Mechanistic dissection of substrate selectivity of protein palmitoyltransferases

Authors

• T Mondal
• A Banerjee

Abstract

Protein palmitoylation is the most prevalent form among all forms of protein lipidation and recognition of its importance in cellular physiology has been emerging in recent years. Approximately 6000 proteins are known to be targets of protein palmitoylation, which is catalyzed by 23 members of the zDHHC family of integral membrane enzymes and plays critical roles in numerous pathologies such as neurodegenerative diseases, cancer. Many substrates can be palmitoylated by more than one zDHHC enzymes in cell-based experiments, raising the question whether there are any substrate-selective interactions for each zDHHC-substrate pair at all. However, the role of complex cellular factors cannot be eliminated in these experiments and therefore there is a dire need for biochemical reconstitution experiments to study protein palmitoylation. I have developed an in vitro reconstitution assay for palmitoylation of several substrates using purified zDHHC enzymes and synthesized peptide fragments of the substrates. Remarkably, all the zDHHC enzymes showed distinct preferred activity for substrates over non-substrates. This is the first in vitro reconstitution of substrate discrimination of any zDHHC enzyme with a biochemical assay. Using this assay, I examined how nearby residues around the target cysteine on the substrate impact substrate palmitoylation, yet another completely unexplored aspect of protein palmitoylation. This work also builds the groundwork for future structural studies to understand the atomic basis of substrate palmitoylation by zDHHC enzymes.

Scientific Focus Area: Chemical Biology

This page was last updated on Monday, September 25, 2023