Background:SidA is a flavin-dependent monooxygenase that catalyzes the hydroxylation of ornithine and is essential for virulence in Aspergillus fumigatus. Results: Mutation of Ser-257 leads to destabilization of the C4a-hydroperoxyflavin and a faster hydride transfer step. Conclusion: Ser-257 is important for positioning NADP ϩ in the correct orientation for stabilization of the C4a-

The high mortality rate associated with pancreatic ductal adenocarcinoma (PDAC) is in part due to lack of effective therapy for this highly chemoresistant tumor. Cancer stem cells, a subset of cancer cells responsible for tumor initiation and metastasis, are not targeted by conventional cytotoxic agents, which renders the identification of factors that facilitate cancer stem cell activation useful in defining targetable mechanisms. We determined that bioactive sphingolipid induced migration of pancreatic cancer stem cells (PCSC) and signaling was specific to ceramide-1-phosphate (C1P). Furthermore, PDAC cells were identified as a rich source of C1P. Importantly, PDAC cells express the C1P converting enzyme ceramide kinase (CerK), secrete C1P-containing extracellular vesicles that mediate PCSC migration, and when co-injected with PCSC reduce animal survival in a PDAC peritoneal dissemination model. Our findings suggest that PDAC secrete C1P-containing extracellular vesicles as a means of recruiting PCSC to sustain tumor growth therefore making C1P release a mechanism that could facilitate tumor progression.

To reduce costs of lipid-binding assays, allow for multiple lipids to be screened for protein binding simultaneously, and to make lipid binding more user friendly, lipids have been dotted onto membranes to investigate lipid-protein interactions. These assays are similar to a western blot where the membrane is blocked, incubated with a protein of interest and detected using antibodies. Although the assay is inexpensive and straightforward, problems with promiscuous or poor binding, as well as insufficient blocking occur frequently. In this technical note, we share several specific improvements to ensure lipid-protein overlay assays are of high quality and contain proper controls.

Ceramide-1-phosphate (C1P) is an important signaling sphingolipid and a metabolite of ceramide. C1P contains an anionic phosphomonoester head group and has been shown to regulate physiological and pathophysiological processes such as cell proliferation, inflammation, apoptosis, phagocytosis, and macrophage chemotaxis. Despite this mechanistic information on its role in intra- and intercellular communication, little information is available on the biophysical properties of C1P in biological membranes and how it interacts with effector proteins. Fluorescently labeled lipids have been a useful tool to understand the membrane behavior properties of lipids such as phosphatidylserine, cholesterol, and some phosphoinositides. However, to the best of our knowledge, fluorescently labeled C1P hasn’t been implemented to investigate its ability to serve as a mimetic of endogenous C1P in cells or untagged C1P in in vitro experiments. Cellular and in vitro assays demonstrate TopFluor-C1P harbors a fluorescent group that is fully buried in the hydrocarbon core and fluoresces across the spectrum of physiological pH values. Moreover, TopFluor-C1P didn’t affect cellular toxicity at concentrations employed, was as effective as unlabeled C1P in recruiting an established protein effector to intracellular membranes, and its subcellular localization recapitulated what is known for endogenous C1P. Notably, the diffusion coefficient of TopFluor-C1P was slower than that of TopFluor-phosphatidylserine or TopFluor-cholesterol in the plasma membrane and similar to that of other fluorescently labeled sphingolipids including ceramide and sphingomyelin. These studies demonstrate that TopFluor-C1P should be a reliable mimetic of C1P to study C1P membrane biophysical properties and C1P interactions with proteins.

The Ebola virus (EBOV) is a genetically simple negative sense RNA virus with only 7 genes yet it causes severe hemorrhagic fever in humans. The matrix protein VP40 of EBOV is the main driver of viral budding through binding to host plasma membrane lipids and formation of the filamentous, pleomorphic virus particles. To better understand this dynamic and complex process we have asked what the role of two highly conserved cysteine residues are in the C-terminal domain of VP40.Here we report that the mutation of Cys 311 to alanine increases VP40 membrane binding affinity for phosphatidylserine containing membranes. C311A has a significant increase in binding to PS compared to WT, has longer virus like particles, and displays evidence of increased budding. C314A also has an increase in PS binding compared to WT, however to a lesser extent. The double Cys mutant shares the phenotypes of the single mutants with increased binding to PS. Computational studies demonstrate these Cys residues, Cys 311 in particular, restrain a loop segment containing Lys residues that interact with the plasma membrane. Mutation of Cys 311 promotes membrane binding loop flexibility, alters internal VP40 H-bonding, and increases PS binding. To the best of our knowledge, this is the first evidence of mutations that increase VP40 affinity for biological membranes and the length of EBOV virus like particles. Together, our findings indicate these residues are important for membrane dynamics at the plasma membrane via the interaction with phosphatidylserine.

No abstract

Sphingolipids are a class of biomolecules that play key roles in cellular signaling and membrane trafficking, with main players including sphingosine, sphingosine‐1‐phosphate (S1P), ceramide, and ceramide‐1‐phosphate (C1P). Specifically, C1P has been shown to play a role in important intra‐ and extracellular events such as cell proliferation, phagocytosis, inflammation, tumor metastasis, and migration of macrophages. Despite the identification of several C1P binding proteins such as cytosolic phospholipase A2α (cPLA2α), Annexin A2, diacylglycerol kinase γ, tumor necrosis factor‐α converting enzyme (TACE), and prostaglandin D synthase, there are still many gaps to fill in our understanding of C1P binding proteins and how these cellular interactions are facilitated. Therefore, we characterized a fluorescently labeled C1P for its in vitro and cellular activity, and sought to identify more C1P binding proteins. Here, we investigated a new fluorescently labeled C1P, called TopFluor‐C1P, to see if it could mimic C1P naturally found in mammalian cells. TopFluor‐C1P proved to be non‐toxic to cells, localize to the trans‐Golgi Network and plasma membrane, and successfully recruit the translocation of cPLA2α, as previous work has shown with native C1P. We also used fluorescence recovery after photobleaching (FRAP) to determine the mobile fraction and diffusion coefficient of TopFluor‐C1P at the plasma membrane to understand C1P membrane dynamics. This revealed the diffusion coefficient of C1P is less than other signaling lipids such as phosphatidylserine and cholesterol, and more similar to that of other sphingolipids. From our studies we determined TopFluor‐C1P is a valuable mimetic that can be further utilized to study many important biophysical and cellular questions regarding C1P properties in membranes and its role in lipid‐lipid and lipid‐protein interactions. In addition we aimed to discover the C1P binding proteome. Therefore, we performed an IP assay from A549 lung adenocarcinoma cells using lipid‐coated beads. The mass spectrometry analysis of lipid phosphomonoesters identified 212 C1P, 280 ceramide, 588 S1P, and 449 phosphatidic acid binding proteins. From this data we were were able to identify proteins that were found among all four structurally similar lipids, or were specific to only one lipid. We also used the Gene Ontology consortium and MetaCore pathways analysis software to further identify the molecular function, cellular localization and pathways involved in C1P binding proteins. Identifying these direct effectors of C1P will aid us in discovering C1P lipid‐protein binding characteristics, and help us better understand the role of C1P within the cell and in disease and cancer metabolism.Support or Funding InformationThis material is based upon work supported by the National Science Foundation Graduate Research Fellowship under Grant No. DGE‐1313583.