Protein-Protein Project Overview:  Protein-Protein Interactions are the basis for metabolon formation.  That is the channeling of metabolites between sequential enzymes in a metabolic pathway.  For some time the interaction of mitochondrial MDH with CS and other TCA enzymes has been shown.  Its regulation and the specific residues involved in the interface with MDH is not fully established.  Like its mitochondrial counterpart, cytosolic MDH has several potential interactions with a number of enzymes.  Interactions of both isoforms of MDH and their metabolon partners are currently involved in CUREs using MDH from both human and parasite sources.

Research Ideas/Gaps of Knowledge:  While the interaction between mitochondrial MDH and CS has been established,  the identity of which residues of MDH interact with which residues of CS is not known.  Also unknown are if other evolutionarily diverse organisms such as plants or parasites have the same interactions.  Cytosolic MDH is predicted and very likely to interact with substrate/product sharing enzymes such as malic enzyme, PEPCK and other enzymes.  Docking and interaction prediction software support this potential new area of metabolomics opening a novel area of research.  Finally it is highly likely that these interactions are not static and instead are dynamically regulated by metabolites, redox state or post translational modifications.  Thus phosphorylation, acetylation or methylation may all impact the interactions of cytosolic and mitochondrial MDH interactions.  Only a few publications have investigated the impact of metabolites on the interactions and there are no publications describing phosphorylation or other modification on MDH protein-protein interactions.  .

 MDH Interaction Hypothesis Development

More Information & Resources

•      Protein-Protein Interaction Introduction

•      Link to MDH Protein-Protein Interaction

•      Research ideas for both mito and cyto MDH

•      Video background on mito MDH-CS interaction

•      Video background on cyto MDH interactions

•      Protein-Protein Interaction Introduction

•      Link to MDH Protein-Protein Interaction

•      Video background on mito MDH-CS interaction

•      Video background on cyto MDH interactions

•      Docking prediction software

•      String

Relevant Publications

•       Substrate Channeling of Oxalacetate in Solid-state Complexes of Malate Dehydrogenase and Citrate Synthase

•       Quantitation of the interaction between citrate synthase and malate dehydrogenase

•       Regulation of malate dehydrogenase activity by glutamate, citrate, alpha-ketoglutarate, and multienzyme interaction

•       The versatility of plant organic acid metabolism in leaves is underpinned by mitochondrial malate-citrate exchange

•       Protein-protein interactions and metabolite channelling in the plant tricarboxylic acid cycle

•       Complex formation between malate dehydrogenase and isocitrate dehydrogenase from Bacillus subtilis is regulated by tricarboxylic acid cycle metabolites

•        Association of the malate dehydrogenase-citrate synthase metabolon is modulated by intermediates of the Krebs tricarboxylic acid cycle

•       The PEP-pyruvate-oxaloacetate node: variation at the heart of metabolism

•       Krebs Cycle Metabolon: Structural Evidence of Substrate Channeling Revealed by Cross-linking and Mass Spectrometry. 

•       Direct Evidence for Metabolon Formation and Substrate Channeling in Recombinant TCA Cycle Enzymes

•       Association of the malate dehydrogenase-citrate synthase metabolon is modulated by intermediates of the Krebs tricarboxylic acid cycle

List of Skills/Techniques Needed:  Purification, protein assay, site directed mutagenesis (if generating new sites to study), His tag removal by TEV protease, enzyme assay to identify changes in the function after modification and protein-protein interaction detection (competitive MDH assay with GOT, pull down interaction assay, fast thermal melt or CD interaction, docking analysis, cross-linking, yeast or bacterial two-hybrid assay, far-western blot).

Current adopters (links to slack or Hubs)  Joseph Provost (Univ San Diego), Celest Peterson (Suffolk Univ), David Hecht (Southwestern Community College), Kate Huisinga (Malone Univ), Amy Parente (Mercyhurst Univ).

MDH Clones to use: Human cytosolic MDH, human mitochondrial MDH, parasitic MDH, CS, fumarase, PEPCK, mitochondrial GOT, cytosolic GOT, malic enzyme