Dissecting substrate interactions of zDHHC enzymes by in vitro biochemical reconstitution

Authors

• T Mondal
• A Banerjee

Abstract

Protein palmitoylation is the most prevalent form of protein lipidation with 23 zDHHC enzymes catalyzing the palmitoylation of 6000 substrates. In cellulo experiments raise the question of substrate-selective interactions for each zDHHC-substrate pair. There is a dire lack of biochemical reconstitution to study palmitoylation. I have developed in vitro reconstitution assay for palmitoylation of several substrates using purified zDHHC enzymes and synthesized peptide fragments of substrates. Results from in vitro assay agreed well with literature as to what the cognate zDHHC enzyme/s are for each substrate. This is the first in vitro biochemical reconstitution of substrate discrimination by any zDHHC enzyme and demonstrates zDHHC enzymes have clear, distinct preferences for substrates. I further interrogated the role of residues around target cysteine on substrate-palmitoylation in detergent-micelles and lipid-nanodiscs which led to the first insights into how neighboring residues around target cysteine affects palmitoylation of SARS-CoV-2 Spike protein as well as PSD-95.

Scientific Focus Area: Chemical Biology

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