Biology subjects

Reed, A.

Publications and source records attributed to Reed, A..

9 recordsLinked to original sources

bioRxiv2026

Lipid acyl chain remodeling governs KRAS nanoclustering and oncogenic functions through membrane composition control

KRAS signaling is spatially organized within the dynamic proteolipid nanoclusters on the plasma membrane (PM) that selectively concentrate anionic phospholipids, yet the molecular determinants governing this lipid sorting remain incompletely defined. Here we identify phospholipid acyl chain composition as a critical regulator of KRAS spatial organization and signal output. Using genetic and pharmacological perturbation of lysophosphatidylcholine acyltransferase 3 (LPCAT3), a key enzyme that incorporates polyunsaturated fatty acids into phospholipids, we demonstrate that remodeling of membrane acyl chain unsaturation alters the phosphatidylserine (PS) lipidome without changing total PS abundance. Loss of LPCAT3 depletes polyunsaturated PS species, disrupts KRAS PM association and nanoclustering, and attenuates downstream MAPK signaling and oncogenic activity across multiple KRAS mutant contexts. Acute restoration of polyunsaturated, but not saturated, lipids rescue KRAS nanoclustering, establishing a direct requirement for lipid unsaturation in sustaining signaling-competent assemblies. Mechanistically, these findings define membrane acyl chain composition as a determinant of proteolipid organization that encodes KRAS signaling capacity. Consistent with this model, LPCAT3 inhibition selectively impairs KRAS-dependent proliferation and sensitizes tumor cells to direct KRAS inhibition. Together, this work uncovers lipid acyl chain remodeling as a fundamental regulator of membrane-associated signaling and identifies membrane composition as an exploitable vulnerability in oncogenic KRAS systems.

cancer biology↗

bioRxiv2026

Mapping ortholog-restricted ligandable cysteines in the wheat pathogen Zymoseptoria tritici

Zymoseptoria (Z.) tritici is the fungal phytopathogen responsible for Septoria tritici leaf blotch (STB), the main foliar disease of wheat. Fungicides mitigate crop loss caused by STB, but multidrug-resistant Z. tritici strains pose a major threat to the global food supply and underscore the need to identify new fungicidal targets. Here, we use activity-based protein profiling to generate a covalent ligandability map of Z. tritici, leading to the discovery of cysteines in a diverse set of essential proteins that are targeted by stereochemically defined, electrophilic small molecules (stereoprobes). We identify stereoprobes that display nucleotide-dependent reactivity with an ortholog-restricted cysteine in the GTPase SAR1, leading to accumulation of this protein and other COPII components at apparent ER exit sites and corresponding impairments in Z. tritici growth. Our findings thus expand the scope of ligandable, essential proteins in Z. tritici and illuminate opportunities to selectively target this destructive plant pathogen.

biochemistry↗

bioRxiv2025

Genetic background influences the effects of RNAi-by-feeding for DNA methyltransferases in Daphnia pulex.

DNA methyltransferases (DNMTs) are well-characterized epigenetic enzymes responsible for transferring methyl groups to and from DNA. Three main DNMT orthologues differ in function and methylation capability. They each are evolutionarily conserved across diverse taxa, but few studies investigate them jointly. We did so in Daphnia, freshwater microcrustaceans used extensively in research on genetic diversity, phenotypic plasticity, and maternal effects. Most Daphnia reproduce asexually through cyclic parthenogenesis, making the Daphnia system an ideal choice for studying epigenetic phenomena. Advances in gene expression control techniques, including RNA interference (RNAi), have increased the versatility and power of the Daphnia system. RNAi is a post-transcriptional gene silencing mechanism that operates through sequence-specific cleavage of endogenous messenger RNA (mRNA) transcripts. Here, we used an RNAi bacterial feeding regime to target the three DNA methyltransferase genes in two clones of Daphnia pulex. We observed significant genotypic differences in response to the RNAi feeding regime, namely, the mortality of one clone. In the other, DNMT expression significantly increased in five of the six experimental treatments, with the highest level observed in animals treated with the GFP double-stranded RNA bacterial vector control. However, DNMT expression was reduced in all three DNMT RNAi treatments relative to the GFP control. Furthermore, we found strong cross-reactivity, where targeting one DNMT resulted in the reduction of the other two. This response may be associated with known immune pathways involving signal transduction that can be stimulated by viral and bacterial signals, or it may result from previously unknown aspects of DNMT biology.

genetics↗

bioRxiv2025

Proteomic Ligandability Maps of Phosphorus(V) Stereoprobes Iden-tify Covalent TLCD1 Inhibitors

Activity-based protein profiling (ABPP) of stereoisomerically defined sets of electrophilic compounds ( stereoprobes) offers a versatile way to discover covalent ligands for proteins in native biological systems. Here we report the synthesis and chemical proteomic characterization of stereoprobes bearing a P(V)-oxathiaphospholane (OTP) reactive group. ABPP experiments identified numerous proteins in human cancer cells that showed stereoselective reactivity with OTP stereoprobes, and we confirmed several of these liganding events with recombinant proteins. OTP stereoprobes engaging the poorly characterized transmembrane protein TLCD1 impaired the incorporation of monounsaturated fatty acids into phosphatidylethanolamine lipids in cells, a lipidomic phenotype that mirrored genetic disruption of this protein. Using AlphaFold2, we found that TLCD1 structurally resembles the ceramide synthase and fatty acid elongase families of coenzyme A-dependent lipid processing enzymes. This structural similarity included conservation of catalytic histidine residues, the mutation of which blocked the OTP stereoprobe reactivity and lipid remodeling activity of recombinant TLCD1. Taken together, these data indicate that TLCD1 acts as a lipid acyltransferase in cells, and that OTP stereoprobes function as inhibitors of this enzymatic activity. Our findings thus illuminate how the chemical proteomic analysis of electrophilic compounds can facilitate the functional annotation and chemical inhibition of a key lipid metabolic enzyme in human cells.

biochemistry↗

bioRxiv2024

Dietary control of peripheral adipose storage capacity through membrane lipid remodelling

Complex genetic and dietary cues contribute to the development of obesity, but how these are integrated on a molecular level is incompletely understood. Here, we show that PPAR{gamma} supports hypertrophic expansion of adipose tissue via transcriptional control of LPCAT3, a membrane-bound O-acyltransferase that enriches diet-derived omega-6 (n-6) polyunsaturated fatty acids (PUFAs) in the phospholipidome. In high-fat diet-fed mice, lowering membrane n-6 PUFA levels by adipocyte-specific Lpcat3 knockout (Lpcat3AKO) or by dietary lipid manipulation leads to dysfunctional triglyceride (TG) storage, ectopic fat deposition and insulin resistance. Aberrant lipolysis of stored TGs in Lpcat3AKO adipose tissues instigates a non-canonical adaptive response that engages a futile lipid cycle to increase energy expenditure and limit further body weight gain. Mechanistically, we find that adipocyte LPCAT3 activity promotes TG storage by selectively enriching n-6 arachidonoyl-phosphatidylethanolamine at the ER-lipid droplet interface, which in turn favours the budding of large droplets that exhibit greater resistance to ATGL-dependent hydrolysis. Thus, our study highlights the PPAR{gamma}-LPCAT3 pathway as a molecular link between dietary n-6 PUFA intake, adipose expandability and systemic energy balance.

physiology↗

bioRxiv2023

Dietary protein shapes the profile and repertoire of intestinal CD4+ T cells

The intestinal immune system must tolerate food antigens to avoid allergy, a process requiring CD4+ T cells. Combining antigenically defined diets with gnotobiotic models, we show that food and microbiota distinctly influence the profile and T cell receptor repertoire of intestinal CD4+ T cells. Independent of the microbiota, dietary proteins contributed to accumulation and clonal selection of antigen-experienced CD4+ T cells at the intestinal epithelium, imprinting a tissue specialized transcriptional program including cytotoxic genes on both conventional and regulatory CD4+ T cells (Tregs). This steady state CD4+ T cell response to food was disrupted by inflammatory challenge, and protection against food allergy in this context was associated with Treg clonal expansion and decreased pro-inflammatory gene expression. Finally, we identified both steady state epithelium-adapted CD4+ T cells and tolerance-induced Tregs that recognize dietary antigens, suggesting that both cell types may be critical for preventing inappropriate immune responses to food.

immunology↗

bioRxiv2023

A thin-film optogenetic visual prosthesis

Retinitis pigmentosa and macular degeneration lead to photoreceptor death and loss of visual perception. Despite recent progress, restorative technologies for photoreceptor degeneration remain largely unavailable. Here, we describe a novel optogenetic visual prosthesis (FlexLED) based on a combination of a thin-film retinal display and optogenetic activation of retinal ganglion cells (RGCs). The FlexLED implant is a 30 {micro}m thin, flexible, wireless {micro}LED display with 8,192 pixels, each with an emission area of 66 {micro}m2. The display is affixed to the retinal surface, and the electronics package is mounted under the conjunctiva in the form factor of a conventional glaucoma drainage implant. In a rabbit model of photoreceptor degeneration, optical stimulation of the retina using the FlexLED elicits activity in visual cortex. This technology is readily scalable to hundreds of thousands of pixels, providing a route towards an implantable optogenetic visual prosthesis capable of generating vision by stimulating RGCs at near-cellular resolution.

neuroscience↗

bioRxiv2022

TMEM164 is an acyltransferase that forms ferroptotic polyunsaturated ether phospholipids

Ferroptosis is an iron-dependent form of cell death driven by the oxidation of polyunsaturated (PUFA) phospholipids. Large-scale genetic screens have pointed to a specialized role for PUFA ether phospholipids (ePLs) in promoting ferroptosis. Our understanding of the enzymes involved in PUFA ePL production, however, remains incomplete. Here we show using a combination of pathway mining of genetic dependency maps, AlphaFold-guided structure predictions, and targeted lipidomics that the uncharacterized transmembrane protein TMEM164 - genetic ablation of which has been shown to protect cells from ferroptosis - is a cysteine active-center enzyme that selectively transfers C20:4 acyl chains from phosphatidylcholine to lyso-ePLs to furnish PUFA-ePLs. TMEM164-null cells show substantial reductions in PUFA-ePLs, but not PUFA ester phospholipids, supporting that the selective suppression of PUFA-ePLs is sufficient to protect cells from ferroptosis and designating TMEM164 as a key enzyme specifically responsible for regulating this class of lipids.

biochemistry↗

bioRxiv2021

Vγ usage distinguishes pro- and anti-tumor intestinal γδ T cell subsets

{gamma}{delta} T cells physiologically scan the intestinal epithelium, representing a substantial fraction of infiltrating lymphocytes in colorectal cancer (CRC), albeit their role in CRC remains unclear. Using murine CRC models, we found that most {gamma}{delta} T cells in pre- or non-tumor colon express V{gamma}1+ or V{gamma}7+ and exhibit a cytotoxic profile. Targeting these {gamma}{delta} T cell subsets, as well as conditionally interfering with {gamma}{delta} T cell function at early stages of tumorigenesis led to heightened tumor development, suggesting anti-CRC functions for V{gamma}1+ and V{gamma}7+ subsets. In contrast, ROR{gamma}t+ {gamma}{delta} T cell subsets, including V{gamma}4+ and microbiotadependent V{gamma}6+, accumulated during CRC progression. Conditional deletion of ROR{gamma}t or V{gamma} chains revealed redundant roles for IL-17-producing V{gamma}4+ and V{gamma}6+ {gamma}{delta} T cells in promoting tumor growth. Our results uncover pro- and anti-tumor roles for {gamma}{delta} T cell subsets.

immunology↗