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1.
Exp Cell Res ; 357(1): 40-50, 2017 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-28442266

RESUMO

The Neuronal ceroid lipofuscinoses (NCLs) are a group of recessive disorders of childhood with overlapping symptoms including vision loss, ataxia, cognitive regression and premature death. 14 different genes have been linked to NCLs (CLN1-CLN14), but the functions of the proteins encoded by the majority of these genes have not been fully elucidated. Mutations in the CLN5 gene are responsible for the Finnish variant late-infantile form of NCL (Finnish vLINCL). CLN5 is translated as a 407 amino acid transmembrane domain containing protein that is heavily glycosylated, and subsequently cleaved into a mature soluble protein. Functionally, CLN5 is implicated in the recruitment of the retromer complex to endosomes, which is required to sort the lysosomal sorting receptors from endosomes to the trans-Golgi network. The mechanism that processes CLN5 into a mature soluble protein is currently not known. Herein, we demonstrate that CLN5 is initially translated as a type II transmembrane protein and subsequently cleaved by SPPL3, a member of the SPP/SPPL intramembrane protease family, into a mature soluble protein consisting of residues 93-407. The remaining N-terminal fragment is then cleaved by SPPL3 and SPPL2b and degraded in the proteasome. This work further characterizes the biology of CLN5 in the hopes of identifying a novel therapeutic strategy for affected children.


Assuntos
Ácido Aspártico Endopeptidases/metabolismo , Endossomos/metabolismo , Proteínas de Membrana/metabolismo , Lipofuscinoses Ceroides Neuronais/metabolismo , Linhagem Celular , Humanos , Lisossomos/metabolismo , Transporte Proteico , Solubilidade , Rede trans-Golgi/metabolismo
2.
Biochem Biophys Res Commun ; 433(1): 90-5, 2013 Mar 29.
Artigo em Inglês | MEDLINE | ID: mdl-23485461

RESUMO

Sortilin is a transmembrane domain protein that has been implicated in the sorting of prosaposin and other soluble cargo from the Golgi to the lysosomal compartment. While the majority of the receptor is recycled back to the Golgi from endosomes, it is known that upon successive rounds of transport, a proportion of sortilin is degraded in lysosomes. Recently, it was shown that sortilin is palmitoylated and that this post-translational modification prevents its degradation and enables sortilin to efficiently traffic back to the Golgi. Thus palmitoylation can be used to modulate the amount of receptor and hence cargo reaching the lysosome. In this work, we demonstrate that non-palmitoylated sortilin is ubiquitinated and internalized into the lysosomal compartment via the ESCRT pathway for degradation. Furthermore, we identified Nedd4 as an E3 ubiquitin ligase that mediates this post-translational modification. We propose a model where palmitoylation and ubiquitination play opposite roles in the stability and turnover of sortilin and serve as a control mechanism that balances the amount of lysosomal sorting and trafficking in cells.


Assuntos
Proteínas Adaptadoras de Transporte Vesicular/metabolismo , Proteínas Adaptadoras de Transporte Vesicular/química , Proteínas Adaptadoras de Transporte Vesicular/genética , Sequência de Aminoácidos , Complexos Endossomais de Distribuição Requeridos para Transporte/genética , Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , Complexo de Golgi/metabolismo , Células HeLa , Humanos , Lipoilação , Lisina/química , Lisossomos/metabolismo , Modelos Biológicos , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Ubiquitina-Proteína Ligases Nedd4 , Processamento de Proteína Pós-Traducional , Estabilidade Proteica , Transporte Proteico , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitinação
3.
Mol Cell Biol ; 32(10): 1855-66, 2012 May.
Artigo em Inglês | MEDLINE | ID: mdl-22431521

RESUMO

Mutations in the gene encoding CLN5 are the cause of Finnish variant late infantile Neuronal Ceroid Lipofuscinosis (NCL), and the gene encoding CLN5 is 1 of 10 genes (encoding CLN1 to CLN9 and cathepsin D) whose germ line mutations result in a group of recessive disorders of childhood. Although CLN5 localizes to the lysosomal compartment, its function remains unknown. We have uncovered an interaction between CLN5 and sortilin, the lysosomal sorting receptor. However, CLN5, unlike prosaposin, does not require sortilin to localize to the lysosomal compartment. We demonstrate that in CLN5-depleted HeLa cells, the lysosomal sorting receptors sortilin and cation-independent mannose 6-phosphate receptor (CI-MPR) are degraded in lysosomes due to a defect in recruitment of the retromer (an endosome-to-Golgi compartment trafficking component). In addition, we show that the retromer recruitment machinery is also affected by CLN5 depletion, as we found less loaded Rab7, which is required to recruit retromer. Taken together, our results support a role for CLN5 in controlling the itinerary of the lysosomal sorting receptors by regulating retromer recruitment at the endosome.


Assuntos
Endossomos/metabolismo , Lipofuscinoses Ceroides Neuronais/metabolismo , Proteínas Adaptadoras de Transporte Vesicular/metabolismo , Complexo de Golgi , Células HeLa , Humanos , Lisossomos/metabolismo , Ligação Proteica , Transporte Proteico , Receptor IGF Tipo 2/metabolismo , Proteínas de Transporte Vesicular/metabolismo
4.
J Cell Physiol ; 227(5): 1911-22, 2012 May.
Artigo em Inglês | MEDLINE | ID: mdl-21732362

RESUMO

Ovarian follicle development is a process regulated by various endocrine, paracrine and autocrine factors that act coordinately to promote follicle growth. However, the vast majority of follicles does not reach the pre-ovulatory stage but instead, undergo atresia by apoptosis. We have recently described a role for the somatic hyaluronidases (Hyal-1, Hyal-2, and Hyal-3) in ovarian follicular atresia and induction of granulosa cell apoptosis. Herein, we show that Hyal-1 but not Hyal-3 null mice have decreased apoptotic granulosa cells after the induction of atresia and an increased number of retrieved oocytes after stimulation of ovulation. Furthermore, young Hyal-1 null mice had a significantly higher number of primordial follicles than age matched wild-type animals. Recruitment of these follicles at puberty resulted in an increased number of primary and healthy preantral follicles in Hyal-1 null mice. Consequently, older Hyal-1 deficient female mice have prolonged fertility. At the molecular level, immature Hyal-1 null mice have decreased mRNA expression of follistatin and higher levels of phospho-Smad3 protein, resulting in increased levels of phospho-Akt in pubertal mice. Hyal-1 null ovarian follicles did not exhibit hyaluronan accumulation. For Hyal-3 null mice, compensation by Hyal-1 or Hyal-2 might be related to the lack of an ovarian phenotype. In conclusion, our results demonstrate that Hyal-1 plays a key role in the early phases of folliculogenesis by negatively regulating ovarian follicle growth and survival. Our findings add Hyal-1 as an ovarian regulator factor for follicle development, showing for the first time an interrelationship between this enzyme and the follistatin/activin/Smad3 pathway.


Assuntos
Ativinas/metabolismo , Apoptose/fisiologia , Fertilidade/fisiologia , Folistatina/metabolismo , Hialuronoglucosaminidase/deficiência , Folículo Ovariano/crescimento & desenvolvimento , Proteína Smad3/metabolismo , Animais , Feminino , Atresia Folicular/metabolismo , Células da Granulosa/citologia , Células da Granulosa/fisiologia , Humanos , Hialuronoglucosaminidase/genética , Isoenzimas/genética , Isoenzimas/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Oócitos/citologia , Folículo Ovariano/citologia , Folículo Ovariano/patologia , Transdução de Sinais/fisiologia , Fator de Crescimento Transformador beta/genética , Fator de Crescimento Transformador beta/metabolismo
5.
J Cell Sci ; 123(Pt 13): 2273-80, 2010 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-20530568

RESUMO

Sorting from the Golgi apparatus requires the recruitment of cytosolic coat proteins to package cargo into trafficking vesicles. An important early step in the formation of trafficking vesicles is the activation of Arf1 by the guanine nucleotide exchange factor GBF1. To activate Arf1, GBF1 must be recruited to and bound to Golgi membranes, a process that requires Rab1b. However, the mechanistic details of how Rab1 is implicated in GBF1 recruitment are not known. In this study, we demonstrate that the recruitment of GBF1 also requires phosphatidylinositol 4-phosphate [PtdIns(4)P]. Inhibitors of PtdIns(4)P synthesis or depletion of PI4KIIIalpha, a phosphatidylinositol 4-kinase localized to the endoplasmic reticulum and Golgi, prevents the recruitment of GBF1 to Golgi membranes. Interestingly, transfection of dominant-active Rab1 increased the amount of PtdIns(4)P at the Golgi, as detected by GFP-PH, a PtdIns(4)P-sensing probe. We propose that Rab1 contributes to the specificity and timing of GBF1 recruitment by activating PI4KIIIalpha. The PtdIns(4)P produced then allows GBF1 to bind to Golgi membranes and activate Arf1.


Assuntos
1-Fosfatidilinositol 4-Quinase/metabolismo , Complexo de Golgi/ultraestrutura , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Membranas Intracelulares/metabolismo , Isoenzimas/metabolismo , 1-Fosfatidilinositol 4-Quinase/genética , Fator 1 de Ribosilação do ADP/metabolismo , Animais , Células COS , Chlorocebus aethiops , Inibidores Enzimáticos/metabolismo , Complexo de Golgi/metabolismo , Fatores de Troca do Nucleotídeo Guanina/genética , Células HeLa , Humanos , Isoenzimas/genética , Fosfatos de Fosfatidilinositol/metabolismo , Interferência de RNA , Proteínas rab1 de Ligação ao GTP/genética , Proteínas rab1 de Ligação ao GTP/metabolismo
6.
Traffic ; 9(11): 1984-97, 2008 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-18817523

RESUMO

For the efficient trafficking of lysosomal proteins, the cationic-dependent and -independent mannose 6-phosphate receptors and sortilin must bind cargo in the Golgi apparatus, be packaged into clathrin-coated trafficking vesicles and traffic to the endosomes. Once in the endosomes, the receptors release their cargo into the endosomal lumen and recycle back to the Golgi for another round of trafficking, a process that requires retromer. In this study, we demonstrate that palmitoylation is required for the efficient retrograde trafficking of sortilin, and the cationic-independent mannose 6-phosphate as palmitoylation-deficient receptors remain trapped in the endosomes. Importantly, we also show that palmitoylation is required for receptor interaction with retromer as nonpalmitoylated receptor did not interact with retromer. In addition, we have identified DHHC-15 as the palmitoyltransferase responsible for this modification. In summary, we have shown the functional significance of palmitoylation in lysosomal receptor sorting and trafficking.


Assuntos
Endocitose , Lisossomos/metabolismo , Ácido Palmítico/metabolismo , Proteínas Adaptadoras de Transporte Vesicular/metabolismo , Células HeLa , Humanos , Hidrólise , Frações Subcelulares/metabolismo
7.
Endocrinology ; 149(11): 5835-47, 2008 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-18653706

RESUMO

During ovarian folliculogenesis, the vast majority of follicles will undergo atresia by apoptosis, allowing a few dominant follicles to mature. Mammalian hyaluronidases comprise a family of six to seven enzymes sharing the same catalytic domain responsible for hyaluronan hydrolysis. Interestingly, some of these enzymes have been shown to induce apoptosis. In the ovary, expression of three hyaluronidases (Hyal-1, Hyal-2, and Hyal-3) has been documented. However, their precise cellular localization and role in ovarian regulation have not yet been defined. We herein investigated the possible involvement of these enzymes in ovarian atresia. First, we established a mouse model for ovarian atresia (gonadotropin withdrawal by anti-equine chorionic gonadotropin treatment) and showed that the mRNA levels of Hyal-1, Hyal-2, and Hyal-3 were significantly increased in apoptotic granulosa cells as well as in atretic follicles. Second, using ovaries of normally cycling mice, we demonstrated the correlation of Hyal-1 mRNA and protein expression with cleavage of caspase-3. In addition, we showed that expression of all three hyaluronidases induced apoptosis in transfected granulosa cells. Significantly, the induction of apoptosis by hyaluronidases was independent of catalytic activity, because enzymatically inactive Hyal-1 mutant (D157A/E159A) was as efficient as the wild-type enzyme in apoptosis induction. The activation of the extrinsic apoptotic signaling pathway was involved in this induction, because increased levels of cleaved caspase-8, caspase-3, and poly-ADP-ribose polymerase (PARP) were observed upon hyaluronidase ectopic expression. Our present findings provide a better understanding of the role of hyaluronidases in ovarian functions, showing for the first time their involvement in follicular atresia.


Assuntos
Apoptose/genética , Atresia Folicular/genética , Células da Granulosa/fisiologia , Hialuronoglucosaminidase/fisiologia , Animais , Anticorpos/farmacologia , Apoptose/efeitos dos fármacos , Caspase 3/metabolismo , Caspase 8/metabolismo , Ciclo Estral/genética , Ciclo Estral/metabolismo , Feminino , Atresia Folicular/metabolismo , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Gonadotropinas Equinas/imunologia , Células da Granulosa/efeitos dos fármacos , Células da Granulosa/enzimologia , Células da Granulosa/metabolismo , Hialuronoglucosaminidase/genética , Hialuronoglucosaminidase/metabolismo , Masculino , Mamíferos/metabolismo , Camundongos , Camundongos Endogâmicos ICR , Poli(ADP-Ribose) Polimerases/metabolismo
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