Remodeling of Proteostasis Upon Transition to Adulthood is Linked to Reproduction Onset

Protein folding and clearance networks sense and respond to misfolded and aggregation-prone proteins by activating cytoprotective cell stress responses that safeguard the proteome against damage, maintain the health of the cell, and enhance lifespan. Surprisingly, cellular proteostasis undergoes a sudden and widespread failure early in Caenorhabditis elegans adulthood, with marked consequences on proteostasis functions later in life. These changes in the regulation of quality control systems, such as chaperones, the ubiquitin proteasome system and cellular stress responses, are controlled cell-nonautonomously by the proliferation of germline stem cells. Here, we review recent studies examining changes in proteostasis upon transition to adulthood and how proteostasis is modulated by reproduction onset, focusing on C. elegans. Based on these and our own findings, we propose that the regulation of quality control systems is actively remodeled at the point of transition between development and adulthood to influence the subsequent course of aging.     

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.     

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.     

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.     

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     

Heat Shock Protein Reports on Proteome Stress

Proper regulation of protein homeostasis (proteostasis) is essential to maintain cellular fitness. Proteome stress causes imbalance of the proteostasis, leading to various diseases represented by neurodegenerative diseases, cancers, and metabolic disorders. The biosensor community recently embarked on the development of proteome stress sensors to report on the integrity of proteostasis in live cells. While most of these sensors are based on metastable mutants of specific client proteins, a recent sensor takes advantage of the specific association of heat shock protein 27 with protein aggregates and exhibits a diffusive to punctate fluorescent change in cells that are subjected to stress conditions. Thus, heat shock proteins can be also used as a family of sensors to monitor proteome stress.     

The endoplasmic reticulum proteostasis network profoundly shapes the protein sequence space accessible to HIV envelope

The sequence space accessible to evolving proteins can be enhanced by cellular chaperones that assist biophysically defective clients in navigating complex folding landscapes. It is also possible, at least in theory, for proteostasis mechanisms that promote strict quality control to greatly constrain accessible protein sequence space. Unfortunately, most efforts to understand how proteostasis mechanisms influence evolution rely on artificial inhibition or genetic knockdown of specific chaperones. The few experiments that perturb quality control pathways also generally modulate the levels of only individual quality control factors. Here, we use chemical genetic strategies to tune proteostasis networks via natural stress response pathways that regulate the levels of entire suites of chaperones and quality control mechanisms. Specifically, we upregulate the unfolded protein response (UPR) to test the hypothesis that the host endoplasmic reticulum (ER) proteostasis network shapes the sequence space accessible to human immunodeficiency virus-1 (HIV-1) envelope (Env) protein. Elucidating factors that enhance or constrain Env sequence space is critical because Env evolves extremely rapidly, yielding HIV strains with antibody- and drug-escape mutations. We find that UPR-mediated upregulation of ER proteostasis factors, particularly those controlled by the IRE1-XBP1s UPR arm, globally reduces Env mutational tolerance. Conserved, functionally important Env regions exhibit the largest decreases in mutational tolerance upon XBP1s induction. Our data indicate that this phenomenon likely reflects strict quality control endowed by XBP1s-mediated remodeling of the ER proteostasis environment. Intriguingly, and in contrast, specific regions of Env, including regions targeted by broadly neutralizing antibodies, display enhanced mutational tolerance when XBP1s is induced, hinting at a role for host proteostasis network hijacking in potentiating antibody escape. These observations reveal a key function for proteostasis networks in decreasing instead of expanding the sequence space accessible to client proteins, while also demonstrating that the host ER proteostasis network profoundly shapes the mutational tolerance of Env in ways that could have important consequences for HIV adaptation.

The host cell’s endoplasmic reticulum proteostasis network has a profound, constraining impact on the protein sequence space accessible to HIV’s envelope protein, which is a major target of the host’s adaptive immune system; in particular, upregulation of stringent quality control pathways appears to restrict the viability of destabilizing envelope variants.     

Modeling general proteostasis: proteome balance in health and disease

Protein function is generated and maintained by the proteostasis network (PN) (Balch et al. (2008) Science, 319:916). The PN is a modular, yet integrated system unique to each cell type that is sensitive to signaling pathways that direct development and aging, and respond to folding stress. Mismanagement of protein folding and function triggered by genetic, epigenetic and environmental causes poses a major challenge to human health and lifespan. Herein, we address the impact of proteostasis defined by the FoldFx model on our understanding of protein folding and function in biology. FoldFx describes how general proteostasis control (GPC) enables the polypeptide chain sequence to achieve functional balance in the context of the cellular proteome. By linking together the chemical and energetic properties of the protein fold with the composition of the PN we discuss the principle of the proteostasis boundary (PB) as a key component of GPC. The curved surface of the PB observed in 3-dimensional space suggests that the polypeptide chain sequence and the PN operate as an evolutionarily conserved functional unit to generate and sustain protein dynamics required for biology. Modeling general proteostasis provides a rational basis for tackling some of the most challenging diseases facing mankind in the 21st century.     

LRSAM1 E3 ubiquitin ligase promotes proteasomal clearance of E6-AP protein

Numerous proteins participate and actively contribute to the various cellular mechanisms, where several of them are crucial for regular metabolism, including survival. Thus, to maintain optimal cellular physiology, cells govern protein quality control functions with the assistance of comprehensive actions of molecular chaperones, the ubiquitin-proteasome system, and autophagy. In the ubiquitin-proteasome pathway, few quality control E3 ubiquitin ligases actively participate against misfolded protein aggregation generated via stress conditions. But how these quality control E3s active expression levels returned to basal levels when cells achieved re-establishment of proteostasis is still poorly understood. Our current study demonstrated that LRSAM1 E3 ubiquitin ligase promotes the proteasomal degradation of quality control E3 ubiquitin ligase E6-AP. We have observed the co-localization and recruitment of LRSAM1 with E6-AP protein and noticed that LRSAM1 induces the endogenous turnover of E6-AP. Partial depletion of LRSAM1 elevates the levels of E6-AP and affects overall cell cycle regulatory proteins (p53 and p27) expression, including the rate of cellular proliferation. The current finding also provides an excellent opportunity to better understand the basis of the E6-AP associated pathomechanism of Angelman Syndrome disorder. Additionally, this study touches upon the novel potential molecular strategy to regulate the levels of one quality control E3 ubiquitin ligase with another E3 ubiquitin ligase and restore proteostasis and provide a possible therapeutic approach against abnormal protein aggregation diseases.     

Mitochondrial HSP90s and tumor cell metabolism

The control of protein homeostasis, or proteostasis, has been traditionally viewed through the lenses of a general housekeeping function that all cells need, regardless of pathway specification or link to defined cellular responses. A more updated perspective considers proteostasis as an essential adaptive mechanism, taking place in specialized subcellular organelles, and maintaining the functionality of defined cellular networks. Fresh experimental evidence now identifies heat shock protein 90 (HSP90) chaperones as pivotal regulators of proteostasis in mitochondria, selectively in tumor cells. This function connects to a global network of cellular compensation, linking autophagy, endoplasmic reticulum (ER) stress and metabolic reprogramming in a single adaptive continuum, and offers prime opportunities for novel cancer therapeutics.     

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.     

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.     

Proteostasis failure and cellular senescence in long‐term cultured postmitotic rat neurons

Cellular senescence, a stress‐induced irreversible cell cycle arrest, has been defined for mitotic cells and is implicated in aging of replicative tissues. Age‐related functional decline in the brain is often attributed to a failure of protein homeostasis (proteostasis), largely in postmitotic neurons, which accordingly is a process distinct by definition from senescence. It is nevertheless possible that proteostasis failure and cellular senescence have overlapping molecular mechanisms. Here, we identify postmitotic cellular senescence as an adaptive stress response to proteostasis failure. Primary rat hippocampal neurons in long‐term cultures show molecular changes indicative of both senescence (senescence‐associated β‐galactosidase, p16, and loss of lamin B1) and proteostasis failure relevant to Alzheimer's disease. In addition, we demonstrate that the senescent neurons exhibit resistance to stress. Importantly, treatment of the cultures with an mTOR antagonist, protein synthesis inhibitor, or chemical compound that reduces the amount of protein aggregates relieved the proteotoxic stresses as well as the appearance of senescence markers. Our data propose mechanistic insights into the pathophysiological brain aging by establishing senescence as a primary cell‐autonomous neuroprotective response.     

Integrating Protein Homeostasis Strategies in Prokaryotes

Bacterial cells are frequently exposed to dramatic fluctuations in their environment, which cause perturbation in protein homeostasis and lead to protein misfolding. Bacteria have therefore evolved powerful quality control networks consisting of chaperones and proteases that cooperate to monitor the folding states of proteins and to remove misfolded conformers through either refolding or degradation. The levels of the quality control components are adjusted to the folding state of the cellular proteome through the induction of compartment specific stress responses. In addition, the activities of several quality control components are directly controlled by these stresses, allowing for fast activation. Severe stress can, however, overcome the protective function of the proteostasis network leading to the formation of protein aggregates, which are sequestered at the cell poles. Protein aggregates are either solubilized by AAA+ chaperones or eliminated through cell division, allowing for the generation of damage-free daughter cells.     

Autophagy machinery promotes the chaperone-mediated formation and compartmentalization of protein aggregates during appressorium development by the rice blast fungus

The chaperone-mediated sequestration of misfolded proteins into specialized quality control compartments represents an important strategy for maintaining protein homeostasis in response to stress. However, precisely how this process is controlled in time and subcellular space and integrated with the cell''s protein refolding and degradation pathways remains unclear. We set out to understand how aggregated proteins are managed during infection-related development by a globally devastating plant pathogenic fungus and to determine how impaired protein quality control impacts cellular differentiation and pathogenesis in this system. Here we show that in the absence of Hsp104 disaggregase activity, aggregated proteins are spatially sequestered into quality control compartments within conidia, but not within terminally differentiated infection cells, and thus spatial protein quality control is cell type–dependent. We demonstrate that impaired aggregate resolution results in a short-term developmental penalty but has no significant impact upon appressorium function. Finally, we show that, somewhat unexpectedly, the autophagy machinery is necessary for the normal formation and compartmentalization of protein aggregates. Taken together, our findings provide important new insight into spatial protein quality control during the process of terminal cellular differentiation by a globally important model eukaryote and reveal a new level of interplay between major proteostasis pathways.     

Proteasome dysfunction in Drosophila signals to an Nrf2‐dependent regulatory circuit aiming to restore proteostasis and prevent premature aging

The ubiquitin–proteasome system is central to the regulation of cellular proteostasis. Nevertheless, the impact of in vivo proteasome dysfunction on the proteostasis networks and the aging processes remains poorly understood. We found that RNAi‐mediated knockdown of 20S proteasome subunits in Drosophila melanogaster resulted in larval lethality. We therefore studied the molecular effects of proteasome dysfunction in adult flies by developing a model of dose‐dependent pharmacological proteasome inhibition. Impaired proteasome function promoted several ‘old‐age’ phenotypes and markedly reduced flies' lifespan. In young somatic tissues and in gonads of all ages, loss of proteasome activity induced higher expression levels and assembly rates of proteasome subunits. Proteasome dysfunction was signaled to the proteostasis network by reactive oxygen species that originated from malfunctioning mitochondria and triggered an Nrf2‐dependent upregulation of the proteasome subunits. RNAi‐mediated Nrf2 knockdown reduced proteasome activities, flies' resistance to stress, as well as longevity. Conversely, inducible activation of Nrf2 in transgenic flies upregulated basal proteasome expression and activity independently of age and conferred resistance to proteotoxic stress. Interestingly, prolonged Nrf2 overexpression reduced longevity, indicating that excessive activation of the proteostasis pathways can be detrimental. Our in vivo studies add new knowledge on the proteotoxic stress‐related regulation of the proteostasis networks in higher metazoans. Proteasome dysfunction triggers the activation of an Nrf2‐dependent tissue‐ and age‐specific regulatory circuit aiming to adjust the cellular proteasome activity according to temporal and/or spatial proteolytic demands. Prolonged deregulation of this proteostasis circuit accelerates aging.     

Inhibition of endoplasmic reticulum associated degradation reduces endoplasmic reticulum stress and alters lysosomal morphology and distribution

Disturbances in proteostasis are observed in many neurodegenerative diseases. This leads to activation of protein quality control to restore proteostasis, with a key role for the removal of aberrant proteins by proteolysis. The unfolded protein response (UPR) is a protein quality control mechanism of the endoplasmic reticulum (ER) that is activated in several neurodegenerative diseases. Recently we showed that the major proteolytic pathway during UPR activation is via the autophagy/lysosomal system. Here we investigate UPR induction if the other major proteolytic pathway of the ER -ER associated degradation (ERAD)-is inhibited. Surprisingly, impairment of ERAD results in decreased UPR activation and protects against ER stress toxicity. Autophagy induction is not affected under these conditions, however, a striking relocalization of the lysosomes is observed. Our data suggest that a protective UPR-modulating mechanism is activated if ERAD is inhibited, which involves lysosomes. Our data provide insight in the cross-talk between proteolytic pathways involved in ER proteostasis. This has implications for neurodegenerative diseases like Alzheimer’s disease where disturbed ER proteostasis and proteolytic impairment are early phenomena in the pathology.