RESUMO
BACKGROUND: The dystonias are a heterogeneous group of hyperkinetic disorders characterized by sustained or intermittent muscle contractions that cause abnormal movements and/or postures. Although more than 200 causal genes are known, many cases of primary dystonia have no clear genetic cause. OBJECTIVES: To identify the causal gene in a consanguineous family with three siblings affected by a complex persistent generalized dystonia, generalized epilepsy, and mild intellectual disability. METHODS: We performed exome sequencing in the parents and two affected siblings and characterized the expression of the identified gene by immunohistochemistry in control human and zebrafish brains. RESULTS: We identified a novel missense variant (c.142G>A (NM_032192); p.Glu48Lys) in the protein phosphatase 1 regulatory inhibitor subunit 1B gene (PPP1R1B) that was homozygous in all three siblings and heterozygous in the parents. This gene is also known as dopamine and cAMP-regulated neuronal phosphoprotein 32 (DARPP-32) and has been involved in the pathophysiology of abnormal movements. The uncovered variant is absent in public databases and modifies the conserved glutamate 48 localized close to the serine 45 phosphorylation site. The PPP1R1B protein was shown to be expressed in cells and regions involved in movement control, including projection neurons of the caudate-putamen, substantia nigra neuropil, and cerebellar Purkinje cells. The latter cells were also confirmed to be positive for PPP1R1B expression in the zebrafish brain. CONCLUSIONS: We report the association of a PPP1R1B/DARPP-32 variant with generalized dystonia in man. It might be relevant to include the sequencing of this new gene in the diagnosis of patients with otherwise unexplained movement disorders. © 2021 International Parkinson and Movement Disorder Society.
Assuntos
Fosfoproteína 32 Regulada por cAMP e Dopamina/genética , Distonia , Distúrbios Distônicos , Animais , Distúrbios Distônicos/genética , Homozigoto , Humanos , Peixe-ZebraRESUMO
COPI (coatomer complex I) coated vesicles are involved in Golgi-to-ER and intra-Golgi trafficking pathways, and mediate retrieval of ER resident proteins. Functions and components of the COPI-mediated trafficking pathways, beyond the canonical set of Sec/Arf proteins, are constantly increasing in number and complexity. In mammalian cells, GORAB, SCYL1 and SCYL3 proteins regulate Golgi morphology and protein glycosylation in concert with the COPI machinery. Here, we show that Cex1, homologous to the mammalian SCYL proteins, is a component of the yeast COPI machinery, by interacting with Sec27, Sec28 and Sec33 (Ret1/Cop1) proteins of the COPI coat. Cex1 was initially reported to mediate channeling of aminoacylated tRNA outside of the nucleus. Our data show that Cex1 localizes at membrane compartments, on structures positive for the Sec33 α-COP subunit. Moreover, the Wbp1 protein required for N-glycosylation and interacting via its di-lysine motif with the Sec27 ß'-COP subunit is mis-targeted in cex1Δ deletion mutant cells. Our data point to the possibility of developing Cex1 yeast-based models to study neurodegenerative disorders linked to pathogenic mutations of its human homologue SCYL1.
Assuntos
Complexo I de Proteína do Envoltório/metabolismo , Proteínas Fúngicas/metabolismo , Proteínas de Transporte Nucleocitoplasmático/metabolismo , Proteínas de Ligação a RNA/metabolismo , Cromatografia Líquida , Complexo I de Proteína do Envoltório/genética , Retículo Endoplasmático/metabolismo , Proteínas Fúngicas/genética , Deleção de Genes , Complexo de Golgi/metabolismo , Espaço Intracelular , Espectrometria de Massas , Mutação , Proteínas de Transporte Nucleocitoplasmático/genética , Ligação Proteica , Transporte Proteico , Proteômica/métodos , Proteínas de Ligação a RNA/genéticaRESUMO
The Nck-associated protein 1-like (NCKAP1L) gene, alternatively called hematopoietic protein 1 (HEM-1), encodes a hematopoietic lineage-specific regulator of the actin cytoskeleton. Nckap1l-deficient mice have anomalies in lymphocyte development, phagocytosis, and neutrophil migration. Here we report, for the first time, NCKAP1L deficiency cases in humans. In two unrelated patients of Middle Eastern origin, recessive mutations in NCKAP1L abolishing protein expression led to immunodeficiency, lymphoproliferation, and hyperinflammation with features of hemophagocytic lymphohistiocytosis. Immunophenotyping showed an inverted CD4/CD8 ratio with a major shift of both CD4+ and CD8+ cells toward memory compartments, in line with combined RNA-seq/proteomics analyses revealing a T cell exhaustion signature. Consistent with the core function of NCKAP1L in the reorganization of the actin cytoskeleton, patients' T cells displayed impaired early activation, immune synapse morphology, and leading edge formation. Moreover, knockdown of nckap1l in zebrafish led to defects in neutrophil migration. Hence, NCKAP1L mutations lead to broad immune dysregulation in humans, which could be classified within actinopathies.
Assuntos
Síndromes de Imunodeficiência/complicações , Inflamação/complicações , Transtornos Linfoproliferativos/complicações , Proteínas de Membrana/metabolismo , Actinas/metabolismo , Animais , Degranulação Celular , Proliferação de Células , Criança , Citotoxicidade Imunológica , Família , Feminino , Homozigoto , Humanos , Síndromes de Imunodeficiência/imunologia , Sinapses Imunológicas/metabolismo , Lactente , Inflamação/imunologia , Inflamação/patologia , Ativação Linfocitária/imunologia , Transtornos Linfoproliferativos/imunologia , Masculino , Proteínas de Membrana/química , Proteínas de Membrana/deficiência , Proteínas de Membrana/genética , Mutação/genética , Linhagem , Fenótipo , Síndrome , Peixe-ZebraRESUMO
Mutations in genes encoding aminoacyl-tRNA synthetases have been reported in several neurological disorders. KARS is a dual localized lysyl-tRNA synthetase and its cytosolic isoform belongs to the multiple aminoacyl-tRNA synthetase complex (MSC). Biallelic mutations in the KARS gene were described in a wide phenotypic spectrum ranging from nonsyndromic deafness to complex impairments. Here, we report on a patient with severe neurological and neurosensory disease investigated by whole-exome sequencing and found to carry biallelic mutations c.683C>T (p.Pro228Leu) and c.871T>G (p.Phe291Val), the second one being novel, in the KARS gene. The patient presented with an atypical clinical presentation with an optic neuropathy not previously reported. At the cellular level, we show that cytoplasmic KARS was expressed at a lower level in patient cells and displayed decreased interaction with MSC. In vitro, these two KARS variants have a decreased aminoacylation activity compared with wild-type KARS, the p.Pro228Leu being the most affected. Our data suggest that dysfunction of cytoplasmic KARS resulted in a decreased level of translation of the nuclear-encoded lysine-rich proteins belonging to the respiratory chain complex, thus impairing mitochondria functions.
Assuntos
Aminoacil-tRNA Sintetases/genética , Lisina-tRNA Ligase/genética , Mutação , Doenças do Sistema Nervoso/complicações , Doenças do Sistema Nervoso/genética , Doenças do Nervo Óptico/complicações , Transtornos de Sensação/complicações , Transtornos de Sensação/genética , Alelos , Sequência de Aminoácidos , Aminoacil-tRNA Sintetases/química , Aminoacil-tRNA Sintetases/metabolismo , Complexo I de Transporte de Elétrons/genética , Complexo I de Transporte de Elétrons/metabolismo , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Fibroblastos/metabolismo , Estudos de Associação Genética , Predisposição Genética para Doença , Humanos , Lisina-tRNA Ligase/química , Lisina-tRNA Ligase/metabolismo , Imageamento por Ressonância Magnética , Modelos Moleculares , Doenças do Sistema Nervoso/diagnóstico , Doenças do Nervo Óptico/diagnóstico , Linhagem , Ligação Proteica , Conformação Proteica , Transtornos de Sensação/diagnóstico , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
OBJECTIVE: To describe adult-onset limb-girdle-type muscular dystrophy caused by biallelic variants in the PYROXD1 gene, which has been recently linked to early-onset congenital myofibrillar myopathy. METHODS: Whole exome sequencing was performed for adult-onset neuromuscular disease patients with no molecular diagnosis. Patients with PYROXD1 variants underwent clinical characterization, lower limb muscle MRI, muscle biopsy and spirometry. A yeast complementation assay was used to determine the biochemical consequences of the genetic variants. RESULTS: We identified four patients with biallelic PYROXD1 variants. Three patients, who had symptom onset in their 20s or 30s, were homozygous for the previously described p.Asn155Ser. The fourth patient, with symptom onset at age 49, was compound heterozygous for p.Asn155Ser variant and previously unknown p.Tyr354Cys. All patients presented with a LGMD-type phenotype of symmetric muscle weakness and wasting. Symptoms started in proximal muscles of the lower limbs, and progressed slowly to involve also upper limbs in a proximal-predominant fashion. All patients remained ambulant past the age of 60. They had restrictive lung disease but no cardiac impairment. Muscle MRI showed strong involvement of anterolateral thigh muscles. Muscle biopsy displayed chronic myopathic changes. Yeast complementation assay demonstrated the p.Tyr354Cys mutation to impair PYROXD1 oxidoreductase ability. CONCLUSION: PYROXD1 variants can cause an adult-onset slowly progressive LGMD-type phenotype.
Assuntos
Distrofia Muscular do Cíngulo dos Membros/genética , Distrofia Muscular do Cíngulo dos Membros/fisiopatologia , Oxirredutases/genética , Idoso , Feminino , Finlândia , Genes Recessivos , Humanos , Masculino , Distrofia Muscular do Cíngulo dos Membros/patologia , Linhagem , Sequenciamento do ExomaRESUMO
Regulation of skeletal muscle development and organization is a complex process that is not fully understood. Here, we focused on amphiphysin 2 (BIN1, also known as bridging integrator-1) and dynamin 2 (DNM2), two ubiquitous proteins implicated in membrane remodeling and mutated in centronuclear myopathies (CNMs). We generated Bin1-/- Dnm2+/- mice to decipher the physiological interplay between BIN1 and DNM2. While Bin1-/- mice die perinatally from a skeletal muscle defect, Bin1-/- Dnm2+/- mice survived at least 18 months, and had normal muscle force and intracellular organization of muscle fibers, supporting BIN1 as a negative regulator of DNM2. We next characterized muscle-specific isoforms of BIN1 and DNM2. While BIN1 colocalized with and partially inhibited DNM2 activity during muscle maturation, BIN1 had no effect on the isoform of DNM2 found in adult muscle. Together, these results indicate that BIN1 and DNM2 regulate muscle development and organization, function through a common pathway, and define BIN1 as a negative regulator of DNM2 in vitro and in vivo during muscle maturation. Our data suggest that DNM2 modulation has potential as a therapeutic approach for patients with CNM and BIN1 defects. As BIN1 is implicated in cancers, arrhythmia, and late-onset Alzheimer disease, these findings may trigger research directions and therapeutic development for these common diseases.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Dinamina II/metabolismo , Fibras Musculares Esqueléticas/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Dinamina II/genética , Camundongos , Camundongos Knockout , Fibras Musculares Esqueléticas/patologia , Miopatias Congênitas Estruturais/genética , Miopatias Congênitas Estruturais/metabolismo , Miopatias Congênitas Estruturais/patologia , Miopatias Congênitas Estruturais/terapia , Proteínas do Tecido Nervoso/genética , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Proteínas Supressoras de Tumor/genéticaRESUMO
The family of WD40-repeat (WDR) proteins is one of the largest in eukaryotes, but little is known about their function in brain development. Among 26 WDR genes assessed, we found 7 displaying a major impact in neuronal morphology when inactivated in mice. Remarkably, all seven genes showed corpus callosum defects, including thicker (Atg16l1, Coro1c, Dmxl2, and Herc1), thinner (Kif21b and Wdr89), or absent corpus callosum (Wdr47), revealing a common role for WDR genes in brain connectivity. We focused on the poorly studied WDR47 protein sharing structural homology with LIS1, which causes lissencephaly. In a dosage-dependent manner, mice lacking Wdr47 showed lethality, extensive fiber defects, microcephaly, thinner cortices, and sensory motor gating abnormalities. We showed that WDR47 shares functional characteristics with LIS1 and participates in key microtubule-mediated processes, including neural stem cell proliferation, radial migration, and growth cone dynamics. In absence of WDR47, the exhaustion of late cortical progenitors and the consequent decrease of neurogenesis together with the impaired survival of late-born neurons are likely yielding to the worsening of the microcephaly phenotype postnatally. Interestingly, the WDR47-specific C-terminal to LisH (CTLH) domain was associated with functions in autophagy described in mammals. Silencing WDR47 in hypothalamic GT1-7 neuronal cells and yeast models independently recapitulated these findings, showing conserved mechanisms. Finally, our data identified superior cervical ganglion-10 (SCG10) as an interacting partner of WDR47. Taken together, these results provide a starting point for studying the implications of WDR proteins in neuronal regulation of microtubules and autophagy.
Assuntos
Autofagia/fisiologia , Encéfalo/crescimento & desenvolvimento , Encéfalo/metabolismo , Proteínas dos Microfilamentos/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Repetições WD40/fisiologia , Animais , Movimento Celular/fisiologia , Proliferação de Células/fisiologia , Células Cultivadas , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Microtúbulos/metabolismo , Microtúbulos/fisiologia , Neurogênese/fisiologia , Neurônios/metabolismo , Neurônios/fisiologia , Fenótipo , Células-Tronco/metabolismo , Células-Tronco/fisiologiaRESUMO
Myotubularins (MTMs) are active or dead phosphoinositides phosphatases defining a large protein family conserved through evolution and implicated in different neuromuscular diseases. Loss-of-function mutations in MTM1 cause the severe congenital myopathy called myotubular myopathy (or X-linked centronuclear myopathy) while mutations in the MTM1-related protein MTMR2 cause a recessive Charcot-Marie-Tooth peripheral neuropathy. Here we aimed to determine the functional specificity and redundancy of MTM1 and MTMR2, and to assess their abilities to compensate for a potential therapeutic strategy. Using molecular investigations and heterologous expression of human MTMs in yeast cells and in Mtm1 knockout mice, we characterized several naturally occurring MTMR2 isoforms with different activities. We identified the N-terminal domain as responsible for functional differences between MTM1 and MTMR2. An N-terminal extension observed in MTMR2 is absent in MTM1, and only the short MTMR2 isoform lacking this N-terminal extension behaved similarly to MTM1 in yeast and mice. Moreover, adeno-associated virus-mediated exogenous expression of several MTMR2 isoforms ameliorates the myopathic phenotype owing to MTM1 loss, with increased muscle force, reduced myofiber atrophy, and reduction of the intracellular disorganization hallmarks associated with myotubular myopathy. Noteworthy, the short MTMR2 isoform provided a better rescue when compared with the long MTMR2 isoform. In conclusion, these results point to the molecular basis for MTMs functional specificity. They also provide the proof-of-concept that expression of the neuropathy-associated MTMR2 gene improves the MTM1-associated myopathy, thus identifying MTMR2 as a novel therapeutic target for myotubular myopathy.
Assuntos
Miopatias Congênitas Estruturais/genética , Proteínas Tirosina Fosfatases não Receptoras/metabolismo , Animais , Humanos , Masculino , Camundongos , Camundongos Knockout , Mutação , Miopatias Congênitas Estruturais/enzimologia , Miopatias Congênitas Estruturais/metabolismo , Fenótipo , Domínios Proteicos , Isoformas de Proteínas , Proteínas Tirosina Fosfatases não Receptoras/genéticaRESUMO
Ent3 and Ent5 are yeast epsin N-terminal homology (ENTH) domain-containing proteins involved in protein trafficking between the Golgi and late endosomes. They interact with clathrin, clathrin adaptors at the Golgi (AP-1 and GGA) and different SNAREs (Vti1, Snc1, Pep12 and Syn8) required for vesicular transport at the Golgi and endosomes. To better understand the role of these epsins in membrane trafficking, we performed a protein-protein interaction screen. We identified Btn3 (also known as Tda3), a putative oxidoreductase, as a new partner of both Ent3 and Ent5. Btn3 is a negative regulator of the Batten-disease-linked protein Btn2 involved in the retrieval of specific SNAREs (Vti1, Snc1, Tlg1 and Tlg2) from the late endosome to the Golgi. We show that Btn3 endosomal localization depends on the epsins Ent3 and Ent5. We demonstrated that in btn3Δ mutant cells, endosomal sorting of ubiquitylated cargos and endosomal recycling of the Snc1 SNARE are delayed. We thus propose that Btn3 regulates the sorting function of two adaptors for SNARE proteins, the epsin Ent3 and the Batten-disease-linked protein Btn2.
Assuntos
Proteínas Adaptadoras de Transporte Vesicular/metabolismo , Sistemas de Transporte de Aminoácidos/metabolismo , Endossomos/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Proteínas Adaptadoras de Transporte Vesicular/genética , Sistemas de Transporte de Aminoácidos/genética , Complexo de Golgi/metabolismo , Análise Serial de Proteínas , Mapeamento de Interação de Proteínas , Transporte Proteico/fisiologia , Proteínas SNARE/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Transporte Vesicular/genéticaRESUMO
The formation and budding of endoplasmic reticulum ER-derived vesicles depends on the COPII coat protein complex that was first identified in yeast Saccharomyces cerevisiae. The ER-associated Sec12 and the Sar1 GTPase initiate the COPII coat formation by recruiting the Sec23-Sec24 heterodimer following the subsequent recruitment of the Sec13-Sec31 heterotetramer. In yeast, there is usually one gene encoding each COPII protein and these proteins are essential for yeast viability, whereas the plant genome encodes multiple isoforms of all COPII subunits. Here, we used a systematic yeast complementation assay to assess the functionality of Arabidopsis thaliana COPII proteins. In this study, the different plant COPII subunits were expressed in their corresponding temperature-sensitive yeast mutant strain to complement their thermosensitivity and secretion phenotypes. Secretion was assessed using two different yeast cargos: the soluble α-factor pheromone and the membranous v-SNARE (vesicle-soluble NSF (N-ethylmaleimide-sensitive factor) attachment protein receptor) Snc1 involved in the fusion of the secretory vesicles with the plasma membrane. This complementation study allowed the identification of functional A. thaliana COPII proteins for the Sec12, Sar1, Sec24 and Sec13 subunits that could represent an active COPII complex in plant cells. Moreover, we found that AtSec12 and AtSec23 were co-immunoprecipitated with AtSar1 in total cell extract of 15 day-old seedlings of A. thaliana. This demonstrates that AtSar1, AtSec12 and AtSec23 can form a protein complex that might represent an active COPII complex in plant cells.
Assuntos
Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Arabidopsis/genética , Teste de Complementação Genética , Mutação , Saccharomyces cerevisiae/genética , Vesículas Revestidas pelo Complexo de Proteína do Envoltório/metabolismo , Fenótipo , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/fisiologia , TemperaturaRESUMO
The spatial and temporal regulation of actin polymerization is crucial for various cellular processes. Members of the Wiskott-Aldrich syndrome protein (WASP) family activate the Arp2/3-complex leading to actin polymerization. The yeast Saccharomyces cerevisiae contains only one WASP homolog, Las17, that requires additional factors for its regulation. Lsb1 and Lsb2/Pin3 are two yeast homologous proteins bearing an SH3 domain that were identified as Las17-binding proteins. Lsb2/Pin3 that promotes prion induction was suggested to link this prion formation to the actin cytoskeleton. However, the cellular role of Lsb1 and the molecular function of both Lsb1 and Lsb2 remain unknown. In this study, we show that Lsb1 and/or Lsb2 full-length proteins inhibit Las17-mediated actin polymerization in vitro, Lsb2 being a less potent inhibitor of Las17 activity compared to Lsb1. Addition of Lsb1 or Lsb2 to the corresponding full-length Lsb1/2 further inhibits Las17 activity. Lsb1 and Lsb2 form homo- and hetero-oligomeric complexes suggesting that these two proteins could regulate Las17 activity via dimerization or cooperative binding. In vivo, overexpressed Lsb1 and Lsb2 proteins cluster Las17-CFP in few cytoplasmic punctate structures that are also positive for other Arp2/3-dependent actin polymerization effectors like Sla1 or Abp1. But, only Lsb1 overexpression blocks the internalization step of receptor-mediated endocytosis. This shows a specific function of Lsb1 in endocytosis.
Assuntos
Actinas/química , Proteínas de Transporte/metabolismo , Endocitose , Multimerização Proteica , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/metabolismo , Proteína da Síndrome de Wiskott-Aldrich/metabolismo , Complexo 2-3 de Proteínas Relacionadas à Actina/metabolismo , Sistemas de Transporte de Aminoácidos Básicos/metabolismo , Proteínas de Transporte/química , Estrutura Quaternária de Proteína , Transporte Proteico , Proteínas de Saccharomyces cerevisiae/químicaRESUMO
Myotubularin MTM1 is a phosphoinositide (PPIn) 3-phosphatase mutated in X-linked centronuclear myopathy (XLCNM; myotubular myopathy). We investigated the involvement of MTM1 enzymatic activity on XLCNM phenotypes. Exogenous expression of human MTM1 in yeast resulted in vacuolar enlargement, as a consequence of its phosphatase activity. Expression of mutants from patients with different clinical progression and determination of PtdIns3P and PtdIns5P cellular levels confirmed the link between vacuolar morphology and MTM1 phosphatase activity, and showed that some disease mutants retain phosphatase activity. Viral gene transfer of phosphatase-dead myotubularin mutants (MTM1(C375S) and MTM1(S376N)) significantly improved most histological signs of XLCNM displayed by a Mtm1-null mouse, at similar levels as wild-type MTM1. Moreover, the MTM1(C375S) mutant improved muscle performance and restored the localization of nuclei, triad alignment, and the desmin intermediate filament network, while it did not normalize PtdIns3P levels, supporting phosphatase-independent roles of MTM1 in maintaining normal muscle performance and organelle positioning in skeletal muscle. Among the different XLCNM signs investigated, we identified only triad shape and fiber size distribution as being partially dependent on MTM1 phosphatase activity. In conclusion, this work uncovers MTM1 roles in the structural organization of muscle fibers that are independent of its enzymatic activity. This underlines that removal of enzymes should be used with care to conclude on the physiological importance of their activity.
Assuntos
Miopatias Congênitas Estruturais/genética , Fenótipo , Proteínas Tirosina Fosfatases não Receptoras/genética , Animais , Desmina/metabolismo , Modelos Animais de Doenças , Ativação Enzimática/genética , Expressão Gênica , Humanos , Masculino , Camundongos , Camundongos Knockout , Força Muscular/genética , Músculo Esquelético/metabolismo , Músculo Esquelético/patologia , Músculo Esquelético/ultraestrutura , Mutação , Miopatias Congênitas Estruturais/metabolismo , Fosfatos de Fosfatidilinositol/metabolismo , Monoéster Fosfórico Hidrolases/metabolismo , Proteínas Tirosina Fosfatases não Receptoras/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismoRESUMO
Multivesicular endosomes (MVBs) are major sorting platforms for membrane proteins and participate in plasma membrane protein turnover, vacuolar/lysosomal hydrolase delivery, and surface receptor signal attenuation. MVBs undergo unconventional inward budding, which results in the formation of intraluminal vesicles (ILVs). MVB cargo sorting and ILV formation are achieved by the concerted function of endosomal sorting complex required for transport (ESCRT)-0 to ESCRT-III. The ESCRT-0 subunit Vps27 is a key player in this pathway since it recruits the other complexes to endosomes. Here we show that the Pkh1/Phk2 kinases, two yeast orthologues of the 3-phosphoinositide-dependent kinase, phosphorylate directly Vps27 in vivo and in vitro. We identify the phosphorylation site as the serine 613 and demonstrate that this phosphorylation is required for proper Vps27 function. Indeed, in pkh-ts temperature-sensitive mutant cells and in cells expressing vps27(S613A), MVB sorting of the carboxypeptidase Cps1 and of the α-factor receptor Ste2 is affected and the Vps28-green fluorescent protein ESCRT-I subunit is mainly cytoplasmic. We propose that Vps27 phosphorylation by Pkh1/2 kinases regulates the coordinated cascade of ESCRT complex recruitment at the endosomal membrane.
Assuntos
Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , Endossomos/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas Quinases Dependentes de 3-Fosfoinositídeo , Complexos Endossomais de Distribuição Requeridos para Transporte/química , Proteínas de Fluorescência Verde/metabolismo , Corpos Multivesiculares/metabolismo , Mutação/genética , Fosforilação , Fosfosserina/metabolismo , Transporte Proteico , Proteínas Recombinantes de Fusão/metabolismo , Saccharomyces cerevisiae/citologia , Proteínas de Saccharomyces cerevisiae/químicaRESUMO
A parallel 5'-d(TGGGGT)-3' quadruplex was formed in Na(+) solution and crystallized using lithium sulfate as the main precipitating agent. The X-ray structure was determined to 1.5 A resolution in space group P2(1) by molecular replacement. The asymmetric unit consists of a characteristic motif of two quadruplexes stacked at their 5' ends. All nucleotides are clearly defined in the density and could be positioned. A single bound Li(+) ion is observed at the surface of the column formed by the two joined molecules. Thus, this small alkali metal ion appears to be unsuitable as a replacement for the Na(+) ion in the central channel of G-quartets, unlike K(+) or Tl(+) ions. A well conserved constellation of water molecules is observed in the grooves of the dimeric structure.
Assuntos
Desoxirribonucleotídeos/química , Sequência de Bases , Cristalização , Cristalografia por Raios X , Desoxirribonucleotídeos/genética , Lítio/química , Modelos Moleculares , Conformação de Ácido Nucleico , Sódio/química , Eletricidade Estática , Água/químicaRESUMO
Nucleolin is a multidomain phosphoprotein involved in ribosome biogenesis. In vitro selection and binding studies with pre-rRNA fragments have shown that the first two RNA-binding domains (RBDs) in nucleolin (RBD12) recognize the consensus sequence (U/G)CCCG(A/G) in the context of a stem-loop structure (nucleolin-recognition element = NRE). Structural studies of nucleolin RBD12 in complex with an in vitro selected NRE (sNRE) and a natural pre-rRNA NRE (b2NRE) have revealed that sequence-specific binding of the consensus NRE is achieved in a similar manner in both complexes using residues in both RBDs as well as the linker connecting them. Using fluorescence anisotropy (FA) and nuclear magnetic resonance (NMR), we demonstrate the importance of the linker for NRE affinity by showing that only the individual RBDs with the linker attached retain the ability to specifically bind, albeit weakly, to sNRE and b2NRE. Binding of RBD1 and RBD2 to the NREs in trans is not detected even when one of the RBDs has the linker attached, which suggests that the linker also contributes to the affinity by tethering the two RBDs. To determine if binding of nucleolin RBD12 to natural NREs is dependent on a specific RNA stem-loop structure, as was the case for the sNRE, we conducted FA and NMR binding assays with nucleolin RBD12 and a single-stranded NRE. The results show that nucleolin RBD12 sequence-specifically binds a single-stranded NRE with an affinity similar to that for b2NRE, indicating that a stem-loop structure is not required for the nucleolin RBD12/pre-rRNA NRE interaction.
Assuntos
Fosfoproteínas/metabolismo , Precursores de RNA/metabolismo , Proteínas de Ligação a RNA/metabolismo , RNA/química , Sequência de Aminoácidos , Animais , Sequência de Bases , Sítios de Ligação , Sequência Consenso , Cricetinae , Polarização de Fluorescência , Cinética , Espectroscopia de Ressonância Magnética , Dados de Sequência Molecular , Conformação de Ácido Nucleico , Fosfoproteínas/química , Fosfoproteínas/genética , Ligação Proteica , Conformação Proteica , Estrutura Terciária de Proteína , RNA/genética , RNA/metabolismo , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/genética , NucleolinaRESUMO
The DNA damage dependence of poly(ADP-ribose) polymerase-2 (PARP-2) activity is suggestive of its implication in genome surveillance and protection. Here we show that the PARP-2 gene, mainly expressed in actively dividing tissues follows, but to a smaller extent, that of PARP-1 during mouse development. We found that PARP-2 and PARP-1 homo- and heterodimerize; the interacting interfaces, sites of reciprocal modification, have been mapped. PARP-2 was also found to interact with three other proteins involved in the base excision repair pathway: x-ray cross complementing factor 1 (XRCC1), DNA polymerase beta, and DNA ligase III, already known as partners of PARP-1. XRCC1 negatively regulates PARP-2 activity, as it does for PARP-1, while being a polymer acceptor for both PARP-1 and PARP-2. To gain insight into the physiological role of PARP-2 in response to genotoxic stress, we developed by gene disruption mice deficient in PARP-2. Following treatment by the alkylating agent N-nitroso-N-methylurea (MNU), PARP-2-deficient cells displayed an important delay in DNA strand breaks resealing, similar to that observed in PARP-1 deficient cells, thus confirming that PARP-2 is also an active player in base excision repair despite its low capacity to synthesize ADP-ribose polymers.