Heat shock response and autophagy—cooperation and control

Protein quality control (proteostasis) depends on constant protein degradation and resynthesis, and is essential for proper homeostasis in systems from single cells to whole organisms. Cells possess several mechanisms and processes to maintain proteostasis. At one end of the spectrum, the heat shock proteins modulate protein folding and repair. At the other end, the proteasome and autophagy as well as other lysosome-dependent systems, function in the degradation of dysfunctional proteins. In this review, we examine how these systems interact to maintain proteostasis. Both the direct cellular data on heat shock control over autophagy and the time course of exercise-associated changes in humans support the model that heat shock response and autophagy are tightly linked. Studying the links between exercise stress and molecular control of proteostasis provides evidence that the heat shock response and autophagy coordinate and undergo sequential activation and downregulation, and that this is essential for proper proteostasis in eukaryotic systems.     

Heat shock factor 2 is required for maintaining proteostasis against febrile-range thermal stress and polyglutamine aggregation

Heat shock response is characterized by the induction of heat shock proteins (HSPs), which facilitate protein folding, and non-HSP proteins with diverse functions, including protein degradation, and is regulated by heat shock factors (HSFs). HSF1 is a master regulator of HSP expression during heat shock in mammals, as is HSF3 in avians. HSF2 plays roles in development of the brain and reproductive organs. However, the fundamental roles of HSF2 in vertebrate cells have not been identified. Here we find that vertebrate HSF2 is activated during heat shock in the physiological range. HSF2 deficiency reduces threshold for chicken HSF3 or mouse HSF1 activation, resulting in increased HSP expression during mild heat shock. HSF2-null cells are more sensitive to sustained mild heat shock than wild-type cells, associated with the accumulation of ubiquitylated misfolded proteins. Furthermore, loss of HSF2 function increases the accumulation of aggregated polyglutamine protein and shortens the lifespan of R6/2 Huntington's disease mice, partly through αB-crystallin expression. These results identify HSF2 as a major regulator of proteostasis capacity against febrile-range thermal stress and suggest that HSF2 could be a promising therapeutic target for protein-misfolding diseases.     

Firefly luciferase mutants as sensors of proteome stress

Maintenance of cellular protein homeostasis (proteostasis) depends on a complex network of molecular chaperones, proteases and other regulatory factors. Proteostasis deficiency develops during normal aging and predisposes individuals for many diseases, including neurodegenerative disorders. Here we describe sensor proteins for the comparative measurement of proteostasis capacity in different cell types and model organisms. These sensors are increasingly structurally destabilized versions of firefly luciferase. Imbalances in proteostasis manifest as changes in sensor solubility and luminescence activity. We used EGFP-tagged constructs to monitor the aggregation state of the sensors and the ability of cells to solubilize or degrade the aggregated proteins. A set of three sensor proteins serves as a convenient toolkit to assess the proteostasis status in a wide range of experimental systems, including cell and organism models of stress, neurodegenerative disease and aging.     

Endoplasmic reticulum proteostasis impairment in aging

Perturbed neuronal proteostasis is a salient feature shared by both aging and protein misfolding disorders. The proteostasis network controls the health of the proteome by integrating pathways involved in protein synthesis, folding, trafficking, secretion, and their degradation. A reduction in the buffering capacity of the proteostasis network during aging may increase the risk to undergo neurodegeneration by enhancing the accumulation of misfolded proteins. As almost one‐third of the proteome is synthetized at the endoplasmic reticulum (ER), maintenance of its proper function is fundamental to sustain neuronal function. In fact, ER stress is a common feature of most neurodegenerative diseases. The unfolded protein response (UPR) operates as central player to maintain ER homeostasis or the induction of cell death of chronically damaged cells. Here, we discuss recent evidence placing ER stress as a driver of brain aging, and the emerging impact of neuronal UPR in controlling global proteostasis at the whole organismal level. Finally, we discuss possible therapeutic interventions to improve proteostasis and prevent pathological brain aging.     

Identification of Proteins Sensitive to Thermal Stress in Human Neuroblastoma and Glioma Cell Lines

Heat-shock is an acute insult to the mammalian proteome. The sudden elevation in temperature has far-reaching effects on protein metabolism, leads to a rapid inhibition of most protein synthesis, and the induction of protein chaperones. Using heat-shock in cells of neuronal (SH-SY5Y) and glial (CCF-STTG1) lineage, in conjunction with detergent extraction and sedimentation followed by LC-MS/MS proteomic approaches, we sought to identify human proteins that lose solubility upon heat-shock. The two cell lines showed largely overlapping profiles of proteins detected by LC-MS/MS. We identified 58 proteins in detergent insoluble fractions as losing solubility in after heat shock; 10 were common between the 2 cell lines. A subset of the proteins identified by LC-MS/MS was validated by immunoblotting of similarly prepared fractions. Ultimately, we were able to definitively identify 3 proteins as putatively metastable neural proteins; FEN1, CDK1, and TDP-43. We also determined that after heat-shock these cells accumulate insoluble polyubiquitin chains largely linked via lysine 48 (K-48) residues. Collectively, this study identifies human neural proteins that lose solubility upon heat-shock. These proteins may represent components of the human proteome that are vulnerable to misfolding in settings of proteostasis stress.     

The nascent polypeptide‐associated complex is a key regulator of proteostasis

The adaptation of protein synthesis to environmental and physiological challenges is essential for cell viability. Here, we show that translation is tightly linked to the protein‐folding environment of the cell through the functional properties of the ribosome bound chaperone NAC (nascent polypeptide‐associated complex). Under non‐stress conditions, NAC associates with ribosomes to promote translation and protein folding. When proteostasis is imbalanced, NAC relocalizes from a ribosome‐associated state to protein aggregates in its role as a chaperone. This results in a functional depletion of NAC from the ribosome that diminishes translational capacity and the flux of nascent proteins. Depletion of NAC from polysomes and re‐localisation to protein aggregates is observed during ageing, in response to heat shock and upon expression of the highly aggregation‐prone polyglutamine‐expansion proteins and Aβ‐peptide. These results demonstrate that NAC has a central role as a proteostasis sensor to provide the cell with a regulatory feedback mechanism in which translational activity is also controlled by the folding state of the cellular proteome and the cellular response to stress.     

Interrelation Between Protein Synthesis,Proteostasis and Life Span

The production of newly synthesized proteins is a key process of protein homeostasis that initiates the biosynthetic flux of proteins and thereby determines the composition, stability and functionality of the proteome. Protein synthesis is highly regulated on multiple levels to adapt the proteome to environmental and physiological challenges such as aging and proteotoxic conditions. Imbalances of protein folding conditions are sensed by the cell that then trigger a cascade of signaling pathways aiming to restore the protein folding equilibrium. One regulatory node to rebalance proteostasis upon stress is the control of protein synthesis itself. Translation is reduced as an immediate response to perturbations of the protein folding equilibrium that can be observed in the cytosol as well as in the organelles such as the endoplasmatic reticulum and mitochondria. As reduction of protein synthesis is linked to life span increase, the signaling pathways regu-lating protein synthesis might be putative targets for treatments of age-related diseases. Eukaryotic cells have evolved a complex system for protein synthesis regulation and this review will summarize cellular strategies to regulate mRNA translation upon stress and its impact on longevity.     

Build-UPS and break-downs: metabolism impacts on proteostasis and aging

Perturbation of metabolism elicits cellular stress which profoundly modulates the cellular proteome and thus protein homeostasis (proteostasis). Consequently, changes in the cellular proteome due to metabolic shift require adaptive mechanisms by molecular protein quality control. The mechanisms vitally controlling proteostasis embrace the entire life cycle of a protein involving translational control at the ribosome, chaperone-assisted native folding, and subcellular sorting as well as proteolysis by the proteasome or autophagy. While metabolic imbalance and proteostasis decline have been recognized as hallmarks of aging and age-associated diseases, both processes are largely considered independently. Here, we delineate how proteome stability is governed by insulin/IGF1 signaling (IIS), mechanistic target of Rapamycin (TOR), 5′ adenosine monophosphate-activated protein kinase (AMPK), and NAD-dependent deacetylases (Sir2-like proteins known as sirtuins). This comprehensive overview is emphasizing the regulatory interconnection between central metabolic pathways and proteostasis, indicating the relevance of shared signaling nodes as targets for future therapeutic interventions.Subject terms: Protein quality control, Metabolic pathways, Ageing     

The Stress of Protein Misfolding: From Single Cells to Multicellular Organisms

Organisms survive changes in the environment by altering their rates of metabolism, growth, and reproduction. At the same time, the system must ensure the stability and functionality of its macromolecules. Fluctuations in the environment are sensed by highly conserved stress responses and homeostatic mechanisms, and of these, the heat shock response (HSR) represents an essential response to acute and chronic proteotoxic damage. However, unlike the strategies employed to maintain the integrity of the genome, protection of the proteome must be tailored to accommodate the normal flux of nonnative proteins and the differences in protein composition between cells, and among individuals. Moreover, adult cells are likely to have significant differences in the rates of synthesis and clearance that are influenced by intrinsic errors in protein expression, genetic polymorphisms, and fluctuations in physiological and environmental conditions. Here, we will address how protein homeostasis (proteostasis) is achieved at the level of the cell and organism, and how the threshold of the stress response is set to detect and combat protein misfolding. For metazoans, the requirement for coordinated function and growth imposes additional constraints on the detection, signaling, and response to misfolding, and requires that the HSR is integrated into various aspects of organismal physiology, such as lifespan. This is achieved by hierarchical regulation of heat shock factor 1 (HSF1) by the metabolic state of the cell and centralized neuronal control that could allow optimal resource allocation between cells and tissues. We will examine how protein folding quality control mechanisms in individual cells may be integrated into a multicellular level of control, and further, even custom-designed to support individual variability and impose additional constraints on evolutionary adaptation.     

The intrinsic chaperone network of Arabidopsis stem cells confers protection against proteotoxic stress

The biological purpose of plant stem cells is to maintain themselves while providing new pools of differentiated cells that form organs and rejuvenate or replace damaged tissues. Protein homeostasis or proteostasis is required for cell function and viability. However, the link between proteostasis and plant stem cell identity remains unknown. In contrast to their differentiated counterparts, we find that root stem cells can prevent the accumulation of aggregated proteins even under proteotoxic stress conditions such as heat stress or proteasome inhibition. Notably, root stem cells exhibit enhanced expression of distinct chaperones that maintain proteome integrity. Particularly, intrinsic high levels of the T‐complex protein‐1 ring complex/chaperonin containing TCP1 (TRiC/CCT) complex determine stem cell maintenance and their remarkable ability to suppress protein aggregation. Overexpression of CCT8, a key activator of TRiC/CCT assembly, is sufficient to ameliorate protein aggregation in differentiated cells and confer resistance to proteotoxic stress in plants. Taken together, our results indicate that enhanced proteostasis mechanisms in stem cells could be an important requirement for plants to persist under extreme environmental conditions and reach extreme long ages. Thus, proteostasis of stem cells can provide insights to design and breed plants tolerant to environmental challenges caused by the climate change.     

Emerging fluorescence tools for the study of proteostasis in cells

Understanding how cells maintain the functional proteome and respond to stress conditions is critical for deciphering molecular pathogenesis and developing treatments for conditions such as neurodegenerative diseases. Efforts towards finer quantification of cellular proteostasis machinery efficiency, phase transitions and local environment changes remain a priority. Herein, we describe recent developments in fluorescence-based strategy and methodology, building on the experimental toolkit, for the study of proteostasis (protein homeostasis) in cells. We hope this review can assist in bridging gaps between a multitude of research disciplines and promote interdisciplinary collaboration to address the crucial topic of proteostasis.     

Accumulation of “Old Proteins” and the Critical Need for MS‐based Protein Turnover Measurements in Aging and Longevity

Aging and age‐related diseases are accompanied by proteome remodeling and progressive declines in cellular machinery required to maintain protein homeostasis (proteostasis), such as autophagy, ubiquitin‐mediated degradation, and protein synthesis. While many studies have focused on capturing changes in proteostasis, the identification of proteins that evade these cellular processes has recently emerged as an approach to studying the aging proteome. With advances in proteomic technology, it is possible to monitor protein half‐lives and protein turnover at the level of individual proteins in vivo. For large‐scale studies, these technologies typically include the use of stable isotope labeling coupled with MS and comprehensive assessment of protein turnover rates. Protein turnover studies have revealed groups of highly relevant long‐lived proteins (LLPs), such as the nuclear pore complexes, extracellular matrix proteins, and protein aggregates. Here, the role of LLPs during aging and age‐related diseases and the methods used to identify and quantify their changes are reviewed. The methods available to conduct studies of protein turnover, used in combination with traditional proteomic methods, will enable the field to perform studies in a systems biology context, as changes in proteostasis may not be revealed in studies that solely measure differential protein abundances.     

Protein stress and stress proteins: implications in aging and disease

Environmantal stress induces damage that activates an adaptive response in any organism. The cellular stress response is based on the induction of cytoprotective proteins, the so called stress or heat shock proteins. The stress response as well as stress proteins are ubiquitous, highly conserved mechanism, and genes, respectively, already present in prokaryotes. Chaperones protect the proteome against conformational damage, promoting the function of protein networks. Protein damage takes place during aging and in several degenerative diseases, and presents a threat to overload the cellular defense mechanisms. The preservation of a robust stress response and protein disposal is indispensable for health and longevity. This review summarizes the present knowledge of protein damage, turnover, and the stress response in aging and degenerative diseases.     

Heat shock proteins in neurodegenerative disorders and aging

Many members of the heat shock protein family act in unison to refold or degrade misfolded proteins. Some heat shock proteins also directly interfere with apoptosis. These homeostatic functions are especially important in proteinopathic neurodegenerative diseases, in which specific proteins misfold, aggregate, and kill cells through proteotoxic stress. Heat shock protein levels may be increased or decreased in these disorders, with the direction of the response depending on the individual heat shock protein, the disease, cell type, and brain region. Aging is also associated with an accrual of proteotoxic stress and modulates expression of several heat shock proteins. We speculate that the increase in some heat shock proteins in neurodegenerative conditions may be partly responsible for the slow progression of these disorders, whereas the increase in some heat shock proteins with aging may help delay senescence. The protective nature of many heat shock proteins in experimental models of neurodegeneration supports these hypotheses. Furthermore, some heat shock proteins appear to be expressed at higher levels in longer-lived species. However, increases in heat shock proteins may be insufficient to override overwhelming proteotoxic stress or reverse the course of these conditions, because the expression of several other heat shock proteins and endogenous defense systems is lowered. In this review we describe a number of stress-induced changes in heat shock proteins as a function of age and neurodegenerative pathology, with an emphasis on the heat shock protein 70 (Hsp70) family and the two most common proteinopathic disorders of the brain, Alzheimer’s and Parkinson’s disease.