Modulation of anti-cancer drug sensitivity through the regulation of mitochondrial activity by adenylate kinase 4.
Fujisawa Koichi,Terai Shuji,Takami Taro,Yamamoto Naoki,Yamasaki Takahiro,Matsumoto Toshihiko,Yamaguchi Kazuhito,Owada Yuji,Nishina Hiroshi,Noma Takafumi,Sakaida Isao
Journal of experimental & clinical cancer research : CR
BACKGROUND:Adenylate kinase is a key enzyme in the high-energy phosphoryl transfer reaction in living cells. An isoform of this enzyme, adenylate kinase 4 (AK4), is localized in the mitochondrial matrix and is believed to be involved in stress, drug resistance, malignant transformation in cancer, and ATP regulation. However, the molecular basis for the AK4 functions remained to be determined. METHODS:HeLa cells were transiently transfected with an AK4 small interfering RNA (siRNA), an AK4 short hairpin RNA (shRNA) plasmid, a control shRNA plasmid, an AK4 expression vector, and a control expression vector to examine the effect of the AK4 expression on cell proliferation, sensitivity to anti-cancer drug, metabolome, gene expression, and mitochondrial activity. RESULTS:AK4 knockdown cells treated with short hairpin RNA increased ATP production and showed greater sensitivity to hypoxia and anti-cancer drug, cis-diamminedichloro-platinum (II) (CDDP). Subcutaneous grafting AK4 knockdown cells into nude mice revealed that the grafted cells exhibited both slower proliferation and reduced the tumor sizes in response to CDDP. AK4 knockdown cell showed a increased oxygen consumption rate with FCCP treatment, while AK4 overexpression lowered it. Metabolome analysis showed the increased levels of the tricarboxylic acid cycle intermediates, fumarate and malate in AK4 knockdown cells, while AK4 overexpression lowered them. Electron microscopy detected the increased mitochondrial numbers in AK4 knockdown cells. Microarray analysis detected the increased gene expression of two key enzymes in TCA cycle, succinate dehydrogenase A (SDHA) and oxoglutarate dehydrogenease L (OGDHL), which are components of SDH complex and OGDH complex, supporting the metabolomic results. CONCLUSIONS:We found that AK4 was involved in hypoxia tolerance, resistance to anti-tumor drug, and the regulation of mitochondrial activity. These findings provide a new potential target for efficient anticancer therapies by controlling AK4 expression.
Hippocampal proteomic changes of susceptibility and resilience to depression or anxiety in a rat model of chronic mild stress.
Tang Min,Huang Haojun,Li Shuiming,Zhou Mi,Liu Zhao,Huang Rongzhong,Liao Wei,Xie Peng,Zhou Jian
Chronic stressful occurrences are documented as a vital cause of both depression and anxiety disorders. However, the stress-induced molecular mechanisms underlying the common and distinct pathophysiology of these disorders remains largely unclear. We utilized a chronic mild stress (CMS) rat model to differentiate and subgroup depression-susceptible, anxiety-susceptible, and insusceptible rats. The hippocampus was analyzed for differential proteomes by combining mass spectrometry and the isobaric tags for relative and absolute quantitation (iTRAQ) labeling technique. Out of 2593 quantified proteins, 367 were aberrantly expressed. These hippocampal protein candidates might be associated with susceptibility to stress-induced depression or anxiety and stress resilience. They provide the potential protein systems involved in various metabolic pathways as novel investigative protein targets. Further, independent immunoblot analysis identified changes in Por, Idh2 and Esd; Glo1, G6pdx, Aldh2, and Dld; Dlat, Ogdhl, Anxal, Tpp2, and Sdha that were specifically associated to depression-susceptible, anxiety-susceptible, or insusceptible groups respectively, suggesting that identical CMS differently impacted the mitochondrial and metabolic processes in the hippocampus. Collectively, the observed alterations to protein abundance profiles of the hippocampus provided significant and novel insights into the stress regulation mechanism in a CMS rat model. This might serve as the molecular basis for further studies that would contributed to a better understanding of the similarities and differences in pathophysiologic mechanisms underlying stress-induced depression or anxiety, and stress resiliency.
Loss of Nardilysin, a Mitochondrial Co-chaperone for α-Ketoglutarate Dehydrogenase, Promotes mTORC1 Activation and Neurodegeneration.
Yoon Wan Hee,Sandoval Hector,Nagarkar-Jaiswal Sonal,Jaiswal Manish,Yamamoto Shinya,Haelterman Nele A,Putluri Nagireddy,Putluri Vasanta,Sreekumar Arun,Tos Tulay,Aksoy Ayse,Donti Taraka,Graham Brett H,Ohno Mikiko,Nishi Eiichiro,Hunter Jill,Muzny Donna M,Carmichael Jason,Shen Joseph,Arboleda Valerie A,Nelson Stanley F,Wangler Michael F,Karaca Ender,Lupski James R,Bellen Hugo J
We previously identified mutations in Nardilysin (dNrd1) in a forward genetic screen designed to isolate genes whose loss causes neurodegeneration in Drosophila photoreceptor neurons. Here we show that NRD1 is localized to mitochondria, where it recruits mitochondrial chaperones and assists in the folding of α-ketoglutarate dehydrogenase (OGDH), a rate-limiting enzyme in the Krebs cycle. Loss of Nrd1 or Ogdh leads to an increase in α-ketoglutarate, a substrate for OGDH, which in turn leads to mTORC1 activation and a subsequent reduction in autophagy. Inhibition of mTOR activity by rapamycin or partially restoring autophagy delays neurodegeneration in dNrd1 mutant flies. In summary, this study reveals a novel role for NRD1 as a mitochondrial co-chaperone for OGDH and provides a mechanistic link between mitochondrial metabolic dysfunction, mTORC1 signaling, and impaired autophagy in neurodegeneration.
Novel isoenzyme of 2-oxoglutarate dehydrogenase is identified in brain, but not in heart.
Bunik Victoria,Kaehne Thilo,Degtyarev Dmitry,Shcherbakova Tatiana,Reiser Georg
The FEBS journal
2-Oxoglutarate dehydrogenase (OGDH) is the first and rate-limiting component of the multienzyme OGDH complex (OGDHC) whose malfunction is associated with neurodegeneration. The essential role of this complex in the degradation of glucose and glutamate, which have specific significance in brain, raises questions about the existence of brain-specific OGDHC isoenzyme(s). We purified OGDHC from extracts of brain or heart mitochondria using the same procedure of poly(ethylene glycol) fractionation, followed by size-exclusion chromatography. Chromatographic behavior and the insufficiency of mitochondrial disruption to solubilize OGDHC revealed functionally significant binding of the complex to membrane. Components of OGDHC from brain and heart were identified using nano-high performance liquid chromatography electrospray tandem mass spectrometry after trypsinolysis of the electrophoretically separated proteins. In contrast to the heart complex, where only the known OGDH was determined, the band corresponding to the brain OGDH component was found to also include the novel 2-oxoglutarate dehydrogenase-like (OGDHL) protein. The ratio of identified peptides characteristic of OGDH and OGDHL was preserved during purification and indicated comparable quantities of the two proteins in brain. Brain OGDHC also differed from the heart complex in the abundance of the components, lower apparent molecular mass and decreased stability upon size-exclusion chromatography. The functional competence of the novel brain isoenzyme and different regulation of OGDH and OGDHL by 2-oxoglutarate are inferred from the biphasic dependence of the overall reaction rate versus 2-oxoglutarate concentration. OGDHL may thus participate in brain-specific control of 2-oxoglutarate distribution between energy production and synthesis of the neurotransmitter glutamate.
Exclusive neuronal detection of KGDHC-specific subunits in the adult human brain cortex despite pancellular protein lysine succinylation.
Dobolyi Arpad,Bago Attila,Palkovits Miklos,Nemeria Natalia S,Jordan Frank,Doczi Judit,Ambrus Attila,Adam-Vizi Vera,Chinopoulos Christos
Brain structure & function
The ketoglutarate dehydrogenase complex (KGDHC) consists of three different subunits encoded by OGDH (or OGDHL), DLST, and DLD, combined in different stoichiometries. DLD subunit is shared between KGDHC and pyruvate dehydrogenase complex, branched-chain alpha-keto acid dehydrogenase complex, and the glycine cleavage system. Despite KGDHC's implication in neurodegenerative diseases, cell-specific localization of its subunits in the adult human brain has never been investigated. Here, we show that immunoreactivity of all known isoforms of OGDHL, OGDH, and DLST was detected exclusively in neurons of surgical human cortical tissue samples identified by their morphology and visualized by double labeling with fluorescent Nissl, while being absent from glia expressing GFAP, Aldhl1, myelin basic protein, Olig2, or IBA1. In contrast, DLD immunoreactivity was evident in both neurons and glia. Specificity of anti-KGDHC subunits antisera was verified by a decrease in staining of siRNA-treated human cancer cell lines directed against the respective coding gene products; furthermore, immunoreactivity of KGDHC subunits in human fibroblasts co-localized > 99% with mitotracker orange, while western blotting of 63 post-mortem brain samples and purified recombinant proteins afforded further assurance regarding antisera monospecificity. KGDHC subunit immunoreactivity correlated with data from the Human Protein Atlas as well as RNA-Seq data from the Allen Brain Atlas corresponding to genes coding for KGDHC components. Protein lysine succinylation, however, was immunohistochemically evident in all cortical cells; this was unexpected, because this posttranslational modification requires succinyl-CoA, the product of KGDHC. In view of the fact that glia of the human brain cortex lack succinate-CoA ligase, an enzyme producing succinyl-CoA when operating in reverse, protein lysine succinylation in these cells must exclusively rely on propionate and/or ketone body metabolism or some other yet to be discovered pathway encompassing succinyl-CoA.