Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 11 de 11
Filtrar
1.
Int J Mol Sci ; 24(16)2023 Aug 14.
Artigo em Inglês | MEDLINE | ID: mdl-37628938

RESUMO

Protein misfolding is a common feature of aging, various diseases and stresses. Recent work has revealed that misfolded proteins can be gathered into specific compartments, which can limit their deleterious effects. Chaperones play a central role in the formation of these misfolded protein deposits and can also be used to mark them. While studying chimeric yeast Hsp70 (Ssa1-GFP), we discovered that this protein was prone to the formation of large insoluble deposits during growth on non-fermentable carbon sources under mild heat stress. This was mitigated by the addition of antioxidants, suggesting that either Ssa1 itself or some other proteins were affected by oxidative damage. The protein deposits colocalized with a number of other chaperones, as well as model misfolded proteins, and could be disassembled by the Hsp104 chaperone. Notably, the wild-type protein, as well as a fusion protein of Ssa1 to the fluorescent protein Dendra2, were much less prone to forming similar foci, indicating that this phenomenon was related to the perturbation of Ssa1 function by fusion to GFP. This was also confirmed by monitoring Hsp104-GFP aggregates in the presence of known Ssa1 point mutants. Our data indicate that impaired Ssa1 function can favor the formation of large misfolded protein deposits under various conditions.


Assuntos
Proteínas de Choque Térmico HSP70 , Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Proteínas de Choque Térmico HSP70/genética , Estresse Oxidativo , Causalidade
2.
Int J Mol Sci ; 23(10)2022 May 20.
Artigo em Inglês | MEDLINE | ID: mdl-35628548

RESUMO

Amyloids are protein aggregates with a specific filamentous structure that are related to a number of human diseases, and also to some important physiological processes in animals and other kingdoms of life. Amyloids in yeast can stably propagate as heritable units, prions. Yeast prions are of interest both on their own and as a model for amyloids and prions in general. In this review, we consider the structure of yeast prions and its variation, how such structures determine the balance of aggregated and soluble prion protein through interaction with chaperones and how the aggregated state affects the non-prion functions of these proteins.


Assuntos
Príons , Proteínas de Saccharomyces cerevisiae , Amiloide/metabolismo , Chaperonas Moleculares/metabolismo , Príons/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo
3.
Int J Mol Sci ; 22(4)2021 Feb 03.
Artigo em Inglês | MEDLINE | ID: mdl-33546497

RESUMO

Amyloid formation is associated with many incurable diseases. For some of these, sporadic cases are much more common than familial ones. Some reports point to the role of somatic cell mosaicism in these cases via origination of amyloids in a limited number of cells, which can then spread through tissues. However, specific types of sporadic mutations responsible for such effects are unknown. In order to identify mutations capable of increasing the de novo appearance of amyloids, we searched for such mutants in the yeast prionogenic protein Sup35. We introduced to yeast cells an additional copy of the SUP35 gene with mutated amyloidogenic domain and observed that some nonsense mutations increased the incidence of prions by several orders of magnitude. This effect was related to exposure at the C-terminus of an internal amyloidogenic region of Sup35. We also discovered that SUP35 mRNA could undergo splicing, although inefficiently, causing appearance of a shortened Sup35 isoform lacking its functional domain, which was also highly prionogenic. Our data suggest that truncated forms of amyloidogenic proteins, resulting from nonsense mutations or alternative splicing in rare somatic cells, might initiate spontaneous localized formation of amyloids, which can then spread, resulting in sporadic amyloid disease.


Assuntos
Amiloide/metabolismo , Códon sem Sentido , Príons/genética , Príons/metabolismo , Amiloidose/genética , Amiloidose/metabolismo , Amiloidose/patologia , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Espectrometria de Massas , Príons/química , Agregados Proteicos , Splicing de RNA
4.
Int J Mol Sci ; 20(11)2019 May 29.
Artigo em Inglês | MEDLINE | ID: mdl-31146333

RESUMO

The yeast [PSI+] prion, formed by the Sup35 (eRF3) protein, has multiple structural variants differing in the strength of nonsense suppressor phenotype. Structure of [PSI+] and its variation are characterized poorly. Here, we mapped Sup35 amyloid cores of 26 [PSI+] ex vivo prions of different origin using proteinase K digestion and mass spectrometric identification of resistant peptides. In all [PSI+] variants the Sup35 amino acid residues 2-32 were fully resistant and the region up to residue 72 was partially resistant. Proteinase K-resistant structures were also found within regions 73-124, 125-153, and 154-221, but their presence differed between [PSI+] isolates. Two distinct digestion patterns were observed for region 2-72, which always correlated with the "strong" and "weak" [PSI+] nonsense suppressor phenotypes. Also, all [PSI+] with a weak pattern were eliminated by multicopy HSP104 gene and were not toxic when combined with multicopy SUP35. [PSI+] with a strong pattern showed opposite properties, being resistant to multicopy HSP104 and lethal with multicopy SUP35. Thus, Sup35 prion cores can be composed of up to four elements. [PSI+] variants can be divided into two classes reliably distinguishable basing on structure of the first element and the described assays.


Assuntos
Fatores de Terminação de Peptídeos/metabolismo , Príons/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Endopeptidase K/metabolismo , Proteínas de Choque Térmico/genética , Proteínas de Choque Térmico/metabolismo , Fatores de Terminação de Peptídeos/química , Fatores de Terminação de Peptídeos/genética , Príons/química , Príons/genética , Domínios Proteicos , Multimerização Proteica , Proteólise , Saccharomyces cerevisiae , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética
5.
Int J Mol Sci ; 19(11)2018 Nov 20.
Artigo em Inglês | MEDLINE | ID: mdl-30463309

RESUMO

The [PSI⁺] nonsense-suppressor determinant of Saccharomyces cerevisiae is based on the formation of heritable amyloids of the Sup35 (eRF3) translation termination factor. [PSI⁺] amyloids have variants differing in amyloid structure and in the strength of the suppressor phenotype. The appearance of [PSI⁺], its propagation and manifestation depend primarily on chaperones. Besides chaperones, the Upf1/2/3, Siw14 and Arg82 proteins restrict [PSI⁺] formation, while Sla2 can prevent [PSI⁺] toxicity. Here, we identify two more non-chaperone proteins involved in [PSI⁺] detoxification. We show that simultaneous lack of the Pub1 and Upf1 proteins is lethal to cells harboring [PSI⁺] variants with a strong, but not with a weak, suppressor phenotype. This lethality is caused by excessive depletion of the Sup45 (eRF1) termination factor due to its sequestration into Sup35 polymers. We also show that Pub1 acts to restrict excessive Sup35 prion polymerization, while Upf1 interferes with Sup45 binding to Sup35 polymers. These data allow consideration of the Pub1 and Upf1 proteins as a novel [PSI⁺] detoxification system.


Assuntos
Proteínas de Ligação a Poli(A)/metabolismo , Príons/toxicidade , RNA Helicases/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Cromossomos Fúngicos/genética , Códon sem Sentido/genética , Deleção de Genes , Modelos Biológicos , Fatores de Terminação de Peptídeos/metabolismo , Plasmídeos/metabolismo , Polimerização , Saccharomyces cerevisiae/efeitos dos fármacos , Mutações Sintéticas Letais
6.
Cell Biochem Funct ; 31(1): 30-5, 2013 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-22763713

RESUMO

It is known that ouabain, a selective inhibitor of Na/K-ATPase, not only can cause the activation of signal cascades, which regulate the cell viability, but also can cause the accumulation of free radicals, which can evoke the oxidative stress. We have shown that the nanomolar concentrations of ouabain result in the temporary increase in the level of intracellular free radicals, but the millimolar concentration of ouabain induces a stable intracellular accumulation of free radicals in rat thymocytes. The increasing level of free radicals resulting from both low and high concentrations of ouabain can be attenuated by the antioxidant, carnosine. Moreover, the long-term incubation with ouabain leads to the cell death by necrosis and apoptosis. Ouabain-mediated apoptosis and necrosis were also abolished by carnosine.


Assuntos
Carnosina/farmacologia , Ouabaína/antagonistas & inibidores , ATPase Trocadora de Sódio-Potássio/efeitos dos fármacos , Timócitos/efeitos dos fármacos , Animais , Apoptose/efeitos dos fármacos , Sinalização do Cálcio/efeitos dos fármacos , Meios de Cultura/farmacologia , Relação Dose-Resposta a Droga , Ativação Enzimática/efeitos dos fármacos , Radicais Livres/metabolismo , Necrose , Óxido Nítrico Sintase/metabolismo , Nitroarginina/farmacologia , Ouabaína/farmacologia , Isoformas de Proteínas/efeitos dos fármacos , Ratos , Ratos Endogâmicos WKY , Transdução de Sinais/efeitos dos fármacos , Timócitos/citologia , Timócitos/enzimologia
7.
Biomolecules ; 11(12)2021 12 15.
Artigo em Inglês | MEDLINE | ID: mdl-34944528

RESUMO

Amyloids are filamentous protein aggregates that are associated with a number of incurable diseases, termed amyloidoses. Amyloids can also manifest as infectious or heritable particles, known as prions. While just one prion is known in humans and animals, more than ten prion amyloids have been discovered in fungi. The propagation of fungal prion amyloids requires the chaperone Hsp104, though in excess it can eliminate some prions. Even though Hsp104 acts to disassemble prion fibrils, at normal levels it fragments them into multiple smaller pieces, which ensures prion propagation and accelerates prion conversion. Animals lack Hsp104, but disaggregation is performed by the same complement of chaperones that assist Hsp104 in yeast-Hsp40, Hsp70, and Hsp110. Exogenous Hsp104 can efficiently cooperate with these chaperones in animals and promotes disaggregation, especially of large amyloid aggregates, which indicates its potential as a treatment for amyloid diseases. However, despite the significant effects, Hsp104 and its potentiated variants may be insufficient to fully dissolve amyloid. In this review, we consider chaperone mechanisms acting to disassemble heritable protein aggregates in yeast and animals, and their potential use in the therapy of human amyloid diseases.


Assuntos
Amiloide/metabolismo , Fungos/metabolismo , Proteínas de Choque Térmico/metabolismo , Príons/metabolismo , Amiloide/química , Animais , Proteínas Fúngicas/química , Proteínas Fúngicas/metabolismo , Proteínas de Choque Térmico/química , Humanos , Modelos Moleculares , Príons/química , Agregados Proteicos , Conformação Proteica
8.
Prion ; 14(1): 11-19, 2020 12.
Artigo em Inglês | MEDLINE | ID: mdl-31876447

RESUMO

Amyloids and their infectious subset, prions, represent fibrillary aggregates with regular structure. They are formed by proteins that are soluble in their normal state. In amyloid form, all or part of the polypeptide sequence of the protein is resistant to treatment with proteinase K (PK). Amyloids can have structural variants, which can be distinguished by the patterns of their digestion by PK. In this review, we describe and compare studies of the resistant cores of various amyloids from different organisms. These data provide insight into the fine structure of amyloids and their variants as well as raise interesting questions, such as those concerning the differences between amyloids obtained ex vivo and in vitro, as well as the manner in which folding of one region of the amyloid can affect other regions.


Assuntos
Amiloide/metabolismo , Endopeptidase K/metabolismo , Príons/metabolismo , Amiloide/química , Animais , Humanos , Príons/química , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , alfa-Sinucleína/metabolismo
9.
MethodsX ; 6: 329-332, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30847282

RESUMO

Microscopy of multiple samples using slides and coverslips is time consuming and good images are sometimes difficult to obtain due to cell movement. Our method involves manual spotting of multiple cell samples onto solid-medium pads, which creates the following benefits: •Rapid, high-quality imaging of multiple samples (hundreds per day) by visible and fluorescence microscopy.•No need for expensive automated equipment, multi-well plates or large amounts of consumables.•Wide range of working cell concentrations. The method was implemented for S. cerevisiae yeast, however it is likely to be applicable to all types of microorganisms and possibly other microscopic samples such as pollen. The lack of need for automated equipment may also make this method useful for field work.

10.
Biol Open ; 8(7)2019 Jul 10.
Artigo em Inglês | MEDLINE | ID: mdl-31285266

RESUMO

Proteins can aggregate in response to stresses, including hyperosmotic shock. Formation and disassembly of aggregates is a relatively slow process. We describe a novel instant response of the cell to hyperosmosis, during which chaperones and other proteins form numerous foci with properties uncharacteristic of classical aggregates. These foci appeared/disappeared seconds after shock onset/removal, in close correlation with cell volume changes. Genome-wide and targeted testing revealed chaperones, metabolic enzymes, P-body components and amyloidogenic proteins in the foci. Most of these proteins can form large assemblies and for some, the assembled state was pre-requisite for participation in foci. A genome-wide screen failed to identify genes whose absence prevented foci participation by Hsp70. Shapes of and interconnections between foci, revealed by super-resolution microscopy, indicated that the foci were compressed between other entities. Based on our findings, we suggest a new model of cytosol architecture as a collection of numerous gel-like regions suspended in a liquid network. This network is reduced in volume in response to hyperosmosis and forms small pockets between the gel-like regions.

11.
PLoS One ; 9(12): e116003, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-25549323

RESUMO

Despite extensive study, progress in elucidation of biological functions of amyloids and their role in pathology is largely restrained due to the lack of universal and reliable biochemical methods for their discovery. All biochemical methods developed so far allowed only identification of glutamine/asparagine-rich amyloid-forming proteins or proteins comprising amyloids that form large deposits. In this article we present a proteomic approach which may enable identification of a broad range of amyloid-forming proteins independently of specific features of their sequences or levels of expression. This approach is based on the isolation of protein fractions enriched with amyloid aggregates via sedimentation by ultracentrifugation in the presence of strong ionic detergents, such as sarkosyl or SDS. Sedimented proteins are then separated either by 2D difference gel electrophoresis or by SDS-PAGE, if they are insoluble in the buffer used for 2D difference gel electrophoresis, after which they are identified by mass-spectrometry. We validated this approach by detection of known yeast prions and mammalian proteins with established capacity for amyloid formation and also revealed yeast proteins forming detergent-insoluble aggregates in the presence of human huntingtin with expanded polyglutamine domain. Notably, with one exception, all these proteins contained glutamine/asparagine-rich stretches suggesting that their aggregates arose due to polymerization cross-seeding by human huntingtin. Importantly, though the approach was developed in a yeast model, it can easily be applied to any organism thus representing an efficient and universal tool for screening for amyloid proteins.


Assuntos
Proteínas Amiloidogênicas/isolamento & purificação , Proteínas Amiloidogênicas/metabolismo , Proteômica/métodos , Animais , Detergentes/farmacologia , Eletroforese em Gel Bidimensional , Proteínas Fúngicas/isolamento & purificação , Humanos , Proteína Huntingtina , Espectrometria de Massas/métodos , Proteínas do Tecido Nervoso/metabolismo , Sarcosina/análogos & derivados , Sarcosina/farmacologia
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA