Eukaryotic Initiation Translation Factor 2A activation by cannabidiolic acid alters the protein homeostasis balance in glioblastoma cells.

Eukaryotic Initiation Translation Factor 2A (EIF2A) is considered to be primarily responsible for the initiation of translation when a cell is subjected to stressful conditions. However, information regarding this protein is still incomplete. Using a combination of proteomic approaches, we demonstrated that EIF2A is the molecular target of the naturally occurring bioactive compound cannabidiolic acid (CBDA) within human glioblastoma cells. This finding allowed us to undertake a study aimed at obtaining further information on the functions that EIF2A plays in tumor cells. Indeed, our data showed that CBDA is able to activate EIF2A when the cells are in no-stress conditions. It induces conformational changes in the protein structure, thus increasing EIF2A affinity towards the proteins participating in the Eukaryotic Translation Machinery. Consequently, following glioblastoma cells incubation with CBDA we observed an enhanced neosynthesis of proteins involved in the stress response, nucleic acid translation and organization, and protein catabolism. These changes in gene expression resulted in increased levels of ubiquitinated proteins and accumulation of the autophagosome. Our results, in addition to shedding light on the molecular mechanism underlying the biological effect of a phytocannabinoid in cancer cells, demonstrated that EIF2A plays a critical role in regulation of protein homeostasis.

A Metabolomic Approach to Investigate the Effect of Phytonutrients on Proteostasis and Metabolic Pathways in Drosophila melanogaster.

The use of Drosophila melanogaster as a biological platform to study the effect of diet and food bioactives on the metabolome remains a highly unexplored subject. Aiming to establish alternative solutions for the investigation of nutritional interventions with bioactive natural products by employing LC-MS-based metabolomics approaches, we assessed the effect of a phytonutrient-rich extract from the endemic Mediterranean plant Cichorium spinosum (stamnagkàthi) on a Drosophila population. The extract's modulating effect on the proteostasis network and metabolism of young D. melanogaster flies was evaluated. Furthermore, an untargeted metabolomics approach, employing a C18 UPLC-ESI-Orbitrap-HRMS/MS platform, permitted the detection of several biomarkers in the metabolic profile of Drosophila's tissues; while targeted amino acid quantification in Drosophila tissue was simultaneously performed by employing aTRAQ labeling and an ion-pairing UPLC-ESI-SWATH-HRMS/MS platform. The detected metabolites belong to different chemical classes, and statistical analysis with chemometrics tools was utilized to reveal patterns and trends, as well as to uncover potential class-distinguishing features and possible biomarkers. Our findings suggest that Drosophila can serve as a valuable in vivo model for investigating the role of bioactive phytoconstituents, like those found in C. spinosum's decoction, on diverse metabolic processes. Additionally, the fruit fly represents a highly effective platform to investigate the molecular mechanisms underlying sex differences in diverse aspects of nutrition and physiology in higher metazoans.

The role of labile iron on brain proteostasis; could it be an early event of neurodegenerative disease?

Iron deposits in the brain are a natural consequence of aging. Iron accumulation, especially in the form of labile iron, can trigger a cascade of adverse effects, eventually leading to neurodegeneration and cognitive decline. Aging also increases the dysfunction of cellular proteostasis. The question of whether iron alters proteostasis is now being pondered. Herein, we investigated the effect of ferric citrate, considered as labile iron, on various aspects of proteostasis of neuronal cell lines, and also established an animal model having a labile iron diet in order to evaluate proteostasis alteration in the brain along with behavioral effects. According to an in vitro study, labile iron was found to activate lysosome formation but inhibits lysosomal clearance function. Furthermore, the presence of labile iron can alter autophagic flux and can also induce the accumulation of protein aggregates. RNA-sequencing analysis further reveals the upregulation of various terms related to proteostasis along with neurodegenerative disease-related terms. According to an in vivo study, a labile iron-rich diet does not induce iron overload conditions and was not detrimental to the behavior of male Wistar rats. However, an iron-rich diet can promote iron accumulation in a region-dependent manner. By staining for autophagic markers and misfolding proteins in the cerebral cortex and hippocampus, an iron-rich diet was actually found to alter autophagy and induce an accumulation of misfolding proteins. These findings emphasize the importance of labile iron on brain cell proteostasis, which could be implicated in developing of neurological diseases.

Modulating ß-catenin homeostasis for cancer therapy.

ß-Catenin is a well-established driver of many cancers; however, there are challenges in developing agents targeting ß-catenin for clinical use. Recent progress has indicated that most of the pathological changes in ß-catenin may be commonly caused by loss of protein homeostasis. Modulation of ß-catenin homeostasis, especially by hyperactivation of ß-catenin, potentially leads to robust antitumor outcomes. Here, we comprehensively dissect the protein homeostasis of ß-catenin in terms of time, compartmentalization, supramolecular assemblies, and dynamics, with emphasis on changes in ß-catenin homeostasis upon oncogenic mutations. We propose that altered ß-catenin homeostasis could be deleterious for ß-catenin-dependent cancers and that modulation of ß-catenin homeostasis offers a novel avenue for targeting ß-catenin for cancer therapy.

Dendrobium officinale phenolic extract maintains proteostasis by regulating autophagy in a Caenorhabditis elegans model of Alzheimer's disease.

Alzheimer's disease (AD) is a progressive neurodegenerative disease, and accumulating evidence suggested that proteostatic imbalance is a key feature of the disease. Traditional Chinese medicine exhibits a multi-target therapeutic effect, making it highly suitable for addressing protein homeostasis imbalance in AD. Dendrobium officinale is a traditional Chinese herbs commonly used as tonic agent in China. In this study, we investigated protection effects of D. officinale phenolic extract (SH-F) and examined its underlying mechanisms by using transgenic Caenorhabditis elegans models. We found that treatment with SH-F (50 µg/mL) alleviated Aß and tau protein toxicity in worms, and also reduced aggregation of polyglutamine proteins to help maintain proteostasis. RNA sequencing results showed that SH-F treatment significantly affected the proteolytic process and autophagy-lysosomal pathway. Furthermore, we confirmed that SH-F showing maintainance of proteostasis was dependent on bec-1 by qRT-PCR analysis and RNAi methods. Finally, we identified active components of SH-F by LC-MS method, and found the five major compounds including koaburaside, tyramine dihydroferulate, N-p-trans-coumaroyltyramine, naringenin and isolariciresinol are the main bioactive components responsible for the anti-AD activity of SH-F. Our findings provide new insights to develop a treatment strategy for AD by targeting proteostasis, and SH-F could be an alternative drug for the treatment of AD.

Fisetin alleviates cerebral ischemia/reperfusion injury by regulating Sirt1/Foxc1/Ubqln1 pathway-mediated proteostasis.

BACKGROUND: Cerebral ischemia/reperfusion injury (IRI) is pathologically associated with protein damage. The flavonoid fisetin has good therapeutic effects on cerebral IRI. However, the role of fisetin in regulating protein damage during cerebral IRI development remains unclear. This study investigated the pharmacological effects of fisetin on protein damage during cerebral IRI progression and defined the underlying mechanism of action. METHODS: In vivo and in vitro models of cerebral IRI were established by middle cerebral artery occlusion/reperfusion (MACO/R) and oxygen-glucose deprivation/reperfusion (OGD/R) treatment, respectively. Triphenyl tetrazolium chloride staining was performed to detect cerebral infarct size, and the modified neurologic severity score was used to examine neurological deficits. LDH activity and protein damage were assessed using kits. HT22 cell vitality and apoptosis were examined using CCK-8 assay and TUNEL staining, respectively. Interactions between Foxc1, Ubqln1, Sirt1, and Ezh2 were analyzed using CoIP, ChIP and/or dual-luciferase reporter gene assays. RESULTS: Fisetin alleviated protein damage and ubiquitinated protein aggregation and neuronal death caused by MCAO/R and OGD/R. Ubqln1 knockdown abrogated the inhibitory effect of fisetin on OGD/R-induced protein damage, ubiquitinated protein aggregation, and neuronal death in HT22 cells. Further experiments demonstrated that Foxc1 functions as a transcriptional activator of Ubqln1 and that Sirt1 promotes Foxc1 expression by deacetylating Ezh2 and inhibiting its activity. Furthermore, Sirt1 knockdown abrogated fisetin-mediated biological effects on OGD/R-treated HT22 cells. CONCLUSION: Fisetin improved proteostasis during cerebral IRI by regulating the Sirt1/Foxc1/Ubqln1 signaling axis. Our findings strongly suggest that fisetin-mediated inhibition of protein damage after ischemic stroke is a part of the mechanism through which fisetin is neuroprotective in cerebral IRI.

Elexacaftor/VX-445-mediated CFTR interactome remodeling reveals differential correction driven by mutation-specific translational dynamics.

Cystic fibrosis (CF) is one of the most prevalent lethal genetic diseases with over 2000 identified mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. Pharmacological chaperones such as lumacaftor (VX-809), tezacaftor (VX-661), and elexacaftor (VX-445) treat mutation-induced defects by stabilizing CFTR and are called correctors. These correctors improve proper folding and thus facilitate processing and trafficking to increase the amount of functional CFTR on the cell surface. Yet, CFTR variants display differential responses to each corrector. Here, we report that variants P67L and L206W respond similarly to VX-809 but divergently to VX-445 with P67L exhibiting little rescue when treated with VX-445. We investigate the underlying cellular mechanisms of how CFTR biogenesis is altered by correctors in these variants. Affinity purification-mass spectrometry multiplexed with isobaric tandem mass tags was used to quantify CFTR protein-protein interaction changes between variants P67L and L206W. VX-445 facilitates unique proteostasis factor interactions especially in translation, folding, and degradation pathways in a CFTR variant-dependent manner. A number of these interacting proteins knocked down by siRNA, such as ribosomal subunit proteins, moderately rescued fully glycosylated P67L. Importantly, these knockdowns sensitize P67L to VX-445 and further enhance the trafficking correction of this variant. Partial inhibition of protein translation also mildly sensitizes P67L CFTR to VX-445 correction, supporting a role for translational dynamics in the rescue mechanism of VX-445. Our results provide a better understanding of VX-445 biological mechanism of action and reveal cellular targets that may sensitize nonresponsive CFTR variants to known and available correctors.

Proteostasis Regulators in Cystic Fibrosis: Current Development and Future Perspectives.

In cystic fibrosis (CF), the deletion of phenylalanine 508 (F508del) in the CF transmembrane conductance regulator (CFTR) leads to misfolding and premature degradation of the mutant protein. These defects can be targeted with pharmacological agents named potentiators and correctors. During the past years, several efforts have been devoted to develop and approve new effective molecules. However, their clinical use remains limited, as they fail to fully restore F508del-CFTR biological function. Indeed, the search for CFTR correctors with different and additive mechanisms has recently increased. Among them, drugs that modulate the CFTR proteostasis environment are particularly attractive to enhance therapy effectiveness further. This Perspective focuses on reviewing the recent progress in discovering CFTR proteostasis regulators, mainly describing the design, chemical structure, and structure-activity relationships. The opportunities, challenges, and future directions in this emerging and promising field of research are discussed, as well.

ISRIB plus bortezomib triggers paraptosis in breast cancer cells via enhanced translation and subsequent proteotoxic stress.

Despite the success of proteasome inhibitors (PIs) in treating hematopoietic malignancies, including multiple myeloma (MM), their clinical efficacy is limited in solid tumors. In this study, we investigated the involvement of the integrated stress response (ISR), a central cellular adaptive program that responds to proteostatic defects by tuning protein synthesis rates, in determining the fates of cells treated with PI, bortezomib (Bz). We found that Bz induces ISR, and this can be reversed by ISRIB, a small molecule that restores eIF2B-mediated translation during ISR, in both Bz-sensitive MM cells and Bz-insensitive breast cancer cells. Interestingly, while ISRIB protected MM cells from Bz-induced apoptosis, it enhanced Bz sensitivity in breast cancer cells by inducing paraptosis, the cell death mode that is accompanied by dilation of the endoplasmic reticulum (ER) and mitochondria. Combined treatment with ISRIB and Bz may shift the fate of Bz-insensitive cancer cells toward paraptosis by inducing translational rescue, leading to irresolvable proteotoxic stress.

Identifying Interaction Partners of Yeast Protein Disulfide Isomerases Using a Small Thiol-Reactive Cross-Linker: Implications for Secretory Pathway Proteostasis.

Protein disulfide isomerases (PDIs) function in forming the correct disulfide bonds in client proteins, thereby aiding the folding of proteins that enter the secretory pathway. Recently, several PDIs have been identified as targets of organic electrophiles, yet the client proteins of specific PDIs remain largely undefined. Here, we report that PDIs expressed in Saccharomyces cerevisiae are targets of divinyl sulfone (DVSF) and other thiol-reactive protein cross-linkers. Using DVSF, we identified the interaction partners that were cross-linked to Pdi1 and Eug1, finding that both proteins form cross-linked complexes with other PDIs, as well as vacuolar hydrolases, proteins involved in cell wall biosynthesis and maintenance, and many ER proteostasis factors involved ER stress signaling and ER-associated protein degradation (ERAD). The latter discovery prompted us to examine the effects of DVSF on ER quality control, where we found that DVSF inhibits the degradation of the ERAD substrate CPY*, in addition to covalently modifying Ire1 and blocking the activation of the unfolded protein response. Our results reveal that DVSF targets many proteins within the ER proteostasis network and suggest that these proteins may be suitable targets for covalent therapeutic development in the future.

Anakinra restores cellular proteostasis by coupling mitochondrial redox balance to autophagy.

Autophagy selectively degrades aggregation-prone misfolded proteins caused by defective cellular proteostasis. However, the complexity of autophagy may prevent the full appreciation of how its modulation could be used as a therapeutic strategy in disease management. Here, we define a molecular pathway through which recombinant IL-1 receptor antagonist (IL-1Ra, anakinra) affects cellular proteostasis independently from the IL-1 receptor (IL-1R1). Anakinra promoted H2O2-driven autophagy through a xenobiotic sensing pathway involving the aryl hydrocarbon receptor that, activated through the indoleamine 2,3-dioxygenase 1-kynurenine pathway, transcriptionally activated NADPH oxidase 4 independent of the IL-1R1. By coupling the mitochondrial redox balance to autophagy, anakinra improved the dysregulated proteostasis network in murine and human cystic fibrosis. We anticipate that anakinra may represent a therapeutic option in addition to its IL-1R1-dependent antiinflammatory properties by acting at the intersection of mitochondrial oxidative stress and autophagy with the capacity to restore conditions in which defective proteostasis leads to human disease.

An update on the unfolded protein response in brain ischemia: Experimental evidence and therapeutic opportunities.

After ischemic stroke or cardiac arrest, brain ischemia occurs. Currently, no pharmacologic intervention that targets cellular processes has proven effective in improving neurologic outcome in patients after brain ischemia. Recent experimental research has identified the crucial role of proteostasis in survival and recovery of cells after ischemia. In particular, the unfolded protein response (UPR), a key signaling pathway that safeguards cellular proteostasis, is emerging as a promising therapeutic target for brain ischemia. For some time, the UPR has been known to play a critical role in the pathophysiology of brain ischemia; however, only in the recent years has the field grown substantially, largely due to the extensive use of UPR-specific mouse genetic models and the rapidly expanding availability of pharmacologic tools that target the UPR. In this review, we provide a timely update on the progress in our understanding of the UPR in experimental brain ischemia, and discuss the therapeutic implications of targeting the UPR in ischemic stroke and cardiac arrest.

Erythemal and vitamin D weighted solar UV dose-rates and doses estimated from measurements in mainland France and on Réunion Island.

Solar UV radiation causes beneficial and detrimental changes in human health. International and national Health agencies recommend avoiding sun exposure when the solar rays are strongest (typically 2 h before and after solar noon). In this study we detail and refine such recommendations. We estimated biologically-effective radiation (inductive of erythema and pre-vitamin D) using spectral solar UV radiation measurements on a horizontal plane at three French sites equipped with spectroradiometers: Villeneuve d'Ascq (VDA) (North of France); Observatoire de Haute-Provence (OHP) (French Southern Alps); and Saint-Denis de La Réunion (SDR) on Réunion Island, in the Indian Ocean. These sites are very different: VDA is a semi-urban site in a flat region, OHP a rural mountainous site and SDR a coastal urban site on a small mountainous island. Biologically active radiation was analyzed by studying erythema induction and measuring pre-vitamin D synthesis. Dose-rates, doses and times for sunburn induction and vitamin D production were derived. Regarding the level of vitamin D dose considered here (1000 IU), we found that at mainland sites time required for vitamin D synthesis was relatively long, even around solar noon, in winter months this could be 2-3 h for phototype II individuals exposing their face and hands. In the tropics vitamin D could always be synthesized in a reasonable time (e.g. 20 min in winter). By contrast, in summer, the required duration times (exposing face, hands, arms and legs) are very short, approximately 2-4 min on the mainland and 1 min in the tropics for phototype II individuals. In all skin phototypes the duration of sun exposure required to induce erythema was generally longer than that to produce vitamin D. These quantitative results, obtained using an instrument measuring on a horizontal plane and with an unobstructed view, do not represent realistic values for human exposure. To account for realistic human body exposure, received doses and times of exposure were adjusted. Our study shows that, mostly in summer, the time periods where limited solar exposure is recommended should be extended, especially at low latitude locations.

Proteasome Based Molecular Strategies Against Improper Cellular Proliferation.

Cells contain several proteins that routinely fulfill multiple requirements for normal physiological survival. Proteostasis dysfunction is linked with different complex human disorders, like cancer, neuron degeneration, and imperfect aging. The ubiquitin proteasome system (UPS) forms the primary proteostasis mechanism taking part in cytoprotection. Cancer cells are well known to possess enhanced cytoprotective properties, and different UPS elements are implicated to be dysregulated at several stages of tumor progression. Furthermore, many studies have found tumor cells to exhibit higher levels of various UPS components, possibly contributing to their robust endurance. In this article, we have presented different cellular protein quality control strategies, essential for maintaining healthy proteome. Here, we have also discussed key contributions and functions of UPS involved in molecular pathomechanisms for establishing cancer conditions. Along with this, the emerging different therapeutic strategies against defective proteome linked with improper cellular proliferation and cancer progression are also reviewed. UPS performs critical regulatory functions in modulating the cellular apoptotic pathways. The proteasomal system involvement as probable therapeutic targets influencing cancer cell apoptosis is also discussed. Our article summarizes the recent developments in proteasome-associated pathways regulating tumor cell proteome and survival. Additionally, how the engagement and cross functions of these physiological processes can induce apoptosis and may develop regulation over tumor progression. A better understanding of multifaceted protein quality control pathways may inform therapeutic interventions based on cellular proteostasis response determined against complex diseases.

Inhibitor Combinations Reveal Wiring of the Proteostasis Network in Prostate Cancer Cells.

The protein homeostasis (proteostasis) network is composed of multiple pathways that work together to balance protein folding, stability, and turnover. Cancer cells are particularly reliant on this network; however, it is hypothesized that inhibition of one node might lead to compensation. To better understand these connections, we dosed 22Rv1 prostate cancer cells with inhibitors of four proteostasis targets (Hsp70, Hsp90, proteasome, and p97), either alone or in binary combinations, and measured the effects on cell growth. The results reveal a series of additive, synergistic, and antagonistic relationships, including strong synergy between inhibitors of p97 and the proteasome and striking antagonism between inhibitors of Hsp90 and the proteasome. Based on RNA-seq, these relationships are associated, in part, with activation of stress pathways. Together, these results suggest that cocktails of proteostasis inhibitors might be a powerful way of treating some cancers, although antagonism that blunts the efficacy of both molecules is also possible.

Mitochondria-affecting small molecules ameliorate proteostasis defects associated with neurodegenerative diseases.

Macroautophagic recycling of dysfunctional mitochondria, known as mitophagy, is essential for mitochondrial homeostasis and cell viability. Accumulation of defective mitochondria and impaired mitophagy have been widely implicated in many neurodegenerative diseases, and loss-of-function mutations of PINK1 and Parkin, two key regulators of mitophagy, are amongst the most common causes of heritable parkinsonism. This has led to the hypothesis that pharmacological stimulation of mitophagy may be a feasible approach to combat neurodegeneration. Toward this end, we screened ~ 45,000 small molecules using a high-throughput, whole-organism, phenotypic screen that monitored accumulation of PINK-1 protein, a key event in mitophagic activation, in a Caenorhabditis elegans strain carrying a Ppink-1::PINK-1::GFP reporter. We obtained eight hits that increased mitochondrial fragmentation and autophagosome formation. Several of the compounds also reduced ATP production, oxygen consumption, mitochondrial mass, and/or mitochondrial membrane potential. Importantly, we found that treatment with two compounds, which we named PS83 and PS106 (more commonly known as sertraline) reduced neurodegenerative disease phenotypes, including delaying paralysis in a C. elegans ß-amyloid aggregation model in a PINK-1-dependent manner. This report presents a promising step toward the identification of compounds that will stimulate mitochondrial turnover.

Citrulline, Biomarker of Enterocyte Functional Mass and Dietary Supplement. Metabolism, Transport, and Current Evidence for Clinical Use.

L-Citrulline is a non-essential but still important amino acid that is released from enterocytes. Because plasma levels are reduced in case of impaired intestinal function, it has become a biomarker to monitor intestinal integrity. Moreover, oxidative stress induces protein citrullination, and antibodies against anti-citrullinated proteins are useful to monitor rheumatoid diseases. Citrullinated histones, however, may even predict a worse outcome in cancer patients. Supplementation of citrulline is better tolerated compared to arginine and might be useful to slightly improve muscle strength or protein balance. The following article shall provide an overview of L-citrulline properties and functions, as well as the current evidence for its use as a biomarker or as a therapeutic supplement.

A heat-shock response regulated by the PfAP2-HS transcription factor protects human malaria parasites from febrile temperatures.

Periodic fever is a characteristic clinical feature of human malaria, but how parasites survive febrile episodes is not known. Although the genomes of Plasmodium species encode a full set of chaperones, they lack the conserved eukaryotic transcription factor HSF1, which activates the expression of chaperones following heat shock. Here, we show that PfAP2-HS, a transcription factor in the ApiAP2 family, regulates the protective heat-shock response in Plasmodium falciparum. PfAP2-HS activates the transcription of hsp70-1 and hsp90 at elevated temperatures. The main binding site of PfAP2-HS in the entire genome coincides with a tandem G-box DNA motif in the hsp70-1 promoter. Engineered parasites lacking PfAP2-HS have reduced heat-shock survival and severe growth defects at 37 °C but not at 35 °C. Parasites lacking PfAP2-HS also have increased sensitivity to imbalances in protein homeostasis (proteostasis) produced by artemisinin, the frontline antimalarial drug, or the proteasome inhibitor epoxomicin. We propose that PfAP2-HS contributes to the maintenance of proteostasis under basal conditions and upregulates specific chaperone-encoding genes at febrile temperatures to protect the parasite against protein damage.

Therapeutic Approaches Targeting Proteostasis in Kidney Disease and Fibrosis.

Pathological insults usually disturb the folding capacity of cellular proteins and lead to the accumulation of misfolded proteins in the endoplasmic reticulum (ER), which leads to so-called "ER stress". Increasing evidence indicates that ER stress acts as a trigger factor for the development and progression of many kidney diseases. The unfolded protein responses (UPRs), a set of molecular signals that resume proteostasis under ER stress, are thought to restore the adaptive process in chronic kidney disease (CKD) and renal fibrosis. Furthermore, the idea of targeting UPRs for CKD treatment has been well discussed in the past decade. This review summarizes the up-to-date literature regarding studies on the relationship between the UPRs, systemic fibrosis, and renal diseases. We also address the potential therapeutic possibilities of renal diseases based on the modulation of UPRs and ER proteostasis. Finally, we list some of the current UPR modulators and their therapeutic potentials.

Nuclear envelope budding is a response to cellular stress.

Nuclear envelope budding (NEB) is a recently discovered alternative pathway for nucleocytoplasmic communication distinct from the movement of material through the nuclear pore complex. Through quantitative electron microscopy and tomography, we demonstrate how NEB is evolutionarily conserved from early protists to human cells. In the yeast Saccharomyces cerevisiae, NEB events occur with higher frequency during heat shock, upon exposure to arsenite or hydrogen peroxide, and when the proteasome is inhibited. Yeast cells treated with azetidine-2-carboxylic acid, a proline analog that induces protein misfolding, display the most dramatic increase in NEB, suggesting a causal link to protein quality control. This link was further supported by both localization of ubiquitin and Hsp104 to protein aggregates and NEB events, and the evolution of these structures during heat shock. We hypothesize that NEB is part of normal cellular physiology in a vast range of species and that in S. cerevisiae NEB comprises a stress response aiding the transport of protein aggregates across the nuclear envelope.