RESUMO
Numerous studies have demonstrated that people with type 2 diabetes mellitus (associated with IAPP peptide aggregation) show an increased incidence of Alzheimer's disease (associated with Aß aggregation), but the mechanism responsible for this correlation is presently unknown. Here, we applied a yeast-based model to study the interactions of IAPP with PrP (associated with TSEs) and with the Aß42 peptide. We demonstrated that fluorescently tagged IAPP forms detergent-resistant aggregates in yeast cells. Using the FRET approach, we showed that IAPP and Aß aggregates co-localize and physically interact in yeast cells. We also showed that this interaction is specific and that there is no interaction between IAPP and PrP in the yeast system. Our data confirmed a direct physical interaction between IAPP and Aß42 aggregates in a living cell. Based on these findings, we hypothesize that this interaction may play a crucial role in seeding Aß42 aggregation in T2DM patients, thereby promoting the development of AD.
Assuntos
Doença de Alzheimer , Diabetes Mellitus Tipo 2 , Humanos , Peptídeos beta-Amiloides , Saccharomyces cerevisiae , Polipeptídeo Amiloide das Ilhotas PancreáticasRESUMO
Understanding the aggregation mechanism of amyloid proteins, such as Sup35NM, is essential to understanding amyloid diseases. Significant recent work has focused on using the fluorescence of thioflavin T (ThT), which undergoes a red shift when bound to amyloid aggregates, to monitor amyloid fibril formation. In the present study, the progression of the total mass of aggregates during fibril formation is monitored for initial monomer concentrations in order to infer the relevant aggregation mechanisms. This workflow was implemented using the amyloid-forming fragment Sup35NM under different agitation conditions and for initial monomer concentrations spanning 2 orders of magnitude. The analysis suggests that primary nucleation, monomeric elongation, secondary nucleation, and fragmentation might all be relevant, but their relative importance could not be determined unambiguously, despite the large set of high-quality data. Discriminating between the fibril-generating processes is shown to require additional information, such as a fibril length distribution. Using Sup35NM as a case study, a framework for fitting the parameters of arbitrary amyloid aggregation kinetics is developed based on a population balance model (PBM), which resolves not only the total aggregate mass (monitored experimentally via ThT fluorescence) but the entire fibril length distribution over time. In addition to the rich new set of ThT fluorescence data, we have reanalyzed a previously published aggregate size distribution using this method. With the size distribution, it was determined that in the reanalyzed in vitro experiment, secondary nucleation generated significantly fewer new Sup35NM fibrils than fragmentation. The proposed strategy of applying the same PBM to a combination of kinetic data from fluorescence monitoring and experimental fibril length distributions will allow the inference of aggregation mechanisms with far greater confidence than fluorescence studies alone.
Assuntos
Amiloide , Amiloidose , Proteínas Amiloidogênicas , Humanos , CinéticaRESUMO
Amyloids are highly ordered fibrous cross-ß protein aggregates that are notorious primarily because of association with a variety of incurable human and animal diseases (termed amyloidoses), including Alzheimer's disease (AD), Parkinson's disease (PD), type 2 diabetes (T2D), and prion diseases. Some amyloid-associated diseases, in particular T2D and AD, are widespread and affect hundreds of millions of people all over the world. However, recently it has become evident that many amyloids, termed "functional amyloids," are involved in various activities that are beneficial to organisms. Functional amyloids were discovered in diverse taxa, ranging from bacteria to mammals. These amyloids are involved in vital biological functions such as long-term memory, storage of peptide hormones and scaffolding melanin polymerization in animals, substrate attachment, and biofilm formation in bacteria and fungi, etc. Thus, amyloids undoubtedly are playing important roles in biological and pathological processes. This review is focused on functional amyloids in mammals and summarizes approaches used for identifying new potentially amyloidogenic proteins and domains.
RESUMO
Amyloids are fibrous cross-ß protein aggregates that are capable of proliferation via nucleated polymerization. Amyloid conformation likely represents an ancient protein fold and is linked to various biological or pathological manifestations. Self-perpetuating amyloid-based protein conformers provide a molecular basis for transmissible (infectious or heritable) protein isoforms, termed prions. Amyloids and prions, as well as other types of misfolded aggregated proteins are associated with a variety of devastating mammalian and human diseases, such as Alzheimer's, Parkinson's and Huntington's diseases, transmissible spongiform encephalopathies (TSEs), amyotrophic lateral sclerosis (ALS) and transthyretinopathies. In yeast and fungi, amyloid-based prions control phenotypically detectable heritable traits. Simplicity of cultivation requirements and availability of powerful genetic approaches makes yeast Saccharomyces cerevisiae an excellent model system for studying molecular and cellular mechanisms governing amyloid formation and propagation. Genetic techniques allowing for the expression of mammalian or human amyloidogenic and prionogenic proteins in yeast enable researchers to capitalize on yeast advantages for characterization of the properties of disease-related proteins. Chimeric constructs employing mammalian and human aggregation-prone proteins or domains, fused to fluorophores or to endogenous yeast proteins allow for cytological or phenotypic detection of disease-related protein aggregation in yeast cells. Yeast systems are amenable to high-throughput screening for antagonists of amyloid formation, propagation and/or toxicity. This review summarizes up to date achievements of yeast assays in application to studying mammalian and human disease-related aggregating proteins, and discusses both limitations and further perspectives of yeast-based strategies.
Assuntos
Peptídeos beta-Amiloides/metabolismo , Amiloidose/metabolismo , Doenças Priônicas/metabolismo , Príons/metabolismo , Saccharomyces cerevisiae/metabolismo , alfa-Sinucleína/metabolismo , Proteínas tau/metabolismo , Doença de Alzheimer/genética , Doença de Alzheimer/metabolismo , Peptídeos beta-Amiloides/química , Peptídeos beta-Amiloides/genética , Amiloidose/genética , Esclerose Lateral Amiotrófica/genética , Esclerose Lateral Amiotrófica/metabolismo , Animais , Humanos , Doença de Parkinson/genética , Doença de Parkinson/metabolismo , Doenças Priônicas/genética , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Proteínas tau/genéticaRESUMO
Previous studies have reported an inverse association between cancer and Alzheimer's disease (AD), which are leading causes of human morbidity and mortality. We analyzed the SEER (Surveillance, Epidemiology, and End Results) data to estimate the risk of AD death in (i) cancer patients relative to reference populations stratified on demographic and clinical variables, and (ii) female breast cancer (BC) patients treated with chemotherapy or radiotherapy, relative to those with no/unknown treatment status. Our results demonstrate the impact of race, cancer type, age and time since cancer diagnosis on the risk of AD death in cancer patients. While the risk of AD death was decreased in white patients diagnosed with various cancers at 45 or more years of age, it was increased in black patients diagnosed with cancers before 45 years of age (likely due to early onset AD). Chemotherapy decreased the risk of AD death in white women diagnosed with BC at the age of 65 or more, however radiotherapy displayed a more complex pattern with early decrease and late increase in the risk of AD death during a prolonged time interval after the treatment. Our data point to links between molecular mechanisms involved in cancer and AD, and to the potential applicability of some anti-cancer treatments against AD.
RESUMO
Biomolecular assembly is a key driving force in nearly all life processes, providing structure, information storage, and communication within cells and at the whole organism level. These assembly processes rely on precise interactions between functional groups on nucleic acids, proteins, carbohydrates, and small molecules, and can be fine-tuned to span a range of time, length, and complexity scales. Recognizing the power of these motifs, researchers have sought to emulate and engineer biomolecular assemblies in the laboratory, with goals ranging from modulating cellular function to the creation of new polymeric materials. In most cases, engineering efforts are inspired or informed by understanding the structure and properties of naturally occurring assemblies, which has in turn fueled the development of predictive models that enable computational design of novel assemblies. This Review will focus on selected examples of protein assemblies, highlighting the story arc from initial discovery of an assembly, through initial engineering attempts, toward the ultimate goal of predictive design. The aim of this Review is to highlight areas where significant progress has been made, as well as to outline remaining challenges, as solving these challenges will be the key that unlocks the full power of biomolecules for advances in technology and medicine.
Assuntos
Peptídeos/síntese química , Polímeros/síntese química , Proteínas/síntese química , Modelos Moleculares , Peptídeos/química , Polímeros/química , Proteínas/químicaRESUMO
Endogenous yeast amyloids that control heritable traits and are frequently used as models for human amyloid diseases are termed yeast prions. Yeast prions, including the best studied ones ([PSI +] and [URE3]), propagate via intimate interactions with molecular chaperones. Different yeast prions exhibit differential responses to changes in levels, functionality or localization of the components of chaperone machinery. Here, we provide additional data confirming differential effects of chaperones (and specifically, Hsp40s) on yeast prions and summarize current knowledge of the mechanisms underlying chaperone specificities. Contrary to frequent statements in literature, overproduction of the Hsp104 chaperone antagonizes both [PSI +] and [URE3] prions, while overproduction of the Hsp70-Ssa1 chaperone antagonizes [URE3] prion only in some, but not in all strains. Recently, we demonstrated that the relocalization of a fraction of the Hsp40 chaperone Sis1 from the cytosol to the nucleus by the chaperone-sorting factor Cur1 exhibits opposite effects on [PSI +] and [URE3] prions. We suggest that the response of prions to changes in Sis1 localization represents a combination of the effects of Sis1 shortage on fragmentation of prion aggregates and on malpartition of prion aggregates during a cell division. Differences in sensitivity of prion fragmentation to Sis1 and in relative inputs of fragmentation and malpartition in prion propagation result in opposite effects of Sis1 relocalization on [PSI +] and [URE3].
Assuntos
Glutationa Peroxidase/genética , Proteínas de Choque Térmico HSP40/genética , Chaperonas Moleculares/genética , Príons/genética , Proteínas de Saccharomyces cerevisiae/genética , Adenosina Trifosfatases/genética , Proteínas de Choque Térmico HSP70/genética , Saccharomyces cerevisiae/genéticaRESUMO
Prions are infectious proteins that cause fatal neurodegenerative disorders including Creutzfeldt-Jakob and bovine spongiform encephalopathy (mad cow) diseases. The yeast [PSI+] prion is formed by the translation-termination factor Sup35, is the best-studied prion, and provides a useful model system for studying such diseases. However, despite recent progress in the understanding of prion diseases, the cellular defense mechanism against prions has not been elucidated. Here, we report that proteolytic cleavage of Sup35 suppresses spontaneous de novo generation of the [PSI+] prion. We found that during yeast growth in glucose media, a maximum of 40% of Sup35 is cleaved at its N-terminal prion domain. This cleavage requires the vacuolar proteases PrA-PrB. Cleavage occurs in a manner dependent on translation but independently of autophagy between the glutamine/asparagine-rich (Q/N-rich) stretch critical for prion formation and the oligopeptide-repeat region required for prion maintenance, resulting in the removal of the Q/N-rich stretch from the Sup35 N terminus. The complete inhibition of Sup35 cleavage, by knocking out either PrA (pep4Δ) or PrB (prb1Δ), increased the rate of de novo formation of [PSI+] prion up to â¼5-fold, whereas the activation of Sup35 cleavage, by overproducing PrB, inhibited [PSI+] formation. On the other hand, activation of the PrB pathway neither cleaved the amyloid conformers of Sup35 in [PSI+] strains nor eliminated preexisting [PSI+]. These findings point to a mechanism antagonizing prion generation in yeast. Our results underscore the usefulness of the yeast [PSI+] prion as a model system to investigate defense mechanisms against prion diseases and other amyloidoses.
Assuntos
Proteínas Fúngicas/metabolismo , Fatores de Terminação de Peptídeos/metabolismo , Proteínas Priônicas/metabolismo , Príons/antagonistas & inibidores , Proteólise , Proteínas de Saccharomyces cerevisiae/metabolismo , Peptídeo Hidrolases/metabolismo , Príons/metabolismo , Vacúolos/enzimologia , Leveduras/metabolismoRESUMO
Cotranslational degradation of polypeptide nascent chains plays a critical role in quality control of protein synthesis and the rescue of stalled ribosomes. In eukaryotes, ribosome stalling triggers release of 60S subunits with attached nascent polypeptides, which undergo ubiquitination by the E3 ligase Ltn1 and proteasomal degradation facilitated by the ATPase Cdc48. However, the identity of factors acting upstream in this process is less clear. Here, we examined how the canonical release factors Sup45-Sup35 (eRF1-eRF3) and their paralogs Dom34-Hbs1 affect the total population of ubiquitinated nascent chains associated with yeast ribosomes. We found that the availability of the functional release factor complex Sup45-Sup35 strongly influences the amount of ubiquitinated polypeptides associated with 60S ribosomal subunits, while Dom34-Hbs1 generate 60S-associated peptidyl-tRNAs that constitute a relatively minor fraction of Ltn1 substrates. These results uncover two separate pathways that target nascent polypeptides for Ltn1-Cdc48-mediated degradation and suggest that in addition to canonical termination on stop codons, eukaryotic release factors contribute to cotranslational protein quality control.
Assuntos
Terminação Traducional da Cadeia Peptídica/genética , Ribossomos/metabolismo , Saccharomyces cerevisiae/genética , Modelos Biológicos , Peso Molecular , Peptídeos/metabolismo , Subunidades Ribossômicas Maiores de Eucariotos/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Ubiquitina/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , UbiquitinaçãoRESUMO
The aggresome is an organelle that recruits aggregated proteins for storage and degradation. We performed an siRNA screen for proteins involved in aggresome formation and identified novel mammalian AAA+ protein disaggregases RuvbL1 and RuvbL2. Depletion of RuvbL1 or RuvbL2 suppressed aggresome formation and caused buildup of multiple cytoplasmic aggregates. Similarly, downregulation of RuvbL orthologs in yeast suppressed the formation of an aggresome-like body and enhanced the aggregate toxicity. In contrast, their overproduction enhanced the resistance to proteotoxic stress independently of chaperone Hsp104. Mammalian RuvbL associated with the aggresome, and the aggresome substrate synphilin-1 interacted directly with the RuvbL1 barrel-like structure near the opening of the central channel. Importantly, polypeptides with unfolded structures and amyloid fibrils stimulated the ATPase activity of RuvbL. Finally, disassembly of protein aggregates was promoted by RuvbL. These data indicate that RuvbL complexes serve as chaperones in protein disaggregation.
Assuntos
Amiloide/metabolismo , Proteínas de Transporte/metabolismo , DNA Helicases/metabolismo , Organelas/metabolismo , ATPases Associadas a Diversas Atividades Celulares , Amiloide/genética , Proteínas de Transporte/genética , DNA Helicases/genética , Células HEK293 , Células HeLa , Proteínas de Choque Térmico/genética , Proteínas de Choque Térmico/metabolismo , Humanos , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Organelas/genética , Organelas/patologiaRESUMO
Yeast prions are self-propagating amyloid-like aggregates of Q/N-rich protein that confer heritable traits and provide a model of mammalian amyloidoses. [PSI(+)] is a prion isoform of the translation termination factor Sup35. Propagation of [PSI(+)] during cell division under normal conditions and during the recovery from damaging environmental stress depends on cellular chaperones and is influenced by ubiquitin proteolysis and the actin cytoskeleton. The paralogous yeast proteins Lsb1 and Lsb2 bind the actin assembly protein Las17 (a yeast homolog of human Wiskott-Aldrich syndrome protein) and participate in the endocytic pathway. Lsb2 was shown to modulate maintenance of [PSI(+)] during and after heat shock. Here, we demonstrate that Lsb1 also regulates maintenance of the Sup35 prion during and after heat shock. These data point to the involvement of Lsb proteins in the partitioning of protein aggregates in stressed cells. Lsb1 abundance and cycling between actin patches, endoplasmic reticulum, and cytosol is regulated by the Guided Entry of Tail-anchored proteins pathway and Rsp5-dependent ubiquitination. Heat shock-induced proteolytic processing of Lsb1 is crucial for prion maintenance during stress. Our findings identify Lsb1 as another component of a tightly regulated pathway controlling protein aggregation in changing environments.
Assuntos
Actinas/metabolismo , Proteínas de Transporte/metabolismo , Resposta ao Choque Térmico , Fatores de Terminação de Peptídeos/metabolismo , Príons/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/fisiologia , Proteínas de Transporte/genética , Citoesqueleto/genética , Citoesqueleto/metabolismo , Fatores de Terminação de Peptídeos/genética , Príons/genética , Proteólise , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genéticaRESUMO
Polyglutamine expansion causes diseases in humans and other mammals. One example is Huntington's disease. Fragments of human huntingtin protein having an expanded polyglutamine stretch form aggregates and cause cytotoxicity in yeast cells bearing endogenous QN-rich proteins in the aggregated (prion) form. Attachment of the proline(P)-rich region targets polyglutamines to the large perinuclear deposit (aggresome). Aggresome formation ameliorates polyglutamine cytotoxicity in cells containing only the prion form of Rnq1 protein. Here we show that expanded polyglutamines both with (poly-QP) or without (poly-Q) a P-rich stretch remain toxic in the presence of the prion form of translation termination (release) factor Sup35 (eRF3). A Sup35 derivative that lacks the QN-rich domain and is unable to be incorporated into aggregates counteracts cytotoxicity, suggesting that toxicity is due to Sup35 sequestration. Increase in the levels of another release factor, Sup45 (eRF1), due to either disomy by chromosome II containing the SUP45 gene or to introduction of the SUP45-bearing plasmid counteracts poly-Q or poly-QP toxicity in the presence of the Sup35 prion. Protein analysis confirms that polyglutamines alter aggregation patterns of Sup35 and promote aggregation of Sup45, while excess Sup45 counteracts these effects. Our data show that one and the same mode of polyglutamine aggregation could be cytoprotective or cytotoxic, depending on the composition of other aggregates in a eukaryotic cell, and demonstrate that other aggregates expand the range of proteins that are susceptible to sequestration by polyglutamines.
Assuntos
Dosagem de Genes , Fatores de Terminação de Peptídeos , Peptídeos , Príons/genética , Proteínas de Saccharomyces cerevisiae , Epigênese Genética , Humanos , Proteína Huntingtina , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Fatores de Terminação de Peptídeos/genética , Fatores de Terminação de Peptídeos/metabolismo , Peptídeos/genética , Peptídeos/toxicidade , Príons/química , Príons/metabolismo , Ligação Proteica/genética , Biossíntese de Proteínas , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Deleção de SequênciaRESUMO
Yeast prions are self-perpetuating, QN-rich amyloids that control heritable traits and serve as a model for mammalian amyloidoses. De novo prion formation by overproduced prion protein is facilitated by other aggregated QN-rich protein(s) and is influenced by alterations of protein homeostasis. Here we explore the mechanism by which the Las17-binding protein Lsb2 (Pin3) promotes conversion of the translation termination factor Sup35 into its prion form, [PSI(+)]. We show that Lsb2 localizes with some Sup35 aggregates and that Lsb2 is a short-lived protein whose levels are controlled via the ubiquitin-proteasome system and are dramatically increased by stress. Loss of Lsb2 decreases stability of [PSI(+)] after brief heat shock. Mutations interfering with Lsb2 ubiquitination increase prion induction, while a mutation eliminating association of Lsb2 with the actin cytoskeleton blocks its aggregation and prion-inducing ability. These findings directly implicate the UPS and actin cytoskeleton in regulating prions via a stress-inducible QN-rich protein.
Assuntos
Actinas/metabolismo , Proteínas de Transporte/genética , Citoesqueleto/metabolismo , Príons/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Ubiquitinação/fisiologia , Proteínas de Transporte/metabolismo , Mutação , Fatores de Terminação de Peptídeos/genética , Fatores de Terminação de Peptídeos/metabolismo , Príons/genética , Complexo de Endopeptidases do Proteassoma/genética , Complexo de Endopeptidases do Proteassoma/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Estresse FisiológicoRESUMO
The glutamine/asparagine (Q/N)-rich yeast prion protein Sup35 has a low intrinsic propensity to spontaneously self-assemble into ordered, beta-sheet-rich amyloid fibrils. In yeast cells, de novo formation of Sup35 aggregates is greatly facilitated by high protein concentrations and the presence of preformed Q/N-rich protein aggregates that template Sup35 polymerization. Here, we have investigated whether aggregation-promoting polyglutamine (polyQ) tracts can stimulate the de novo formation of ordered Sup35 protein aggregates in the absence of Q/N-rich yeast prions. Fusion proteins with polyQ tracts of different lengths were produced and their ability to spontaneously self-assemble into amlyloid structures was analyzed using in vitro and in vivo model systems. We found that Sup35 fusions with pathogenic (>or=54 glutamines), as opposed to non-pathogenic (19 glutamines) polyQ tracts efficiently form seeding-competent protein aggregates. Strikingly, polyQ-mediated de novo assembly of Sup35 protein aggregates in yeast cells was independent of pre-existing Q/N-rich protein aggregates. This indicates that increasing the content of aggregation-promoting sequences enhances the tendency of Sup35 to spontaneously self-assemble into insoluble protein aggregates. A similar result was obtained when pathogenic polyQ tracts were linked to the yeast prion protein Rnq1, demonstrating that polyQ sequences are generic inducers of amyloidogenesis. In conclusion, long polyQ sequences are powerful molecular tools that allow the efficient production of seeding-competent amyloid structures.
Assuntos
Amiloide/genética , Fatores de Terminação de Peptídeos/genética , Peptídeos/genética , Príons/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Choque Térmico/química , Microscopia Eletrônica de Transmissão/métodos , Microscopia de Fluorescência/métodos , Chaperonas Moleculares/metabolismo , Príons/química , Ligação Proteica , Proteínas de Saccharomyces cerevisiae/químicaRESUMO
In mammalian cells, abnormal proteins that escape proteasome-dependent degradation form small aggregates that can be transported into a centrosome-associated structure, called an aggresome. Here we demonstrate that in yeast a single aggregate formed by the huntingtin exon 1 with an expanded polyglutamine domain (103QP) represents a bona fide aggresome that colocalizes with the spindle pole body (the yeast centrosome) in a microtubule-dependent fashion. Since a polypeptide lacking the proline-rich region (P-region) of huntingtin (103Q) cannot form aggresomes, this domain serves as an aggresome-targeting signal. Coexpression of 103Q with 25QP, a soluble polypeptide that also carries the P-region, led to the recruitment of 103Q to the aggresome via formation of hetero-oligomers, indicating the aggresome targeting in trans. To identify additional factors involved in aggresome formation and targeting, we purified 103QP aggresomes and 103Q aggregates and identified the associated proteins using mass spectrometry. Among the aggresome-associated proteins we identified, Cdc48 (VCP/p97) and its cofactors, Ufd1 and Nlp4, were shown genetically to be essential for aggresome formation. The 14-3-3 protein, Bmh1, was also found to be critical for aggresome targeting. Its interaction with the huntingtin fragment and its role in aggresome formation required the huntingtin N-terminal N17 domain, adjacent to the polyQ domain. Accordingly, the huntingtin N17 domain, along with the P-region, plays a role in aggresome targeting. We also present direct genetic evidence for the protective role of aggresomes by demonstrating genetically that aggresome targeting of polyglutamine polypeptides relieves their toxicity.
Assuntos
Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Transdução de Sinais , Proteínas 14-3-3/genética , Proteínas 14-3-3/metabolismo , Adenosina Trifosfatases/genética , Adenosina Trifosfatases/metabolismo , Sequência de Aminoácidos , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Dados de Sequência Molecular , Peptídeos/genética , Peptídeos/metabolismo , Ligação Proteica , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Especificidade por Substrato , Ubiquitinação , Proteína com ValosinaRESUMO
The cell's failure to refold or break down abnormal polypeptides often leads to their aggregation, which could cause toxicity and various pathologies. Here we investigated cellular factors involved in protein aggregation in yeast and mammalian cells using model polypeptides containing polyglutamine domains. In yeast, a number of mutations affecting the complex responsible for formation of the endocytic vesicle reduced the aggregation. Components of the endocytic complex (EC) Sla1, Sla2, and Pan1 were seen as clusters in the polyglutamine aggregates. These proteins associate with EC at the later stages of its maturation. In contrast, Ede1 and Ent1, the elements of EC at the earlier stages, were not found in the aggregates, suggesting that late ECs are involved in polyglutamine aggregation. Indeed, stabilization of the late complexes by inhibition of actin polymerization enhanced aggregation of polypeptides with polyglutamine domains. Similarly, in mammalian cells, inhibitors of actin polymerization, as well as depletion of a mediator of actin polymerization, Arp2, strongly enhanced the aggregation. In contrast, destabilization of EC by depletion or inhibition of a scaffolding protein N-WASP effectively suppressed the aggregation. Therefore, EC appears to play a pivotal role in aggregation of cytosolic polypeptides with polyglutamine domains in both yeast and mammalian cells.
Assuntos
Endocitose , Complexos Multiproteicos/metabolismo , Peptídeos/metabolismo , Proteínas/química , Proteínas/metabolismo , Actinas/antagonistas & inibidores , Linhagem Celular , Citosol , Dimerização , Humanos , LevedurasAssuntos
Envelhecimento , Doenças Priônicas/genética , Doenças Priônicas/metabolismo , Príons/genética , Príons/metabolismo , Dobramento de Proteína , Animais , Pesquisa Biomédica/tendências , Genes Neoplásicos , Humanos , Pessoa de Meia-Idade , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Neoplasias/genética , Neoplasias/metabolismo , Neoplasias/terapia , Doenças Priônicas/embriologia , Doenças Priônicas/terapia , Doenças Priônicas/transmissãoRESUMO
A DNA fragment enhancing efficiency of [PSI+]-dependent termination suppressor, sup111, was isolated from a genomic library of Saccharomyces cerevisiae and its function was attributed to an ORF of 1272 bp. This ORF, designated ESU1 (enhancer of termination suppression), corresponded to the 3'-terminal portion of GAL11. Contrasting to ESU1, GAL11 lowered the suppression efficiency of [PSI+] sup111. ESU1 possesses a TATA-like sequence of its own and three ATG codons following it within a distance of about 70 bp and all in the same reading frame as GAL11. A 52.7 kDa protein corresponding in size to the predicted Esu1 protein is detected by western blot analysis using anti-Gal11 antiserum. We therefore conclude that ESU1 is the gene that encodes a polypeptide corresponding to the C-terminal 424 amino acids of Gal11. It was further found that ESU1 increases the level of GAL11 mRNA and probably also of its own mRNA. Moreover, ESU1 increased the cellular level of mRNA transcribed from the leu2-1(UAA) mutant gene, while GAL11 did not. Based on these findings, we propose the following scheme for the events taking place in the [PSI+] sup111 cell that is transformed with an ESU1-bearing plasmid: (a) ESU1 stimulates transcription of leu2-1; (b) leu2-1 mRNA is not effectively degraded because of the possession of sup111, which belongs to the upf group; (c) [PSI+] causes increased mis-termination due to depletion of eRF3; (d) functional Leu2 product is made using leu2-1 mRNA; and (d) suppression of leu2-1 is eventually accomplished.
Assuntos
Galactose/metabolismo , Proteínas de Saccharomyces cerevisiae/fisiologia , Supressão Genética , Transativadores/genética , Fatores de Transcrição/fisiologia , Regulação Fúngica da Expressão Gênica , Genes Fúngicos , Complexo Mediador , Saccharomyces cerevisiae , Proteínas de Saccharomyces cerevisiae/genética , Fatores de Transcrição/metabolismo , Transcrição GênicaRESUMO
In yeast, aggregation and toxicity of the expanded polyglutamine fragment of human huntingtin strictly depend on the presence of the endogenous self-perpetuating aggregated proteins (prions), which contain glutamine/asparagine-rich domains. Some chaperones of the Hsp100/70/40 complex, modulating propagation of yeast prions, were also reported to influence polyglutamine aggregation in yeast, but it was not clear whether they do it directly or via affecting prions. Our data show that although some chaperone alterations indeed act on polyglutamines via curing endogenous prions, other alterations decrease size and ameliorate toxicity of polyglutamine aggregates without affecting prion propagation. Therefore, the role of yeast chaperones in polyglutamine aggregation and toxicity is not restricted only to their effects on the endogenous prions. Moreover, chaperone interactions with prion and polyglutamine aggregates appear to be of a highly specific nature. One and the same chaperone alteration, substitution A503V in the middle region of the chaperone Hsp104, exhibited opposite effects on one of the endogenous prions ([PSI(+)], the prion form of Sup35) and on polyglutamines, increasing aggregate size and toxicity in the former case and decreasing them in the latter case. On the other hand, different members of a single chaperone family exhibited opposite effects on one and the same type of aggregates: excess of the Hsp40 chaperone Ydj1 increased polyglutamine aggregate size and toxicity, whereas excess of the other Hsp40 chaperone, Sis1, decreased them. As many stress-defense proteins are conserved between yeast and mammals, these data shed light on possible mechanisms modulating polyglutamine aggregation and toxicity in mammalian cells.
Assuntos
Peptídeos/química , Príons/química , Alelos , Centrifugação , Endocitose , Proteínas de Fluorescência Verde/química , Proteínas de Choque Térmico HSP40 , Proteínas de Choque Térmico HSP70/química , Proteínas de Choque Térmico/química , Microscopia de Fluorescência , Microscopia de Contraste de Fase , Chaperonas Moleculares/química , Mutação , Plasmídeos/metabolismo , Polirribonucleotídeos , Estrutura Terciária de Proteína , Saccharomyces cerevisiae/metabolismo , Fatores de TempoRESUMO
[PSI(+)] is a prion isoform of the yeast release factor Sup35. In some assays, the cytosolic chaperones Ssa1 and Ssb1/2 of the Hsp70 family were previously shown to exhibit "pro-[PSI(+)]" and "anti-[PSI(+)]" effects, respectively. Here, it is demonstrated for the first time that excess Ssa1 increases de novo formation of [PSI(+)] and that pro-[PSI(+)] effects of Ssa1 are shared by all other Ssa proteins. Experiments with chimeric constructs show that the peptide-binding domain is a major determinant of differences in the effects of Ssa and Ssb proteins on [PSI(+)]. Surprisingly, overproduction of either chaperone increases loss of [PSI(+)] when Sup35 is simultaneously overproduced. Excess Ssa increases both the average size of prion polymers and the proportion of monomeric Sup35 protein. Both in vivo and in vitro experiments uncover direct physical interactions between Sup35 and Hsp70 proteins. The proposed model postulates that Ssa stimulates prion formation and polymer growth by stabilizing misfolded proteins, which serve as substrates for prion conversion. In the case of very large prion aggregates, further increase in size may lead to the loss of prion activity. In contrast, Ssb either stimulates refolding into nonprion conformation or targets misfolded proteins for degradation, in this way counteracting prion formation and propagation.