Customizing Functionalized Cofactor Mimics to Study the Human Pyridoxal 5'-Phosphate-Binding Proteome

Cell Chem Biol. 2019 Oct 17;26(10):1461-1468.e7. doi: 10.1016/j.chembiol.2019.08.003. Epub 2019 Aug 22.

Abstract

Pyridoxal 5'-phosphate (PLP) is a versatile cofactor that catalyzes a plethora of chemical transformations within a cell. Although many human PLP-dependent enzymes (PLP-DEs) with crucial physiological and pathological roles are known, a global method enabling their cellular profiling is lacking. Here, we demonstrate the utility of a cofactor probe for the identification of human PLP-binding proteins in living cells. Striking selectivity of human pyridoxal kinase led to a customized labeling strategy covering a large fraction of known PLP-binding proteins across various cancer-derived cell lines. Labeling intensities of some PLP-DEs varied depending on the cell type while the overall protein expression levels of these proteins remained constant. In addition, we applied the methodology for in situ screening of PLP-antagonists and unraveled known binders as well as unknown off-targets. Taken together, our proteome-wide method to study PLP-DEs in human cancer-derived cells enables global understanding of the interactome of this important cofactor.

Keywords: chemical proteomics; cofactor; enzyme; mass spectrometry; pyridoxal 5′-phosphate.

Publication types

  • Research Support, Non-U.S. Gov't