In vitro reconstitution reveals substrate selectivity of protein S-acyltransferases

Area of Science:

• Biochemistry
• Molecular Biology
• Cell Biology

Background:

• Protein S-acylation (palmitoylation) is a prevalent lipidation affecting thousands of proteins.
• The zDHHC enzyme family catalyzes palmitoylation, crucial for cellular functions and diseases.
• zDHHC-substrate interactions are complex and poorly understood due to limited in vitro studies.

Purpose of the Study:

• To develop and utilize an in vitro assay for studying zDHHC enzyme-substrate interactions.
• To investigate substrate specificity and hierarchy among zDHHC enzymes.
• To elucidate the role of neighboring residues in substrate S-acylation.

Main Methods:

• Developed the Pep-PAT assay using purified zDHHC enzymes and substrate peptide fragments.
• Assayed S-acylation of seven substrates by three zDHHC enzymes in vitro.
• Investigated the impact of residues flanking the target cysteine on S-acylation of PSD-95 and SARS-CoV-2 Spike protein.
• Validated findings using in cellulo assays.

Main Results:

• Demonstrated distinct substrate preferences and hierarchies for different zDHHC enzymes.
• Identified specific neighboring residues that modulate substrate S-acylation.
• Confirmed the influence of these residues on S-acylation in cellular contexts.
• Established a framework for understanding zDHHC enzyme-substrate recognition.

Conclusions:

• zDHHC enzymes exhibit preferred substrate hierarchies.
• Neighboring residues critically influence substrate S-acylation by zDHHC enzymes.
• The Pep-PAT assay provides a powerful tool for dissecting palmitoylation mechanisms.