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1.
Cell ; 156(1-2): 146-57, 2014 Jan 16.
Artigo em Inglês | MEDLINE | ID: mdl-24439374

RESUMO

Misfolded proteins are often cytotoxic, unless cellular systems prevent their accumulation. Data presented here uncover a mechanism by which defects in secretory proteins lead to a dramatic reduction in their mRNAs and protein expression. When mutant signal sequences fail to bind to the signal recognition particle (SRP) at the ribosome exit site, the nascent chain instead contacts Argonaute2 (Ago2), and the mutant mRNAs are specifically degraded. Severity of signal sequence mutations correlated with increased proximity of Ago2 to nascent chain and mRNA degradation. Ago2 knockdown inhibited degradation of the mutant mRNA, while overexpression of Ago2 or knockdown of SRP54 promoted degradation of secretory protein mRNA. The results reveal a previously unappreciated general mechanism of translational quality control, in which specific mRNA degradation preemptively regulates aberrant protein production (RAPP).


Assuntos
Biossíntese de Proteínas , Dobramento de Proteína , Estabilidade de RNA , Partícula de Reconhecimento de Sinal/metabolismo , Sequência de Aminoácidos , Animais , Proteínas Argonautas/metabolismo , Cães , Células HeLa , Humanos , Dados de Sequência Molecular , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Alinhamento de Sequência
2.
Traffic ; 10(8): 1157-67, 2009 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-19522758

RESUMO

TRPML3 is an inward rectifying Ca(2+) channel that is regulated by extracytosolic H(+). Although gain-of-function mutation in TRPML3 causes the varitint-waddler phenotype, the role of TRPML3 in cellular physiology is not known. In this study, we report that TRPML3 is a prominent regulator of endocytosis, membrane trafficking and autophagy. Gradient fractionation and confocal localization reveal that TRPML3 is expressed in the plasma membrane and multiple intracellular compartments. However, expression of TRPML3 is dynamic, with accumulation of TRPML3 in the plasma membrane upon inhibition of endocytosis, and recruitment of TRPML3 to autophagosomes upon induction of autophagy. Accordingly, overexpression of TRPML3 leads to reduced constitutive and regulated endocytosis, increased autophagy and marked exacerbation of autophagy evoked by various cell stressors with nearly complete recruitment of TRPML3 into the autophagosomes. Importantly, both knockdown of TRPML3 by siRNA and expression of the channel-dead dominant negative TRPML3(D458K) have a reciprocal effect, reducing endocytosis and autophagy. These findings reveal a prominent role for TRPML3 in regulating endocytosis, membrane trafficking and autophagy, perhaps by controlling the Ca(2+) in the vicinity of cellular organelles that is necessary to regulate these cellular events.


Assuntos
Autofagia/fisiologia , Transporte Biológico/fisiologia , Canais de Potencial de Receptor Transitório/metabolismo , Animais , Cálcio/metabolismo , Linhagem Celular , Endocitose/fisiologia , Fator de Crescimento Epidérmico/metabolismo , Técnicas de Silenciamento de Genes , Humanos , Proteínas de Membrana Lisossomal/metabolismo , Técnicas de Patch-Clamp , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Canais de Cátion TRPM/genética , Canais de Cátion TRPM/metabolismo , Transferrina/metabolismo , Canais de Potencial de Receptor Transitório/genética , Proteínas de Transporte Vesicular/metabolismo
3.
EMBO J ; 27(8): 1197-205, 2008 Apr 23.
Artigo em Inglês | MEDLINE | ID: mdl-18369318

RESUMO

TRPML3 belongs to the TRPML subfamily of the transient receptor potential (TRP) channels. The A419P mutation in TRPML3 causes the varitint-waddler phenotype as a result of gain-of-function mutation (GOF). Regulation of the channels and the mechanism by which the A419P mutation leads to GOF are not known. We report here that TRPML3 is a Ca(2+)-permeable channel with a unique form of regulation by extracytosolic (luminal) H(+) (H(+)(e-cyto)). Regulation by H(+)(e-cyto) is mediated by a string of three histidines (H252, H273, H283) in the large extracytosolic loop between transmembrane domains (TMD) 1 and 2. Each of the histidines has a unique role, whereby H252 and H273 retard access of H(+)(e-cyto) to the inhibitory H283. Notably, the H283A mutation has the same phenotype as A419P and locks the channel in an open state, whereas the H283R mutation inactivates the channel. Accordingly, A419P eliminates regulation of TRPML3 by H(+)(e-cyto), and confers full activation to TRPML3(H283R). Activation of TRPML3 and regulation by H(+)(e-cyto) are altered by both the alpha-helix-destabilizing A419G and the alpha-helix-favouring A419M and A419K. These findings suggest that regulation of TRPML3 by H(+)(e-cyto) is due to an effect of the large extracytosolic loop on the orientation of fifth TMD and thus pore opening and show that the GOF of TRPML3(A419P) is due to disruption of this communication.


Assuntos
Citosol/fisiologia , Fenótipo , Canais de Potencial de Receptor Transitório/metabolismo , Substituição de Aminoácidos/genética , Cálcio/metabolismo , Linhagem Celular , Humanos , Concentração de Íons de Hidrogênio , Mucolipidoses/genética , Mucolipidoses/metabolismo , Estrutura Terciária de Proteína , Canais de Potencial de Receptor Transitório/química , Canais de Potencial de Receptor Transitório/genética
4.
J Biol Chem ; 282(50): 36138-42, 2007 Dec 14.
Artigo em Inglês | MEDLINE | ID: mdl-17962195

RESUMO

TRPML3 is a member of the TRPML subfamily of the transient receptor potential cation channel superfamily. The TRPML3(A419P) mutation causes a severe form, whereas the TRPML3(I362T/A419P) mutation results in a mild form of the varitint-waddler phenotype. The channel properties of TRPML3 and how the mutations cause each phenotype are not known. In this study, we report the first channel properties of TRPML3 as a strongly inward rectifying cation channel with a novel regulation by extracytosolic Na+. Preincubating the extracytosolic face of TRPML3 in Na+-free medium is required for channel activation, but then the channel slowly inactivates. The A419P mutation locks the channel in an open unregulated state. Similar gain of function was observed with the A419G mutation, which, like A419P, is expected to destabilize the alpha-helical fifth transmembrane domain of TRPML3. The I362T mutation results in an inactive channel, but the channel properties of TRPML3(I362T/A419P) are similar to those of TRPML3(A419P). However, the surface expression and current density of TRPML3(I362T/A419P) are lower than those of TRPML3(A419P). The A419P mutation also affects channel glycosylation and causes massive cell death. These findings show that the varitint-waddler phenotype is due to a gain of function of TRPML3(A419P) that is reduced by the TRPML3(I362T/A419P) mutant, resulting in a milder phenotype.


Assuntos
Mucolipidoses/metabolismo , Mutação de Sentido Incorreto , Processamento de Proteína Pós-Traducional , Sódio/metabolismo , Canais de Potencial de Receptor Transitório/metabolismo , Substituição de Aminoácidos , Animais , Morte Celular/genética , Linhagem Celular , Glicosilação , Humanos , Transporte de Íons/genética , Camundongos , Mucolipidoses/genética , Fenótipo , Processamento de Proteína Pós-Traducional/genética , Estrutura Secundária de Proteína/genética , Estrutura Terciária de Proteína/genética , Canais de Potencial de Receptor Transitório/genética
5.
J Biol Chem ; 281(11): 7294-301, 2006 Mar 17.
Artigo em Inglês | MEDLINE | ID: mdl-16361256

RESUMO

Mucolipidosis type IV (MLIV) is caused by mutations in the ion channel mucolipin 1 (TRP-ML1). MLIV is typified by accumulation of lipids and membranous materials in intracellular organelles, which was hypothesized to be caused by the altered membrane fusion and fission events. How mutations in TRP-ML1 lead to aberrant lipolysis is not known. Here we present evidence that MLIV is a metabolic disorder that is not associated with aberrant membrane fusion/fission events. Thus, measurement of lysosomal pH revealed that the lysosomes in TRP-ML1(-/-) cells obtained from the patients with MLIV are over-acidified. TRP-ML1 can function as a H(+) channel, and the increased lysosomal acidification in TRP-ML1(-/-) cells is likely caused by the loss of TRP-ML1-mediated H(+) leak. Measurement of lipase activity using several substrates revealed a marked reduction in lipid hydrolysis in TRP-ML1(-/-) cells, which was rescued by the expression of TRP-ML1. Cell fractionation indicated specific loss of acidic lipase activity in TRP-ML1(-/-) cells. Furthermore, dissipation of the acidic lysosomal pH of TRP-ML1(-/-) cells by nigericin or chloroquine reversed the lysosomal storage disease phenotype. These findings provide a new mechanism to account for the pathogenesis of MLIV.


Assuntos
Lisossomos/metabolismo , Esterol Esterase/química , Canais de Cátion TRPM/química , Laranja de Acridina/farmacologia , Cálcio/química , Cálcio/metabolismo , Sinalização do Cálcio , Ácidos Carboxílicos/farmacologia , Linhagem Celular , Membrana Celular/metabolismo , Cloroquina/química , Cromatografia em Camada Fina , Humanos , Concentração de Íons de Hidrogênio , Hidrolases/química , Hidrólise , Ionóforos/química , Lipase/química , Lipídeos/química , Doenças por Armazenamento dos Lisossomos/metabolismo , Lisossomos/química , Magnésio/química , Fusão de Membrana , Microscopia Eletrônica , Microscopia de Fluorescência , Modelos Biológicos , Mutação , Fenótipo , Prótons , Frações Subcelulares/metabolismo , Canais de Cátion TRPM/metabolismo , Canais de Potencial de Receptor Transitório
6.
J Biol Chem ; 280(52): 43218-23, 2005 Dec 30.
Artigo em Inglês | MEDLINE | ID: mdl-16257972

RESUMO

Mutations in the gene MCOLN1 coding for the TRP (transient receptor potential) family ion channel TRP-ML1 lead to the lipid storage disorder mucolipidosis type IV (MLIV). The function and role of TRP-ML1 are not well understood. We report here that TRP-ML1 is a lysosomal monovalent cation channel. Both native and recombinant TRP-ML1 are cleaved resulting in two products. Recombinant TRP-ML1 is detected as the full-length form and as short N- and C-terminal forms, whereas in native cells mainly the cleaved N and C termini are detected. The N- and C-terminal fragments of TRP-ML1 were co-immunoprecipitated from cell lysates and co-eluted from a Ni2+ column. TRP-ML1 undergoes proteolytic cleavage that is inhibited by inhibitors of cathepsin B (CatB) and is altered when TRP-ML1 is expressed in CatB-/- cells. N-terminal sequencing of purified C-terminal fragment of TRP-ML1 expressed in Sf9 cells indicates a cleavage site at Arg200 downward arrow Pro201. Consequently, the conserved R200H mutation changed the cleavage pattern of TRP-ML1. The cleavage inhibited TRP-ML1 channel activity. This work provides the first example of inactivation by cleavage of a TRP channel. The significance of the cleavage to the function of TRP-ML1 is under investigation.


Assuntos
Lisossomos/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/fisiologia , Mutação , Canais de Potencial de Receptor Transitório/fisiologia , Animais , Arginina/química , Sítios de Ligação , Cátions , Linhagem Celular , Eletrofisiologia , Fibroblastos/citologia , Células HeLa , Humanos , Immunoblotting , Imuno-Histoquímica , Imunoprecipitação , Insetos , Lipídeos/química , Proteínas de Membrana/química , Microscopia de Fluorescência , Mutagênese Sítio-Dirigida , Níquel/química , Prolina/química , Estrutura Terciária de Proteína , Proteínas Recombinantes/química , Pele/citologia , Canais de Cátion TRPM , Canais de Potencial de Receptor Transitório/metabolismo
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