Peptide array-based screening reveals a large number of proteins interacting with the ankyrin-repeat domain of the zDHHC17 S-acyltransferase

Abstract

zDHHC S-acyltransferases are enzymes catalyzing protein S-acylation, acommonpost-translational modificationonproteins frequently affecting their membrane targeting and trafficking. The ankyrin repeat (AR) domain of zDHHC17 (HIP14) and zDHHC13 (HIP14L) S-acyltransferases, which is involved in both substrate recruitment and S-acylation-independent functions, was recently shown to bind at least six proteins, by specific recognition of a consensus sequence in them. To further refine the rules governing binding to theARof zDHHC17, we employed peptide arrays based onzDHHCAR-binding motif(zDABM)sequences of synaptosomal- associated protein 25 (SNAP25) and cysteine string protein α (CSPα). Quantitative comparisons of the binding preferences of 400 peptides allowed us to construct a position-specific scoring matrix(PSSM)forzDHHC17ARbinding, with whichwepredicted and subsequently validated many putative zDHHC17 interactors. We identified 95 human zDABM sequences with unexpected versatility in amino acid usage; these sequences were distributed among 90 proteins, of which 62 have not been previously implicated in zDHHC17/13 binding. These zDABM-containing proteins included all family members of the SNAP25, sprouty, cornifelin, ankyrin, and SLAIN-motif containing families; seven endogenous Gag polyproteins sharing the same binding sequence; and several proteins involved in cytoskeletal organization, cell communication, and regulation of signaling. A dozen of the zDABM-containing proteins had more than one zDABM sequence, whereas isoform-specific binding to the AR of zDHHC17 was identified for the Ena/VASP-like protein. The large number of zDABM sequences within the human proteome suggests that zDHHC17 may be an interaction hub regulating many cellular processes.