of CCDC91, enabling researchers to recapitulate disease-specific mutations or create complete knockout models for comprehensive functional studies.

What immunofluorescence protocols best visualize CCDC91-dependent cellular phenotypes?

Based on published research, optimal immunofluorescence protocols for CCDC91 studies include:

• Sample Preparation:

  • Grow cells on glass coverslips

  • Fix in 4% formaldehyde for 10 minutes

  • Wash with PBS

  • Block for 30 minutes in 1% BSA

• Primary Antibodies:

  • Anti-human CCDC91 (sc-514452, Santa Cruz)

  • Anti-GM130 (12480T, Cell Signaling Technology) for Golgi visualization

  • Anti-tropoelastin (PR398, Elastin Products Company) for elastin trafficking

  • Anti-human aortic alpha elastin (PR533, EPC)

  • Anti-human fibrillin-1 (NBP1-84722, NOVUS biologicals)

• Secondary Antibodies:

  • For HSF cells: Anti-mouse IgG Alexa Fluor 488 and anti-rabbit IgG Alexa Fluor 568Cy3

  • For HEK293T cells: Anti-mouse IgG Alexa Fluor 488 and anti-rabbit IgG Alexa Fluor 594Cy3

• Nuclear Counterstaining: DAPI for DNA visualization

• Imaging: Laser scanning microscopy (LSM880, Carl Zeiss) for high-resolution analysis of subcellular structures

This approach enables clear visualization of Golgi morphology alterations, vesicle accumulation, and trafficking defects resulting from CCDC91 dysfunction.

How does CCDC91 participate in elastin trafficking pathways?

Recent functional studies have revealed CCDC91's specific role in elastin processing:

• Golgi Exit Regulation: CCDC91 appears to be critical for the export of tropoelastin from the Golgi apparatus to the extracellular space. When CCDC91 function is compromised, tropoelastin accumulates within the Golgi .

• Selective Cargo Specificity: Importantly, CCDC91 dysfunction affects elastin trafficking while having minimal impact on other extracellular matrix components such as fibrillin-1, suggesting a selective cargo recognition mechanism .

• Post-Golgi Vesicle Formation: The presence of cytoplasmic vesicle accumulation in CCDC91-deficient cells suggests its involvement in vesicle budding or trafficking from the Golgi to the cell surface .

• Extracellular Matrix Organization: The formation of abnormal extracellular elastin aggregates in CCDC91-deficient models indicates that proper CCDC91 function is necessary for normal elastin deposition and organization in the extracellular matrix .

This specific role in elastin trafficking may explain the link between CCDC91 mutations and certain connective tissue phenotypes observed in clinical studies.

What are the current limitations in understanding CCDC91's molecular interactions?

Despite progress in understanding CCDC91 function, several significant challenges remain:

• Limited Interactome Data: The complete set of CCDC91's protein-protein interactions remains poorly characterized, hampering our understanding of its position within cellular signaling networks.

• Tissue-Specific Functions: While CCDC91's role in elastin trafficking has been established in certain cell types, its function in other tissues, particularly the central nervous system, remains underexplored despite its association with neurodevelopmental disorders .

• Structural Information Gaps: The three-dimensional structure of CCDC91 has not been fully determined, limiting structure-based drug design approaches and mechanistic understanding of how mutations disrupt function.

• Regulatory Mechanisms: The transcriptional, post-transcriptional, and post-translational regulation of CCDC91 remains largely uncharacterized.

Addressing these knowledge gaps represents a critical opportunity for researchers to advance understanding of this protein's biological significance.

How do CCDC91 variants contribute to diverse phenotypic outcomes?

The diverse phenotypic manifestations associated with CCDC91 mutations present an intriguing research challenge:

• Neurodevelopmental vs. Connective Tissue Phenotypes: CCDC91 variants have been associated with both autism spectrum disorders and elastin trafficking defects , raising questions about tissue-specific functions.

• Inheritance Patterns: Both de novo and inherited variants have been identified , suggesting potential differences in pathogenic mechanisms or penetrance.

• Genotype-Phenotype Correlations: Different mutation types (copy number variations, missense mutations, splicing variants) may impact cellular function through distinct mechanisms, contributing to phenotypic variability.

• Modifier Genes: The influence of genetic background and modifier genes on CCDC91-associated phenotypes remains largely unexplored but may explain variable expressivity.

Future research incorporating multi-omics approaches, patient-derived cellular models, and comprehensive phenotypic characterization will be essential to unravel these complex genotype-phenotype relationships.