RESUMO
Oocytes are among the longest-lived cells in the body and need to preserve their cytoplasm to support proper embryonic development. Protein aggregation is a major threat for intracellular homeostasis in long-lived cells. How oocytes cope with protein aggregation during their extended life is unknown. Here, we find that mouse oocytes accumulate protein aggregates in specialized compartments that we named endolysosomal vesicular assemblies (ELVAs). Combining live-cell imaging, electron microscopy, and proteomics, we found that ELVAs are non-membrane-bound compartments composed of endolysosomes, autophagosomes, and proteasomes held together by a protein matrix formed by RUFY1. Functional assays revealed that in immature oocytes, ELVAs sequester aggregated proteins, including TDP-43, and degrade them upon oocyte maturation. Inhibiting degradative activity in ELVAs leads to the accumulation of protein aggregates in the embryo and is detrimental for embryo survival. Thus, ELVAs represent a strategy to safeguard protein homeostasis in long-lived cells.
Assuntos
Vesículas Citoplasmáticas , Oócitos , Agregados Proteicos , Animais , Feminino , Camundongos , Autofagossomos , Vesículas Citoplasmáticas/metabolismo , Lisossomos/metabolismo , Oócitos/citologia , Oócitos/metabolismo , Complexo de Endopeptidases do Proteassoma , ProteóliseRESUMO
Synucleinopathies are characterized by the accumulation of α-synuclein (α-Syn) aggregates in the brain. Positron emission tomography (PET) imaging of synucleinopathies requires radiopharmaceuticals that selectively bind α-Syn deposits. We report the identification of a brain permeable and rapid washout PET tracer [18F]-F0502B, which shows high binding affinity for α-Syn, but not for Aß or Tau fibrils, and preferential binding to α-Syn aggregates in the brain sections. Employing several cycles of counter screenings with in vitro fibrils, intraneuronal aggregates, and neurodegenerative disease brain sections from several mice models and human subjects, [18F]-F0502B images α-Syn deposits in the brains of mouse and non-human primate PD models. We further determined the atomic structure of the α-Syn fibril-F0502B complex by cryo-EM and revealed parallel diagonal stacking of F0502B on the fibril surface through an intense noncovalent bonding network via inter-ligand interactions. Therefore, [18F]-F0502B is a promising lead compound for imaging aggregated α-Syn in synucleinopathies.
Assuntos
Doenças Neurodegenerativas , Sinucleinopatias , Animais , Humanos , alfa-Sinucleína/metabolismo , Sinucleinopatias/diagnóstico por imagem , Sinucleinopatias/metabolismo , Doenças Neurodegenerativas/metabolismo , Tomografia por Emissão de Pósitrons , Encéfalo/diagnóstico por imagem , Encéfalo/metabolismoRESUMO
Decades of research have identified genetic factors and biochemical pathways involved in neurodegenerative diseases (NDDs). We present evidence for the following eight hallmarks of NDD: pathological protein aggregation, synaptic and neuronal network dysfunction, aberrant proteostasis, cytoskeletal abnormalities, altered energy homeostasis, DNA and RNA defects, inflammation, and neuronal cell death. We describe the hallmarks, their biomarkers, and their interactions as a framework to study NDDs using a holistic approach. The framework can serve as a basis for defining pathogenic mechanisms, categorizing different NDDs based on their primary hallmarks, stratifying patients within a specific NDD, and designing multi-targeted, personalized therapies to effectively halt NDDs.
Assuntos
Doenças Neurodegenerativas , Humanos , Doenças Neurodegenerativas/patologia , Proteostase , Agregação Patológica de Proteínas/metabolismo , Morte Celular , Citoesqueleto/metabolismoRESUMO
Cryoelectron microscopy (cryo-EM) has provided unprecedented insights into amyloid fibril structures, including those associated with disease. However, these structures represent the endpoints of long assembly processes, and their relationship to fibrils formed early in assembly is unknown. Consequently, whether different fibril architectures, with potentially different pathological properties, form during assembly remains unknown. Here, we used cryo-EM to determine structures of amyloid fibrils at different times during in vitro fibrillation of a disease-related variant of human islet amyloid polypeptide (IAPP-S20G). Strikingly, the fibrils formed in the lag, growth, and plateau phases have different structures, with new forms appearing and others disappearing as fibrillation proceeds. A time course with wild-type hIAPP also shows fibrils changing with time, suggesting that this is a general property of IAPP amyloid assembly. The observation of transiently populated fibril structures has implications for understanding amyloid assembly mechanisms with potential new insights into amyloid progression in disease.
Assuntos
Amiloide , Polipeptídeo Amiloide das Ilhotas Pancreáticas , Humanos , Amiloide/química , Microscopia Crioeletrônica , Polipeptídeo Amiloide das Ilhotas Pancreáticas/química , Proteínas AmiloidogênicasRESUMO
Protein aggregation is a hallmark of multiple human pathologies. Autophagy selectively degrades protein aggregates via aggrephagy. How selectivity is achieved has been elusive. Here, we identify the chaperonin subunit CCT2 as an autophagy receptor regulating the clearance of aggregation-prone proteins in the cell and the mouse brain. CCT2 associates with aggregation-prone proteins independent of cargo ubiquitination and interacts with autophagosome marker ATG8s through a non-classical VLIR motif. In addition, CCT2 regulates aggrephagy independently of the ubiquitin-binding receptors (P62, NBR1, and TAX1BP1) or chaperone-mediated autophagy. Unlike P62, NBR1, and TAX1BP1, which facilitate the clearance of protein condensates with liquidity, CCT2 specifically promotes the autophagic degradation of protein aggregates with little liquidity (solid aggregates). Furthermore, aggregation-prone protein accumulation induces the functional switch of CCT2 from a chaperone subunit to an autophagy receptor by promoting CCT2 monomer formation, which exposes the VLIR to ATG8s interaction and, therefore, enables the autophagic function.
Assuntos
Chaperonina com TCP-1 , Macroautofagia , Agregados Proteicos , Animais , Camundongos , Proteínas Reguladoras de Apoptose/metabolismo , Autofagia/fisiologia , Proteínas de Transporte/metabolismo , Chaperonina com TCP-1/metabolismo , Proteína Sequestossoma-1/metabolismoRESUMO
Components of the proteostasis network malfunction in aging, and reduced protein quality control in neurons has been proposed to promote neurodegeneration. Here, we investigate the role of chaperone-mediated autophagy (CMA), a selective autophagy shown to degrade neurodegeneration-related proteins, in neuronal proteostasis. Using mouse models with systemic and neuronal-specific CMA blockage, we demonstrate that loss of neuronal CMA leads to altered neuronal function, selective changes in the neuronal metastable proteome, and proteotoxicity, all reminiscent of brain aging. Imposing CMA loss on a mouse model of Alzheimer's disease (AD) has synergistic negative effects on the proteome at risk of aggregation, thus increasing neuronal disease vulnerability and accelerating disease progression. Conversely, chemical enhancement of CMA ameliorates pathology in two different AD experimental mouse models. We conclude that functional CMA is essential for neuronal proteostasis through the maintenance of a subset of the proteome with a higher risk of misfolding than the general proteome.
Assuntos
Envelhecimento/metabolismo , Doença de Alzheimer/metabolismo , Encéfalo/metabolismo , Autofagia Mediada por Chaperonas/fisiologia , Neurônios/metabolismo , Proteostase , Envelhecimento/patologia , Doença de Alzheimer/patologia , Animais , Encéfalo/patologia , Caseína Quinase I/genética , Autofagia Mediada por Chaperonas/genética , Modelos Animais de Doenças , Feminino , Masculino , Camundongos , Neurônios/patologia , ProteomaRESUMO
Biological processes are regulated by intermolecular interactions and chemical modifications that do not affect protein levels, thus escaping detection in classical proteomic screens. We demonstrate here that a global protein structural readout based on limited proteolysis-mass spectrometry (LiP-MS) detects many such functional alterations, simultaneously and in situ, in bacteria undergoing nutrient adaptation and in yeast responding to acute stress. The structural readout, visualized as structural barcodes, captured enzyme activity changes, phosphorylation, protein aggregation, and complex formation, with the resolution of individual regulated functional sites such as binding and active sites. Comparison with prior knowledge, including other 'omics data, showed that LiP-MS detects many known functional alterations within well-studied pathways. It suggested distinct metabolite-protein interactions and enabled identification of a fructose-1,6-bisphosphate-based regulatory mechanism of glucose uptake in E. coli. The structural readout dramatically increases classical proteomics coverage, generates mechanistic hypotheses, and paves the way for in situ structural systems biology.
Assuntos
Proteínas de Escherichia coli/metabolismo , Imageamento Tridimensional , Proteoma/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Regulação Alostérica , Sequência de Aminoácidos , Escherichia coli/enzimologia , Escherichia coli/metabolismo , Espectrometria de Massas , Simulação de Dinâmica Molecular , Pressão Osmótica , Fosforilação , Proteólise , Reprodutibilidade dos Testes , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Estresse FisiológicoRESUMO
Defects in translation lead to changes in the expression of proteins that can serve as drivers of cancer formation. Here, we show that cytosolic NAD+ synthesis plays an essential role in ovarian cancer by regulating translation and maintaining protein homeostasis. Expression of NMNAT-2, a cytosolic NAD+ synthase, is highly upregulated in ovarian cancers. NMNAT-2 supports the catalytic activity of the mono(ADP-ribosyl) transferase (MART) PARP-16, which mono(ADP-ribosyl)ates (MARylates) ribosomal proteins. Depletion of NMNAT-2 or PARP-16 leads to inhibition of MARylation, increased polysome association and enhanced translation of specific mRNAs, aggregation of their translated protein products, and reduced growth of ovarian cancer cells. Furthermore, MARylation of the ribosomal proteins, such as RPL24 and RPS6, inhibits polysome assembly by stabilizing eIF6 binding to ribosomes. Collectively, our results demonstrate that ribosome MARylation promotes protein homeostasis in cancers by fine-tuning the levels of protein synthesis and preventing toxic protein aggregation.
Assuntos
ADP-Ribosilação , Neoplasias Ovarianas/metabolismo , Biossíntese de Proteínas , Proteostase , Ribossomos/metabolismo , Regiões 3' não Traduzidas/genética , Animais , Sequência de Bases , Linhagem Celular Tumoral , Proliferação de Células , Estresse do Retículo Endoplasmático , Tubas Uterinas/metabolismo , Feminino , Humanos , Camundongos Endogâmicos NOD , Camundongos SCID , NAD/metabolismo , Nicotinamida-Nucleotídeo Adenililtransferase , Conformação de Ácido Nucleico , Neoplasias Ovarianas/patologia , Poli(ADP-Ribose) Polimerases/metabolismo , Polirribossomos/metabolismo , RNA Mensageiro/química , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/metabolismo , Proteínas Ribossômicas/metabolismoRESUMO
To elucidate the role of Tau isoforms and post-translational modification (PTM) stoichiometry in Alzheimer's disease (AD), we generated a high-resolution quantitative proteomics map of 95 PTMs on multiple isoforms of Tau isolated from postmortem human tissue from 49 AD and 42 control subjects. Although Tau PTM maps reveal heterogeneity across subjects, a subset of PTMs display high occupancy and frequency for AD, suggesting importance in disease. Unsupervised analyses indicate that PTMs occur in an ordered manner, leading to Tau aggregation. The processive addition and minimal set of PTMs associated with seeding activity was further defined by analysis of size-fractionated Tau. To summarize, features in the Tau protein critical for disease intervention at different stages of disease are identified, including enrichment of 0N and 4R isoforms, underrepresentation of the C terminus, an increase in negative charge in the proline-rich region (PRR), and a decrease in positive charge in the microtubule binding domain (MBD).
Assuntos
Doença de Alzheimer/metabolismo , Doença de Alzheimer/patologia , Processamento de Proteína Pós-Traducional , Proteínas tau/metabolismo , Estudos de Casos e Controles , Estudos de Coortes , Progressão da Doença , Humanos , Análise de Componente Principal , Isoformas de Proteínas/metabolismoRESUMO
As the world's population ages, neurodegenerative disorders are poised to become the commonest cause of death. Despite this, they remain essentially untreatable. Characterized pathologically both by the aggregation of disease-specific misfolded proteins and by changes in cellular stress responses, to date, therapeutic approaches have focused almost exclusively on reducing misfolded protein load-notably amyloid beta (Aß) in Alzheimer's disease. The repeated failure of clinical trials has led to despondency over the possibility that these disorders will ever be treated. We argue that this is in fact a time for optimism: Targeting various generic stress responses is emerging as an increasingly promising means of modifying disease progression across these disorders. New treatments are approaching clinical trials, while novel means of targeting aggregates could eventually act preventively in early disease.
Assuntos
Doenças Neurodegenerativas/terapia , Agregados Proteicos , Estresse Fisiológico , Animais , Autofagossomos/metabolismo , Humanos , Lisossomos/metabolismo , Resposta a Proteínas não DobradasRESUMO
A healthy proteome is essential for cell survival. Protein misfolding is linked to a rapidly expanding list of human diseases, ranging from neurodegenerative diseases to aging and cancer. Many of these diseases are characterized by the accumulation of misfolded proteins in intra- and extracellular inclusions, such as amyloid plaques. The clear link between protein misfolding and disease highlights the need to better understand the elaborate machinery that manages proteome homeostasis, or proteostasis, in the cell. Proteostasis depends on a network of molecular chaperones and clearance pathways involved in the recognition, refolding, and/or clearance of aberrant proteins. Recent studies reveal that an integral part of the cellular management of misfolded proteins is their spatial sequestration into several defined compartments. Here, we review the properties, function, and formation of these compartments. Spatial sequestration plays a central role in protein quality control and cellular fitness and represents a critical link to the pathogenesis of protein aggregation-linked diseases.
Assuntos
Envelhecimento/metabolismo , Chaperonas Moleculares/metabolismo , Doenças Neurodegenerativas/metabolismo , Agregação Patológica de Proteínas/metabolismo , Deficiências na Proteostase/metabolismo , Envelhecimento/genética , Envelhecimento/patologia , Proteínas Amiloidogênicas/química , Proteínas Amiloidogênicas/genética , Proteínas Amiloidogênicas/metabolismo , Compartimento Celular , Regulação da Expressão Gênica , Humanos , Chaperonas Moleculares/genética , Doenças Neurodegenerativas/genética , Doenças Neurodegenerativas/patologia , Proteínas Priônicas/química , Proteínas Priônicas/genética , Proteínas Priônicas/metabolismo , Agregação Patológica de Proteínas/genética , Agregação Patológica de Proteínas/patologia , Biossíntese de Proteínas , Conformação Proteica , Dobramento de Proteína , Redobramento de Proteína , Proteólise , Deficiências na Proteostase/genética , Deficiências na Proteostase/patologiaRESUMO
Peptides and proteins have been found to possess an inherent tendency to convert from their native functional states into intractable amyloid aggregates. This phenomenon is associated with a range of increasingly common human disorders, including Alzheimer and Parkinson diseases, type II diabetes, and a number of systemic amyloidoses. In this review, we describe this field of science with particular reference to the advances that have been made over the last decade in our understanding of its fundamental nature and consequences. We list the proteins that are known to be deposited as amyloid or other types of aggregates in human tissues and the disorders with which they are associated, as well as the proteins that exploit the amyloid motif to play specific functional roles in humans. In addition, we summarize the genetic factors that have provided insight into the mechanisms of disease onset. We describe recent advances in our knowledge of the structures of amyloid fibrils and their oligomeric precursors and of the mechanisms by which they are formed and proliferate to generate cellular dysfunction. We show evidence that a complex proteostasis network actively combats protein aggregation and that such an efficient system can fail in some circumstances and give rise to disease. Finally, we anticipate the development of novel therapeutic strategies with which to prevent or treat these highly debilitating and currently incurable conditions.
Assuntos
Doença de Alzheimer/história , Amiloide/química , Amiloidose/história , Diabetes Mellitus Tipo 2/história , Doença de Parkinson/história , Deficiências na Proteostase/história , Doença de Alzheimer/tratamento farmacológico , Doença de Alzheimer/metabolismo , Doença de Alzheimer/patologia , Amiloide/genética , Amiloide/metabolismo , Amiloidose/tratamento farmacológico , Amiloidose/metabolismo , Amiloidose/patologia , Diabetes Mellitus Tipo 2/tratamento farmacológico , Diabetes Mellitus Tipo 2/metabolismo , Diabetes Mellitus Tipo 2/patologia , Drogas em Investigação , Regulação da Expressão Gênica , História do Século XXI , Humanos , Amiloidose de Cadeia Leve de Imunoglobulina , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , Terapia de Alvo Molecular , Doença de Parkinson/tratamento farmacológico , Doença de Parkinson/metabolismo , Doença de Parkinson/patologia , Agregação Patológica de Proteínas/história , Agregação Patológica de Proteínas/metabolismo , Agregação Patológica de Proteínas/patologia , Agregação Patológica de Proteínas/prevenção & controle , Conformação Proteica , Dobramento de Proteína , Deficiências na Proteostase/tratamento farmacológico , Deficiências na Proteostase/metabolismo , Deficiências na Proteostase/patologia , Deficiências na Proteostase/prevenção & controleRESUMO
The majority of protein molecules must fold into defined three-dimensional structures to acquire functional activity. However, protein chains can adopt a multitude of conformational states, and their biologically active conformation is often only marginally stable. Metastable proteins tend to populate misfolded species that are prone to forming toxic aggregates, including soluble oligomers and fibrillar amyloid deposits, which are linked with neurodegeneration in Alzheimer and Parkinson disease, and many other pathologies. To prevent or regulate protein aggregation, all cells contain an extensive protein homeostasis (or proteostasis) network comprising molecular chaperones and other factors. These defense systems tend to decline during aging, facilitating the manifestation of aggregate deposition diseases. This volume of the Annual Review of Biochemistry contains a set of three articles addressing our current understanding of the structures of pathological protein aggregates and their associated disease mechanisms. These articles also discuss recent insights into the strategies cells have evolved to neutralize toxic aggregates by sequestering them in specific cellular locations.
Assuntos
Envelhecimento/metabolismo , Doença de Alzheimer/metabolismo , Doença de Parkinson/metabolismo , Agregação Patológica de Proteínas/metabolismo , Deficiências na Proteostase/metabolismo , Envelhecimento/genética , Envelhecimento/patologia , Doença de Alzheimer/genética , Doença de Alzheimer/patologia , Amiloide/química , Amiloide/genética , Amiloide/metabolismo , Regulação da Expressão Gênica , Humanos , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , Doença de Parkinson/genética , Doença de Parkinson/patologia , Agregação Patológica de Proteínas/genética , Agregação Patológica de Proteínas/patologia , Conformação Proteica , Dobramento de Proteína , Deficiências na Proteostase/genética , Deficiências na Proteostase/patologiaRESUMO
Protein aggregation is a hallmark of many diseases but also underlies a wide range of positive cellular functions. This phenomenon has been difficult to study because of a lack of quantitative and high-throughput cellular tools. Here, we develop a synthetic genetic tool to sense and control protein aggregation. We apply the technology to yeast prions, developing sensors to track their aggregation states and employing prion fusions to encode synthetic memories in yeast cells. Utilizing high-throughput screens, we identify prion-curing mutants and engineer "anti-prion drives" that reverse the non-Mendelian inheritance pattern of prions and eliminate them from yeast populations. We extend our technology to yeast RNA-binding proteins (RBPs) by tracking their propensity to aggregate, searching for co-occurring aggregates, and uncovering a group of coalescing RBPs through screens enabled by our platform. Our work establishes a quantitative, high-throughput, and generalizable technology to study and control diverse protein aggregation processes in cells.
Assuntos
Técnicas Genéticas , Príons/genética , Engenharia Genética , Técnicas Genéticas/economia , Proteínas de Ligação a RNA/metabolismo , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Biologia Sintética/métodos , Fatores de Poliadenilação e Clivagem de mRNA/metabolismoRESUMO
Eukaryotic cells have evolved extensive protein quality-control mechanisms to remove faulty translation products. Here, we show that yeast cells continually produce faulty mitochondrial polypeptides that stall on the ribosome during translation but are imported into the mitochondria. The cytosolic protein Vms1, together with the E3 ligase Ltn1, protects against the mitochondrial toxicity of these proteins and maintains cell viability under respiratory conditions. In the absence of these factors, stalled polypeptides aggregate after import and sequester critical mitochondrial chaperone and translation machinery. Aggregation depends on C-terminal alanyl/threonyl sequences (CAT-tails) that are attached to stalled polypeptides on 60S ribosomes by Rqc2. Vms1 binds to 60S ribosomes at the mitochondrial surface and antagonizes Rqc2, thereby facilitating import, impeding aggregation, and directing aberrant polypeptides to intra-mitochondrial quality control. Vms1 is a key component of a rescue pathway for ribosome-stalled mitochondrial polypeptides that are inaccessible to ubiquitylation due to coupling of translation and translocation.
Assuntos
Proteínas de Transporte/metabolismo , Mitocôndrias/fisiologia , Ribossomos/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/citologia , Citosol/metabolismo , Transporte de Elétrons , Homeostase , Saccharomyces cerevisiae/fisiologia , Ubiquitina-Proteína Ligases/metabolismoRESUMO
Expression of many disease-related aggregation-prone proteins results in cytotoxicity and the formation of large intracellular inclusion bodies. To gain insight into the role of inclusions in pathology and the in situ structure of protein aggregates inside cells, we employ advanced cryo-electron tomography methods to analyze the structure of inclusions formed by polyglutamine (polyQ)-expanded huntingtin exon 1 within their intact cellular context. In primary mouse neurons and immortalized human cells, polyQ inclusions consist of amyloid-like fibrils that interact with cellular endomembranes, particularly of the endoplasmic reticulum (ER). Interactions with these fibrils lead to membrane deformation, the local impairment of ER organization, and profound alterations in ER membrane dynamics at the inclusion periphery. These results suggest that aberrant interactions between fibrils and endomembranes contribute to the deleterious cellular effects of protein aggregation. VIDEO ABSTRACT.
Assuntos
Doença de Huntington/patologia , Corpos de Inclusão/patologia , Neurônios/patologia , Neurônios/ultraestrutura , Peptídeos/metabolismo , Amiloide/química , Animais , Microscopia Crioeletrônica , Retículo Endoplasmático/metabolismo , Retículo Endoplasmático/patologia , Feminino , Células HeLa , Humanos , Proteína Huntingtina/genética , Proteína Huntingtina/metabolismo , Corpos de Inclusão/química , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Microscopia Eletrônica de Transmissão , Mutação , Agregação Patológica de Proteínas , Tomografia/métodosRESUMO
J-domain proteins (JDPs) constitute a large family of molecular chaperones that bind a broad spectrum of substrates, targeting them to Hsp70, thus determining the specificity of and activating the entire chaperone functional cycle. The malfunction of JDPs is therefore inextricably linked to myriad human disorders. Here, we uncover a unique mechanism by which chaperones recognize misfolded clients, present in human class A JDPs. Through a newly identified ß-hairpin site, these chaperones detect changes in protein dynamics at the initial stages of misfolding, prior to exposure of hydrophobic regions or large structural rearrangements. The JDPs then sequester misfolding-prone proteins into large oligomeric assemblies, protecting them from aggregation. Through this mechanism, class A JDPs bind destabilized p53 mutants, preventing clearance of these oncoproteins by Hsp70-mediated degradation, thus promoting cancer progression. Removal of the ß-hairpin abrogates this protective activity while minimally affecting other chaperoning functions. This suggests the class A JDP ß-hairpin as a highly specific target for cancer therapeutics.
Assuntos
Neoplasias , Proteína Supressora de Tumor p53 , Humanos , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , Proteínas de Choque Térmico HSP70/metabolismo , Dobramento de ProteínaRESUMO
Aggregation of proteins containing expanded polyglutamine (polyQ) repeats is the cytopathologic hallmark of a group of dominantly inherited neurodegenerative diseases, including Huntington's disease (HD). Huntingtin (Htt), the disease protein of HD, forms amyloid-like fibrils by liquid-to-solid phase transition. Macroautophagy has been proposed to clear polyQ aggregates, but the efficiency of aggrephagy is limited. Here, we used cryo-electron tomography to visualize the interactions of autophagosomes with polyQ aggregates in cultured cells in situ. We found that an amorphous aggregate phase exists next to the radially organized polyQ fibrils. Autophagosomes preferentially engulfed this amorphous material, mediated by interactions between the autophagy receptor p62/SQSTM1 and the non-fibrillar aggregate surface. In contrast, amyloid fibrils excluded p62 and evaded clearance, resulting in trapping of autophagic structures. These results suggest that the limited efficiency of autophagy in clearing polyQ aggregates is due to the inability of autophagosomes to interact productively with the non-deformable, fibrillar disease aggregates.
Assuntos
Amiloide , Autofagossomos , Autofagia , Proteína Huntingtina , Doença de Huntington , Peptídeos , Agregados Proteicos , Proteína Sequestossoma-1 , Peptídeos/metabolismo , Peptídeos/química , Peptídeos/genética , Humanos , Proteína Huntingtina/metabolismo , Proteína Huntingtina/genética , Proteína Huntingtina/química , Autofagossomos/metabolismo , Autofagossomos/ultraestrutura , Proteína Sequestossoma-1/metabolismo , Proteína Sequestossoma-1/genética , Amiloide/metabolismo , Amiloide/química , Amiloide/genética , Doença de Huntington/metabolismo , Doença de Huntington/genética , Doença de Huntington/patologia , Microscopia Crioeletrônica , Animais , Agregação Patológica de Proteínas/metabolismo , Agregação Patológica de Proteínas/genéticaRESUMO
Ribosome assembly requires precise coordination between the production and assembly of ribosomal components. Mutations in ribosomal proteins that inhibit the assembly process or ribosome function are often associated with ribosomopathies, some of which are linked to defects in proteostasis. In this study, we examine the interplay between several yeast proteostasis enzymes, including deubiquitylases (DUBs) Ubp2 and Ubp14, and E3 ligases Ufd4 and Hul5, and we explore their roles in the regulation of the cellular levels of K29-linked unanchored polyubiquitin (polyUb) chains. Accumulating K29-linked unanchored polyUb chains associate with maturing ribosomes to disrupt their assembly, activate the ribosome assembly stress response (RASTR), and lead to the sequestration of ribosomal proteins at the intranuclear quality control compartment (INQ). These findings reveal the physiological relevance of INQ and provide insights into mechanisms of cellular toxicity associated with ribosomopathies.
Assuntos
Poliubiquitina , Proteínas Ribossômicas , Ribossomos , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Proteínas Ribossômicas/metabolismo , Proteínas Ribossômicas/genética , Ribossomos/metabolismo , Ribossomos/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Poliubiquitina/metabolismo , Poliubiquitina/genética , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitina-Proteína Ligases/genética , Ubiquitinação , Proteostase , Núcleo Celular/metabolismoRESUMO
The ability to sense and respond to proteotoxic insults declines with age, leaving cells vulnerable to chronic and acute stressors. Reproductive cues modulate this decline in cellular proteostasis to influence organismal stress resilience in Caenorhabditis elegans We previously uncovered a pathway that links the integrity of developing embryos to somatic health in reproductive adults. Here, we show that the nuclear receptor NHR-49, an ortholog of mammalian peroxisome proliferator-activated receptor α (PPARα), regulates stress resilience and proteostasis downstream from embryo integrity and other pathways that influence lipid homeostasis and upstream of HSF-1. Disruption of the vitelline layer of the embryo envelope, which activates a proteostasis-enhancing intertissue pathway in somatic cells, triggers changes in lipid catabolism gene expression that are accompanied by an increase in fat stores. NHR-49, together with its coactivator, MDT-15, contributes to this remodeling of lipid metabolism and is also important for the elevated stress resilience mediated by inhibition of the embryonic vitelline layer. Our findings indicate that NHR-49 also contributes to stress resilience in other pathways known to change lipid homeostasis, including reduced insulin-like signaling and fasting, and that increased NHR-49 activity is sufficient to improve proteostasis and stress resilience in an HSF-1-dependent manner. Together, our results establish NHR-49 as a key regulator that links lipid homeostasis and cellular resilience to proteotoxic stress.