Sphingolipids: Less Enigmatic but Still Many Questions about the Role(s) of Ceramide in the Synthesis/Function of the Ganglioside Class of Glycosphingolipids.

While much has been learned about sphingolipids, originally named for their sphinx-like enigmatic properties, there are still many unanswered questions about the possible effect(s) of the composition of ceramide on the synthesis and/or behavior of a glycosphingolipid (GSL). Over time, studies of their ceramide component, the sphingoid base containing the lipid moiety of GSLs, were frequently distinct from those performed to ascertain the roles of the carbohydrate moieties. Due to the number of classes of GSLs that can be derived from ceramide, this review focuses on the possible role(s) of ceramide in the synthesis/function of just one GSL class, derived from glucosylceramide (Glc-Cer), namely sialylated ganglio derivatives, initially characterized and named gangliosides (GGs) due to their presence in ganglion cells. While much is known about their synthesis and function, much is still being learned. For example, it is only within the last 15-20 years or so that the mechanism by which the fatty acyl component of ceramide affected its transport to different sites in the Golgi, where it is used for the synthesis of Glu- or galactosyl-Cer (Gal-Cer) and more complex GSLs, was defined. Still to be fully addressed are questions such as (1) whether ceramide composition affects the transport of partially glycosylated GSLs to sites where their carbohydrate chain can be elongated or affects the activity of glycosyl transferases catalyzing that elongation; (2) what controls the differences seen in the ceramide composition of GGs that have identical carbohydrate compositions but vary in that of their ceramide and vice versa; (3) how alterations in ceramide composition affect the function of membrane GGs; and (4) how this knowledge might be applied to the development of therapies for treating diseases that correlate with abnormal expression of GGs. The availability of an updatable data bank of complete structures for individual classes of GSLs found in normal tissues as well as those associated with disease would facilitate research in this area.

A Membrane-Assisted Mechanism for the Release of Ceramide from the CERT START Domain.

Ceramide transfer protein CERT is the mediator of nonvesicular transfer of ceramide from the ER to Golgi. In CERT, START is the domain responsible for the binding and transport of ceramide. A wealth of structural data has revealed a helix-grip fold surrounding a large hydrophobic cavity holding the ceramide. Yet, little is known about the mechanisms by which START releases the ceramide through the polar region and into the packed environment of cellular membranes. As such events do not lend themselves easily to experimental investigations, we used multiple unbiased microsecond-long molecular simulations. We propose a membrane-assisted mechanism in which the membrane acts as an allosteric effector initiating the release of ceramide and where the passage of the ceramide acyl chains is facilitated by the intercalation of a single phosphatidylcholine lipid in the cavity, practically greasing the ceramide way out. We verify using free energy calculation and experimental lipidomics data that CERT forms stable complexes with phosphatidylcholine lipids, in addition to ceramide, thus providing validation for the proposed mechanism.

Engineered nanomicelles targeting proliferation and angiogenesis inhibit tumour progression by impairing the synthesis of ceramide-1-phosphate.

Tumour cells secrete various proangiogenic factors like VEGF, PDGF, and EGF that result in the formation of highly vascularized tumours with an immunosuppressive tumour microenvironment. As tumour growth and metastasis are highly dependent on angiogenesis, targeting tumour vasculature along with rapidly dividing tumour cells is a potential approach for cancer treatment. Here, we specifically engineered sub-100 sized nanomicelles (DTX-CA4 NMs) targeting proliferation and angiogenesis using an esterase-sensitive phosphocholine-tethered docetaxel conjugate of lithocholic acid (LCA) (PC-LCA-DTX) and a poly(ethylene glycol) (PEG) derivative of an LCA-combretastatin A4 conjugate (PEG-LCA-CA4). DTX-CA4 NMs effectively inhibit the tumour growth in syngeneic (CT26) and xenograft (HCT116) colorectal cancer models, inhibit tumour recurrence, and enhance the percentage survival in comparison with individual drug-loaded NMs. DTX-CA4 NMs enhance the T cell-mediated anti-tumour immune response and DTX-CA4 NMs in combination with an immune checkpoint inhibitor, anti-PDL1 antibody, enhance the anti-tumour response. We additionally showed that DTX-CA4 NMs effectively attenuate the production of ceramide-1-phosphate, a key metabolite of the sphingolipid pathway, by downregulating the expression of ceramide kinase at both transcriptional and translational levels. Therefore, this study presents the engineering of effective DTX-CA4 NMs for targeting the tumour microenvironment that can be explored further for clinical applications.

Design, synthesis of ceramide 1-phosphate analogs and their affinity for cytosolic phospholipase A2 as evidenced by surface plasmon resonance.

Ceramide 1-phosphate (C1P) is a lipid mediator that specifically binds and activates cytosolic phospholipase A2α (cPLA2α). To elucidate the structure-activity relationship of the affinity of C1P for cPLA2α in lipid environments, we prepared a series of C1P analogs containing structural modifications in the hydrophilic parts and subjected them to surface plasmon resonance (SPR). The results suggested the presence of a specific binding site for cPLA2α on the amide, 3-OH and phosphate groups in C1P structure. Especially, dihydro-C1P exhibited enhanced affinity for cPLA2α, suggesting the hydrogen bonding ability of 3-hydroxy group is important for interactions with cPLA2α. This study helps to understand the influence of specific structural moieties of C1P on the interaction with cPLA2α at the atomistic level and may lead to the design of drugs that regulate cPLA2α activation.

Molecular Dynamics Investigation into CerENP's Effect on the Lipid Matrix of Stratum Corneum.

The extracellular lipid matrix in the stratum corneum (SC) plays a critical role in skin barrier functionality, comprising three primary components: ceramides, cholesterol, and free fatty acids. The diverse ceramides, differentiated by molecular structures such as hydroxylations and varying chain lengths, are essential for the lipid matrix's structural integrity. Recently, a new subclass of ceramide, 1-O-acylceramide NP (CerENP), has been identified; however, its precise role in the lipid matrix of the SC is still elusive. Herein, we investigate the role of CerENP on the structure and permeability of the SC using molecular dynamics simulations. Our findings indicate that CerENP contributes to a compact lipid matrix in the lateral dimension of our SC model with a repeat distance of about 13 nm. Additionally, ethanol permeability assessments show that CerENP effectively reduces molecular penetration through the lipid matrix. This study provides an insight into the role of a new subclass of ceramide in the SC, enhancing our understanding of skin structure and the mechanisms behind barrier dysfunction in skin diseases.

Dynamic Simulations of Interaction of the PEG-DPPE Micelle-Encapsulated Short-Chain Ceramides with the Raft-Included Membrane.

The lipid raft subdomains in cancer cell membranes play a key role in signal transduction, biomolecule recruitment, and drug transmembrane transport. Augmented membrane rigidity due to the formation of a lipid raft is unfavorable for the entry of drugs, a limiting factor in clinical oncology. The short-chain ceramide (CER) has been reported to promote drug entry into membranes and disrupt lipid raft formation, but the underlying mechanism is not well understood. We recently explored the carrier-membrane fusion dynamics of PEG-DPPE micelles in delivering doxorubicin (DOX). Based on the phase-segregated membrane model composed of DPPC/DIPC/CHOL/GM1/PIP2, we aim to explore the dynamic mechanism of the PEG-DPPE micelle-encapsulating DOXs in association with the raft-included cell membrane modulated by C8 acyl tail CERs. The results show that the lipid raft remains integrated and DOX-resistant subjected to free DOXs and the micelle-encapsulating ones. Addition of CERs disorganizes the lipid raft by pushing CHOL aside from DPPC. It subsequently allows for a good permeability for PEG-DPPE micelle-encapsulated DOXs, which penetrate deeper as CER concentration increases. GM1 is significant in guiding drugs' redistributing between bilayer phases, and the anionic PIP2 further helps DOXs attain the inner bilayer surface. These results elaborate on the perturbing effect of CERs on lipid raft stability, which provides a new comprehensive approach for further design of drug delivery systems.

Models of the Three-Component Bilayer of Stratum Corneum: A Molecular Simulation Study.

The construction of the stratum corneum (SC) is crucial to the problems of transdermal drug delivery. SC consists of the keratinocyte layers and the lipid matrix surrounding it. Among them, the lipid matrix is the barrier for many exogenous molecules, mainly composed of ceramides (CERs), free fatty acids (FFA), and cholesterol (CHOL). In this work, we developed single-component (CERs, CER-NS, and CER-EOS) and six three-component models, and each model was simulated by using the GROMOS-54A7 force field. Short-period phase (SPP) and long-period phase (LPP) systems were established separately, and area per lipid (APL), thickness, order of carbon chain (SCD), and density distribution were analyzed. The transition of CER-NS and CER-EOS in LPP was observed. The results of hydrogen bonds in the lipid systems indicated that a strong hydrogen-bond network was formed between the skin-lipid bilayers. Umbrella sampling method simulations were performed to calculate the free energy change of ethanol moving into the skin-lipid bilayer. The results revealed that ethanol molecules pulled some water molecules into the membrane when they passed through SPP-1. Our findings provided some insights and models of the stratum corneum that could be used for the subsequent mechanism of macromolecule permeation through membranes in drugs, cosmetics, and so on.

The molecular arrangement of ceramides in the unit cell of the long periodicity phase of stratum corneum models shows a high adaptability to different ceramide head group structures.

The stratum corneum (SC) lipid matrix, composed primarily of ceramides (CERs), cholesterol and free fatty acids (FFA), has an important role for the skin barrier function. The presence of the long periodicity phase (LPP), a unique lamellar phase, is characteristic for the SC. Insight into the lipid molecular arrangement within the LPP unit cell is imperative for understanding the relationship between the lipid subclasses and the skin barrier function. In this study, the impact of the CER head group structure on the lipid arrangement and barrier functionality was investigated using lipid models forming the LPP. The results demonstrate that the positions of CER N-(tetracosanoyl)-sphingosine (CER NS) and CER N-(tetracosanoyl)-phytosphingosine (CER NP), two essentials CER subclasses, are not influenced by the addition of another CER subclass (N-(tetracosanoyl)-dihydrosphingosine (CER NdS), N-(2R-hydroxy-tetracosanoyl)-sphingosine (CER AS) or D-(2R-hydroxy-tetracosanoyl)-phytosphingosine (CER AP)). However, differences are observed in the lipid organization and the hydrogen bonding network of the three different models. A similar localization of CER NP and CER NS is also observed in a more complex lipid model, with the CER subclass composition mimicking that of human SC. These studies show the adaptability and insensitivity of the LPP unit cell structure to changes in the lipid head group structures of the CER subclasses.

A Novel Non-Psychoactive Fatty Acid from a Marine Snail, Conus inscriptus, Signals Cannabinoid Receptor 1 (CB1) to Accumulate Apoptotic C16:0 and C18:0 Ceramides in Teratocarcinoma Cell Line PA1.

The cannabinoid-type I (CB1) receptor functions as a double-edged sword to decide cell fate: apoptosis/survival. Elevated CB1 receptor expression is shown to cause acute ceramide accumulation to meet the energy requirements of fast-growing cancers. However, the flip side of continual CB1 activation is the initiation of a second ceramide peak that leads to cell death. In this study, we used ovarian cancer cells, PA1, which expressed CB1, which increased threefold when treated with a natural compound, bis(palmitoleic acid) ester of a glycerol (C2). This novel compound is isolated from a marine snail, Conus inscriptus, using hexane and the structural details are available in the public domain PubChem database (ID: 14275348). The compound induced two acute ceramide pools to cause G0/G1 arrest and killed cells by apoptosis. The compound increased intracellular ceramides (C:16 to 7 times and C:18 to 10 times), both of which are apoptotic inducers in response to CB1 signaling and thus the compound is a potent CB1 agonist. The compound is not genotoxic because it did not induce micronuclei formation in non-cancerous Chinese hamster ovarian (CHO) cells. Since the compound induced the cannabinoid pathway, we tested if there was a psychotropic effect in zebrafish models, however, it was evident that there were no observable neurobehavioral changes in the treatment groups. With the available data, we propose that this marine compound is safe to be used in non-cancerous cells as well as zebrafish. Thus, this anticancer compound is non-toxic and triggers the CB1 pathway without causing psychotropic effects.

Targeting dihydroceramide desaturase 1 (Des1): Syntheses of ceramide analogues with a rigid scaffold, inhibitory assays, and AlphaFold2-assisted structural insights reveal cyclopropenone PR280 as a potent inhibitor.

Dihydroceramide desaturase 1 (Des1) catalyzes the formation of a CC double bond in dihydroceramide to furnish ceramide. Inhibition of Des1 is related to cell cycle arrest and programmed cell death. The lack of the Des1 crystalline structure, as well as that of a close homologue, hampers the detailed understanding of its inhibition mechanism and difficults the design of new inhibitors, thus making Des1 a strategic target. Based on previous structure-activity studies, different ceramides containing rigid scaffolds were designed. The synthesis and evaluation of these compounds as Des1 inhibitors allowed the identification of PR280 as a better Des 1 inhibitor in vitro (IC50 = 700 nM) than GT11 and XM462, the current reference inhibitors. This cyclopropenone ceramide was obtained in a 6-step synthesis with a 24 % overall yield. The highly confident 3D structure of Des1, recently predicted by AlphaFold2, served as the basis for conducting docking studies of known Des1 inhibitors and the ceramide derivatives synthesized by us in this study. For this purpose, a complete holoprotein structure was previously constructed. This study has allowed a better knowledge of key ligand-enzyme interactions for Des1 inhibitory activity. Furthermore, it sheds some light on the inhibition mechanism of GT11.

Cryo-EM structure of human sphingomyelin synthase and its mechanistic implications for sphingomyelin synthesis.

Sphingomyelin (SM) has key roles in modulating mammalian membrane properties and serves as an important pool for bioactive molecules. SM biosynthesis is mediated by the sphingomyelin synthase (SMS) family, comprising SMS1, SMS2 and SMS-related (SMSr) members. Although SMS1 and SMS2 exhibit SMS activity, SMSr possesses ceramide phosphoethanolamine synthase activity. Here we determined the cryo-electron microscopic structures of human SMSr in complexes with ceramide, diacylglycerol/phosphoethanolamine and ceramide/phosphoethanolamine (CPE). The structures revealed a hexameric arrangement with a reaction chamber located between the transmembrane helices. Within this structure, a catalytic pentad E-H/D-H-D was identified, situated at the interface between the lipophilic and hydrophilic segments of the reaction chamber. Additionally, the study unveiled the two-step synthesis process catalyzed by SMSr, involving PE-PLC (phosphatidylethanolamine-phospholipase C) hydrolysis and the subsequent transfer of the phosphoethanolamine moiety to ceramide. This research provides insights into the catalytic mechanism of SMSr and expands our understanding of sphingolipid metabolism.

ReTimeML: a retention time predictor that supports the LC-MS/MS analysis of sphingolipids.

The analysis of ceramide (Cer) and sphingomyelin (SM) lipid species using liquid chromatography-tandem mass spectrometry (LC-MS/MS) continues to present challenges as their precursor mass and fragmentation can correspond to multiple molecular arrangements. To address this constraint, we developed ReTimeML, a freeware that automates the expected retention times (RTs) for Cer and SM lipid profiles from complex chromatograms. ReTimeML works on the principle that LC-MS/MS experiments have pre-determined RTs from internal standards, calibrators or quality controls used throughout the analysis. Employed as reference RTs, ReTimeML subsequently extrapolates the RTs of unknowns using its machine-learned regression library of mass-to-charge (m/z) versus RT profiles, which does not require model retraining for adaptability on different LC-MS/MS pipelines. We validated ReTimeML RT estimations for various Cer and SM structures across different biologicals, tissues and LC-MS/MS setups, exhibiting a mean variance between 0.23 and 2.43% compared to user annotations. ReTimeML also aided the disambiguation of SM identities from isobar distributions in paired serum-cerebrospinal fluid from healthy volunteers, allowing us to identify a series of non-canonical SMs associated between the two biofluids comprised of a polyunsaturated structure that confers increased stability against catabolic clearance.

IL-10 constrains sphingolipid metabolism to limit inflammation.

Interleukin-10 (IL-10) is a key anti-inflammatory cytokine that can limit immune cell activation and cytokine production in innate immune cell types1. Loss of IL-10 signalling results in life-threatening inflammatory bowel disease in humans and mice-however, the exact mechanism by which IL-10 signalling subdues inflammation remains unclear2-5. Here we find that increased saturated very long chain (VLC) ceramides are critical for the heightened inflammatory gene expression that is a hallmark of IL-10 deficiency. Accordingly, genetic deletion of ceramide synthase 2 (encoded by Cers2), the enzyme responsible for VLC ceramide production, limited the exacerbated inflammatory gene expression programme associated with IL-10 deficiency both in vitro and in vivo. The accumulation of saturated VLC ceramides was regulated by a decrease in metabolic flux through the de novo mono-unsaturated fatty acid synthesis pathway. Restoring mono-unsaturated fatty acid availability to cells deficient in IL-10 signalling limited saturated VLC ceramide production and the associated inflammation. Mechanistically, we find that persistent inflammation mediated by VLC ceramides is largely dependent on sustained activity of REL, an immuno-modulatory transcription factor. Together, these data indicate that an IL-10-driven fatty acid desaturation programme rewires VLC ceramide accumulation and aberrant activation of REL. These studies support the idea that fatty acid homeostasis in innate immune cells serves as a key regulatory node to control pathologic inflammation and suggests that 'metabolic correction' of VLC homeostasis could be an important strategy to normalize dysregulated inflammation caused by the absence of IL-10.

Emerging roles and therapeutic potentials of sphingolipids in pathophysiology: emphasis on fatty acyl heterogeneity.

Sphingolipids not only exert structural roles in cellular membranes, but also act as signaling molecules in various physiological and pathological processes. A myriad of studies have shown that abnormal levels of sphingolipids and their metabolic enzymes are associated with a variety of human diseases. Moreover, blood sphingolipids can also be used as biomarkers for disease diagnosis. This review summarizes the biosynthesis, metabolism, and pathological roles of sphingolipids, with emphasis on the biosynthesis of ceramide, the precursor for the biosynthesis of complex sphingolipids with different fatty acyl chains. The possibility of using sphingolipids for disease prediction, diagnosis, and treatment is also discussed. Targeting endogenous ceramides and complex sphingolipids along with their specific fatty acyl chain to promote future drug development will also be discussed.

Syntheses of the ω-pyridinium-containing very-long-chain ceramides PyrCer(24:1(15Z)) and PyrCer(24:0) and their anticancer activity.

Ceramides, crucial sphingolipids in cellular biology, play various roles ranging from structural membrane integrity to signaling pathway regulation. Structurally, a ceramide consists of a fatty acid connected to a sphingoid base. The characteristics of the fatty acid chain, including length and saturation, determine the physiological properties of the ceramide. Ceramides typically fall into the following categories based on chain length: medium, long, very-long, and ultra-long. Among them, two very-long-chain ceramides, Cer(24:1(15Z)) and Cer(24:0), have been extensively studied, and they are known for their regulatory functions. However, the hydrophobic natures of ceramides, arising from their long hydrocarbon chain impedes their solubilities and levels of cellular delivery. Although ω-pyridinium ceramide analogs (ω-PyrCers) have been developed to address this issue, ω-PyrCers with very-long fatty acid chains or unsaturation have not been developed, presumably due to limited access to the corresponding ω-bromo fatty acids required in their syntheses. In this study, we prepared the ω-PyrCers of Cer(24:1(15Z)) and Cer(24:0), PyrCer(24:1(15Z)) and PyrCer(24:0), respectively. The key in the synthesis is the Wittig reaction to prepare the ω-bromo fatty acid with an appropriate chain length and (Z)-double bond position. Preliminary evaluation of the PyrCer(24:1(15Z)) and PyrCer(24:0) revealed their potential in hepatocellular carcinoma treatment.

Lipidomic analysis of Porphyromonas gingivalis reveals novel glycerol bisphosphoceramide, phosphatidyl-, and phosphoglycerol dipeptide lipid families.

Porphyromonas gingivalis, like other members of the phylum Bacteroidetes (synonym Bacteroidota), synthesizes several classes of dihydroceramides and peptidolipids. Using a similar strategy as that recently used to delimit the lipidome of its close relative Bacteroides fragilis, we applied linear ion trap multiple-stage mass spectrometry (linear ion trap MSn) with high-resolution mass spectrometry, to structurally characterize the complete lipidome of P. gingivalis and compare it to B. fragilis. This analysis discovered that the P. gingivalis lipidome consists of several previously unidentified lipid families, including dihydroceramide-1-phosphophate, acylated dihydroceramide-1-phosphophate, phosphoglycerol glycylserine lipid, and bis(phosphodihydroceramide) glycerol. Interestingly, we also found a novel sphingolipid family containing a polyunsaturated long-chain base, and a new lipoglycylserine phosphatic acid containing unsaturated acyl chains not reported for the lipid family. The comprehensive coverage of the lipidome of P. gingivalis conducted in this study has revealed more than 140 lipid species including several novel lipids in over 20 lipid families/subfamilies.

Trajectory of stratum corneum lipid subclasses in the first year of life and associations with atopic dermatitis: A prospective cohort study.

BACKGROUND: Trajectories of stratum corneum (SC) lipid subclasses and their associations with infant atopic dermatitis (AD) are unclear. This study aimed to quantify the trajectories of 15 SC subclasses and carbon chain lengths and their associations with AD within 12 months. METHODS: In total, 213 newborns were enrolled at birth with nonlesional skin samples collected from the inner forearm at birth, 42 days, 3, 6, and 12 months, respectively. Lesional skin samples were collected from 120 AD patients at clinic with the disease onset within the first year of life. Mass spectrometry was applied to assess relative contents of 12 ceramide (CER), three free fatty acid (FFA) subclasses, and average carbon chain length (CCL). AD incident within 1 year old was diagnosed by dermatologists according to UK criteria. RESULTS: Sixty-four (30.0%) cases of ADs occurred in the cohort. All SC lipid subclasses and CCLs, but EOP varied significantly during the first year. AD infants showed lower NP but higher NS, NH, AP, hydroxy FFA, and CCL of FFAs compared with nonaffected infants. After normalization by age, the differences remained and were more pronounced in lesional skin of clinical AD infants compared with non-ADs. NS, NH, and CCL of FFAs in lesional skin of AD infants showed positive and significant correlations with the levels of transepidermal water loss at 3 month; some evidence supports a negative correlation for NP. CONCLUSIONS: We provide an overview of developmental trajectories of 15 CER and FFA subclasses across the first year of healthy infants and a link between the imbalance of some subclasses with the development of AD.

Neutral ceramidase-active site inhibitor chemotypes and binding modes.

Ceramides impact a diverse array of biological functions and have been implicated in disease pathogenesis. The enzyme neutral ceramidase (nCDase) is a zinc-containing hydrolase and mediates the metabolism of ceramide to sphingosine (Sph), both in cells and in the intestinal lumen. nCDase inhibitors based on substrate mimetics, for example C6-urea ceramide, have limited potency, aqueous solubility, and micelle-free fraction. To identify non-ceramide mimetic nCDase inhibitors, hit compounds from an HTS campaign were evaluated in biochemical, cell based and in silico modeling approaches. A majority of small molecule nCDase inhibitors contained pharmacophores capable of zinc interaction but retained specificity for nCDase over zinc-containing acid and alkaline ceramidases, as well as matrix metalloprotease-3 and histone deacetylase-1. nCDase inhibitors were refined by SAR, were shown to be substrate competitive and were active in cellular assays. nCDase inhibitor compounds were modeled by in silico DOCK screening and by molecular simulation. Modeling data supports zinc interaction and a similar compound binding pose with ceramide. nCDase inhibitors were identified with notably improved activity and solubility in comparison with the reference lipid-mimetic C6-urea ceramide.

The Interplay between Bioactive Sphingolipids in the Psoriatic Skin and the Severity of the Disease.

Psoriasis is a complex chronic immunologically mediated disease that may involve skin, nails, and joints. It is characterized by hyperproliferation, deregulated differentiation, and impaired apoptosis of keratinocytes. Sphingolipids, namely ceramide, sphingosine-1-phosphate, sphingosine, sphingomyelin, and sphinganine-1-phosphate, are signal molecules that may regulate cell growth, immune reactions, and apoptosis. Fifteen patients with psoriasis and seventeen healthy persons were enrolled in the study. Skin samples were taken from psoriatic lesions and non-lesional areas. Tissue concentration of ceramides, sphingosine-1-phosphate, sphingosine, sphingomyelin, and sphinganine-1-phosphate was measured by liquid chromatography. We assessed that all levels of ceramides, sphingosine-1-phosphate, sphingosine, sphingomyelin, and sphinganine-1-phosphate were higher in lesioned psoriatic skin than in non-affected skin. The profile of bioactive lipids in the lesional skin of patients with psoriasis differed significantly from non-involved psoriatic skin and skin in healthy subjects.

Detection of Bacterial Neutral Ceramidase in Diabetic Foot Ulcers with an Optimized Substrate and Chemoenzymatic Probes.

Ceramidases (CDases) are important in controlling skin barrier integrity by regulating ceramide composition and affording downstream signal molecules. While the functions of epidermal CDases are known, roles of neutral CDases secreted by skin-residing microbes are undefined. Here, we developed a one-step fluorogenic substrate, S-B, for specific detection of bacterial CDase activity and inhibitor screening. We identified a non-hydrolyzable substrate mimic, C6, as the best hit. Based on C6, we designed a photoaffinity probe, JX-1, which efficiently detects bacterial CDases. Using JX-1, we identified endogenous low-abundance PaCDase in a P. aeruginosa monoculture and in a mixed skin bacteria culture. Harnessing both S-B and JX-1, we found that CDase activity positively correlates with the relative abundance of P. aeruginosa and is negatively associated with wound area reduction in clinical diabetic foot ulcer patient samples. Overall, our study demonstrates that bacterial CDases are important regulators of skin ceramides and potentially play a role in wound healing.