Identification of Azalamellarin N as a Pyroptosis Inhibitor.

Pyroptosis is a form of regulated cell death that promotes inflammation; it attracts much attention because its dysregulation leads to various inflammatory diseases. To help explore the precise mechanisms by which pyroptosis is regulated, in this study, we searched for chemical compounds that inhibit pyroptosis. From our original compound library, we identified azalamellarin N (AZL-N), a hexacyclic pyrrole alkaloid, as an inhibitor of pyroptosis induced by R837 (also called imiquimod), which is an agonist of the intracellular multiprotein complex nucleotide-binding and oligomerization domain-like receptor (NLR) family pyrin domain containing 3 (NLRP3) inflammasome. However, whereas the effect of AZL-N on R837-induced pyroptosis was relatively weak, AZL-N strongly inhibited pyroptosis induced by extracellular ATP or nigericin, which are different types of NLRP3 inflammasome agonists. This was in contrast with the results that MCC950, a well-established NLRP3 inhibitor, consistently inhibited pyroptosis irrespective of the type of stimulus. We also found that AZL-N inhibited activation of caspase-1 and apoptosis-associated speck-like proteins containing a caspase activation and recruitment domain (ASC), which are components of the NLRP3 inflammasome. Analysis of the structure-activity relationship revealed that a lactam ring of AZL-N, which has been shown to contribute to the strong binding of AZL-N to its known target protein kinases, is required for its inhibitory effects on pyroptosis. These results suggest that AZL-N inhibits pyroptosis by targeting molecule(s), which may be protein kinase(s), that act upstream of NLRP3 inflammasome activation, rather than by directly targeting the components of the NLRP3 inflammasome. Further identification and analysis of target molecule(s) of AZL-N will shed light on the regulatory mechanisms of pyroptosis, particularly those depending on proinflammatory stimuli.

Screening and Synthesis of Tetrazole Derivatives that Inhibit the Growth of Cryptococcus Species.

Cryptococcosis has become a major health problem worldwide and caused morbidity and mortality in immunocompromised patients, especially those infected with human immunodeficiency virus (HIV). Despite the global distribution of cryptococcosis, the number and types of the available antifungals are limited, and the treatment outcomes in HIV patients are generally poor. In this study, we screened a compound library and identified one tetrazole derivative as an efficient inhibitor of Cryptococcus neoformans and Cryptococcus gattii. We further designed and synthesized a series of tetrazole derivatives and determined their structure-activity relationship, demonstrating that tetrazole backbone-containing compounds could be developed as novel antifungal drugs with distinct mechanisms against Cryptococcus spp. Our findings provide a starting point for novel target identification and structural optimization to develop a distinct class of therapeutics for patients with cryptococcosis.

Marine-derived microbes and molecules for drug discovery.

Increasing attention has been paid to marine-derived biomolecules as sources of therapeutics for autoimmune diseases. Nagasaki Prefecture has many islands and is surrounded by seas, straits, gulfs, bays, and coves, giving it the second longest coastline in Japan after Hokkaido. We have collected more than 20,000 marine microbes and have been preparing an original marine microbial extract library, which contains small and mid-size biomolecules that may penetrate cell membranes and interfere with the intracellular protein-protein interaction involved in the development of autoinflammatory diseases such as familial Mediterranean fever. In addition, we have been developing an indoor shark farming system to prepare shark nanobodies that could be developed as potential therapeutic agents for autoimmune diseases. Sharks produce heavy-chain antibodies, called immunoglobulin new antigen receptors (IgNARs), consisting of one variable domain (VNAR) and five constant domains (CNAR); of these, VNAR can recognize a variety of foreign antigens. A VNAR single domain fragment, called a nanobody, can be expressed in Escherichia coli and has the properties of an ideal therapeutic candidate for autoimmune diseases. Shark nanobodies contain complementarity-determining regions that are formed through the somatic rearrangement of variable, diversity, and joining segments, with the segment end trimming and the N- and P-additions, as found in the variable domains of mammalian antibodies. The affinity and diversity of shark nanobodies are thus expected to be comparable to those of mammalian antibodies. In addition, shark nanobodies are physically robust and can be prepared inexpensively; as such, they may lead to the development of highly specific, stable, effective, and inexpensive biotherapeutics in the future. In this review, we first summarize the history of the development of conventional small molecule drugs and monoclonal antibody therapeutics for autoimmune diseases, and then introduce our drug discovery system at Nagasaki University, including the preparation of an original marine microbial extract library and the development of shark nanobodies.

ASK1 signaling regulates phase-specific glial interactions during neuroinflammation.

Neuroinflammation is well known to be associated with neurodegenerative diseases. Apoptosis signal-regulating kinase 1 (ASK1) is a mitogen-activated protein kinase kinase kinase that has been implicated in neuroinflammation, but its precise cellular and molecular mechanisms remain unknown. In this study, we generated conditional knockout (CKO) mice that lack ASK1 in T cells, dendritic cells, microglia/macrophages, microglia, or astrocytes, to assess the roles of ASK1 during experimental autoimmune encephalomyelitis (EAE). We found that neuroinflammation was reduced in both the early and later stages of EAE in microglia/macrophage-specific ASK1 knockout mice, whereas only the later-stage neuroinflammation was ameliorated in astrocyte-specific ASK1 knockout mice. ASK1 deficiency in T cells and dendritic cells had no significant effects on EAE severity. Further, we found that ASK1 in microglia/macrophages induces a proinflammatory environment, which subsequently activates astrocytes to exacerbate neuroinflammation. Microglia-specific ASK1 deletion was achieved using a CX3CR1CreER system, and we found that ASK1 signaling in microglia played a major role in generating and maintaining disease. Activated astrocytes produce key inflammatory mediators, including CCL2, that further activated and recruited microglia/macrophages, in an astrocytic ASK1-dependent manner. Astrocyte-specific analysis revealed CCL2 expression was higher in the later stage compared with the early stage, suggesting a greater proinflammatory role of astrocytes in the later stage. Our findings demonstrate cell-type-specific roles of ASK1 and suggest phase-specific ASK1-dependent glial cell interactions in EAE pathophysiology. We propose glial ASK1 as a promising therapeutic target for reducing neuroinflammation.

Lead Optimization of Influenza Virus RNA Polymerase Inhibitors Targeting PA-PB1 Interaction.

Influenza viruses are responsible for contagious respiratory illnesses in humans and cause seasonal epidemics and occasional pandemics worldwide. Previously, we identified a quinolinone derivative PA-49, which inhibited the influenza virus RNA-dependent RNA polymerase (RdRp) by targeting PA-PB1 interaction. This paper reports the structure optimization of PA-49, which resulted in the identification of 3-((dibenzylamino)methyl)quinolinone derivatives with more potent anti-influenza virus activity. During the optimization, the hit compound 89, which was more active than PA-49, was identified. Further optimization and scaffold hopping of 89 led to the most potent compounds 100 and a 1,8-naphthyridinone derivative 118, respectively. We conclusively determined that compounds 100 and 118 suppressed the replication of influenza virus and exhibited anti-influenza virus activity against both influenza virus types A and B in the range of 50% effective concentration (EC50) = 0.061-0.226 µM with low toxicity (50% cytotoxic concentration (CC50) >10 µM).

ERAD components Derlin-1 and Derlin-2 are essential for postnatal brain development and motor function.

Derlin family members (Derlins) are primarily known as components of the endoplasmic reticulum-associated degradation pathway that eliminates misfolded proteins. Here we report a function of Derlins in the brain development. Deletion of Derlin-1 or Derlin-2 in the central nervous system of mice impaired postnatal brain development, particularly of the cerebellum and striatum, and induced motor control deficits. Derlin-1 or Derlin-2 deficiency reduced neurite outgrowth in vitro and in vivo and surprisingly also inhibited sterol regulatory element binding protein 2 (SREBP-2)-mediated brain cholesterol biosynthesis. In addition, reduced neurite outgrowth due to Derlin-1 deficiency was rescued by SREBP-2 pathway activation. Overall, our findings demonstrate that Derlins sustain brain cholesterol biosynthesis, which is essential for appropriate postnatal brain development and function.

Cleaved PGAM5 dephosphorylates nuclear serine/arginine-rich proteins during mitophagy.

PGAM5 is a protein phosphatase located in the inner mitochondrial membrane through its transmembrane (TM) domain and is cleaved within the TM domain upon mitochondrial dysfunction. We found previously that cleaved PGAM5 is released from mitochondria, following proteasome-mediated rupture of the outer mitochondrial membrane during mitophagy, a selective form of autophagy specific to mitochondria. Here, we examined the role of cleaved PGAM5 outside mitochondria. Deletion mutants that mimic cleaved PGAM5 existed not only in the cytosol but also in the nucleus, and a fraction of cleaved PGAM5 translocated to the nucleus during mitophagy induced by the uncoupler CCCP. We identified serine/arginine-related nuclear matrix protein of 160 kDa (SRm160)/SRRM1, which contains a highly phosphorylated domain rich in arginine/serine dipeptides, called the RS domain, as a nuclear protein that interacts with PGAM5. PGAM5 dephosphorylated SRm160, and incubation of lysates from WT cells, but not of those from PGAM5-deficient cells, induced dephosphorylation of SRm160 and another RS domain-containing protein SRSF1, one of the most characterized serine/arginine-rich (SR) proteins. Moreover, phosphorylation of these proteins and other SR proteins, which are commonly reactive toward the 1H4 monoclonal antibody that detects phosphorylated SR proteins, decreased during mitophagy, largely because of PGAM5 activity. These results suggest that PGAM5 regulates phosphorylation of these nuclear proteins during mitophagy. Because SRm160 and SR proteins play critical roles in mRNA metabolism, PGAM5 may coordinate cellular responses to mitochondrial stress at least in part through post-transcriptional and pre-translational events.

Screening of Inhibitors Targeting Heat Shock Protein 47 Involved in the Development of Idiopathic Pulmonary Fibrosis.

Heat shock protein 47 (HSP47), a collagen-specific molecular chaperone, is causally related to fibrotic diseases, including idiopathic pulmonary fibrosis. The identification of Compounds that interfere with the HSP47-collagen interaction is essential for the development of relevant therapeutics. Herein, we prepared human HSP47 as a soluble fusion protein expressed in E. coli and established an assay system for HSP47 inhibitor screening. We screened a natural and synthetic Compound library established at Nagasaki University. Among 1023 Compounds, 13 exhibited inhibitory activity against human HSP47, of which three inhibited its function in a dose-dependent manner. Epigallocatechin-3-O-gallate, one of these three Compounds, is a typical polyphenol Compound derived from tea leaves. Structurally related Compounds were synthesized and examined for their activity, revealing a hydroxyl group at A-ring position 5 as important for its activity. The present findings provide valuable insight for the development of natural product-derived therapeutics for fibrotic diseases, including idiopathic pulmonary fibrosis.

ATP exposure stimulates glutathione efflux as a necessary switch for NLRP3 inflammasome activation.

The NLRP3 inflammasome is a multiprotein complex responsible for the maturation of precursor forms of interleukin (IL)-1ß and IL-18 into active proinflammatory cytokines. Increasing evidence suggests that modulation of redox homeostasis contributes to the activation of the NLRP3 inflammasome. However, specific mechanistic details remain unclear. We demonstrate here that ATP exposure evoked a sharp decrease in glutathione (GSH) levels in macrophages, which led to NLRP3 inflammasome activation. We detected an increase in GSH levels in culture supernatants that was comparable to the GSH decrease in macrophages, which suggests that exposure to ATP stimulated GSH efflux. Exogenous addition of P2X7 receptor antagonist, GSH, or the oxidized form GSSG attenuated this efflux. Also, exogenous GSH or GSSG strongly inhibited NLRP3 inflammasome activation in vitro and in vivo. These data suggest that GSH efflux controls NLRP3 inflammasome activation, which may lead to development of novel therapeutic strategies for NLRP3 inflammasome-associated disorders.

ASK1 suppresses NK cell-mediated intravascular tumor cell clearance in lung metastasis.

Tumor metastasis is the leading cause of death worldwide and involves an extremely complex process composed of multiple steps. Our previous study demonstrated that apoptosis signal-regulating kinase 1 (ASK1) deficiency in mice attenuates tumor metastasis in an experimental lung metastasis model. However, the steps of tumor metastasis regulated by ASK1 remain unclear. Here, we showed that ASK1 deficiency in mice promotes natural killer (NK) cell-mediated intravascular tumor cell clearance in the initial hours of metastasis. In response to tumor inoculation, ASK1 deficiency upregulated immune response-related genes, including interferon-gamma (IFNγ). We also revealed that NK cells are required for these anti-metastatic phenotypes. ASK1 deficiency augmented cytokine production chemoattractive to NK cells possibly through induction of the ligand for NKG2D, a key activating receptor of NK cells, leading to further recruitment of NK cells into the lung. These results indicate that ASK1 negatively regulates NK cell-dependent anti-tumor immunity and that ASK1-targeted therapy can provide a new tool for cancer immunotherapy to overcome tumor metastasis.

SH3P2 suppresses osteoclast differentiation through restricting membrane localization of myosin 1E.

Osteoclasts are multinucleated cells responsible for bone resorption. Src homology 3 (SH3) domain-containing protein-2 (SH3P2)/osteoclast-stimulating factor-1 regulates osteoclast differentiation, but its exact role remains elusive. Here, we show that SH3P2 suppresses osteoclast differentiation. SH3P2 knockout (KO) mice displayed decreased femoral trabecular bone mass and enhanced localization of osteoclasts on the tibial trabecular bone surface, suggesting that SH3P2 suppresses bone resorption by osteoclasts. Osteoclast differentiation based on cellular multinuclearity induced by macrophage colony-stimulating factor and receptor activator of nuclear factor-κB ligand (RANKL) was enhanced in bone marrow-derived macrophages lacking SH3P2. RANKL induced SH3P2 dephosphorylation, which increased the association of actin-dependent motor protein myosin 1E (Myo1E) with SH3P2 and thereby prevented Myo1E localization to the plasma membrane. Consistent with this, Myo1E in the membrane fraction increased in SH3P2-KO cells. Together with the attenuated osteoclast differentiation in Myo1E knocked down cells, SH3P2 may suppress osteoclast differentiation by preventing their cell-to-cell fusion depending on Myo1E membrane localization.

Cleaved PGAM5 is released from mitochondria depending on proteasome-mediated rupture of the outer mitochondrial membrane during mitophagy.

PGAM5 is a unique type of protein phosphatase that exists in mitochondria. It has been shown to exist in the inner mitochondrial membrane through its transmembrane domain and to be cleaved within the transmembrane domain upon mitochondrial dysfunction. However, its submitochondrial localization remains controversial; many researchers claim that PGAM5 localizes to the outer mitochondrial membrane based on the findings that PGAM5 associates with many cytoplasmic proteins. Here, we found that cleaved PGAM5 was released from mitochondria during mitophagy, a selective form of autophagy specific for mitochondria, and that the release was inhibited by proteasome inhibitors in HeLa cells stably expressing the E3 ubiquitin ligase Parkin. However, treatment of parental HeLa cells lacking Parkin with mitophagy-inducing agents caused PGAM5 cleavage but did not cause its release from mitochondria. Thus, cleaved PGAM5 appears to be released from mitochondria depending on proteasome-mediated rupture of the outer membrane during mitophagy, which has been previously shown to precede autophagy-mediated degradation of whole mitochondria. This study suggests that PGAM5 senses mitochondrial dysfunction in the inner mitochondrial membrane and serves as a signalling intermediate that regulates the cellular response to mitochondrial stress upon its cleavage and release from mitochondria.

ß-TrCP-dependent degradation of ASK1 suppresses the induction of the apoptotic response by oxidative stress.

Apoptosis signal-regulating kinase 1 (ASK1) is a key player in the homeostatic response of many organisms. Of the many functions of ASK1, it is most well-known for its ability to induce canonical caspase 3-dependent apoptosis through the MAPK pathways in response to reactive oxygen species (ROS). As ASK1 is a regulator of apoptosis, its proper regulation is critical for the well-being of an organism. To date, several E3 ubiquitin ligases have been identified that are capable of degrading ASK1, signifying the importance of maintaining ASK1 expression levels during stress responses. ASK1 protein regulation under unstimulated conditions, however, is still largely unknown. Using tandem mass spectrometry, we have identified beta-transducin repeat containing protein (ß-TrCP), an E3 ubiquitin ligase, as a novel interacting partner of ASK1 that is capable of ubiquitinating and subsequently degrading ASK1 through the ubiquitin-proteasome system (UPS). This interaction requires the seven WD domains of ß-TrCP and the C-terminus of ASK1. By silencing the ß-TrCP genes, we observed a significant increase in caspase 3 activity in response to oxidative stress, which could subsequently be suppressed by silencing ASK1. These findings suggest that ß-TrCP is capable of suppressing oxidative stress-induced caspase 3-dependent apoptosis through suppression of ASK1, assisting in the organism's ability to maintain homeostasis in an unstable environment.

ERK signalling as a regulator of cell motility.

Cell motility is regulated by multiple processes, including cell protrusion, cell retraction, cell-matrix adhesion, polarized exocytosis and polarized vesicle trafficking, each of which is spatiotemporally controlled by various intracellular signalling pathways. Dysregulation of cell motility leads to pathological conditions, such as tumour invasion and metastasis. Accumulating evidence has revealed that extracellular signal-regulated kinase (ERK) signalling is one of the critical regulators of cell motility, although it is classically known as an important regulator of cell proliferation, differentiation and survival through regulation of gene expression. ERK and its downstream kinase, p90 ribosomal S6 kinase (RSK), dynamically regulate cell motility mainly through direct phosphorylation of various molecules that are not necessarily involved in the regulation of gene transcription and translation. In this review, we summarize how ERK signalling regulates cell motility by focusing on the components of the cell motility machinery that are directly regulated by ERK or RSK.

ASK1 facilitates tumor metastasis through phosphorylation of an ADP receptor P2Y12 in platelets.

Tumor metastasis is the major cause of deaths in cancer patients and is modulated by intertwined stress-responsive signaling cascades. Here we demonstrate that deletion of stress-responsive apoptosis signal-regulating kinase 1 (Ask1) in platelets results in unstable hemostasis and drastic attenuation of tumor lung metastasis, both of which are attributable to platelet dysfunction. Platelet-specific deletion of Ask1 in mice leads to defects in ADP-dependent platelet aggregation, unstable hemostasis and subsequent attenuation of tumor metastasis. We also revealed that activating phosphorylation of Akt is attenuated in Ask1-deficient platelets, contrary to the previous reports suggesting that Akt is negatively regulated by ASK1. Mechanistically, ASK1-JNK/p38 axis phosphorylates an ADP receptor P2Y12 at Thr345, which is required for the ADP-dependent sustained Akt activity that is vital to normal platelet functions. Our findings offer insight into positive regulation of Akt signaling through P2Y12 phosphorylation as well as MAPK signaling in platelets by ASK1 and suggest that ASK1-JNK/p38 axis provides a new therapeutic opportunity for tumor metastasis.

Structures of PGAM5 Provide Insight into Active Site Plasticity and Multimeric Assembly.

PGAM5 is a mitochondrial membrane protein that functions as an atypical Ser/Thr phosphatase and is a regulator of oxidative stress response, necroptosis, and autophagy. Here we present several crystal structures of PGAM5 including the activating N-terminal regulatory sequences, providing a model for structural plasticity, dimerization of the catalytic domain, and the assembly into an enzymatically active dodecameric form. Oligomeric states observed in structures were supported by hydrogen exchange mass spectrometry, size-exclusion chromatography, and analytical ultracentrifugation experiments in solution. We report that the catalytically important N-terminal WDPNWD motif acts as a structural integrator assembling PGAM5 into a dodecamer, allosterically activating the phosphatase by promoting an ordering of the catalytic loop. Additionally the observed active site plasticity enabled visualization of essential conformational rearrangements of catalytic elements. The comprehensive biophysical characterization offers detailed structural models of this key mitochondrial phosphatase that has been associated with the development of diverse diseases.

Mitochondrial function is required for extracellular ATP-induced NLRP3 inflammasome activation.

The NLRP3 inflammasome plays a critical role in the processing and release of inflammatory cytokines, such as interleukin-1ß (IL-1ß) and IL-18. Accumulating evidence suggests that mitochondria are common mediators of NLRP3 inflammasome activation induced by a wide range of inflammatory stimuli; however, the precise role of mitochondria is still not fully understood. Here, we show that mitochondrial function is required for extracellular ATP-induced NLRP3 inflammasome activation. Extracellular ATP induced the loss of mitochondrial membrane potential and mitochondrial fragmentation in a different manner than other stimuli in primary mouse macrophages. CCCP, an uncoupler and antimycin A, an inhibitor of the mitochondrial electron transport chain, inhibited IL-1ß release induced by ATP but not by other stimuli. CCCP did not inhibit the ATP-induced generation of reactive oxygen species and cell death, both of which are known to promote IL-1ß release, but did inhibit the ATP-induced activation of caspase-1, a component of the NLRP3 inflammasome. These results suggest that mitochondrial function is required somewhat specifically for ATP-induced NLRP3 inflammasome activation. In contrast to many previous reports that dysfunctional mitochondria promote NLRP3 inflammasome activation, the function of intact mitochondria appears to be required for NLRP3 inflammasome activation, depending on the stimulus.

WP1066 suppresses macrophage cell death induced by inflammasome agonists independently of its inhibitory effect on STAT3.

The compound WP1066 was originally synthesized by modifying the structure of AG490, which inhibits the activation of signal transducer and activator of transcription 3 (STAT3) by directly targeting Janus kinases (JAKs). WP1066 exhibits stronger anti-cancer activity than AG490 against malignant glioma and other cancer cells and is regarded as a promising therapeutic agent. By screening a small library of target-known compounds, we identified WP1066 as an inhibitor of macrophage cell death induced by agonists of the NLRP3 inflammasome, an intracellular protein complex required for the processing of the proinflammatory cytokine interleukin (IL)-1ß. WP1066 strongly inhibited cell death as well as extracellular release of IL-1ß induced by inflammasome agonists in mouse peritoneal exudate cells and human leukemia monocytic THP-1 cells that were differentiated into macrophagic cells by treatment with PMA. However, inflammasome agonists did not increase STAT3 phosphorylation, and another JAK inhibitor, ruxolitinib, did not inhibit cell death, although it strongly inhibited basal STAT3 phosphorylation. Thus, WP1066 appears to suppress macrophage cell death independently of its inhibitory effect on STAT3. In contrast, WP1066 itself induced the death of undifferentiated THP-1 cells, suggesting that WP1066 differentially modulates cell death in a context-dependent manner. Consistent with previous findings, WP1066 induced the death of human glioma A172 and T98G cells. However, neither ruxolitinib nor AG490, the former of which completely suppressed STAT3 phosphorylation, induced the death of these glioma cells. These results suggest that WP1066 targets cell death-modulating molecules other than those involved in JAK-STAT3 signaling.

ERK signaling promotes cell motility by inducing the localization of myosin 1E to lamellipodial tips.

Signaling by extracellular signal-regulated kinase (ERK) plays an essential role in the induction of cell motility, but the precise mechanism underlying such regulation has remained elusive. We recently identified SH3P2 as a negative regulator of cell motility whose function is inhibited by p90 ribosomal S6 kinase (RSK)-mediated phosphorylation downstream of ERK. We here show that myosin 1E (Myo1E) is a binding partner of SH3P2 and that the interaction of the two proteins in the cytosol prevents the localization of Myo1E to the plasma membrane. Serum-induced phosphorylation of SH3P2 at Ser(202) by RSK results in dissociation of Myo1E from SH3P2 in the cytosol and the subsequent localization of Myo1E to the tips of lamellipodia mediated by binding of its TH2 domain to F-actin. This translocation of Myo1E is essential for lamellipodium extension and consequent cell migration. The ERK signaling pathway thus promotes cell motility through regulation of the subcellular localization of Myo1E.

The Ablation of Mitochondrial Protein Phosphatase Pgam5 Confers Resistance Against Metabolic Stress.

Phosphoglycerate mutase family member 5 (PGAM5) is a mitochondrial protein phosphatase that has been reported to be involved in various stress responses from mitochondrial quality control to cell death. However, its roles in vivo are largely unknown. Here, we show that Pgam5-deficient mice are resistant to several metabolic insults. Under cold stress combined with fasting, Pgam5-deficient mice better maintained body temperature than wild-type mice and showed an extended survival rate. Serum triglycerides and lipid content in brown adipose tissue (BAT), a center of adaptive thermogenesis, were severely reduced in Pgam5-deficient mice. Moreover, although Pgam5 deficiency failed to maintain proper mitochondrial integrity in BAT, it reciprocally resulted in the dramatic induction of fibroblast growth factor 21 (FGF21) that activates various functions of BAT including thermogenesis. Thus, the enhancement of lipid metabolism and FGF21 may contribute to the cold resistance of Pgam5-deficient mice under fasting condition. Finally, we also found that Pgam5-deficient mice are resistant to high-fat-diet-induced obesity. Our study uncovered that PGAM5 is involved in the whole-body metabolism in response to stresses that impose metabolic challenges on mitochondria.