Segregation of nascent GPCRs in the ER-to-Golgi transport by CCHCR1 via direct interaction.

G protein-coupled receptors (GPCRs) constitute the largest superfamily of cell surface signaling proteins that share a common structural topology. When compared with agonist-induced internalization, how GPCRs are sorted and delivered to functional destinations after synthesis in the endoplasmic reticulum (ER) is much less well understood. Here, we demonstrate that depletion of coiled-coil α-helical rod protein 1 (CCHCR1) by siRNA and CRISPR-Cas9 significantly inhibits surface expression and signaling of α2A-adrenergic receptor (α2A-AR; also known as ADRA2A), without affecting α2B-AR. Further studies show that CCHCR1 depletion specifically impedes α2A-AR export from the ER to the Golgi, but not from the Golgi to the surface. We also demonstrate that CCHCR1 selectively interacts with α2A-AR. The interaction is mediated through multiple domains of both proteins and is ionic in nature. Moreover, mutating CCHCR1-binding motifs significantly attenuates ER-to-Golgi export, surface expression and signaling of α2A-AR. Collectively, these data reveal a novel function for CCHCR1 in intracellular protein trafficking, indicate that closely related GPCRs can be sorted into distinct ER-to-Golgi transport routes by CCHCR1 via direct interaction, and provide important insights into segregation and anterograde delivery of nascent GPCR members.

Clinical Findings, Response to Steroid Treatment, and Recurrence Rate in Alopecia Areata Patients with or without a Nonsynonymous Variant of Coiled-Coil Alpha-Helical Rod Protein 1.

BACKGROUND: Alopecia areata (AA) is considered complex genetic and tissue-specific autoimmune disease. We recently discovered a nonsynonymous variant in the coiled-coil alpha-helical rod protein 1 (CCHCR1) gene within the AA risk haplotype. And a water avoidance stress test on CCHCR1 knockout mice induced AA-like lesions. OBJECTIVE: To investigate the difference clinical findings of AA in patients with the CCHCR1 variant and without. METHODS: We conducted a retrospective analysis of the data from 142 AA patients. Among these patients, 20 (14.1%) had a variant of CCHCR1. We evaluated the sex distribution of the patients, age at onset, distribution of the clinical types, prevalence of a positive family history of AA, prevalence of association of AA with atopic dermatitis, response to steroid therapy, and recurrence rate. We used multivariate logistic regression analysis and Fisher's exact test for statistical analysis. We also investigate electron microscopic observations of hair samples with the CCHCR1 variant and without. RESULTS: The results showed a significant correlation between the CCHCR1 variant and the recurrence rate compared with the variant-negative group (p=0.0072). Electron microscopy revealed abnormalities in the hair shaft structure and hair cuticle in patients with the CCHCR1 variant (p=0.00174). CONCLUSION: Our results suggest that AA with CCHCR1 variant is clinically characterized by a high recurrence rate and hair morphological abnormality.

CCHCR1-astrin interaction promotes centriole duplication through recruitment of CEP72.

BACKGROUND: The centrosome is one of the most important non-membranous organelles regulating microtubule organization and progression of cell mitosis. The coiled-coil alpha-helical rod protein 1 (CCHCR1, also known as HCR) gene is considered to be a psoriasis susceptibility gene, and the protein is suggested to be localized to the P-bodies and centrosomes in mammalian cells. However, the exact cellular function of HCR and its potential regulatory role in the centrosomes remain unexplored. RESULTS: We found that HCR interacts directly with astrin, a key factor in centrosome maturation and mitosis. Immunoprecipitation assays showed that the coiled-coil region present in the C-terminus of HCR and astrin respectively mediated the interaction between them. Astrin not only recruits HCR to the centrosome, but also protects HCR from ubiquitin-proteasome-mediated degradation. In addition, depletion of either HCR or astrin significantly reduced centrosome localization of CEP72 and subsequent MCPH proteins, including CEP152, CDK5RAP2, and CEP63. The absence of HCR also caused centriole duplication defects and mitotic errors, resulting in multipolar spindle formation, genomic instability, and DNA damage. CONCLUSION: We conclude that HCR is localized and stabilized at the centrosome by directly binding to astrin. HCR are required for the centrosomal recruitment of MCPH proteins and centriolar duplication. Both HCR and astrin play key roles in keeping normal microtubule assembly and maintaining genomic stability.

The Alopecia Areata Phenotype Is Induced by the Water Avoidance Stress Test In cchcr1-Deficient Mice.

We recently discovered a nonsynonymous variant in the coiled-coil alpha-helical rod protein 1 (CCHCR1) gene within the alopecia areata (AA) risk haplotype. We also reported that the engineered mice with this risk allele exhibited. To investigate more about the involvement of the CCHCR1 gene in AA pathogenesis, we developed an AA model using C57BL/6N cchcr1 gene knockout mice. In this study, mice (6-8 weeks) were divided into two groups: cchcr1-/- mice and wild-type (WT) littermates. Both groups were subjected to a water avoidance stress (WAS) test. Eight weeks after the WAS test, 25% of cchcr1-/- mice exhibited non-inflammatory foci of alopecia on the dorsal skin. On the other hand, none of wild-type littermates cause hair loss. The foci resembled human AA in terms of gross morphology, trichoscopic findings and histological findings. Additionally, gene expression microarray analysis of cchcr1-/- mice revealed abnormalities of hair related genes compared to the control. Our results strongly suggest that CCHCR1 is associated with AA pathogenesis and that cchcr1-/- mice are a good model for investigating AA.

Identification of protein/mRNA network involving the PSORS1 locus gene CCHCR1 and the PSORS4 locus gene HAX1.

CCHCR1 (Coiled-Coil alpha-Helical Rod 1), maps to chromosomal region 6p21.3, within the major psoriasis susceptibility locus PSORS1. CCHCR1 itself is a plausible psoriasis candidate gene, however its role in psoriasis pathogenesis remains unclear. We previously demonstrated that CCHCR1 protein acts as a cytoplasmic docking site for RNA polymerase II core subunit 3 (RPB3) in cycling cells, suggesting a role for CCHCR1 in vesicular trafficking between cellular compartments. Here, we report a novel interaction between CCHCR1 and the RNA binding protein HAX1. HAX1 maps to chromosomal region 1q21.3 within the PSORS4 locus and is over-expressed in psoriasis. Both CCHCR1 and HAX1 share subcellular co-localization with mitochondria, nuclei and cytoplasmic vesicles as P-bodies. By a series of ribonucleoprotein immunoprecipitation (RIP) assays, we isolated a pool of mRNAs complexed with HAX1 and/or CCHCR1 proteins. Among the mRNAs complexed with both CCHCR1 and HAX1 proteins, there are Vimentin mRNA, previously described to be bound by HAX1, and CAMP/LL37 mRNA, whose gene product is over-expressed in psoriasis.

Intracellular signalling pathways and cytoskeletal functions converge on the psoriasis candidate gene CCHCR1 expressed at P-bodies and centrosomes.

BACKGROUND: CCHCR1 (Coiled-Coil α-Helical Rod protein 1) is a putative psoriasis candidate gene with the risk alleles CCHCR1*WWCC and *Iso3, the latter inhibiting the translation of isoform 1. CCHCR1 was recently shown to be a centrosomal protein, as well as a component of cytoplasmic processing bodies (P-bodies) that regulate mRNA turnover. The function of CCHCR1 has remained unsettled, partly because of the inconsistent findings; it has been shown to play a wide variety of roles in divergent processes, e.g., cell proliferation and steroidogenesis. Here we utilized RNA sequencing (RNAseq) using HEK293 cells overexpressing isoforms 1 or 3 (Iso1, Iso3 cells), in combination with the coding non-risk or risk (*WWCC) haplotype of CCHCR1. Our aim was to study the overall role of CCHCR1 and the effects of its variants. RESULTS: The overexpression of CCHCR1 variants in HEK293 cells resulted in cell line-specific expression profiles though several similarities were observable. Overall the Iso1 and Iso3 cells showed a clear isoform-specific clustering as two separate groups, and the Non-risk and Risk cells often exhibited opposite effects. The RNAseq supported a role for CCHCR1 in the centrosomes and P-bodies; the most highlighted pathways included regulation of cytoskeleton, adherens and tight junctions, mRNA surveillance and RNA transport. Interestingly, both the RNAseq and immunofluorescent localization revealed variant-specific differences for CCHCR1 within the P-bodies. CONCLUSIONS: CCHCR1 influenced a wide variety of signaling pathways, which could reflect its active role in the P-bodies and centrosomes that both are linked to the cytoskeleton; as a centrosomal P-body protein CCHCR1 may regulate diverse cytoskeleton-mediated functions, such as cell adhesion and -division. The present findings may explain the previous inconsistent observations about the functions of CCHCR1.

CCHCR1 interacts with EDC4, suggesting its localization in P-bodies.

Coiled-coil alpha-helical rod protein 1 (CCHCR1) is suggested as a candidate biomarker for psoriasis for more than a decade but its function remains poorly understood because of the inconsistent findings in the literature. CCHCR1 protein is suggested to be localized in the cytoplasm, nucleus, mitochondria, or centrosome and to regulate various cellular functions, including steroidogenesis, proliferation, differentiation, and cytoskeleton organization. In this study, we attempted to find a consensus between these findings by identifying the interaction partners of CCHCR1 using co-immunoprecipiation with a stable cell line expressing EGFP-tagged CCHCR1. Out of more than 100 co-immunoprecipitants identified by liquid chromatography-tandem mass spectrometry (LC-MS/MS), the enhancer of mRNA-decapping protein 4 (EDC4), which is a processing body (P-body) component, was particularly found to be the major interacting partner of CCHCR1. Confocal imaging confirmed the localization of CCHCR1 in P-bodies and its N-terminus is required for this subcellular localization, suggesting that CCHCR1 is a novel P-body component. As P-bodies are the site for mRNA metabolism, our findings provide a molecular basis for the function of CCHCR1, any disruption of which may affect the transcriptome of the cell, and causing abnormal cell functions.