RESUMO
The membrane of the primary cilium is a highly specialized compartment that organizes proteins to achieve spatially ordered signaling. Disrupting ciliary organization leads to diseases called ciliopathies, with phenotypes ranging from retinal degeneration and cystic kidneys to neural tube defects. How proteins are selectively transported to and organized in the primary cilium remains unclear. Using a proteomic approach, we identified the ARL3 effector UNC119 as a binding partner of the myristoylated ciliopathy protein nephrocystin-3 (NPHP3). We mapped UNC119 binding to the N-terminal 200 residues of NPHP3 and found the interaction requires myristoylation. Creating directed mutants predicted from a structural model of the UNC119-myristate complex, we identified highly conserved phenylalanines within a hydrophobic ß sandwich to be essential for myristate binding. Furthermore, we found that binding of ARL3-GTP serves to release myristoylated cargo from UNC119. Finally, we showed that ARL3, UNC119b (but not UNC119a), and the ARL3 GAP Retinitis Pigmentosa 2 (RP2) are required for NPHP3 ciliary targeting and that targeting requires UNC119b myristoyl-binding activity. Our results uncover a selective, membrane targeting GTPase cycle that delivers myristoylated proteins to the ciliary membrane and suggest that other myristoylated proteins may be similarly targeted to specialized membrane domains.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal , Caenorhabditis elegans , Cílios/metabolismo , GTP Fosfo-Hidrolases/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular , Cinesinas , Proteínas Monoméricas de Ligação ao GTP , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Sequência de Aminoácidos , Animais , Caenorhabditis elegans/enzimologia , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/química , Proteínas de Caenorhabditis elegans/metabolismo , Linhagem Celular , Cílios/enzimologia , GTP Fosfo-Hidrolases/genética , Técnicas de Silenciamento de Genes , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Cinesinas/genética , Cinesinas/metabolismo , Proteínas Monoméricas de Ligação ao GTP/genética , Proteínas Monoméricas de Ligação ao GTP/metabolismo , Mutação , Fenótipo , Ligação Proteica , Estrutura Terciária de Proteína , Transporte Proteico , Alinhamento de Sequência , Peixe-Zebra/embriologia , Peixe-Zebra/genética , Peixe-Zebra/metabolismo , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismoRESUMO
Congenital heart malformations, including those of the great vessels, are among the most common human birth defects. The goal of this study was to identify the significance of a de novo mosaic PTPN12 partial deletion identified in a newborn with an interrupted aortic arch type A, ventricular septal defect, and pyloric stenosis. PTPN12, a downstream target of the RAS pathway, has a known role in endothelial cell adhesion and migration. Neither genetic nor genomic variants in PTPN12 have been described in a human patient; therefore, we evaluated the effect of ptpn12 in a mouse conditional knockout and zebrafish knockdown model to determine the significance of a loss in gene expression. Observed loss of ptpn12 expression in zebrafish resulted in abnormal branchial arch and tail vasculature patterns, with reduced blood flow throughout the animal. This phenotype was supported by anomalous vasculature in a conditional Ptpn12 mouse knockout. Given the novel co-occurrence of interrupted aortic arch type A, ventricular septal defect, and partial deletion of PTPN12 in the patient, as well as vascular phenotypes in Ptpn12 mouse and ptpn12 zebrafish models, it is likely that PTPN12 has a significant role in cardiovascular development and vessel formation during human embryonic development. Furthermore, the partial deletion of PTPN12 lead to interrupted aortic arch type A in this child and may represent a novel condition caused by a null mutation in the RAS pathway.
Assuntos
Aorta Torácica/anormalidades , Aorta Torácica/patologia , Mosaicismo , Proteína Tirosina Fosfatase não Receptora Tipo 12/genética , Deleção de Sequência , Adulto , Sequência de Aminoácidos , Angiografia , Animais , Aorta Torácica/diagnóstico por imagem , Aorta Torácica/embriologia , Criança , Hibridização Genômica Comparativa , Sequência Conservada , Desenvolvimento Embrionário , Técnicas de Silenciamento de Genes , Humanos , Hibridização in Situ Fluorescente , Lactente , Recém-Nascido , Larva , Masculino , Camundongos , Dados de Sequência Molecular , Neovascularização Fisiológica , Fenótipo , Proteína Tirosina Fosfatase não Receptora Tipo 12/química , Alinhamento de Sequência , Tomografia Computadorizada por Raios X , Peixe-Zebra/embriologiaRESUMO
Bardet-Biedl syndrome (BBS) is a syndromic form of retinal degeneration. Recently, homozygosity mapping with a consanguineous family with isolated retinitis pigmentosa identified a missense mutation in BBS3, a known BBS gene. The mutation in BBS3 encodes a single amino acid change at position 89 from alanine to valine. Since this amino acid is conserved in a wide range of vertebrates, we utilized the zebrafish model system to functionally characterize the BBS3 A89V mutation. Knockdown of bbs3 in zebrafish alters intracellular transport, a phenotype observed with knockdown of all BBS genes in the zebrafish, as well as visual impairment. Here, we find that BBS3 A89V is sufficient to rescue the transport delays induced by the loss of bbs3, indicating that this mutation does not affect the function of BBS3 as it relates to syndromic disease. BBS3L A89V, however, was unable to rescue vision impairment, highlighting a role for a specific amino acid within BBS3 that is necessary for visual function, but dispensable in other cell types. These data aid in our understanding of why patients with the BBS3 A89V missense mutation only present with isolated retinitis pigmentosa.
Assuntos
Fatores de Ribosilação do ADP/genética , Síndrome de Bardet-Biedl/genética , Proteínas Recombinantes/genética , Retinose Pigmentar/genética , Proteínas de Peixe-Zebra/genética , Peixe-Zebra/embriologia , Fatores de Ribosilação do ADP/biossíntese , Motivos de Aminoácidos , Sequência de Aminoácidos , Animais , Inativação Gênica , Melanossomas/metabolismo , Dados de Sequência Molecular , Mutação de Sentido Incorreto , Proteínas Recombinantes/biossíntese , Reflexo de Sobressalto , Alinhamento de Sequência , Peixe-Zebra/genética , Proteínas de Peixe-Zebra/biossínteseRESUMO
Bardet-Biedl Syndrome (BBS) is a heterogeneous syndromic form of retinal degeneration. We have identified a novel transcript of a known BBS gene, BBS3 (ARL6), which includes an additional exon. This transcript, BBS3L, is evolutionally conserved and is expressed predominantly in the eye, suggesting a specialized role in vision. Using antisense oligonucleotide knockdown in zebrafish, we previously demonstrated that bbs3 knockdown results in the cardinal features of BBS in zebrafish, including defects to the ciliated Kupffer's Vesicle and delayed retrograde melanosome transport. Unlike bbs3, knockdown of bbs3L does not result in Kupffer's Vesicle or melanosome transport defects, rather its knockdown leads to impaired visual function and mislocalization of the photopigment green cone opsin. Moreover, BBS3L RNA, but not BBS3 RNA, is sufficient to rescue both the vision defect as well as green opsin localization in the zebrafish retina. In order to demonstrate a role for Bbs3L function in the mammalian eye, we generated a Bbs3L-null mouse that presents with disruption of the normal photoreceptor architecture. Bbs3L-null mice lack key features of previously published Bbs-null mice, including obesity. These data demonstrate that the BBS3L transcript is required for proper retinal function and organization.
Assuntos
Fatores de Ribosilação do ADP/metabolismo , Visão Ocular , Proteínas de Peixe-Zebra/metabolismo , Fatores de Ribosilação do ADP/química , Fatores de Ribosilação do ADP/deficiência , Fatores de Ribosilação do ADP/genética , Sequência de Aminoácidos , Animais , Síndrome de Bardet-Biedl/complicações , Síndrome de Bardet-Biedl/genética , Síndrome de Bardet-Biedl/patologia , Síndrome de Bardet-Biedl/fisiopatologia , Anormalidades do Olho/complicações , Anormalidades do Olho/patologia , Anormalidades do Olho/fisiopatologia , Gânglios/efeitos dos fármacos , Gânglios/metabolismo , Gânglios/patologia , Regulação da Expressão Gênica/efeitos dos fármacos , Técnicas de Silenciamento de Genes , Humanos , Camundongos , Camundongos Mutantes , Dados de Sequência Molecular , Oligonucleotídeos Antissenso/farmacologia , Especificidade de Órgãos/efeitos dos fármacos , Fenótipo , Células Fotorreceptoras de Vertebrados/efeitos dos fármacos , Células Fotorreceptoras de Vertebrados/metabolismo , Células Fotorreceptoras de Vertebrados/patologia , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Transporte Proteico/efeitos dos fármacos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Reflexo de Sobressalto/efeitos dos fármacos , Opsinas de Bastonetes/metabolismo , Visão Ocular/efeitos dos fármacos , Peixe-Zebra , Proteínas de Peixe-Zebra/química , Proteínas de Peixe-Zebra/genéticaRESUMO
Phosphoribosyl pyrophosphate synthetase-1 (PRPS1) is a key enzyme in nucleotide biosynthesis, and mutations in PRPS1 are found in several human diseases including nonsyndromic sensorineural deafness, Charcot-Marie-Tooth disease-5, and Arts Syndrome. We utilized zebrafish as a model to confirm that mutations in PRPS1 result in phenotypic deficiencies in zebrafish similar to those in the associated human diseases. We found two paralogs in zebrafish, prps1a and prps1b and characterized each paralogous mutant individually as well as the double mutant fish. Zebrafish prps1a mutants and prps1a;prps1b double mutants showed similar morphological phenotypes with increasingly severe phenotypes as the number of mutant alleles increased. Phenotypes included smaller eyes and reduced hair cell numbers, consistent with the optic atrophy and hearing impairment observed in human patients. The double mutant also showed abnormal development of primary motor neurons, hair cell innervation, and reduced leukocytes, consistent with the neuropathy and recurrent infection of the human patients possessing the most severe reductions of PRPS1 activity. Further analyses indicated the phenotypes were associated with a prolonged cell cycle likely resulting from reduced nucleotide synthesis and energy production in the mutant embryos. We further demonstrated the phenotypes were caused by delays in the tissues most highly expressing the prps1 genes.