Identification of the palmitoyltransferase DHHC4 interactome using proximity-dependent biotinylation by BioID2

ROCÍO MEINERO; JAVIER VALDEZ TAUBAS

Mendoza

Congreso; 58 SAIB; 2022

SAIB-SAMIGE

Identification of protein-protein associations is a fundamental approach to the study of protein function and biological mechanisms. BioID (proximity-dependent biotin identification) it?s a novel and powerful tool for studying protein-protein interactions in vivo. This system relies on fusing a bait protein of interest with a mutant promiscuous biotin ligase enzyme capable of biotinylation of the amine groups of the neighboring proteins over biotin. After, biotinylated proteins can be selectively isolated by conventional biotin capture methods and identified using liquid chromatography-mass spectrometry (LC-MS). One of the greatest benefits of BioID2 is that is not limited to direct binders because can detect weak and transient interactions between proteins over a while. Also, this approach does not require toxic reagents, and it is simple to use.S-acylation is a common post-translational modification of cellular proteins involving the attachment of fatty acids onto cysteine residue. This modification is reversible and regulates processes of great biological importance and many diseases are linked to perturbations in S-acylation. A family of enzymes named Palmitoyltransferases (PATs) catalyzed this modification. There are 23 members of this family (DHHC1-23) in the human genome and the information about them reminds limited and still growing. Here, we focus our research on DHHC4, a not-well-characterized PAT. The present study explored the interactome of DHHC4 looking for new interacting proteins, using BioID2 in cultured human cells HeLa. We electroporated HeLa with DHHC4-BioID2-HA or BioID2-HA only as a control. Differences in protein biotinylation between treatments were confirmed by immunoblotting from HeLa cells. Next, we confirm the presence of the constructs in the endoplasmic reticulum by indirect immunofluorescence microscopy. In the LC-MS experiment, our preliminary results detected x interacting proteins that may represent new possible regulators or targets of DHHC4. In an attempt to identify possible substrates in the list of interacting proteins found, we have searched for predicted palmitoylation sites in all identified proteins in the SwissPalm software. We are currently confirming these results increasing the replicates and validating the palmitoylation status of some of these proteins. Thus, new possible regulators or targets of DHHC4 may have been identified, contributing to future palmitoylation research.