Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 10 de 10
Filtrar
1.
Cell ; 177(3): 711-721.e8, 2019 04 18.
Artigo em Inglês | MEDLINE | ID: mdl-30982603

RESUMO

Yeast ataxin-2, also known as Pbp1, senses the activity state of mitochondria in order to regulate TORC1. A domain of Pbp1 required to adapt cells to mitochondrial activity is of low sequence complexity. The low-complexity (LC) domain of Pbp1 forms labile, cross-ß polymers that facilitate phase transition of the protein into liquid-like or gel-like states. Phase transition for other LC domains is reliant upon widely distributed aromatic amino acids. In place of tyrosine or phenylalanine residues prototypically used for phase separation, Pbp1 contains 24 similarly disposed methionine residues. Here, we show that the Pbp1 methionine residues are sensitive to hydrogen peroxide (H2O2)-mediated oxidation in vitro and in living cells. Methionine oxidation melts Pbp1 liquid-like droplets in a manner reversed by methionine sulfoxide reductase enzymes. These observations explain how reversible formation of labile polymers by the Pbp1 LC domain enables the protein to function as a sensor of cellular redox state.


Assuntos
Proteínas de Transporte/metabolismo , Metionina/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Sequência de Aminoácidos , Proteínas de Transporte/química , Proteínas de Transporte/genética , Peróxido de Hidrogênio/farmacologia , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Metionina/metabolismo , Metionina Sulfóxido Redutases/metabolismo , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Mutagênese Sítio-Dirigida , Oxirredução , Estresse Oxidativo/efeitos dos fármacos , Transição de Fase , Domínios Proteicos , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética
2.
Cell ; 177(3): 697-710.e17, 2019 04 18.
Artigo em Inglês | MEDLINE | ID: mdl-30982600

RESUMO

Yeast ataxin-2, also known as Pbp1 (polyA binding protein-binding protein 1), is an intrinsically disordered protein implicated in stress granule formation, RNA biology, and neurodegenerative disease. To understand the endogenous function of this protein, we identify Pbp1 as a dedicated regulator of TORC1 signaling and autophagy under conditions that require mitochondrial respiration. Pbp1 binds to TORC1 specifically during respiratory growth, but utilizes an additional methionine-rich, low complexity (LC) region to inhibit TORC1. This LC region causes phase separation, forms reversible fibrils, and enables self-association into assemblies required for TORC1 inhibition. Mutants that weaken phase separation in vitro exhibit reduced capacity to inhibit TORC1 and induce autophagy. Loss of Pbp1 leads to mitochondrial dysfunction and reduced fitness during nutritional stress. Thus, Pbp1 forms a condensate in response to respiratory status to regulate TORC1 signaling.


Assuntos
Proteínas de Transporte/metabolismo , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Transdução de Sinais , Sequência de Aminoácidos , Autofagia/efeitos dos fármacos , Proteínas de Transporte/química , Proteínas de Transporte/genética , Alvo Mecanístico do Complexo 1 de Rapamicina/antagonistas & inibidores , Metionina/metabolismo , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Mutagênese Sítio-Dirigida , Fosforilação , Ligação Proteica , Domínios Proteicos , Saccharomyces cerevisiae/crescimento & desenvolvimento , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Transdução de Sinais/efeitos dos fármacos , Sirolimo/farmacologia
3.
PLoS Genet ; 19(5): e1010774, 2023 05.
Artigo em Inglês | MEDLINE | ID: mdl-37216416

RESUMO

Pbp1 (poly(A)-binding protein-binding protein 1) is a cytoplasmic stress granule marker that is capable of forming condensates that function in the negative regulation of TORC1 signaling under respiratory conditions. Polyglutamine expansions in its mammalian ortholog ataxin-2 lead to spinocerebellar dysfunction due to toxic protein aggregation. Here, we show that loss of Pbp1 in S. cerevisiae leads to decreased amounts of mRNAs and mitochondrial proteins which are targets of Puf3, a member of the PUF (Pumilio and FBF) family of RNA-binding proteins. We found that Pbp1 supports the translation of Puf3-target mRNAs in respiratory conditions, such as those involved in the assembly of cytochrome c oxidase and subunits of mitochondrial ribosomes. We further show that Pbp1 and Puf3 interact through their respective low complexity domains, which is required for Puf3-target mRNA translation. Our findings reveal a key role for Pbp1-containing assemblies in enabling the translation of mRNAs critical for mitochondrial biogenesis and respiration. They may further explain prior associations of Pbp1/ataxin-2 with RNA, stress granule biology, mitochondrial function, and neuronal health.


Assuntos
Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Animais , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Ataxina-2/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Biogênese de Organelas , Proteínas de Ligação a RNA/metabolismo , Mamíferos/genética , Proteínas de Transporte/genética
4.
J Biomed Sci ; 22: 33, 2015 May 16.
Artigo em Inglês | MEDLINE | ID: mdl-25981436

RESUMO

BACKGROUND: MSP58 is a nucleolar protein associated with rRNA transcription and cell proliferation. Its mechanism of translocation into the nucleus or the nucleolus, however, is not entirely known. In order to address this lack, the present study aims to determine a crucial part of this mechanism: the nuclear localization signal (NLS) and the nucleolar localization signal (NoLS) associated with the MSP58 protein. RESULTS: We have identified and characterized two NLSs in MSP58. The first is located between residues 32 and 56 (NLS1) and constitutes three clusters of basic amino acids (KRASSQALGTIPKRRSSSRFIKRKK); the second is situated between residues 113 and 123 (NLS2) and harbors a monopartite signal (PGLTKRVKKSK). Both NLS1 and NLS2 are highly conserved among different vertebrate species. Notably, one bipartite motif within the NLS1 (residues 44-56) appears to be absolutely necessary for MSP58 nucleolar localization. By yeast two-hybrid, pull-down, and coimmunoprecipitation analysis, we show that MSP58 binds to importin α1 and α6, suggesting that nuclear targeting of MSP58 utilizes a receptor-mediated and energy-dependent import mechanism. Functionally, our data show that both nuclear and nucleolar localization of MSP58 are crucial for transcriptional regulation on p21 and ribosomal RNA genes, and context-dependent effects on cell proliferation. CONCLUSIONS: Results suggest that MSP58 subnuclear localization is regulated by two nuclear import signals, and that proper subcellular localization of MSP58 is critical for its role in transcriptional regulation. Our study reveals a molecular mechanism that controls nuclear and nucleolar localization of MSP58, a finding that might help future researchers understand the MSP58 biological signaling pathway.


Assuntos
Núcleo Celular/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Transporte Ativo do Núcleo Celular , Sequência de Aminoácidos , Nucléolo Celular/metabolismo , Humanos , Sinais de Localização Nuclear/química , Sinais de Localização Nuclear/genética , Sinais de Localização Nuclear/metabolismo , Proteínas Nucleares/química , Estrutura Terciária de Proteína , Proteínas de Ligação a RNA/química , Alinhamento de Sequência
5.
Neuron ; 110(22): 3711-3726.e16, 2022 11 16.
Artigo em Inglês | MEDLINE | ID: mdl-36087583

RESUMO

Axon degeneration is an early pathological event in many neurological diseases. The identification of the nicotinamide adenine dinucleotide (NAD) hydrolase SARM1 as a central metabolic sensor and axon executioner presents an exciting opportunity to develop novel neuroprotective therapies that can prevent or halt the degenerative process, yet limited progress has been made on advancing efficacious inhibitors. We describe a class of NAD-dependent active-site SARM1 inhibitors that function by intercepting NAD hydrolysis and undergoing covalent conjugation with the reaction product adenosine diphosphate ribose (ADPR). The resulting small-molecule ADPR adducts are highly potent and confer compelling neuroprotection in preclinical models of neurological injury and disease, validating this mode of inhibition as a viable therapeutic strategy. Additionally, we show that the most potent inhibitor of CD38, a related NAD hydrolase, also functions by the same mechanism, further underscoring the broader applicability of this mechanism in developing therapies against this class of enzymes.


Assuntos
Proteínas do Domínio Armadillo , NAD , Proteínas do Domínio Armadillo/genética , Proteínas do Domínio Armadillo/metabolismo , NAD/metabolismo , Neuroproteção , Proteínas do Citoesqueleto/metabolismo , Axônios/metabolismo , Hidrolases/metabolismo
6.
J Biol Chem ; 285(28): 21567-80, 2010 Jul 09.
Artigo em Inglês | MEDLINE | ID: mdl-20448040

RESUMO

Protein phosphatase 2A (PP2A) is a heterotrimeric enzyme consisting of a scaffold subunit (A), a catalytic subunit (C), and a variable regulatory subunit (B). The regulatory B subunits determine the substrate specificity and subcellular localization of the PP2A holoenzyme. Here, we demonstrate that the subcellular localization of the B56gamma3 regulatory subunit is regulated in a cell cycle-specific manner. Notably, B56gamma3 becomes enriched in the nucleus at the G(1)/S border and in S phase. The S phase-specific nuclear enrichment of B56gamma3 is accompanied by increases of nuclear A and C subunits and nuclear PP2A activity. Overexpression of B56gamma3 promotes nuclear localization of the A and C subunits, whereas silencing both B56gamma2 and B56gamma3 blocks the S phase-specific increase in the nuclear localization and activity of PP2A. In NIH3T3 cells, B56gamma3 overexpression reduces p27 phosphorylation at Thr-187, concomitantly elevates p27 protein levels, delays the G(1) to S transition, and retards cell proliferation. Consistently, knockdown of endogenous B56gamma3 expression reduces p27 protein levels and increases cell proliferation in HeLa cells. These findings demonstrate that the dynamic nuclear distribution of the B56gamma3 regulatory subunit controls nuclear PP2A activity, which regulates cell cycle controllers, such as p27, to restrain cell cycle progression, and may be responsible for the tumor suppressor function of PP2A.


Assuntos
Regulação Enzimológica da Expressão Gênica , Proteína Fosfatase 2/metabolismo , Transporte Ativo do Núcleo Celular , Animais , Domínio Catalítico , Ciclo Celular , Proliferação de Células , Fase G1 , Células HeLa , Humanos , Camundongos , Células NIH 3T3 , Fosforilação , Proteína Fosfatase 2/química , Proteína Fosfatase 2/fisiologia , Fase S , Frações Subcelulares/metabolismo
8.
Cell Rep ; 32(5): 107999, 2020 08 04.
Artigo em Inglês | MEDLINE | ID: mdl-32755591

RESUMO

The NADase SARM1 is a central switch in injury-activated axon degeneration, an early hallmark of many neurological diseases. Here, we present cryo-electron microscopy (cryo-EM) structures of autoinhibited (3.3 Å) and active SARM1 (6.8 Å) and provide mechanistic insight into the tight regulation of SARM1's function by the local metabolic environment. Although both states retain an octameric core, the defining feature of the autoinhibited state is a lock between the autoinhibitory Armadillo/HEAT motif (ARM) and catalytic Toll/interleukin-1 receptor (TIR) domains, which traps SARM1 in an inactive state. Mutations that break this lock activate SARM1, resulting in catastrophic neuronal death. Notably, the mutants cannot be further activated by the endogenous activator nicotinamide mononucleotide (NMN), and active SARM1 is product inhibited by Nicotinamide (NAM), highlighting SARM1's functional dependence on key metabolites in the NAD salvage pathway. Our studies provide a molecular understanding of SARM1's transition from an autoinhibited to an injury-activated state and lay the foundation for future SARM1-based therapies to treat axonopathies.


Assuntos
Proteínas do Domínio Armadillo/química , Proteínas do Domínio Armadillo/metabolismo , Proteínas do Citoesqueleto/química , Proteínas do Citoesqueleto/metabolismo , NAD/metabolismo , Animais , Morte Celular , Linhagem Celular Tumoral , Microscopia Crioeletrônica , Feminino , Células HEK293 , Humanos , Camundongos Endogâmicos C57BL , Modelos Moleculares , Neurônios/citologia , Mononucleotídeo de Nicotinamida/metabolismo , Domínios Proteicos
9.
Oncotarget ; 7(4): 4542-58, 2016 Jan 26.
Artigo em Inglês | MEDLINE | ID: mdl-26684356

RESUMO

The B56γ-containing protein phosphatase 2A (PP2A-B56γ) has been postulated to have tumor suppressive functions. Here, we report regulation of p27KIP1 subcellular localization by PP2A-B56γ3. B56γ3 overexpression enhanced nuclear localization of p27KIP1, whereas knockdown of B56γ3 decreased p27KIP1 nuclear localization. B56γ3 overexpression decreased phosphorylation at Thr157 (phospho-Thr157), whose phosphorylation promotes cytoplasmic localization of p27KIP1, whereas B56γ3 knockdown significantly increased the level of phospho-Thr157. In vitro, PP2A-B56γ3 catalyzed dephosphorylation of phospho-Thr157 in a dose-dependent and okadaic acid-sensitive manner. B56γ3 did not increase p27KIP1 nuclear localization by down-regulating the upstream kinase Akt activity and outcompeted a myristoylated constitutively active Akt (Aktca) in regulating Thr157 phosphorylation and subcellular localization of p27KIP1. In addition, results of interaction domain mapping revealed that both the N-terminal and C-terminal domains of p27 and a domain at the C-terminus of B56γ3 are required for interaction between p27 and B56γ3. Furthermore, we demonstrated that p27KIP1 levels are positively correlated with B56γ levels in both non-tumor and tumor parts of a set of human colon tissue specimens. However, positive correlation between nuclear p27KIP1 levels and B56γ levels was found only in the non-tumor parts, but not in tumor parts of these tissues, implicating a dysregulation in PP2A-B56γ3-regulated p27KIP1 nuclear localization in these tumor tissues. Altogether, this study provides a new mechanism by which the PP2A-B56γ3 holoenzyme plays its tumor suppressor role.


Assuntos
Biomarcadores Tumorais/metabolismo , Neoplasias do Colo/metabolismo , Inibidor de Quinase Dependente de Ciclina p27/metabolismo , Regulação Neoplásica da Expressão Gênica , Proteína Fosfatase 2/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Treonina/metabolismo , Animais , Western Blotting , Núcleo Celular/metabolismo , Células Cultivadas , Citoplasma/metabolismo , Imunofluorescência , Células HeLa , Humanos , Camundongos , Células NIH 3T3 , Fosforilação , Frações Subcelulares
10.
J Biol Chem ; 283(4): 1882-92, 2008 Jan 25.
Artigo em Inglês | MEDLINE | ID: mdl-18042541

RESUMO

Akt is a protein serine/threonine kinase that is involved in the regulation of diverse cellular processes. Phosphorylation of Akt at regulatory residues Thr-308 and Ser-473 leads to its full activation. The protein phosphatase 2A (PP2A) has long been known to negatively regulate Akt activity. The PP2A holoenzyme consists of the structural subunit (A), catalytic subunit (C), and a variable regulatory subunit (B). Here we report the identification of the specific B regulatory subunit that targets the PP2A holoenzyme to Akt. We found endogenous association of PP2A AB55C holoenzymes with Akt by co-immunoprecipitation analyses in pro-lymphoid FL5.12 cells. Akt was shown to associate with ectopically expressed B55alpha subunit in NIH3T3 cells. The direct interaction between B55alpha subunit and Akt was confirmed using in vitro pulldown analyses. Intriguingly, we found that overexpression of B55alpha subunit significantly impaired phosphorylation at Thr-308, but to a lesser extent at Ser-473 of Akt in both FL5.12 and NIH3T3 cells. Concomitantly, phosphorylation of a subset of Akt substrates, including FoxO3a, was substantially decreased by B55alpha overexpression in these cells. Silencing of B55alpha expression markedly increased phosphorylation at Thr-308 but not at Ser-473 in both FL5.12 cells and NIH3T3 cells. Consistently, PP2A AB55alphaC holoenzymes preferentially dephosphorylated phospho-Thr-308 rather than phospho-Ser-473 in in vitro dephosphorylation assays. Furthermore, B55alpha overexpression retarded proliferation of NIH3T3 cells, and knockdown of B55alpha expression increased survival of FL5.12 cells upon interleukin-3 deprivation. Together, our data demonstrate that B55alpha-dependent targeting of the PP2A holoenzyme to Akt selectively regulates Akt phosphorylation at Thr-308 to regulate cell proliferation and survival.


Assuntos
Proliferação de Células , Proteína Fosfatase 2/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Animais , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/fisiologia , Inativação Gênica , Holoenzimas/genética , Holoenzimas/metabolismo , Humanos , Interleucina-3/metabolismo , Interleucina-3/farmacologia , Camundongos , Células NIH 3T3 , Fosforilação/efeitos dos fármacos , Proteína Fosfatase 2/genética , Proteínas Proto-Oncogênicas c-akt/genética
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA