Abstract
zDHHC S-acyltransferases are enzymes catalyzing protein S-acylation, a common post-translational modification on proteins 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 the AR of zDHHC17, we employed peptide arrays based on zDHHC AR-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) for zDHHC17 AR binding, with which we predicted 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.
Highlights
ZDHHC S-acyltransferases are enzymes catalyzing protein S-acylation, a common post-translational modification on proteins frequently affecting their membrane targeting and trafficking
To further refine the rules governing binding to the ankyrin repeat (AR) of zDHHC17, we employed peptide arrays based on zDHHC AR-binding motif sequences of synaptosomal-associated protein 25 (SNAP25) and cysteine string protein ␣ (CSP␣)
Binding of ARzD17-His was more sensitive to alterations of amino acids at positions 2–3 and 6 –7 for both SNAP25 and CSP␣ peptides; amino acid preferences at position 2 for the ARzD17His binding were slightly different for SNAP25 and CSP␣, whereas additional sensitivity at position 9 was observed for SNAP25 peptides (Fig. 1)
Summary
Establishment of sequence rules governing binding of peptides to the AR of zDHHC17. Peptide arrays consisting of 15-mers of SNAP25 and CSP␣ peptides were constructed, with all amino acids within a 10-amino acid region containing the zDABM serially substituted to any possible amino acid. Binding of purified His6-tagged AR domain (residues 51–288) of zDHHC17 (ARzD17-His) to each of these peptides was subsequently assessed by far-Western blotting using a histidine tag antibody (Fig. 1). Binding of ARzD17-His was more sensitive to alterations of amino acids at positions 2–3 and 6 –7 for both SNAP25 and CSP␣ peptides; amino acid preferences at position 2 for the ARzD17His binding were slightly different for SNAP25 and CSP␣ (e.g. preference for proline in CSP␣), whereas additional sensitivity at position 9 was observed for SNAP25 peptides (Fig. 1). As derived from both SNAP25 and CSP␣ arrays, no amino acid preference for ARzD17-binding was observed at position 1; amino acids glycine and proline were strongly disfavored at positions 4 and 5; and negatively charged (Asp and Glu) amino acids were disfavored at positions 8 –10
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