Biology subjects

Thomas, G. M.

Publications and source records attributed to Thomas, G. M..

8 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↗

bioRxiv2025

Targeted RNAi screen reveals novel regulators of RNA-binding protein phase transitions in Caenorhabditis elegans oocytes

The ability of oocytes to maintain their quality is essential for successful reproduction. One critical aspect of oocyte quality and successful embryogenesis after fertilization is the proper regulation of the stores of maternal mRNA by RNA-binding proteins. Many RNA-binding proteins undergo regulated phase transitions during oogenesis, and alterations of the protein phase can disrupt its ability to regulate mRNA stability and translation. In C. elegans, genetic screens have identified regulators of RNA-binding protein condensation in arrested oocytes of females and in embryos, but less attention has focused on phase transitions in maturing oocytes of young adult hermaphrodites. Interestingly, of the relatively few regulators of RNA-binding protein phase transitions identified to date in maturing oocytes, several genes overlap with those required for clearance of protein aggregates in maturing oocytes. To determine the extent to which the temporally linked processes of clearance of damaged proteins and maintenance of RNP complexes are coordinated at a molecular level, we conducted a targeted RNAi screen of genes required for removal of protein aggregates in maturing oocytes. We identified six novel regulators of phase transitions of the KH-domain protein MEX-3 and obtained strong evidence that the regulatory network of protein aggregate clearance overlaps with, but is distinct from, the regulation of MEX-3 phase transitions in the oocyte.

genetics↗

bioRxiv2024

Short-Term Warming Induces Cyanobacterial Blooms and Antibiotic Resistance in a Freshwater Lake as Revealed by Metagenomics Analysis

Climate change poses significant risks to freshwater ecosystems, potentially exacerbating harmful cyanobacterial blooms and antibiotic resistance. We investigated these dual threats in Cosseys Reservoir, New Zealand, by simulating short-term warming scenarios and assessing the role of oxidative stress. Microcosms were subjected to Base (22{degrees}C), Normal (24{degrees}C), and projected Future (27{degrees}C) temperatures, with additional treatments including reactive oxygen species (ROS). Metagenomic analysis revealed substantial restructuring of microbial communities under warming conditions. Cyanobacterial abundance increased from 6.11% (initial) to 20.53% at 24{degrees}C and 10.66% at 27{degrees}C. Notably, ROS addition mimicked the effects of temperature increase on cyanobacterial proliferation. Toxin-producing families, including Microcystaceae and Nostocaceae, proliferated significantly. The microcystin synthesis gene (mcy) showed a strong positive association (R{superscript 2} = 0.88) with cyanobacterial abundance. Moreover, cyanobacteria exhibited enhanced nutrient acquisition (pstS gene, R{superscript 2} = 0.69) and upregulated nitrogen metabolism pathways under warming conditions. Concurrently, we observed a marked increase in antibiotic resistance gene (ARG) abundance with rising temperatures. The relative abundance of multidrug resistance genes was consistently high across all conditions (50.82 % of total ARGs). ROS stress further intensified ARG proliferation, particularly for efflux pump genes (e.g., acrB, adeJ, ceoB, emrB, MexK, muxB). Co-association network analysis identified key antibiotic-resistant bacteria (e.g., Streptococcus pneumoniae, Acinetobacter baylyi) and ARGs (e.g., acrB, MexK, rpoB2, bacA) central to resistance dissemination under warming conditions. This study demonstrates that even modest temperature increases (2-5{degrees}C) can promote both cyanobacterial blooms and antibiotic resistance in freshwater ecosystems over short time scales. The synergistic effects of temperature and oxidative stress underscore the complex challenges posed by climate change to water quality and public health. HighlightsO_LIShort-term warming promotes toxic cyanobacteria and antibiotic resistance in freshwater. C_LIO_LIHarmful cyanobacteria and their metabolic potential increase under warming conditions. C_LIO_LIReactive oxygen species mimic and exacerbate temperature effects. C_LIO_LIOxidative stress response genes strongly correlate with ARG abundance. C_LIO_LICo-association networks identify key host pathogen central to resistance spread. C_LI O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=114 SRC="FIGDIR/small/602497v1_ufig1.gif" ALT="Figure 1"> View larger version (31K): org.highwire.dtl.DTLVardef@157fc25org.highwire.dtl.DTLVardef@16b1103org.highwire.dtl.DTLVardef@29fa6eorg.highwire.dtl.DTLVardef@1f88650_HPS_FORMAT_FIGEXP M_FIG C_FIG

genomics↗

bioRxiv2024

The CCT chaperonin and actin modulate the ER and RNA-binding protein condensation during oogenesis to maintain translational repression of maternal mRNA and oocyte quality

The regulation of maternal mRNAs is essential for proper oogenesis, the production of viable gametes, and to avoid birth defects and infertility. Many oogenic RNA-binding proteins have been identified with roles in mRNA metabolism, some of which localize to dynamic ribonucleoprotein granules and others that appear dispersed. Here, we use a combination of in vitro condensation assays and the in vivo C. elegans oogenesis model to determine the intrinsic properties of the conserved KH-domain MEX-3 protein and to identify novel regulators of MEX-3 and the Lsm protein, CAR-1. We demonstrate that MEX-3 undergoes liquid-liquid phase separation and appears to have intrinsic gel-like properties in vitro. We also identify novel roles for the CCT chaperonin and actin in preventing ectopic RNA-binding protein condensates in maturing oocytes that appear to be independent of MEX-3 folding. CCT and actin also oppose the expansion of ER sheets that may promote ectopic condensation of RNA-binding proteins that are associated with de-repression of maternal mRNA. This regulatory network is essential to preserve oocyte quality, prevent infertility, and may have implications for understanding the role of hMex3 phase transitions in cancer. Significance statementO_LIThe molecular mechanisms that regulate phase transitions of oogenic RNA-binding proteins are critical to elucidate but are not fully understood. C_LIO_LIWe identify novel regulators of RNA-binding protein phase transitions in maturing oocytes that are required to maintain translational repression of maternal mRNAs and oocyte quality. C_LIO_LIThis study is the first to elucidate a regulatory network involving the CCT chaperonin, actin, and the ER for phase transitions of RNA-binding proteins during oogenesis. Our findings for the conserved MEX-3 protein may also be applicable to better understanding the role of hMex3 phase transitions in cancer. C_LI

developmental biology↗

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↗

bioRxiv2020

The Atr-Chek1 pathway inhibits axon regeneration in response to Piezo-dependent mechanosensation

Atr is a serine/threonine kinase, known to sense single-stranded DNA breaks and activate the DNA damage checkpoint by phosphorylating Chek1, which inhibits Cdc25, causing cell cycle arrest. This pathway has not been implicated in neuroregeneration. We show that in Drosophila sensory neurons, removing Atr or Chek1, or overexpressing Cdc25 promotes regeneration, whereas Atr or Chek1 overexpression, or Cdc25 knockdown impedes regeneration. Inhibiting the Atr-associated checkpoint complex in neurons promotes regeneration and improves synapse/behavioral recovery after CNS injury. Independent of DNA damage, Atr responds to the mechanical stimulus elicited during regeneration, via the mechanosensitive ion channel Piezo and its downstream NO signaling. Sensory neuron-specific knockout of Atr in adult mice, or pharmacological inhibition of Atr-Chek1 in mammalian neurons in vitro and in flies in vivo enhance regeneration. Our findings reveal the Piezo-Atr-Chek1-Cdc25 axis as an evolutionarily conserved inhibitory mechanism for regeneration, and identify potential therapeutic targets for treating nervous system trauma.

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↗