The proteostasis network and its decline in ageing

Ageing is a major risk factor for the development of many diseases, prominently including neurodegenerative disorders such as Alzheimer disease and Parkinson disease. A hallmark of many age-related diseases is the dysfunction in protein homeostasis (proteostasis), leading to the accumulation of protein aggregates. In healthy cells, a complex proteostasis network, comprising molecular chaperones and proteolytic machineries and their regulators, operates to ensure the maintenance of proteostasis. These factors coordinate protein synthesis with polypeptide folding, the conservation of protein conformation and protein degradation. However, sustaining proteome balance is a challenging task in the face of various external and endogenous stresses that accumulate during ageing. These stresses lead to the decline of proteostasis network capacity and proteome integrity. The resulting accumulation of misfolded and aggregated proteins affects, in particular, postmitotic cell types such as neurons, manifesting in disease. Recent analyses of proteome-wide changes that occur during ageing inform strategies to improve proteostasis. The possibilities of pharmacological augmentation of the capacity of proteostasis networks hold great promise for delaying the onset of age-related pathologies associated with proteome deterioration and for extending healthspan

Individual and Collective Contributions of Chaperoning and Degradation to Protein Homeostasis in E. coli

SummaryThe folding fate of a protein in vivo is determined by the interplay between a protein’s folding energy landscape and the actions of the proteostasis network, including molecular chaperones and degradation enzymes. The mechanisms of individual components of the E. coli proteostasis network have been studied extensively, but much less is known about how they function as a system. We used an integrated experimental and computational approach to quantitatively analyze the folding outcomes (native folding versus aggregation versus degradation) of three test proteins biosynthesized in E. coli under a variety of conditions. Overexpression of the entire proteostasis network benefited all three test proteins, but the effect of upregulating individual chaperones or the major degradation enzyme, Lon, varied for proteins with different biophysical properties. In sum, the impact of the E. coli proteostasis network is a consequence of concerted action by the Hsp70 system (DnaK/DnaJ/GrpE), the Hsp60 system (GroEL/GroES), and Lon

Exploring the Impact of the E. coli Proteostasis Network on the Folding Fate of Proteins with Different Intrinsic Biophysical Properties

The three-dimensional (3D) native structure of most proteins is crucial for their functions. Despite the complex cellular environment and the variety of challenges that proteins experience as they fold, proteins can still fold to their native states with high fidelity. The reason for this is the presence of the cellular proteostasis network (PN), consisting of molecular chaperones and degradation enzymes, that collaborates to maintain proteostasis, in which the necessary levels of functional proteins are optimized. Although extensive research has been carried out on the mechanisms of individual components of the proteostasis network, little is known about how these components contribute to the functioning of the network as a whole. A new protein can have three folding fates: natively folded, aggregated, or degraded. The fate is determined by both a protein’s intrinsic biophysical properties and the cellular proteostasis network through kinetic partitioning. To understand the interplay between a protein’s intrinsic biophysical properties and the cellular proteostasis network, an integrated computational and experimental approach was used. The folding fates of model proteins with different intrinsic biophysical properties under varying conditions of the proteostasis network were determined. Using FoldEco, the effects of the kinetic and thermodynamic properties of proteins on their folding fates were investigated systematically, and predictions were consistent with wet lab experiments. The folding fate of a protein is under a thermo-kinetic limitation, which indicates that the fate depends on either the kinetics or thermodynamics, but (for the most part) not on both at the same time. Different proteins behave according to the values of their limiting properties. Furthermore, up-regulation of the entire proteostasis network through the σ32 transcription factor has beneficial effects on model proteins with low stabilities and high aggregation propensities. However, the effects of up-regulation of individual chaperones or the major degradation enzyme, Lon are substrate-dependent and are related to their biophysical properties. Furthermore, KJE, GroELS, and Lon form an efficacious triad for maintaining proteostasis, and their contributions depend on the biophysical properties of their substrates, and on the concentrations of these PN components and substrates at any given time

Protein quality control:from mechanism to disease EMBO Workshop, Costa de la Calma (Mallorca), Spain, April 28-May 03, 2019

The cellular protein quality control machinery with its central constituents of chaperones and proteases is vital to maintain protein homeostasis under physiological conditions and to protect against acute stress conditions. Imbalances in protein homeostasis also are keys to a plethora of genetic and acquired, often age-related, diseases as well as aging in general. At the EMBO Workshop, speakers covered all major aspects of cellular protein quality control, from basic mechanisms at the molecular, cellular, and organismal level to medical translation. In this report, the highlights of the meeting will be summarized

Redox sensing by yeast Hsp70 facilitates modulation of protein quality control and the cytoprotective response

Neurodegenerative disease affects millions of Americans every year, through diagnoses such as Alzheimer’s, Parkinson’s, and Huntington’s diseases. One factor linked to formation of these aggregates is damage sustained to proteins by oxidative stress. Cellular protein homeostasis (proteostasis) relies on the ubiquitous Hsp70 chaperone family. Hsp70 activity has been previously shown to be modulated by modification of two key cysteines in the ATPase domain by oxidizing or thiol-modifying compounds. To investigate the biological consequences of cysteine modification on the Hsp70 Ssa1 in budding yeast, I generated cysteine null (cysteine to serine) and oxidomimetic (cysteine to aspartic acid) mutant variants of both C264 and C303 and demonstrate reduced ATP binding, hydrolysis and protein folding properties in both the oxidomimetic as well as hydrogen peroxide-treated Ssa1. In contrast, cysteine nullification rendered Ssa1 insensitive to oxidative inhibition. The oxidomimetic ssa1-2CD (C264D, C303D) allele was unable to function as the sole Ssa1 isoform in yeast cells and also exhibited negative effects on cell growth and viability. Ssa1 binds to and represses Hsf1, the major transcription factor controlling the heat shock response, and the oxidomimetic Ssa1 failed to stably interact with Hsf1, resulting in constitutive activation of the heat shock response. Consistent with the in vitro findings, ssa1-2CD cells were compromised for de novo folding, post-stress protein refolding and in regulated degradation of a model terminally misfolded protein. Together these findings pinpoint Hsp70 as a key link between oxidative stress and proteostasis, information critical to understanding cytoprotective systems that prevent and manage cellular insults underlying complex disease states

Investigating the impact of small molecule ligands and the proteostasis network on protein folding inside the cell

The folded forms of most proteins are critical to their functions. Despite the complexity of the cellular milieu and the presence of high-risk deleterious interactions, there is a high level of fidelity observed in the folding process for entire proteomes. Two important reasons for this are the presence of the quality control machinery consisting of chaperones and degradation enzymes that work jointly to optimize the population of the folded state and interaction partners that re-enforce the functional state and add to the competitive advantage of an organism. While substantial effort has been directed to understand protein folding and interactions in vitro, comparatively little of these processes are explored inside the cell. This work examines two important aspects of protein folding inside the cell; first, the impact of small molecule ligands on protein folding; and second, the impact of the proteostasis network on the folding of an obligatory chaperone client. We deploy a combination of experiments and mathematical modeling based on the principle of kinetic partitioning to understand how these phenomena sculpt the protein folding landscape inside the cell. We find that ligands specifically deplete unfolded and aggregation- or degradation - prone protein populations by favoring the folded state and the chaperone and degradation proteins work to minimize off-pathway species thus reducing the population of aggregated protein inside the cell

Cytoplasmic protein aggregates interfere with nucleo-cytoplasmic transport of protein and RNA

Protein misfolding and aggregation are linked to various forms of dementia and amyloidoses, such as Alzheimer’s, Parkinson’s, and Creutzfeldt Jakob diseases. Although the primary misfolding proteins are disease-specific and structurally diverse, the related disorders share numerous symptoms and cellular malfunctions. A sustainable cure remains so far out of reach. The highly complex nature of the associated cellular deficiencies challenges our understanding of primary causes and consequences in the disease progression. To focus on the toxic properties and pathogenic gain-of-function mechanisms of misfolded structures in cells, we applied a set of artificial β proteins directly folding into amyloid-like oligomers and fibrils. Amyloid-related proteotoxicity appeared sequence-dependently in human, murine neuronal, fungal, and bacterial cells. The interplay between elevated surface hydrophobicity and structural disorder among the β proteins and their cellular interactors was critical for toxicity. Small distributed oligomers correlated to elevated toxicity. Protein-rich plaques or misfolded assemblies appear in patients often simultaneously in different cellular compartments and in the extracellular space. To analyze site-specific toxicities and vulnerabilities, we targeted the β proteins specifically into distinct compartments. Aggregation in the cytoplasm was highly toxic and interfered with active nucleo-cytoplamsic transport in both directions, including the translocation of NF-κB and mRNA. We compared our results to human disease-associated mutants of Huntingtin, TDP-43, and Parkin, causing comparable transport defects. Remarkably, toxicity of the β proteins was strongly reduced when targeted to the nucleus. Aggregates localized in dense nucleolar foci caused no transport inhibition. Only protein aggregation in the cytoplasm led to sequestration and mislocalization of numerous proteins with extended disordered regions, including factors involved in nucleo-cytoplasmic transport of proteins and mRNA (importin α and THOC proteins). Nuclear β proteins in contrast behaved very inert, potentially being shielded by nucleolar factors such as nucleophosmin (NPM-1). In presence of cytoplasmic aggregation vital signaling processes were impaired, further destabilizing cellular homoeostasis. The mRNA accumulated in enlarged “nuclear RNA bodies”. Depletion of cytoplasmic mRNA consequently resulted in a reduction of protein synthesis. Impairment of nucleo-cytoplasmic transport caused by cytoplasmic protein aggregation may thus seriously aggravate the cellular pathology initiated by misfolding and aggregation in human amyloid diseases. Our findings suggest that novel therapeutic strategies may improve nucleocytoplasmic transport, utilize the nuclear proteostasis for aggregate removal, or increase the cellular resilience towards misfolded structures in general.Proteinmissfaltung und -aggregation wird mit neurodegenerativen Krankheiten wie Alzheimer, Parkinson und der Creutzfeldt-Jakob-Krankheit, sowie mit systemischen Amyloidosen in Verbindung gebracht. Auch wenn sich anfangs die Hauptbestandteile der Proteinaggregate krankheitsspezifisch unterscheiden, so kommt es bei den verschiedenen Demenzerkrankungen doch oft zu ähnlichen Symptomen und zellulären Fehlfunktionen. Eine nachhaltige Heilung ist bisher nicht möglich. Die Komplexität der auftretenden zellulären Fehlfunktionen erschwert eine klare Unterscheidung von primären Ursachen sowie deren Folgen und Nebenwirkungen. Um uns auf die toxischen Eigenschaften und die toxische Wirkung von missgefalteten Strukturen in Zellen zu konzentrieren, setzen wir eine Reihe von künstlichen β Proteinen ein, welche direkt amyloide Oligomere und Aggregate bilden. Die Toxizität der β Proteine trat sequenzabhängig in menschlichen, neuronalen, Pilz- und Bakterienzellen auf. Erhöhte Hydrophobie an der Proteinoberfläche und unstrukturierte Sequenzbereiche wurden als kritische strukturelle Merkmale der β Proteine und ihrer zellulären Interaktionspartner im Zusammenhang zur Toxizität identifiziert. Auch korrelierten kleinere, über das Zytoplasma verteilte Oligomere mit hoher Toxizität. Proteinaggregate treten in Patienten in verschiedenen Kompartimenten der Zelle und im extrazellulären Raum auf, oft an mehreren Stellen gleichzeitig. Um die Toxizität in verschiedenen Kompartimenten und deren Sensibilitäten zu untersuchen, schickten wir die β Proteine mittels Signalsequenzen gezielt in bestimmte zelluläre Kompartimente. Aggregation im Zytoplasma war hochtoxisch und störte den aktiven Transport zwischen Zytoplasma und Zellkern, einschließlich der Translokation von NF-κB und mRNA. Wir reproduzierten unsere Ergebnisse mit krankheitsassoziierten Mutanten von Huntingtin, TDP-43 und Parkin, welche vergleichbare Transportdefekte verursachten. Bemerkenswerterweise reduzierte sich die Toxizität der β Proteine stark, wenn sie in den Zellkern geschickt wurden. Hier sammelten sich die β Proteine in dichten Aggregaten in den Nukleoli. Dabei traten keine Transportprobleme auf. Nur Proteinaggregation im Zytoplasma verursachte (Ko-)Aggregation und Fehllokalisation zahlreicher zellulärer Proteine, besonders von solchen mit längeren unstrukturierten Bereichen. Dazu zählten auch Faktoren, welche den Transport von Proteinen und mRNA zwischen Zytoplasma und Zellkern vermitteln (Importin α und THOC Proteine). Die β Proteine im Zellkern verhielten sich im Gegensatz sehr unauffällig. Anscheinend wurden sie zusätzlich durch nukleoläre Faktoren wie Nukleophosmin (NPM-1) abgeschirmt. Aggregation im Zytoplasma beeinträchtigte die Übermittlung lebenswichtiger zellulärer Signale, was die zelluläre Homöostase weiter destabilisierte. Die mRNA hat sich dabei in vergrößerten „nukleären RNA Körperchen“ angesammelt. Die fehlende mRNA im Zytoplasma führte zu einer Abnahme der Proteinsynthese. Die von Proteinaggregaten verursachten Defekte im molekularen Transport zwischen Zytoplasma und Zellkern könnten so ernsthaft zur Verschlimmerung der zellulären Funktionsfähigkeit in neurodegenerativen und anderen Proteinfehlfaltungserkrankungen beitragen. Neue Therapieansätze könnten in einer Verbesserung des Kerntransports, in einer Verminderung von Aggregaten durch Proteostasissysteme im Zellkern, oder in einer generellen Stärkung der zellulären Resilienz gegenüber fehlgefalteten Proteinen zu finden sein

Single-molecule spectroscopy: investigations of protein folding to multi-laboratory consistencies on proteins

The investigation of complex biological processes has been challenging and require a variety of sophisticated tools to interpret the underlying processes. The study of the folding process in proteins is one of the focuses of this thesis work. To this end, both spontaneous and chaperone- assisted folding mechanisms were investigated. Single-molecule fluorescence spectroscopy has been extensively applied to the study of biomolecular bindings, conformational changes, and their dynamics due to its high sensitivity, time resolution, and its ability to differentiate between homogenous and heterogenous populations. Specifically, single-molecule Förster Resonance Energy Transfer (smFRET) studies on protein folding have elucidated the basic mechanisms of spontaneous protein folding, and properties of the chaperone-substrate interactions. The possibility to measure at low concentrations making it possible to avoid the aggregation, which is difficult to avoid in ensemble experiments. To investigate the spontaneous folding mechanisms in large multi-domain proteins, two-color smFRET studies were carried out on a slowly folding version of the two-domain Maltose- binding protein (MBP). Three-color smFRET, an extension of typical two-color smFRET to three-colors, was applied on specifically labeled MBP to visualize the co-ordination between the domains as they fold. Chaperone-substrate interactions are crucial to process the substrates and thus enable them to carry out their physiological function. Cavity confinement effect of GroEL/ES, a bacterial Hsp60 on MBP folding landscape was demonstrated. Another substrate protein, p53-DNA-binding domain was probed concerning the combined action of Hsp70 and Hsp90 chaperone on its folding. To conclude the thesis work, a smFRET comparison study on proteins involving 16 laboratories was undertaken to assess the accuracy and precision of smFRET measurements as well as to determine a detection limit for dynamic motions in proteins