BBS8蛋白参与衣藻鞭毛膜蛋白的运输

真核细胞的纤毛(也称鞭毛)是一种突出于细胞表面的极性细胞器,纤毛不仅参与细胞运动,还参与信号传导等过程,其结构或功能异常引发的一系列人类疾病称为"纤毛相关性疾病"。纤毛相关性疾病巴德-毕德氏综合征(Bardet-Biedl syndrome,简称BBS)由BBS相关基因缺陷导致,为了研究致病基因BBS8的生理作用和功能,构建模式生物莱茵衣藻BBS8基因缺陷突变体,利用性状观测和生化分析检测突变体的表现型和生理功能。免疫荧光表明BBS8蛋白是一种鞭毛蛋白且在基体有特异性定位;bbs8突变体感光极性运动消失,并在解聚诱导实验中鞭毛解聚缓慢;鞭毛的银染和质谱结果表明突变体的鞭毛膜蛋白在鞭毛内异常积累。文中通过实验证据说明BBS8蛋白在参与鞭毛内膜蛋白运输中起到重要作用,并极可能通过介导膜蛋白反向运输发挥生理功能。     

莱茵衣藻BBS8蛋白的原核表达、纯化及多克隆抗体的制备

为制备一支高特异性的莱茵衣藻BBS8兔源多克隆抗体, 研究首先在大肠杆菌中表达N-端6×His标签标记的BBS8融合蛋白(6×His::BBS8)并对其进行镍柱纯化, 而后将纯化所得6×His::BBS8蛋白免疫新西兰大白兔。免疫3次后采集少量抗血清, 利用间接ELISA法测定其效价为1﹕102400。然后, 利用protein A纯化珠对所得BBS8抗血清进行IgG亚型抗体富集, 接着利用大肠杆菌表达和纯化所得N-端MBP标签标记的BBS8(MBP::BBS8)对IgG抗血清进行抗原抗体亲和纯化。利用纯化后的anti-BBS8多克隆抗体对莱茵衣藻野生型CC-125和bbs8突变体藻种的全细胞蛋白提取物进行免疫印迹鉴定, 所得anti-BBS8多克隆抗体特异性较高, 适合用于后续莱茵衣藻BBS8蛋白功能的研究。     

Intrinsic protein-protein interaction-mediated and chaperonin-assisted sequential assembly of stable bardet-biedl syndrome protein complex, the BBSome

The pleiotropic features of obesity, retinal degeneration, polydactyly, kidney abnormalities, cognitive impairment, hypertension, and diabetes found in Bardet-Biedl syndrome (BBS) make this disorder an important model disorder for identifying molecular mechanisms involved in common human diseases. To date, 16 BBS genes have been reported, seven of which (BBS1, 2, 4, 5, 7, 8, and 9) code for proteins that form a complex known as the BBSome. The function of the BBSome involves ciliary membrane biogenesis. Three additional BBS genes (BBS6, BBS10, and BBS12) have homology to type II chaperonins and interact with CCT/TRiC proteins and BBS7 to form a complex termed the BBS-chaperonin complex. This complex is required for BBSome assembly. Little is known about the process and the regulation of BBSome formation. We utilized point mutations and null alleles of BBS proteins to disrupt assembly of the BBSome leading to the accumulation of BBSome assembly intermediates. By characterizing BBSome assembly intermediates, we show that the BBS-chaperonin complex plays a role in BBS7 stability. BBS7 interacts with BBS2 and becomes part of a BBS7-BBS2-BBS9 assembly intermediate referred to as the BBSome core complex because it forms the core of the BBSome. BBS1, BBS5, BBS8, and finally BBS4 are added to the BBSome core to form the complete BBSome.     

Antenatal presentation of Bardet-Biedl syndrome may mimic Meckel syndrome

Bardet-Biedl syndrome (BBS) is a multisystemic disorder characterized by postaxial polydactyly, progressive retinal dystrophy, obesity, hypogonadism, renal dysfunction, and learning difficulty. Other manifestations include diabetes mellitus, heart disease, hepatic fibrosis, and neurological features. The condition is genetically heterogeneous, and eight genes (BBS1-BBS8) have been identified to date. A mutation of the BBS1 gene on chromosome 11q13 is observed in 30%-40% of BBS cases. In addition, a complex triallelic inheritance has been established in this disorder--that is, in some families, three mutations at two BBS loci are necessary for the disease to be expressed. The clinical features of BBS that can be observed at birth are polydactyly, kidney anomaly, hepatic fibrosis, and genital and heart malformations. Interestingly, polydactyly, cystic kidneys, and liver anomalies (hepatic fibrosis with bile-duct proliferation) are also observed in Meckel syndrome, along with occipital encephalocele. Therefore, we decided to sequence the eight BBS genes in a series of 13 antenatal cases presenting with cystic kidneys and polydactyly and/or hepatic fibrosis but no encephalocele. These fetuses were mostly diagnosed as having Meckel or "Meckel-like" syndrome. In six cases, we identified a recessive mutation in a BBS gene (three in BBS2, two in BBS4, and one in BBS6). We found a heterozygous BBS6 mutation in three additional cases. No BBS1, BBS3, BBS5, BBS7, or BBS8 mutations were identified in our series. These results suggest that the antenatal presentation of BBS may mimic Meckel syndrome.     

The First Nationwide Survey and Genetic Analyses of Bardet-Biedl Syndrome in Japan

Bardet-Biedl syndrome (BBS) is an autosomal recessive disorder characterized by central obesity, mental impairment, rod-cone dystrophy, polydactyly, hypogonadism in males, and renal abnormalities. The causative genes have been identified as BBS1-19. In Western countries, this disease is often reported, but remains undiagnosed in many patients until later in life, while only a few patients with no mutations identified have been reported in Japan. We thus conducted the first nationwide survey of BBS in Japan by sending questionnaires to 2,166 clinical departments with board-certified specialists and found 7 patients with clinically definite BBS. We performed exome analyses combined with analyses of mRNA and protein in these patients. We identified 2 novel mutations in the BBS5 gene (p.R89X and IVS7-27 T>G) in 2 sibling patients. The latter mutation that resided far from the authentic splicing site was associated with skipping of exon 8. We also found 3 previously reported mutations in the BBS2 (p.R413X and p.R480X) and BBS7 (p.C243Y) genes in 2 patients. To our knowledge, a nationwide survey of BBS has not been reported in any other country. In addition, this is the first study to identify genetic alterations in Japanese patients with BBS. Our results indicate that BBS in Japan is genetically heterogeneous and at least partly shares genetic features with BBS in other countries.     

Genetic and mutational analyses of a large multiethnic Bardet-Biedl cohort reveal a minor involvement of BBS6 and delineate the critical intervals of other loci

Bardet-Biedl syndrome (BBS) is a rare autosomal recessive disorder characterized primarily by obesity, polydactyly, retinal dystrophy, and renal disease. The significant genetic and clinical heterogeneity of this condition have substantially hindered efforts to positionally clone the numerous BBS genes, because the majority of available pedigrees are small and the disorder cannot be assigned to any of the six known BBS loci. Consequently, the delineation of critical BBS intervals, which would accelerate the discovery of the underlying genetic defect(s), becomes difficult, especially for loci with minor contributions to the syndrome. We have collected a cohort of 163 pedigrees from diverse ethnic backgrounds and have evaluated them for mutations in the recently discovered BBS6 gene (MKKS) on chromosome 20 and for potential assignment of the disorder to any of the other known BBS loci in the human genome. Using a combination of mutational and haplotype analysis, we describe the spectrum of BBS6 alterations that are likely to be pathogenic; propose substantially reduced critical intervals for BBS2, BBS3, and BBS5; and present evidence for the existence of at least one more BBS locus. Our data also suggest that BBS6 is a minor contributor to the syndrome and that some BBS6 alleles may act in conjunction with mutations at other BBS loci to cause or modify the BBS phenotype.     

Cloning and characterization of a splice variant of human Bardet-Biedl syndrome 4 gene (BBS4).

Bardet-Biedl syndrome (BBS) is a heterogeneous multisystemic disorder characterized primarily by five cardinal features of retinal degeneration, obesity, polydactyly, hypogenitalism and mental retardation. To date, six distinct BBS loci that have been identified on different chromosomes. BBS4 gene is mapped to 15q22.2-23, which when mutated can cause BBS4. Its protein shows strong homology to O-linked N-acetylglucosamine (O-GlcNAc) transferase. Here we report a splice variant of BBS4, which is 2556 bp in length and has an open reading frame coding a predicted 527 amino-acids protein. RT-PCR shows that the cDNA is widely expressed while it has higher expression levels in pancreas, liver and prostate.     

Novel interaction partners of Bardet-Biedl syndrome proteins

Bardet-Biedl syndrome (BBS) is a rare, developmental disorder characterized by six major symptoms: rod-cone dystrophy, obesity, polydactyly, renal abnormalities, learning difficulties, and hypogonadism. Secondary features include cardiac and hepatic anomalies, metabolic disturbancies, and hearing loss. BBS is genetically heterogeneous with 12 disease genes (BBS1-BBS12) described thus far. Current data suggest a functional disturbance in ciliary function and intraflagellar transport being associated with the phenotype. However, the precise functions of the BBS proteins have yet to be elucidated. This study focuses on the detection of protein factors interacting with BBS proteins. Applying yeast two-hybrid (Y2H) technology we found a series of novel, functionally potentially plausible binding partners of BBS1, BBS2, BBS4, and BBS7. Protein interactions were supported by coimmunoprecipitation analyses (ALDOB, EPAS1) and substantiated by colocalization studies at the subcellular level (ALDOB, EXOC7, FLOT1, KRT18, PAX2). Our work provides new insights into the understanding of BBS interactions and thus their biological function.     

A core complex of BBS proteins cooperates with the GTPase Rab8 to promote ciliary membrane biogenesis

Primary cilium dysfunction underlies the pathogenesis of Bardet-Biedl syndrome (BBS), a genetic disorder whose symptoms include obesity, retinal degeneration, and nephropathy. However, despite the identification of 12 BBS genes, the molecular basis of BBS remains elusive. Here we identify a complex composed of seven highly conserved BBS proteins. This complex, the BBSome, localizes to nonmembranous centriolar satellites in the cytoplasm but also to the membrane of the cilium. Interestingly, the BBSome is required for ciliogenesis but is dispensable for centriolar satellite function. This ciliogenic function is mediated in part by the Rab8 GDP/GTP exchange factor, which localizes to the basal body and contacts the BBSome. Strikingly, Rab8(GTP) enters the primary cilium and promotes extension of the ciliary membrane. Conversely, preventing Rab8(GTP) production blocks ciliation in cells and yields characteristic BBS phenotypes in zebrafish. Our data reveal that BBS may be caused by defects in vesicular transport to the cilium.     

Identification of a novel BBS gene (BBS12) highlights the major role of a vertebrate-specific branch of chaperonin-related proteins in Bardet-Biedl syndrome

Bardet-Biedl syndrome (BBS) is primarily an autosomal recessive ciliopathy characterized by progressive retinal degeneration, obesity, cognitive impairment, polydactyly, and kidney anomalies. The disorder is genetically heterogeneous, with 11 BBS genes identified to date, which account for ~70% of affected families. We have combined single-nucleotide-polymorphism array homozygosity mapping with in silico analysis to identify a new BBS gene, BBS12. Patients from two Gypsy families were homozygous and haploidentical in a 6-Mb region of chromosome 4q27. FLJ35630 was selected as a candidate gene, because it was predicted to encode a protein with similarity to members of the type II chaperonin superfamily, which includes BBS6 and BBS10. We found pathogenic mutations in both Gypsy families, as well as in 14 other families of various ethnic backgrounds, indicating that BBS12 accounts for approximately 5% of all BBS cases. BBS12 is vertebrate specific and, together with BBS6 and BBS10, defines a novel branch of the type II chaperonin superfamily. These three genes are characterized by unusually rapid evolution and are likely to perform ciliary functions specific to vertebrates that are important in the pathophysiology of the syndrome, and together they account for about one-third of the total BBS mutational load. Consistent with this notion, suppression of each family member in zebrafish yielded gastrulation-movement defects characteristic of other BBS morphants, whereas simultaneous suppression of all three members resulted in severely affected embryos, possibly hinting at partial functional redundancy within this protein family.     

The molecular genetics of Bardet-Biedl syndrome

Bardet-Biedl syndrome (BBS) has been shown to be a genetically heterogeneous disorder involving genes mapping to at least six known loci. One BBS gene (MKKS) has been identified and the form of the disorder caused by this gene is allelic to McKusick-Kaufman syndrome. MKKS codes for a putative chaperonin, suggesting that other BBS genes may also code for components of chaperone complexes or be substrates of chaperone function.     

Identification of a novel Bardet-Biedl syndrome protein,BBS7, that shares structural features with BBS1 and BBS2

Bardet-Biedl syndrome (BBS) is a genetically heterogeneous disorder, the primary features of which include obesity, retinal dystrophy, polydactyly, hypogenitalism, learning difficulties, and renal malformations. Conventional linkage and positional cloning have led to the mapping of six BBS loci in the human genome, four of which (BBS1, BBS2, BBS4, and BBS6) have been cloned. Despite these advances, the protein sequences of the known BBS genes have provided little or no insight into their function. To delineate functionally important regions in BBS2, we performed phylogenetic and genomic studies in which we used the human and zebrafish BBS2 peptide sequences to search dbEST and the translation of the draft human genome. We identified two novel genes that we initially named "BBS2L1" and "BBS2L2" and that exhibit modest similarity with two discrete, overlapping regions of BBS2. In the present study, we demonstrate that BBS2L1 mutations cause BBS, thereby defining a novel locus for this syndrome, BBS7, whereas BBS2L2 has been shown independently to be BBS1. The motif-based identification of a novel BBS locus has enabled us to define a potential functional domain that is present in three of the five known BBS proteins and, therefore, is likely to be important in the pathogenesis of this complex syndrome.     

The cloning and developmental expression of unconventional myosin IXA (MYO9A) a gene in the Bardet-Biedl syndrome (BBS4) region at chromosome 15q22-q23.

Bardet-Biedl Syndrome (BBS) is a heterogeneous, autosomal recessive disorder characterized by mental retardation, obesity, retinitis pigmentosa, syndactyly and/or polydactyly, short stature, and hypogenitalism and is caused by mutations at a number of distinct loci. Using a positional cloning approach for identifying the BBS4 (chromosome 15) gene, we identified and cloned an unconventional myosin gene, myosin IXA (HGMW-approved symbol MYO9A). Since mutations in unconventional myosins are known to cause several human diseases, and since mutations of unconventional myosin VIIa cause retinal degeneration, we evaluated myosin IXA as a candidate for BBS. We exploited PCR-based techniques to clone a 8473-nt cDNA for myosin IXA. A 7644-bp open reading frame predicts a protein with all the hallmarks of class IX unconventional myosins. Human Northern blot analysis and in situ hybridization of mouse embryos reveal that myosin IXA is expressed in many tissues consistent with BBS. Intron/exon boundaries were identified, and myosin IXA DNA and RNA from BBS4 patients were evaluated for mutation.     

Genetic interaction of BBS1 mutations with alleles at other BBS loci can result in non-Mendelian Bardet-Biedl syndrome

Bardet-Biedl syndrome is a genetically and clinically heterogeneous disorder caused by mutations in at least seven loci (BBS1-7), five of which are cloned (BBS1, BBS2, BBS4, BBS6, and BBS7). Genetic and mutational analyses have indicated that, in some families, a combination of three mutant alleles at two loci (triallelic inheritance) is necessary for pathogenesis. To date, four of the five known BBS loci have been implicated in this mode of oligogenic disease transmission. We present a comprehensive analysis of the spectrum, distribution, and involvement in non-Mendelian trait transmission of mutant alleles in BBS1, the most common BBS locus. Analyses of 259 independent families segregating a BBS phenotype indicate that BBS1 participates in complex inheritance and that, in different families, mutations in BBS1 can interact genetically with mutations at each of the other known BBS genes, as well as at unknown loci, to cause the phenotype. Consistent with this model, we identified homozygous M390R alleles, the most frequent BBS1 mutation, in asymptomatic individuals in two families. Moreover, our statistical analyses indicate that the prevalence of the M390R allele in the general population is consistent with an oligogenic rather than a recessive model of disease transmission. The distribution of BBS oligogenic alleles also indicates that all BBS loci might interact genetically with each other, but some genes, especially BBS2 and BBS6, are more likely to participate in triallelic inheritance, suggesting a variable ability of the BBS proteins to interact genetically with each other.     

Evaluation of complex inheritance involving the most common Bardet-Biedl syndrome locus (BBS1)

Bardet-Biedl syndrome (BBS) is a genetic disorder with the primary features of obesity, pigmentary retinopathy, polydactyly, renal malformations, mental retardation, and hypogenitalism. Patients with BBS are also at increased risk for diabetes mellitus, hypertension, and congenital heart disease. BBS is known to map to at least six loci: 11q13 (BBS1), 16q21 (BBS2), 3p13-p12 (BBS3), 15q22.3-q23 (BBS4), 2q31 (BBS5), and 20p12 (BBS6). Although these loci were all mapped on the basis of an autosomal recessive mode of inheritance, it has recently been suggested-on the basis of mutation analysis of the identified BBS2, BBS4, and BBS6 genes-that BBS displays a complex mode of inheritance in which, in some families, three mutations at two loci are necessary to manifest the disease phenotype. We recently identified BBS1, the gene most commonly involved in Bardet-Biedl syndrome. The identification of this gene allows for further evaluation of complex inheritance. In the present study we evaluate the involvement of the BBS1 gene in a cohort of 129 probands with BBS and report 10 novel BBS1 mutations. We demonstrate that a common BBS1 missense mutation accounts for approximately 80% of all BBS1 mutations and is found on a similar genetic background across populations. We show that the BBS1 gene is highly conserved between mice and humans. Finally, we demonstrate that BBS1 is inherited in an autosomal recessive manner and is rarely, if ever, involved in complex inheritance.     

BBS4 is a minor contributor to Bardet-Biedl syndrome and may also participate in triallelic inheritance

Bardet-Biedl syndrome (BBS) is an uncommon multisystemic disorder characterized primarily by retinal dystrophy, obesity, polydactyly, and renal dysfunction. BBS has been modeled historically as an autosomal recessive trait, under which premise six independent BBS loci (BBS1-BBS6) have been mapped in the human genome. However, extended mutational analyses of BBS2 and BBS6, the first two BBS genes cloned, suggest that BBS exhibits a more complex pattern of inheritance, in which three mutations at two loci simultaneously are necessary and sufficient in some families to manifest the phenotype. We evaluated the spectrum of mutations in the recently identified BBS4 gene with a combination of haplotype analysis and mutation screening on a multiethnic cohort of 177 families. Consistent with predictions from previous genetic analyses, our data suggest that mutations in BBS4 contribute to BBS in <3% of affected families. Furthermore, integrated mutational data from all three currently cloned BBS genes raise the possibility that BBS4 may participate in triallelic inheritance with BBS2 and BBS1, but not the other known loci. Establishment of the loci pairing in triallelism is likely to be important for the elucidation of the functional relationships among the different BBS proteins.     

Characterization of CCDC28B reveals its role in ciliogenesis and provides insight to understand its modifier effect on Bardet–Biedl syndrome

Bardet–Biedl syndrome (BBS) is a genetically heterogeneous disorder that is generally inherited in an autosomal recessive fashion. However, in some families, trans mutant alleles interact with the primary causal locus to modulate the penetrance and/or the expressivity of the phenotype. CCDC28B (MGC1203) was identified as a second site modifier of BBS encoding a protein of unknown function. Here we report the first functional characterization of this protein and show it affects ciliogenesis both in cultured cells and in vivo in zebrafish. Consistent with this biological role, our in silico analysis shows that the presence of CCDC28B homologous sequences is restricted to ciliated metazoa. Depletion of Ccdc28b in zebrafish results in defective ciliogenesis and consequently causes a number of phenotypes that are characteristic of BBS and other ciliopathy mutants including hydrocephalus, left–right axis determination defects and renal function impairment. Thus, this work reports CCDC28B as a novel protein involved in the process of ciliogenesis whilst providing functional insight into the cellular basis of its modifier effect in BBS patients.     

A founder effect in the newfoundland population reduces the Bardet-Biedl syndrome I (BBS1) interval to 1 cM

Bardet-Biedl syndrome (BBS) is a rare, autosomal recessive disorder; major phenotypic findings include dysmorphic extremities, retinal dystrophy, obesity, male hypogenitalism, and renal anomalies. In the majority of northern European families with BBS, the syndrome is linked to a 26-cM region on chromosome 11q13. However, the finding, so far, of five distinct BBS loci (BBS1, 1q; BBS2, 16q; BBS3, 3p; BBS4, 15q; BBS5, 2q) has hampered the positional cloning of these genes. We use linkage disequilibrium (LD) mapping in an isolated founder population in Newfoundland to significantly reduce the BBS1 critical region. Extensive haplotyping in several unrelated BBS families of English descent revealed that the affected members were homozygous for overlapping portions of a rare, disease-associated ancestral haplotype on chromosome 11q13. The LD data suggest that the BBS1 gene lies in a 1-Mb, sequence-ready region on chromosome 11q13, which should enable its identification.