Coiled-coil Protein Research Articles

Fasudil, a ROCK inhibitor, preserves limb integrity in a mouse model of unilateral critical limb ischemia: Possible interplay of inflammatory and angiogenic signaling pathways

Peripheral arterial diseases (PAD) had a great attention owing to devastating consequences of disability and cardiovascular morbidity and mortality. Yet, current therapeutic options are limited to surgical revascularization with no effective pharmacotherapy available. Excessive activity of Rho-associated coiled-coil protein kinase (ROCK) is implicated with several vascular diseases, rendering ROCK inhibition as a potential therapeutic strategy for patients suffering vascular disorders. AimThe current study was dedicated to investigating the vascular protective potential of Fasudil, a ROCK inhibitor, on an experimentally induced unilateral critical limb ischemia (CLI) model in mice and demonstrated the possible underlying mechanisms. MethodsUnilateral CLI was induced by ligation and excision of femoral artery followed by daily i.p. injection of Fasudil (10 mg/kg or 25 mg/kg) up to two weeks post-surgery. Key findingsMice underwent CLI showed decreased antioxidant capacity and increased inflammatory signal, evident by elevation of ERK1/2 in both serum and GC muscles that coincided with increases in VEGFA, HIF-1α and CD34+ cells of GC muscles. CLI resulted in structural damage of GC muscle fibers, with marked apoptosis, declined proliferation and deteriorated peripheral limb function. Treatment with Fasudil restored antioxidant capacity and attenuated VEGFA, HIF-1α, CD34+ cells and inflammatory markers in ischemic limbs. Furthermore, Fasudil preserved histological integrity of ischemic GC muscles, with amelioration of apoptosis, preserved proliferation rate and improvement in peripheral limb function. SignificanceFasudil could protect against experimentally induced unilateral CLI, in a dose-dependent manner, which could pave the way for future clinical application of Fasudil in patients suffering PAD.

Emerging roles of centrosome cohesion.

The centrosome, consisting of centrioles and the associated pericentriolar material, is the main microtubule-organizing centre (MTOC) in animal cells. During most of interphase, the two centrosomes of a cell are joined together by centrosome cohesion into one MTOC. The most dominant element of centrosome cohesion is the centrosome linker, an interdigitating, fibrous network formed by the protein C-Nap1 anchoring a number of coiled-coil proteins including rootletin to the proximal end of centrioles. Alternatively, centrosomes can be kept together by the action of the minus end directed kinesin motor protein KIFC3 that works on interdigitating microtubules organized by both centrosomes and probably by the actin network. Although cells connect the two interphase centrosomes by several mechanisms into one MTOC, the general importance of centrosome cohesion, particularly for an organism, is still largely unclear. In this article, we review the functions of the centrosome linker and discuss how centrosome cohesion defects can lead to diseases.

The coiled-coil protein gene WPRb confers recessive resistance to Cucumber green mottle mosaic virus.

Cucumber green mottle mosaic virus (CGMMV) is one of the major global quarantine viruses and causes severe symptoms in Cucurbit crops, particularly with regard to fruit decay. However, the genetic mechanisms that control plant resistance to CGMMV have yet to be elucidated. Here, we found that WPRb, a weak chloroplast movement under blue light 1 and plastid movement impaired 2-related protein family gene, is recessively associated with CGMMV resistance in watermelon (Citrullus lanatus). We developed a reproducible marker based on a single non-synonymous substitution (G1282A) in WPRb, which can be used for marker-assisted selection for CGMMV resistance in watermelon. Editing of WPRb conferred greater tolerance to CGMMV. We found WPRb targets to the plasmodesmata (PD) and biochemically interacts with the CGMMV movement protein, facilitating viral intercellular movement by affecting the permeability of PD. Our findings enable us to genetically control CGMMV resistance in planta by using precise genome editing techniques targeted to WPRb.

Novel compound heterozygous CCDC40 mutations in a familial case of primary ciliary dyskinesia.

Primary ciliary dyskinesia (PCD) is a rare genetic disorder characterized by motile ciliary dysfunction and impaired ultrastructure. Despite numerous studies, the genetic basis for about 30% of PCD cases remains to be elucidated. Here, we present the identification and functional analysis of two novel mutations in the gene encoding coiled-coil domain-containing protein 40 (CCDC40), which are found in a familial case of PCD. These novel CCDC40 mutations, NM_017950.4: c.2236-2delA and c.2042_2046delTCACA, NP_060420.2: p.(Ile681fs), were identified by whole-exome sequencing (WES). Sanger sequencing was then performed to confirm the WES results and determine the CCDC40 gene sequences of the proband’s parents. The c.2042_2046delTCACA mutation disrupts the reading frame of the protein and is therefore predicted to produce a non-functional protein. Using a minigene assay with the pcDNA3.1(+) plasmid, we further investigated the potential pathogenic effects of the c.2236-2delA mutation and found that this mutation leads to formation of a truncated protein via splicing disruption. Thus, in summary, we identified two mutations of the CCDC40 gene that can be considered pathogenic compound heterozygous mutations in a case of familial PCD, thereby expanding the known mutational spectrum of the CCDC40 gene in this disease.

POLLEN WALL ABORTION 1 is essential for pollen wall development in rice.

The integrity of pollen wall structures is essential for pollen development and maturity in rice (Oryza sativa L.). In this study, we isolated and characterized the rice male-sterile mutant pollen wall abortion 1 (pwa1), which exhibits a defective pollen wall (DPW) structure and has sterile pollen. Map-based cloning, genetic complementation, and gene knockout experiments revealed that PWA1 corresponds to the gene LOC_Os01g55094 encoding a coiled-coil domain-containing protein. PWA1 localized to the nucleus, and PWA1 was expressed in the tapetum and microspores. PWA1 interacted with the transcription factor TAPETUM DEGENERATION RETARDATION (TDR)-INTERACTING PROTEIN2 (TIP2, also named bHLH142) in vivo and in vitro. The tip2-1 mutant, which we obtained by clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9-mediated gene editing, showed delayed tapetum degradation, sterile pollen, and DPWs. We determined that TIP2/bHLH142 regulates PWA1 expression by binding to its promoter. Analysis of the phenotype of the tip2-1 pwa1 double mutant indicated that TIP2/bHLH142 functions upstream of PWA1. Further studies suggested that PWA1 has transcriptional activation activity and participates in pollen intine development through the β-glucosidase Os12BGlu38. Therefore, we identified a sterility factor, PWA1, and uncovered a regulatory network underlying the formation of the pollen wall and mature pollen in rice.

MNG-tagged fusion proteins and nanobodies to visualize tropomyosins in yeast and mammalian cells.

Tropomyosins are structurally conserved α-helical coiled-coil proteins that bind along the length of filamentous actin (F-actin) in fungi and animals. Tropomyosins play essential roles in the stability of actin filaments and in regulating myosin II contractility. Despite the crucial role of tropomyosin in actin cytoskeletal regulation, in vivo investigations of tropomyosin are limited, mainly due to the suboptimal live-cell imaging tools currently available. Here, we report on an mNeonGreen (mNG)-tagged tropomyosin, with native promoter and linker length configuration, that clearly reports tropomyosin dynamics in Schizosaccharomyces pombe (Cdc8), Schizosaccharomyces japonicus (Cdc8) and Saccharomyces cerevisiae (Tpm1 and Tpm2). We also describe a fluorescent probe to visualize mammalian tropomyosin (TPM2 isoform). Finally, we generated a camelid nanobody against S. pombe Cdc8, which mimics the localization of mNG-Cdc8 in vivo. Using these tools, we report the presence of tropomyosin in previously unappreciated patch-like structures in fission and budding yeasts, show flow of tropomyosin (F-actin) cables to the cytokinetic actomyosin ring and identify rearrangements of the actin cytoskeleton during mating. These powerful tools and strategies will aid better analyses of tropomyosin and F-actin cables in vivo.

Implication of non-coding RNA-mediated ROCK1 regulation in various diseases.

Rho Associated Coiled-Coil Containing Protein Kinase 1 (ROCK1) is a protein serine/threonine kinase which is activated upon binding with the GTP-bound form of Rho. This protein can modulate actin-myosin contraction and stability. Moreover, it has a crucial role in the regulation of cell polarity. Therefore, it participates in modulation of cell morphology, regulation of expression of genes, cell proliferation and differentiation, apoptotic processes as well as oncogenic processes. Recent studies have highlighted interactions between ROCK1 and several non-coding RNAs, namely microRNAs, circular RNAs and long non-coding RNAs. Such interactions can be a target of medications. In fact, it seems that the interactions are implicated in therapeutic response to several medications. In the current review, we aimed to explain the impact of these interactions in the pathoetiology of cancers as well as non-malignant disorders.

In vivo characterization of Drosophila golgins reveals redundancy and plasticity of vesicle capture at the Golgi apparatus

The Golgi is the central sorting station in the secretory pathway and thus the destination of transport vesicles arriving from the endoplasmic reticulum and endosomes and from within the Golgi itself. Cell viability, therefore, requires that the Golgi accurately receives multiple classes of vesicle. One set of proteins proposed to direct vesicle arrival at the Golgi are the golgins, long coiled-coil proteins localized to specific parts of the Golgi stack. In mammalian cells, three of the golgins, TMF, golgin-84, and GMAP-210, can capture intra-Golgi transport vesicles when placed in an ectopic location. However, the individual golgins are not required for cell viability, and mouse knockout mutants only have defects in specific tissues. To further illuminate this system, we examine the Drosophila orthologs of these three intra-Golgi golgins. We show that ectopic forms can capture intra-Golgi transport vesicles, but strikingly, the cargo present in the vesicles captured by each golgin varies between tissues. Loss-of-function mutants show that the golgins are individually dispensable, although the loss of TMF recapitulates the male fertility defects observed in mice. However, the deletion of multiple golgins results in defects in glycosylation and loss of viability. Examining the vesicles captured by a particular golgin when another golgin is missing reveals that the vesicle content in one tissue changes to resemble that of a different tissue. This reveals a plasticity in Golgi organization between tissues, providing an explanation for why the Golgi is sufficiently robust to tolerate the loss of many of the individual components of its membrane traffic machinery.

Two New Variants in FYCO1 Are Responsible for Autosomal Recessive Congenital Cataract in Iranian Population.

The purpose of this experimental study was to investigate the genetic etiology of congenital cataract (CC) manifesting an autosomal recessive pattern of inheritance in four Iranian families. Affected individuals and their normal first-degree relatives in each family were included in the present study. The genomic DNA of the blood samples was extracted from all participants, and one affected member belonging to each family was subjected to Whole Exome Sequencing (WES). Using bidirectional Sanger sequencing, the identified variants were validated by co-segregation analysis. Two different mutations were detected in the FYCO1 gene encoding FYVE and coiled-coil domain-containing protein. A previously reported missense mutation, c.265C>T (p.Arg89Cys), was found in one Iranian family for the first time, and a combination of two variants in a single codon, c.[265C>T;267C>A] (p.Arg89X), was identified in the three other families. On the other hand, accompanying the c.265C>T mutation, the presence of the c.267C>A polymorphism leads to a premature stop codon. In-Silico Analysis of FYCO1 protein demonstrated that RUN domain will be interrupted so that the large part of functional protein will be eliminated due to this novel variant. FYCO1 has been proved to be involved in human lens development and transparency. Its mutations, therefore, result in CC. Herein, we reported the first autosomal recessive CC patients with c.265C>T (p.Arg89Cys) or c.[265C>T;267C>A] variant in Iranian population for the FYCO1 gene. FYCO1 mutations could be tracked for preventive objectives or even be targeted as therapeutic candidates via treatment approaches in the future.

Abnormal TACC3 Expression is an Independent Prognostic Biomarker in Lung Carcinoma.

Abnormal expression of transforming acidic coiled-coil protein 3 (TACC3) has been reported in many types of human malignancies. However, the expression of TACC3 and its clinical significance have not been well characterized in lung carcinoma (LUAD). The aim of this study was to investigate possible associations between tumor expression of TACC3 and the clinicopathological characteristics and prognosis of LUAD patients. The expression of TACC3 in LUAD patients was determined using the Oncomine, Gene Expression Profiling Interactive Analysis (GEPIA), UALCAN, and Cancer Genome Atlas (TCGA) databases. The expression of TACC3 in LUAD tissues was also determined by qRT-PCR. TACC3 was found to be significantly overexpressed in LUAD tumors compared with non-tumor tissue in the above public databases. Receiver operating characteristic (ROC) curve analysis indicated that TACC3 could have diagnostic value in LUAD patients. Kaplan-Meier analysis further indicated that high TACC3 expression in tumors was significantly associated with worse overall survival (OS) in LUAD patients. In addition, univariate and multivariate Cox regression analyses showed that high TACC3 expression was an independent factor for worse OS in LUAD patients. Finally, based on gene set enrichment analysis (GSEA 3.0), we identified several potential pathways related to TACC3 that were enriched in the high TACC3 expression phenotype. The present study provides evidence that TACC3 expression is upregulated in LUAD and may be an independent risk factor for worse prognosis in these patients.

Acute nitrite exposure-induced oxidative damage, endoplasmic reticulum stress, autophagy and apoptosis caused gill tissue damage of grass carp (Ctenopharyngodon idella): Relieved by dietary protein

Nitrite poses a serious threat to intensive aquaculture. Protein, as a major nutrient in animals, is vital for protecting animal tissues from damage. In this study, we investigated the protective effect of dietary protein on gill tissue structure and the underlying mechanisms in sub-adult grass carp (Ctenopharyngodon idella) exposed to nitrite stress. Six iso-energetic semi-purified diets containing different protein levels (16–31 %) were formulated, and fed to fish for 60 d. The fish were then exposed to a nitrite solution for 4 d. Histopathological observation and determination of related indices (serum glucose, serum cortisol, nitric oxide, peroxynitrite, reactive oxygen species, malondialdehyde, and protein carbonyl) showed that 22–25 % dietary protein significantly alleviated the nitrite-induced stress response, gill tissue damage and oxidative damage. Further research found that a suitable dietary protein suppressed the nitrite-induced endoplasmic reticulum stress (ERS) 78 kDa glucose-regulated protein (GRP78) related signaling pathway which possibly activated autophagy and apoptosis. Interestingly, we discovered that proper dietary protein reduced autophagy, probably through unc-51-like kinase 1 (Ulk1), BCL-2-interacting myosin-like coiled-coil protein (Beclin1), autophagy-related gene 5 (Atg5), Atg12, microtubule-associated protein1 light chain 3 (LC3), BCL-2 interacting protein 3 (BNIP3) and autophagy receptor P62 (p62). We also found that an appropriate dietary protein inhibited nitrite-induced apoptosis via mitochondrial and death receptor pathways. In summary, our findings are the first to demonstrate that 22–25 % of dietary protein levels can play a protective role against nitrite-induced gill injury.

Characterization of an experimental model to determine streptococcal M protein–induced autoimmune cardiac and neurobehavioral abnormalities

Group A streptococcal (GAS) infection is associated with a spectrum of autoimmune diseases including acute rheumatic fever/rheumatic heart disease (ARF/RHD) and neurobehavioral abnormalities. Antibodies against GAS M proteins cross‐react with host tissue proteins in the heart and brain leading to the symptomatology observed in ARF/RHD. As throat carriage of Streptococcus dysgalactiae subspecies equisimilis (SDSE) has been reported to be relatively high in some ARF/RHD endemic regions compared with GAS, and both SDSE and GAS express coiled‐coil surface protein called M protein, we hypothesized that streptococci other than GAS can also associated with ARF/RHD and neurobehavioral abnormalities. Neurobehavioral assessments and electrocardiography were performed on Lewis rats before and after exposure to recombinant GAS and SDSE M proteins. Histological assessments were performed to confirm inflammatory changes in cardiac and neuronal tissues. ELISA and Western blot analysis were performed to determine the cross‐reactivity of antibodies with host connective, cardiac and neuronal tissue proteins. Lewis rats injected with M proteins either from GAS or SDSE developed significant cardiac functional and neurobehavioral abnormalities in comparison to control rats injected with phosphate‐buffered saline. Antibodies against GAS and SDSE M proteins cross‐reacted with cardiac, connective and neuronal proteins. Serum from rats injected with streptococcal antigens showed higher immunoglobulin G binding to the striatum and cortex of the brain. Cardiac and neurobehavioral abnormalities observed in our experimental model were comparable to the cardinal symptoms observed in patients with ARF/RHD. Here for the first time, we demonstrate in an experimental model that M proteins from different streptococcal species could initiate and drive the autoimmune‐mediated cardiac tissue damage and neurobehavioral abnormalities.

Exercise-derived peptide protects against pathological cardiac remodeling.

SummaryBackgroundExercise training protects the heart against pathological cardiac remodeling and confers cardioprotection from heart failure. However, the underlying mechanism is still elusive.MethodsAn integrative analysis of multi-omics data of the skeletal muscle in response to exercise is performed to search for potential exerkine. Then, CCDC80tide is examined in humans after acute exercise. The role of CCDC80tide is assessed in a mouse model of hypertensive cardiac remodeling and in hypertension-mediated cell injury models. The transcriptomic analysis and immunoprecipitation assay are conducted to explore the mechanism.FindingsThe coiled-coil domain-containing protein 80 (CCDC80) is found strongly positively associated with exercise. Interestingly, exercise stimuli induce the secretion of C-terminal CCDC80 (referred as CCDC80tide hereafter) via EVs-encapsulated CCDC80tide into the circulation. Importantly, cardiac-specific expression of CCDC80tide protects against angiotensin II (Ang II)-induced cardiac hypertrophy and fibrosis in mice. In in vitro studies, the expression of CCDC80tide reduces Ang II-induced cardiomyocyte hypertrophy, cardiac microvascular endothelial cell (CMEC) inflammation, and mitigated vascular smooth muscle cell (VSMC) proliferation and collagen formation. To understand the cardioprotective effect of CCDC80tide, a transcriptomic analysis reveals a dramatic inhibition of the STAT3 (Signal transducer and activator of transcription 3) signaling pathway in CCDC80tide overexpressing cells. Mechanistically, CCDC80tide selectively interacts with the kinase-active form of JAK2 (Janus kinase 2) and consequently inhibits its kinase activity to phosphorylate and activate STAT3.InterpretationThe results provide new insights into exercise-afforded cardioprotection in pathological cardiac remodeling and highlight the therapeutic potential of CCDC80tide in heart failure treatment.FundingThis work was supported by the National Natural Science Foundation of China [Grant/Award Numbers: 81770428, 81830010, 82130012, 81900438, 82100447); Shanghai Science and Technology Committee [Grant/Award Numbers: 21S11903000, 19JC1415702]; Emerging and Advanced Technology Programs of Hospital Development Center of Shanghai [Grant/Award Number: SHDC12018129]; China Postdoctoral Science Foundation [2021M692108]; and China National Postdoctoral Program for Innovative Talents [BX20200211].

Revealing the species-specific genotype of the edible bird’s nest-producing swiftlet, Aerodramus fuciphagus and the proteome of edible bird’s nest

Only a few species of swiftlets in the Aerodramus and Collocalia genera can produce edible bird's nests (EBN). These saliva-cemented nests have been consumed as delicacies for centuries in Asia. Many researches have reported the aqueous extract of EBN has epidermal growth factor-like (EGF-like) activity. However, no standalone EGF has been identified in EBN. Moreover, proteome of EBN remained unclear due to lack of genomic data base of an EBN-producing swiftlet to support proteomic analysis of EBN. To address this, the first genome of the EBN-producing swiftlet, Aerodramus fuciphagus, was constructed. Orthology comparison of A. fuciphagus with 10 other avian species were conducted. The results revealed that the number of predicted paralogous coiled-coil domain-containing protein 63 (CCDC63) coding sequences (CDSs) in A. fuciphagus was found to be significantly expanded in comparison to Gallus gallus. There were 3 paralogous CCDC63 genes in the genome of A.fuciphagus. The CDSs predicted from the genome of A. fuciphagus were used to construct a database for proteomic analysis of EBN. In total, 398 proteins have been identified in EBN. The proteome of EBN was significant enriched with extracellular proteins as well as proteins related to extracellular matrix (ECM) organization and immune response. A few proteins with Ca2+-binding EGF-like domains were found in the proteome of EBN, like fibrillin-1, protocadherin fat 4 and coagulation factor X. No standalone EGF protein was identified. This indicated that the proteins with EGF-like domains might be responsible for the EGF-like activity of EBN. In addition, acidic mammalian chitinase and lysyl oxidase in EBN were found to be active when extracting with distilled water at room temperature. The current study has not just revealed the species-specific genotype of the EBN-producing swiftlet, A. fuciphagus, but also revealed the proteome of EBN. This established an important foundation for subsequently studies on efficacies of EBN.

Female meiosis II and pronuclear fusion require the microtubule transport factor Bicaudal D.

Bicaudal D (BicD) is a dynein adaptor that transports different cargoes along microtubules. Reducing the activity of BicD specifically in freshly laid Drosophila eggs by acute protein degradation revealed that BicD is needed to produce normal female meiosis II products, to prevent female meiotic products from re-entering the cell cycle, and for pronuclear fusion. Given that BicD is required to localize the spindle assembly checkpoint (SAC) components Mad2 and BubR1 to the female meiotic products, it appears that BicD functions to localize these components to control metaphase arrest of polar bodies. BicD interacts with Clathrin heavy chain (Chc), and both proteins localize to centrosomes, mitotic spindles and the tandem spindles during female meiosis II. Furthermore, BicD is required to localize clathrin and the microtubule-stabilizing factors transforming acidic coiled-coil protein (D-TACC/Tacc) and Mini spindles (Msps) correctly to the meiosis II spindles, suggesting that failure to localize these proteins may perturb SAC function. Furthermore, immediately after the establishment of the female pronucleus, D-TACC and Caenorhabditis elegans BicD, tacc and Chc are also needed for pronuclear fusion, suggesting that the underlying mechanism might be more widely used across species.

The beginning and the end of SNARE-induced membrane fusion.

Membrane fusion is not a spontaneous process. Physiologically, the formation of coiled-coil protein complexes, the SNAREpins, bridges the membrane of a vesicle and a target membrane, brings them in close contact, and provides the energy necessary for their fusion. In this review, we utilize results from invitro experiments and simple physics and chemistry models to dissect the kinetics and energetics of the fusion process from the encounter of the two membranes to the full expansion of a fusion pore. We find three main energy barriers that oppose the fusion process: SNAREpin initiation, fusion pore opening, and expansion. SNAREpin initiation is inherent to the proteins and makes invitro fusion kinetic experiments rather slow. The kinetics are physiologically accelerated by effectors. The energy barriers that precede pore opening and pore expansion can be overcome by several SNAREpins acting in concert.

Circ_0004712 promotes endometriotic epithelial cell proliferation, migration and invasion by regulating miR-488-3p/ROCK1 axis in vitro

Recent evidence indicates that circular RNAs (circRNAs) play crucial regulatory roles in the pathogenesis and development of endometriosis. Circ_0004712 was found to be differentially expressed in endometriosis. However, the detailed function and mechanism of circ_0004712 in endometriosis are still unclear. Quantitative real-time polymerase chain reaction and Western blot were used for the detection of circ_0004712, miR-488-3p and ROCK1 (Rho Associated Coiled-Coil Containing Protein Kinase 1) levels. In vitro experiments in endometrial endothelial cells were performed by cell counting kit-8, EdU, transwell, wound healing assays, and flow cytometry, respectively. The molecular mechanism of circ_0004712 function was investigated using bioinformatics target predication, dual-luciferase reporter, and RNA immunoprecipitation (RIP) assays. The expression of circ_0004712 was higher in endometriotic endometrial tissues and epithelial cells. Knockdown of circ_0004712 suppressed cell proliferation, migration, invasion, EMT process and induced apoptosis in ectopic endometrial epithelial cells in vitro. Mechanistically, circ_0004712 acted as a ceRNA to sponge miR-488-3p, thus elevating the expression of ROCK1, which was confirmed to be a target of miR-488-3p. Rescue experiments suggested that miR-488-3p inhibition reversed the inhibitory effects of circ_0004712 silencing on cell growth and metastasis. Moreover, miR-488-3p restoration restrained the proliferation and metastasis in ectopic endometrial epithelial cells, which were attenuated by ROCK1 overexpression. Circ_0004712 knockdown suppressed the proliferation and metastasis of ectopic endometrial epithelial cells via miR-488-3p/ROCK1 axis in vitro, suggesting a new insight into the pathogenesis of endometriosis.

Deficiency of proline/serine-rich coiled-coil protein 1 (PSRC1) accelerates trimethylamine N-oxide-induced atherosclerosis in ApoE−/− mice

AimsThe main therapeutic strategies for coronary artery disease (CAD) are mainly based on the correction of abnormal cholesterol levels; however, residual risks remain. The newly proven gut microbial metabolite trimethylamine N-oxide (TMAO) linked with CAD has broadened our horizons. In this study, we determined the role of proline/serine-rich coiled-coil protein 1 (PSRC1) in TMAO-driven atherosclerosis. Methods and resultsWe first analyzed the levels of TMAO and PSRC1 in patients with or without atherosclerosis with a target LDL-C < 1.8 mmol/L. Plasma TMAO levels were increased and negatively associated with decreased PSRC1 in peripheral blood mononuclear cells. Animals and in vitro studies showed that TMAO inhibited macrophage PSRC1 expression due to DNA hypermethylation of CpG islands. ApoE−/− mice fed a choline-supplemented diet exhibited reduced PSRC1 expression accompanied by increased atherosclerotic lesions and plasma TMAO levels. We further deleted PSRC1 in apoE−/− mice and PSRC1 deficiency significantly accelerated choline-induced atherogenesis, characterized by increased macrophage infiltration, foam cell formation and M1 macrophage polarization. Mechanistically, we overexpressed and knocked out PSRC1 in cultured macrophages to explore the mechanisms underlying TMAO-induced cholesterol accumulation and inflammation. PSRC1 deletion impaired reverse cholesterol transport and enhanced cholesterol uptake and inflammation, while PSRC1 overexpression rescued the proatherogenic phenotype observed in TMAO-stimulated macrophages, which was partially attributed to sulfotransferase 2B1b (SULT2B1b) inhibition. ConclusionsHerein, clinical data provide evidence that TMAO may participate in the development of CAD beyond well-controlled LDL-C levels. Our work also suggests that PSRC1 is a negative regulator mediating the unfavorable effects of TMAO-containing diets. Therefore, PSRC1 overexpression and reduced choline consumption may further alleviate atherosclerosis.

Generation and characterization of Ccdc28b mutant mice links the Bardet-Biedl associated gene with mild social behavioral phenotypes.

CCDC28B (coiled-coil domain-containing protein 28B) was identified as a modifier in the ciliopathy Bardet-Biedl syndrome (BBS). Our previous work in cells and zebrafish showed that CCDC28B plays a role regulating cilia length in a mechanism that is not completely understood. Here we report the generation of a Ccdc28b mutant mouse using CRISPR/Cas9 (Ccdc28b mut). Depletion of CCDC28B resulted in a mild phenotype. Ccdc28b mut animals i) do not present clear structural cilia affectation, although we did observe mild defects in cilia density and cilia length in some tissues, ii) reproduce normally, and iii) do not develop retinal degeneration or obesity, two hallmark features of reported BBS murine models. In contrast, Ccdc28b mut mice did show clear social interaction defects as well as stereotypical behaviors. This finding is indeed relevant regarding CCDC28B as a modifier of BBS since behavioral phenotypes have been documented in BBS. Overall, this work reports a novel mouse model that will be key to continue evaluating genetic interactions in BBS, deciphering the contribution of CCDC28B to modulate the presentation of BBS phenotypes. In addition, our data underscores a novel link between CCDC28B and behavioral defects, providing a novel opportunity to further our understanding of the genetic, cellular, and molecular basis of these complex phenotypes.

MTCL2 promotes asymmetric microtubule organization by crosslinking microtubules on the Golgi membrane.

The Golgi complex plays an active role in organizing asymmetric microtubule arrays, which are essential for polarized vesicle transport. The coiled-coil protein MTCL1 stabilizes microtubules nucleated from the Golgi membrane. Here, we report an MTCL1 paralog, MTCL2, which preferentially acts on the perinuclear microtubules accumulated around the Golgi. MTCL2 associates with the Golgi membrane through the N-terminal coiled-coil region and directly binds microtubules through the conserved C-terminal domain without promoting microtubule stabilization. Knockdown of MTCL2 significantly impaired microtubule accumulation around the Golgi, as well as the compactness of the Golgi ribbon assembly structure. Given that MTCL2 forms parallel oligomers through homo-interaction of the central coiled-coil motifs, our results indicate that MTCL2 promotes asymmetric microtubule organization by crosslinking microtubules on the Golgi membrane. Results of in vitro wound healing assays further suggest that this function of MTCL2 enables integration of the centrosomal and Golgi-associated microtubules on the Golgi membrane, supporting directional migration. Additionally, the results demonstrated the involvement of CLASPs and giantin in mediating the Golgi association of MTCL2.