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Unraveling the activity of glycosyltransferases and other PTM enzymes with bioluminescent biochemical and cell based assays
Author(s) -
Zegzouti Hicham,
Engel Laurie,
Hwang Brian Byounghoon,
Alves Juliano,
Goueli Said
Publication year - 2019
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.2019.33.1_supplement.799.4
Subject(s) - phosphorylation , enzyme , glycosyltransferase , biology , biochemistry , glycosylation , computational biology , kinase , microbiology and biotechnology
Post translational modifications (PTMs) are central to all aspects of biological regulation. PTMs amplify the diverse functions of the proteome by covalently adding functional groups to proteins. These modifications include phosphorylation, methylation, glycosylation, and ubiquitination, and influence many aspects of normal cell biology and pathogenesis. Examples of post translational modification enzymes include but are not limited to, kinases/phosphatases, methyltransferases/demethylases, and glycosyltransferases/glucanases. Under normal physiological conditions, the regulation of PTM enzymes is tightly regulated. However, under pathological conditions, these enzyme activities can be dysregulated, and the disruption of the intracellular networks they govern leads to an array of diseases including cancer and inflammation. Consequently, PTM enzymes have become important targets for drug discovery creating a need for development of activity detection assays. Current assays to analyze cellular or biochemical PTM enzyme activities can be tedious, non‐homogeneous, and not easily adaptable to HTS. They can also suffer from generation of false hits due to compound interference or require special instruments for detection. To overcome these shortcomings, we developed an array of biochemical and cellular bioluminescent assays to detect these enzyme activities. This presentation will showcase how cellular mechanisms driven by PTM in general and phosphorylation and glcnacylation in particular can be addressed by analyzing these enzyme's cellular activity using the NanoLuc Binary Technology (NanoBiT), a two‐subunit complementation system based on NanoLuc luciferase. We will also discuss how the bioluminescent nucleotide assays that detect UDP, GDP, UMP and CMP as means to measure glycosyltransferase activities can streamline studies on GT specificity of transfer of different sugars to different acceptors. We will show their use in screening for specific inhibitors as well as in studies of their modes of action. Development of these bioluminescent detection assays will enable the investigation of key signaling pathways and the study of many PTM enzymes in general and GTs in particular, which may have significant impact on diverse areas of glycobiology research. This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .