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.     

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.     

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.     

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.     

The Chlamydomonas reinhardtii BBSome is an IFT cargo required for export of specific signaling proteins from flagella

In humans, seven evolutionarily conserved genes that cause the cilia-related disorder Bardet-Biedl syndrome (BBS) encode proteins that form a complex termed the BBSome. The function of the BBSome in the cilium is not well understood. We purified a BBSome-like complex from Chlamydomonas reinhardtii flagella and found that it contains at least BBS1, -4, -5, -7, and -8 and undergoes intraflagellar transport (IFT) in association with a subset of IFT particles. C. reinhardtii insertional mutants defective in BBS1, -4, and -7 assemble motile, full-length flagella but lack the ability to phototax. In the bbs4 mutant, the assembly and transport of IFT particles are unaffected, but the flagella abnormally accumulate several signaling proteins that may disrupt phototaxis. We conclude that the BBSome is carried by IFT but is an adapter rather than an integral component of the IFT machinery. C. reinhardtii BBS4 may be required for the export of signaling proteins from the flagellum via IFT.     

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.     

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。     

The centriolar satellite proteins Cep72 and Cep290 interact and are required for recruitment of BBS proteins to the cilium

Defects in centrosome and cilium function are associated with phenotypically related syndromes called ciliopathies. Centriolar satellites are centrosome-associated structures, defined by the protein PCM1, that are implicated in centrosomal protein trafficking. We identify Cep72 as a PCM1-interacting protein required for recruitment of the ciliopathy-associated protein Cep290 to centriolar satellites. Loss of centriolar satellites by depletion of PCM1 causes relocalization of Cep72 and Cep290 from satellites to the centrosome, suggesting that their association with centriolar satellites normally restricts their centrosomal localization. We identify interactions between PCM1, Cep72, and Cep290 and find that disruption of centriolar satellites by overexpression of Cep72 results in specific aggregation of these proteins and the BBSome component BBS4. During ciliogenesis, BBS4 relocalizes from centriolar satellites to the primary cilium. This relocalization occurs normally in the absence of centriolar satellites (PCM1 depletion) but is impaired by depletion of Cep290 or Cep72, resulting in defective ciliary recruitment of the BBSome subunit BBS8. We propose that Cep290 and Cep72 in centriolar satellites regulate the ciliary localization of BBS4, which in turn affects assembly and recruitment of the BBSome. Finally, we show that loss of centriolar satellites in zebrafish leads to phenotypes consistent with cilium dysfunction and analogous to those observed in human ciliopathies.     

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.     

The Centriolar Satellite Protein AZI1 Interacts with BBS4 and Regulates Ciliary Trafficking of the BBSome

Bardet-Biedl syndrome (BBS) is a well-known ciliopathy with mutations reported in 18 different genes. Most of the protein products of the BBS genes localize at or near the primary cilium and the centrosome. Near the centrosome, BBS proteins interact with centriolar satellite proteins, and the BBSome (a complex of seven BBS proteins) is believed to play a role in transporting ciliary membrane proteins. However, the precise mechanism by which BBSome ciliary trafficking activity is regulated is not fully understood. Here, we show that a centriolar satellite protein, AZI1 (also known as CEP131), interacts with the BBSome and regulates BBSome ciliary trafficking activity. Furthermore, we show that AZI1 interacts with the BBSome through BBS4. AZI1 is not involved in BBSome assembly, but accumulation of the BBSome in cilia is enhanced upon AZI1 depletion. Under conditions in which the BBSome does not normally enter cilia, such as in BBS3 or BBS5 depleted cells, knock down of AZI1 with siRNA restores BBSome trafficking to cilia. Finally, we show that azi1 knockdown in zebrafish embryos results in typical BBS phenotypes including Kupffer''s vesicle abnormalities and melanosome transport delay. These findings associate AZI1 with the BBS pathway. Our findings provide further insight into the regulation of BBSome ciliary trafficking and identify AZI1 as a novel BBS candidate gene.     

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.     

Bombesin stimulates prolactin secretion from cultured rat pituitary tumour cells (GH4C1) via activation of phospholipase C

Bombesin (BBS) stimulated prolactin (PRL) secretion from monolayer cultures of rat pituitary tumour cells (GH4C1) in a dose-dependent manner with half maximal and maximal effect at 2 nM and 100 nM, respectively. No additional stimulatory effect on PRL secretion was seen when BBS was combined with thyroliberin (TRH) used in concentrations known to give maximal effects, while the effects of BBS and vasoactive intestinal peptide (VIP) were additive. Using a parafusion system, BBS (1 microM) was found to increase PRL secretion within 4 s and the secretion profiles elicited by BBS and TRH (1 microM) were similar. Both BBS and TRH increased inositoltrisphosphate (IP3) as well as inositolbisphosphate (IP2) formation within 2 s. BBS also induced the same biphasic changes in the electrical membrane properties of GH4C1 cells as TRH, and both peptides caused a rapid and sustained increase in intracellular [Ca2+]. These results suggest that BBS stimulates PRL secretion from the GH4C1 cells via a mechanism involving the immediate formation of IP3 thus resembling the action of TRH.     

Establishing a connection between cilia and Bardet-Biedl Syndrome

Bardet-Biedl Syndrome (BBS) is a gentic disorder with primary features of retinal dystrophy, obesity, polydactyly, structural and functional renal abnormalities, and learning disabilities. In addition to displaying remarkable pleiotropy, BBS is a heterogeneous disorder with linkage to at least eight loci. The identification of the first five BBS genes provided little insight into BBS protein function. Ansley at al. have now identified a sixth BBS gene (BBS8) and provide evidence that the BBS8 protein and other BBS proteins localize to the basal body of ciliated cells, suggesting that BBS is a ciliary dysfunction disorder.     

ATG7(2) Interacts With Metabolic Proteins and Regulates Central Energy Metabolism

Macroautophagy/autophagy is an essential catabolic process that targets a wide variety of cellular components including proteins, organelles, and pathogens. ATG7, a protein involved in the autophagy process, plays a crucial role in maintaining cellular homeostasis and can contribute to the development of diseases such as cancer. ATG7 initiates autophagy by facilitating the lipidation of the ATG8 proteins in the growing autophagosome membrane. The noncanonical isoform ATG7(2) is unable to perform ATG8 lipidation; however, its cellular regulation and function are unknown. Here, we uncovered a distinct regulation and function of ATG7(2) in contrast with ATG7(1), the canonical isoform. First, affinity-purification mass spectrometry analysis revealed that ATG7(2) establishes direct protein–protein interactions (PPIs) with metabolic proteins, whereas ATG7(1) primarily interacts with autophagy machinery proteins. Furthermore, we identified that ATG7(2) mediates a decrease in metabolic activity, highlighting a novel splice-dependent function of this important autophagy protein. Then, we found a divergent expression pattern of ATG7(1) and ATG7(2) across human tissues. Conclusively, our work uncovers the divergent patterns of expression, protein interactions, and function of ATG7(2) in contrast to ATG7(1). These findings suggest a molecular switch between main catabolic processes through isoform-dependent expression of a key autophagy gene.     

Ectopic Expression of Human BBS4 Can Rescue Bardet-Biedl Syndrome Phenotypes in Bbs4 Null Mice

Bardet-Biedl syndrome (BBS) is a genetically heterogeneous autosomal recessive disorder characterized by obesity, retinal degeneration, polydactyly, hypogenitalism and renal defects. Recent findings have associated the etiology of the disease with cilia, and BBS proteins have been implicated in trafficking various ciliary cargo proteins. To date, 17 different genes have been reported for BBS among which BBS1 is the most common cause of the disease followed by BBS10, and BBS4. A murine model of Bbs4 is known to phenocopy most of the human BBS phenotypes, and it is being used as a BBS disease model. To better understand the in vivo localization, cellular function, and interaction of BBS4 with other proteins, we generated a transgenic BBS4 mouse expressing the human BBS4 gene under control of the beta actin promoter. The transgene is expressed in various tissues including brain, eye, testis, heart, kidney, and adipose tissue. These mice were further bred to express the transgene in Bbs4 null mice, and their phenotype was characterized. Here we report that despite tissue specific variable expression of the transgene, human BBS4 was able to complement the deficiency of Bbs4 and rescue all the BBS phenotypes in the Bbs4 null mice. These results provide an encouraging prospective for gene therapy for BBS related phenotypes and potentially for other ciliopathies.     

Sheep gene mapping: assignment of ALDOB, CYP19, WT and SOX2 by somatic cell hybrid analysis

Twenty-four hamster-sheep hybrid cell lines representing eleven ovine synteny groups were used to make syntenic assignments for seven loci ALDOB (aldolase B, fructose biphosphate); AMH (anti-Müllerian hormone); CYP19 [cytochrome P₄₅₀ aromatase, subfamily XIX (aromatization of androgens)]; WT (Wilms' tumour gene); SOX2 (SRY-related HMG-box gene 2); FSHB (follicle-stimulating hormone, beta polypeptide); and SRY (sex region of Y chromosome). These loci were assigned to synteny groups U11(chr2) ( ALDOB ); U19 ( AMH ); U3(chr7) ( CYP19 ); and to chromosomes 15 ( WT ) and 1 ( SOX2 ). SRY defines the hybrids containing the Y chromosome.     

Genetic Heterogeneity of Bardet–Biedl Syndrome in a Distinct Canadian Population: Evidence for a Fifth Locus

Bardet–Biedl syndrome (BBS) is a rare, autosomal recessive disease characterized by retinal dystrophy, renal structural abnormalities, obesity, dysmorphic extremities, and hypogenitalism in males. BBS is genetically heterogeneous with four known loci: BBS1 (11q), BBS2 (16q), BBS3 (3p), and BBS4 (15q). The prevalence of BBS in Newfoundland is approximately 10-fold greater than in Switzerland (1:160,000) and similar to the prevalence among the Bedouin of Kuwait (1:13,500). A population-based genetic survey was performed on 17 BBS families from the island portion of the province of Newfoundland, a comparatively isolated region of Canada. The families in the study had a total of 36 well-documented, affected individuals with 12 families having 2 or more affected individuals. Linkage at each of the four known loci was tested with two-point linkage and haplotype analysis. Three of the 17 kindreds showed linkage to 11q, 1 to 16q, and 1 to 3p. The latter is the first BBS3 family identified in a population of northern European descent. Six families remain undetermined because of poor pedigree structure or inconclusive haplotype analyses. Six families were excluded from all four known BBS loci, indicating that there is at least a fifth BBS locus (BBS5).     

Knockdown of Bardet-Biedl syndrome gene BBS9/PTHB1 leads to cilia defects

Bardet-Biedl Syndrome (BBS, MIM#209900) is a genetically heterogeneous disorder with pleiotropic phenotypes that include retinopathy, mental retardation, obesity and renal abnormalities. Of the 15 genes identified so far, seven encode core proteins that form a stable complex called BBSome, which is implicated in trafficking of proteins to cilia. Though BBS9 (also known as PTHB1) is reportedly a component of BBSome, its direct function has not yet been elucidated. Using zebrafish as a model, we show that knockdown of bbs9 with specific antisense morpholinos leads to developmental abnormalities in retina and brain including hydrocephaly that are consistent with the core phenotypes observed in syndromic ciliopathies. Knockdown of bbs9 also causes reduced number and length of cilia in Kupffer's vesicle. We also demonstrate that an orthologous human BBS9 mRNA, but not one carrying a missense mutation identified in BBS patients, can rescue the bbs9 morphant phenotype. Consistent with these findings, knockdown of Bbs9 in mouse IMCD3 cells results in the absence of cilia. Our studies suggest a key conserved role of BBS9 in biogenesis and/or function of cilia in zebrafish and mammals.