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

Area of Science:

• Biochemistry
• Cell Biology
• Proteomics

Background:

• Pyridoxal 5'-phosphate (PLP) is a crucial cofactor for numerous enzymes involved in vital cellular processes.
• Existing methods for profiling human PLP-dependent enzymes (PLP-DEs) in cells are limited.
• Understanding PLP-DEs is critical due to their roles in physiology and pathology, including cancer.

Purpose of the Study:

• To develop and validate a global method for profiling human PLP-binding proteins in living cells.
• To investigate the cellular variability of PLP-DE labeling and its relationship to protein expression.
• To apply the methodology for screening potential PLP antagonists and identifying off-target interactions.

Main Methods:

• Development of a selective cofactor probe for pyridoxal 5'-phosphate.
• Application of the probe for labeling PLP-binding proteins in various human cancer cell lines.
• Quantitative analysis of labeling intensities and comparison with overall protein expression levels.
• In situ screening of small molecules for PLP-antagonist activity.

Main Results:

• The cofactor probe successfully identified a large fraction of known PLP-binding proteins across different cancer cell lines.
• Labeling intensities of specific PLP-DEs varied by cell type, independent of total protein expression.
• The method identified known and novel off-targets during in situ screening of PLP antagonists.

Conclusions:

• A novel proteome-wide method enables global profiling of human PLP-DEs in cancer cells.
• The findings provide insights into the cellular interactome of the PLP cofactor.
• This methodology facilitates the study of PLP-DEs and the screening of their modulators.