Lysosomes mediate the mitochondrial UPR via mTORC1-dependent ATF4 phosphorylation.

Lysosomes are central platforms for not only the degradation of macromolecules but also the integration of multiple signaling pathways. However, whether and how lysosomes mediate the mitochondrial stress response (MSR) remain largely unknown. Here, we demonstrate that lysosomal acidification via the vacuolar H+-ATPase (v-ATPase) is essential for the transcriptional activation of the mitochondrial unfolded protein response (UPRmt). Mitochondrial stress stimulates v-ATPase-mediated lysosomal activation of the mechanistic target of rapamycin complex 1 (mTORC1), which then directly phosphorylates the MSR transcription factor, activating transcription factor 4 (ATF4). Disruption of mTORC1-dependent ATF4 phosphorylation blocks the UPRmt, but not other similar stress responses, such as the UPRER. Finally, ATF4 phosphorylation downstream of the v-ATPase/mTORC1 signaling is indispensable for sustaining mitochondrial redox homeostasis and protecting cells from ROS-associated cell death upon mitochondrial stress. Thus, v-ATPase/mTORC1-mediated ATF4 phosphorylation via lysosomes links mitochondrial stress to UPRmt activation and mitochondrial function resilience.

Mitochondrial and NAD+ metabolism predict recovery from acute kidney injury in a diverse mouse population.

Acute kidney failure and chronic kidney disease are global health issues steadily rising in incidence and prevalence. Animal models on a single genetic background have so far failed to recapitulate the clinical presentation of human nephropathies. Here, we used a simple model of folic acid-induced kidney injury in 7 highly diverse mouse strains. We measured plasma and urine parameters, as well as renal histopathology and mRNA expression data, at 1, 2, and 6 weeks after injury, covering the early recovery and long-term remission. We observed an extensive strain-specific response ranging from complete resistance of the CAST/EiJ to high sensitivity of the C57BL/6J, DBA/2J, and PWK/PhJ strains. In susceptible strains, the severe early kidney injury was accompanied by the induction of mitochondrial stress response (MSR) genes and the attenuation of NAD+ synthesis pathways. This is associated with delayed healing and a prolonged inflammatory and adaptive immune response 6 weeks after insult, heralding a transition to chronic kidney disease. Through a thorough comparison of the transcriptomic response in mouse and human disease, we show that critical metabolic gene alterations were shared across species, and we highlight the PWK/PhJ strain as an emergent model of transition from acute kidney injury to chronic disease.

V-ATPase/TORC1-mediated ATFS-1 translation directs mitochondrial UPR activation in C. elegans.

To adapt mitochondrial function to the ever-changing intra- and extracellular environment, multiple mitochondrial stress response (MSR) pathways, including the mitochondrial unfolded protein response (UPRmt), have evolved. However, how the mitochondrial stress signal is sensed and relayed to UPRmt transcription factors, such as ATFS-1 in Caenorhabditis elegans, remains largely unknown. Here, we show that a panel of vacuolar H+-ATPase (v-ATPase) subunits and the target of rapamycin complex 1 (TORC1) activity are essential for the cytosolic relay of mitochondrial stress to ATFS-1 and for the induction of the UPRmt. Mechanistically, mitochondrial stress stimulates v-ATPase/Rheb-dependent TORC1 activation, subsequently promoting ATFS-1 translation. Increased translation of ATFS-1 upon mitochondrial stress furthermore relies on a set of ribosomal components but is independent of GCN-2/PEK-1 signaling. Finally, the v-ATPase and ribosomal subunits are required for mitochondrial surveillance and mitochondrial stress-induced longevity. These results reveal a v-ATPase-TORC1-ATFS-1 signaling pathway that links mitochondrial stress to the UPRmt through intimate crosstalks between multiple organelles.

Multi-omics analysis identifies essential regulators of mitochondrial stress response in two wild-type C. elegans strains.

The mitochondrial unfolded protein response (UPRmt) is a promising pharmacological target for aging and age-related diseases. However, the integrative analysis of the impact of UPRmt activation on different signaling layers in animals with different genetic backgrounds is lacking. Here, we applied systems approaches to investigate the effect of UPRmt induced by doxycycline (Dox) on transcriptome, proteome, and lipidome in two genetically divergent worm strains, named N2 and CB4856. From the integrated omics datasets, we found that Dox prolongs lifespan of both worm strains through shared and strain-specific mechanisms. Specifically, Dox strongly impacts mitochondria, upregulates defense response, and lipid metabolism, while decreasing triglycerides. We further validated that lipid genes acs-2/20 and fat-7/6 were required for Dox-induced UPRmt and longevity in N2 and CB4856 worms, respectively. Our data have translational value as they indicate that the beneficial effects of Dox-induced UPRmt on lifespan are consistent across different genetic backgrounds through different regulators.

Pleiotropic effects of mitochondria in aging.

Aging is typified by a progressive decline in mitochondrial activity and stress resilience. Here, we review how mitochondrial stress pathways have pleiotropic effects on cellular and systemic homeostasis, which can comprise protective or detrimental responses during aging. We describe recent evidence arguing that defects in these conserved adaptive pathways contribute to aging and age-related diseases. Signaling pathways regulating the mitochondrial unfolded protein response, mitochondrial membrane dynamics, and mitophagy are discussed, emphasizing how their failure contributes to heteroplasmy and de-regulation of key metabolites. Our current understanding of how these processes are controlled and interconnected explains how mitochondria can widely impact fundamental aspects of aging.

The transcriptional coactivator CBP/p300 is an evolutionarily conserved node that promotes longevity in response to mitochondrial stress.

Organisms respond to mitochondrial stress by activating multiple defense pathways including the mitochondrial unfolded protein response (UPRmt). However, how UPRmt regulators are orchestrated to transcriptionally activate stress responses remains largely unknown. Here we identified CBP-1, the worm ortholog of the mammalian acetyltransferases CBP/p300, as an essential regulator of the UPRmt, as well as mitochondrial stress-induced immune response, reduction of amyloid-ß aggregation and lifespan extension in Caenorhabditis elegans. Mechanistically, CBP-1 acts downstream of histone demethylases, JMJD-1.2/JMJD-3.1, and upstream of UPRmt transcription factors including ATFS-1, to systematically induce a broad spectrum of UPRmt genes and execute multiple beneficial functions. In mouse and human populations, transcript levels of CBP/p300 positively correlate with UPRmt transcripts and longevity. Furthermore, CBP/p300 inhibition disrupts, while forced expression of p300 is sufficient to activate, the UPRmt in mammalian cells. These results highlight an evolutionarily conserved mechanism that determines mitochondrial stress response, and promotes health and longevity through CBP/p300.

Proto-oncogene Src links lipogenesis via lipin-1 to breast cancer malignancy.

Increased lipogenesis has been linked to an increased cancer risk and poor prognosis; however, the underlying mechanisms remain obscure. Here we show that phosphatidic acid phosphatase (PAP) lipin-1, which generates diglyceride precursors necessary for the synthesis of glycerolipids, interacts with and is a direct substrate of the Src proto-oncogenic tyrosine kinase. Obesity-associated microenvironmental factors and other Src-activating growth factors, including the epidermal growth factor, activate Src and promote Src-mediated lipin-1 phosphorylation on Tyr398, Tyr413 and Tyr795 residues. The tyrosine phosphorylation of lipin-1 markedly increases its PAP activity, accelerating the synthesis of glycerophospholipids and triglyceride. Alteration of the three tyrosine residues to phenylalanine (3YF-lipin-1) disables lipin-1 from mediating Src-enhanced glycerolipid synthesis, cell proliferation and xenograft growth. Re-expression of 3YF-lipin-1 in PyVT;Lpin1-/- mice fails to promote progression and metastasis of mammary tumours. Human breast tumours exhibit increased p-Tyr-lipin-1 levels compared to the adjacent tissues. Importantly, statistical analyses show that levels of p-Tyr-lipin-1 correlate with tumour sizes, lymph node metastasis, time to recurrence and survival of the patients. These results illustrate a direct lipogenesis-promoting role of the pro-oncogenic Src, providing a mechanistic link between obesity-associated mitogenic signaling and breast cancer malignancy.

Identifying gene function and module connections by the integration of multispecies expression compendia.

The functions of many eukaryotic genes are still poorly understood. Here, we developed and validated a new method, termed GeneBridge, which is based on two linked approaches to impute gene function and bridge genes with biological processes. First, Gene-Module Association Determination (G-MAD) allows the annotation of gene function. Second, Module-Module Association Determination (M-MAD) allows predicting connectivity among modules. We applied the GeneBridge tools to large-scale multispecies expression compendia-1700 data sets with over 300,000 samples from human, mouse, rat, fly, worm, and yeast-collected in this study. G-MAD identifies novel functions of genes-for example, DDT in mitochondrial respiration and WDFY4 in T cell activation-and also suggests novel components for modules, such as for cholesterol biosynthesis. By applying G-MAD on data sets from respective tissues, tissue-specific functions of genes were identified-for instance, the roles of EHHADH in liver and kidney, as well as SLC6A1 in brain and liver. Using M-MAD, we identified a list of module-module associations, such as those between mitochondria and proteasome, mitochondria and histone demethylation, as well as ribosomes and lipid biosynthesis. The GeneBridge tools together with the expression compendia are available as an open resource, which will facilitate the identification of connections linking genes, modules, phenotypes, and diseases.

Tip60-mediated lipin 1 acetylation and ER translocation determine triacylglycerol synthesis rate.

Obesity is characterized by excessive fatty acid conversion to triacylglycerols (TAGs) in adipose tissues. However, how signaling networks sense fatty acids and connect to the stimulation of lipid synthesis remains elusive. Here, we show that homozygous knock-in mice carrying a point mutation at the Ser86 phosphorylation site of acetyltransferase Tip60 (Tip60 SA/SA ) display remarkably reduced body fat mass, and Tip60 SA/SA females fail to nurture pups to adulthood due to severely reduced milk TAGs. Mechanistically, fatty acids stimulate Tip60-dependent acetylation and endoplasmic reticulum translocation of phosphatidic acid phosphatase lipin 1 to generate diacylglycerol for TAG synthesis, which is repressed by deacetylase Sirt1. Inhibition of Tip60 activity strongly blocks fatty acid-induced TAG synthesis while Sirt1 suppression leads to increased adiposity. Genetic analysis of loss-of-function mutants in Saccharomyces cerevisiae reveals a requirement of ESA1, yeast ortholog of Tip60, in TAG accumulation. These findings uncover a conserved mechanism linking fatty acid sensing to fat synthesis.

AIDA Selectively Mediates Downregulation of Fat Synthesis Enzymes by ERAD to Retard Intestinal Fat Absorption and Prevent Obesity.

The efficiency of intestinal absorption of dietary fat constitutes a primary determinant accounting for individual vulnerability to obesity. However, how fat absorption is controlled and contributes to obesity remains unclear. Here, we show that inhibition of endoplasmic-reticulum-associated degradation (ERAD) increases the abundance of triacylglycerol synthesis enzymes and fat absorption in small intestine. The C2-domain protein AIDA acts as an essential factor for the E3-ligase HRD1 of ERAD to downregulate rate-limiting acyltransferases GPAT3, MOGAT2, and DGAT2. Aida-/- mice, when grown in a thermal-neutral condition or fed high-fat diet, display increased intestinal fatty acid re-esterification, circulating and tissue triacylglycerol, accompanied with severely increased adiposity without enhancement of adipogenesis. Intestine-specific knockout of Aida largely phenocopies its whole-body knockout, strongly indicating that increased intestinal TAG synthesis is a primary impetus to obesity. The AIDA-mediated ERAD system may thus represent an anti-thrifty mechanism impinging on the enzymes for intestinal fat absorption and systemic fat storage.

Liver-specific deficiency of unc-51 like kinase 1 and 2 protects mice from acetaminophen-induced liver injury.

unc-51-like autophagy activating kinase 1 and 2 (Ulk1/2) regulate autophagy initiation under various stress conditions. However, the physiological functions of these Ser/Thr kinases are not well characterized. Here, we show that mice with liver-specific double knockout (LDKO) of Ulk1 and Ulk2 (Ulk1/2 LDKO) are viable, but exhibit overt hepatomegaly phenotype. Surprisingly, Ulk1/2 LDKO mice display normal autophagic activity in hepatocytes upon overnight fasting, but are strongly resistant to acetaminophen (APAP)-induced liver injury. Further studies revealed that Ulk1/2 are also dispensable for APAP-induced autophagy process, but are essential for the maximum activation of c-Jun N-terminal kinase (JNK) signaling both in vivo and in isolated primary hepatocytes during APAP treatment. Mechanistically, APAP-induced inhibition of mechanistic target of rapamycin complex 1 releases Ulk1 from an inactive state. Activated Ulk1 then directly phosphorylates and increases the kinase activity of mitogen-activated protein kinase kinase 4 and 7 (MKK4/7), the upstream kinases and activator of JNK, and mediates APAP-induced liver injury. Ulk1-dependent phosphorylation of MKK7 was further confirmed by a context-dependent phosphorylation antibody. Moreover, activation of JNK and APAP-induced cell death was markedly attenuated in Mkk4/7 double knockdown hepatocytes reconstituted with an Ulk1-unphosphorylatable mutant of MKK7 compared to those in cells rescued with wild-type MKK7. CONCLUSION: Together, these findings reveal an important role of Ulk1/2 for APAP-induced JNK activation and liver injury, and understanding of this regulatory mechanism may offer us new strategies for prevention and treatment of human APAP hepatotoxicity. (Hepatology 2018;67:2397-2413).

Fructose-1,6-bisphosphate and aldolase mediate glucose sensing by AMPK.

The major energy source for most cells is glucose, from which ATP is generated via glycolysis and/or oxidative metabolism. Glucose deprivation activates AMP-activated protein kinase (AMPK), but it is unclear whether this activation occurs solely via changes in AMP or ADP, the classical activators of AMPK. Here, we describe an AMP/ADP-independent mechanism that triggers AMPK activation by sensing the absence of fructose-1,6-bisphosphate (FBP), with AMPK being progressively activated as extracellular glucose and intracellular FBP decrease. When unoccupied by FBP, aldolases promote the formation of a lysosomal complex containing at least v-ATPase, ragulator, axin, liver kinase B1 (LKB1) and AMPK, which has previously been shown to be required for AMPK activation. Knockdown of aldolases activates AMPK even in cells with abundant glucose, whereas the catalysis-defective D34S aldolase mutant, which still binds FBP, blocks AMPK activation. Cell-free reconstitution assays show that addition of FBP disrupts the association of axin and LKB1 with v-ATPase and ragulator. Importantly, in some cell types AMP/ATP and ADP/ATP ratios remain unchanged during acute glucose starvation, and intact AMP-binding sites on AMPK are not required for AMPK activation. These results establish that aldolase, as well as being a glycolytic enzyme, is a sensor of glucose availability that regulates AMPK.

ULK1/2 Constitute a Bifurcate Node Controlling Glucose Metabolic Fluxes in Addition to Autophagy.

Metabolic reprogramming is fundamental to biological homeostasis, enabling cells to adjust metabolic routes after sensing altered availability of fuels and growth factors. ULK1 and ULK2 represent key integrators that relay metabolic stress signals to the autophagy machinery. Here, we demonstrate that, during deprivation of amino acid and growth factors, ULK1/2 directly phosphorylate key glycolytic enzymes including hexokinase (HK), phosphofructokinase 1 (PFK1), enolase 1 (ENO1), and the gluconeogenic enzyme fructose-1,6-bisphosphatase (FBP1). Phosphorylation of these enzymes leads to enhanced HK activity to sustain glucose uptake but reduced activity of FBP1 to block the gluconeogenic route and reduced activity of PFK1 and ENO1 to moderate drop of glucose-6-phosphate and to repartition more carbon flux to pentose phosphate pathway (PPP), maintaining cellular energy and redox homeostasis at cellular and organismal levels. These results identify ULK1/2 as a bifurcate-signaling node that sustains glucose metabolic fluxes besides initiation of autophagy in response to nutritional deprivation.

RHOBTB3 promotes proteasomal degradation of HIFα through facilitating hydroxylation and suppresses the Warburg effect.

Hypoxia-inducible factors (HIFs) are master regulators of adaptive responses to low oxygen, and their α-subunits are rapidly degraded through the ubiquitination-dependent proteasomal pathway after hydroxylation. Aberrant accumulation or activation of HIFs is closely linked to many types of cancer. However, how hydroxylation of HIFα and its delivery to the ubiquitination machinery are regulated remains unclear. Here we show that Rho-related BTB domain-containing protein 3 (RHOBTB3) directly interacts with the hydroxylase PHD2 to promote HIFα hydroxylation. RHOBTB3 also directly interacts with the von Hippel-Lindau (VHL) protein, a component of the E3 ubiquitin ligase complex, facilitating ubiquitination of HIFα. Remarkably, RHOBTB3 dimerizes with LIMD1, and constructs a RHOBTB3/LIMD1-PHD2-VHL-HIFα complex to effect the maximal degradation of HIFα. Hypoxia reduces the RHOBTB3-centered complex formation, resulting in an accumulation of HIFα. Importantly, the expression level of RHOBTB3 is greatly reduced in human renal carcinomas, and RHOBTB3 deficiency significantly elevates the Warburg effect and accelerates xenograft growth. Our work thus reveals that RHOBTB3 serves as a scaffold to organize a multi-subunit complex that promotes the hydroxylation, ubiquitination and degradation of HIFα.

The lysosomal v-ATPase-Ragulator complex is a common activator for AMPK and mTORC1, acting as a switch between catabolism and anabolism.

AMPK and mTOR play principal roles in governing metabolic programs; however, mechanisms underlying the coordination of the two inversely regulated kinases remain unclear. In this study we found, most surprisingly, that the late endosomal/lysosomal protein complex v-ATPase-Ragulator, essential for activation of mTORC1, is also required for AMPK activation. We also uncovered that AMPK is a residential protein of late endosome/lysosome. Under glucose starvation, the v-ATPase-Ragulator complex is accessible to AXIN/LKB1 for AMPK activation. Concurrently, the guanine nucleotide exchange factor (GEF) activity of Ragulator toward RAG is inhibited by AXIN, causing dissociation from endosome and inactivation of mTORC1. We have thus revealed that the v-ATPase-Ragulator complex is also an initiating sensor for energy stress and meanwhile serves as an endosomal docking site for LKB1-mediated AMPK activation by forming the v-ATPase-Ragulator-AXIN/LKB1-AMPK complex, thereby providing a switch between catabolism and anabolism. Our current study also emphasizes a general role of late endosome/lysosome in controlling metabolic programs.

Mechanism and physiological significance of growth factor-related autophagy.

Growth factors, typically defined as natural substances capable of stimulating cell growth and differentiation, are vital regulators for the survival of metazoan cells. In this review, we will focus on growth factor signaling pathways that are closely related to autophagy induction and discuss the critical roles of this fascinating cellular process in intracellular energy homeostasis, cell fate determination, and pathophysiological regulation.

The orphan nuclear receptor Nur77 regulates LKB1 localization and activates AMPK.

Liver kinase B1 (LKB1) has important roles in governing energy homeostasis by regulating the activity of the energy sensor kinase AMP-activated protein kinase (AMPK). The regulation of LKB1 function, however, is still poorly understood. Here we demonstrate that the orphan nuclear receptor Nur77 binds and sequesters LKB1 in the nucleus, thereby attenuating AMPK activation. This Nur77 function is antagonized by the chemical compound ethyl 2-[2,3,4-trimethoxy-6-(1-octanoyl)phenyl]acetate (TMPA), which interacts with Nur77 with high affinity and at specific sites. TMPA binding of Nur77 results in the release and shuttling of LKB1 to the cytoplasm to phosphorylate AMPKα. Moreover, TMPA effectively reduces blood glucose and alleviates insulin resistance in type II db/db and high-fat diet- and streptozotocin-induced diabetic mice but not in diabetic littermates with the Nur77 gene knocked out. This study attains a mechanistic understanding of the regulation of LKB1-AMPK axis and implicates Nur77 as a new and amenable target for the design and development of therapeutics to treat metabolic diseases.

Protein phosphorylation-acetylation cascade connects growth factor deprivation to autophagy.

Different from unicellular organisms, metazoan cells require the presence of extracellular growth factors to utilize environmental nutrients. However, the underlying mechanism was unclear. We have delineated a pathway, in which glycogen synthase kinase 3 (GSK3) in cells deprived of growth factors phosphorylates and activates the acetyltransferase KAT5/TIP60, which in turn stimulates the protein kinase ULK1 to elicit autophagy. Cells with the Kat5/Tip60 gene replaced with Kat5(S86A) that cannot be phosphorylated by GSK3 are resistant to serum starvation-induced autophagy. Acetylation sites on ULK1 were mapped to K162 and K606, and the acetylation-defective mutant ULK1(K162,606R) displays reduced kinase activity and fails to rescue autophagy in Ulk1(-/-) mouse embryonic fibroblasts, indicating that acetylation is vital to the activation of ULK1. The GSK3-KAT5-ULK1 cascade seems to be specific for cells to sense growth factors, as KAT5 phosphorylation is not enhanced under glucose deprivation. Distinct from the glucose starvation-autophagy pathway that is conserved in all eukaryotic organisms, the growth factor deprivation response pathway is perhaps unique to metazoan organisms.

GSK3-TIP60-ULK1 signaling pathway links growth factor deprivation to autophagy.

In metazoans, cells depend on extracellular growth factors for energy homeostasis. We found that glycogen synthase kinase-3 (GSK3), when deinhibited by default in cells deprived of growth factors, activates acetyltransferase TIP60 through phosphorylating TIP60-Ser(86), which directly acetylates and stimulates the protein kinase ULK1, which is required for autophagy. Cells engineered to express TIP60(S86A) that cannot be phosphorylated by GSK3 could not undergo serum deprivation-induced autophagy. An acetylation-defective mutant of ULK1 failed to rescue autophagy in ULK1(-/-) mouse embryonic fibroblasts. Cells used signaling from GSK3 to TIP60 and ULK1 to regulate autophagy when deprived of serum but not glucose. These findings uncover an activating pathway that integrates protein phosphorylation and acetylation to connect growth factor deprivation to autophagy.