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Summer Research Fellowship Programme of India's Science Academies

Establishing SUMOylation Assay (In-vivo and In-vitro) to understand the dynamics of filamentous proteins

Jyoti K

KLE College of Pharmacy, Belagavi 590010

Dr. Ram Kumar Mishra

Indian Institute of Science Education and Research, Bhopal 462066

Abstract ​​

 The formation of senile plaques and neurofibrillary tangles are the main hallmarks of neurodegenerative disorder, whereas synaptic loss best correlates to progressive cognitive decline. Several molecular, genetic, pathophysiological, pharmacological and therapeutical studies support the hypothesis of aggregation and misfolding of certain proteins like beta (amyloid, tau) and alpha (synuclein). Interestingly, some of these proteins involved in these pathophysiological processes have been reported to be subject to post-translational modification by ubiquitin and/or the small ubiquitin-like modifier (SUMO ) by modulating the function of human disease-related misfolded proteins Tau and alpha-synuclein. A multi-enzyme catalyzed SUMOylation process thus holds potential to be a therapeutic target in psychiatric diseases and neurodegenerative disorders. A better understanding of SUMO regulatory mechanisms will lead to improved approaches for analyzing the function of SUMO and substrate conjugation. Thus, the cornerstone of the project is to understand the mechanism of action of SUMO modification and to apprehend the knowledge of how the structure and function of the target proteins are modulated through SUMOylation pathway, which is best understood by in-vitro and in-vivo experiments. The in-vivo experiment involves molecular cloning of the gene which undergoes SUMOylation and the in-vitro experiment uses glutathione S-transferase (GST) gene fusion proteins as a method for inducible, high-level protein expression and purification from bacterial cell lysates (in which the protein is expressed in a pGEX vector, with the GST moiety located at the N-terminus followed by the target protein). Purified proteins have been used successfully in SUMOylation studies and other biochemical analyses.

 Keywords: SUMO proteins, molecular cloning, post-translational modification(PTMs), misfolding proteins, GST tagged purification, biochemical reactions

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