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In biological systems, efficient folding and assembly is necessary for cell health and longevity. Nascent proteins are initially targeted to the endoplasmic reticulum (ER), where they are correctly folded and assembled before being delivered to their final cellular destinations. When proteins are not folded correctly, the proteostasis network (PN) acts to degrade the aberrant proteins, and thus, prevent cellular stress. The PN is a complex network of different proteins, multiple organelles, including the ER, Golgi, endolysosomal system, lipids, and degradation pathways that work in concert to maintain protein homeostasis.An ER-resident protein and member of the rhomboid protein family, Dfm1, has been found to be required for the identification and degradation of misfolded membrane proteins at the endoplasmic reticulum (ER), along with having various other functions including substrate binding, lipid thinning, and AAA-ATPase recruitment. Dfm1 is a well-studied protein; however, not all its biological functions are known. Using genetic, protein, and biochemical approaches in Saccharomyces cerevisiae, this dissertation describes our original work in demonstrating Dfm1 as a chaperone-like protein that does not require recruitment of the ATPase Cdc48, a finding distinct from Dfm1’s previously identified function in dislocating misfolded membrane proteins to the cytosol for degradation in ER‐associated degradation (ERAD). Additionally, we establish another independent and novel role of Dfm1 in the sphingolipid biosynthesis pathway. Here, Dfm1 facilitates sphingolipid homeostasis by exporting phosphorylated Orm2, a key sphingolipid biosynthesis regulating enzyme, from the ER which results in the degradation of Orm2 through Endosome and Golgi-Associated Degradation (EGAD). Additionally, it has been found that lipid and protein homeostasis are tightly linked, so this work aims to develop a holistic system for further investigation of Dfm1’s role in regulating sphingolipid biosynthetic enzymes. Taken together, this dissertation highlights Dfm1’s extensive role in chaperone-like regulation of membrane protein aggregation stress and its novel role in sphingolipid biosynthesis. Given that the accumulation of aberrant misfolded proteins and dysregulation of sphingolipids are associated with various pathologies, this study serves as a molecular foothold for understanding how dysregulation of proteins and sphingolipid metabolism leads to various diseases.

The innate immune system is critical in protecting against foreign invaders and can be activated by detection of microbe-associated molecules, as well as detection of the consequences of pathogen infection. In the nematode C. elegans, the molecularly diverse intracellular pathogens of microsporidia and virus can induce a common host transcriptional response called the Intracellular Pathogen Response (IPR). The IPR can also be induced by proteotoxic stress, which is often a consequence of intracellular infection. In parallel, the IPR is regulated by antagonistic paralogs in C. elegans called pals-22 and pals-25. The wild-type function of pals-22 is to repress IPR gene expression and increase thermotolerance and pathogen resistance, while the wild-type function of pals-25 is to activate these phenotypes, acting downstream of pals-22. To investigate how pals genes control the IPR, I further characterized the role of pals-22/pals-25, as well as two additional pals pairs: pals-17/pals-20 and pals-23/pals-24. First, I investigated the role of pals-22 and pals-25 in mediating the induction of IPR genes upon proteotoxic stress. Next, I characterized pals-17 as another negative regulator of IPR genes and found that pals-17 may work with an antagonistic paralog pals-20 as an activator. Finally, I knocked out another pals gene pair, pals-23 and pals-24, and found that they do not regulate expression of a GFP reporter for the IPR gene pals-5. Altogether, these findings demonstrate general principles of host gene regulation by antagonistic paralogs, which control expression of genes that promote resistance against natural intracellular pathogens and tolerance of proteotoxic stress.

Cell competition allows winner cells to eliminate less fit loser cells in tissues. In Minute cell competition, cells with a heterozygous mutation in ribosome genes, such as RpS3+/− cells, are eliminated by wild-type cells. How cells are primed as losers is partially understood and it has been proposed that reduced translation underpins the loser status of ribosome mutant, or Minute, cells. Here, using Drosophila, we show that reduced translation does not cause cell competition. Instead, we identify proteotoxic stress as the underlying cause of the loser status for Minute competition and competition induced by mahjong, an unrelated loser gene. RpS3+/− cells exhibit reduced autophagic and proteasomal flux, accumulate protein aggregates and can be rescued from competition by improving their proteostasis. Conversely, inducing proteotoxic stress is sufficient to turn otherwise wild-type cells into losers. Thus, we propose that tissues may preserve their health through a proteostasis-based mechanism of cell competition and cell selection. Baumgartner et al. identify proteotoxic stress as the underlying cause of the loser status in a cell competition model caused by reduced autophagic, proteasomal flux and accumulation of protein aggregates.

Non-proteinogenic amino acids, such as the proline analog L-azetidine-2-carboxylic acid (AZC), are detrimental to cells because they are mis-incorporated into proteins and lead to proteotoxic stress. Our goal was to identify genes that show chemical-genetic interactions with AZC in Saccharomyces cerevisiae and thus also potentially define the pathways cells use to cope with amino acid mis-incorporation. Screening the yeast deletion and temperature sensitive collections, we found 72 alleles with negative chemical-genetic interactions with AZC treatment and 12 alleles that suppress AZC toxicity. Many of the genes with negative chemical-genetic interactions are involved in protein quality control pathways through the proteasome. Genes involved in actin cytoskeleton organization and endocytosis also had negative chemical-genetic interactions with AZC. Related to this, the number of actin patches per cell increases upon AZC treatment. Many of the same cellular processes were identified to have interactions with proteotoxic stress caused by two other amino acid analogs, canavanine and thialysine, or a mistranslating tRNA variant that mis-incorporates serine at proline codons. Alleles that suppressed AZC-induced toxicity functioned through the amino acid sensing TOR pathway or controlled amino acid permeases required for AZC uptake. Further suggesting the potential of genetic changes to influence the cellular response to proteotoxic stress, overexpressing many of the genes that had a negative chemical-genetic interaction with AZC suppressed AZC toxicity.

The mitochondrial unfolded protein response (UPRmt) is characterized by the transcriptional induction of mitochondrial chaperone and protease genes in response to impaired mitochondrial proteostasis and is regulated by ATF5 and CHOP in mammalian cells. However, the detailed mechanisms underlying the UPRmt are currently unclear. Here, we show that HSF1 is required for activation of mitochondrial chaperone genes, including HSP60, HSP10, and mtHSP70, in mouse embryonic fibroblasts during inhibition of matrix chaperone TRAP1, protease Lon, or electron transfer complex 1 activity. HSF1 bound constitutively to mitochondrial chaperone gene promoters, and we observed that its occupancy was remarkably enhanced at different levels during the UPRmt. Furthermore, HSF1 supported the maintenance of mitochondrial function under the same conditions. These results demonstrate that HSF1 is required for induction of mitochondrial chaperones during the UPRmt, and thus, it may be one of the guardians of mitochondrial function under conditions of impaired mitochondrial proteostasis., The mitochondrial unfolded protein response (UPRmt) is characterized by the transcriptional induction of mitochondrial chaperone and protease genes in response to impaired mitochondrial proteostasis. Here, we show that heat shock transcription factor (HSF1) is required for activation of mitochondrial chaperone genes and supports the maintenance of mitochondrial function in mouse cells during the UPRmt.

The daily organisation of most mammalian cellular functions is attributed to circadian regulation of clock‐controlled protein expression, driven by daily cycles of CRYPTOCHROME‐dependent transcriptional feedback repression. To test this, we used quantitative mass spectrometry to compare wild‐type and CRY‐deficient fibroblasts under constant conditions. In CRY‐deficient cells, we found that temporal variation in protein, phosphopeptide, and K+ abundance was at least as great as wild‐type controls. Most strikingly, the extent of temporal variation within either genotype was much smaller than overall differences in proteome composition between WT and CRY‐deficient cells. This proteome imbalance in CRY‐deficient cells and tissues was associated with increased susceptibility to proteotoxic stress, which impairs circadian robustness, and may contribute to the wide‐ranging phenotypes of CRY‐deficient mice. Rather than generating large‐scale daily variation in proteome composition, we suggest it is plausible that the various transcriptional and post‐translational functions of CRY proteins ultimately act to maintain protein and osmotic homeostasis against daily perturbation., CRYPTOCHROMES are not essential for circadian proteome oscillations but play a crucial role in the temporal regulation of protein and osmotic homeostasis.

Arabidopsis cargo receptor NBR1 contributes to protein quality control by promoting the formation of protein aggregates and mediating their clearance via selective autophagy., Aggrephagy, a type of selective autophagy that sequesters protein aggregates for degradation in the vacuole, is an important protein quality control mechanism, particularly during cell stress. In mammalian cells, aggrephagy and several other forms of selective autophagy are mediated by dedicated cargo receptors such as NEIGHBOR OF BRCA1 (NBR1). Although plant NBR1 homologs have been linked to selective autophagy during biotic stress, it remains unclear how they impact selective autophagy under non-stressed and abiotic stress conditions. Through microscopic and biochemical analysis of nbr1 mutants expressing autophagy markers and an aggregation-prone reporter, we tested the connection between NBR1 and aggrephagy in Arabidopsis. Although NBR1 is not essential for general autophagy, or for the selective clearance of peroxisomes, mitochondria, or the ER, we found that NBR1 is required for the heat-induced formation of autophagic vesicles. Moreover, cytoplasmic puncta containing aggregation-prone proteins, which were rarely observed in wild-type plants, were found to accumulate in nbr1 mutants under both control and heat stress conditions. Given that NBR1 co-localizes with these cytoplasmic puncta, we propose that Arabidopsis NBR1 is a plant aggrephagy receptor essential for maintaining proteostasis under both heat stress and non-stress conditions.

Small heat shock proteins (sHsps) are an evolutionarily conserved class of ATP-independent chaperones that form the first line of defence during proteotoxic stress. sHsps are defined not only by their relatively low molecular weight, but also by the presence of a conserved α-crystallin domain, which is flanked by less conserved, mostly unstructured, N- and C-terminal domains. sHsps form oligomers of different sizes which deoligomerize upon stress conditions into smaller active forms. Activated sHsps bind to aggregation-prone protein substrates to form assemblies that keep substrates from irreversible aggregation. Formation of these assemblies facilitates subsequent Hsp70 and Hsp100 chaperone-dependent disaggregation and substrate refolding into native species. This mini review discusses what is known about the role and place of bacterial sHsps in the chaperone network.

Simple Summary Resistance to therapy and subsequent relapse of the disease are common in patients with cancers in the head and neck region (HNSCC). Recent technological advancements have revitalized the concept of combining hyperthermia (HT) with radio(chemo)therapy for treating these patients. Heat inherently affects multiple cellular components and destroys protein structures, thereby influencing the DNA damage response. However, the plethora of adverse mechanisms in HT-induced radiosensitization is still not fully elucidated. We uniquely evaluated the radiosensitizing potential of HT in HNSCC cells using a sophisticated spheroid assay platform, which turned out as a powerful tool to compare different treatment modalities and gain new mechanistic insight. We show that HT disrupts vital cellular proteostasis and affects global stress response signaling. This triggers massive heat shock and proteotoxic stress responses contributing to the cancer cells’ protection against HT-induced radiosensitization. Selected molecules in this scenario may serve as new targets for combination with hyperthermia and radiotherapy. Abstract Hyperthermia (HT) combined with irradiation is a well-known concept to improve the curative potential of radiotherapy. Technological progress has opened new avenues for thermoradiotherapy, even for recurrent head and neck squamous cell carcinomas (HNSCC). Preclinical evaluation of the curative radiosensitizing potential of various HT regimens remains ethically, economically, and technically challenging. One key objective of our study was to refine an advanced 3-D assay setup for HT + RT research and treatment testing. For the first time, HT-induced radiosensitization was systematically examined in two differently radioresponsive HNSCC spheroid models using the unique in vitro “curative” analytical endpoint of spheroid control probability. We further investigated the cellular stress response mechanisms underlying the HT-related radiosensitization process with the aim to unravel the impact of HT-induced proteotoxic stress on the overall radioresponse. HT disrupted the proteome’s thermal stability, causing severe proteotoxic stress. It strongly enhanced radiation efficacy and affected paramount survival and stress response signaling networks. Transcriptomics, q-PCR, and western blotting data revealed that HT + RT co-treatment critically triggers the heat shock response (HSR). Pre-treatment with chemical chaperones intensified the radiosensitizing effect, thereby suppressing HT-induced Hsp27 expression. Our data suggest that HT-induced radiosensitization is adversely affected by the proteotoxic stress response. Hence, we propose the inhibition of particular heat shock proteins as a targeting strategy to improve the outcome of combinatorial HT + RT.

To maintain proteostasis, cells must integrate information and activities that supervise protein synthesis, protein folding, conformational stability, and also protein degradation. Extrinsic and intrinsic conditions can both impact normal proteostasis, causing the appearance of proteotoxic stress. Initially, proteotoxic stress elicits adaptive responses aimed at restoring proteostasis, allowing cells to survive the stress condition. However, if the proteostasis restoration fails, a permanent and sustained proteotoxic stress can be deleterious, and cell death ensues. Many cancer cells convive with high levels of proteotoxic stress, and this condition could be exploited from a therapeutic perspective. Understanding the cell death pathways engaged by proteotoxic stress is instrumental to better hijack the proliferative fate of cancer cells.

Heat shock proteins (HSPs) constitute a major part of the molecular chaperone system and play a fundamental role in cell proteostasis. The HSPA (HSP70) family groups twelve highly homologous HSPA proteins. Certain HSPAs are regarded as important cancer-related proteins, prospective therapeutic targets for cancer treatment, and also as potential cancer biomarkers. Heat Shock Protein A2 (HSPA2), a testis-enriched chaperone and one of the least characterized members of the HSPA family, has recently emerged as an important cancer-relevant protein with potential biomarker significance. Nevertheless, conflicting conclusions have been recently drawn both according to HSPA2 role in cancer cells, as well as to its prognostic value. In this work we have shown that one of the serious limitations in HSPA2 protein research is cross-reactivity of antibodies marketed as specific for HSPA2 with one or more other HSPA(s). Among non-specific antibodies were also those recently used for HSPA2 detection in functional and biomarker studies. We showed how using non-specific antibodies can generate misleading conclusions on HSPA2 expression in non-stressed cancer cells and tumors, as well as in cancer cells exposed to proteotoxic stress. Our findings addressed concerns on some published studies dealing with HSPA2 as a cancer-related protein.

The unfolded protein response (UPR) is central to endoplasmic reticulum (ER) homeostasis by controlling its size and protein folding capacity. When activated by unfolded proteins in the ER-lumen or aberrant lipid compositions, the UPR adjusts the expression of hundreds of target genes to counteract ER stress. The proteotoxic drugs dithiothreitol (DTT) and tunicamycin (TM) are commonly used to induce misfolding of proteins in the ER and to study the UPR. However, their potential impact on the cellular lipid composition has never been systematically addressed. Here, we report the quantitative, cellular lipid composition of Saccharomyces cerevisiae during acute, proteotoxic stress in both rich and synthetic media. We show that DTT causes rapid remodeling of the lipidome when used in rich medium at growth-inhibitory concentrations, while TM has only a marginal impact on the lipidome under our conditions of cultivation. We formulate recommendations on how to study UPR activation by proteotoxic stress without interferences from a perturbed lipid metabolism. Furthermore, our data suggest an intricate connection between the cellular growth rate, the abundance of the ER, and the metabolism of fatty acids. We show that Saccharomyces cerevisiae can produce asymmetric lipids with two saturated fatty acyl chains differing substantially in length. These observations indicate that the pairing of saturated fatty acyl chains is tightly controlled and suggest an evolutionary conservation of asymmetric lipids and their biosynthetic machineries.

Malignant transformation is accompanied by alterations in the key cellular pathways that regulate development, metabolism, proliferation and motility as well as stress resilience. The members of the transcription factor family, called heat shock factors (HSFs), have been shown to play important roles in all of these biological processes, and in the past decade it has become evident that their activities are rewired during tumorigenesis. This review focuses on the expression patterns and functions of HSF1, HSF2, and HSF4 in specific cancer types, highlighting the mechanisms by which the regulatory functions of these transcription factors are modulated. Recently developed therapeutic approaches that target HSFs are also discussed.

Background The protein homeostasis (proteostasis) network maintains balanced protein synthesis, folding, transport, and degradation within a cell. Failure to maintain proteostasis is associated with aging and disease, leading to concerted efforts to study how the network responds to various proteotoxic stresses. This is often accomplished using ectopic overexpression of well-characterized, model misfolded protein substrates. However, how cells tolerate large-scale, diverse burden to the proteostasis network is not understood. Aneuploidy, the state of imbalanced chromosome content, adversely affects the proteostasis network by dysregulating the expression of hundreds of proteins simultaneously. Using aneuploid haploid yeast cells as a model, we address whether cells can tolerate large-scale, diverse challenges to the proteostasis network. Results Here we characterize several aneuploid Saccharomyces cerevisiae strains isolated from a collection of stable, randomly generated yeast aneuploid cells. These strains exhibit robust growth and resistance to multiple drugs which induce various forms of proteotoxic stress. Whole genome re-sequencing of the strains revealed this was not the result of genetic mutations, and transcriptome profiling combined with ribosome footprinting showed that genes are expressed and translated in accordance to chromosome copy number. In some strains, various facets of the proteostasis network are mildly upregulated without chronic activation of environmental stress response or heat shock response pathways. No severe defects were observed in the degradation of misfolded proteins, using model misfolded substrates of endoplasmic reticulum-associated degradation or cytosolic quality control pathways, and protein biosynthesis capacity was not impaired. Conclusions We show that yeast strains of some karyotypes in the genetic background studied here can tolerate the large aneuploidy-associated burden to the proteostasis machinery without genetic changes, dosage compensation, or activation of canonical stress response pathways. We suggest that proteotoxic stress, while common, is not always an obligate consequence of aneuploidy, but rather certain karyotypes and genetic backgrounds may be able to tolerate the excess protein burden placed on the protein homeostasis machinery. This may help clarify how cancer cells are paradoxically both highly aneuploid and highly proliferative at the same time.

International audience; The ubiquitin-like molecule NEDD8 controls several biological processes and is a promising target for therapeutic intervention. NEDDylation occurs through specific NEDD8 enzymes (canonical) or enzymes of the ubiquitin system (atypical). Identification of NEDD8 sites on substrates is critical for delineating the processes controlled by NEDDylation. By combining the use of the NEDD8 R74K mutant with anti-di-glycine (anti-diGly) antibodies, we identified 1,101 unique NEDDylation sites in 620 proteins. Bioinformatics analysis reveals that canonical and atypical NEDDylation have distinct proteomes; the spliceosome/mRNA surveillance/DNA replication and ribosome/proteasome, respectively. The data also reveal the formation of poly-NEDD8, hybrid NEDD8-ubiquitin, and NEDD8-SUMO-2 chains as potential molecular signals. In particular, NEDD8-SUMO-2 chains are induced upon proteotoxic stress (atypical) through NEDDylation of K11 in SUMO-2, and conjugates accumulate in previously described nucleolus-related inclusions. The study uncovers a diverse proteome for NEDDylation and is consistent with the concept of extensive cross-talk between ubiquitin and Ubls under proteotoxic stress conditions.

Initially discovered as a protease responsible for degradation of misfolded or damaged proteins, the mitochondrial Lon protease (Lonp1) turned out to be a multifaceted enzyme, that displays at least three different functions (proteolysis, chaperone activity, binding of mtDNA) and that finely regulates several cellular processes, within and without mitochondria. Indeed, LONP1 in humans is ubiquitously expressed, and is involved in regulation of response to oxidative stress and, heat shock, in the maintenance of mtDNA, in the regulation of mitophagy. Furthermore, its proteolytic activity can regulate several biochemical pathways occurring totally or partially within mitochondria, such as TCA cycle, oxidative phosphorylation, steroid and heme biosynthesis and glutamine production. Because of these multiple activities, Lon protease is highly conserved throughout evolution, and mutations occurring in its gene determines severe diseases in humans, including a rare syndrome characterized by Cerebral, Ocular, Dental, Auricular and Skeletal anomalies (CODAS). Finally, alterations of LONP1 regulation in humans can favor tumor progression and aggressiveness, further highlighting the crucial role of this enzyme in mitochondrial and cellular homeostasis.

Human cancer cells display extensive heterogeneity in their sensitivities to the proteasome inhibitor bortezomib (Velcade). The molecular mechanisms underlying this heterogeneity remain unclear, and strategies to overcome resistance are limited. Here, we discover that inherent differences in eIF2α phosphorylation among a panel of ten human pancreatic cancer cell lines significantly impacts bortezomib sensitivity, and implicate the HRI (heme-regulated inhibitor) eIF2α kinase as a novel therapeutic target. Within our panel, we identified a subset of cell lines with defective induction of eIF2α phosphorylation, conferring a high degree of sensitivity to bortezomib. These bortezomib-sensitive cells exhibited impaired translation attenuation followed by toxic accumulation of protein aggregates and reactive oxygen species (ROS), whereas the bortezomib-resistant cell lines displayed increased phosphorylation of eIF2α, decreased translation, few protein aggregates, and minimal ROS production. Importantly, we identified HRI as the primary bortezomib-activated eIF2α kinase, and demonstrated that HRI knockdown promoted cell death in the bortezomib-resistant cells. Overall, our data implicate inducible HRI-mediated phosphorylation of eIF2α as a central cytoprotective mechanism following exposure to bortezomib and provide proof-of-concept for the development of HRI inhibitors to overcome proteasome inhibitor resistance.

Numerous cellular processes are controlled by the proteasome, a multicatalytic protease in the cytosol and nucleus of all eukaryotic cells, through regulated protein degradation. The immunoproteasome is a special type of proteasome which is inducible under inflammatory conditions and constitutively expressed in hematopoietic cells. MECL-1 (β2i), LMP2 (β1i), and LMP7 (β5i) are the proteolytically active subunits of the immunoproteasome (IP), which is known to shape the antigenic repertoire presented on major histocompatibility complex (MHC) class I molecules. Furthermore, the immunoproteasome is involved in T cell expansion and inflammatory diseases. In recent years, targeting the immunoproteasome in cancer, autoimmune diseases, and transplantation proved to be therapeutically effective in preclinical animal models. However, the prime function of standard proteasomes and immunoproteasomes is the control of protein homeostasis in cells. To maintain protein homeostasis in cells, proteasomes remove proteins which are not properly folded, which are damaged by stress conditions such as reactive oxygen species formation, or which have to be degraded on the basis of regular protein turnover. In this review we summarize the latest insights on how the immunoproteasome influences protein homeostasis.

Summary DDX3X is a ubiquitously expressed RNA helicase involved in multiple stages of RNA biogenesis. DDX3X is frequently mutated in Burkitt lymphoma, but the functional basis for this is unknown. Here, we show that loss-of-function DDX3X mutations are also enriched in MYC-translocated diffuse large B cell lymphoma and reveal functional cooperation between mutant DDX3X and MYC. DDX3X promotes the translation of mRNA encoding components of the core translational machinery, thereby driving global protein synthesis. Loss-of-function DDX3X mutations moderate MYC-driven global protein synthesis, thereby buffering MYC-induced proteotoxic stress during early lymphomagenesis. Established lymphoma cells restore full protein synthetic capacity by aberrant expression of DDX3Y, a Y chromosome homolog, the expression of which is normally restricted to the testis. These findings show that DDX3X loss of function can buffer MYC-driven proteotoxic stress and highlight the capacity of male B cell lymphomas to then compensate for this loss by ectopic DDX3Y expression.

The nucleolus, a dynamic nuclear compartment long regarded as the cell ribosome factory, is emerging as an important player in the regulation of cell survival and recovery from stress. In larger eukaryotes, the stress-induced transcriptional response is mediated by a family of heat-shock transcription factors. Among these, HSF1, considered the master regulator of stress-induced transcriptional responses, controls the expression of cytoprotective heat shock proteins (HSPs), molecular chaperones/cochaperones constituting a major component of the cell protein quality control machinery essential to circumvent stress-induced degradation and aggregation of misfolded proteins. Herein we identify human NF-KB repressing factor (NKRF) as a nucleolar HSP essential for nucleolus homeostasis and cell survival under proteotoxic stress. NKRF acts as a thermosensor translocating from the nucleolus to the nucleoplasm during heat stress; nucleolar pools are replenished during recovery upon HSF1-mediated NKRF resynthesis. Silencing experiments demonstrate that NKRF is an unconventional HSP crucial for correct ribosomal RNA (rRNA) processing and preventing aberrant rRNA precursors and discarded fragment accumulation. These effects are mediated by NKRF interaction with the 5?-to-3? exoribonuclease XRN2, a key coordinator of multiple pre-rRNA cleavages, driving mature rRNA formation and discarded rRNA decay. Under stress conditions, NKRF directs XRN2 nucleolus/nucleoplasm trafficking, controlling 5?-to-3? exoribonuclease nucleolar levels and regulating rRNA processing. Our study reveals a different aspect of rRNA biogenesis control in human cells and sheds light on a sophisticated mechanism of nucleolar homeostasis surveillance during stress.

Oxidative stress placed on tissues that involved in pathogenesis of a disease activates compensatory metabolic changes, such as DNA damage repair that in turn causes intracellular accumulation of detritus and ‘proteotoxic stress’, leading to emergence of ‘senescent’ cellular phenotypes, which express high levels of inflammatory mediators, resulting in degradation of tissue function. Proteotoxic stress resulting from hyperactive inflammation following reperfusion of ischaemic tissue causes accumulation of proteinaceous debris in cells of the heart in ways that cause potentially fatal arrhythmias, in particular ventricular fibrillation (VF). An adaptive response to VF is occurrence of autophagy, an intracellular bulk degradation of damaged macromolecules and organelles that may restore cellular and tissue homoeostasis, improving chances for recovery. Nevertheless, depending on the type and intensity of stressors and inflammatory responses, autophagy may become pathological, resulting in excessive cell death. The present review examines the multilayered defences that cells have evolved to reduce proteotoxic stress by degradation of potentially toxic material beginning with endoplasmic reticulum‐associated degradation, and the unfolded protein response, which are mechanisms for removal from the endoplasmic reticulum of misfolded proteins, and then progressing through the stages of autophagy, including descriptions of autophagosomes and related vesicular structures which process material for degradation and autophagy‐associated proteins including Beclin‐1 and regulatory complexes. The physiological roles of each mode of proteotoxic defence will be examined along with consideration of how emerging understanding of autophagy, along with a newly discovered regulatory cell type called telocytes, may be used to augment existing strategies for the prevention and management of cardiovascular disease.

Ribosomes are multicomponent molecular machines that synthesize all of the proteins of living cells. Most of the genes that encode the protein components of ribosomes are therefore essential. A reduction in gene dosage is often viable albeit deleterious and is associated with human syndromes, which are collectively known as ribosomopathies1–3. The cell biological basis of these pathologies has remained unclear. Here, we model human ribosomopathies in Drosophila and find widespread apoptosis and cellular stress in the resulting animals. This is not caused by insufficient protein synthesis, as reasonably expected. Instead, ribosomal protein deficiency elicits proteotoxic stress, which we suggest is caused by the accumulation of misfolded proteins that overwhelm the protein degradation machinery. We find that dampening the integrated stress response4 or autophagy increases the harm inflicted by ribosomal protein deficiency, suggesting that these activities could be cytoprotective. Inhibition of TOR activity—which decreases ribosomal protein production, slows down protein synthesis and stimulates autophagy5—reduces proteotoxic stress in our ribosomopathy model. Interventions that stimulate autophagy, combined with means of boosting protein quality control, could form the basis of a therapeutic strategy for this class of diseases. Recasens-Alvarez et al. model human ribosomopathies and find that apoptosis and cellular stress result from proteotoxic stress that overwhelms the degradation machinery.

Summary Mechanisms establishing higher-order chromosome structures and their roles in gene regulation are elusive. We analyzed chromosome architecture during nematode X chromosome dosage compensation, which represses transcription via a dosage-compensation condensin complex (DCC) that binds hermaphrodite Xs and establishes megabase-sized topologically associating domains (TADs). We show that DCC binding at high-occupancy sites (rex sites) defines eight TAD boundaries. Single rex deletions disrupted boundaries, and single insertions created new boundaries, demonstrating that a rex site is necessary and sufficient to define DCC-dependent boundary locations. Deleting eight rex sites (8rexΔ) recapitulated TAD structure of DCC mutants, permitting analysis when chromosome-wide domain architecture was disrupted but most DCC binding remained. 8rexΔ animals exhibited no changes in X expression and lacked dosage-compensation mutant phenotypes. Hence, TAD boundaries are neither the cause nor the consequence of DCC-mediated gene repression. Abrogating TAD structure did, however, reduce thermotolerance, accelerate aging, and shorten lifespan, implicating chromosome architecture in stress responses and aging.

Heat shock protein (HSP) synthesis is switched on in a remarkably wide range of tumor cells, in both experimental animal systems and in human cancer, in which these proteins accumulate in high levels. In each case, elevated HSP concentrations bode ill for the patient, and are associated with a poor outlook in terms of survival in most cancer types. The significance of elevated HSPs is underpinned by their essential roles in mediating tumor cell intrinsic traits such as unscheduled cell division, escape from programmed cell death and senescence, de novo angiogenesis, and increased invasion and metastasis. An increased HSP expression thus seems essential for tumorigenesis. Perhaps of equal significance is the pronounced interplay between cancer cells and the tumor milieu, with essential roles for intracellular HSPs in the properties of the stromal cells, and their roles in programming malignant cells and in the release of HSPs from cancer cells to influence the behavior of the adjacent tumor and infiltrating the normal cells. These findings of a triple role for elevated HSP expression in tumorigenesis strongly support the targeting of HSPs in cancer, especially given the role of such stress proteins in resistance to conventional therapies. Fil: Lang, Benjamin J.. Harvard Medical School; Estados Unidos Fil: Guerrero Gimenez, Martin Eduardo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Medicina y Biología Experimental de Cuyo; Argentina Fil: Prince, Thomas L.. Geisinger Medical Center; Estados Unidos Fil: Ackerman, Andrew. Geisinger Medical Center; Estados Unidos Fil: Bonorino, Cristina. Universidade Federal de Ciências da Saúde de Porto Alegre; Brasil. University of California; Estados Unidos Fil: Calderwood, Stuart K.. Harvard Medical School; Estados Unidos

Proteins are secreted throughout the mycelium of Aspergillus niger except for the sporulating zone. A link between sporulation and repression of protein secretion was underlined by the finding that inactivation of the sporulation gene flbA results in mycelial colonies that secrete proteins throughout the colony. However, ΔflbA strain hyphae also lyse and have thinner cell walls. This pleiotropic phenotype is associated with differential expression of 36 predicted transcription factor genes, one of which, rpnR, was inactivated in this study. Sporulation, biomass, and secretome complexity were not affected in the ΔrpnR deletion strain of the fungus. In contrast, ribosomal subunit expression and protein secretion into the medium were reduced when A. niger was grown on xylose. Moreover, the ΔrpnR strain showed decreased resistance to H2O2 and the proteotoxic stress-inducing agent dithiothreitol. Taking the data together, RpnR is involved in proteotoxic stress resistance and impacts protein secretion when A. niger is grown on xylose. IMPORTANCEAspergillus niger secretes a large amount and diversity of industrially relevant enzymes into the culture medium. This makes the fungus a widely used industrial cell factory. For instance, carbohydrate-active enzymes of A. niger are used in biofuel production from lignocellulosic feedstock. These enzymes represent a major cost factor in this process. Higher production yields could substantially reduce these costs and therefore contribute to a more sustainable economy and less dependence on fossil fuels. Enzyme secretion is inhibited in A. niger by asexual reproduction. The sporulation protein FlbA is involved in this process by impacting the expression of 36 predicted transcription factor genes. Here, we show that one of these predicted transcriptional regulators, RpnR, regulates protein secretion and proteotoxic stress resistance. The gene is thus an interesting target to improve enzyme production in A. niger.

Nuclear factor (erythroid-derived 2)-like 2 (NRF2) is a central regulator of cellular stress responses and its transcriptional activation promotes multiple cellular defense and survival mechanisms. The loss of NRF2 has been shown to increase oxidative and proteotoxic stress, two key pathological features of neurodegenerative disorders such as Parkinson’s disease (PD). Moreover, compromised redox homeostasis and protein quality control can cause the accumulation of pathogenic proteins, including alpha-synuclein (α-Syn) which plays a key role in PD. However, despite this link, the precise mechanisms by which NRF2 may regulate PD pathology is not clear. In this study, we generated a humanized mouse model to study the importance of NRF2 in the context of α-Syn-driven neuropathology in PD. Specifically, we developed NRF2 knockout and wild-type mice that overexpress human α-Syn (hα-Syn+/Nrf2-/- and hα-Syn+/Nrf2+/+ respectively) and tested changes in their behavior through nest building, challenging beam, and open field tests at three months of age. Cellular and molecular alterations in α-Syn, including phosphorylation and subsequent oligomerization, as well as changes in oxidative stress, inflammation, and autophagy were also assessed across multiple brain regions. It was observed that although monomeric α-Syn levels did not change, compared to their wild-type counterparts, hα-Syn+/Nrf2-/- mice exhibited increased phosphorylation and oligomerization of α-Syn. This was associated with a loss of tyrosine hydroxylase expressing dopaminergic neurons in the substantia nigra, and more pronounced behavioral deficits reminiscent of early-stage PD, in the hα-Syn+/Nrf2-/- mice. Furthermore, hα-Syn+/Nrf2-/- mice showed significantly amplified oxidative stress, greater expression of inflammatory markers, and signs of increased autophagic burden, especially in the midbrain, striatum and cortical brain regions. These results support an important role for NRF2, early in PD progression. More broadly, it indicates NRF2 biology as fundamental to PD pathogenesis and suggests that targeting NRF2 activation may delay the onset and progression of PD.

Aging is characterized by the progressive decline of physiology at the cell, tissue and organism level, leading to an increased risk of mortality. Proteotoxic stress, mitochondrial dysfunction and genomic instability are considered major universal drivers of cell aging, and accumulating evidence establishes clear biunivocal relationships among these key hallmarks. In this regard, the finite lifespan of the budding yeast, together with the extensive armamentarium of available analytical tools, has made this single cell eukaryote a key model to study aging at molecular and cellular levels. Here we review the current data that link proteostasis to cell cycle progression in the budding yeast, focusing on senescence as an inherent phenotype displayed by aged cells. Recent advances in high-throughput systems to study yeast mother cells while they replicate are providing crucial information on aging-related processes and their temporal interdependencies at a systems level. In our view, the available data point to the existence of multiple feedback mechanisms among the major causal factors of aging, which would converge into the loss of proteostasis as a nodal driver of cell senescence and death., This work was funded by grants from the Ministry of Economy and Competitiveness of Spain and the European Union (FEDER) to M.A. D.F.M. received a FI fellowship of Generalitat de Catalunya.

Summary The role of stress-induced increases in SUMO2/3 conjugation during the heat shock response (HSR) has remained enigmatic. We investigated SUMO signal transduction at the proteomic and functional level during the HSR in cells depleted of proteostasis network components via chronic heat shock factor 1 inhibition. In the recovery phase post heat shock, high SUMO2/3 conjugation was prolonged in cells lacking sufficient chaperones. Similar results were obtained upon inhibiting HSP90, indicating that increased chaperone activity during the HSR is critical for recovery to normal SUMO2/3 levels post-heat shock. Proteasome inhibition likewise prolonged SUMO2/3 conjugation, indicating that stress-induced SUMO2/3 targets are subsequently degraded by the ubiquitin-proteasome system. Functionally, we suggest that SUMOylation can enhance the solubility of target proteins upon heat shock, a phenomenon that we experimentally observed in vitro. Collectively, our results implicate SUMO2/3 as a rapid response factor that coordinates proteome degradation and assists the maintenance of proteostasis upon proteotoxic stress., Graphical Abstract, Highlights • Chaperone depletion delays recovery of SUMOylation in response to heat shock • SUMOylated proteins are targeted to the proteasome during heat shock recovery • Chaperone depletion impairs clearance of Ub/SUMO co-modified proteins after stress • SUMO is a rapid response factor likely increasing protein solubility under heat shock, Functional consequences of SUMO2/3 conjugation during proteotoxic stress remain unclear. Liebelt et al. show that SUMO2/3 acts synergistically with the proteostasis network to reduce aggregation during acute heat shock and facilitate target degradation by the proteasome during stress recovery. SUMO2/3, therefore, acts as a first protective response to proteotoxic stress.

The anti-apoptotic protein Mcl-1 is highly expressed in castration-resistant prostate cancer (CRPC), resulting in resistance to apoptosis and association with poor prognosis. Although predominantly localized in the cytoplasm, there is evidence that Mcl-1 exhibits nuclear localization where it is thought to protect against DNA damage-induced cell death. The role of Mcl-1 in mediating resistance to chemotherapy-induced DNA damage in prostate cancer (PCa) is not known. We show in human PCa cell lines and in TRAMP, a transgenic mouse model of PCa, that the combination of the antimitotic agent ENMD-1198 (analog of 2-methoxyestradiol) with betulinic acid (BA, increases proteotoxic stress) targets Mcl-1 by increasing its proteasomal degradation, resulting in increased γH2AX (DNA damage) and apoptotic/necrotic cell death. Knockdown of Mcl-1 in CRPC cells leads to elevated γH2AX, DNA strand breaks, and cell death after treatment with 1198 + BA- or doxorubicin. Additional knockdowns in PC3 cells suggests that cytoplasmic Mcl-1 protects against DNA damage by blocking the mitochondrial release of apoptosis-inducing factor and thereby preventing its nuclear translocation and subsequent interaction with the cyclophilin A endonuclease. Overall, our results suggest that chemotherapeutic agents that target Mcl-1 will promote cell death in response to DNA damage, particularly in CRPC.

Sequestration of protein aggregates in inclusion bodies and their subsequent degradation prevents proteostasis imbalance, cytotoxicity, and proteinopathies. The underlying molecular mechanisms controlling the turnover of protein aggregates are mostly uncharacterized. Herein, we show that a TRIM family protein, TRIM16, governs the process of stress‐induced biogenesis and degradation of protein aggregates. TRIM16 facilitates protein aggregate formation by positively regulating the p62‐NRF2 axis. We show that TRIM16 is an integral part of the p62‐KEAP1‐NRF2 complex and utilizes multiple mechanisms for stabilizing NRF2. Under oxidative and proteotoxic stress conditions, TRIM16 activates ubiquitin pathway genes and p62 via NRF2, leading to ubiquitination of misfolded proteins and formation of protein aggregates. We further show that TRIM16 acts as a scaffold protein and, by interacting with p62, ULK1, ATG16L1, and LC3B, facilitates autophagic degradation of protein aggregates. Thus, TRIM16 streamlines the process of stress‐induced aggregate clearance and protects cells against oxidative/proteotoxic stress‐induced toxicity in vitro and in vivo . Taken together, this work identifies a new mechanism of protein aggregate turnover, which could be relevant in protein aggregation‐associated diseases such as neurodegeneration.

Summary The sole nonredundant role of the thymic medulla is to induce central tolerance, a vital process that depends on promiscuous gene expression (pGE), a unique feature of medullary thymic epithelial cells (mTECs). Although pGE enhances transcription of >3,000 genes in mTECs, its impact on the regulation of protein homeostasis remains unexplored. Here, we report that, because of pGE, mature mTECs synthesize substantially more proteins than other cell types and are exquisitely sensitive to loss of immunoproteasomes (IPs). Indeed, IP deficiency causes proteotoxic stress in mTECs and leads to exhaustion of postnatal mTEC progenitors. Moreover, IP-deficient mice show accelerated thymic involution, which is characterized by a selective loss of mTECs and multiorgan autoimmune manifestations. We conclude that pGE, the quintessential feature of mTECs, is a major burden for the maintenance of proteostasis, which is alleviated by the constitutive expression of IPs in mTECs.

Background Proteotoxic stress and transforming growth factor (TGFβ)- induced epithelial-mesenchymal transition (EMT) are two main contributors of intraocular fibrotic disorders, including proliferative vitreoretinopathy (PVR) and proliferative diabetic retinopathy (PDR). However, how these two factors communicate with each other is not well-characterized. Objective The aim was to investigate the regulatory role of proteotoxic stress on TGFβ signaling in retinal pigment epithelium. Methods ARPE-19 cells and primary human retinal pigment epithelial (RPE) cells were treated with proteasome inhibitor MG132 and TGFβ. Cell proliferation was analyzed by CCK-8 assay. The levels of mesenchymal markers α-SMA, fibronectin, and vimentin were analyzed by real-time polymerase chain reaction (PCR), western blot, and immunofluorescence. Cell migration was analyzed by scratch wound assay. The levels of p-Smad2, total Smad2, p-extracellular signal-regulated kinase 1/2 (ERK1/2), total ERK1/2, p-focal adhesion kinase (FAK), and total FAK were analyzed by western blot. The mRNA and protein levels of TGFβ receptor-II (TGFβR-II) were measured by realtime PCR and western blot, respectively. Results MG132-induced proteotoxic stress resulted in reduced cell proliferation. MG132 significantly suppressed TGFβ-induced upregulation of α-SMA, fibronectin, and vimentin, as well as TGFβ-induced cell migration. The phosphorylation levels of Smad2, ERK1/2, and FAK were also suppressed by MG132. Additionally, the mRNA level and protein level of TGFβR-II decreased upon MG132 treatment. Conclusion Proteotoxic stress suppressed TGFβ-induced EMT through downregulation of TGFβR-II and subsequent blockade of Smad2, ERK1/2, and FAK activation.

Molecular chaperones are required to maintain the proteome in a folded and functional state. When challenges to intracellular folding occur, the heat shock response is triggered, leading to increased synthesis of a class of inducible chaperones known as heat shock proteins (HSP). Although HSP synthesis is known to undergo a general decline in most cells with aging, the extent of this process varies quite markedly in some of the diseases associated with advanced age. In Alzheimer's disease (AD), a prevalent protein folding disorder in the brain, the heat shock response of some critical classes of neurons becomes reduced. The resulting decline in HSP expression may be a consequence of the general enfeeblement of many aspects of cell physiology with aging and/or a response to the pathological changes in metabolism observed specifically in AD. Cancer cells, in contrast to normal aging cells, undergo de novo increases in HSP levels. This expansion in HSP expression has been attributed to increases in folding demand in cancer or to the evolution of new mechanisms for induction of the heat shock response in rapidly adapting cancer cells. As the predominant pathway for regulation of HSP synthesis involves transcription factor HSF1, it has been suggested that dysregulation of this factor may play a decisive role in the development of each disease. We will discuss what is known of the mechanisms of HSF1 regulation in regard to the HSP dysregulation seen in in AD and cancer.

Cells robustly reprogram gene expression during stress generated by protein misfolding and aggregation. In this condition, cells assemble the bulk of mRNAs into translationally silent stress granules (SGs), while they sustain the translation of specific mRNAs coding for proteins that are needed to overcome cellular stress. Alterations of this process are deeply associated to neurodegeneration. This is the case of amyotrophic lateral sclerosis (ALS), a neurodegenerative disorder caused by a selective loss of motor neurons. Indeed, impairment of protein homeostasis as well as alterations of RNA metabolism are now recognized as major players in the pathogenesis of ALS. In particular, evidence shows that defective mRNA transport and translation are implicated. Here, we provide a review of what is currently known about altered mRNA translation in ALS and how this impacts on the ability of affected cells to cope with proteotoxic stress.

Cancer cells are addicted to numerous non-oncogenic traits that enable them to thrive. Proteotoxic stress is one such non-oncogenic trait that is experienced by all tumor cells, owing to increased genomic abnormalities and the resulting synthesis and accumulation of non-stoichiometric amounts of cellular proteins. This imbalance in the amounts of proteins ultimately culminates in proteotoxic stress. p97, or valosin containing protein (VCP) is an ATP-ase whose function is essential to restore protein homeostasis in the cells. Working in concert with the ubiquitin proteasome system, p97 promotes the retrotranslocation from cellular organelles and/or degradation of misfolded proteins. Consequently, p97 inhibition has emerged as a novel therapeutic target in cancer cells, especially those that have a highly secretory phenotype. This review summarizes our current understanding of the function of p97 in maintaining protein homeostasis and its inhibition with small molecule inhibitors as an emerging strategy to target cancer cells.

Cellular toxicity introduced by protein misfolding threatens cell fitness and viability. Failure to eliminate these polypeptides is associated with numerous aggregation diseases. Several protein quality control mechanisms degrade non-native proteins by the ubiquitin-proteasome system. Here, we use quantitative mass spectrometry to demonstrate that heat-shock triggers a large increase in the level of ubiquitylation associated with misfolding of cytosolic proteins. We discover that the Hul5 HECT ubiquitin ligase participates in this heat-shock stress response. Hul5 is required to maintain cell fitness after heat-shock and to degrade short-lived misfolded proteins. In addition, localization of Hul5 in the cytoplasm is important for its quality control function. We identify potential Hul5 substrates in heat-shock and physiological conditions to reveal that Hul5 is required for ubiquitylation of low-solubility cytosolic proteins including the Pin3 prion-like protein. These findings indicate that Hul5 is involved in a cytosolic protein quality control pathway that targets misfolded proteins for degradation.

Although nearly ubiquitous in cancer, aneuploidy exerts detrimental effects on human cells. We recently demonstrated that aneuploid human cells exhibit impaired heat shock factor protein 1 (HSF1) and HSP90 function, suggesting a functional link between two recurring features of cancer cells: aneuploidy and proteotoxic stress. Further, our findings implicate HSF1 as a key factor in mitigating the effects of aneuploidy.

Oxidative stress is known to impair architecture and function of cells, which may lead to various chronic diseases, and therefore therapeutic and nutritional interventions to reduce oxidative damages represent a viable strategy in the amelioration of oxidative stress-related disorders, including neurodegenerative diseases. Over the past decade, a variety of natural polysaccharides from functional and medicinal foods have attracted great interest due to their antioxidant functions such as scavenging free radicals and reducing oxidative damages. Interestingly, these antioxidant polysaccharides are also found to attenuate neuronal damages and alleviate cognitive and motor decline in a range of neurodegenerative models. It has recently been established that the neuroprotective mechanisms of polysaccharides are related to oxidative stress-related pathways, including mitochondrial function, antioxidant defense system and pathogenic protein aggregation. Here, we first summarize the current status of antioxidant function of food-derived polysaccharides and then attempt to appraise their anti-neurodegeneration activities.

Mutations in genes that encode tRNAs, aminoacyl-tRNA syntheases, tRNA modifying enzymes and other tRNA interacting partners are associated with neuropathies, cancer, type-II diabetes and hearing loss, but how these mutations cause disease is unclear. We have hypothesized that levels of tRNA decoding error (mistranslation) that do not fully impair embryonic development can accelerate cell degeneration through proteome instability and saturation of the proteostasis network. To test this hypothesis we have induced mistranslation in zebrafish embryos using mutant tRNAs that misincorporate Serine (Ser) at various non-cognate codon sites. Embryo viability was affected and malformations were observed, but a significant proportion of embryos survived by activating the unfolded protein response (UPR), the ubiquitin proteasome pathway (UPP) and downregulating protein biosynthesis. Accumulation of reactive oxygen species (ROS), mitochondrial and nuclear DNA damage and disruption of the mitochondrial network, were also observed, suggesting that mistranslation had a strong negative impact on protein synthesis rate, ER and mitochondrial homeostasis. We postulate that mistranslation promotes gradual cellular degeneration and disease through protein aggregation, mitochondrial dysfunction and genome instability. published

Background Organisms use highly accurate molecular processes to transcribe their genes and a variety of mRNA quality control and ribosome proofreading mechanisms to maintain intact the fidelity of genetic information flow. Despite this, low level gene translational errors induced by mutations and environmental factors cause neurodegeneration and premature death in mice and mitochondrial disorders in humans. Paradoxically, such errors can generate advantageous phenotypic diversity in fungi and bacteria through poorly understood molecular processes. Results In order to clarify the biological relevance of gene translational errors we have engineered codon misreading in yeast and used profiling of total and polysome-associated mRNAs, molecular and biochemical tools to characterize the recombinant cells. We demonstrate here that gene translational errors, which have negligible impact on yeast growth rate down-regulate protein synthesis, activate the unfolded protein response and environmental stress response pathways, and down-regulate chaperones linked to ribosomes. Conclusions We provide the first global view of transcriptional and post-transcriptional responses to global gene translational errors and we postulate that they cause gradual cell degeneration through synergistic effects of overloading protein quality control systems and deregulation of protein synthesis, but generate adaptive phenotypes in unicellular organisms through activation of stress cross-protection. We conclude that these genome wide gene translational infidelities can be degenerative or adaptive depending on cellular context and physiological condition.

International audience; Ectotherms from sunny and hot environments need to cope with solar radiation. Mediterranean land snails of the superfamily Helicoidea feature a behavioural strategy to escape from solar radiation-induced excessive soil heating by climbing up vertical objects. The height of climbing, and also other parameters like shell colouration pattern, shell orientation, shell size, body mass, actual internal and shell surface temperature, and the interactions between those factors may be expected to modulate proteotoxic effects in snails exposed to solar radiation and, thus, their stress response. Focussing on natural populations of Xeropicta derbentina, we conducted a 'snapshot' field study using the individual Hsp70 level as a proxy for proteotoxic stress. In addition to correlation analyses, an IT-model selection approach based on Akaike's Information Criterion was applied to evaluate a set of models with respect to their explanatory power and to assess the relevance of each of the above-mentioned parameters for individual stress, by model averaging and parameter estimation. The analysis revealed particular importance of the individuals' shell size, height above ground, the shell colouration pattern and the interaction height x orientation. Our study showed that a distinct set of behavioural traits and intrinsic characters define the Hsp70 level and that environmental factors and individual features strongly interact.

Autophagy, a highly conserved proteolytic mechanism of quality control, is essential for the maintenance of metabolic and cellular homoeostasis and for an efficient cellular response to stress. Autophagy declines with aging and is believed to contribute to different aspects of the aging phenotype. The nutrient-sensing pathways PKA (protein kinase A), Sch9 and TOR (target of rapamycin), involved in the regulation of yeast lifespan, also converge on a common targeted process: autophagy. The molecular mechanisms underlying the regulation of autophagy and aging by these signalling pathways in yeast, with special attention to theTOR pathway, are discussed in the present paper. The question of whether or not autophagy could contribute to yeast cell death occurring during CLS (chronological lifespan) is discussed in the light of our findings obtained after autophagy activation promoted by proteotoxic stress. Autophagy progressively increases in cells expressing the aggregation-prone protein α-synuclein and seems to participate in the early cell death and shortening of CLS under these conditions, highlighting that autophagic activity should be maintained below physiological levels to exert its promising anti-aging effects., This work was supported by a grant from the Fundação para a Ciência e Tecnologia [grant number PTDC/BIA-MIC/114116/2009]; A.S. and B.S.M. received fellowships from the Fundação para a Ciência e Tecnologia [grant numbers SFRH/BD/33125/2007 and SRFH/BD/41674/2007 respectively].

Heat shock transcription factor (HSF), a key regulator in the expression of heat shock protein (HSP) chaperones, is involved in the maintenance of protein homeostasis. However, the impact of HSF-mediated transcription of each HSP gene on this process is not fully understood. We show that Saccharomyces cerevisiae cells containing mutations in the HSF-binding sequences of chromosomal HSP90 promoters exhibit various phenotypes, including slow growth, proteotoxic stress sensitivity, and reduced chronological lifespan. Similar phenotypes were observed when HSF-binding sequences in five mitochondrial HSP promoters were mutated. Therefore, HSF-regulated changes in expression of these chaperone genes are necessary to maintain cell viability under various growth conditions.

Within tumor cells the heat shock factor 1 (HSF1)-mediated stress response is constitutively mobilized to counter persistent proteotoxic stress, hence sustaining their fragile proteome homeostasis and supporting robust malignant phenotypes. Intriguingly, our new studies reveal that metabolic stressors, such as metformin, inactivate HSF1 and provoke proteomic chaos, thereby impeding tumorigenesis.

International audience; Clusterin is a secreted protein chaperone up-regulated in several pathologies, including cancer and neurodegenerative diseases. The present study shows that accumulation of aberrant proteins, caused by the proteasome inhibitor MG132 or the incorporation of the amino acid analogue AZC (L-azetidine-2-carboxylic acid), increased both clusterin protein and mRNA levels in the human glial cell line U-251 MG. Consistently, MG132 treatment was capable of stimulating a 1.3 kb clusterin gene promoter. Promoter deletion and mutation studies revealed a critical MG132-responsive region between -218 and -106 bp, which contains a particular heat-shock element, named CLE for 'clusterin element'. Gel mobility-shift assays demonstrated that MG132 and AZC treatments induced the formation of a protein complex that bound to CLE. As shown by supershift and chromatin-immunoprecipitation experiments, CLE is bound by HSF1 (heat-shock factor 1) and HSF2 upon proteasome inhibition. Furthermore, co-immunoprecipitation assays indicated that these two transcription factors interact. Gel-filtration analyses revealed that the HSF1-HSF2 heterocomplexes bound to CLE after proteasome inhibition have the same apparent mass as HSF1 homotrimers after heat shock, suggesting that HSF1 and HSF2 could heterotrimerize. Therefore these studies indicate that the clusterin is a good candidate to be part of a cellular defence mechanism against neurodegenerative diseases associated with misfolded protein accumulation or decrease in proteasome activity

The dysfunction of the ubiquitin proteasome system has been related to a broad array of neurodegenerative disorders in which the accumulation of misfolded protein aggregates causes proteotoxicity. The ability of proteasome inhibitors to induce cell cycle arrest and apoptosis has emerged as a powerful strategy for cancer therapy. Bortezomib is a proteasome inhibitor used as an antineoplastic drug, although its neurotoxicity frequently causes a severe sensory peripheral neuropathy. In this study we used a rat model of bortezomib treatment to study the nucleolar and Cajal body responses to the proteasome inhibition in sensory ganglion neurons that are major targets of bortezomib-induced neurotoxicity. Treatment with bortezomib induced dose-dependent dissociation of protein synthesis machinery (chromatolysis) and nuclear retention of poly(A) RNA granules resulting in neuronal dysfunction. However, as a compensatory response to the proteotoxic stress, both nucleoli and Cajal bodies exhibited reactive changes. These include an increase in the number and size of nucleoli, strong nucleolar incorporation of the RNA precursor 5′-fluorouridine, and increased expression of both 45S rRNA and genes encoding nucleolar proteins UBF, fibrillarin and B23. Taken together, these findings appear to reflect the activation of the nucleolar transcription in response to proteotoxic stress Furthermore, the number of Cajal bodies, a parameter related to transcriptional activity, increases upon proteasome inhibition. We propose that nucleoli and Cajal bodies are important targets in the signaling pathways that are activated by the proteotoxic stress response to proteasome inhibition. The coordinating activity of these two organelles in the production of snRNA, snoRNA and rRNA may contribute to neuronal survival after proteasome inhibition. This article is part of a Special Issue entitled: Role of the Nucleolus in Human Disease.