MDH1 Research Articles

The disassembly of lipid droplets in Chlamydomonas.

Lipid droplets (LDs) are ubiquitous and specialized organelles in eukaryotic cells. Consisting of a triacylglycerol core surrounded by a monolayer of membrane lipids, LDs are decorated with proteins and have myriad functions, from carbon/energy storage to membrane lipid remodeling and signal transduction. The biogenesis and turnover of LDs are therefore tightly coordinated with cellular metabolic needs in a fluctuating environment. Lipid droplet turnover requires remodeling of the protein coat, lipolysis, autophagy and fatty acid β-oxidation. Several key components of these processes have been identified in Chlamydomonas (Chlamydomonas reinhardtii), including the major lipid droplet protein, a CXC-domain containing regulatory protein, the phosphatidylethanolamine-binding DTH1 (DELAYED IN TAG HYDROLYSIS1), two lipases and two enzymes involved in fatty acid β-oxidation. Here, we review LD turnover and discuss its physiological significance in Chlamydomonas, a major model green microalga in research on algal oil.

Microglial lnc-U90926 facilitates neutrophil infiltration in ischemic stroke via MDH2/CXCL2 axis

Dysregulated long non-coding RNAs (lncRNAs) have been shown to contribute to the pathogenesis of ischemic stroke. However, the potential role of lncRNAs in post-stroke microglial activation remains largely unknown. Here, we uncovered that lncRNA-U90926 was significantly increased in microglia exposed to ischemia/reperfusion both invivo and invitro. In addition, adenovirus-associated virus (AAV)-mediated microglial U90926 silencing alleviated neurological deficits and reduced infarct volume in experimental stroke mice. Microglial U90926 knockdown could reduce the infiltration of neutrophils into ischemic lesion site, which might be attributed to the downregulation of C-X-C motif ligand 2 (CXCL2). Mechanistically, U90926 directly bound to malate dehydrogenase 2 (MDH2) and competitively inhibited the binding of MDH2 to the CXCL2 3' untranslated region (UTR), thus protecting against MDH2-mediated decay of CXCL2 mRNA. Taken together, our study demonstrated that microglial U90926 aggravated ischemic brain injury via facilitating neutrophil infiltration, suggesting that U90926 might be a potential biomarker and therapeutic target for ischemic stroke.

Hypertension due to loss of water

In the current issue of Acta Physiologica, two papers are published investigating the development of arterial hypertension under conditions of pathological loss of free water in two disease models. Kovaric et al 1 studied rats with chronic renal failure, shedding excessive free water via their remnant kidney due to a hypostenuric concentrating defect. Wild et al. 2 investigated a transgenic mouse model for psoriasis, losing free water via the psoriatic skin lesions. In both cases the animals displayed hypertension, cutaneous vasoconstriction, and metabolic adaptations towards enhanced production of urea and organic osmolytes typical for a phylogenetically ancient water conservation response termed aestivation 3 . The international consortia of investigators spearheaded by Titze, Karbach, and Kitada therefore conclude that in both cases the hypertension was due to cutaneous vasoconstriction in an attempt to limit epidermal water loss. Because in either case the hypertension was associated with loss of water, and thus with hypo- rather than hypervolemia, the authors argue, that this concept of pathogenesis of hypertension is antipodal to the classic view that relies on hypervolemia due to sodium retention.

Not gone but forgotten: Tritrichomonas foetus in extensively-managed bulls from Australia’s Northern Territory

Bovine trichomonosis, caused by infection with the protozoan parasite Tritrichomonas foetus, is globally recognised as a cause of reproductive failure in cattle. Maintained in clinically normal bulls, T. foetus infection results in infertility and abortion in infected cows. In Australia’s Northern Territory (NT), logistical limitations associated with extensive livestock production inhibit wide-scale testing and diagnosis, allowing the parasite to persist undetected. In the present study, T. foetus was detected in 18/109 preputial cultures collected from bulls on a property in the NT with a history of low birth rates and reproductive failure using real-time PCR testing. Of the T. foetus-positive samples, 13/18 were genotyped using the internal transcribed spacer regions (ITS1 and ITS2) and the 5.8S rDNA unit. Selected samples were further characterised using the protein-coding genes of cysteine proteases (CP-1, 2, 4–9) and cytosolic malate dehydrogenase 1 (MDH-1) to determine if the isolates were ‘bovineʼ, ‘felineʼ or ‘Southern Africaʼ genotypes. All samples were 100% identical to the T. foetus ‘bovine’ genotype across all markers. This is the first reported case of trichomonosis in Australian cattle since 1988 and is a reminder that T. foetus should be considered whenever reproductive failure occurs in extensive cattle systems.

A Hydride Transfer Complex Reprograms NAD Metabolism Preventing Senescence

Metabolic rewiring and redox balance play pivotal roles in cancer. Cellular senescence is a barrier for tumorigenesis circumvented in cancer cells by poorly understood mechanisms. We report a novel multi-enzymatic complex that reprograms NAD metabolism by transferring reducing equivalents from NADH to NADP +. This hydride transfer complex (HTC) is assembled by Malate Dehydrogenase-1, Malic Enzyme-1 and cytosolic Pyruvate Carboxylase. HTC enzymes are found in phase-separated bodies in the cytosol of cancer cells and can be assembled in vitro from purified proteins. They are repressed in senescent cells but induced by p53 inactivation. HTC enzymes are highly expressed in mouse and human prostate cancer models and their inactivation triggers senescence. Exogenous expression of HTC is sufficient to bypass senescence, rescue cells from complex I inhibitors and cooperate with oncogenic RAS to transform primary cells. We provide evidence for a new multi-enzymatic complex that reprograms metabolism and prevents cellular senescence.

Abstract 2083: Alternative splicing regulation by the androgen receptor in prostate cancer cells

Abstract The androgen receptor (AR) is a transcription factor that drives prostate cancer (PCa) by modulating the expression of thousands of genes to promote proliferation and survival and to reprogram metabolism. However, how AR activation affects alternative splicing is mostly unknown. Our objective was to define its role in the transcriptome-wide regulation of alternative splicing. Three human PCa models–LNCaP, LAPC4, and 22Rv1 cells–were treated with and without androgens, and RNA was purified for deep-sequencing analyses (RNA-seq). Several bio-informatic tools were then used to study alternative splicing. We demonstrate that in the absence of androgens, alternative splicing complexity is similar among AR-positive PCa cells, with 48% of all transcripts having various levels of alternative splicing. We also describe splicing differences among cell lines, such as specific splicing of AR, REST, TSC2, and CTBP1. Interestingly, AR activation changed the alternative splicing of thousands of transcripts in all the cell lines tested. Overlap between AR-sensitive alternative splicing events revealed that genes linked to PCa metabolism are major targets for this specific modulation. These genes encode metabolic enzymes such as the prostate-specific membrane antigen (FOLH1), malate dehydrogenase 1 (MDH1), and malic enzyme 2 (ME2). Enzymatic assays revealed that AR alters their activities without changing their total gene expression levels, demonstrating that AR-driven regulation of alternative splicing has a direct effect on cancer cell metabolism. Overall, our study presents a comprehensive analysis of PCa cell transcriptome and its modulation by AR, revealing cell metabolism as a key target for AR-dependent regulation of alternative splicing. Citation Format: Lucas Germain, Camille Lafront, Jolyane Beaudette, Raghavendra Tejo Karthik Poluri, Cindy Weidmann, Etienne Audet-Walsh. Alternative splicing regulation by the androgen receptor in prostate cancer cells [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2083.

Alternative splicing regulation by the androgen receptor in prostate cancer cells

The androgen receptor (AR) is a transcription factor that drives prostate cancer (PCa) by modulating the expression of thousands of genes to promote proliferation and survival and to reprogram metabolism. However, how AR activation controls alternative splicing is mostly unknown. Our objective was to define its role in the transcriptome-wide regulation of alternative splicing. Three human PCa models—LNCaP, LAPC4, and 22Rv1 cells—were treated with and without androgens, and RNA was purified for deep-sequencing analyses (RNA-seq). Several bioinformatic tools were then used to study alternative splicing. We demonstrate that in the absence of androgens, alternative splicing complexity is similar among AR-positive PCa cells, with 48 % of all transcripts having various levels of alternative splicing. We also describe alternative splicing differences among cell lines, such as specific splicing of AR, REST, TSC2, and CTBP1. Interestingly, AR activation changed the alternative splicing of thousands of genes in all the PCa cell lines tested. Overlap between AR-sensitive alternative splicing events revealed that genes linked to cell metabolism are major targets for this specific modulation. These genes encode metabolic enzymes such as the prostate-specific membrane antigen, encoded by FOLH1, and the malate dehydrogenase 1 (MDH1). Overall, our study presents a comprehensive analysis of the PCa cell transcriptome and its modulation by AR, revealing a significant enrichment of metabolic genes in this AR-dependent regulation of alternative splicing.

Label-free quantitative proteomics analysis of skin of yellow drum (Nibea albiflora) reveals immune mechanism against Cryptocaryon irritans

To explore the resistance mechanism of locally infected skin of yellow drum (Nibea albiflora) against Cryptocaryon irritans infection, N. albiflora were infected with C. irritans at a median lethal concentration of 2050 theronts/g fish. Then, the skin of the infected group (24 hT and 72 hT) and the control group (24 hC and 72 hC) were sampled at 24 h and 72 h for quantitative proteomics analysis. A total of 643 proteins were identified, of which 61 proteins were significantly affected by interaction between time and infection, 83 and 119 proteins were significantly affected by the infection and time, respectively. In addition, 17, 61, 81 and 45 differentially expressed proteins (DEPs) were obtained from pairwise comparison (24 hT vs 24 hC, 72 hT vs 72 hC, 72 hT vs 24 hT and 72 hC vs 24 hC), respectively. DEPs in 24 hT vs 24 hC and 72 hT vs 72 hC were mainly enriched in Gene Ontology terms (transferase activity, protein folding and isomerase activity) and Kyoto Encyclopedia of Genes and Genomes pathways (biosynthesis of antibiotics, carbon metabolism and Citrate cycle). Among them, enriched DEPs were malate dehydrogenase 2 (MDH2), malate dehydrogenase 1 ab (MDH 1 ab), citrate synthase, etc. Immune-related DEPs such as complement component C3 and Cell division cycle 42 were involved in response to stimulus and signal transduction, etc. Also, DEPs such as collagen, heat shock protein 75 and MDH2 play a role in helping fish skin wounds to heal and provide energy. Furthermore, protein-protein interaction analysis indicated that 18 proteins such as MDH2, MDH 1 ab, complement C3 and collagen were interrelated. In conclusion, this study found that many proteins in N. albiflora contribute to resist against C. irritans and promote fish recovery.

Characterization of Amplified Malate Dehydrogenase 1 (MDH1) in the Context of Squamous Cell Lung Cancer

Cancer cells often reroute or stimulate their metabolism to support proliferation, which requires a change in the activity or regulation of key metabolic residues. Malate dehydrogenase 1 (MDH1) catalyzes the NADH‐dependent conversion of oxaloacetate to malate, which can help drive glycolysis through the production of NAD+. MDH1 is amplified in many squamous cell lung carcinomas, the second leading cause of cancer related death, and this amplification is associated with a poor prognosis of 50% reduced survival. The precise role of MDH1 in driving squamous cell lung cancer and the mechanisms that regulate this protein are not yet well understood.Here, we develop a tool to examine the effect of MDH1 amplification in an in vitro model. We aim to examine MDH1 amplification in squamous cell lung carcinoma with an isogenic control. It has been previously shown that MDH1 methylation can suppress glutamine metabolism in pancreatic cancers, and that acetylation of MDH1 alters its activity. We will examine how MDH1 amplification affects squamous cell carcinomas by using biochemical kinetics and cellular methods. We have found that MDH1 amplification influences the production of metabolites in the TCA cycle. Through monoclonal single cell isolation, we will generate a squamous epithelial lung cancer cell line stably overexpressing MDH1. We hypothesize that MDH1 has cysteine residues that can be sensitive to oxidative stress which can alter the normal enzymatic activities of MDH1. Through site‐directed mutagenesis, we will mutate a cysteine residue to a serine residue at the 137 position to examine the possible effect of oxidative stress on MDH1. This residue has been shown to be sensitive to oxidation in the mouse form of MDH1. We will also heterologously express and purify MDH1 mutants and perform kinetic assays to examine the impact of amplified MDH1. By understanding the regulatory strategies of MDH1, we hope to identify new pathways that may be targeted therapeutically.Support or Funding InformationThis work was funded by a Research Scholar Grant, RSG‐19‐075‐01‐TBE, from the American Cancer Society (C.D.S.), National Institutes of Health R00 CA187594 (C.D.S.), U54CA132384 (SDSU) & U54CA132379 (UC San Diego), and IMSD 5R25GM058906 (SDSU), as well as the California Metabolic Research Foundation (SDSU).

Catalytic Characterization of Human Malate Dehydrogenase 1 (MDH1)

Lung cancer is the leading cause of cancer death. Non‐small cell lung cancer (NSCLC) makes up 85% of all types of lung cancer, and includes squamous cancer, adenocarcinomas, and large cell cancer. Malate dehydrogenase (MDH) exists as two forms, MDH1 (cytosolic) and MDH2 (mitochondrial). Both forms are elevated in tumor cells, but only high expression of MDH1 is associated with poor prognosis. Patients with amplified MDH1 have a 50% reduction rate of survival. MDH1 appears to support proliferation of tumor cells by fueling glycolysis with the regeneration of nicotinamide adenine dinucleotide (NAD+). However, studies of the kinetics of human MDH1 is limited. Our goal is to characterize the catalytic features and regulation of MDH1. We hypothesized that MDH1 activity is inhibited in the presence of reactive oxygen species (ROS). Human MDH1 was expressed heterogenously from Escherichia coli and purified using immobilized cobalt affinity chromatography. The enzyme catalytic activity was determined in the optimal conditions of salt, pH, temperature, and buffer. In the assay, MDH1 activity was calculated from the oxidation rate of NADH to NAD+ at 340 nm using a spectrophotometer after incubating MDH1 with oxaloacetate and NADH under steady‐state conditions. To test our hypothesis under oxidative environment, we repeated this assay using oxidized glutathione. The enzyme activity was 6‐fold lower than that measured in reducing conditions. In the absence of the enzyme, oxidized glutathione does not convert NADH to NAD+, which implied the enzyme’s conformation was affected and hence the observed loss in enzyme activity. In the future, we will employ site‐directed mutagenesis to gain an insight whether our observed inhibition was due to the sensitivity of cysteine residues to oxidation.Support or Funding InformationThis work was funded by a Research Scholar Grant, RSG‐19‐075‐01‐TBE, from the American Cancer Society (C.D.S.), National Institutes of Health R00 CA187594 (C.D.S.), U54CA132384 (SDSU) & U54CA132379 (UC San Diego), MARC 5T34GM008303 (SDSU), and IMSD 5R25GM058906 (SDSU), as well as the California Metabolic Research Foundation (SDSU).

Impact of Phosphorylation on MDH2 Structure, Function and Protein

Mitochondrial malate dehydrogenase (MDH2) is an enzyme utilizing the NAD/NADH cofactor system in the citric acid cycle to reversibly oxidize malate to oxaloacetate, where citrate synthase (CS) uses oxaloacetate to produce citrate. Coupling of the MDH2‐CS of the two reactions is critical as the thermodynamics of MDH2 alone would not drive the cycle forward. However, shuttling of carbon through the cycle during gluconeogenesis necessitates an uncoupling of the two proteins, allowing for the reversal of the reaction. Thus, a mechanism that favors the known interaction and disrupts substrate channeling through these two enzymes must exist. We performed an analysis of putative MDH2 phosphorylation sites, identified the potential kinases, and conducted a meta‐analysis of mass spectrometry data bases of phosphorylation. We have identified 13 predicted, and 30 identified phosphorylated sites of mammalian MDH2. Further analysis of possible interaction sites and mapping functional domains with these phosphorylation sites highlight the important yet unknown role of phosphorylation to regulate MDH structure and function. In order to better understand MDH2‐CS interactions and MDH function, we synthesized four phosphomimics (S/T‐D) using site directed mutagenesis on a codon optimized human MDH2 gene in a His‐tagged, pET28a construct for bacterial expression. Using purified protein, we show impact of each mutation structure with circular dichroism and the thermal melting profile of the phosphomimics and the wildtype hMDH2. The impact of phosphorylation on MHD function was also studied by comparing kinetic values. Lastly the impact of the mutations on MDH‐CS binding was analyzed using a micro‐protein thermal shift assay.Support or Funding InformationNSF EHR‐IUSE Project support NSF‐1726932

A Novel Role for Malate Dehydrogenase 1 in Acute Myeloid Leukemia Progression

The tumor suppressor role of the metabolic genes is rarely reported. Here, we report that malate dehydrogenase 1 (MDH1) acts as an anti-oncogene in acute myeloid leukemia (AML) progression. In a screen of various leukemia cell lines, as well as peripheral blood mononuclear cells from patients with AML and healthy individuals, we observed that patients with AML had lower mRNA expression of MDH1 than healthy individuals. In addition, we found that overexpression of MDH1 in the AML cell lines MV4-11 and THP-1 in vitro promoted their differentiation, reduced their growth, induced apoptosis, and increased the proportion of cells in S phase, which were more sensitive to the chemotherapeutic drugs cytarabine and daunorubicin. Using an in vivo xenograft mouse model of AML, we found that disruption of MDH1 expression influenced tumor burden and mouse survival. These findings were consistent with an inverse relationship between MDH1 expression and overall survival in two independent cohorts of AML patients. We also identified changes in the expression of other genes, concomitant with reduced MDH1 expression in patients with AML, that largely explain the anti-oncogenic role of MDH1 in AML progression. Notably, downregulation of P53 and upregulation of the MAPK pathway in low MDH1-expressing MV4-11 cells were reversed upon overexpression of MDH1. In conclusion, we demonstrated that MDH1 plays a tumor-suppressing role in AML and can be used as a novel mimic drug therapy in the future. Disclosures No relevant conflicts of interest to declare.

A Novel Role for Malate Dehydrogenase 1 in Acute Myeloid Leukemia Progression

The tumor suppressor role of the metabolic genes is rarely reported. Here, we report that malate dehydrogenase 1 (MDH1) acts as an anti-oncogene in acute myeloid leukemia (AML) progression. In a screen of various leukemia cell lines, as well as peripheral blood mononuclear cells from patients with AML and healthy individuals, we observed that patients with AML had lower mRNA expression of MDH1 than healthy individuals. In addition, we found that overexpression of MDH1 in the AML cell lines MV4-11 and THP-1 in vitro promoted their differentiation, reduced their growth, induced apoptosis, and increased the proportion of cells in S phase, which were more sensitive to the chemotherapeutic drugs cytarabine and daunorubicin. Using an in vivo xenograft mouse model of AML, we found that disruption of MDH1 expression influenced tumor burden and mouse survival. These findings were consistent with an inverse relationship between MDH1 expression and overall survival in two independent cohorts of AML patients. We also identified changes in the expression of other genes, concomitant with reduced MDH1 expression in patients with AML, that largely explain the anti-oncogenic role of MDH1 in AML progression. Notably, downregulation of P53 and upregulation of the MAPK pathway in low MDH1-expressing MV4-11 cells were reversed upon overexpression of MDH1. In conclusion, we demonstrated that MDH1 plays a tumor-suppressing role in AML and can be used as a novel drug target in the future. Funding Statement: This work was supported by grants from the National Natural Science Foundation of China grants (81820108004 and 81470305-H0812) and Zhejiang Province Natural Science Foundation of China (LY19H080009). Declaration of Interests: The authors declare that they have no competing interests. Ethics Approval Statement: Our animal study was approved by Ethics Committee for Laboratory Animals of the First Affiliated Hospital, College of Medicine, Zhejiang University (Hangzhou, China) and were conducted in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals.

MDH1 and MPP7 Regulate Autophagy in Pancreatic Ductal Adenocarcinoma.

Pancreatic ductal adenocarcinoma (PDAC) is driven by metabolic changes in pancreatic cells caused by oncogenic mutations and dysregulation of p53. PDAC cell lines and PDAC-derived xenografts grow as a result of altered metabolic pathways, changes in stroma, and autophagy. Selective targeting and inhibition of one of these may open avenues for the development of new therapeutic strategies. In this study, we performed a genome-wide siRNA screen in a PDAC cell line using endogenous autophagy as a readout and identified several regulators of autophagy that were required for autophagy-dependent PDAC cell survival. Validation of two promising candidates, MPP7 (MAGUK p55 subfamily member 7, a scaffolding protein involved in cell-cell contacts) and MDH1 (cytosolic Malate dehydrogenase 1), revealed their role in early stages of autophagy during autophagosome formation. MPP7 was involved in the activation of YAP1 (a transcriptional coactivator in the Hippo pathway), which in turn promoted autophagy, whereas MDH1 was required for maintenance of the levels of the essential autophagy initiator serine-threonine kinase ULK1, and increased in the activity upon induction of autophagy. Our results provide a possible explanation for how autophagy is regulated by MPP7 and MDH1, which adds to our understanding of autophagy regulation in PDAC. SIGNIFICANCE: This study identifies and characterizes MPP7 and MDH1 as novel regulators of autophagy, which is thought to be responsible for pancreatic cancer cell survival.

Functional mechanism of malate dehydrogenase 2 in tumors

The role of malate dehydrogenase 2 (MDH2) in tumors is double-sided, it has a cancer-promoting effect in some tumors and an inhibitory effect in other tumors. The function of MDH2 is related to energy metabolism, tumor resistance and its pseudo hypoxia. MDH2 plays an important role in the occurrence, development, invasion and metastasis of tumors. An in-depth understanding of the functional mechanism of MDH2 in tumors can provide new molecular targets for tumor intervention in the clinic. Key words: Oxidoreductases; Neoplasms; Malate dehydrogenase 2

Alternol eliminates excessive ATP production by disturbing Krebs cycle in prostate cancer.

Alternol is a natural compound isolated from fermentation products of a mutant fungus. Our previous studies demonstrated that Alternol specifically kills cancer cells but spares benign cells. To investigate the mechanism underlying alternol-induced cancer cell-specific killing effect, we took a comprehensive strategy to identify Alternol's protein targets in prostate cancer cells, including PC-3, C4-2, and 22RV1, plus benign BPH1 cell lines. Major experimental techniques included biotin-streptavidin pulldown assay coupled with mass-spectrometry, in vitro enzyme activity assay for Krebs cycle enzymes and gas chromatography-mass spectrometry (GC-MS) for metabolomic analysis. Among 14 verified protein targets, four were Krebs cycle enzymes, fumarate hydratase (FH), malate dehydrogenase-2 (MDH2), dihydrolipoamide acetyltransferase (DLAT) in pyruvate dehydrogenase complex (PDHC) and dihydrolipoamide S-succinyltransferase (DLST) in a-ketoglutarate dehydrogenase complex (KGDHC). Functional assays revealed that PDHC and KGDHC activities at the basal level were significantly higher in prostate cancer cells compared to benign prostate BPH1 cells, while alternol treatment reduced their activities in cancer cells close to the levels in BPH1 cells. Although FH and MDH2 activities were comparable among prostate cancer and benign cell lines at the basal level, Alternol treatment largely increased their activities in cancer cells. Metabolomic analysis revealed that Alternol treatment remarkably reduced the levels of malic acid, fumaric acid, and isocitric acid and mitochondrial respiration in prostate cancer cells. Alternol also drastically reduced mitochondrial respiration and ATP production in PC-3 cells in vitro or in xenograft tissues but not in BPH1 cells or host liver tissues. Alternol interacts with multiple Krebs cycle enzymes, resulting in reduced mitochondrial respiration and ATP production in prostate cancer cells and xenograft tissues, providing a novel therapeutic strategy for prostate cancer treatment.

The age-related changes and differences in energy metabolism and glutamate-glutamine recycling in the d-gal-induced and naturally occurring senescent astrocytes in vitro.

Previously, we successfully established a d-galactose (d-gal)-induced astrocyte aging model in vitro. However, whether the changes in the aged astrocytes induced by d-gal are similar to those occurred in naturally are unknown. Therefore, in current study, we simultaneously established d-gal-induced and naturally aged astrocyte aging models in vitro to explore the age-related changes and to compare the differences in these two astrocyte aging models. The Seahorse Extracellular Flux Analyzer was used to examine the mitochondrial metabolism and glycolysis activities of the young and senescent astrocytes. The results showed that the mitochondrial ATP-linked oxygen consumption rates (OCRs) were decreased markedly both in the d-gal-induced and naturally occurring senescent astrocytes. The basal glycolysis activity was increased in the naturally occurring senescent astrocytes, whereas it was decreased in the d-gal-induced senescent astrocytes. Western blot analysis showed that isocitrate dehydrogenase 3 (IDH3), succinate dehydrogenase (SDH) and malate dehydrogenase 2 (MDH2) were markedly decreased both in these two aging models, whereas the iron‑sulfur cluster assembly enzyme (ISCU) was up-regulated in the naturally occurring senescent astrocytes but was down-regulated in the d-gal-induced senescent astrocytes. The expression levels of glial glutamate transporter-1 (GLT-1), glutamine synthetase (GS) and γ-aminobutyric acid type B receptor subunit 2 (GABABR2) were also markedly decreased in these two aging models. In addition, the PI3K/AKT signaling pathway was to be inactivated both in the d-gal-induced and naturally occurring senescent astrocytes. These results indicate that the age-related changes in d-gal-induced senescent astrocytes are not fully consistent with those in naturally occurring senescent astrocytes, and it may be not suitable to use d-gal-induced senescent astrocytes to replace the naturally occurring senescent astrocytes to explore the aging mechanisms under some circumstances.

Biochemical and Epigenetic Insights into L-2-Hydroxyglutarate, a Potential Therapeutic Target in Renal Cancer.

Elevation of L-2-hydroxylgutarate (L-2-HG) in renal cell carcinoma (RCC) is due in part to reduced expression of L-2-HG dehydrogenase (L2HGDH). However, the contribution of L-2-HG to renal carcinogenesis and insight into the biochemistry and targets of this small molecule remains to be elucidated. Genetic and pharmacologic approaches to modulate L-2-HG levels were assessed for effects on in vitro and in vivo phenotypes. Metabolomics was used to dissect the biochemical mechanisms that promote L-2-HG accumulation in RCC cells. Transcriptomic analysis was utilized to identify relevant targets of L-2-HG. Finally, bioinformatic and metabolomic analyses were used to assess the L-2-HG/L2HGDH axis as a function of patient outcome and cancer progression. L2HGDH suppresses both in vitro cell migration and in vivo tumor growth and these effects are mediated by L2HGDH's catalytic activity. Biochemical studies indicate that glutamine is the predominant carbon source for L-2-HG via the activity of malate dehydrogenase 2 (MDH2). Inhibition of the glutamine-MDH2 axis suppresses in vitro phenotypes in an L-2-HG-dependent manner. Moreover, in vivo growth of RCC cells with basal elevation of L-2-HG is suppressed by glutaminase inhibition. Transcriptomic and functional analyses demonstrate that the histone demethylase KDM6A is a target of L-2-HG in RCC. Finally, increased L-2-HG levels, L2HGDH copy loss, and lower L2HGDH expression are associated with tumor progression and/or worsened prognosis in patients with RCC. Collectively, our studies provide biochemical and mechanistic insight into the biology of this small molecule and provide new opportunities for treating L-2-HG-driven kidney cancers.

Cold modulated nuclear S-nitrosoproteome analysis indicates redox modulation of novel Brassicaceae specific, myrosinase and napin in Brassica juncea

Stress leads to production of reactive oxygen species (ROS) and reactive nitrogen species (RNS). Among the RNS, nitric oxide (NO) is an important signaling molecule that mediates its effect majorly via a reversible post-translational modification (PTM) S-nitrosylation. However, information on the NO mediated cold stress signaling is still missing for nucleus. Therefore, in the present study an attempt was made to analyze NO signaling, particularly, S-nitrosylation in the nuclei of Brassica juncea seedlings. Cold stress (4 °C) induced nitrate reductase (NR) mediated NO production along with the accumulation of available protein thiols (5 fold, at 96 h, 4 °C) and low molecular weight thiols (1.2 fold at 12 h, 4 °C). S-nitrosoglutathione (GSNO, a NO donor) and cold (endogenous, S-nitrosylation without the addition of NO donor) treated, neutravidin-agarose affinity chromatography purified S-nitrosylated proteins were resolved as 55- and 32-spots respectively on the 2-D gels. Functional categorization after MALDI-TOF/TOF identification showed 67% targets to be stress/defense/redox related and 33% to be metabolic, suggesting the potential role(s) of S-nitrosylation in these responses. Cold stress increased S-nitrosylation of Cu/Zn SOD, l-ascorbate oxidase, fructose bisphosphate aldolase (FBA2) and malate dehydrogenase 1 (MDH1), therefore, indicating NO mediated regulation of redox and metabolism related pathways. Identification of targets of glucosinolate hydrolysis pathway (myrosinase and myrosinase-binding protein 2-like) indicated the role of S-nitrosylation in regulating mustard oil bomb. Presence of myrosinase was confirmed by activity measurements and western blotting. Increased S-nitrosylation of myrosinase in cold stress in the nucleus is reported for the first time. Here, we proposed S-nitrosylation mediated nuclear trafficking of non-nuclear targets such as beta-d-xylosidase, daikon cysteine protease, and beta-glucosidase to the nucleus. Similarly, identification of FBA2 and MDH1 in the nucleus, indicated the role of NO in modulating their moonlighting function via S-nitrosylation in stress. Present study provided the first repertoire of cold modulated S-nitrosylated proteins in the nucleus.

Interorganelle Communication: Peroxisomal MALATE DEHYDROGENASE2 Connects Lipid Catabolism to Photosynthesis through Redox Coupling in Chlamydomonas.

Plants and algae must tightly coordinate photosynthetic electron transport and metabolic activities given that they often face fluctuating light and nutrient conditions. The exchange of metabolites and signaling molecules between organelles is thought to be central to this regulation but evidence for this is still fragmentary. Here, we show that knocking out the peroxisome-located MALATE DEHYDROGENASE2 (MDH2) of Chlamydomonas reinhardtii results in dramatic alterations not only in peroxisomal fatty acid breakdown but also in chloroplast starch metabolism and photosynthesis. mdh2 mutants accumulated 50% more storage lipid and 2-fold more starch than the wild type during nitrogen deprivation. In parallel, mdh2 showed increased photosystem II yield and photosynthetic CO2 fixation. Metabolite analyses revealed a >60% reduction in malate, together with increased levels of NADPH and H2O2 in mdh2 Similar phenotypes were found upon high light exposure. Furthermore, based on the lack of starch accumulation in a knockout mutant of the H2O2-producing peroxisomal ACYL-COA OXIDASE2 and on the effects of H2O2 supplementation, we propose that peroxisome-derived H2O2 acts as a regulator of chloroplast metabolism. We conclude that peroxisomal MDH2 helps photoautotrophs cope with nitrogen scarcity and high light by transmitting the redox state of the peroxisome to the chloroplast by means of malate shuttle- and H2O2-based redox signaling.