Biology subjects

Majcher, A.

Publications and source records attributed to Majcher, A..

4 recordsLinked to original sources

bioRxiv2026

CYP4F2-mediated ω-hydroxylation of 1-deoxysphingolipids reveals a new hepatic detoxification pathway

1-deoxysphingolipids (1-deoxySLs) are atypical, cytotoxic sphingolipids (SL) formed by the serine palmitoyltransferase through the alternative use of L-Alanine over its canonical substrate L-Serine. Elevated plasma levels of 1-deoxySLs have been implicated in metabolic and neurodegenerative diseases. Due to the missing C1 hydroxyl group, 1-deoxySLs cannot be converted into complex sphingolipids nor degraded via the canonical SL catabolic pathways. However, previous reports suggested a cytochrome P450 mediated {omega}-hydroxylation of 1-deoxySLs as a potential detoxification mechanism although the exacts downstream metabolism of these lipids remained unclear. We combined genome-wide association analysis with targeted lipid analysis to identify genes involved in 1-deoxySL metabolism. Functional validation was performed in cell culture models, enzyme assays, and through quantitative high-resolution mass spectrometry using isotope labelled synthetic standards.We identified a strong association between the CYP4F2 rs2108622 variant and plasma 1-deoxySL, implicating CYP4F2 is involved in 1-deoxySL metabolism. We demonstrated that CYP4F2 catalyzes the {omega}-hydroxylation of 1-deoxysphinganine, forming a previously uncharacterized hydroxylated sphingoid base. In liver cells, this metabolite was further metabolized via three distinct pathways: one forming the N-acyl, a second involving omega acylation and third resulting in omega carboxylation. All reactions generated a new spectrum of 1-deoxysphingolipids that are based on {omega}-hydroxylated 1-deoxySA as a precursor. The metabolic steps were confirmed by structural validation using synthetically prepared external standards. Importantly, {omega}-hydroxylation significantly attenuated the acute cytotoxicity of 1-deoxySLs in liver cells, indicating that this modification is the initiating step of a multi-branched metabolic clearance pathway. This study identifies CYP4F2 as a key enzyme initiating the hepatic clearance of atypical 1-deoxySLs, mitigating their cellular toxicity and revealing multiple downstream metabolic fates. Our findings highlight a previously unrecognized clearance mechanism for atypical sphingolipids with relevance to metabolic disease.

biochemistry↗

bioRxiv2025

Very long chain fatty acids drive 1-deoxy-Sphingolipid toxicity

1-deoxy-sphingolipids (1-deoxySLs) are atypical sphingolipids synthesized by the serine palmitoyltransferase (SPT) when L-alanine is used instead of its canonical substrate L-serine. Increased 1-deoxySLs are associated with sensory neuropathies such as Hereditary Sensory and Autonomic Neuropathy type 1 (HSAN1) and diabetic polyneuropathy (DPN). Despite their known cellular, mitochondrial, and neurotoxic effects, the mechanisms underlying their toxicity remain poorly understood. Using a CRISPR interference (CRISPRi) screening approach, we identified CERS2, ELOVL1, ACACA, HSD17B12, and PTPLB as key mediators of 1-deoxySL-induced toxicity. All genes are integral to the biosynthesis of very long-chain (VLC) fatty acids and VLC-ceramides. We validated these findings through genetic knockdown experiments, cytotoxicity assays, and stable isotope-resolved lipidomics via LC-MS/MS. Pharmacological inhibition of ELOVL1 using a preclinical tested compound alleviated the cellular, mitochondrial, and neuronal toxicity induced by 1-deoxySLs. Supplementation experiments combining 1-deoxySLs with various VLC fatty acids revealed that 1-deoxyDHceramide conjugated to nervonic acid (m18:0/24:1) is the principal toxic specie. Further mechanistic studies showed that m18:0/24:1 induces apoptosis through the mitochondrial permeability transition pore (mPTP) formation. Inhibition of BAX or blocking mPTP formation with cyclosporin A effectively prevented toxicity. In conclusion, our findings demonstrate that 1-deoxyDHCeramides conjugated to nervonic acid are the primary mediators of 1-deoxySL toxicity, acting through mitochondrial dysfunction and BAX-dependent apoptosis.

biochemistry↗

bioRxiv2025

Metabolic Origins of Neurotoxic 1-deoxySphingolipids in Type 2 Diabetes

Type 2 diabetes (T2D) and diabetic peripheral neuropathy (DPN) are associated with disruptions in sphingolipid (SL) metabolism, including an increased formation of neurotoxic 1-deoxysphingolipids (1-deoxySL). Here we report data from an untargeted proteomics, lipidomics and metabolomics profiling in plasma and skin samples of a carefully characterized T2D cohort and age-matched healthy controls. We investigated the association between plasma and skin amino acids and the sphingolipidome in blood and skin of T2D patients and several diabetic rodent models. We developed a hypothesis on how changes in the metabolism of the two amino acids relates to SL formation and DPN. To test this hypothesis and identify key enzymes responsible for the 1-deoxySL formation we developed stable isotope based SL flux assays, using UC13Glucose, 15NGlutamine, D4-Palmitic acid, D4-Alanine and D3N15-Serine as tracers. In combination with genetic interference approaches, we identified pathways that are responsible for shifting between the formation of 1-deoxySL and canonical SL in T2D. Furthermore, we verified these findings in vivo in several rodent models. This study links disturbances in amino acids, lipids, and protein homeostasis to DPN in T2D.

neuroscience↗

bioRxiv2025

Disrupted Sphingosine-1-Phosphate Homeostasis Drives Nephrotoxicity in Sphingosine-1-Phosphate Lyase Insufficiency Syndrome (SPLIS)

Sphingosine-1-phosphate lyase insufficiency syndrome (SPLIS), also known as nephrotic syndrome type 14 (NPHS14), is an autosomal recessive disorder characterized by renal, neurological, dermatological, endocrine, and immunological symptoms. This condition is caused by loss-of-function mutations in the SGPL1 gene, which encodes sphingosine-1-phosphate lyase (SGPL1p/SPL), the enzyme responsible for the terminal degradation of sphingosine-1-phosphate (S1P) in sphingolipid catabolism. We investigated a novel case of SPLIS associated with a recently reported SGPL1 mutation (c.1084T>A; p.Ser362Thr). Using stable isotope flux analyses, we demonstrated in patient-derived fibroblasts and HEK293T SGPL1 knockout models that SGPL1p deficiency does not consistently result in pathological S1P accumulation. Instead, SPL-deficient cells are able to maintain steady-state S1P levels through two compensatory mechanisms: O_LIRegulation of de novo sphingolipid synthesis via the ORMDL-ceramide axis. C_LIO_LIIncreased conversion of excess ceramides into glycosphingolipids. C_LI However, when steady-state conditions are disrupted--either by external sphingolipid supplementation or by impairing homeostatic control--a pathological increase in intracellular S1P occurs in SPL-deficient cells. In vivo, Sgpl1-/-mice exhibited significant urinary excretion of S1P and marked S1P enrichment in the kidneys. This pathological accumulation of S1P dysregulates cytoskeletal homeostasis, impairing renal epithelial formation. Based on these findings, we hypothesize that the reabsorption of urinary S1P contributes to toxic renal accumulation, providing an explanation for the nephrotoxicity observed in SPLIS and its association with nephrotic syndrome. Importantly, we found that the cytoskeletal disruptions could be mitigated by inhibiting the Rho-ROCK signaling pathway using the clinically approved inhibitor Fasudil. These findings illuminate the pathophysiological basis of SPLIS nephrotoxicity and propose a promising pharmacological intervention strategy. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=144 SRC="FIGDIR/small/634100v1_ufig1.gif" ALT="Figure 1"> View larger version (24K): org.highwire.dtl.DTLVardef@e70e68org.highwire.dtl.DTLVardef@1630198org.highwire.dtl.DTLVardef@fde277org.highwire.dtl.DTLVardef@1f2b8e_HPS_FORMAT_FIGEXP M_FIG C_FIG

physiology↗