Biology subjects

Don, A. S.

Publications and source records attributed to Don, A. S..

12 recordsLinked to original sources

bioRxiv2026

Mechanism of phospholipid transport to the bacterial outer membrane by TAM.

Gram-negative bacteria transport phospholipids from the inner membrane (IM) to the outer membrane (OM) via poorly understood processes. These processes are essential for cell growth and the establishment of an antibiotic-resistant barrier. Here, we conducted single-particle cryo-electron microscopy, in vivo functional assays, and lipidomics to investigate the role of the "translocation and assembly module" (TAM) in lipid transport. We found that the OM-embedded subunit TamA anchors the IM-embedded bridge-like subunit TamB to the OM by forming a functional stable hybrid-barrel structure with the highly conserved C-terminal domain of unknown function 490 (DUF490). Using in vivo disulfide-tethering experiments we found that a highly conserved amphipathic helix within TamB DUF490 is important for TAM to function in OM maintenance. We also found that TamB DUF490 forms a {beta}-taco channel containing lipid-like densities and that the lipophilic property of the channel is important for TAM to maintain the levels of cardiolipin in the OM. Not only do our data support a novel model in which TAM acts to direct specific lipid classes into the OM, but it also supports the notion that TamB is a bacterial evolutionary prototype of a structurally homologous superfamily of eukaryotic bridge-like lipid transfer proteins.

biochemistry↗

bioRxiv2026

In vivo deuteration reveals pronounced variation in myelin lipid turnover rates and reduced myelin renewal with ageing

Myelin turnover is essential for its structural and functional integrity, yet how this particularly lipid-rich membrane is renewed and why it deteriorates with ageing remain unresolved. Combining deuterium oxide administration in mice with high resolution lipidomics, we establish that brain lipid turnover rates are highly heterogeneous, differ by brain region, and depend primarily on lipid class. Half-lives of common glycerophospholipids in purified myelin were under 2 months whereas many sphingolipids exhibited half-lives exceeding 8 months, dependent on acyl chain length and saturation. Myelin sphingolipid and cholesterol replacement rates in the corpus callosum decreased markedly between 3 and 12 months of age, while disrupting lipid trafficking through ApoE ablation preferentially impaired cholesterol turnover and incorporation into myelin. Our results establish that myelin renewal occurs through continual replacement of individual lipid constituents in a manner that depends on lipid class, hydrophobicity, and ApoE-dependent trafficking, and that this process slows significantly with ageing.

biochemistry↗

bioRxiv2025

Quantification and Localisation of New Brain Lipid Synthesis Using Deuterium Oxide and High Resolution Mass Spectrometry

Myelin is the lipid-rich membrane that surrounds neuronal axons and is essential for neurological function in vertebrates. The development of therapeutics that stimulate myelin repair to treat demyelinating disorders such as multiple sclerosis is hampered by the inability to distinguish newly-synthesised from pre-existing myelin. This study aimed to develop a method to quantify and localise new myelin lipid synthesis in vivo. Deuterium oxide was administered for two weeks in the drinking water of mice fed normal chow, chow containing the demyelinating toxin cuprizone, or during spontaneous remyelination following cuprizone withdrawal. Liquid chromatography-tandem mass spectrometry and mass spectrometry imaging were used to quantify and localise the newly synthesised, deuterated lipids. While most glycerophospholipids were constitutively deuterated, deuteration of myelin-enriched sulfatides, hexosylceramides, and phosphatidylethanolamine plasmalogens was only apparent during remyelination. Deuterated hexosylceramide and phosphatidylethanolamine plasmalogen species were localised primarily to the corpus callosum, the white matter tract that is most heavily affected by cuprizone. Most deuterium atoms were found in the fatty acyl chains, indicative of de novo lipid synthesis. These methods provide the means to quantify and spatially profile dynamic lipid synthesis across diverse biological contexts, including understanding myelin homeostasis and preclinical evaluation of remyelinating therapeutics.

biochemistry↗

bioRxiv2025

Very long-chain ceramides in muscle associate with insulin resistance independent of obesity

Lipids, in particular ceramides and diacylglycerols (DAGs), are implicated in insulin resistance, however their precise roles remain unclear. We leverage natural genetic variation to examine muscle lipids and systemic insulin resistance (IR) in 399 Diversity Outbred Australia mice. Adipose mass was associated with 55% of muscle lipids and IR, with DAGs as the only enriched lipid class. To disentangle adiposity and muscle lipid contributions, we used two approaches: (1) linear modelling of muscle lipids corrected for adipose mass on systemic IR, and (2) stratifying mice into insulin sensitivity quartiles within adiposity bins. Both revealed that very long-chain ceramides, but not DAGs, were linked to IR. Transcriptomic and proteomics further associated these ceramides with cellular and mitochondrial stress. DAGs correlated with leptin expression in muscle, suggesting they originate from muscle-residing adipocytes. We propose that many muscle lipids, including DAGs, associate with IR due to adipose accumulation rather than directly influencing muscle insulin sensitivity. By addressing the relationship between adiposity and metabolic state, we identified very long-chain muscle ceramides as highly associated with IR independently of adiposity.

systems biology↗

bioRxiv2025

Reduced plasma hexosylceramides in frontotemporal dementia are a biomarker of white matter integrity

INTRODUCTIONBlood biomarkers are needed to facilitate new therapeutic trials and improve management of behavioural variant frontotemporal dementia (bvFTD). Since altered white matter integrity is characteristic of bvFTD, this study aimed to determine if plasma levels of myelin-enriched glycolipids are altered in bvFTD and correlate with white matter integrity. METHODSNineteen glycolipids were quantified in bvFTD (n=31) and control (n=26) plasma samples. White matter integrity was assessed using MRI-derived fibre tract density and cross-section (FDC). RESULTSEleven lipids were significantly lower in bvFTD compared to control subjects, seven were inversely correlated with disease duration, and twelve were positively correlated with cognitive performance, with C22:0 hexosylceramide most strongly correlated. FDC was lower in frontotemporal white matter tracts of bvFTD compared to control subjects, and plasma C22:0 hexosylceramide was significantly correlated with FDC in these tracts. DISCUSSIONCirculating glycolipids may be a valuable biomarker of myelin integrity and disease progression in FTD.

neuroscience↗

bioRxiv2025

Histone methyltransferase PRDM9 promotes survival of drug-tolerant persister cells in glioblastoma

Chemotherapy often kills a large fraction of cancer cells but leaves behind a small population of drug- tolerant persister cells. These persister cells survive drug treatments through reversible, non-genetic mechanisms and cause tumour recurrence upon cessation of therapy. Here, we report a drug tolerance mechanism regulated by the germ-cell-specific H3K4 methyltransferase PRDM9. Through histone proteomic, transcriptomic, lipidomic, and ChIP-sequencing studies combined with CRISPR knockout and phenotypic drug screen, we identified that chemotherapy-induced PRDM9 upregulation promotes metabolic rewiring in glioblastoma stem cells, leading to chemotherapy tolerance. Mechanistically, PRDM9-dependent H3K4me3 at cholesterol biosynthesis genes enhances cholesterol biosynthesis, which persister cells rely on to maintain homeostasis under chemotherapy- induced oxidative stress and lipid peroxidation. PRDM9 inhibition, combined with chemotherapy, resulted in strong anti-cancer efficacy in preclinical glioblastoma models, significantly enhancing the magnitude and duration of the antitumor response by eliminating persisters. These findings demonstrate a previously unknown role of PRDM9 in promoting metabolic reprogramming that enables the survival of drug-tolerant persister cells.

cancer biology↗

bioRxiv2023

Acetyl-CoA carboxylase 1-dependent lipogenesis drives breast cancer progression

Dysregulation of cellular energetics, including lipid synthesis mediated through de novo lipogenesis, is a feature of many cancers. Here we report that acetyl-coenzyme A carboxylase (ACC) 1, the rate-limiting enzyme of de novo lipogenesis, is a key regulator of breast cancer progression and cancer cell phenotype. Mammary epithelial-specific deletion of ACC1 impaired tumour progression and decreased cancer cell proliferation in the PyMT model of breast cancer in vivo. ACC1 knockout in human breast cancer cell lines resulted in decreased cell number and altered cell and membrane morphology. Lipidomic profiling demonstrated reduced levels of acyl-carnitines (CARs) and several phospholipid (PL) classes, whilst also shifting the lipid profiles to exhibit more elongated and less saturated lipids in ACC1 knockout breast cancer cells. Palmitate rescue of ACC1 deletion phenotypes demonstrated a critical role for ACC1 driven de novo lipogenesis in breast cancer cell function. Analysis of human breast tumour-microarrays identified strong ACC1 expression at all breast cancer stages, grade and metastasis, compared to normal adjacent tissue. Together our data demonstrate a novel role for ACC1 in breast cancer progression and cancer cell function, mediated through its lipogenic role, that together with its expression profile, identify ACC1 as a potential therapeutic target in breast cancer. Statement of significanceThis study investigates the impact of ACC1 deletion in breast cancer progression, revealing the importance of ACC1-derived lipids in breast cancer cell phenotypes and identifies ACC1 as a potential novel therapeutic target.

cancer biology↗

bioRxiv2023

Mitochondrial electron transport chain, ceramide and Coenzyme Q are linked in a pathway that drives insulin resistance in skeletal muscle

Insulin resistance (IR) is a complex metabolic disorder that underlies several human diseases, including type 2 diabetes and cardiovascular disease. Despite extensive research, the precise mechanisms underlying IR development remain poorly understood. Here, we provide new insights into the mechanistic connections between cellular alterations associated with IR, including increased ceramides, deficiency of coenzyme Q (CoQ), mitochondrial dysfunction, and oxidative stress. We demonstrate that elevated levels of ceramide in the mitochondria of skeletal muscle cells results in CoQ depletion and loss of mitochondrial respiratory chain components, leading to mitochondrial dysfunction and IR. Further, decreasing mitochondrial ceramide levels in vitro and in animal models (under chow and high fat diet) increased CoQ levels and was protective against IR. CoQ supplementation also rescued ceramide-associated IR. Examination of the mitochondrial proteome from human muscle biopsies revealed a strong correlation between the respirasome system and mitochondrial ceramide as key determinants of insulin sensitivity. Our findings highlight the mitochondrial Ceramide-CoQ-respiratory chain nexus as a potential foundation of an IR pathway that may also play a critical role in other conditions associated with ceramide accumulation and mitochondrial dysfunction, such as heart failure, cancer, and aging. These insights may have important clinical implications for the development of novel therapeutic strategies for the treatment of IR and related metabolic disorders.

cell biology↗

bioRxiv2022

Early microglial response, myelin deterioration and lethality in mice deficient for very long chain ceramide synthesis in oligodendrocytes

The sphingolipids galactosylceramide (GalCer), sulfatide (ST) and sphingomyelin (SM) are essential for myelin stability and function. GalCer and ST are synthesized mostly from C22-C24 ceramides, generated by Ceramide Synthase 2 (CerS2). To clarify the requirement for C22-C24 sphingolipid synthesis in myelin lipid biosynthesis and stability, we generated mice lacking CerS2 specifically in myelinating cells (CerS2{Delta}O/{Delta}O). At 6 weeks of age, normal-appearing myelin had formed in CerS2{Delta}O/{Delta}O mice, however there was a reduction in myelin thickness and the percentage of myelinated axons. Pronounced loss of C22-C24 sphingolipids in myelin of CerS2{Delta}O/{Delta}O mice was compensated by greatly increased levels of C18 sphingolipids. A distinct microglial population expressing high levels of activation and phagocytic markers such as CD64, CD11c, MHC class II, and CD68 was apparent at 6 weeks of age in CerS2{Delta}O/{Delta}O mice, and had increased by 10 weeks. Increased staining for denatured myelin basic protein was also apparent in 6-week-old CerS2{Delta}O/{Delta}O mice. By 16 weeks, CerS2{Delta}O/{Delta}O mice showed pronounced myelin atrophy, motor deficits, and axon beading, a hallmark of axon stress. 90% of CerS2{Delta}O/{Delta}O mice died between 16 and 26 weeks of age. This study highlights the importance of sphingolipid acyl chain length for the structural integrity of myelin, demonstrating how a modest reduction in lipid chain length causes exposure of a denatured myelin protein epitope and expansion of phagocytic microglia, followed by axon pathology, myelin degeneration, and motor deficits. Understanding the molecular trigger for microglial activation should aid the development of therapeutics for demyelinating and neurodegenerative diseases. Main PointsO_LIOligodendrocytes lacking CerS2 produce myelin using sphingolipids with C16/C18 instead of C22/C24 N-acyl chains C_LIO_LIC22/C24 myelin sphingolipids are essential for myelin stability, microglial quiescence, and survival beyond young adulthood C_LI Table of Contents Image O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=172 SRC="FIGDIR/small/493337v2_ufig1.gif" ALT="Figure 1"> View larger version (52K): org.highwire.dtl.DTLVardef@1125540org.highwire.dtl.DTLVardef@9aeebdorg.highwire.dtl.DTLVardef@1afbeb4org.highwire.dtl.DTLVardef@11156c2_HPS_FORMAT_FIGEXP M_FIG C_FIG

neuroscience↗

bioRxiv2021

Sphingosine kinase 2 is essential for remyelination following cuprizone intoxication

Therapeutics that promote oligodendrocyte survival and remyelination are needed to restore neurological function in demyelinating diseases. Sphingosine 1-phosphate (S1P) is an essential lipid metabolite that signals through five G-protein coupled receptors. S1P receptor agonists such as Fingolimod are valuable immunosuppressants used to treat multiple sclerosis, and promote oligodendrocyte survival. However, the role for endogenous S1P, synthesized by the enzyme sphingosine kinase 2 (SphK2), in oligodendrocyte survival and myelination has not been established. This study investigated the requirement for SphK2 in oligodendrocyte survival and remyelination using the cuprizone mouse model of acute demyelination, followed by spontaneous remyelination. Oligodendrocyte density did not differ between untreated wild- type (WT) and SphK2 knockout (SphK2-/-) mice. However, cuprizone treatment caused significantly greater loss of mature oligodendrocytes in SphK2-/- compared to WT mice. Following cuprizone withdrawal, spontaneous remyelination occurred in WT but not SphK2-/- mice, even though progenitor and mature oligodendrocyte density increased in both genotypes. Levels of cytotoxic sphingosine and ceramide were higher in the corpus callosum of SphK2-/- mice, and in contrast to WT mice, did not decline following cuprizone withdrawal in SphK2-/- mice. We also observed a significant reduction in myelin thickness with ageing in SphK2-/- compared to WT mice. These results provide the first evidence that SphK2, the dominant enzyme catalysing S1P synthesis in the adult brain, is essential for remyelination following a demyelinating insult and myelin maintenance with ageing. We propose that persistently high levels of sphingosine and ceramide, a direct consequence of SphK2 deficiency, may block remyelination. Table of Contents Image O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=121 SRC="FIGDIR/small/431183v2_ufig1.gif" ALT="Figure 1"> View larger version (25K): org.highwire.dtl.DTLVardef@e01edcorg.highwire.dtl.DTLVardef@9fdf6org.highwire.dtl.DTLVardef@6012org.highwire.dtl.DTLVardef@a3513b_HPS_FORMAT_FIGEXP M_FIG C_FIG Main PointsO_LIThe lipid kinase sphingosine kinase 2 (SphK2) is essential for remyelination following a demyelinating insult (cuprizone). C_LIO_LISphK2 protects against cuprizone-mediated oligodendrocyte loss. C_LIO_LISphK2 deficiency leads to thinner myelin with ageing. C_LI

neuroscience↗

bioRxiv2020

A novel function of sphingosine kinase 2 in the metabolism of sphinga-4,14-diene lipids.

The number, position, and configuration of double bonds in lipid acyl chains affects membrane packing, fluidity, and recruitment of signalling proteins. Studies on mammalian sphingolipids have focused on those with a saturated sphinganine or mono-unsaturated sphingosine long chain base. Sphingolipids with a diunsaturated sphingadiene base have been reported but are poorly characterised. Employing high-resolution untargeted mass spectrometry, we observed marked accumulation of lipids containing a sphingadiene base, but not those with a more common sphingosine backbone, in the hippocampus of mice lacking the metabolic enzyme sphingosine kinase 2 (SphK2). Applying ultraviolet photodissociation tandem mass spectrometry (UVPD-MS/MS) the double bonds were confidently assigned to the C4-C5 and C14-C15 positions of the sphingoid base. Sphingosine kinases are involved in lysosomal catabolism of all sphingolipids, producing sphingoid base phosphates that are irreversibly degraded by sphingosine 1-phosphate lyase. Both SphK1 and SphK2 phosphorylated sphinga-4,14-diene as efficiently as sphingosine, however deuterated tracer experiments demonstrated that ceramides with a sphingosine base are more rapidly metabolised in cultured cells than those with a sphingadiene base. SphK2 silencing significantly impeded the catabolism of both sphingosine- and sphingadiene-based sphingolipids. Since SphK2 is the dominant sphingosine kinase in brain, we propose that accumulation of sphingadiene lipids in SphK2-deficient brains results from the intrinsically slower catabolism of sphingadiene lipids, combined with a bottleneck in the catabolic pathway created by the absence of SphK2. We speculate that accumulation of these lipids in the absence of SphK2 function may affect the fluidity and signalling properties of cell membranes.

biochemistry↗

bioRxiv2019

DECR1 is an androgen-repressed survival factor that regulates PUFA oxidation to protect prostate tumor cells from ferroptosis

Fatty acid {beta}-oxidation (FAO) is the main bioenergetic pathway in prostate cancer (PCa) and a promising novel therapeutic vulnerability. Here we demonstrate therapeutic efficacy of targeting FAO in clinical prostate tumors cultured ex vivo, and identify DECR1, which encodes the rate-limiting enzyme for oxidation of polyunsaturated fatty acids (PUFAs), as robustly overexpressed in PCa tissues and associated with shorter relapse-free survival. DECR1 is a negatively-regulated androgen receptor (AR) target gene and, therefore, may promote PCa cell survival and resistance to AR targeting therapeutics. DECR1 knockdown in PCa cells selectively inhibited {beta}-oxidation of PUFAs, inhibited proliferation and migration of PCa cells, including treatment resistant lines, and suppressed tumor cell proliferation in vivo. Mechanistically, targeting of DECR1 caused cellular accumulation of linoleic acid, enhanced mitochondrial oxidative stress and lipid peroxidation, and ferroptosis. These findings implicate PUFA oxidation via DECR1 as a previously unexplored facet of FAO that promotes survival of PCa cells.

cancer biology↗