RESUMO
Cilia are highly specialized microtubule-based organelles that have pivotal roles in numerous biological processes, including transducing sensory signals. Defects in cilia biogenesis and transport cause pleiotropic human ciliopathies. Mutations in over 30 different genes can lead to cilia defects, and complex interactions exist among ciliopathy-associated proteins. Mutations of the centrosomal protein 290 kDa (CEP290) lead to distinct clinical manifestations, including Leber congenital amaurosis (LCA), a hereditary cause of blindness due to photoreceptor degeneration. Mice homozygous for a mutant Cep290 allele (Cep290rd16 mice) exhibit LCA-like early-onset retinal degeneration that is caused by an in-frame deletion in the CEP290 protein. Here, we show that the domain deleted in the protein encoded by the Cep290rd16 allele directly interacts with another ciliopathy protein, MKKS. MKKS mutations identified in patients with the ciliopathy Bardet-Biedl syndrome disrupted this interaction. In zebrafish embryos, combined subminimal knockdown of mkks and cep290 produced sensory defects in the eye and inner ear. Intriguingly, combinations of Cep290rd16 and Mkksko alleles in mice led to improved ciliogenesis and sensory functions compared with those of either mutant alone. We propose that altered association of CEP290 and MKKS affects the integrity of multiprotein complexes at the cilia transition zone and basal body. Amelioration of the sensory phenotypes caused by specific mutations in one protein by removal of an interacting domain/protein suggests a possible novel approach for treating human ciliopathies.
Assuntos
Antígenos de Neoplasias/genética , Síndrome de Bardet-Biedl/genética , Cílios/ultraestrutura , Regulação da Expressão Gênica no Desenvolvimento , Chaperoninas do Grupo II/genética , Amaurose Congênita de Leber/genética , Proteínas de Neoplasias/genética , Proteínas Nucleares/genética , Transtornos de Sensação/genética , Alelos , Sequência de Aminoácidos , Animais , Proteínas de Ciclo Celular , Chaperoninas/deficiência , Chaperoninas/genética , Chaperoninas/fisiologia , Proteínas do Citoesqueleto , Análise Mutacional de DNA , Orelha/anormalidades , Orelha/embriologia , Anormalidades do Olho/embriologia , Anormalidades do Olho/genética , Teste de Complementação Genética , Chaperoninas do Grupo II/deficiência , Chaperoninas do Grupo II/fisiologia , Células HEK293 , Células Ciliadas Auditivas/ultraestrutura , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Proteínas Associadas aos Microtúbulos/deficiência , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/fisiologia , Dados de Sequência Molecular , Proteínas Nucleares/deficiência , Proteínas Nucleares/fisiologia , Neurônios Receptores Olfatórios/ultraestrutura , Cílio Conector dos Fotorreceptores/ultraestrutura , Mapeamento de Interação de Proteínas , Transtornos de Sensação/patologia , Transtornos de Sensação/prevenção & controle , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Peixe-Zebra/embriologia , Peixe-Zebra/genética , Proteínas de Peixe-Zebra/deficiência , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/fisiologiaRESUMO
Bardet-Biedl syndrome (BBS) is a genetically heterogeneous, autosomal-recessive disorder associated with several clinical features including obesity, hypertension, and cardiovascular abnormalities. BBS proteins play an important role in the function of cilia, a mechanosensory organelle in endothelial cells, but whether these proteins are directly involved in the regulation of vascular function is unclear. Here, we show that Bbs genes (1-12) are expressed in endothelial and smooth muscle cell lines and tissues enriched in endothelial (lung) and smooth muscle (stomach) cells as well as the aorta. Next, we used aortic rings to examine the vascular function of two BBS mouse models that recapitulate the human phenotype, namely Bbs2(-/-) (obese and normotensive) and Bbs6(-/-) (obese and hypertensive) mice. Interestingly, the endothelium-dependent relaxation (induced by ACh) was significantly enhanced in Bbs2(-/-) but not Bbs6(-/-) mice. In contrast, the endothelium-independent relaxation (induced by sodium nitroprusside) was unaltered in both BBS mouse models. In addition, the contractile responses to serotonin and endothelin-1 were attenuated in Bbs2(-/-) but not Bbs6(-/-) mice. Of note, the NO-producing enzymes (eNOS and iNOS) were upregulated in the aorta of Bbs2(-/-) but not Bbs6(-/-) mice. On the other hand, the expression level of membrane subunits of NADPH oxidase (p22(phox) and p47(phox)) in the aorta was decreased in Bbs2(-/-) mice but increased in Bbs6(-/-) mice. In conclusion, these data implicate Bbs genes in the regulation of vascular function and demonstrate that disrupting Bbs2 and Bbs6 genes affect differentially the vascular function.
Assuntos
Síndrome de Bardet-Biedl/fisiopatologia , Endotélio Vascular/fisiopatologia , Chaperoninas do Grupo II/deficiência , Hipertensão/fisiopatologia , Músculo Liso Vascular/fisiopatologia , Obesidade/fisiopatologia , Proteínas/metabolismo , Vasoconstrição , Vasodilatação , Animais , Aorta/metabolismo , Aorta/fisiopatologia , Síndrome de Bardet-Biedl/genética , Síndrome de Bardet-Biedl/metabolismo , Linhagem Celular , Relação Dose-Resposta a Droga , Endotélio Vascular/efeitos dos fármacos , Endotélio Vascular/metabolismo , Genótipo , Chaperoninas do Grupo II/genética , Hipertensão/genética , Hipertensão/metabolismo , Pulmão/irrigação sanguínea , Camundongos , Camundongos Knockout , Músculo Liso Vascular/efeitos dos fármacos , Músculo Liso Vascular/metabolismo , NADPH Oxidases/metabolismo , Óxido Nítrico/metabolismo , Óxido Nítrico Sintase Tipo II/metabolismo , Óxido Nítrico Sintase Tipo III/metabolismo , Obesidade/genética , Obesidade/metabolismo , Fenótipo , Proteínas/genética , Estômago/irrigação sanguínea , Vasoconstrição/efeitos dos fármacos , Vasoconstritores/farmacologia , Vasodilatação/efeitos dos fármacos , Vasodilatadores/farmacologiaRESUMO
Bardet-Biedl syndrome (BBS) is a human genetic disorder resulting in obesity, retinal degeneration, polydactyly, and nephropathy. Recent studies indicate that trafficking defects to the ciliary membrane are involved in this syndrome. Here, we show that a novel complex composed of three chaperonin-like BBS proteins (BBS6, BBS10, and BBS12) and CCT/TRiC family chaperonins mediates BBSome assembly, which transports vesicles to the cilia. Chaperonin-like BBS proteins interact with a subset of BBSome subunits and promote their association with CCT chaperonins. CCT activity is essential for BBSome assembly, and knockdown of CCT chaperonins in zebrafish results in BBS phenotypes. Many disease-causing mutations found in BBS6, BBS10, and BBS12 disrupt interactions among these BBS proteins. Our data demonstrate that BBS6, BBS10, and BBS12 are necessary for BBSome assembly, and that impaired BBSome assembly contributes to the etiology of BBS phenotypes associated with the loss of function of these three BBS genes.
