PCK2 maintains intestinal homeostasis and prevents colitis by protecting antibody-secreting cells from oxidative stress.

Maintaining intracellular redox balance is essential for the survival, antibody secretion, and mucosal immune homeostasis of immunoglobulin A (IgA) antibody-secreting cells (ASCs). However, the relationship between mitochondrial metabolic enzymes and the redox balance in ASCs has yet to be comprehensively studied. Our study unveils the pivotal role of mitochondrial enzyme PCK2 in regulating ASCs' redox balance and intestinal homeostasis. We discover that PCK2 loss, whether globally or in B cells, exacerbates dextran sodium sulphate (DSS)-induced colitis due to increased IgA ASC cell death and diminished antibody production. Mechanistically, the absence of PCK2 diverts glutamine into the TCA cycle, leading to heightened TCA flux and excessive mitochondrial reactive oxygen species (mtROS) production. In addition, PCK2 loss reduces glutamine availability for glutathione (GSH) synthesis, resulting in a decrease of total glutathione level. The elevated mtROS and reduced GSH expose ASCs to overwhelming oxidative stress, culminating in cell apoptosis. Crucially, we found that the mitochondria-targeted antioxidant Mitoquinone (Mito-Q) can mitigate the detrimental effects of PCK2 deficiency in IgA ASCs, thereby alleviating colitis in mice. Our findings highlight PCK2 as a key player in IgA ASC survival and provide a potential new target for colitis treatment.

Toll-like receptor 4 deficiency in mice impairs venous thrombus resolution.

Objective: Toll-like receptor 4 (TLR4) is crucial to the development of sterile inflammatory responses. The deep venous thrombosis resolution (DVT) is similar to sterile inflammation, so we hypothesize that TLR4 is involved. Methods and Results: We evaluated the effects of TLR4 deficiency on thrombus lysis in vivo, and explored the mechanisms in vitro. DVT mouse model was established by inferior vena cava (IVC) ligation. After the IVC ligation (1, 3, and 7 d), the mice were euthanized to collect the venous thrombus. The Tlr4-/- mice had significantly elevated weight/length ratios of thrombi at 3 and 7 d and increased collagen content at 3 d after IVC ligation, in addition to significantly lesser intrathrombus infiltration of neutrophils and macrophages, lower monocyte chemoattractant protein-1 (MCP-1) and matrix metalloproteinase-9 (MMP-9) expression in thrombus tissue sections and homogenates, and lower pro-MMP-9 activity at 3 d after IVC ligation than wild-type mice. After 7 days of IVC ligation, VEGF, IFNß, and MCP-5 protein expression were decreased in venous thrombus from Tlr4-/- mice. 2 ml of 3% thioglycolate was injected intraperitoneally and peritoneal exudate was collected 3 days later from Tlr4-/- and wild type mice respectively. The intraperitoneal macrophages were isolated from adherent culture after centrifugation. Lipopolysaccharide (LPS) can activate TLR4/NF-κB signalling pathway in a concentration-dependent manner, initiated p65 nuclear translocation, IκBα phosphorylation and degradation, MMP-9 and MCP-1 transcription in WT intraperitoneal macrophages but not in Tlr4-/- intraperitoneal macrophages. Conclusion: TLR4 is involved in venous thrombosis resolution through NF-κB pathway. Loss of TLR4 in mice impairs the process.

Tanshinone functions as a coenzyme that confers gain of function of NQO1 to suppress ferroptosis.

Ferroptosis is triggered by the breakdown of cellular iron-dependent redox homeostasis and the abnormal accumulation of lipid ROS. Cells have evolved defense mechanisms to prevent lipid ROS accumulation and ferroptosis. Using a library of more than 4,000 bioactive compounds, we show that tanshinone from Salvia miltiorrhiza (Danshen) has very potent inhibitory activity against ferroptosis. Mechanistically, we found that tanshinone functions as a coenzyme for NAD(P)H:quinone oxidoreductase 1 (NQO1), which detoxifies lipid peroxyl radicals and inhibits ferroptosis both in vitro and in vivo. Although NQO1 is recognized as an oxidative stress response gene, it does not appear to have a direct role in ferroptosis inhibition in the absence of tanshinone. Here, we demonstrate a gain of function of NQO1 induced by tanshinone, which is a novel mechanism for ferroptosis inhibition. Using mouse models of acute liver injury and ischemia/reperfusion heart injury, we observed that tanshinone displays protective effects in both the liver and the heart in a manner dependent on NQO1. Our results link the clinical use of tanshinone to its activity in ferroptosis inhibition.

Integration analysis identifies the role of metallothionein in the progression from hepatic steatosis to steatohepatitis.

Background: Non-alcoholic fatty liver disease (NAFLD), a metabolic disorder that develops from non-alcoholic fatty liver (NAFL) to non-alcoholic steatohepatitis (NASH), has become an epidemic of chronic liver dysfunction worldwide. However, mechanisms that govern the transition from NAFL to NASH have not been fully elucidated. Methods: Gene expression profile data of NAFLD liver tissues were obtained from Gene Expression Omnibus (GEO), including three microarray datasets with 60 NAFL and 44 NASH patients. Integrative differentially expressed genes (DEGs) between NAFL and NASH patients were identified using robust rank aggregation (RRA) analysis. Hub genes were identified combined with gene ontology functional annotation and protein-protein interaction network construction and validated using a sequencing dataset. Huh-7 cells with palmitate-induced lipid overload and NAFLD-diet mouse model of different stages were used to verify our findings. Results: RRA analysis determined 70 robust DEGs between NAFL and NASH. The most robustly upregulated genes were SPP1, AKR1B10, CHST9, and ANXA2, while the most robustly downregulated DEGs were SNORD94, SCARNA10, SNORA20, and MT1M. Cellular response to zinc ion (GO: 0071294) ranked first in GO analysis of downregulated genes, and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment showed that mineral absorption (hsa04978) was significantly enriched. The involvement of the metallothionein pathway was further validated by the decrease of Mt1 expression during NAFL to NASH progression in NAFLD mice and the protection from lipotoxicity in liver cells by overexpressing MT1M. Conclusions: Our integrated analysis identified novel gene signatures and provided comprehensive molecular mechanisms underlying the transition from NAFL to NASH. Metallothionein might be a potential intervention target for NAFLD progression.

TET2 deficiency sensitizes tumor cells to statins by reducing HMGCS1 expression.

TET2 (ten-eleven-translocation) protein is a Fe(II)- and α-ketoglutarate-dependent dioxygenase that catalyzes DNA demethylation to regulate gene expression. While TET2 gene is frequently mutated in hematological cancer, its enzymatic activity is also compromised in various solid tumors. Whether TET2 deficiency creates vulnerability for cancer cells has not been studied. Here we reported that TET2 deficiency is associated with the change of lipid metabolism processes in acute myeloid leukemia (AML) patient. We demonstrate that statins, the inhibitors of ß-Hydroxy ß-methylglutaryl-CoA (HMG-CoA) reductase and commonly used cholesterol-lowering medicines, significantly sensitize TET2 deficient tumor cells to apoptosis. TET2 directly regulates the expression of HMG-CoA synthase (HMGCS1) by catalyzing demethylation on its promoter region, and conversely TET2 deficiency leads to significant down-regulation of HMGCS1 expression and the mevalonate pathway. Consistently, overexpression of HMGCS1 in TET2-deficient cells rescues statin-induced apoptosis. We further reveal that decrease of geranylgeranyl diphosphate (GGPP), an intermediate metabolite in the mevalonate pathway, is responsible for statin-induced apoptosis. GGPP shortage abolishes normal membrane localization and function of multiple small GTPases, leading to cell dysfunction. Collectively, our study reveals a vulnerability in TET2 deficient tumor and a potential therapeutic strategy using an already approved safe medicine.

A Class of Disulfide Compounds Suppresses Ferroptosis by Stabilizing GPX4.

Ferroptosis is a nonapoptotic form of cell death characterized by iron-dependent lipid peroxidation and has been implicated in multiple pathological conditions. Glutathione peroxidase 4 (GPX4) plays an essential role in inhibiting ferroptosis by eliminating lipid peroxide using glutathione (GSH) as a reductant. In this study, we found Ellman's reagent DTNB and a series of disulfide compounds, including disulfiram (DSF), an FDA-approved drug, which protect cells from erastin-induced ferroptosis. Mechanistically, DTNB or DSF is conjugated to multiple cysteine residues in GPX4 and disrupts GPX4 interaction with HSC70, an adaptor protein for chaperone mediated autophagy, thus preventing GPX4 degradation induced by erastin. In addition, DSF ameliorates concanavalin A induced acute liver injury by suppressing ferroptosis in a mouse model. Our work reveals a novel regulatory mechanism for GPX4 protein stability control. We also discover disulfide compounds as a new class of ferroptosis inhibitors and suggest therapeutic repurposing of DSF in treating ferroptosis-related diseases.

ALK fusion promotes metabolic reprogramming of cancer cells by transcriptionally upregulating PFKFB3.

Anaplastic lymphoma kinase (ALK), a receptor tyrosine kinase of the insulin receptor kinase subfamily, is activated in multiple cancer types through translocation or overexpression. Although several generations of ALK tyrosine kinase inhibitors (TKIs) have been developed for clinic use, drug resistance remains a major challenge. In this study, by quantitative proteomic approach, we identified the glycolytic regulatory enzyme, 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3 (PFKFB3), as a new target of ALK. Expression of PFKFB3 is highly dependent on ALK activity in ALK+ anaplastic large cell lymphoma and non-small-cell lung cancer (NSCLC) cells. Notably, ALK and PFKFB3 expressions exhibit significant correlation in clinic ALK+ NSCLC samples. We further demonstrated that ALK promotes PFKFB3 transcription through the downstream transcription factor STAT3. Upregulation of PFKFB3 by ALK is important for high glycolysis level as well as oncogenic activity of ALK+ lymphoma cells. Finally, targeting PFKFB3 by its inhibitor can overcome drug resistance in cells bearing TKI-resistant mutants of ALK. Collectively, our studies reveal a novel ALK-STAT3-PFKFB3 axis to promote cell proliferation and tumorigenesis, providing an alternative strategy for the treatment of ALK-positive tumors.

WWC proteins mediate LATS1/2 activation by Hippo kinases and imply a tumor suppression strategy.

YAP and TAZ (YAP/TAZ), two major effectors of the Hippo signaling pathway, are frequently activated in human cancers. The activity of YAP/TAZ is strictly repressed upon phosphorylation by LATS1/2 tumor suppressors. However, it is unclear how LATS1/2 are precisely regulated by upstream factors such as Hippo kinases MST1/2. Here, we show that WWC proteins (WWC1/2/3) directly interact with LATS1/2 and SAV1, and SAV1, in turn, brings in MST1/2 to phosphorylate and activate LATS1/2. Hence, WWC1/2/3 play an organizer role in a signaling module that mediates LATS1/2 activation by MST1/2. Moreover, we have defined a minimum protein interaction interface on WWC1/2/3 that is sufficient to activate LATS1/2 in a robust and specific manner. The corresponding minigene, dubbed as SuperHippo, can effectively suppress tumorigenesis in multiple tumor models. Our study has uncovered a molecular mechanism underlying LATS1/2 regulation and provides a strategy for treating diverse malignancies related to Hippo pathway dysregulation.

Elevated nuclear localization of glycolytic enzyme TPI1 promotes lung adenocarcinoma and enhances chemoresistance.

Increased glycolysis is a hallmark of tumor, which can provide tumor cells with energy and building blocks to promote cell proliferation. Recent studies have shown that not only the expression of glycolytic genes but also their subcellular localization undergoes a variety of changes to promote development of different types of tumors. In this study, we performed a comprehensive analysis of glycolysis and gluconeogenesis genes based on data from TCGA to identify those with significant tumor-promoting potential across 14 types of tumors. This analysis not only confirms genes that are known to be involved in tumorigenesis, but also reveals a significant correlation of triosephosphate isomerase 1 (TPI1) with poor prognosis, especially in lung adenocarcinoma (LUAD). TPI1 is a glycolytic enzyme that interconverts dihydroxyacetone phosphate (DHAP) to glyceraldehyde 3-phosphate (GAP). We confirm the upregulation of TPI1 expression in clinical LUAD samples and an inverse correlation with the overall patient survival. Knocking down of TPI1 in lung cancer cells significantly reduced cell migration, colony formation, and xenograft tumor growth. Surprisingly, we found that the oncogenic function of TPI1 depends on its translocation to cell nucleus rather than its catalytic activity. Significant accumulation of TPI1 in cell nucleus was observed in LUAD tumor tissues compared with the cytoplasm localization in adjacent normal tissues. Moreover, nuclear translocation of TPI1 is induced by extracellular stress (such as chemotherapy agents and peroxide), which facilitates the chemoresistance of cancer cells. Our study uncovers a novel function of the glycolytic enzyme TPI1 in the LUAD.

Elaiophylin reduces body weight and lowers glucose levels in obese mice by activating AMPK.

Obesity is an epidemic affecting 13% of the global population and increasing the risk of many chronic diseases. However, only several drugs are licensed for pharmacological intervention for the treatment of obesity. As a master regulator of metabolism, the therapeutic potential of AMPK is widely recognized and aggressively pursued for the treatment of metabolic diseases. We found that elaiophylin (Ela) rapidly activates AMPK in a panel of cancer-cell lines, as well as primary hepatocytes and adipocytes. Meanwhile, Ela inhibits the mTORC1 complex, turning on catabolism and turning off anabolism together with AMPK. In vitro and in vivo studies showed that Ela does not activate AMPK directly, instead, it increases cellular AMP/ATP and ADP/ATP ratios, leading to AMPK phosphorylation in a LKB1-dependent manner. AMPK activation induced by Ela caused changes in diverse metabolic genes, thereby promoting glucose consumption and fatty acid oxidation. Importantly, Ela activates AMPK in mouse liver and adipose tissue. As a consequence, it reduces body weight and blood glucose levels and improves glucose and insulin tolerance in both ob/ob and high-fat diet-induced obese mouse models. Our study has identified a novel AMPK activator as a candidate drug for the treatment of obesity and its associated chronic diseases.

CBFB-MYH11 Fusion Sequesters RUNX1 in Cytoplasm to Prevent DNMT3A Recruitment to Target Genes in AML.

A growing number of human diseases have been found to be associated with aberrant DNA methylation, including cancer. Mutations targeting genes encoding DNA methyltransferase (DNMT), TET family of DNA demethylases, and isocitrate dehydrogenase (IDH1, IDH2) that produce TET inhibitory metabolite, 2-hyoxyglutarate (2-HG), are found in more than half of acute myeloid leukemia (AML). To gain new insights into the regulation of DNA de/methylation and consequence of its alteration in cancer development, we searched for genes which are mutated in a manner that is linked with gene mutations involved in DNA de/methylation in multiple cancer types. We found that recurrent CBFB-MYH11 fusions, which result in the expression of fusion protein comprising core-binding factor ß (CBFB) and myosin heavy chain 11 (MYH11) and are found in 6∼8% of AML patients, occur mutually exclusively with DNMT3A mutations. Tumors bearing CBFB-MYH11 fusion show DNA hypomethylation patterns similar to those with loss-of-function mutation of DNMT3A. Expression of CBFB-MYH11 fusion or inhibition of DNMT3A similarly impairs the methylation and expression of target genes of Runt related transcription factor 1 (RUNX1), a functional partner of CBFB. We demonstrate that RUNX1 directly interacts with DNMT3A and that CBFB-MYH11 fusion protein sequesters RUNX1 in the cytoplasm, thereby preventing RUNX1 from interacting with and recruiting DNMT3A to its target genes. Our results identify a novel regulation of DNA methylation and provide a molecular basis how CBFB-MYH11 fusion contributes to leukemogenesis.

Structural insights into TSC complex assembly and GAP activity on Rheb.

Tuberous sclerosis complex (TSC) integrates upstream stimuli and regulates cell growth by controlling the activity of mTORC1. TSC complex functions as a GTPase-activating protein (GAP) towards small GTPase Rheb and inhibits Rheb-mediated activation of mTORC1. Mutations in TSC genes cause tuberous sclerosis. In this study, the near-atomic resolution structure of human TSC complex reveals an arch-shaped architecture, with a 2:2:1 stoichiometry of TSC1, TSC2, and TBC1D7. This asymmetric complex consists of two interweaved TSC1 coiled-coil and one TBC1D7 that spans over the tail-to-tail TSC2 dimer. The two TSC2 GAP domains are symmetrically cradled within the core module formed by TSC2 dimerization domain and central coiled-coil of TSC1. Structural and biochemical analyses reveal TSC2 GAP-Rheb complimentary interactions and suggest a catalytic mechanism, by which an asparagine thumb (N1643) stabilizes γ-phosphate of GTP and accelerate GTP hydrolysis of Rheb. Our study reveals mechanisms of TSC complex assembly and GAP activity.

Effectiveness and clinical benefit of a therapy of combined non-pharmaceutical Traditional Chinese Medicine for knee osteoarthritis: a randomized controlled study.

OBJECTIVE: To compare the efficacy of honey mouthwash 12.5% and chlorhexidine solution 0.2% to reduce the rate of oropharyngeal bacterial colonization in mechanically-ventilated patients. METHODS: This study was a randomized, single blind, phase Ⅲ controlled clinical trial. Sixty patients newly admitted to internal and trauma Intensive Care Units of the two educational hospitals of Sanandaj city affiliated with Kurdistan University of Medical Sciences were selected by convenience sampling and allocated to two groups of 30 patients using random blocks design. In each group, the mouthwash was applied twice a day for four consecutive days. Swab samples were taken from the mouth and throat of all patients three times a day (pre- intervention, two days, and four days after the intervention) and then the samples were transferred onto the blood agar and eosin methylene blue (EMB) culture plates and investigated for bacterial growth and colonization after 24-48 h. RESULTS: The findings showed that oropharyngeal colonization was not significantly different between the two groups, pre-intervention, two days, and four days after the intervention (P > 0.05). Rinsing with honey mouthwash 12.5% led to the inhibition of Staphylococcus aureus and Pseudomonas aeruginosa on the fourth day of the intervention in all samples. CONCLUSION: None of the studied solutions contributed to the reduction of oropharyngeal bacterial colonization. It seems that the growth inhibition of Staphylococcus aureus and Pseudomonas aeruginosa by the honey 12.5% mouthwash in mechanically-ventilated patients need further investigation.

Targeting ferroptosis alleviates methionine-choline deficient (MCD)-diet induced NASH by suppressing liver lipotoxicity.

BACKGROUND: NASH is one of the fastest growing liver diseases that leads to severe steatosis, inflammation and ultimately liver injury. However, the pathophysiological mechanisms of NASH remain unclear and pharmacological treatment against the disease is unavailable currently. Ferroptosis is a non-apoptotic form of cell death induced by iron-dependent lipid peroxidation. Since NASH progression is accompanied by massive lipid accumulation, which generates lipotoxic species, we investigated the role of ferroptosis in NASH progression. METHOD: Mice were fed on MCD-diet to mimic NASH progression and gene expression in liver was analysed by RNA-seq. The occurrence of hepatic ferroptosis was measured by lipid ROS level, electron microscopy and in vivo PI staining. The beneficial effects of ferroptosis inhibitors on NASH was evaluated by liver pathology analysis. The mechanism of lipid ROS induced lipid droplets accumulation was investigated by in vitro cell culture. RESULTS: RNA-seq analysis suggested that elevated arachidonic acid metabolism promotes ferroptosis in MCD-diet fed mouse livers, which was further demonstrated by lipid ROS accumulation, morphological change of mitochondria and increased cell death. Iron accumulation was detected in the liver and the serum of MCD-fed mice. Scavenging of ferroptosis-linked lipid peroxides reduced lipid accumulation both in vivo and in vitro. Importantly, ferroptosis inhibitors alleviated MCD-diet induced inflammation, fibrogenesis and liver injury. Finally, lipid ROS promotes liver steatosis by boosting lipid droplets formation. CONCLUSION: Our results demonstrate an important role of ferroptosis in the progression of MCD-diet induced NASH and suggest that ferroptosis may serve as a therapeutic target for NASH treatment.

Scopoletin Activates Adenosine Monophosphate-Activated Protein Kinase/Mammalian Target of Rapamycin Signaling Pathway and Improves Functional Recovery after Spinal Cord Injury in Rats.

BACKGROUND: Scopoletin (SPT) is known to exert neuroprotective autophagy effect. However, the efficacy of SPT in the treatment of spinal cord injury (SCI) has yet not been explored. The investigation was intended to elucidate whether SPT can exert neuroprotective effect by triggering neuronal autophagy after SCI. The study was also directed to investigate the role of adenosine monophosphate-activated protein kinase (AMPK)/mammalian target of rapamycin (mTOR) signaling pathway in the autophagy facilitated by SPT. MATERIALS AND METHODS: The SCI was developed in female Sprague-Dawley rats by damaging the T10 spinal level using an impounder impact. Three animals groups were investigated - Sham group, SCI group, and SCI + SPT group. The SCI + SPT group was administered with SPT (100 mg/kg) intraperitoneally. Basso, Beattie, and Bresnahan scores and angle of incline test revealed that SPT administration improved the movement of hind limbs after SCI induction. RESULTS: Results indicated that SPT imparted neuronal protection, alleviated neuronal apoptosis, and improved neuronal autophagy. SPT-induced autophagy was identified by increased Beclin-1 expression and LC3B-positive neuronal cells. Further investigations revealed that SPT triggers the pathway involving AMPK/mTOR signaling, thereby stimulating autophagy in SCI-induced rat model. CONCLUSION: The findings of the present investigation strongly advocate the beneficial effects of SPT in the treatment of the SCI. SPT ameliorates the AMPK/mTOR signaling-induced autophagy and thereby improves functional recovery in SCI-induced rats.

A clinical study on the association of clinical outcome and acute systolic blood pressure in cerebral hemorrhage patients
.

OBJECTIVE: To investigate the outcome of the rapid lowering of elevated blood pressure in patients with intracerebral hemorrhage and to understand its association with clinical outcome. MATERIALS AND METHODS: Between July 2014 and June 2018, a total of 1,500 patients diagnosed with cerebral hemorrhage were randomized and assessed for their neurological symptoms and diagnosed with CT scan. 1,500 (42%) patients received intensive treatment, while 1,645 (58%) patients were assigned the guideline-recommended therapy. The systolic blood pressure of these patients was measured every half hour during the first day of admission. The intensive-treatment group was further categorized into five different subgroups in 10-mmHg intervals. On the other hand, the clinical outcome, as represented by the volume of hematoma, adverse events, modified Rankin scale etc., was measured and analyzed. RESULTS: The volume of hematoma varied with a p-value of 0.014 among the investigated groups. There was no direct correlation among the five groups based on the systolic blood pressure groups and modified Rankin scale 4 - 6. The 140 - 150 mmHg group observed an elevated risk compared to the 120 - 130 mmHg group in the modified Rankin scale ((OR = 1.59; 95% CI (0.98 - 2.61)). The hematoma enlargement increased significantly with a p-value of 0.012. There was no direct association or statistical significance between the occurrence of the clinical outcome and the multivariate relationship between the five groups based on the multivariates (p = 0.513). CONCLUSION: Systolic blood pressure ranging between 120 and 130 mmHg serves as an optimal goal for acute intracerebral hemorrhage by reducing the hematoma enlargement. It is also evident that the lowering of high mean systolic blood pressure after blood pressure-lowering therapy usually leads to cardiorenal injury.

The oncometabolite 2-hydroxyglutarate produced by mutant IDH1 sensitizes cells to ferroptosis.

Ferroptosis is a non-apoptotic form of cell death characterized by the iron-dependent lipid peroxidation and is implicated in several human pathologies, such as tissue ischemia, neurodegeneration, and cancer. Ferroptosis appears to be high cell-context dependent and the regulation of ferroptosis by physiological or pathological conditions are unclear. Here, we report that tumor-derived IDH1 mutation sensitizes cells to ferroptosis. Deletion of the mutant IDH1 allele in IDH1 heterozygous tumor cells or pharmacological inhibition of mutant IDH1 to produce the oncometabolite D-2-hydroxyglutarate (D-2-HG) confers resistance to erastin-induced ferroptosis. Conversely, ectopic expression of mutant IDH1 or treatment of cells with cell-permeable D-2-HG promotes the accumulation of lipid reactive oxygen species (ROS) and subsequently ferroptosis. Mechanistically, mutant IDH1 reduces the protein level of the glutathione peroxidase 4 (GPX4), a key enzyme in removing lipid ROS and ferroptosis, and promotes depletion of glutathione. Our results uncover a new role of mutant IDH1 and 2-HG in ferroptosis.

ELP3 Acetyltransferase is phosphorylated and regulated by the oncogenic anaplastic lymphoma kinase (ALK).

Protein lysine acetylation is one of the major posttranslational modifications (PTMs) with several thousands of proteins identified to be acetylated in mammalian tissues. Mechanistic studies have revealed important functions of acetylation in the regulation of protein function. Much less is known on how the acetyltransferases themselves are regulated. In the current study, we discover that the Elongator protein 3 (ELP3) acetyltransferase is modified by tyrosine phosphorylation. We demonstrate that the anaplastic lymphoma kinase (ALK) is the major tyrosine kinase responsible for ELP3 tyrosine phosphorylation. ELP3 is phosphorylated in tumor cells expressing oncogenic NPM-ALK fusion protein. We further identify Tyr202 as the major ALK phosphorylation site in ELP3. Importantly, the introduction of Y202 phosphorylation mutant ELP3 into ALK-positive tumor cells reduced cell growth and impaired gene expression. Collectively, our study reveals a novel regulatory mechanism for ELP3, provides an example that acetyltransferase itself can be regulated by PTM, and suggests a potential target for ALK-positive cancer therapies.

GPCR signaling inhibits mTORC1 via PKA phosphorylation of Raptor.

The mammalian target of rapamycin complex 1 (mTORC1) regulates cell growth, metabolism, and autophagy. Extensive research has focused on pathways that activate mTORC1 like growth factors and amino acids; however, much less is known about signaling cues that directly inhibit mTORC1 activity. Here, we report that G-protein coupled receptors (GPCRs) paired to Gαs proteins increase cyclic adenosine 3'5' monophosphate (cAMP) to activate protein kinase A (PKA) and inhibit mTORC1. Mechanistically, PKA phosphorylates the mTORC1 component Raptor on Ser 791, leading to decreased mTORC1 activity. Consistently, in cells where Raptor Ser 791 is mutated to Ala, mTORC1 activity is partially rescued even after PKA activation. Gαs-coupled GPCRs stimulation leads to inhibition of mTORC1 in multiple cell lines and mouse tissues. Our results uncover a signaling pathway that directly inhibits mTORC1, and suggest that GPCRs paired to Gαs proteins may be potential therapeutic targets for human diseases with hyperactivated mTORC1.