Investigation of isoAsp accumulation to antioxidative enzymes during thermal stress and their repair by PROTEIN L-ISOASPARTYL METHYLTRANSFERASE (PIMT).

ABSTRACT: The formation of isoAsp residues from Asp and Asn residues in proteins and peptides is accelerated under stressful environments and has detrimental effects on protein structure and function. PROTEIN L-ISOASPARTYL METHYLTRANSFERASE (PIMT) is a protein repairing enzyme which revert such deleterious isoAsp residues to normal aspartyl residues as a protein repair process. In present work, we aim to study the isoaspartyl formation of antioxidative enzymes, superoxide dismutase (SOD) and catalase (CAT) under stressful environments and their repair by PIMT in Arabidopsis. The role of PIMT in repairing these enzymes for the plant survival under heat and oxidative stress were investigated through molecular, biochemical approaches in Arabidopsis. We demonstrated that isoAsp accumulation in proteins increase upon heat and oxidative stress, and PIMT activity is essential to restrict such isoAsp accumulation. We demonstrated that PIMT play an important role under heat and oxidative stress by restricting stress-induced deleterious isoAsp accumulation in proteins. Analyses of PIMT overexpression and RNAi lines suggest that PIMT maintains ROS homeostasis by protecting the functionality of antioxidative enzymes from isoAsp mediated damage under stressful environments. To confirm further whether Asn residues of antioxidative enzymes indeed susceptible to isoAsp formation, we used bacterially expressed and purified recombinant proteins for MS analyses. Purified proteins were subjected to thermal insult and then MS/MS analyses were carried out to identify isoAsp modification. Our MS analyses suggest that these antioxidative enzymes are indeed susceptible to isoAsp formation under thermal stress.

INSTRUMENT(S): Q TRAP

ORGANISM(S): Arabidopsis Thaliana (mouse-ear Cress)

SUBMITTER: Manoj Majee  

LAB HEAD: Dr. Manoj Majee

PROVIDER: PXD016240 | Pride | 2019-12-18

REPOSITORIES: Pride