Dataset Information

Protein-metabolite interactomics of carbohydrate metabolism reveal regulation of lactate dehydrogenase

ABSTRACT:

Metabolic networks are interconnected and influence diverse cellular processes. The protein-metabolite interactions that mediate these networks are frequently low affinity and challenging to systematically discover. We developed mass spectrometry integrated with equilibrium dialysis for the discovery of allostery systematically (MIDAS) to identify such interactions. Analysis of 33 enzymes from human carbohydrate metabolism identified 830 protein-metabolite interactions, including known regulators, substrates and products as well as previously unreported interactions. We functionally validated a subset of interactions, including the isoform-specific inhibition of lactate dehydrogenase by long-chain acyl-coenzyme A. Cell treatment with fatty acids caused a loss of pyruvate-lactate interconversion dependent on lactate dehydrogenase isoform expression. These protein-metabolite interactions may contribute to the dynamic, tissue-specific metabolic flexibility that enables growth and survival in an ever-changing nutrient environment.

INSTRUMENT(S): Flow injection analysis MS - positive, Flow injection analysis MS - negative

SUBMITTER: Jordan Berg

PROVIDER: MTBLS7237 | MetaboLights | 2023-08-07

REPOSITORIES: MetaboLights

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Dataset's files

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			Action	DRS
	MTBLS7237	Other
	FILES	Other
	a_MTBLS7237_FIA-MS_negative__metabolite_profiling.txt	Txt
	a_MTBLS7237_FIA-MS_positive__metabolite_profiling.txt	Txt
	files-all.json	Other

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Publications

Protein-metabolite interactomics of carbohydrate metabolism reveal regulation of lactate dehydrogenase.

Hicks Kevin G KG Cluntun Ahmad A AA Schubert Heidi L HL Hackett Sean R SR Berg Jordan A JA Leonard Paul G PG Ajalla Aleixo Mariana A MA Zhou Youjia Y Bott Alex J AJ Salvatore Sonia R SR Chang Fei F Blevins Aubrie A Barta Paige P Tilley Samantha S Leifer Aaron A Guzman Andrea A Arok Ajak A Fogarty Sarah S Winter Jacob M JM Ahn Hee-Chul HC Allen Karen N KN Block Samuel S Cardoso Iara A IA Ding Jianping J Dreveny Ingrid I Gasper William C WC Ho Quinn Q Matsuura Atsushi A Palladino Michael J MJ Prajapati Sabin S Sun Pengkai P Tittmann Kai K Tolan Dean R DR Unterlass Judith J VanDemark Andrew P AP Vander Heiden Matthew G MG Webb Bradley A BA Yun Cai-Hong CH Zhao Pengkai P Wang Bei B Schopfer Francisco J FJ Hill Christopher P CP Nonato Maria Cristina MC Muller Florian L FL Cox James E JE Rutter Jared J

Science (New York, N.Y.) 20230309 6636

PMID: 36893255

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Project description:Caldicellulosiruptor saccharolyticus is an extremely thermophilic, gram-positive anaerobe which ferments a broad range of substrates to mainly acetate, CO2, and hydrogen gas (H2). Its high hydrogen-producing capacity make this bacterium an attractive candidate for microbial biohydrogen production. However, increased H2 levels tend to inhibit hydrogen formation and leads to the formation of other reduced end products like lactate and ethanol. To investigate the organismM-bM-^@M-^Ys strategy for dealing with elevated H2 levels and to identify alternative pathways involved in the disposal of the reducing equivalents, the effect of the hydrogen partial pressure (PH2) on fermentation performance was studied. For this purpose cultures were grown under high and low PH2 in a glucose limited chemostat setup. Transcriptome analysis revealed the up-regulation of genes involved in the disposal of reducing equivalents under high PH2, like lactate dehydrogenase and alcohol dehydrogenase as well as the NADH-dependent and ferredoxin-dependent hydrogenases. These findings were in line with the observed shift in fermentation profiles from acetate production under low PH2 to a mixed production of acetate, lactate and ethanol under high PH2. In addition, differential transcription was observed for genes involved in carbon metabolism, fatty acid biosynthesis and several transport systems. The presented transcription data provides experimental evidence for the involvement of the redox sensing Rex protein in gene regulation under high PH2 cultivation conditions. Overall, these findings indicate that the PH2 dependent changes in the fermentation pattern of C. saccharolyticus are, in addition to the known regulation at the enzyme/metabolite level, also regulated at the transcription level. Two conditions: low H2 partial pressure and high H2 partial pressure, both at steady state growth were harvested for a dye-flip microarray experimental design. Biological replicates were harvested for both conditions and combined prior to cDNA synthesis. Both conditions were labeled with cy3 and cy5 dyes allowing for a technical replicate of hybridization in addition to the biological replicates.