Institutional Publications

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PROTEIN L-ISOASPARTYL METHYLTRANSFERASE (PIMT) in plants: regulations and functions
(Portland Press, 2020) Kamble, Nitin Uttam; Majee, Manoj
Proteins are essential molecules that carry out key functions in a cell. However, as a result of aging or stressful environments, the protein undergoes a range of spontaneous covalent modifications, including the formation of abnormal l-isoaspartyl residues from aspartyl or asparaginyl residues, which can disrupt the protein's inherent structure and function. PROTEIN l-ISOASPARTYL METHYLTRANSFERASE (PIMT: EC 2.1.1.77), an evolutionarily conserved ancient protein repairing enzyme (PRE), converts such abnormal l-isoaspartyl residues to normal l-aspartyl residues and re-establishes the protein's native structure and function. Although originally discovered in animals as a PRE, PIMT emerged as a key PRE in plants, particularly in seeds, in which PIMT plays a predominant role in preserving seed vigor and viability for prolonged periods of time. Interestingly, higher plants encode a second PIMT (PIMT2) protein which possesses a unique N-terminal extension, and exhibits several distinct features and far more complexity than non-plant PIMTs. Recent studies indicate that the role of PIMT is not restricted to preserving seed vigor and longevity but is also implicated in enhancing the growth and survivability of plants under stressful environments. Furthermore, expression studies indicate the tantalizing possibility that PIMT is involved in various physiological processes apart from its role in seed vigor, longevity and plant's survivability under abiotic stress. This review article particularly describes new insights and emerging interest in all facets of this enzyme in plants along with a concise comparative overview on isoAsp formation, and the role and regulation of PIMTs across evolutionary diverse species. Additionally, recent methods and their challenges in identifying isoaspartyl containing proteins (PIMT substrates) are highlighted.
Arabidopsis Protein L-Isoaspartyl Methyltransferase repairs isoaspartyl damage to antioxidant enzymes and increases heat and oxidative stress tolerance
(American Society for Biochemistry and Molecular Biology, 2020) Ghosh, Shraboni; Kamble, Nitin Uttam; Verma, Pooja; Salvi, Prafull; Petla, Bhanu Prakash; Roy, Shweta; Rao, Venkateswara; Hazra, Abhijit; Varshney, Vishal; Kaur, Harmeet; Majee, Manoj
Stressful environments accelerate the formation of isoaspartyl (isoAsp) residues in proteins, which detrimentally affect protein structure and function. The enzyme Protein L-Isoaspartyl Methyltransferase (PIMT) repairs other proteins by reverting deleterious isoAsp residues to functional aspartyl residues. PIMT function previously has been elucidated in seeds, but its role in plant survival under stress conditions remains undefined. Herein, we used molecular, biochemical, and genetic approaches, including protein overexpression and knockdown experiments, in Arabidopsis to investigate the role of PIMTs in plant growth and survival during heat and oxidative stresses. We demonstrate that these stresses increase isoAsp accumulation in plant proteins, that PIMT activity is essential for restricting isoAsp accumulation, and that both PIMT1 and PIMT2 play an important role in this restriction and Arabidopsis growth and survival. Moreover, we show that PIMT improves stress tolerance by facilitating efficient reactive oxygen species (ROS) scavenging and thereby protecting the functionality of antioxidant enzymes from isoAsp-mediated damage during stress. Specifically, biochemical and MS/MS analyses revealed that antioxidant enzymes acquire deleterious isoAsp residues during stress, which adversely affect their catalytic activities, and that PIMT repairs the isoAsp residues and thereby restores antioxidant enzyme function. Collectively, our results suggest that the PIMT-mediated protein repair system is an integral part of the stress tolerance mechanism in plants, in which PIMTs protect antioxidant enzymes that maintain proper ROS homeostasis against isoAsp-mediated damage in stressful environments.
Arabidopsis SKP1-like protein 13 (ASK13) positively regulates seed germination and seedling growth under abiotic stresses
(Oxford University Press, 2018) Rao, Venkateswara; Petla, Bhanu Prakash; Verma, Pooja; Salvi, Prafull; Kamble, Nitin Uttam; Ghosh, Sharboni; Kaur, Harmeet; Saxena, Saurabh C; Majee, Manoj
SKP1 (S-Phase Kinase Associated Protein1) proteins are key members of the SCF (SKP-Cullin-F-box protein) E3 ligase complexes that ubiquitinate the target proteins and play diverse roles in plant biology. However, as compared to other members of the SCF complex, study of SKP1-like proteins is very limited in plants. In the present work, we report that Arabidopsis SKP1-like protein13 (ASK13) is differentially regulated in different organs, during seed development and germination, and is upregulated in response to abiotic stresses. Y2H library screening and subsequent in vivo interaction through BiFC analysis revealed that ASK13 not only interacts with F-box proteins but also with other proteins which are not components of SCF complexes. Biochemical analysis revealed that ASK13 not only exists as a monomer but also as a homo-oligomer or heteromer with other ASK proteins. Functional analysis using ASK13 overexpression and knockdown lines revealed that ASK13 positively influences seed germination and seedling growth particularly under abiotic stresses. Taken together, our data strongly suggests that apart from participation to form SCF complexes, ASK13 interacts with several other proteins and is implicated in different cellular processes distinct from protein degradation. Overall, ASK13 positively regulates seed germination and seedling growth particularly under abiotic stress conditions.

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