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1.
iScience ; 27(6): 109891, 2024 Jun 21.
Artículo en Inglés | MEDLINE | ID: mdl-38832020

RESUMEN

Key to a biologists' capacity to understand data is the ability to make meaningful conclusions about differences in experimental observations. Typically, data are noisy, and conventional methods rely on replicates to average out noise and enable univariate statistical tests to assign p-values. Yet thresholding p-values to determine significance is controversial and often misleading, especially for omics datasets with few replicates. This study introduces PERCEPT, an alternative that transforms data using an ad-hoc scaling factor derived from p-values. By applying this method, low confidence effects are suppressed compared to high confidence ones, enabling clearer patterns to emerge from noisy datasets. The effectiveness of PERCEPT scaling is demonstrated using simulated datasets and published omics studies. The approach reduces the exclusion of datapoints, enhances accuracy, and enables nuanced interpretation of data. PERCEPT is easy to apply for the non-expert in statistics and provides researchers a straightforward way to improve data-driven analyses.

2.
Angew Chem Int Ed Engl ; 63(21): e202317756, 2024 05 21.
Artículo en Inglés | MEDLINE | ID: mdl-38523073

RESUMEN

Hyperphosphorylation and aggregation of the protein tau play key roles in the development of Alzheimer's disease (AD). While the molecular structure of the filamentous tau aggregates has been determined to atomic resolution, there is far less information available about the smaller, soluble aggregates, which are believed to be more toxic. Traditional techniques are limited to bulk measures and struggle to identify individual aggregates in complex biological samples. To address this, we developed a novel single-molecule pull-down-based assay (MAPTau) to detect and characterize individual tau aggregates in AD and control post-mortem brain and biofluids. Using MAPTau, we report the quantity, as well as the size and circularity of tau aggregates measured using super-resolution microscopy, revealing AD-specific differences in tau aggregate morphology. By adapting MAPTau to detect multiple phosphorylation markers in individual aggregates using two-color coincidence detection, we derived compositional profiles of the individual aggregates. We find an AD-specific phosphorylation profile of tau aggregates with more than 80 % containing multiple phosphorylations, compared to 5 % in age-matched non-AD controls. Our results show that MAPTau is able to identify disease-specific subpopulations of tau aggregates phosphorylated at different sites, that are invisible to other methods and enable the study of disease mechanisms and diagnosis.


Asunto(s)
Enfermedad de Alzheimer , Agregado de Proteínas , Proteínas tau , Humanos , Enfermedad de Alzheimer/metabolismo , Enfermedad de Alzheimer/diagnóstico , Proteínas tau/metabolismo , Proteínas tau/química , Proteínas tau/análisis , Fosforilación , Imagen Individual de Molécula/métodos , Encéfalo/metabolismo , Encéfalo/diagnóstico por imagen , Encéfalo/patología
3.
NPJ Syst Biol Appl ; 8(1): 46, 2022 11 28.
Artículo en Inglés | MEDLINE | ID: mdl-36443335

RESUMEN

The correct spatio-temporal organization of the proteome is essential for cellular homeostasis. However, a detailed mechanistic understanding of this organization and how it is altered in response to external stimuli in the intact cellular environment is as-yet unrealized. 'Protein painting methods provide a means to address this gap in knowledge by monitoring the conformational status of proteins within cells at the proteome-wide scale. Here, we demonstrate the ability of a protein painting method employing tetraphenylethene maleimide (TPE-MI) to reveal proteome network remodeling in whole cells in response to a cohort of commonly used pharmacological stimuli of varying specificity. We report specific, albeit heterogeneous, responses to individual stimuli that coalesce on a conserved set of core cellular machineries. This work expands our understanding of proteome conformational remodeling in response to cellular stimuli, and provides a blueprint for assessing how these conformational changes may contribute to disorders characterized by proteostasis imbalance.


Asunto(s)
Proteoma , Proteostasis , Humanos
4.
PLoS Pathog ; 18(8): e1009882, 2022 08.
Artículo en Inglés | MEDLINE | ID: mdl-35930605

RESUMEN

Presentation of the variant antigen, Plasmodium falciparum erythrocyte membrane protein 1 (EMP1), at knob-like protrusions on the surface of infected red blood cells, underpins the parasite's pathogenicity. Here we describe a protein PF3D7_0301700 (PTP7), that functions at the nexus between the intermediate trafficking organelle, the Maurer's cleft, and the infected red blood cell surface. Genetic disruption of PTP7 leads to accumulation of vesicles at the Maurer's clefts, grossly aberrant knob morphology, and failure to deliver EMP1 to the red blood cell surface. We show that an expanded low complexity sequence in the C-terminal region of PTP7, identified only in the Laverania clade of Plasmodium, is critical for efficient virulence protein trafficking.


Asunto(s)
Plasmodium falciparum , Proteínas Protozoarias , Membrana Eritrocítica/metabolismo , Eritrocitos/metabolismo , Orgánulos/metabolismo , Plasmodium falciparum/metabolismo , Transporte de Proteínas , Proteínas Protozoarias/genética , Proteínas Protozoarias/metabolismo
5.
Mol Cell ; 82(17): 3193-3208.e8, 2022 09 01.
Artículo en Inglés | MEDLINE | ID: mdl-35853451

RESUMEN

Aberrant phase separation of globular proteins is associated with many diseases. Here, we use a model protein system to understand how the unfolded states of globular proteins drive phase separation and the formation of unfolded protein deposits (UPODs). We find that for UPODs to form, the concentrations of unfolded molecules must be above a threshold value. Additionally, unfolded molecules must possess appropriate sequence grammars to drive phase separation. While UPODs recruit molecular chaperones, their compositional profiles are also influenced by synergistic physicochemical interactions governed by the sequence grammars of unfolded proteins and cellular proteins. Overall, the driving forces for phase separation and the compositional profiles of UPODs are governed by the sequence grammars of unfolded proteins. Our studies highlight the need for uncovering the sequence grammars of unfolded proteins that drive UPOD formation and cause gain-of-function interactions whereby proteins are aberrantly recruited into UPODs.


Asunto(s)
Chaperonas Moleculares , Pliegue de Proteína , Chaperonas Moleculares/metabolismo
6.
J Biol Chem ; 298(8): 102158, 2022 08.
Artículo en Inglés | MEDLINE | ID: mdl-35724963

RESUMEN

Chaperones and other quality control machinery guard proteins from inappropriate aggregation, which is a hallmark of neurodegenerative diseases. However, how the systems that regulate the "foldedness" of the proteome remain buffered under stress conditions and in different cellular compartments remains incompletely understood. In this study, we applied a FRET-based strategy to explore how well quality control machinery protects against the misfolding and aggregation of "bait" biosensor proteins, made from the prokaryotic ribonuclease barnase, in the nucleus and cytosol of human embryonic kidney 293T cells. We found that those barnase biosensors were prone to misfolding, were less engaged by quality control machinery, and more prone to inappropriate aggregation in the nucleus as compared with the cytosol, and that these effects could be regulated by chaperone Hsp70-related machinery. Furthermore, aggregation of mutant huntingtin exon 1 protein (Httex1) in the cytosol appeared to outcompete and thus prevented the engagement of quality control machinery with the biosensor in the cytosol. This effect correlated with reduced levels of DNAJB1 and HSPA1A chaperones in the cell outside those sequestered to the aggregates, particularly in the nucleus. Unexpectedly, we found Httex1 aggregation also increased the apparent engagement of the barnase biosensor with quality control machinery in the nucleus suggesting an independent implementation of "holdase" activity of chaperones other than DNAJB1 and HSPA1A. Collectively, these results suggest that proteostasis stress can trigger a rebalancing of chaperone abundance in different subcellular compartments through a dynamic network involving different chaperone-client interactions.


Asunto(s)
Técnicas Biosensibles , Agregado de Proteínas , Citosol/metabolismo , Proteínas del Choque Térmico HSP40/metabolismo , Proteínas HSP70 de Choque Térmico/genética , Proteínas HSP70 de Choque Térmico/metabolismo , Humanos , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , Pliegue de Proteína
7.
Nat Commun ; 13(1): 1992, 2022 04 14.
Artículo en Inglés | MEDLINE | ID: mdl-35422070

RESUMEN

Methods that assay protein foldedness with proteomics have generated censuses of apparent protein folding stabilities in biological milieu. However, different censuses poorly correlate with each other. Here, we show that the reason for this is that methods targeting foldedness through monitoring amino acid sidechain reactivity also detect changes in conformation and ligand binding, which can be a substantial fraction of the data. We show that the reactivity of only one quarter of cysteine or methionine sidechains in proteins in a urea denaturation curve of mammalian cell lysate can be confidently explained by a two-state unfolding isotherm. Contrary to that expected from unfolding, up to one third of the cysteines decreased reactivity. These cysteines were enriched in proteins with functions relating to unfolded protein stress. One protein, chaperone HSPA8, displayed changes arising from ligand and cofactor binding. Unmasking this hidden information using the approaches outlined here should improve efforts to understand both folding and the remodeling of protein function directly in complex biological settings.


Asunto(s)
Censos , Proteoma , Animales , Dicroismo Circular , Cisteína , Cinética , Ligandos , Mamíferos , Conformación Proteica , Desnaturalización Proteica , Pliegue de Proteína , Termodinámica , Urea
8.
J Proteome Res ; 21(5): 1251-1261, 2022 05 06.
Artículo en Inglés | MEDLINE | ID: mdl-35388693

RESUMEN

Eukaryotic cells respond to heat shock through several regulatory processes including upregulation of stress responsive chaperones and reversible shutdown of cellular activities through formation of protein assemblies. However, the underlying regulatory mechanisms of the recovery of these heat-induced protein assemblies remain largely elusive. Here, we measured the proteome abundance and solubility changes during recovery from heat shock in the mouse Neuro2a cell line. We found that prefoldins and translation machinery are rapidly down-regulated as the first step in the heat shock response. Analysis of proteome solubility reveals that a rapid mobilization of protein quality control machineries, along with changes in cellular energy metabolism, translational activity, and actin cytoskeleton are fundamental to the early stress responses. In contrast, longer term adaptation to stress involves renewal of core cellular components. Inhibition of the Hsp70 family, pivotal for the heat shock response, selectively and negatively affects the ribosomal machinery and delays the solubility recovery of many nuclear proteins. ProteomeXchange: PXD030069.


Asunto(s)
Censos , Proteoma , Animales , Proteínas HSP70 de Choque Térmico/genética , Proteínas HSP70 de Choque Térmico/metabolismo , Respuesta al Choque Térmico/fisiología , Ratones , Proteoma/análisis , Solubilidad
9.
Methods Mol Biol ; 2428: 261-275, 2022.
Artículo en Inglés | MEDLINE | ID: mdl-35171485

RESUMEN

Proteome solubility contains latent information on the nature of protein interaction networks in cells and changes in solubility can provide information on rewiring of networks. Here, we report a simple one-step ultracentrifugation method to separate the soluble and insoluble fraction of the proteome. The method involves quantitative proteomics and a bioinformatics strategy to analyze the changes that arise. Because protein solubility changes are also associated with protein misfolding and aggregation in neurodegenerative disease, we also include a protocol for isolating disease-associated protein aggregates with pulse shape analysis (PulSA) by flow cytometry as a complementary approach that can be used alongside the more general measure of solubility or as a stand-alone approach.


Asunto(s)
Enfermedades Neurodegenerativas , Proteómica , Humanos , Proteoma , Proteómica/métodos , Solubilidad , Ultracentrifugación
10.
Essays Biochem ; 65(7): 913-923, 2021 12 22.
Artículo en Inglés | MEDLINE | ID: mdl-34897410

RESUMEN

PTEN-induced kinase 1 (PINK1) impacts cell health and human pathology through diverse pathways. The strict processing of full-length PINK1 on the outer mitochondrial membrane populates a cytoplasmic pool of cleaved PINK1 (cPINK1) that is constitutively degraded. However, despite rapid proteasomal clearance, cPINK1 still appears to exert quality control influence over the neuronal protein homeostasis network, including protein synthesis and degradation machineries. The cytoplasmic concentration and activity of this molecule is therefore a powerful sensor that coordinates aspects of mitochondrial and cellular health. In addition, full-length PINK1 is retained on the mitochondrial membrane following depolarisation, where it is a powerful inducer of multiple mitophagic pathways. This function is executed primarily through the phosphorylation of several ubiquitin ligases, including its most widely studied substrate Parkin. Furthermore, the phosphorylation of both pro- and anti-apoptotic proteins by mitochondrial PINK1 acts as a pro-cellular survival signal when faced with apoptotic stimuli. Through these varied roles PINK1 directly influences functions central to cell dysfunction in neurodegenerative disease.


Asunto(s)
Mitofagia , Enfermedades Neurodegenerativas , Humanos , Mitocondrias/metabolismo , Membranas Mitocondriales/metabolismo , Enfermedades Neurodegenerativas/metabolismo , Proteínas Quinasas/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo
11.
PLoS One ; 15(7): e0233583, 2020.
Artículo en Inglés | MEDLINE | ID: mdl-32735619

RESUMEN

Mutations that cause Huntington's Disease involve a polyglutamine (polyQ) sequence expansion beyond 35 repeats in exon 1 of Huntingtin. Intracellular inclusion bodies of mutant Huntingtin protein are a key feature of Huntington's disease brain pathology. We previously showed that in cell culture the formation of inclusions involved the assembly of disordered structures of mHtt exon 1 fragments (Httex1) and they were enriched with translational machinery when first formed. We hypothesized that nascent mutant Httex1 chains co-aggregate during translation by phase separation into liquid-like disordered aggregates and then convert to more rigid, amyloid structures. Here we further examined the mechanisms of inclusion assembly in a human epithelial kidney (AD293) cell culture model. We found mHttex1 did not appear to stall translation of its own nascent chain, or at best was marginal. We also found the inclusions appeared to recruit low levels of RNA but there was no difference in enrichment between early formed and mature inclusions. Proteins involved in translation or ribosome quality control were co-recruited to the inclusions (Ltn1 Rack1) compared to a protein not anticipated to be involved (NACAD), but there was no major specificity of enrichment in the early formed inclusions compared to mature inclusions. Furthermore, we observed co-aggregation with other proteins previously identified in inclusions, including Upf1 and chaperone-like proteins Sgta and Hspb1, which also suppressed aggregation at high co-expression levels. The newly formed inclusions also contained immobile mHttex1 molecules which points to the disordered aggregates being mechanically rigid prior to amyloid formation. Collectively our findings show little evidence that inclusion assembly arises by a discrete clustering of stalled nascent chains and associated quality control machinery. Instead, the machinery appear to be recruited continuously, or secondarily, to the nucleation of inclusion formation.


Asunto(s)
Exones/genética , Proteína Huntingtina/genética , Extensión de la Cadena Peptídica de Translación , Agregado de Proteínas/genética , ARN Mensajero/genética , Ribosomas/metabolismo , Secuencia de Bases , Células Epiteliales , Genes Reporteros , Células HEK293 , Humanos , Proteína Huntingtina/biosíntesis , Cuerpos de Inclusión/genética , Cuerpos de Inclusión/metabolismo , Repeticiones de Minisatélite , Péptidos , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo
12.
J Cell Sci ; 133(15)2020 08 05.
Artículo en Inglés | MEDLINE | ID: mdl-32661089

RESUMEN

Protein aggregates that result in inclusion formation are a pathological hallmark common to many neurodegenerative diseases, including amyotrophic lateral sclerosis, Parkinson's disease and Huntington's disease. Under conditions of cellular stress, activation of the heat shock response (HSR) results in an increase in the levels of molecular chaperones and is a first line of cellular defence against inclusion formation. It remains to be established whether neurodegenerative disease-associated proteins and inclusions are themselves capable of inducing an HSR in neuronal cells. To address this, we generated a neuroblastoma cell line that expresses a fluorescent reporter protein under conditions of heat shock transcription factor 1 (HSF1)-mediated HSR induction. We show that the HSR is not induced by exogenous treatment with aggregated forms of recombinant α-synuclein or the G93A mutant of superoxide dismutase-1 (SOD1G93A) nor intracellular expression of SOD1G93A or a pathogenic form of polyglutamine-expanded huntingtin (Htt72Q). These results suggest that pathogenic proteins evade detection or impair induction of the HSR in neuronal cells. A failure of protein aggregation to induce an HSR might contribute to the development of inclusion pathology in neurodegenerative diseases.This article has an associated First Person interview with the first author of the paper.


Asunto(s)
Enfermedades Neurodegenerativas , Factores de Transcripción del Choque Térmico/genética , Respuesta al Choque Térmico/genética , Humanos , Enfermedades Neurodegenerativas/genética , Agregado de Proteínas , Superóxido Dismutasa-1
13.
J Biol Chem ; 295(29): 9838-9854, 2020 07 17.
Artículo en Inglés | MEDLINE | ID: mdl-32417755

RESUMEN

Small heat-shock proteins (sHSPs) are ubiquitously expressed molecular chaperones that inhibit amyloid fibril formation; however, their mechanisms of action remain poorly understood. sHSPs comprise a conserved α-crystallin domain flanked by variable N- and C-terminal regions. To investigate the functional contributions of these three regions, we compared the chaperone activities of various constructs of human αB-crystallin (HSPB5) and heat-shock 27-kDa protein (Hsp27, HSPB1) during amyloid formation by α-synuclein and apolipoprotein C-II. Using an array of approaches, including thioflavin T fluorescence assays and sedimentation analysis, we found that the N-terminal region of Hsp27 and the terminal regions of αB-crystallin are important for delaying amyloid fibril nucleation and for disaggregating mature apolipoprotein C-II fibrils. We further show that the terminal regions are required for stable fibril binding by both sHSPs and for mediating lateral fibril-fibril association, which sequesters preformed fibrils into large aggregates and is believed to have a cytoprotective function. We conclude that although the isolated α-crystallin domain retains some chaperone activity against amyloid formation, the flanking domains contribute additional and important chaperone activities, both in delaying amyloid formation and in mediating interactions of sHSPs with amyloid aggregates. Both these chaperone activities have significant implications for the pathogenesis and progression of diseases associated with amyloid deposition, such as Parkinson's and Alzheimer's diseases.


Asunto(s)
Amiloide/química , Proteínas de Choque Térmico/química , Chaperonas Moleculares/química , Cadena B de alfa-Cristalina/química , Amiloide/metabolismo , Apolipoproteína C-II/química , Apolipoproteína C-II/metabolismo , Proteínas de Choque Térmico/metabolismo , Humanos , Chaperonas Moleculares/metabolismo , Dominios Proteicos , Cadena B de alfa-Cristalina/metabolismo , alfa-Sinucleína/química , alfa-Sinucleína/metabolismo
14.
Mol Cell Proteomics ; 19(4): 640-654, 2020 04.
Artículo en Inglés | MEDLINE | ID: mdl-32086375

RESUMEN

C9ORF72-associated Motor Neuron Disease patients feature abnormal expression of 5 dipeptide repeat (DPR) polymers. Here we used quantitative proteomics in a mouse neuronal-like cell line (Neuro2a) to demonstrate that the Arg residues in the most toxic DPRS, PR and GR, leads to a promiscuous binding to the proteome compared with a relative sparse binding of the more inert AP and GA. Notable targets included ribosomal proteins, translation initiation factors and translation elongation factors. PR and GR comprising more than 10 repeats appeared to robustly stall on ribosomes during translation suggesting Arg-rich peptide domains can electrostatically jam the ribosome exit tunnel during synthesis. Poly-GR also recruited arginine methylases, induced hypomethylation of endogenous proteins, and induced a profound destabilization of the actin cytoskeleton. Our findings point to arginine in GR and PR polymers as multivalent toxins to translation as well as arginine methylation that may explain the dysfunction of biological processes including ribosome biogenesis, mRNA splicing and cytoskeleton assembly.


Asunto(s)
Arginina/metabolismo , Arginina/toxicidad , Proteína C9orf72/metabolismo , Péptidos/metabolismo , Proteoma/metabolismo , Citoesqueleto de Actina/efectos de los fármacos , Citoesqueleto de Actina/metabolismo , Animales , Proteínas Fluorescentes Verdes/metabolismo , Células HEK293 , Humanos , Metilación/efectos de los fármacos , Ratones , Modelos Biológicos , Unión Proteica/efectos de los fármacos , Biosíntesis de Proteínas/efectos de los fármacos , Ribosomas/metabolismo
15.
Proc Natl Acad Sci U S A ; 117(5): 2422-2431, 2020 02 04.
Artículo en Inglés | MEDLINE | ID: mdl-31964829

RESUMEN

The accumulation of protein deposits in neurodegenerative diseases has been hypothesized to depend on a metastable subproteome vulnerable to aggregation. To investigate this phenomenon and the mechanisms that regulate it, we measured the solubility of the proteome in the mouse Neuro2a cell line under six different protein homeostasis stresses: 1) Huntington's disease proteotoxicity, 2) Hsp70, 3) Hsp90, 4) proteasome, 5) endoplasmic reticulum (ER)-mediated folding inhibition, and 6) oxidative stress. Overall, we found that about one-fifth of the proteome changed solubility with almost all of the increases in insolubility were counteracted by increases in solubility of other proteins. Each stress directed a highly specific pattern of change, which reflected the remodeling of protein complexes involved in adaptation to perturbation, most notably, stress granule (SG) proteins, which responded differently to different stresses. These results indicate that the protein homeostasis system is organized in a modular manner and aggregation patterns were not correlated with protein folding stability (ΔG). Instead, distinct cellular mechanisms regulate assembly patterns of multiple classes of protein complexes under different stress conditions.


Asunto(s)
Proteoma/química , Proteostasis/fisiología , Estrés Fisiológico , Animales , Línea Celular Tumoral , Proteína Huntingtina/química , Proteína Huntingtina/genética , Ligandos , Ratones , Mutación , Agregado de Proteínas , Pliegue de Proteína , Proteoma/metabolismo , Solubilidad
16.
Semin Cell Dev Biol ; 99: 40-54, 2020 03.
Artículo en Inglés | MEDLINE | ID: mdl-29753879

RESUMEN

Maintaining protein homeostasis (proteostasis) is essential for cellular health and is governed by a network of quality control machinery comprising over 800 genes. When proteostasis becomes imbalanced, proteins can abnormally aggregate or become mislocalized. Inappropriate protein aggregation and proteostasis imbalance are two of the central pathological features of common neurodegenerative diseases including Alzheimer, Parkinson, Huntington, and motor neuron diseases. How aggregation contributes to the pathogenic mechanisms of disease remains incompletely understood. Here, we integrate some of the key and emerging ideas as to how protein aggregation relates to imbalanced proteostasis with an emphasis on Huntington disease as our area of main expertise. We propose the term "aggregomics" be coined in reference to how aggregation of particular proteins concomitantly influences the spatial organization and protein-protein interactions of the surrounding proteome. Meta-analysis of aggregated interactomes from various published datasets reveals chaperones and RNA-binding proteins are common components across various disease contexts. We conclude with an examination of therapeutic avenues targeting proteostasis mechanisms.


Asunto(s)
Chaperonas Moleculares/metabolismo , Enfermedades Neurodegenerativas/metabolismo , Enfermedades Neurodegenerativas/patología , Agregado de Proteínas , Agregación Patológica de Proteínas/metabolismo , Proteostasis , Proteínas de Unión al ARN/metabolismo , Animales , Humanos
17.
Cell Rep ; 23(12): 3492-3500, 2018 06 19.
Artículo en Inglés | MEDLINE | ID: mdl-29924993

RESUMEN

The aberrant aggregation of α-synuclein is associated with several human diseases, collectively termed the α-synucleinopathies, which includes Parkinson's disease. The progression of these diseases is, in part, mediated by extracellular α-synuclein oligomers that may exert effects through several mechanisms, including prion-like transfer, direct cytotoxicity, and pro-inflammatory actions. In this study, we show that two abundant extracellular chaperones, clusterin and α2-macroglobulin, directly bind to exposed hydrophobic regions on the surface of α-synuclein oligomers. Using single-molecule fluorescence techniques, we found that clusterin, unlike α2-macroglobulin, exhibits differential binding to α-synuclein oligomers that may be related to structural differences between two previously described forms of αS oligomers. The binding of both chaperones reduces the ability of the oligomers to permeabilize lipid membranes and prevents an oligomer-induced increase in ROS production in cultured neuronal cells. Taken together, these data suggest a neuroprotective role for extracellular chaperones in suppressing the toxicity associated with α-synuclein oligomers.


Asunto(s)
Espacio Extracelular/metabolismo , Multimerización de Proteína , alfa-Sinucleína/química , alfa-Sinucleína/toxicidad , Interacciones Hidrofóbicas e Hidrofílicas , Chaperonas Moleculares/metabolismo , Unión Proteica
18.
J Biol Chem ; 293(12): 4486-4497, 2018 03 23.
Artículo en Inglés | MEDLINE | ID: mdl-29382725

RESUMEN

Proteostasis, or protein homeostasis, encompasses the maintenance of the conformational and functional integrity of the proteome and involves an integrated network of cellular pathways. Molecular chaperones, such as the small heat shock proteins (sHsps), are key elements of the proteostasis network that have crucial roles in inhibiting the aggregation of misfolded proteins. Failure of the proteostasis network can lead to the accumulation of misfolded proteins into intracellular and extracellular deposits. Deposits containing fibrillar forms of α-synuclein (α-syn) are characteristic of neurodegenerative disorders including Parkinson's disease and dementia with Lewy bodies. Here we show that the sHsp Hsp27 (HSPB1) binds to α-syn fibrils, inhibiting fibril growth by preventing elongation. Using total internal reflection fluorescence (TIRF)-based imaging methods, we show that Hsp27 binds along the surface of α-syn fibrils, decreasing their hydrophobicity. Binding of Hsp27 also inhibits cytotoxicity of α-syn fibrils. Our results demonstrate that the ability of sHsps, such as Hsp27, to bind fibrils represents an important mechanism through which they may mitigate cellular toxicity associated with aberrant protein aggregation. Fibril binding may represent a generic mechanism by which chaperone-active sHsps interact with aggregation-prone proteins, highlighting the potential to target sHsp activity to prevent or disrupt the onset and progression of α-syn aggregation associated with α-synucleinopathies.


Asunto(s)
Proteínas de Choque Térmico HSP27/metabolismo , Neuroblastoma/patología , Agregado de Proteínas , alfa-Sinucleína/metabolismo , Animales , Proteínas de Choque Térmico HSP27/genética , Proteínas de Choque Térmico , Humanos , Ratones , Chaperonas Moleculares , Neuroblastoma/metabolismo , Células Tumorales Cultivadas , alfa-Sinucleína/genética
19.
Nat Commun ; 9(1): 287, 2018 01 18.
Artículo en Inglés | MEDLINE | ID: mdl-29348634

RESUMEN

The pool of quality control proteins (QC) that maintains protein-folding homeostasis (proteostasis) is dynamic but can become depleted in human disease. A challenge has been in quantitatively defining the depth of the QC pool. With a new biosensor, flow cytometry-based methods and mathematical modeling we measure the QC capacity to act as holdases and suppress biosensor aggregation. The biosensor system comprises a series of barnase kernels with differing folding stability that engage primarily with HSP70 and HSP90 family proteins. Conditions of proteostasis stimulation and stress alter QC holdase activity and aggregation rates. The method reveals the HSP70 chaperone cycle to be rate limited by HSP70 holdase activity under normal conditions, but this is overcome by increasing levels of the BAG1 nucleotide exchange factor to HSPA1A or activation of the heat shock gene cluster by HSF1 overexpression. This scheme opens new paths for biosensors of disease and proteostasis systems.


Asunto(s)
Técnicas Biosensibles/métodos , Citometría de Flujo/métodos , Modelos Teóricos , Proteostasis , Algoritmos , Western Blotting , Células HEK293 , Proteínas del Choque Térmico HSP72/metabolismo , Proteínas HSP90 de Choque Térmico/metabolismo , Factores de Transcripción del Choque Térmico/metabolismo , Humanos , Proteoma/metabolismo , Proteómica/métodos , Espectrometría de Masas en Tándem/métodos
20.
Cell Stress Chaperones ; 22(4): 589-600, 2017 07.
Artículo en Inglés | MEDLINE | ID: mdl-28337642

RESUMEN

Protein homeostasis, or proteostasis, is the process of maintaining the conformational and functional integrity of the proteome. Proteostasis is preserved in the face of stress by a complex network of cellular machinery, including the small heat shock molecular chaperone proteins (sHsps), which act to inhibit the aggregation and deposition of misfolded protein intermediates. Despite this, the pathogenesis of several neurodegenerative diseases has been inextricably linked with the amyloid fibrillar aggregation and deposition of α-synuclein (α-syn). The sHsps are potent inhibitors of α-syn aggregation in vitro. However, the limited availability of a robust, cell-based model of α-syn aggregation has, thus far, restricted evaluation of sHsp efficacy in the cellular context. As such, this work sought to establish a robust model of intracellular α-syn aggregation using Neuro-2a cells. Aggregation of α-syn was found to be sensitive to inhibition of autophagy and the proteasome, resulting in a significant increase in the proportion of cells containing α-syn inclusions. This model was then used to evaluate the capacity of the sHsps, αB-c and Hsp27, to prevent α-syn aggregation in cells. To do so, we used bicistronic expression plasmids to express the sHsps. Unlike traditional fluorescent fusion constructs, these bicistronic expression plasmids enable only individual transfected cells expressing the sHsps (via expression of the fluorescent reporter) to be analysed, but without the need to tag the sHsp, which can affect its oligomeric structure and chaperone activity. Overexpression of both αB-c and Hsp27 significantly reduced the intracellular aggregation of α-syn. Thus, these findings suggest that overexpressing or boosting the activity of sHsps may be a way of preventing amyloid fibrillar aggregation of α-syn in the context of neurodegenerative disease.


Asunto(s)
Proteínas de Choque Térmico HSP27/metabolismo , Agregado de Proteínas , Cadena B de alfa-Cristalina/metabolismo , alfa-Sinucleína/metabolismo , Amiloide/análisis , Amiloide/metabolismo , Animales , Autofagia , Línea Celular , Células HEK293 , Proteínas de Choque Térmico , Humanos , Ratones , Chaperonas Moleculares , alfa-Sinucleína/análisis
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