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Effect of N‐Terminal Splice Variants and Post‐Translational Modification on Regulation and Activity of Human Cytosolic Malate Dehydrogenase
Author(s) -
Cruz Melanie R.,
Balagtas Aaliyah N.,
Provost Joseph John
Publication year - 2020
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.2020.34.s1.02681
Subject(s) - phosphorylation , splice , malate dehydrogenase , biology , phosphorylation cascade , biochemistry , protein phosphorylation , cytosol , microbiology and biotechnology , enzyme , gene , protein kinase a
Post‐translational modification via phosphorylation is crucial in regulation of proteins, as well as in interactions between proteins. Human cytosolic malate dehydrogenase (hMDH1) is involved in multiple metabolic pathways including amino acid metabolism and gluconeogenesis, yet information on the impact of phosphorylation is very limited. There is however, growing evidence for regulation of hMDH1 in several disorders. The cytosolic hMDH1 is ubiquitously expressed, however several N‐terminal splice variants have been identified. Like regulation by phosphorylation, no work has been published on the effects of the splice variants on the structure or function of hMDH1. We first determined the impact of N‐terminal deletion for the three most common splice variants on kinetic values. We next analyzed the potential for phosphorylation using three phosphorylation prediction programs and performed meta‐analysis with existing mass spectrometry databases documenting phosphorylation. This bioinformatic analysis identified 24 residues with overlapping phosphorylation predictions between databases. Of these sites, the longest hMDH1 variant (hMDH1v3) has a unique phosphorylation site that is predicted in all databases and has documented phosphorylation in the N‐terminal extension. To examine the potential impact on MDH structure and function phosphomimetic mutations were generated. MDH S5E mutant of hMDH1v3, unique of all splice variants was created while additional mutations of the mid‐length, canonical variant of hMDH1 (hMDH1v1) at residues: S89, S188, S241, and T321 were also generated. These sites were selected for their potential effect on catalysis due to location within the catalytic or binding domain (S188 and S89, respectively), and the overlap of all phosphorylation prediction and mass spectrometry databases (S241 and T321). Our goal in creating phosphomimics is to determine the role of post‐translational modification (phosphorylation) on the regulation of hMDH1 in its stability, kinetic activity, effects on structural changes, and open the potential of phosphorylation of MDH on protein‐protein interactions. Support or Funding Information NSF EHR‐IUSE Project support NSF‐1726932