Biology subjects

Sanders, S. S.

Publications and source records attributed to Sanders, S. S..

9 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

Brain and neuronal expression and localization of de-S-acylating enzymes

S-acylation is a reversible posttranslational lipid modification important in the nervous system that dynamically regulates protein localization and function. Aberrant S-acylation has been implicated in several neurological conditions. While several de-S-acylases (deacylases hereafter) have been identified, little is known regarding their expression and localization in the brain and in neurons. Here, we characterized the expression, localization, and S-acylation of cytosolic deacylases, including acyl-protein thioesterases APT1, APT2, and APT1L and /{beta} hydrolase domain-containing proteins ABHD7, ABHD10, ABHD13, ABHD16A, and ABHD17A-C. Mouse brain RNA sequencing data reveal high expression of Lypla1/APT1, Lypla2/APT2, Ephx4/ABHD7, Abhd16a, and Abhd17A-C in the brain, whereas Lyplal1/APT1L, Abhd10, and Abhd13 were expressed at very low levels. At the protein level, APT1 and ABHD16A levels are highest in the cerebellum with ABHD17A levels lowest in this region while APT2 levels are highest in the hippocampus. However, all four are abundant in cultured hippocampal neurons. Deacylases are localized throughout neurons on punctate structures, with APT2 and ABHD17C localized to the Golgi. Finally, all ten cytosolic deacylases are themselves S-acylated. These data characterizing deacylase expression, localization, and S-acylation in neural contexts, provides a foundation for future studies investigating deacylase neuronal functions and potential roles in neurological disease.

neuroscience↗

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↗

bioRxiv2023

Reduced S-acylation of SQSTM1/p62 in Huntington disease is associated with impaired autophagy

Disruption of macroautophagy/autophagy has emerged as a common feature in many neurodegenerative diseases. Autophagy is a membrane-dependent pathway that requires many key regulators to quickly localize on and off membranes during induction promoting membrane fusion. Previously, our bioinformatic approaches have shown that autophagy and Huntington disease (HD) are enriched in S-acylated proteins. S-acylation involves the reversible addition of long chain fatty acids to promote membrane binding. Herein, we show that inhibition of S-acylation regulates the abundance of several key regulators of autophagy and leads to a partial block of autophagic flux. We show that the autophagy receptor SQSTM1/p62 (sequestosome 1) is S-acylated and directed to the lysosome. Importantly, we see that SQSTM1 S-acylation is significantly reduced in HD patient and mouse model brains, thus providing a novel mechanism for the generation of empty autophagosomes previously seen in HD models and patient cells.

neuroscience↗

bioRxiv2021

Rescue of aberrant huntingtin palmitoylation ameliorates mutant huntingtin-induced toxicity

Huntington disease (HD) is a neurodegenerative disorder caused by a CAG expansion in the HTT gene that codes for an elongated polyglutamine tract in the huntingtin (HTT) protein. HTT is subject to multiple post-translational modifications (PTMs) that regulate its cellular function. Mutating specific PTM sites within mutant HTT (mHTT) in HD mouse models can modulate disease phenotypes, highlighting the key role of HTT PTMs in the pathogenesis of HD. These findings have led to increased interest in developing small molecules to modulate HTT PTMs in order to decrease mHTT toxicity. However, the therapeutic efficacy of pharmacological modulation of HTT PTMs in preclinical HD models remains largely unknown. HTT is palmitoylated at cysteine 214 by the huntingtin-interacting protein 14 (HIP14 or ZDHHC17) and 14-like (HIP14L or ZDHHC13) acyltransferases. Here, we assessed if HTT palmitoylation should be regarded as a therapeutic target to treat HD by (1) investigating palmitoylation dysregulation in rodent and human HD model systems, (2) measuring the impact of mHTT-lowering therapy on brain palmitoylation, and (3) evaluating if HTT palmitoylation can be pharmacologically modulate. We show that palmitoylation of mHTT and some HIP14/HIP14L-substrates is decreased early in multiple HD mouse models, and that aging further reduces HTT palmitoylation. Lowering mHTT in the brain of YAC128 mice is not sufficient to rescue aberrant palmitoylation. However, we demonstrate that mHTT palmitoylation can be normalized in COS-7 cells, in YAC128 cortico-striatal primary neurons and HD patient-derived lymphoblasts using an acyl-protein thioesterase (APT) inhibitor. Moreover, we show that modulating palmitoylation reduces mHTT aggregation and mHTT-induced cytotoxicity in COS-7 cells and YAC128 neurons. HighlightsO_LIPalmitoylation of mHTT is reduced in multiple transgenic HD mouse models C_LIO_LIHTT palmitoylation decreases with increasing polyQ length in HD patient cells C_LIO_LImHTT-lowering in mouse brains does not rescue aberrant palmitoylation C_LIO_LImHTT palmitoylation in HD patient-derived cells can be rescued via APT inhibition C_LIO_LIPromoting palmitoylation reduces mHTT aggregation and cytotoxicity in vitro C_LI

neuroscience↗

bioRxiv2020

The Palmitoyl Acyltransferase ZDHHC14 Controls Kv1-Family Potassium Channel Clustering at the Axon Initial Segment

The palmitoyl acyltransferase (PAT) ZDHHC14 is highly expressed in the hippocampus and is the only PAT predicted to bind Type I PDZ domain-containing proteins. However, ZDHHC14s neuronal roles are unknown. Here, we identify the PDZ domain-containing Membrane-associated Guanylate Kinase (MaGUK) PSD93 as a direct ZDHHC14 interactor and substrate. PSD93, but not other MaGUKs, localizes to the Axon Initial Segment (AIS). Using lentiviral-mediated shRNA knockdown in rat hippocampal neurons, we find that ZDHHC14 controls palmitoylation and AIS clustering of PSD93 and also of Kv1 potassium channels, which directly bind PSD93. Neurodevelopmental expression of ZDHHC14 mirrors that of PSD93 and Kv1 channels and, consistent with ZDHHC14s importance for Kv1 channel clustering, loss of ZDHHC14 decreases outward currents and increases action potential firing in hippocampal neurons. To our knowledge, these findings identify the first neuronal roles and substrates for ZDHHC14 and reveal a previously unappreciated role for palmitoylation in control of neuronal excitability. Impact StatementZDHHC14 controls palmitoylation and axon initial segment targeting of PSD93 and Kv1-family potassium channels, events that are essential for normal neuronal excitability.

neuroscience↗

bioRxiv2019

Ankyrin-G-190 palmitoylation mediates dendrite and spine morphogenesis and is altered in response to lithium

AnkG, encoded by the ANK3 gene, is a multifunctional scaffold protein with complex isoform expression: the 480 kDa and 270 kDa isoforms have roles at the axon initial segment and node of Ranvier, whereas the 190 kDa isoform (AnkG-190) has an emerging role in the dendritic shaft and spine heads. All isoforms of AnkG undergo palmitoylation, a post-translational modification regulating protein attachment to lipid membranes. However, palmitoylation of AnkG-190 has not been investigated in dendritic spines. The ANK3 gene and altered expression of AnkG proteins are associated with a variety of neuropsychiatric and neurodevelopmental disorders including bipolar disorders and are implicated in the lithium response, a commonly used mood stabilizer for bipolar disorder patients, although the precise mechanisms involved are unknown. Here, we showed that Cys70 palmitoylation stabilizes the localization of AnkG-190 in spine heads and at dendritic plasma membrane nanodomains. Mutation of Cys70 impairs AnkG-190 function in dendritic spines and alters PSD-95 scaffolding. Interestingly, we find that lithium reduces AnkG-190 palmitoylation thereby increasing its mobility in dendritic spines. Finally, we demonstrate that the palmitoyl acyl transferase ZDHHC8, but not ZDHHC5, increases AnkG-190 stability in spine heads and is inhibited by lithium. Together, our data reveal that palmitoylation is critical for AnkG-190 localization and function and a potential ZDDHC8/AnkG-190 mechanism linking AnkG-190 mobility to the neuronal effects of lithium.

neuroscience↗

bioRxiv2018

Identification of novel inhibitors of DLK palmitoylation by High Content Screening

After axonal insult and injury, Dual leucine-zipper kinase (DLK) conveys retrograde pro-degenerative signals to neuronal cell bodies via its downstream target c-Jun N-terminal kinase (JNK). We recently reported that such signals critically require modification of DLK by the fatty acid palmitate, via a process called palmitoylation. Compounds that inhibit DLK palmitoylation could thus reduce neurodegeneration, but identifying such inhibitors requires a suitable assay. Here we report that DLK subcellular localization in non-neuronal cells is highly palmitoylation-dependent and can be used as a proxy readout to identify inhibitors of DLK palmitoylation by High Content Screening (HCS). We exploited this highly specific localization of DLK-GFP as the basis for a screen of the Prestwick Compound Library. We found that ketoconazole, a Prestwick Library compound that most dramatically affected DLK subcellular localization in our primary screen, inhibited DLK palmitoylation in a dose-dependent manner in follow-up biochemical assays. Moeroever, ketoconazole significantly blunted phosphorylation of c-Jun in primary sensory neurons subjected to Trophic Deprivation, a well known model of DLK-dependent pro-degenerative signaling. These findings suggest that our HCS platform is capable of identifying novel inhibitors of DLK palmitoylation and signalling that may have considerable therapeutic potential.

neuroscience↗