Orthogonal Enzyme–Substrate Design Strategy for Discovery of Human Protein Palmitoyltransferase Substrates

Protein palmitoylation,
with more than 5000 substrates, is the
most prevalent form of protein lipidation. Palmitoylated proteins
participate in almost all areas of cellular physiology and have been
linked to several human diseases. Twenty-three zDHHC enzymes catalyze
protein palmitoylation with extensive overlap among the substrates
of each zDHHC member. Currently, there is no global strategy to delineate
the physiological substrates of individual zDHHC enzymes without perturbing
the natural cellular pool. Here, we outline a general approach to
accomplish this on the basis of synthetic orthogonal substrates that
are only compatible with engineered zDHHC enzymes. We demonstrate
the utility of this strategy by validating known substrates and use
it to identify novel substrates of two human zDHHC enzymes. Finally,
we employ this method to discover and explore conserved palmitoylation
in a family of host restriction factors against pathogenic viruses,
including SARS-CoV-2.