Germline stem cell arrest inhibits the collapse of somatic proteostasis early in Caenorhabditis elegans adulthood

All cells rely on highly conserved protein folding and clearance pathways to detect and resolve protein damage and to maintain protein homeostasis (proteostasis). Because age is associated with an imbalance in proteostasis, there is a need to understand how protein folding is regulated in a multicellular organism that undergoes aging. We have observed that the ability of Caenorhabditis elegans to maintain proteostasis declines sharply following the onset of oocyte biomass production, suggesting that a restricted protein folding capacity may be linked to the onset of reproduction. To test this hypothesis, we monitored the effects of different sterile mutations on the maintenance of proteostasis in the soma of C. elegans. We found that germline stem cell (GSC) arrest rescued protein quality control, resulting in maintenance of robust proteostasis in different somatic tissues of adult animals. We further demonstrated that GSC‐dependent modulation of proteostasis requires several different signaling pathways, including hsf‐1 and daf‐16/kri‐1/tcer‐1, daf‐12, daf‐9, daf‐36, nhr‐80, and pha‐4 that differentially modulate somatic quality control functions, such that each signaling pathway affects different aspects of proteostasis and cannot functionally complement the other pathways. We propose that the effect of GSCs on the collapse of proteostasis at the transition to adulthood is due to a switch mechanism that links GSC status with maintenance of somatic proteostasis via regulation of the expression and function of different quality control machineries and cellular stress responses that progressively lead to a decline in the maintenance of proteostasis in adulthood, thereby linking reproduction to the maintenance of the soma.     

The role of cellular proteostasis in antitumor immunity

Immune checkpoint blockade therapy is perhaps the most important development in cancer treatment in recent memory. It is based on decades of investigation into the biology of immune cells and the role of the immune system in controlling cancer growth. While the molecular circuitry that governs the immune system in general—and antitumor immunity in particular—is intensely studied, far less attention has been paid to the role of cellular stress in this process. Proteostasis, intimately linked to cell stress responses, refers to the dynamic regulation of the cellular proteome and is maintained through a complex network of systems that govern the synthesis, folding, and degradation of proteins in the cell. Disruption of these systems can result in the loss of protein function, altered protein function, the formation of toxic aggregates, or pathologies associated with cell stress. However, the importance of proteostasis extends beyond its role in maintaining proper protein function; proteostasis governs how tolerant cells may be to mutations in protein-coding genes and the overall half-life of proteins. Such gene expression changes may be associated with human diseases including neurodegenerative diseases, metabolic disease, and cancer and manifest at the protein level against the backdrop of the proteostasis network in any given cellular environment. In this review, we focus on the role of proteostasis in regulating immune responses against cancer as well the role of proteostasis in determining immunogenicity of cancer cells.     

Heat Shock and Caloric Restriction Have a Synergistic Effect on the Heat Shock Response in a sir2.1-dependent Manner in Caenorhabditis elegans

The heat shock response (HSR) is responsible for maintaining cellular and organismal health through the regulation of proteostasis. Recent data demonstrating that the mammalian HSR is regulated by SIRT1 suggest that this response may be under metabolic control. To test this hypothesis, we have determined the effect of caloric restriction in Caenorhabditis elegans on activation of the HSR and have found a synergistic effect on the induction of hsp70 gene expression. The homolog of mammalian SIRT1 in C. elegans is Sir2.1. Using a mutated C. elegans strain with a sir2.1 deletion, we show that heat shock and caloric restriction cooperate to promote increased survivability and fitness in a sir2.1-dependent manner. Finally, we show that caloric restriction increases the ability of heat shock to preserve movement in a polyglutamine toxicity neurodegenerative disease model and that this effect is dependent on sir2.1.     

Regulation of cellular protein quality control networks in a multicellular organism

The long-term health of all metazoan cells is linked to protein quality control, which is achieved by proteostasis, a complex network of molecular interactions that determines the health of the proteome under physiological or stress conditions. Studying the regulation of cellular proteostasis in the context of the whole organism has unraveled multiple layers of cell-nonautonomous regulation, including neuronal regulation, cell-to-cell stress signals and endocrine signaling that affect growth, development and aging. Here, we discuss emerging concepts in cell-nonautonomous regulation of protein quality control networks. The identification of organismal modulators of cellular proteostasis may present novel, yet general targets for misfolding disease intervention.     

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.     

Fluc‐EGFP reporter mice reveal differential alterations of neuronal proteostasis in aging and disease

The cellular protein quality control machinery is important for preventing protein misfolding and aggregation. Declining protein homeostasis (proteostasis) is believed to play a crucial role in age‐related neurodegenerative disorders. However, how neuronal proteostasis capacity changes in different diseases is not yet sufficiently understood, and progress in this area has been hampered by the lack of tools to monitor proteostasis in mammalian models. Here, we have developed reporter mice for in vivo analysis of neuronal proteostasis. The mice express EGFP‐fused firefly luciferase (Fluc‐EGFP), a conformationally unstable protein that requires chaperones for proper folding, and that reacts to proteotoxic stress by formation of intracellular Fluc‐EGFP foci and by reduced luciferase activity. Using these mice, we provide evidence for proteostasis decline in the aging brain. Moreover, we find a marked reaction of the Fluc‐EGFP sensor in a mouse model of tauopathy, but not in mouse models of Huntington’s disease. Mechanistic investigations in primary neuronal cultures demonstrate that different types of protein aggregates have distinct effects on the cellular protein quality control. Thus, Fluc‐EGFP reporter mice enable new insights into proteostasis alterations in different diseases.     

YME1L degradation reduces mitochondrial proteolytic capacity during oxidative stress

Mitochondrial proteostasis is maintained by a network of ATP‐dependent quality control proteases including the inner membrane protease YME1L. Here, we show that YME1L is a stress‐sensitive mitochondrial protease that is rapidly degraded in response to acute oxidative stress. This degradation requires reductions in cellular ATP and involves the activity of the ATP‐independent protease OMA1. Oxidative stress‐dependent reductions in YME1L inhibit protective YME1L‐dependent functions and increase cellular sensitivity to oxidative insult. Collectively, our results identify stress‐induced YME1L degradation as a biologic process that attenuates protective regulation of mitochondrial proteostasis and promotes cellular death in response to oxidative stress.     

Autophagy in neuronal cell loss: a road to death

The regulation of ageing has been extensively studied in divergent animal model systems including worms, flies and mice. However, little is known about the cellular pathways that mediate the death of these organisms. Analysing major cellular changes in the ageing nematode Caenorhabditis elegans has revealed a gradual, progressive deterioration of different tissues except for the nervous system, which remarkably preserves its integrity even in advanced old age. In addition, genetic data have shown that, in C. elegans and in the fruit fly Drosophila melanogaster, lifespan is controlled by signals derived from neurons and acting throughout adulthood. Organismal death thus seems to be a consequence of the decline of specific neurons. Accumulating evidence demonstrates that late onset of neuronal cell loss generally occurs via autophagy, a process in which eukaryotic cells self-digest parts of their contents during development or to survive starvation. Here we suggest that overactivation of autophagy in the cells of the nervous system is the eventual cause of "physiological" death.     

Fluorodeoxyuridine Improves Caenorhabditis elegans Proteostasis Independent of Reproduction Onset

Protein homeostasis (proteostasis) networks are dynamic throughout the lifespan of an organism. During Caenorhabditis elegans adulthood, the maintenance of metastable proteins and the activation of stress responses are inversely associated with germline stem cell proliferation. Here, we employed the thymidylate synthase inhibitor 5-fluoro-2′-deoxyuridine (FUdR) to chemically inhibit reproduction, thus allowing for examination of the interplay between reproduction and somatic proteostasis. We found that treatment with FUdR modulates proteostasis decline both before and after reproduction onset, such that effective induction of the heat shock response was maintained during adulthood and that metastable temperature-sensitive mutant phenotypes were rescued under restrictive conditions. However, FUdR treatment also improved the folding capacity of germline- and gonadogenesis-defective mutants, suggesting that proteostasis modulation by FUdR is independent of germline stem cell proliferation or inhibition of reproduction. Our data, therefore, indicate that FUdR converges on alternative regulatory signals that modulate C. elegans proteostasis capacity during development and adulthood.     

Loss of hepatic chaperone‐mediated autophagy accelerates proteostasis failure in aging

Chaperone‐mediated autophagy (CMA), a cellular process that contributes to protein quality control through targeting of a subset of cytosolic proteins to lysosomes for degradation, undergoes a functional decline with age. We have used a mouse model with liver‐specific defective CMA to identify changes in proteostasis attributable to reduced CMA activity in this organ with age. We have found that other proteolytic systems compensate for CMA loss in young mice which helps to preserve proteostasis. However, these compensatory responses are not sufficient for protection against proteotoxicity induced by stress (oxidative stress, lipid challenges) or associated with aging. Livers from old mice with CMA blockage exhibit altered protein homeostasis, enhanced susceptibility to oxidative stress and hepatic dysfunction manifested by a diminished ability to metabolize drugs, and a worsening of the metabolic dysregulation identified in young mice. Our study reveals that while the regulatory function of CMA cannot be compensated for in young organisms, its contribution to protein homeostasis can be handled by other proteolytic systems. However, the decline in the compensatory ability identified with age explains the more severe consequences of CMA impairment in older organisms and the contribution of CMA malfunction to the gradual decline in proteostasis and stress resistance observed during aging.     

GCK-3, a newly identified Ste20 kinase, binds to and regulates the activity of a cell cycle-dependent ClC anion channel

CLH-3b is a Caenorhabditis elegans ClC anion channel that is expressed in the worm oocyte. The channel is activated during oocyte meiotic maturation and in response to cell swelling by serine/threonine dephosphorylation events mediated by the type 1 phosphatases GLC-7alpha and GLC-7beta. We have now identified a new member of the Ste20 kinase superfamily, GCK-3, that interacts with the CLH-3b COOH terminus via a specific binding motif. GCK-3 inhibits CLH-3b in a phosphorylation-dependent manner when the two proteins are coexpressed in HEK293 cells. clh-3 and gck-3 are expressed predominantly in the C. elegans oocyte and the fluid-secreting excretory cell. Knockdown of gck-3 expression constitutively activates CLH-3b in nonmaturing worm oocytes. We conclude that GCK-3 functions in cell cycle- and cell volume-regulated signaling pathways that control CLH-3b activity. GCK-3 inactivates CLH-3b by phosphorylating the channel and/or associated regulatory proteins. Our studies provide new insight into physiologically relevant signaling pathways that control ClC channel activity and suggest novel mechanisms for coupling cell volume changes to cell cycle events and for coordinately regulating ion channels and transporters that control cellular Cl- content, cell volume, and epithelial fluid secretion.     

Proteostasis regulation at the endoplasmic reticulum: a new perturbation site for targeted cancer therapy

To deal with the constant challenge of protein misfolding in the endoplasmic reticulum (ER), eukaryotic cells have evolved an ER protein quality control (ERQC) mechanism that is integrated with an adaptive stress response. The ERQC pathway is comprised of factors residing in the ER lumen that function in the identification and retention of aberrantly folded proteins, factors in the ER membrane for retrotranslocation of misfolded polypeptides, and enzymes in the cytosol that degrade retrotranslocated proteins. The integrated stress response (termed ER stress or unfolded protein response, UPR) contains several signaling branches elicited from the ER membrane, which fine-tune the rate of protein synthesis and entry into the ER to match the ER folding capacity. The fitness of the cell, particularly those bearing a high secretory burden, is critically dependent on functional integrity of the ER, which in turn relies on these stress-attenuating mechanisms to maintain protein homeostasis, or proteostasis. Aberrant proteostasis can trigger cellular apoptosis, making these adaptive stress response systems attractive targets for perturbation in treatment of cell malignancies. Here, we review our current understanding of how the cell preserves ER proteostasis and discuss how we may harness the mechanistic information on this process to develop new cancer therapeutics.     

Sphingolipids and impaired hypoxic stress responses in Huntington disease

Huntington disease (HD) is a debilitating, currently incurable disease. Protein aggregation and metabolic deficits are pathological hallmarks but their link to neurodegeneration and symptoms remains debated.Here, we summarize alterations in the levels of different sphingolipids in an attempt to characterize sphingolipid patterns specific to HD, an additional molecular hallmark of the disease. Based on the crucial role of sphingolipids in maintaining cellular homeostasis, the dynamic regulation of sphingolipids upon insults and their involvement in cellular stress responses, we hypothesize that maladaptations or blunted adaptations, especially following cellular stress due to reduced oxygen supply (hypoxia) contribute to the development of pathology in HD. We review how sphingolipids shape cellular energy metabolism and control proteostasis and suggest how these functions may fail in HD and in combination with additional insults. Finally, we evaluate the potential of improving cellular resilience in HD by conditioning approaches (improving the efficiency of cellular stress responses) and the role of sphingolipids therein.Sphingolipid metabolism is crucial for cellular homeostasis and for adaptations following cellular stress, including hypoxia. Inadequate cellular management of hypoxic stress likely contributes to HD progression, and sphingolipids are potential mediators. Targeting sphingolipids and the hypoxic stress response are novel treatment strategies for HD.     

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     

Using Caenorhabditis elegans as a Model System to Study Protein Homeostasis in a Multicellular Organism

The folding and assembly of proteins is essential for protein function, the long-term health of the cell, and longevity of the organism. Historically, the function and regulation of protein folding was studied in vitro, in isolated tissue culture cells and in unicellular organisms. Recent studies have uncovered links between protein homeostasis (proteostasis), metabolism, development, aging, and temperature-sensing. These findings have led to the development of new tools for monitoring protein folding in the model metazoan organism Caenorhabditis elegans. In our laboratory, we combine behavioral assays, imaging and biochemical approaches using temperature-sensitive or naturally occurring metastable proteins as sensors of the folding environment to monitor protein misfolding. Behavioral assays that are associated with the misfolding of a specific protein provide a simple and powerful readout for protein folding, allowing for the fast screening of genes and conditions that modulate folding. Likewise, such misfolding can be associated with protein mislocalization in the cell. Monitoring protein localization can, therefore, highlight changes in cellular folding capacity occurring in different tissues, at various stages of development and in the face of changing conditions. Finally, using biochemical tools ex vivo, we can directly monitor protein stability and conformation. Thus, by combining behavioral assays, imaging and biochemical techniques, we are able to monitor protein misfolding at the resolution of the organism, the cell, and the protein, respectively.