Biology subjects

Arenz, C.

Publications and source records attributed to Arenz, C..

11 recordsLinked to original sources

bioRxiv2026

KPB82 potently and selectively inhibits ACER2 and protects tumor and primary cells from doxorubicin-induced cytotoxicity

Alkaline ceramidase 2 (ACER2) catalyzes the hydrolysis of ceramides into sphingosine and free fatty acids, playing crucial roles in regulating various biological processes, including but not limited to cell proliferation, survival, and the DNA damage response. However, a pharmacological tool that regulates its catalytic and signaling functions remains to be developed. In this study, N-[(1S,2R)-1-hydroxy-1-phenylpropan-2-yl]dodecane-1-sulfonamide (KPB82) was identified as a potent and selective ACER2 inhibitor. In vitro enzymatic assays showed that KPB82 inhibited ACER2 with an IC50 of 190 nM, while displaying minimal activity against acid and neutral ceramidases (IC50> 100 {micro}M) and no significant inhibition of alkaline ceramidase 1 or alkaline ceramidase 3. Enzymatic kinetic studies revealed that KPB82 is a competitive inhibitor of ACER2. The three-dimensional structure of ACER2 was modeled in the AlphaFold3 server, revealing key Zn2+-coordinating histidine residues and a substrate-binding pocket. Molecular docking studies demonstrated that KPB82 binds ACER2 within this pocket, forming hydrogen bonds with the catalytically important amino residues, Asp93 and His215, and interacting with several hydrophobic residues. Lactate dehydrogenase release assays revealed that KPB82 significantly reduced cytotoxicity in HeLa cells overexpressing ACER2, as well as in HCT116 cells, RT4 cells, and human umbilical vein endothelial cells treated with the DNA-damaging agent doxorubicin. Lipidomic analysis showed that KPB82 inhibited sphingosine production in response to ACER2 overexpression or doxorubicin treatment. Collectively, these findings demonstrate that KPB82 is a potent and selective ACER2 inhibitor capable of suppressing ACER2-dependent biological processes and highlight its potential as a tool for studying ceramide metabolism and ACER2-mediated signaling pathways.

biochemistry↗

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↗

bioRxiv2026

Fine-tuned synthetic transcription factors for production of 3' phosphoadenosine-5'-phosphosulfate in yeast

Technologies developed over the past decade have made Saccharomyces cerevisiae a promising platform for producing various natural products. Balancing multi-enzyme expression, while maintaining robust microbial growth, remains a limiting factor for engineering long biosynthetic pathways in yeast. Here, we improved the transcriptional capacity of our previously developed isopropyl {beta}-D-1-thiogalactopyranoside (IPTG)-inducible synthetic transcription factors (synTFs) derived from the plant JUB1 DNA-binding domain. To this end, at cysteine positions within surface-exposed loop regions of a JUB1-derived DNA-binding scaffold, we introduced a short peptide to enhance loop flexibility while providing local stability and orientation. The generated synTFs, so-called JUB1-X synTFs, varying in strength, have been successfully used to improve the synthesis of 3-phosphoadenosine 5-phosphosulfate (PAPS), a universal sulfate donor necessary for the synthesis of bioactive molecules, including therapeutic glycosaminoglycans and sulfolipids. Using only this engineered yeast strain in simple batch culture, PAPS accumulation of 21.4 {+/-} 5.8 mg g-1 cdw was achieved after only 5 hours of inducing the expression of JUB1-X synTFs. Beyond PAPS production, the design principle demonstrated here provides a generalizable strategy to fine-tune other plant-derived synTFs, expanding the regulatory capabilities of existing synTF collections. Together, this work offers a modular, scalable approach to constructing high-performance gene circuits and supports the development of yeast cell factories for complex metabolic and synthetic biology applications.

synthetic biology↗

bioRxiv2025

Dyslipidemic SPTLC3 Integrates Bile Acid-FXR Signaling with Sphingolipid Remodeling in MASLD

Molecular mechanisms driving metabolic disease pathogenesis remain poorly understood. Genetic and functional studies implicate SPTLC3 with dyslipidemia. SPTLC3 synthesizes atypical long-chain bases (LCB) precursors for sphingolipid production. We demonstrate significant SPTLC3 expression in human liver. ORMDL1-3 regulate SPTLC3 post-translationally in hepatic and non-hepatic cells. Independently, farnesoid X receptor (FXR) represses hepatic SPTLC3 transcription via a negative promoter element. In mice, high-fat diet (HFD) induced whereas bile acids normalized hepatic SPTLC3 transcription. SPTLC3 derived LCBs in plasma originate from the liver and are elevated in HFD-fed mice and in patients with metabolic dysfunction-associated steatotic liver disease (MASLD). Cross-species comparison revealed marked differences in LCB composition between mice and humans. Notably, omega-3-methylsphingosine (meC18SO) was significantly associated with MASLD in humans but undetectable in mice. In Huh7 cells, meC18SO enhanced complex II and IV activity, oxygen consumption, and mitochondrial ROS content. FXR-SPTLC3 axis and presented findings have potential implications for future translational research.

biochemistry↗

bioRxiv2024

A Novel Rapid Host Cell Entry Pathway Determines Intracellular Fate of Staphylococcus aureus

Staphylococcus aureus is an opportunistic pathogen causing severe diseases. Recently, S. aureus was recognized as intracellular pathogen, whereby the intracellular niche promotes immune evasion and antibiotic resistance. Interaction of S. aureus with versatile host cell receptors was described previously, suggesting that internalization of the pathogen can occur via several pathways. It remains elusive whether the pathway of internalization can affect the intracellular fate of the bacteria. Here, we identified a mechanism governing cellular uptake of S. aureus which relies on lysosomal Ca2+, lysosomal exocytosis and occurs concurrently to other well-known entry pathways within the same host cell population. This internalization pathway is rapid and active within only few minutes after bacterial contact with host cells. Compared to slow bacterial internalization, the rapid pathway demonstrates altered phagosomal maturation as well as translocation of the pathogen to the host cytosol and ultimately results in different rates of intracellular bacterial replication and host cell death. We show that these alternative infection outcomes are caused by the mode of bacterial uptake.

microbiology↗

bioRxiv2024

A first-in-Plasmodium study on tRNA intron splicing endonuclease PfTSEN1 and its substrate expression in clinical stage malaria

Mature tRNAs play critical role in several cellular processes including protein translation, post-translational-modifications and programmed-cell-death. Maturation of pre-tRNAs require removal of 5-leaders, 3-trailers, splicing of introns and addition of conserved 3-terminal CCA sequence. The tRNA splicing mechanism, an essential step in tRNA maturation govern by a tRNA splicing endonuclease. While the existence of functional tRNA splicing endonuclease(s) in Plasmodium falciparum has not been identified, its significance in other eukaryotes suggests a potential role in tRNA splicing event. Our study identified total tRNAs in Plasmodium and characterize a PftRNA splicing endonuclease (annotated as PfTSEN1) recognised recently as a component of ribonucleoprotein (RNP) complex, and synthesized a naphthoquinone derivative as a novel anti-malarial compound ( TSENi) targeting the functional activity of this protein. Enzyme activity assays elucidated that PfTSEN1 catalyses splicing of in vitro transcribed pre-tRNAleu, the expression of which was confirmed during the clinical stages of malaria parasite by RT-PCR. Interestingly, TSENi binds to and inhibits enzymatic activity of PfTSEN1, and showed potent anti-malarial activity against chloroquine-sensitive 3D7 and resistant strains Dd2 of P. falciparum. Overall, our study deliver key knowledge towards the functional role of PftRNA splicing endonuclease, and its inhibitor TSENi as potent anti-malarial.

molecular biology↗

bioRxiv2024

Expression of Plasmodium Major Facilitator Superfamily Protein in Transporters-Delta Candida Identifies a Drug Transporter

AimTo assess the functional relevance of a putative Major Facilitator Superfamily (MFS) protein (PF3D7_0210300; "PfMFS_DT") as a drug transporter, using Candida glabrata for orthologous protein expression. MethodsCDS encoding PfMFS_DT was integrated into the genome of genetically engineered C. glabrata strain MSY8 via homologous recombination, followed by assessing its functional relevance as a drug transporter. Results and ConclusionThe modified C. glabrata strain exhibited plasma membrane localization of PfMFS_DT and characteristics of an MFS transporter, conferring resistance to antifungals, ketoconazole and itraconazole. The nanomolar inhibitory effects of the drugs on the intra-erythrocytic growth of P. falciparum highlight their antimalarial properties. This study proposes PfMFS_DT as a drug transporter, expanding the repertoire of the currently known antimalarial "resistome".

cell biology↗

bioRxiv2022

Human malaria parasite cold shock protein plays an essential role in asexual and sexual stage development and presents an excellent druggable target

Cold shock proteins are well characterized in bacteria, plants and humans, however there is no information on their existence and role in malaria parasite. Here, we have delineated the function of a novel cold shock protein of Plasmodium falciparum (Pf) which we have annotated PfCoSP. Our results show that recombinant PfCoSP has both DNA and RNA binding activity, and also interacts with alpha and beta tubulin of Pf. PfCoSP binds with RNA and alpha tubulin simultaneously to form a complex. Expression of PfCoSP was found during asexual blood stages and gametocyte stages of malaria parasite. PfCoSP expression up-regulates many folds upon cold treatment, suggesting its role during hypothermic cold shock. Interestingly, PfCoSP showed binding with human cold shock protein LIN28A inhibitor LI71 that also inhibits PfCSP -alpha/beta tubulin interactions. LI71 showed antimalarial activity against asexual blood stage and gametocyte stage suggesting its multi-stage, transmission-blocking potential. We propose that PfCoSP may form a workbench for translation during cold stress via its interactions with target mRNAs and the cytoskeleton protein tubulin.

molecular biology↗

bioRxiv2021

Discovery and mechanism of action of small molecule inhibitors of ceramidases

Sphingolipid metabolism is tightly controlled by enzymes to regulate essential processes such as energy utilisation and cell proliferation. The central metabolite is ceramide, a pro-apoptotic lipid catabolized by ceramidase enzymes to ultimately produce pro-proliferative sphingosine-1-phosphate. Human ceramidases can be soluble proteins (acid and neutral ceramidase) or integral membrane proteins (alkaline ceramidases). Increasing ceramide levels to increase apoptosis has shown efficacy as a cancer treatment using small molecules inhibiting a soluble ceramidase. Due to the transmembrane nature of alkaline ceramidases, no specific small molecule inhibitors have been reported. Here, we report novel fluorescent substrates (FRETceramides) of ceramidases that can be used to monitor enzyme activity in real-time. We use FRETceramides to discover the first drug-like inhibitors of alkaline ceramidase 3 (ACER3) which are active in cell-based assays. Biophysical characterization of enzyme:inhibitor interactions reveal a new paradigm for inhibition of lipid metabolising enzymes with non-lipidic small molecules. Table of contents summaryUse of synthetic fluorescent ceramide molecules allows the discovery of the first selective drug-like small molecule inhibitors for alkaline ceramidase 3, an intra-membrane enzyme involved in sphingolipid metabolism in health and disease.

biochemistry↗

bioRxiv2021

BRED: bioluminescence energy transfer to dye for monitoring ceramide trafficking in cell

Bioluminescence resonance energy transfer (BRET) is a genetically encoded proximity-based tool to study biomolecular interactions. However, conventional BRET is usually restricted to only a few types of interactions like protein-protein or protein-ligand interactions. We here developed a spatially unbiased resonance energy transfer system, so-called BRED - bioluminescence resonance energy transfer to dye. BRED allows transferring energy from a genetically encoded bright human optimized luciferase to a fluorophore-labelled small molecule. The high efficiency of the system allows RET without specific interaction of donor and acceptor. Here, we applied BRED to monitor the trafficking of the signalling lipid ceramide, to the Golgi. This was enabled by an engineered Golgi-resident luciferase, which was used to sense the influx of BODIPY-labeled ceramide into the surrounding membrane. We demonstrated the implementation of the method via flow cytometry, thereby combining the sensitivity of bulk cell methods with the advantages of single-cell analysis. This toolbox enables simple and robust live-cell analysis of inhibitors of CERT-mediated ceramide transport. The design principle of our optogenetic tool can be applied to study intracellular trafficking of metabolites and screen for inhibitors of their key enzymes.

biochemistry↗

bioRxiv2018

Structure of a human intramembrane ceramidase explains enzymatic dysfunction found in leukodystrophy

Alkaline ceramidases (ACERs) are a class of poorly understood transmembrane enzymes controlling the homeostasis of ceramides. They are implicated in human pathophysiology, including progressive leukodystrophy, colon cancer as well as acute myeloid leukemia. We report here the crystal structure of the human ACER type 3 (ACER3). Together with computational studies, the structure reveals that ACER3 is an intramembrane enzyme with a seven transmembrane domain architecture and a catalytic Zn2+ binding site in its core, similar to adiponectin receptors. Interestingly, we uncover a Ca2+ binding site physically and functionally connected to the Zn2+ providing a structural explanation for the known regulatory role of Ca2+ on ACER3 enzymatic activity and for the loss of function in E33G-ACER3 mutant found in leukodystrophic patients.

biochemistry↗