Nuclear receptor signaling via NHR-49/MDT-15 regulates stress resilience and proteostasis in response to reproductive and metabolic cues.

The ability to sense and respond to proteotoxic insults declines with age, leaving cells vulnerable to chronic and acute stressors. Reproductive cues modulate this decline in cellular proteostasis to influence organismal stress resilience in Caenorhabditis elegans We previously uncovered a pathway that links the integrity of developing embryos to somatic health in reproductive adults. Here, we show that the nuclear receptor NHR-49, an ortholog of mammalian peroxisome proliferator-activated receptor α (PPARα), regulates stress resilience and proteostasis downstream from embryo integrity and other pathways that influence lipid homeostasis and upstream of HSF-1. Disruption of the vitelline layer of the embryo envelope, which activates a proteostasis-enhancing intertissue pathway in somatic cells, triggers changes in lipid catabolism gene expression that are accompanied by an increase in fat stores. NHR-49, together with its coactivator, MDT-15, contributes to this remodeling of lipid metabolism and is also important for the elevated stress resilience mediated by inhibition of the embryonic vitelline layer. Our findings indicate that NHR-49 also contributes to stress resilience in other pathways known to change lipid homeostasis, including reduced insulin-like signaling and fasting, and that increased NHR-49 activity is sufficient to improve proteostasis and stress resilience in an HSF-1-dependent manner. Together, our results establish NHR-49 as a key regulator that links lipid homeostasis and cellular resilience to proteotoxic stress.

Nuclear receptor signaling via NHR-49/MDT-15 regulates stress resilience and proteostasis in response to reproductive and metabolic cues.

The ability to sense and respond to proteotoxic insults declines with age, leaving cells vulnerable to chronic and acute stressors. Reproductive cues modulate this decline in cellular proteostasis to influence organismal stress resilience in C. elegans. We previously uncovered a pathway that links the integrity of developing embryos to somatic health in reproductive adults. Here, we show that the nuclear receptor NHR-49, a functional homolog of mammalian peroxisome proliferator-activated receptor alpha (PPARα), regulates stress resilience and proteostasis downstream of embryo integrity and other pathways that influence lipid homeostasis, and upstream of HSF-1. Disruption of the vitelline layer of the embryo envelope, which activates a proteostasis-enhancing inter-tissue pathway in somatic tissues, also triggers changes in lipid catabolism gene expression that are accompanied by an increase in fat stores. NHR-49 together with its co-activator MDT-15 contributes to this remodeling of lipid metabolism and is also important for the elevated stress resilience mediated by inhibition of the embryonic vitelline layer as well as by other pathways known to change lipid homeostasis, including reduced insulin-like signaling and fasting. Further, we show that increased NHR-49 activity is sufficient to suppress polyglutamine aggregation and improve stress resilience in an HSF-1-dependent manner. Together, our results establish NHR-49 as a key regulator that links lipid homeostasis and cellular resilience to proteotoxic stress.

Protecting the future: balancing proteostasis for reproduction.

The proteostasis network (PN) regulates protein synthesis, folding, and degradation and is critical for the health and function of all cells. The PN has been extensively studied in the context of aging and age-related diseases, and loss of proteostasis is regarded as a major contributor to many age-associated disorders. In contrast to somatic tissues, an important feature of germ cells is their ability to maintain a healthy proteome across generations. Accumulating evidence has now revealed multiple layers of PN regulation that support germ cell function, determine reproductive capacity during aging, and prioritize reproduction at the expense of somatic health. Here, we review recent insights into these different modes of regulation and their implications for reproductive and somatic aging.

Variants in ATP6V0A1 cause progressive myoclonus epilepsy and developmental and epileptic encephalopathy.

The vacuolar H+-ATPase is a large multi-subunit proton pump, composed of an integral membrane V0 domain, involved in proton translocation, and a peripheral V1 domain, catalysing ATP hydrolysis. This complex is widely distributed on the membrane of various subcellular organelles, such as endosomes and lysosomes, and plays a critical role in cellular processes ranging from autophagy to protein trafficking and endocytosis. Variants in ATP6V0A1, the brain-enriched isoform in the V0 domain, have been recently associated with developmental delay and epilepsy in four individuals. Here, we identified 17 individuals from 14 unrelated families with both with new and previously characterized variants in this gene, representing the largest cohort to date. Five affected subjects with biallelic variants in this gene presented with a phenotype of early-onset progressive myoclonus epilepsy with ataxia, while 12 individuals carried de novo missense variants and showed severe developmental and epileptic encephalopathy. The R740Q mutation, which alone accounts for almost 50% of the mutations identified among our cases, leads to failure of lysosomal hydrolysis by directly impairing acidification of the endolysosomal compartment, causing autophagic dysfunction and severe developmental defect in Caenorhabditis elegans. Altogether, our findings further expand the neurological phenotype associated with variants in this gene and provide a direct link with endolysosomal acidification in the pathophysiology of ATP6V0A1-related conditions.

ClpXP-mediated Degradation of the TAC Antitoxin is Neutralized by the SecB-like Chaperone in Mycobacterium tuberculosis.

Bacterial toxin-antitoxin (TA) systems are composed of a deleterious toxin and its antagonistic antitoxin. They are widespread in bacterial genomes and mobile genetic elements, and their functions remain largely unknown. Some TA systems, known as TAC modules, include a cognate SecB-like chaperone that assists the antitoxin in toxin inhibition. Here, we have investigated the involvement of proteases in the activation cycle of the TAC system of the human pathogen Mycobacterium tuberculosis. We show that the deletion of endogenous AAA+ proteases significantly bypasses the need for a dedicated chaperone and identify the mycobacterial ClpXP1P2 complex as the main protease involved in TAC antitoxin degradation. In addition, we show that the ClpXP1P2 degron is located at the extreme C-terminal end of the chaperone addiction (ChAD) region of the antitoxin, demonstrating that ChAD functions as a hub for both chaperone binding and recognition by proteases.

Embryo integrity regulates maternal proteostasis and stress resilience.

The proteostasis network is regulated by transcellular communication to promote health and fitness in metazoans. In Caenorhabditis elegans, signals from the germline initiate the decline of proteostasis and repression of cell stress responses at reproductive maturity, indicating that commitment to reproduction is detrimental to somatic health. Here we show that proteostasis and stress resilience are also regulated by embryo-to-mother communication in reproductive adults. To identify genes that act directly in the reproductive system to regulate somatic proteostasis, we performed a tissue targeted genetic screen for germline modifiers of polyglutamine aggregation in muscle cells. We found that inhibiting the formation of the extracellular vitelline layer of the fertilized embryo inside the uterus suppresses aggregation, improves stress resilience in an HSF-1-dependent manner, and restores the heat-shock response in the somatic tissues of the parent. This pathway relies on DAF-16/FOXO activation in vulval tissues to maintain stress resilience in the mother, suggesting that the integrity of the embryo is monitored by the vulva to detect damage and initiate an organismal protective response. Our findings reveal a previously undescribed transcellular pathway that links the integrity of the developing progeny to proteostasis regulation in the parent.

Publisher Correction: Structural insights into chaperone addiction of toxin-antitoxin systems.

The original version of this Article contained errors in Figures 1 and 4. In Fig. 1b, the Mtb-SecBTA sequence was displayed incorrectly. In the inset panel within Fig. 4c, the y-axis of the graph incorrectly read (Q.Rg)2 × I(Q)//(0), and should have read (Q.Rg)2 × I(Q)/I(0). These errors have been corrected in both the PDF and HTML versions of the Article.

Structural insights into chaperone addiction of toxin-antitoxin systems.

SecB chaperones assist protein export by binding both unfolded proteins and the SecA motor. Certain SecB homologs can also control toxin-antitoxin (TA) systems known to modulate bacterial growth in response to stress. In such TA-chaperone (TAC) systems, SecB assists the folding and prevents degradation of the antitoxin, thus facilitating toxin inhibition. Chaperone dependency is conferred by a C-terminal extension in the antitoxin known as chaperone addiction (ChAD) sequence, which makes the antitoxin aggregation-prone and prevents toxin inhibition. Using TAC of Mycobacterium tuberculosis, we present the structure of a SecB-like chaperone bound to its ChAD peptide. We find differences in the binding interfaces when compared to SecB-SecA or SecB-preprotein complexes, and show that the antitoxin can reach a functional form while bound to the chaperone. This work reveals how chaperones can use discrete surface binding regions to accommodate different clients or partners and thereby expand their substrate repertoire and functions.

An NAD+ Phosphorylase Toxin Triggers Mycobacterium tuberculosis Cell Death.

Toxin-antitoxin (TA) systems regulate fundamental cellular processes in bacteria and represent potential therapeutic targets. We report a new RES-Xre TA system in multiple human pathogens, including Mycobacterium tuberculosis. The toxin, MbcT, is bactericidal unless neutralized by its antitoxin MbcA. To investigate the mechanism, we solved the 1.8 Å-resolution crystal structure of the MbcTA complex. We found that MbcT resembles secreted NAD+-dependent bacterial exotoxins, such as diphtheria toxin. Indeed, MbcT catalyzes NAD+ degradation in vitro and in vivo. Unexpectedly, the reaction is stimulated by inorganic phosphate, and our data reveal that MbcT is a NAD+ phosphorylase. In the absence of MbcA, MbcT triggers rapid M. tuberculosis cell death, which reduces mycobacterial survival in macrophages and prolongs the survival of infected mice. Our study expands the molecular activities employed by bacterial TA modules and uncovers a new class of enzymes that could be exploited to treat tuberculosis and other infectious diseases.

Directed evolution of SecB chaperones toward toxin-antitoxin systems.

SecB chaperones assist protein export in bacteria. However, certain SecB family members have diverged to become specialized toward the control of toxin-antitoxin (TA) systems known to promote bacterial adaptation to stress and persistence. In such tripartite TA-chaperone (TAC) systems, the chaperone was shown to assist folding and to prevent degradation of its cognate antitoxin, thus facilitating inhibition of the toxin. Here, we used both the export chaperone SecB of Escherichia coli and the tripartite TAC system of Mycobacterium tuberculosis as a model to investigate how generic chaperones can specialize toward the control of TA systems. Through directed evolution of SecB, we have identified and characterized mutations that specifically improve the ability of SecB to control our model TA system without affecting its function in protein export. Such a remarkable plasticity of SecB chaperone function suggests that its substrate binding surface can be readily remodeled to accommodate specific clients.

Shaping proteostasis at the cellular, tissue, and organismal level.

The proteostasis network (PN) regulates protein synthesis, folding, transport, and degradation to maintain proteome integrity and limit the accumulation of protein aggregates, a hallmark of aging and degenerative diseases. In multicellular organisms, the PN is regulated at the cellular, tissue, and systemic level to ensure organismal health and longevity. Here we review these three layers of PN regulation and examine how they collectively maintain cellular homeostasis, achieve cell type-specific proteomes, and coordinate proteostasis across tissues. A precise understanding of these layers of control has important implications for organismal health and could offer new therapeutic approaches for neurodegenerative diseases and other chronic disorders related to PN dysfunction.

Chaperone addiction of toxin-antitoxin systems.

Bacterial toxin-antitoxin (TA) systems, in which a labile antitoxin binds and inhibits the toxin, can promote adaptation and persistence by modulating bacterial growth in response to stress. Some atypical TA systems, known as tripartite toxin-antitoxin-chaperone (TAC) modules, include a molecular chaperone that facilitates folding and protects the antitoxin from degradation. Here we use a TAC module from Mycobacterium tuberculosis as a model to investigate the molecular mechanisms by which classical TAs can become 'chaperone-addicted'. The chaperone specifically binds the antitoxin at a short carboxy-terminal sequence (chaperone addiction sequence, ChAD) that is not present in chaperone-independent antitoxins. In the absence of chaperone, the ChAD sequence destabilizes the antitoxin, thus preventing toxin inhibition. Chaperone-ChAD pairs can be transferred to classical TA systems or to unrelated proteins and render them chaperone-dependent. This mechanism might be used to optimize the expression and folding of heterologous proteins in bacterial hosts for biotechnological or medical purposes.

Multitasking SecB chaperones in bacteria.

Protein export in bacteria is facilitated by the canonical SecB chaperone, which binds to unfolded precursor proteins, maintains them in a translocation competent state and specifically cooperates with the translocase motor SecA to ensure their proper targeting to the Sec translocon at the cytoplasmic membrane. Besides its key contribution to the Sec pathway, SecB chaperone tasking is critical for the secretion of the Sec-independent heme-binding protein HasA and actively contributes to the cellular network of chaperones that control general proteostasis in Escherichia coli, as judged by the significant interplay found between SecB and the trigger factor, DnaK and GroEL chaperones. Although SecB is mainly a proteobacterial chaperone associated with the presence of an outer membrane and outer membrane proteins, secB-like genes are also found in Gram-positive bacteria as well as in certain phages and plasmids, thus suggesting alternative functions. In addition, a SecB-like protein is also present in the major human pathogen Mycobacterium tuberculosis where it specifically controls a stress-responsive toxin-antitoxin system. This review focuses on such very diverse chaperone functions of SecB, both in E. coli and in other unrelated bacteria.

Multiple toxin-antitoxin systems in Mycobacterium tuberculosis.

The hallmark of Mycobacterium tuberculosis is its ability to persist for a long-term in host granulomas, in a non-replicating and drug-tolerant state, and later awaken to cause disease. To date, the cellular factors and the molecular mechanisms that mediate entry into the persistence phase are poorly understood. Remarkably, M. tuberculosis possesses a very high number of toxin-antitoxin (TA) systems in its chromosome, 79 in total, regrouping both well-known (68) and novel (11) families, with some of them being strongly induced in drug-tolerant persisters. In agreement with the capacity of stress-responsive TA systems to generate persisters in other bacteria, it has been proposed that activation of TA systems in M. tuberculosis could contribute to its pathogenesis. Herein, we review the current knowledge on the multiple TA families present in this bacterium, their mechanism, and their potential role in physiology and virulence.

TAC from Mycobacterium tuberculosis: a paradigm for stress-responsive toxin-antitoxin systems controlled by SecB-like chaperones.

Bacterial type II toxin-antitoxins (TAs) are two-component systems that modulate growth in response to specific stress conditions, thus promoting adaptation and persistence. The major human pathogen Mycobacterium tuberculosis potentially encodes 75 TAs and it has been proposed that persistence induced by active toxins might be relevant for its pathogenesis. In this work, we focus on the newly discovered toxin-antitoxin-chaperone (TAC) system of M. tuberculosis, an atypical stress-responsive TA system tightly controlled by a molecular chaperone that shows similarity to the canonical SecB chaperone involved in Sec-dependent protein export in Gram-negative bacteria. We performed a large-scale genome screening to reconstruct the evolutionary history of TAC systems and found that TAC is not restricted to mycobacteria and seems to have disseminated in diverse taxonomic groups by horizontal gene transfer. Our results suggest that TAC chaperones are evolutionary related to the solitary chaperone SecB and have diverged to become specialized toward their cognate antitoxins.

SecB-like chaperone controls a toxin-antitoxin stress-responsive system in Mycobacterium tuberculosis.

A major step in the biogenesis of newly synthesized precursor proteins in bacteria is their targeting to the Sec translocon at the inner membrane. In gram-negative bacteria, the chaperone SecB binds nonnative forms of precursors and specifically transfers them to the SecA motor component of the translocase, thus facilitating their export. The major human pathogen Mycobacterium tuberculosis is an unusual gram-positive bacterium with a well-defined outer membrane and outer membrane proteins. Assistance to precursor proteins by chaperones in this bacterium remains largely unexplored. Here we show that the product of the previously uncharacterized Rv1957 gene of M. tuberculosis can substitute for SecB functions in Escherichia coli and prevent preprotein aggregation in vitro. Interestingly, in M. tuberculosis, Rv1957 is clustered with a functional stress-responsive higB-higA toxin-antitoxin (TA) locus of unknown function. Further in vivo experiments in E. coli and in Mycobacterium marinum strains that do not possess the TA-chaperone locus show that the severe toxicity of the toxin was entirely inhibited when the antitoxin and the chaperone were jointly expressed. We found that Rv1957 acts directly on the antitoxin by preventing its aggregation and protecting it from degradation. Taken together, our results show that the SecB-like chaperone Rv1957 specifically controls a stress-responsive TA system relevant for M. tuberculosis adaptive response.