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Extreme acidophilic bacteria like Leptospirillum sp. require an efficient enzyme system to counteract strong oxygen stress conditions in their natural habitat. The genome of Leptospirillum sp. CF-1 encodes the thioredoxin-fold protein TFP2, which exhibits a high structural similarity to the thioredoxin domain of E. coli CnoX. CnoX from Escherichia coli is a chaperedoxin that protects protein substrates from oxidative stress conditions using its holdase function and a subsequent transfer to foldase chaperones for refolding. Recombinantly produced and purified Leptospirillum sp. TFP2 possesses both thioredoxin and chaperone holdase activities in vitro. It can be reduced by thioredoxin reductase (TrxR). The tfp2 gene co-locates with genes for the chaperone foldase GroES/EL on the chromosome. The "tfp2 cluster" (ctpA-groES-groEL-hyp-tfp2-recN) was found between 1.9 and 8.8-fold transcriptionally up-regulated in response to 1 mM hydrogen peroxide (H2O2). Leptospirillum sp. tfp2 heterologously expressed in E. coli wild type and cnoX mutant strains lead to an increased tolerance of these E. coli strains to H2O2 and significantly reduced intracellular protein aggregates. Finally, a proteomic analysis of protein aggregates produced in E. coli upon exposition to oxidative stress with 4 mM H2O2, showed that Leptospirillum sp. tfp2 expression caused a significant decrease in the aggregation of 124 proteins belonging to fifteen different metabolic categories. These included several known substrates of DnaK and GroEL/ES. These findings demonstrate that Leptospirillum sp. TFP2 is a chaperedoxin-like protein, acting as a key player in the control of cellular proteostasis under highly oxidative conditions that prevail in extreme acidic environments.

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The heat shock response (HSR) is an ancient and evolutionarily conserved mechanism designed to restore cellular homeostasis following proteotoxic challenges. However, it has become increasingly evident that disruptions in energy metabolism also trigger the HSR. This interplay between proteostasis and energy regulation is rooted in the fundamental need for ATP to fuel protein synthesis and repair, making the HSR an essential component of cellular energy management. Recent findings suggest that the origins of proteostasis-defending systems can be traced back over 3.6 billion years, aligning with the emergence of sugar kinases that optimized glycolysis around 3.594 billion years ago. This evolutionary connection is underscored by the spatial similarities between the nucleotide-binding domain of HSP70, the key player in protein chaperone machinery, and hexokinases. The HSR serves as a hub that integrates energy metabolism and resolution of inflammation, further highlighting its role in maintaining cellular homeostasis. Notably, 5'-adenosine monophosphate-activated protein kinase emerges as a central regulator, promoting the HSR during predominantly proteotoxic stress while suppressing it in response to predominantly metabolic stress. The complex relationship between 5'-adenosine monophosphate-activated protein kinase and the HSR is finely tuned, with paradoxical effects observed under different stress conditions. This delicate equilibrium, known as caloristasis, ensures that cellular homeostasis is maintained despite shifting environmental and intracellular conditions. Understanding the caloristatic controlling switch at the heart of this interplay is crucial. It offers insights into a wide range of conditions, including glycemic control, obesity, type 2 diabetes, cardiovascular and neurodegenerative diseases, reproductive abnormalities, and the optimization of exercise routines. These findings highlight the profound interconnectedness of proteostasis and energy metabolism in cellular function and adaptation.

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The male gamete is a highly differentiated cell that aims to fuse with the oocyte in fertilization. Sperm have silenced the transcription and translational processes, maintaining proteostasis to guarantee male reproductive health. Despite the information about the implication of molecular chaperones as orchestrators of protein folding or aggregation, and the handling of body homeostasis by the endocannabinoid system, there is still a lack of basic investigation and random controlled clinical trials that deliver more evidence on the involvement of cannabinoids in reproductive function. Besides, we noticed that the information regarding whether recreational marijuana affects male fertility is controversial and requires further investigation. In other cell models, it has recently been evidenced that chaperones and cannabinoids are intimately intertwined. Through a literature review, we aim to explore the interaction between chaperones and cannabinoid signaling in sperm development and function. To untangle how or whether this dialogue happens within the sperm proteostasis. We discuss the action of chaperones, the endocannabinoid system and phytocannabinoids in sperm proteostasis. Reports of some heat shock and lipid proteins interacting with cannabinoid receptors prove that chaperones and the endocannabinoid system are in an intimate dialogue. Meanwhile, advancing the evidence to decipher these mechanisms for introducing innovative interventions into routine clinical settings becomes crucial. We highlight the potential interaction between chaperones and cannabinoid signaling in regulating proteostasis in male reproductive health.

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Alzheimer's disease (AD), the most common neurodegenerative disease and the first cause of dementia worldwide, has no effective treatment, and its pathological mechanisms are not yet fully understood. We conducted this study to explore the proteomic differences associated with Familial Alzheimer's Disease (FAD) in olfactory ecto-mesenchymal stem cells (MSCs) derived from PSEN1 (A431E) mutation carriers compared with healthy donors paired by age and gender through two label-free liquid chromatography-mass spectrometry approaches. The first analysis compared carrier 1 (patient with symptoms, P1) and its control (healthy donor, C1), and the second compared carrier 2 (patient with pre-symptoms, P2) with its respective control cells (C2) to evaluate whether the protein alterations presented in the symptomatic carrier were also present in the pre-symptom stages. Finally, we analyzed the differentially expressed proteins (DEPs) for biological and functional enrichment. These proteins showed impaired expression in a stage-dependent manner and are involved in energy metabolism, vesicle transport, actin cytoskeleton, cell proliferation, and proteostasis pathways, in line with previous AD reports. Our study is the first to conduct a proteomic analysis of MSCs from the Jalisco FAD patients in two stages of the disease (symptomatic and presymptomatic), showing these cells as a new and excellent in vitro model for future AD studies.

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Fungi are a diverse group of eukaryotic organisms that infect humans, animals, and plants. To successfully colonize their hosts, pathogenic fungi must continuously adapt to the host's unique environment, e.g., changes in temperature, pH, and nutrient availability. Appropriate protein folding, assembly, and degradation are essential for maintaining cellular homeostasis and survival under stressful conditions. Therefore, the regulation of proteostasis is crucial for fungal pathogenesis. The heat shock response (HSR) is one of the most important cellular mechanisms for maintaining proteostasis. It is activated by various stresses and regulates the activity of heat shock proteins (HSPs). As molecular chaperones, HSPs participate in the proteostatic network to control cellular protein levels by affecting their conformation, location, and degradation. In recent years, a growing body of evidence has highlighted the crucial yet understudied role of stress response circuits in fungal infections. This review explores the role of protein homeostasis and HSPs in fungal pathogenicity, including their contributions to virulence and host-pathogen interactions, as well as the concerted effects between HSPs and the main proteostasis circuits in the cell. Furthermore, we discuss perspectives in the field and the potential for targeting the components of these circuits to develop novel antifungal therapies.

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Mychael Lourenco is an Assistant Professor of Neuroscience at the Institute of Medical Biochemistry Leopoldo de Meis, Federal University of Rio de Janeiro. Research in his lab focusses on understanding the molecular mechanisms underlying cognitive impairment in neurodegeneration and his research on Alzheimer's disease has been recognized by many awards both in Brazil and internationally. He serves as a Reviews Editor for the Journal of Neurochemistry and led this special issue on Brain Proteostasis as a Guest Editor. Here we interviewed him to hear his thoughts on the future of neuroscience and on career development and training.

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Ischemic stroke is a leading cause of disability worldwide. There is no simple treatment to alleviate ischemic brain injury, as thrombolytic therapy is applicable within a narrow time window. During the last years, the ketogenic diet (KD) and the exogenous administration of the ketone body ß-hydroxybutyrate (BHB) have been proposed as therapeutic tools for acute neurological disorders and both can reduce ischemic brain injury. However, the mechanisms involved are not completely clear. We have previously shown that the D enantiomer of BHB stimulates the autophagic flux in cultured neurons exposed to glucose deprivation (GD) and in the brain of hypoglycemic rats. Here, we have investigated the effect of the systemic administration of D-BHB, followed by its continuous infusion after middle cerebral artery occlusion (MCAO), on the autophagy-lysosomal pathway and the activation of the unfolded protein response (UPR). Results show for the first time that the protective effect of BHB against MCAO injury is enantiomer selective as only D-BHB, the physiologic enantiomer of BHB, significantly reduced brain injury. D-BHB treatment prevented the cleavage of the lysosomal membrane protein LAMP2 and stimulated the autophagic flux in the ischemic core and the penumbra. In addition, D-BHB notably reduced the activation of the PERK/eIF2α/ATF4 pathway of the UPR and inhibited IRE1α phosphorylation. L-BHB showed no significant effect relative to ischemic animals. In cortical cultures under GD, D-BHB prevented LAMP2 cleavage and decreased lysosomal number. It also abated the activation of the PERK/eIF2α/ATF4 pathway, partially sustained protein synthesis, and reduced pIRE1α. In contrast, L-BHB showed no significant effects. Results suggest that protection elicited by D-BHB treatment post-ischemia prevents lysosomal rupture allowing functional autophagy, preventing the loss of proteostasis and UPR activation.

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Cells employ several adaptive mechanisms under conditions of accelerated cell division, such as the unfolded protein response (UPR). The UPR is composed of a tripartite signaling system that involves ATF6, PERK, and IRE1, which maintain protein homeostasis (proteostasis). However, deregulation of protein translation initiation could be associated with breast cancer (BC) chemoresistance. Specifically, eukaryotic initiation factor-4A (eIF4A) is involved in the unfolding of the secondary structures of several mRNAs at the 5' untranslated region (5'-UTR), as well as in the regulation of targets involved in chemoresistance. Importantly, the tumor suppressor gene PDCD4 could modulate this process. This regulation might be disrupted in chemoresistant triple negative-BC (TNBC) cells. Therefore, we characterized the effect of doxorubicin (Dox), a commonly used anthracycline medication, on human breast carcinoma MDA-MB-231 cells. Here, we generated and characterized models of Dox chemoresistance, and chemoresistant cells exhibited lower Dox internalization levels followed by alteration of the IRE1 and PERK arms of the UPR and triggering of the antioxidant Nrf2 axis. Critically, chemoresistant cells exhibited PDCD4 downregulation, which coincided with a reduction in eIF4A interaction, suggesting a sophisticated regulation of protein translation. Likewise, Dox-induced chemoresistance was associated with alterations in cellular migration and invasion, which are key cancer hallmarks, coupled with changes in focal adhesion kinase (FAK) activation and secretion of matrix metalloproteinase-9 (MMP-9). Moreover, eIF4A knockdown via siRNA and its overexpression in chemoresistant cells suggested that eIF4A regulates FAK. Pro-atherogenic low-density lipoproteins (LDL) promoted cellular invasion in parental and chemoresistant cells in an MMP-9-dependent manner. Moreover, Dox only inhibited parental cell invasion. Significantly, chemoresistance was modulated by cryptotanshinone (Cry), a natural terpene purified from the roots of Salvia brandegeei. Cry and Dox co-exposure induced chemosensitization, connected with the Cry effect on eIF4A interaction. We further demonstrated the Cry binding capability on eIF4A and in silico assays suggest Cry inhibition on the RNA-processing domain. Therefore, strategic disruption of protein translation initiation is a druggable pathway by natural compounds during chemoresistance in TNBC. However, plasmatic LDL levels should be closely monitored throughout treatment.

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Aging is a major risk factor to develop neurodegenerative diseases and is associated with decreased buffering capacity of the proteostasis network. We investigated the significance of the unfolded protein response (UPR), a major signaling pathway activated to cope with endoplasmic reticulum (ER) stress, in the functional deterioration of the mammalian brain during aging. We report that genetic disruption of the ER stress sensor IRE1 accelerated age-related cognitive decline. In mouse models, overexpressing an active form of the UPR transcription factor XBP1 restored synaptic and cognitive function, in addition to reducing cell senescence. Proteomic profiling of hippocampal tissue showed that XBP1 expression significantly restore changes associated with aging, including factors involved in synaptic function and pathways linked to neurodegenerative diseases. The genes modified by XBP1 in the aged hippocampus where also altered. Collectively, our results demonstrate that strategies to manipulate the UPR in mammals may help sustain healthy brain aging.

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Background: Proteostasis refers to the processes that regulate the biogenesis, folding, trafficking, and degradation of proteins. Any alteration in these processes can lead to cell malfunction. Protein synthesis, a key proteostatic process, is highly-regulated at multiple levels to ensure adequate adaptation to environmental and physiological challenges such as different stressors, proteotoxic conditions and aging, among other factors. Because alterations in protein translation can lead to protein misfolding, examining how protein translation is regulated may also help to elucidate in part how proteostasis is controlled. Codon usage bias has been implicated in the fine-tuning of translation rate, as more-frequent codons might be read faster than their less-frequent counterparts. Thus, alterations in codon usage due to synonymous mutations may alter translation kinetics and thereby affect the folding of the nascent polypeptide, without altering its primary structure. To date, it has been difficult to predict the effect of synonymous mutations on protein folding and cellular fitness due to a scarcity of relevant data. Thus, the purpose of this work was to assess the effect of synonymous mutations in discrete regions of the gene that encodes the highly-expressed enzyme 3-phosphoglycerate kinase 1 (pgk1) in the fission yeast Schizosaccharomyces pombe. Results: By means of systematic replacement of synonymous codons along pgk1, we found slightly-altered protein folding and activity in a region-specific manner. However, alterations in protein aggregation, heat stress as well as changes in proteasome activity occurred independently of the mutated region. Concomitantly, reduced mRNA levels of the chaperones Hsp9 and Hsp16 were observed. Conclusion: Taken together, these data suggest that codon usage bias of the gene encoding this highly-expressed protein is an important regulator of protein function and proteostasis.

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Protein synthesis is essential for cells to perform life metabolic processes. Pathological alterations of protein content can lead to particular diseases. Cells have an intrinsic array of mechanisms and pathways that are activated when protein misfolding, accumulation, aggregation or mislocalization occur. Some of them (like the unfolded protein response) represent complex interactions between endoplasmic reticulum sensors and elongation factors that tend to increase expression of chaperone proteins and/or repress translation in order to restore protein homeostasis (also known as proteostasis). This is even more important in neurons, as they are very susceptible to harmful effects associated with protein overload and proteostatic mechanisms are less effective with age. Several neurodegenerative pathologies such as Alzheimer's, Parkinson's, and Huntington's diseases, amyotrophic lateral sclerosis and frontotemporal dementia exhibit a particular molecular signature of distinct, unbalanced protein overload. In amyotrophic lateral sclerosis and frontotemporal dementia, the majority of cases present intracellular inclusions of ubiquitinated transactive response DNA-binding protein of 43 kDa (TDP-43). TDP-43 is an RNA binding protein that participates in RNA metabolism, among other functions. Dysregulation of TDP-43 (e.g. aggregation and mislocalization) can dramatically affect neurons, and this has been linked to disease development. Expression of amyotrophic lateral sclerosis/frontotemporal dementia TDP-43-related mutations in cellular and animal models has been shown to recapitulate key features of the amyotrophic lateral sclerosis/frontotemporal dementia disease spectrum. These variants can be causative of degeneration onset and progression. Most neurodegenerative diseases (including amyotrophic lateral sclerosis and frontotemporal dementia) have no cure at the moment; however, modulating translation has recently emerged as an attractive approach that can be performed at several steps (i.e. regulating activation of initiation and elongation factors, inhibiting unfolded protein response activation or inducing chaperone expression and activity). This review focuses on the features of protein imbalance in neurodegenerative disorders and the relevance of developing therapeutical compounds aiming at restoring proteostasis. We strive to highlight the importance of research on drugs that, not only restore protein imbalance without compromising translational activity of cells, but are also as safe as possible for the patients.

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Amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) are two related neurodegenerative disorders that display overlapping features. The hexanucleotide repeat expansion GGGGCC (G4 C2 ) in C9ORF72 gene has been causally linked to both ALS and FTD emergence, thus opening a novel potential therapeutic target for disease intervention. The main driver of C9ORF72 pathology is the disruption of distinct cellular processes involved in the function of the proteostasis network. Here we discuss main findings relating to the induction of neurodegeneration by C9ORF72 mutation and proteostasis deregulation, highlighting the role of the endoplasmic reticulum stress, nuclear transport, and autophagy in the disease process. We further discuss possible points of intervention to target proteostasis mediators to treat C9ORF72-linked ALS/FTD.

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During type 1 diabetes mellitus (T1DM) development, beta-cells undergo intense endoplasmic reticulum (ER) stress that could result in apoptosis through the failure of adaptation to the unfolded protein response (UPR). Islet transplantation is considered an attractive alternative among beta-cell replacement therapies for T1DM. To avoid the loss of beta-cells that will jeopardize the transplant's outcome, several strategies are being studied. We have previously shown that prolactin induces protection against proinflammatory cytokines and redox imbalance-induced beta-cell death by increasing heat-shock protein B1 (HSPB1) levels. Since the role of HSPB1 in beta cells has not been deeply studied, we investigated the mechanisms involved in unbalanced protein homeostasis caused by intense ER stress and overload of the proteasomal protein degradation pathway. We tested whether HSPB1-mediated cytoprotective effects involved UPR modulation and improvement of protein degradation via the ubiquitin-proteasome system. We demonstrated that increased levels of HSPB1 attenuated levels of pro-apoptotic proteins such as CHOP and BIM, as well as increased protein ubiquitination and the speed of proteasomal protein degradation. Our data showed that HSPB1 induced resistance to proteotoxic stress and, thus, enhanced cell survival via an increase in beta-cell proteolytic capacity. These results could contribute to generate strategies aimed at the optimization of beta-cell replacement therapies.

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Antimalarial drugs with novel modes of action and wide therapeutic potential are needed to pave the way for malaria eradication. Violacein is a natural compound known for its biological activity against cancer cells and several pathogens, including the malaria parasite, Plasmodium falciparum (Pf). Herein, using chemical genomic profiling (CGP), we found that violacein affects protein homeostasis. Mechanistically, violacein binds Pf chaperones, PfHsp90 and PfHsp70-1, compromising the latter's ATPase and chaperone activities. Additionally, violacein-treated parasites exhibited increased protein unfolding and proteasomal degradation. The uncoupling of the parasite stress response reflects the multistage growth inhibitory effect promoted by violacein. Despite evidence of proteotoxic stress, violacein did not inhibit global protein synthesis via UPR activation-a process that is highly dependent on chaperones, in agreement with the notion of a violacein-induced proteostasis collapse. Our data highlight the importance of a functioning chaperone-proteasome system for parasite development and differentiation. Thus, a violacein-like small molecule might provide a good scaffold for development of a novel probe for examining the molecular chaperone network and/or antiplasmodial drug design.

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Significance: The systematic investigation of oxidative modification of proteins by reactive oxygen species started in 1980. Later, it was shown that reactive nitrogen species could also modify proteins. Some protein oxidative modifications promote loss of protein function, cleavage or aggregation, and some result in proteo-toxicity and cellular homeostasis disruption. Recent Advances: Previously, protein oxidation was associated exclusively to damage. However, not all oxidative modifications are necessarily associated with damage, as with Met and Cys protein residue oxidation. In these cases, redox state changes can alter protein structure, catalytic function, and signaling processes in response to metabolic and/or environmental alterations. This review aims to integrate the present knowledge on redox modifications of proteins with their fate and role in redox signaling and human pathological conditions. Critical Issues: It is hypothesized that protein oxidation participates in the development and progression of many pathological conditions. However, no quantitative data have been correlated with specific oxidized proteins or the progression or severity of pathological conditions. Hence, the comprehension of the mechanisms underlying these modifications, their importance in human pathologies, and the fate of the modified proteins is of clinical relevance. Future Directions: We discuss new tools to cope with protein oxidation and suggest new approaches for integrating knowledge about protein oxidation and redox processes with human pathophysiological conditions. Antioxid. Redox Signal. 35, 1016-1080.

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Ilex paraguariensis is a plant from South America, used to prepare a tea-like beverage rich in caffeine and polyphenols with antioxidant proprieties. Caffeine consumption is associated with a lower risk of age-associated neuropathologies, besides several extracts that have antioxidant proprieties are known to be neuroprotective, and oxidative stress strongly correlates with Aß-toxicity. This study aims to investigate the neuroprotective effects of the Ilex paraguariensis hydroalcoholic extract (IPHE) and to evaluate if caffeine agent present in IPHE exerts neuroprotective effects in an amyloid beta-peptide (Aß)-induced toxicity in Caenorhabditis elegans. The wild-type and CL2006 worms were treated with IPHE (2 and 4 mg/mL) or caffeine (200 and 400 µM) since larval stage 1 (L1) until they achieved the required age for each assay. IPHE and caffeine increased the lifespan and appeared to act directly by reactive oxygen species (ROS) scavenger in both wild-type and CL2006 worms, also conferred resistance against oxidative stress in wild-type animals. Furthermore, both treatments delayed Aß-induced paralysis and decreased AChE activity in CL2006. The protective effect of IPHE against Aß-induced paralysis was found to be dependent on heat shock factor hsf-1 and FOXO-family transcription factor daf-16, which are respectively involved in aging-related processes and chaperone synthesis, while that of caffeine was dependent only on daf-16. Mechanistically, IPHE and caffeine decreased the levels of Aß mRNA in the CL2006 worms; however, only IPHE induced expression of the heat shock chaperonin hsp-16.2, involved in protein homeostasis. The results were overall better when treated with IPHE than with caffeine.

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Xeroderma pigmentosum complementation group A (XPA), is defective in xeroderma pigmentosum patients, causing pre-disposition to skin cancer and neurological abnormalities, which is not well understood. Here, we analyzed the XPA-deficient cells transcriptional profile under oxidative stress. The imbalance in of ubiquitin-proteasome system (UPS) gene expression was observed in XPA-deficient cells and the involvement of nuclear factor erythroid 2-related factor-2 (NFE2L2) was indicated. Co-immunoprecipitation assays showed the interaction between XPA, apurinic-apyrimidinic endonuclease 1 (APE1) and NFE2L2 proteins. Decreased NFE2L2 protein expression and proteasome activity was also observed in XPA-deficient cells. The data suggest the involvement of the growth arrest and DNA-damage-inducible beta (GADD45ß) in NFE2L2 functions. Similar results were obtained in xpa-1 (RNAi) Caenorhabditis elegans suggesting the conservation of XPA and NFE2L2 interactions. In conclusion, stress response activation occurs in XPA-deficient cells under oxidative stress; however, these cells fail to activate the UPS cytoprotective response, which may contribute to XPA patient's phenotypes.

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