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.

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.

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.

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.

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.

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.

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.

Small-molecule modulators of INAVA cytosolic condensate and cell-cell junction assemblies.

Epithelial cells lining mucosal surfaces distinctively express the inflammatory bowel disease risk gene INAVA. We previously found that INAVA has dual and competing functions: one at lateral membranes where it affects mucosal barrier function and the other in the cytosol where INAVA enhances IL-1ß signal transduction and protein ubiquitination and forms puncta. We now find that IL-1ß-induced INAVA puncta are biomolecular condensates that rapidly assemble and physiologically resolve. The condensates contain ubiquitin and the E3 ligase ßTrCP2, and their formation correlates with amplified ubiquitination, suggesting function in regulation of cellular proteostasis. Accordingly, a small-molecule screen identified ROS inducers, proteasome inhibitors, and inhibitors of the protein folding chaperone HSP90 as potent agonists for INAVA condensate formation. Notably, inhibitors of the p38α and mTOR pathways enhanced resolution of the condensates, and inhibitors of the Rho-ROCK pathway induced INAVA's competing function by recruiting INAVA to newly assembled intercellular junctions in cells where none existed before.

Hsf1 activation by proteotoxic stress requires concurrent protein synthesis.

Heat shock factor 1 (Hsf1) activation is responsible for increasing the abundance of protein-folding chaperones and degradation machinery in response to proteotoxic conditions that give rise to misfolded or aggregated proteins. Here we systematically explored the link between concurrent protein synthesis and proteotoxic stress in the budding yeast, Saccharomyces cerevisiae. Consistent with prior work, inhibiting protein synthesis before inducing proteotoxic stress prevents Hsf1 activation, which we demonstrated across a broad array of stresses and validate using orthogonal means of blocking protein synthesis. However, other stress-dependent transcription pathways remained activatable under conditions of translation inhibition. Titrating the protein denaturant ethanol to a higher concentration results in Hsf1 activation in the absence of translation, suggesting extreme protein-folding stress can induce proteotoxicity independent of protein synthesis. Furthermore, we demonstrate this connection under physiological conditions where protein synthesis occurs naturally at reduced rates. We find that disrupting the assembly or subcellular localization of newly synthesized proteins is sufficient to activate Hsf1. Thus, new proteins appear to be especially sensitive to proteotoxic conditions, and we propose that their aggregation may represent the bulk of the signal that activates Hsf1 in the wake of these insults.

The aminosterol Claramine inhibits ß-secretase 1-mediated insulin receptor cleavage.

The cleavage of the insulin receptor by ß-secretase 1 (BACE1) in the liver increases during diabetes, which contributes to reduce insulin receptor levels and impair insulin signaling. However, the precise signaling events that lead to this increased cleavage are unclear. We showed that BACE1 cleaves the insulin receptor in the early secretory pathway. Indeed, coimmunoprecipitation experiments reveal the interaction of the proforms of the two proteins. Moreover, fragments of insulin receptor are detected in the early secretory pathway and a mutated form of BACE1 that retains its prodomain cleaves an early secretory pathway-resident form of the insulin receptor. We showed that BACE1 proform levels are regulated by proteasome and/or lysosome-dependent degradation systems whose efficiencies are dependent on the O-GlcNacylation process. Our results showed that enhanced O-GlcNacylation reduces the efficiency of intracellular protein degradation systems, leading to the accumulation of the proform of BACE1 in the early secretory pathway where it cleaves the precursor of the insulin receptor. All these dysregulations are found in the livers of diabetic mice. In addition, we performed a screen of molecules according to their ability to increase levels of the insulin receptor at the surface of BACE1-overexpressing cells. This approach identified the aminosterol Claramine, which accelerated intracellular trafficking of the proform of BACE1 and increased autophagy. Both of these effects likely contribute to the reduced amount of the proform of BACE1 in the early secretory pathway, thereby reducing insulin receptor cleavage. These newly described properties of Claramine are consistent with its insulin sensitizing effect.

Proteases and Their Modulators in Cancer Therapy: Challenges and Opportunities.

Proteostasis is the process of regulating intracellular proteins to maintain the balance of the cell proteome, which is crucial for cancer cell survival. Several proteases located in the cytoplasm, mitochondria, lysosome, and extracellular environment have been identified as potential antitumor targets because of their involvement in proteostasis. Although the discovery of small-molecule inhibitors targeting proteases faces particular challenges, rapid advances in chemical biology and structural biology, and the new technology of drug discovery have facilitated the development of promising protease modulators. In this review, the protein structure and function of important tumor-related proteases and their inhibitors are presented. We also provide a prospective on advances and the outlook of new drug strategies that target these proteases.

Neurotoxicity and underlying cellular changes of 21 mitochondrial respiratory chain inhibitors.

Inhibition of complex I of the mitochondrial respiratory chain (cI) by rotenone and methyl-phenylpyridinium (MPP +) leads to the degeneration of dopaminergic neurons in man and rodents. To formally describe this mechanism of toxicity, an adverse outcome pathway (AOP:3) has been developed that implies that any inhibitor of cI, or possibly of other parts of the respiratory chain, would have the potential to trigger parkinsonian motor deficits. We used here 21 pesticides, all of which are described in the literature as mitochondrial inhibitors, to study the general applicability of AOP:3 or of in vitro assays that are assessing its activation. Five cI, three complex II (cII), and five complex III (cIII) inhibitors were characterized in detail in human dopaminergic neuronal cell cultures. The NeuriTox assay, examining neurite damage in LUHMES cells, was used as in vitro proxy of the adverse outcome (AO), i.e., of dopaminergic neurodegeneration. This test provided data on whether test compounds were unspecific cytotoxicants or specifically neurotoxic, and it yielded potency data with respect to neurite degeneration. The pesticide panel was also examined in assays for the sequential key events (KE) leading to the AO, i.e., mitochondrial respiratory chain inhibition, mitochondrial dysfunction, and disturbed proteostasis. Data from KE assays were compared to the NeuriTox data (AO). The cII-inhibitory pesticides tested here did not appear to trigger the AOP:3 at all. Some of the cI/cIII inhibitors showed a consistent AOP activation response in all assays, while others did not. In general, there was a clear hierarchy of assay sensitivity: changes of gene expression (biomarker of neuronal stress) correlated well with NeuriTox data; mitochondrial failure (measured both by a mitochondrial membrane potential-sensitive dye and a respirometric assay) was about 10-260 times more sensitive than neurite damage (AO); cI/cIII activity was sometimes affected at > 1000 times lower concentrations than the neurites. These data suggest that the use of AOP:3 for hazard assessment has a number of caveats: (i) specific parkinsonian neurodegeneration cannot be easily predicted from assays of mitochondrial dysfunction; (ii) deriving a point-of-departure for risk assessment from early KE assays may overestimate toxicant potency.

Proteotoxic stress is a driver of the loser status and cell competition.

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.

Small-molecule endoplasmic reticulum proteostasis regulator acts as a broad-spectrum inhibitor of dengue and Zika virus infections.

Flaviviruses, including dengue and Zika, are widespread human pathogens; however, no broadly active therapeutics exist to fight infection. Recently, remodeling of endoplasmic reticulum (ER) proteostasis by pharmacologic regulators, such as compound 147, was shown to correct pathologic ER imbalances associated with protein misfolding diseases. Here, we establish an additional activity of compound 147 as an effective host-centered antiviral agent against flaviviruses. Compound 147 reduces infection by attenuating the infectivity of secreted virions without causing toxicity in host cells. Compound 147 is a preferential activator of the ATF6 pathway of the ER unfolded protein response, which requires targeting of cysteine residues primarily on protein disulfide isomerases (PDIs). We find that the antiviral activity of 147 is independent of ATF6 induction but does require modification of reactive thiols on protein targets. Targeting PDIs and additional non-PDI targets using RNAi and other small-molecule inhibitors was unable to recapitulate the antiviral effects, suggesting a unique polypharmacology may mediate the activity. Importantly, 147 can impair infection of multiple strains of dengue and Zika virus, indicating that it is suitable as a broad-spectrum antiviral agent.

Bortezomib enhances cytotoxicity of ex vivo-expanded gamma delta T cells against acute myeloid leukemia and T-cell acute lymphoblastic leukemia.

Engagement between the natural killer group 2, member D (NKG2D) receptor and its ligands is one of the main mechanisms used by immune cells to target stressed cells for cell death. NKG2D ligands are known markers of cellular stress and are often upregulated on tumor cells. Certain drugs can further increase NKG2D ligand levels, thereby making tumor cells more susceptible to immune cell detection and destruction. However, the effectiveness of this approach appears to be limited with drug treatment alone, possibly due to immune dysregulation in the setting of malignancies. We hypothesized that a more effective approach would be a combination of NKG2D ligand-inducing drugs, such as the proteasome inhibitor bortezomib, and ex vivo-expanded peripheral blood γδ T cells (i.e., Vγ9Vδ2 T cells). Acute myeloid leukemia (AML) is a high-risk hematologic malignancy, and treatment has shown limited benefit with the addition of bortezomib to standard chemotherapy regimens. Two AML cells lines, Nomo-1 and Kasumi-1, were treated with increasing concentrations of bortezomib, and changes in NKG2D ligand expression were measured. Bortezomib treatment significantly increased expression of the NKG2D ligand UL16 binding protein (ULBP) 2/5/6 in both cell lines. Vγ9Vδ2 T cells were expanded and isolated from peripheral blood of healthy donors to generate a final cellular product with a mean of 96% CD3+/γδ T-cell receptor-positive cells. Combination treatment of the AML cell lines with γδ T cells and bortezomib resulted in significantly greater cytotoxicity than γδ T cells alone, even at lower effector-to-target ratios. Based on the positive results against AML and the generalizable mechanism of this combination approach, it was also tested against T-cell acute lymphoblastic leukemia (T-ALL), another high-risk leukemia. Similarly, bortezomib increased ULBP 2/5/6 expression in T-ALL cell lines, Jurkat and MOLT-4 and improved the cytotoxicity of γδ T cells against each line. Collectively, these results show that bortezomib enhances γδ T-cell-mediated killing of both AML and T-ALL cells in part through increased NKG2D ligand-receptor interaction. Furthermore, proof-of-concept for the combination of ex vivo-expanded γδ T cells with stress ligand-inducing drugs as a therapeutic platform for high-risk leukemias is demonstrated.

BAG3 Proteomic Signature under Proteostasis Stress.

The multifunctional HSP70 co-chaperone BAG3 (BCL-2-associated athanogene 3) represents a key player in the quality control of the cellular proteostasis network. In response to stress, BAG3 specifically targets aggregation-prone proteins to the perinuclear aggresome and promotes their degradation via BAG3-mediated selective macroautophagy. To adapt cellular homeostasis to stress, BAG3 modulates and functions in various cellular processes and signaling pathways. Noteworthy, dysfunction and deregulation of BAG3 and its pathway are pathophysiologically linked to myopathies, cancer, and neurodegenerative disorders. Here, we report a BAG3 proteomic signature under proteostasis stress. To elucidate the dynamic and multifunctional action of BAG3 in response to stress, we established BAG3 interactomes under basal and proteostasis stress conditions by employing affinity purification combined with quantitative mass spectrometry. In addition to the identification of novel potential BAG3 interactors, we defined proteins whose interaction with BAG3 was altered upon stress. By functional annotation and protein-protein interaction enrichment analysis of the identified potential BAG3 interactors, we confirmed the multifunctionality of BAG3 and highlighted its crucial role in diverse cellular signaling pathways and processes, ensuring cellular proteostasis and cell viability. These include protein folding and degradation, gene expression, cytoskeleton dynamics (including cell cycle and transport), as well as granulostasis, in particular.