Biology subjects

Petropavlovskiy, A. A.

Publications and source records attributed to Petropavlovskiy, A. A..

3 recordsLinked to original sources

bioRxiv2026

GAPDH is tethered to axonal transport vesicles by S-acylation

Vesicle movement along axonal microtubules in neurons requires the ATP-dependent molecular motors dynein and kinesin. Fast axonal transport is fueled by ATP, provided by vesicle-associated glycolytic enzymes, but how these predicted soluble enzymes attach to vesicles in unclear. One potential mechanism is the protein lipid modification S-acylation, which involves the addition of long chain fatty acids to protein cysteine residues mediated by the ZDHHC (Asp-His-His-Cys) family of protein S-acyltransferases. Among the many effects this lipid modification imparts is an increase in protein localization to membranes. We found that eight of the ten glycolytic enzymes are S-acylated in the brain. Of the 10 glycolytic enzymes, we focused on glyceraldehyde 3-phosphate dehydrogenase (GAPDH) as it is the first enzyme of the payoff phase of glycolysis. GAPDH is S-acylated on cysteine 247 by ZDHHC5 and ZDHHC17. Importantly, C247 point mutation impairs GAPDH association with vesicles in hippocampal neurons. Investigating the role of S-acylation in glycolytic enzyme localization will lead to novel insights into neuronal transport mechanisms and may also shed light on neurodegenerative disease pathology and potential drug targets.

neuroscience↗

bioRxiv2026

SAPPTree: Identification of an S-Acylation Motif Drives a Novel S-Acylation Prediction Program

S-acylation, the reversible addition of fatty acids to proteins, has emerged as an abundant post-translational modification that drives protein localization and function. With no known consensus sequence, current prediction programs rely on machine learning algorithms that use short peptide sequences and large proteomic datasets. However, current prediction programs often suggest incorrect sites of S-acylation, leading to wasted experimental time and effort following site-directed mutagenesis and low-throughput validation experiments. Using only experimentally confirmed sites of S-acylation, we sought to identify primary sequence, secondary structure, and tertiary structure features common amongst S-acylation sites to aid in developing more robust prediction tools. In doing so, we identified an S-acylation motif including a cysteine cluster flanked by a hydrophobic stretch, and a positively charged polybasic region found within a helical stretch. These features were combined with known or AlphaFold-predicted structures and additional features including residue depth and solvent accessibility into a random forest model to generate a new and more accurate S-acylation prediction program (SAPP), named SAPPTree. All the processed datasets and complete model training pipeline are available at https://github.com/neurdyphagy-lab/palm-prediction-model, while the webserver is available at http://martintools.sci.uwaterloo.ca/.

bioinformatics↗

bioRxiv2026

ZDHHC13 is a likely pseudoenzyme protein S-acyltransferase that functions via a non-canonical mechanism

S-acylation is the addition of fatty acids to cysteine residues to regulate protein function and localization. S-acylation is catalyzed by the ZDHHC (Asp-His-His-Cys) family of protein S-acyltransferases (PATs), which S-acylate protein substrates by first auto-S-acylating the catalytic cysteine of the DHHC active site followed by transfer to the substrate. ZDHHC13 and ZDHHC17 are related ankyrin repeat domain (ANK) PATs that S-acylate multiple neuronal proteins, including huntingtin (HTT), the protein mutated in Huntington disease. However, unlike ZDHHC17 and other human PATs, ZDHHC13 possesses a non-canonical DQHC active site. As the first histidine is essential for auto-S-acylation, it is unclear if ZDHHC13 is catalytically active. Our phylogenetic analysis of eukaryotic ANK-containing PATs shows that ZDHHC13 orthologues are more divergent compared to ZDHHC17. While the ZDHHC17 DHHC is highly conserved, the motif varies among ZDHHC13 orthologues, with some vertebrate lineages containing a serine in place of the catalytic cysteine. Interestingly, we found that the ZDHHC13 S-acylation is lower than that of ZDHHC17, but the ZDHHC13 catalytic cysteine is indeed S-acylated. While expression of wild type (WT) ZDHHC13 in ZDHHC13 deficient HEK293T cells increased S-acylation of a HTT1-588 fragment, surprisingly, expression of catalytically dead DQHS ZDHHC13 was still able to facilitate HTT1-588 S-acylation equally. This suggests the ZDHHC13 catalytic cysteine is not required for S-acylation of target proteins, suggesting ZDHHC13 may coordinate another PAT. Indeed, we identified ZDHHC13 in high-molecular weight complexes. Our results indicate that ZDHHC13 is a likely pseudoenzyme that may function via a non-conventional mechanism reliant on other PATs. This work broadens our understanding of the function of this non-canonical PAT.

biochemistry↗