Energetically economical protective processes can repair some of the spontaneous and deleterious alteration of proteins, occurring in all cells as they age. Non-enzymatic conversion of aspartate and asparagine residues to the abnormal isoaspartate (isoAsp) is a common form of deleterious covalent protein modification in cells. Seeds, comprising 70% of our diet, provoke a cellular environment particularly susceptible to isoAsp formation when they dehydrate as a natural consequence of seed maturation. This project focuses on the enzyme L-isoaspartyl methyltransferase (PIMT), which specifically repairs isoAsp to Asp. The project brings together a group at the University of Kentucky (UKY) with a collaborating team at the University of California Los Angeles (UCLA) to examine enzyme kinetic properties of PIMT1 and PIMT2 from the model plant, Arabidopsis thaliana. In this project, the researchers will study the ability of plant PIMTs to effect the conversion of isoaspartyl residues embedded in a host of amino acid contexts comparing these enzymes with those from other eukaryotes. In addition, the consequences for the plant of altering the expression of either PIMT gene will be elucidated. Cell-sorting and proteomic technology, phage display libraries, and high-affinity PIMT antibodies will be used to identify in vivo PIMT target proteins. Information obtained on enzyme kinetic properties will steer efforts to expose mutants to appropriate stress conditions and to label PIMT targets. This research, examining enzyme kinetic properties of various PIMT proteins produced from alternatively spliced transcripts, will provide insights into PIMT structure and function.
Broader impacts: The identification of isoAsp-susceptible proteins that, when damaged, are inhibitory to the completion of germination of seeds. Work with the plant PIMT enzymes, elucidating their significance in seeds, may lead to improved seed storage, more vigorous germination, and improved nutritional value. Discoveries made during the execution of this project will be disseminated by the web sites, through regional- and international-meetings, and peer-reviewed journals. The project will involve training of students at graduate and undergraduate levels and interactions with a minority-serving institution.
This project is funded jointly by the Biomolecular Systems Cluster and the Experimental Program to Stimulate Competitive Research.
