SUCLA2-coupled regulation of GLS succinylation and activity counteracts oxidative stress in tumor cells

•GLS is upregulated in human pancreatic ductal adenocarcinoma•GLS K311 succinylation enhances the oligomerization and activity of GLS•p38-phosphorylated SUCLA2 dissociates from GLS promotes succinylation•GLS glutaminolysis tumor growth Glutaminase regulates to promote cancer cell proliferation. However, mechanism underlying glutaminase regulation largely unknown. Here, we demonstrate that kidney-type (GLS) highly expressed adenocarcinoma (PDAC) specimens with correspondingly glutamine dependence for PDAC Upon oxidative stress, succinyl-coenzyme A (CoA) synthetase ADP-forming subunit β (SUCLA2) phosphorylated by p38 mitogen-activated protein kinase (MAPK) at S79 GLS, resulting enhanced succinylation, oligomerization, activity. Activated increases production nicotinamide adenine dinucleotide phosphate (NADPH) glutathione, thereby counteracting stress promoting survival mice. In addition, levels pS79 which were mutually correlated, positively associated advanced stages poor prognosis patients. Our findings reveal critical SUCLA2-coupled underscore regulatory role metabolites development. Glutamine (Gln) most enriched amino acid body abundant circulating blood. Many types cells, including use support anabolic processes fuel proliferation (Son et al., 2013Son J. Lyssiotis C.A. Ying H. Wang X. Hua S. Ligorio M. Perera R.M. Ferrone C.R. Mullarky E. Shyh-Chang N. al.Glutamine supports through a KRAS-regulated metabolic pathway.Nature. 2013; 496: 101-105Crossref PubMed Scopus (1188) Google Scholar). plays crucial roles many cellular functions, synthesis maintain mitochondrial metabolism; generation glutathione (GSH) redox homeostasis; nonessential acids, purines, pyrimidines, fatty acids proliferation; signaling (Michalak 2015Michalak K.P. Maćkowska-Kędziora A. Sobolewski B. Woźniak P. Key Roles Pathways Reprogramming Cancer Metabolism.Oxid. Med. Cell. Longev. 2015; 2015: 964321Crossref (45) Scholar; Son Yang 2017Yang L. Venneti Nagrath D. Glutaminolysis: Hallmark Metabolism.Annu. Rev. Biomed. Eng. 2017; 19: 163-194Crossref (267) The process cells convert into α-ketoglutarate, enters tricarboxylic (TCA) cycle, involves multiple enzymes. converts glutamate, then converted α-ketoglutarate via glutamate dehydrogenase (GLUD) or group transaminases, glutamate-oxaloacetate transaminase (GOT), glutamate-pyruvate (GPT), phosphoserine (PSAT) (DeBerardinis Cheng, 2010DeBerardinis R.J. Cheng T. Q’s next: diverse functions metabolism, biology cancer.Oncogene. 2010; 29: 313-324Crossref (848) Michalak There are two isoforms encoded different genes cells: liver-type (also known as GLS2 LGA) only periportal hepatocytes postnatal liver, whereas (known GLS1, KGA) kidney, brain, intestine, fetal lymphocytes, (Curthoys Watford, 1995Curthoys N.P. Watford Regulation metabolism.Annu. Nutr. 1995; 15: 133-159Crossref (499) have structural kinetic properties contribute their regulation. exhibits oncogenic properties, has been described suppressor factor. Selective genomic epigenomic intervention function affects reprogramming (Katt 2017Katt W.P. Lukey M.J. Cerione R.A. tale glutaminases: homologous enzymes distinct tumorigenesis.Future Chem. 9: 223-243Crossref (67) Matés 2018Matés J.M. Campos-Sandoval J.A. Márquez isoenzymes therapy cancer.Biochim. Biophys. Acta Cancer. 2018; 1870: 158-164Crossref (44) whether regulated posttranslationally remains unclear. this report, specimens. Its its association (SUCLA2). Oxidative disrupts interaction between SUCLA2, increasing counteract To examine expression PDAC, performed immunohistochemical (IHC) analyses 54 paired adjacent normal tissues (Figure 1A). We found was overexpressed compared counterpart 1B). Consistent these results, SW1990, AsPC-1, PANC-1 lines higher than duct epithelial (HPDE) 1C). Correspondingly, deprivation exerted greater inhibitory effect on HPDE 1D), suggesting demand high metabolism growth. Consistently, stable isotope-assisted metabolomic analysis using [U-13C5] tracer showed mass isotopolog distributions TCA cycle AsPC-1 1E). line findings, treatment bis-2-(5-phenylacetmido-1,2,4-thiadiazol-2-yl)ethyl sulfide (BPTES) (Figures 1F S1A) glutaminase-IN-1 S1B), specific inhibitors, inhibited degree they similar differential effects obtained expressing short hairpin RNA (shRNA) deplete HPDE, SW1990 1G S1C). These results indicated more dependent addition level, proteins can be modulated posttranslational modifications. determine modified, immunoprecipitated cells. Mass spectrometry succinylated evolutionarily conserved S2A S2B), validated synthetic peptide same sequence S2C). vitro, incubated purified 293T cell-expressed FLAG-GLS S2D) bacterially recombinant S-transferase (GST)-GLS S2E) succinyl-CoA detected increased concentrations within tested range 10–100 μM 2A S2F). Notably, concentration measured matrix, where located (Aledo 1997Aledo J.C. de Pedro Gómez-Fabre P.M. Núñez Castro I. Submitochondrial localization membrane topography Ehrlich ascitic tumour glutaminase.Biochim. Acta. 1997; 1323: 173-184Crossref (27) Scholar), about 26 μM, mitochondria. contrast, tyrosine phosphatase 1 (PTPMT1), also protein, not succinyl-CoA, next mutated K311; K311-adjacent K245, K279, K320; K507 Arg (R) FLAG-tagged mutants Only K311R significantly decreased succinyl-CoA-induced 2B). reconstituted RNAi-resistant (r) wild-type (WT) FLAG-rGLS endogenous GLS-depleted S2G) rGLS mutant considerably lesser extent WT counterpart, an anti-pan-lysine antibody S2H) anti-GLS (K311succ) 2C S2I). K311. forms oligomers, possess (Stalnecker 2015Stalnecker Ulrich S.M. Li Y. Ramachandran McBrayer M.K. DeBerardinis Erickson J.W. Mechanism recently discovered allosteric inhibitor blocks transformed cells.Proc. Natl. Acad. Sci. USA. 112: 394-399Crossref (57) functional examined status depleted expression. mutation substantially dimer tetramer formation, FLAG-rGLS, but 2D), oligomerization. finding, molecular dynamics simulation root-mean-square deviation (RMSD) lower without after 40 ns 2E), supported finding carboxyl oxygen atom amide succinyl formed hydrogen bond side chain H475 monomer, three four residues form 2F). result, H475A reduced 2G) formation 2H). suggested forming monomer interface. report vitro assay inclusion 2I S2D). His-GLS yielded Vmax Km substrate alone S2J). K311R, other lysine mutations led greatly intracellular 2J S2K). demonstrated enzymatic immunoprecipitates revealed ligase [SUCL]) α (SUCLG1) S3A). SUCL heterodimeric enzyme composed invariant SUCLG1 substrate-specific either ATP SUCLG2 guanosine triphosphate (GTP) production. This matrix-located enzyme, component citric catalyzes conversion ADP (or diphosphate [GDP]) CoA, succinate, GTP) S3B) (Lambeth 2004Lambeth D.O. Tews K.N. Adkins Frohlich Milavetz B.I. Expression synthetases nucleotide specificities mammalian tissues.J. Biol. 2004; 279: 36621-36624Abstract Full Text PDF (91) Coimmunoprecipitation 3A), S3C), interacted mRNA those S3D). Immunofluorescence colocalization 3B). interacts colocalizes GLS. Given GLS-regulated contributes homeostasis determined binding dynamically regulated, especially under stress. Treatment H2O2 resulted 3C). alteration modifications often result conformational changes (Lu Hunter, 2009Lu Z. Hunter Degradation activated kinases ubiquitination.Annu. Biochem. 2009; 78: 435-475Crossref (97) H2O2-treated analysis, S3E). S79A blocked H2O2-induced disassociation 3D). phosphorylation-mimicking S79D disrupted even absence 3E). phosphorylation dissociation upon Analyses residue Scansite software show potentially (MAPK). Pretreatment SP600125, PD98059, SB203580 activation JNK, MEK/ERK, p38, respectively, inhibition prevented 4A). His-SUCLA2, ATP-γ-S presence 4B). phosphorylation, anti-pS79-specific S4A), induced S79A, 4C), pretreatment induction 4D). phosphorylates S79, leading 4E). Similarly, differentiated responses elicited menadione-induced S4B). 4F). Lys acetylated ubiquitylated, acetylation ubiquitylation did detect obvious regardless S4C S4D). alter acetylation, S4E S4F). strongly primarily succinylated, treatment. quantification 5% level K311-succinylated 30% S4G). Intriguingly, coimmunoprecipitation S4H). GST pull-down nonsuccinylated ability S4I). induces decreases SUCLA2. Because exert nonmetabolic amounts metabolite products (Jiang 2015Jiang Qian Shen Liu R. Xia Chen Q. Peng G. Lin S.Y. Lu Local fumarate DNA repair histone H3 demethylation.Nat. Cell 17: 1158-1168Crossref (109) 2016bLi L.X. Jiang Hawke D.H. splicing switch ketohexokinase-C ketohexokinase-A drives hepatocellular carcinoma formation.Nat. 2016; 18: 561-571Crossref (96) 2017aLi ACSS2 gene transcription lysosomal biogenesis autophagy.Autophagy. 13: 1790-1791Crossref Scholar, 2017bLi Yu W. Zheng Lee J.H. Lyu Rao Zhang al.Nucleus-Translocated Promotes Gene Transcription Lysosomal Biogenesis Autophagy.Mol. 66: 684-697.e9Abstract (129) 2018aLi Egervari Berger S.L. chromatin metabolites.Nat. Mol. 563-578Crossref (167) 2018bLi Huang B.J. Fang C.N. al.Nuclear PGK1 Alleviates ADP-Dependent Inhibition CDC7 Promote Replication.Mol. 72: 650-660.e8Abstract (31) hypothesized modulates amount SUCL-catalyzed regulating succinylation. Incubation succinyl-CoA-dependent 4G) turn succinate S5A), SUCLA2-mediated reduces depletion comparable elevation total S5B). SUCLG2, 4H). previously reported succinyl-transferase, carnitine palmitoyltransferase 1A (CPT1A) (Kurmi 2018Kurmi K. Hitosugi Wiese E.K. Boakye-Agyeman F. Gonsalves W.I. Lou Karnitz L.M. Goetz M.P. Carnitine Palmitoyltransferase Has Lysine Succinyltransferase Activity.Cell Rep. 22: 1365-1373Abstract (34) failed enhance S5C), indicating CPT1A. Reconstituted had S5D), diminished SUCLA2-depleted 4I). altered V5-rSUCLA2 S5E S5F). oxidative-stress-induced phosphorylation-regulated used label GLS- FLAG-rSUCLA2, FLAG-rSUCLA2 S79A. Stable fractional contribution labeled 5A), SUCLA2-regulated metabolism. NADPH against previous reports ratio NADP+/NADPH 5B S6A) GSH/oxidized (GSSG) 5C S6B) 5C) S6A fully reversed K311R. reactive species (ROS) levels, further 5D S6C). contrast elevated ROS treatment, antioxidative menadione caspase-3 activation, reflected cleavage poly ADP-ribose polymerase (PARP) 5E S6D), apoptosis parental 5F). S6D) rate 5F) acetylation-mimicking K311Q S6E) H2O2-enhanced caspase 5E, 5F, supporting antiapoptotic effects. GSH/GSSG S6F) apoptotic S6G) response Similar S6H), S6I), S6J), PARP S6K) S6L). Collectively, phosphorylation-promoting important GSH protect As expected, 6A) S7A) colony 6B S7B). Compared exhibited rates 6A phosphorylation-promoted development, subcutaneously injected athymic nude 6C) volume 6D) weight 6E), Ki67 6F), 6G) tissues. abrogated 6C–6G). reconstituting significant S7C–S7E) S7F) S7G) mic