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1.
Cell Rep ; 42(11): 113372, 2023 11 28.
Artigo em Inglês | MEDLINE | ID: mdl-37938971

RESUMO

Metacaspases are ancestral homologs of caspases that can either promote cell death or confer cytoprotection. Furthermore, yeast (Saccharomyces cerevisiae) metacaspase Mca1 possesses dual biochemical activity: proteolytic activity causing cell death and cytoprotective, co-chaperone-like activity retarding replicative aging. The molecular mechanism favoring one activity of Mca1 over another remains elusive. Here, we show that this mechanism involves calmodulin binding to the N-terminal pro-domain of Mca1, which prevents its proteolytic activation and promotes co-chaperone-like activity, thus switching from pro-cell death to anti-aging function. The longevity-promoting effect of Mca1 requires the Hsp40 co-chaperone Sis1, which is necessary for Mca1 recruitment to protein aggregates and their clearance. In contrast, proteolytically active Mca1 cleaves Sis1 both in vitro and in vivo, further clarifying molecular mechanism behind a dual role of Mca1 as a cell-death protease versus gerontogene.


Assuntos
Peptídeo Hidrolases , Proteínas de Saccharomyces cerevisiae , Peptídeo Hidrolases/metabolismo , Calmodulina/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Caspases/metabolismo , Saccharomyces cerevisiae/metabolismo , Chaperonas Moleculares/metabolismo
2.
Nat Commun ; 12(1): 4863, 2021 08 11.
Artigo em Inglês | MEDLINE | ID: mdl-34381050

RESUMO

Spreading of aggregate pathology across brain regions acts as a driver of disease progression in Tau-related neurodegeneration, including Alzheimer's disease (AD) and frontotemporal dementia. Aggregate seeds released from affected cells are internalized by naïve cells and induce the prion-like templating of soluble Tau into neurotoxic aggregates. Here we show in a cellular model system and in neurons that Clusterin, an abundant extracellular chaperone, strongly enhances Tau aggregate seeding. Upon interaction with Tau aggregates, Clusterin stabilizes highly potent, soluble seed species. Tau/Clusterin complexes enter recipient cells via endocytosis and compromise the endolysosomal compartment, allowing transfer to the cytosol where they propagate aggregation of endogenous Tau. Thus, upregulation of Clusterin, as observed in AD patients, may enhance Tau seeding and possibly accelerate the spreading of Tau pathology.


Assuntos
Clusterina/metabolismo , Agregação Patológica de Proteínas/metabolismo , Proteínas tau/metabolismo , Animais , Clusterina/genética , Progressão da Doença , Endocitose , Humanos , Camundongos , Doenças Neurodegenerativas/metabolismo , Doenças Neurodegenerativas/patologia , Neurônios/metabolismo , Neurônios/patologia , Agregação Patológica de Proteínas/patologia , Ligação Proteica , alfa-Sinucleína/metabolismo , Proteínas tau/genética
3.
J Mol Biol ; 432(7): 2304-2318, 2020 03 27.
Artigo em Inglês | MEDLINE | ID: mdl-32135190

RESUMO

The cylindrical chaperonin GroEL and its cofactor GroES mediate ATP-dependent protein folding in Escherichia coli by transiently encapsulating non-native substrate in a nano-cage formed by the GroEL ring cavity and the lid-shaped GroES. Mechanistic studies of GroEL/ES with heterologous protein substrates suggested that the chaperonin is inefficient, typically requiring multiple ATP-dependent encapsulation cycles with only a few percent of protein folded per cycle. Here we analyzed the spontaneous and chaperonin-assisted folding of the essential enzyme 5,10-methylenetetrahydrofolate reductase (MetF) of E. coli, an obligate GroEL/ES substrate. We found that MetF, a homotetramer of 33-kDa subunits with (ß/α)8 TIM-barrel fold, populates a kinetically trapped folding intermediate(s) (MetF-I) upon dilution from denaturant that fails to convert to the native state, even in the absence of aggregation. GroEL/ES recognizes MetF-I and catalyzes rapid folding, with ~50% of protein folded in a single round of encapsulation. Analysis by hydrogen/deuterium exchange at peptide resolution showed that the MetF subunit folds to completion in the GroEL/ES nano-cage and binds its cofactor flavin adenine dinucleotide. Rapid folding required the net negative charge character of the wall of the chaperonin cavity. These findings reveal a remarkable capacity of GroEL/ES to catalyze folding of an endogenous substrate protein that would have coevolved with the chaperonin system.


Assuntos
5,10-Metilenotetra-Hidrofolato Redutase (FADH2)/metabolismo , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Proteínas de Choque Térmico/química , Proteínas de Choque Térmico/metabolismo , Dobramento de Proteína , Trifosfato de Adenosina/metabolismo , Catálise , Cinética , Modelos Moleculares , Conformação Proteica , Termodinâmica
4.
Nat Commun ; 11(1): 365, 2020 01 17.
Artigo em Inglês | MEDLINE | ID: mdl-31953415

RESUMO

The ATP-dependent Hsp70 chaperones (DnaK in E. coli) mediate protein folding in cooperation with J proteins and nucleotide exchange factors (E. coli DnaJ and GrpE, respectively). The Hsp70 system prevents protein aggregation and increases folding yields. Whether it also enhances the rate of folding remains unclear. Here we show that DnaK/DnaJ/GrpE accelerate the folding of the multi-domain protein firefly luciferase (FLuc) ~20-fold over the rate of spontaneous folding measured in the absence of aggregation. Analysis by single-pair FRET and hydrogen/deuterium exchange identified inter-domain misfolding as the cause of slow folding. DnaK binding expands the misfolded region and thereby resolves the kinetically-trapped intermediates, with folding occurring upon GrpE-mediated release. In each round of release DnaK commits a fraction of FLuc to fast folding, circumventing misfolding. We suggest that by resolving misfolding and accelerating productive folding, the bacterial Hsp70 system can maintain proteins in their native states under otherwise denaturing stress conditions.


Assuntos
Proteínas de Escherichia coli/metabolismo , Proteínas de Choque Térmico HSP40/metabolismo , Proteínas de Choque Térmico HSP70/metabolismo , Proteínas de Choque Térmico/metabolismo , Domínios Proteicos , Dobramento de Proteína , Animais , Sítios de Ligação , Escherichia coli/metabolismo , Proteínas de Escherichia coli/química , Vaga-Lumes/enzimologia , Vaga-Lumes/genética , Proteínas de Choque Térmico HSP40/química , Proteínas de Choque Térmico HSP70/química , Proteínas de Choque Térmico/química , Cinética , Luciferases de Vaga-Lume/química , Luciferases de Vaga-Lume/genética , Modelos Moleculares , Chaperonas Moleculares/metabolismo , Conformação Proteica
5.
Biotechnol Appl Biochem ; 66(6): 915-923, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31396993

RESUMO

Nicotinamide adenine dinucleotide phosphate (NAD(P)H)-flavin oxidoreductases (flavin reductases) catalyze the reduction of flavin by NAD(P)H and provide the reduced form of flavin mononucleotide (FMN) to flavin-dependent monooxygenases. Based on bioinformatics analysis, we identified a putative flavin reductase gene, sso2055, in the genome of hyperthermophilic archaeon Sulfolobus solfataricus P2, and further cloned this target sequence into an expression vector. The cloned flavin reductase (EC. 1.5.1.30) was purified to homogeneity and characterized further. The purified enzyme exists as a monomer of 17.8 kDa, free of chromogenic cofactors. Homology modeling revealed this enzyme as a TIM barrel, which is also supported by circular dichroism measurements revealing a beta-sheet rich content. The optimal pH for SSO2055 activity was pH 6.5 in phosphate buffer and the highest activity observed was at 120 °C within the measurable temperature. We showed that this enzyme can use FMN and flavin adenine dinucleotide (FAD) as a substrate to generate their reduced forms. The purified enzyme is predicted to be a potential flavin reductase of flavin-dependent monooxygenases that could be involved in the biodesulfurization process of S. solfataricus P2.


Assuntos
Oxirredutases/metabolismo , Sulfolobus solfataricus/enzimologia , Temperatura , Biologia Computacional , Oxirredutases/genética , Oxirredutases/isolamento & purificação
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