bluuebunny/crossref_metadata_2025_split huggingface dataset:
The Characterization of Septoria lycopersici Pathogenicity in Micro Tom Tomatoes
Authors: Allen Katelyn E , Zwiesler‐Vollick Julie 
Published: November 2023

Septoria lycopersici is a foliar fungal pathogen of tomatoes. This pathosystem has been well characterized with respect to the tomato antimicrobial secondary metabolite, alpha‐tomatine and the fungal detoxifying enzyme, tomatinase. However, there are many gaps in our knowledge of other aspects of the S. lycopersici pathogenicity. We know that fungal spores germinate on tomato leaves and grow biotrophically. The hyphae then invade the leaf apoplast via open stomata or wounds. At some point, S. lycopersici begins to damage the surrounding plant tissue. One of the aims of this project is to characterize the process of fungal infection with light, confocal fluorescence and scanning electron microscopy. This will provide the basis for further investigations of the key virulence factors present in S. lycopersici as well as potential defense mechanisms. We will also be assessing the ability of S. lycopersici to infect the dwarf tomato cultivar, Micro Tom. If Micro Tom tomatoes are susceptible, they could become the preferred host cultivar, due to their rapid life cycle. If Micro Tom tomatoes are not susceptible, this provides a source for possible resistance genes. In addition, the closely related wheat fungal pathogen, Mycosphaerella graminicola has been reported to be dimorphic with only the filamentous form able to infect plant hosts. The switch from yeast‐like to filamentous growth is regulated by a mitogen‐activated protein kinase. There are also reports that the switch to filamentous growth can be controlled by temperature. We will be characterizing the growth phase of S. lycopersici at various temperatures to determine if this fungal pathogen is also dimorphic.Support or Funding InformationLawrence Technological University Department of Natural Sciences
Kinetic Characterization of 3‐Hydroxyisobutyrate Dehydrogenase from Arabidopsis thaliana
Authors: Binkley Christiana , McCool Mason , Maurer Taylor , Rouhier Kerry A 
Published: November 2023

Branched‐chain amino acids (BCAAs) initially share a similar metabolic pathway. However, their paths diverge, resulting in unique catabolic pathways for each BCAA. This lab examined valine degradation, as it is the least characterized of all the BCAAs. During valine metabolism, the enzyme 3‐hydroxyisobutyrate dehydrogenase (HIBDH) catalyzes 3‐hydroxyisobutyric acid into methylmalonic semialdehyde as one of the concluding reactions. While HIBDH from Pseudomonas denitrificans had previously been cloned and expressed in Escherichia coli, the goal of this research was to examine the activity of HIBDH in plants. Thus, we cloned the HIBDH gene from Arabidopsis thaliana into E.coli, where its activity could be examined. Two HIBDH constructs were created in order to determine proper exclusion of the mitochondrial leader sequence; these constructs differ only by the length of their amino acid sequences. The expressed proteins were then purified by affinity chromatography and kinetically characterized. The construct composed of nucleotides 97–1044 (denoted At316) resulted in ~9 mg/mL of purified protein, while the construct generated from nucleotides 115–1044 (denoted At310) produced ~1 mg/mL from a similar volume and density of cells at the point of induction. Both constructs resulted in active protein using S‐HIBA (native substrate) and NAD, although very little activity was seen using construct At310. Construct At316 functioned optimally under alkaline conditions similar to HIBDH from P. denitrificans and Homo sapiens. Overall, the biochemical properties of HIBDH in A. thaliana were assessed, offering new insights into both the valine degradation pathway as a whole, as well as the biological and chemical parameters necessary for HIBDH function in plants.Support or Funding InformationThis project was funded by Kenyon College.
Subtle Defects of Four Homocystinuric Variants of Human Cystathionine β‐Synthase
Authors: Hines Judy P , Su Yang , Smith Aaron T. , Burstyn Judith N 
Published: November 2023

In humans, CBS‐deficient homocystinuria (CBSDH) results from mutations in the gene encoding cystathionine β‐synthase (CBS), a heme‐containing PLP‐dependent enzyme that catalyzes condensation of serine and homocysteine to form cystathionine. The role of the heme in CBS is unknown; however, disruption of the native heme iron‐to‐cysteine thiolate bond eliminates enzyme activity even though the heme does not participate in catalysis. Four CBSDH‐associated single amino acid variants within the heme pocket, P49L, P78R, R125Q and R266K, were investigated through studies of their heme environments and catalytic properties. Electronic absorption, resonance Raman, and electron paramagnetic resonance spectroscopic studies revealed that there are subtle changes to the heme environment in these variants, consistent with changes in hydrogen bonding or local heme pocket structure. Most variants exhibited a less stable heme pocket, susceptible to thermal unfolding at lower temperatures than for the wild‐type protein. Variant proteins were often more stable to thermally mediated aggregation resulting from global protein unfolding. Kinetic characterization revealed that the allosteric activator S‐adenosylmethionine (AdoMet) lowered Km for both substrates but had little effect on kcat for the wild‐type protein, a result consistent with the current structural model for AdoMet activation. P49L, P78R and R266K exhibited subtle changes in their kinetic constants and were AdoMet responsive. In contrast, R125Q exhibited a significantly decreased kcat value and was unresponsive to AdoMet activation. The correlation between altered heme environment and catalytic activity suggests that the heme plays an important regulatory role in this protein.Support or Funding InformationUniversity of Wisconsin‐Madison
Characterization of kinetic parameters of the lysophospholipase PldB using a liquid chromatography mass spectrometry based in vitro assay
Authors: Elizalde Gabriel , Garrett Teresa A 
Published: November 2023

Headgroup‐acylated glycerophospholipids (HAGPLs), such as acyl phosphatidylglycerol (acyl PG) and N‐acyl phosphatidylethanolamine, are a class of low‐abundance lipids that may play a role in Escherichia coli cell division. Lysophospholipase L2, also known as PldB, is an acyltransferase found in E. coli known to catalyze the formation of acyl PG. We seek to fully understand the substrate specificity of PldB to ultimately determine what contributes to PldB being an acyltransferase versus a phospholipase. Previous research suggests that PldB catalyzes the transfer of the acyl chain from 2‐acyl lysophospholipids to phosphatidylglycerol to form acyl PG. In order to characterize PldB's substrate specificity, a six‐histidine tagged variant of PldB has been constructed and overexpressed using the pET overexpression system. Cells induced to overexpress PldB are lysed, membranes isolated and PldB solubilized from the membranes with high concentrations of potassium chloride. The solubilized PldB is then purified to homogeneity from the resulting mixture with Ni‐affinity chromatography followed by gel‐filtration chromatography. This purified PldB will be used to determine the Km and kcat for PldB's reaction with a variety of acyl donors and acceptors using a liquid chromatography mass spectrometry (LC/MS) based assay currently under development.Support or Funding InformationNational Science Foundation Research at Undergraduate Institutions Grant #1516805 and #1152463, and National Science Foundation Major Instrumentations Grant #1039659
Structure of human Niemann‐Pick C1 (NPC1) protein and NPC1‐NPC2 complex
Authors: Li Xiaochun 
Published: November 2023

Cholesterol, essential for all animal life, plays a vital role for maintenance of membrane strength and permeability, also serves as a precursor for the biosynthesis of lipoprotein, steroid hormones, bile acid and vitamin D. Our major cholesterol sources are endogenous biosynthesis and diet uptake. Egress of LDL‐derived cholesterol from lysosomes requires two lysosomal proteins, integral membrane protein NPC1 and soluble NPC2. Mutations on NPC1 and NPC2 can cause the inborn errors Niemann‐Pick disease, type C, which lead to the early death of the patients; remarkably. We reported the structure of NPC1 at 3.3Å resolution, which reveals internal two‐fold pseudo symmetry along TMs 2–13, and two structurally homologous domains that protrude 60Å into the lysosomal lumen. The sterol‐sensing domain (SSD) of NPC1 presents an outward‐open conformation and has a very cavity, which might transport/sense the cholesterol through the limiting membrane. Two luminal domains [Middle lumenal Domain (MLD) and C‐terminal Domain (CTD)] shared the similar fold. We also determined the 2.4Å resolution crystal structure of a complex of human NPC1‐MLD and NPC2 bearing bound cholesterol‐3‐O‐sulfate. NPC1‐MLD utilizes two protruding loops to bind NPC2, analogous to its interaction with the primed Ebola virus glycoprotein. These works present the atomic resolution models for cholesterol biogenesis and trafficking, provide the molecular insights into cholesterol transfer in lysosomal lumen and pathogenesis of Niemann‐Pick disease, type C.Support or Funding InformationLife Sciences Research Foundation
Ceramide‐1‐Phosphate: Characterizing a Fluorescent Lipid and Discovering New Binding Proteins
Authors: Shirey Carolyn M. , Stahelin Robert V. 
Published: November 2023

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.
Disruption of Sphingolipid Biosynthesis Blocks Phagocytosis of Candida albicans
Authors: Tafesse Fikadu G. 
Published: November 2023

The ability of phagocytes to clear pathogens is an essential attribute of the innate immune response. The role of signaling lipid molecules such as phosphoinositides is well established, but the role of membrane sphingolipids in phagocytosis is largely unknown. Using a genetic approach and small molecule inhibitors, we show that phagocytosis of Candida albicans requires an intact sphingolipid biosynthetic pathway. Blockade of serine‐palmitoyltransferase (SPT) and ceramide synthase‐enzymes involved in sphingolipid biosynthesis‐by myriocin and fumonisin B1, respectively, impaired phagocytosis by phagocytes. We used CRISPR/Cas9‐mediated genome editing to generate Sptlc2‐deficient DC2.4 dendritic cells, which lack serine palmitoyl transferase activity. Sptlc2−/− DC2.4 cells exhibited a stark defect in phagocytosis, were unable to bind fungal particles and failed to form a normal phagocytic cup to engulf C. albicans. Supplementing the growth media with GM1, the major ganglioside present at the cell surface, restored phagocytic activity of Sptlc2−/− DC2.4 cells. While overall membrane trafficking and endocytic pathways remained functional, Sptlc2−/− DC2.4 cells express reduced levels of the pattern recognition receptors Dectin‐1 and TLR2 at the cell surface. Consistent with the in vitro data, compromised sphingolipid biosynthesis in mice sensitizes the animal to C. albicans infection. Sphingolipid biosynthesis is therefore critical for phagocytosis and in vivo clearance of C. albicans.Support or Funding InformationThis work was funded by The Netherlands Organization for Scientific Research (FGT), and the National Institute of Health grant AI087879 (HLP).
PKC‐δ Mediates Sphk2 Activation And Histone Acetylation In Pseudomonas aeruginosa‐Induced Lung Inflammation
Authors: Ebenezer David Lenin , Fu Panfeng , Ha Alison Wing‐Zi , Natarajan Viswanathan 
Published: November 2023

Pseudomonas aeruginosa (PA), a gram‐negative opportunistic pathogen, the leading cause of inflammation‐driven gradual deterioration of the lung function among cystic fibrotic patients, also causes pneumonia in immunosuppressed and mechanically ventilated patients. Our research intends to understand the role of nuclear Sphingolipids in modifying the epigenetic landscape in PA–induced lung inflammation. Previously, we have shown that Sphingosine kinase 2 (SphK2) generated nuclear Sphingosine‐1‐Phospahate (S1P), a simple bioactive sphingolipid, is an epigenetic co‐regulator of PA‐induced lung inflammation, as evident from our murine model and in vitro studies. Recently, we have identified a novel role of Protein Kinase C‐δ (PKC‐δ) in mediating the PA‐induced phosphorylation of Sphingosine Kinase 2 and histone acetylation. Mouse alveolar Type 2 cells challenged with PA induced phosphorylation of PKC‐δ (S 299), which is necessary for its activation and subsequent translocation to nucleus. PA‐induced activation of PKC‐δ also stimulated nuclear SphK2 phosphorylation in mouse lung type II cells. The PKC inhibitor, bisindolylmaleimide, blocked PA‐induced Histone H3 & H4 acetylation in mouse lung epithelial cell line (MLE‐12). Further, PA‐induced SphK2 phosphorylation and histone acetylation was blocked by small interfering RNA (siRNA) silencing of PKC‐δ in MLE‐12 cells. PA‐induced PKC‐δ activation also resulted in elevated mRNA expression of inflammatory cytokines, IL‐6 and TNF‐α, which was decreased significantly by overexpression of adenoviral dominant negative PKC‐δ mutant in MLE‐12 cells, suggesting a key role for PKC‐δ in regulating inflammatory cytokines. Furthermore, use of dominant negative PKC‐δ mutant also suppressed PA‐induced IL‐6 secretion, suggesting the secondary effector role of PKC‐δ in regulating SphK2 mediated down stream expression of inflammatory cytokines. These studies underscore the role of PKC‐δ in regulating PA‐induced inflammation via SphK2 phosphorylation, and increased histone acetylation suggesting that targeting PKC‐δ could ameliorate the bacterial inflammation of the lung.Support or Funding InformationThis work was supported by NIH P01 HL 98050 to VN
OlyA – A Tool to Study Sphingomyelin‐Cholesterol Interactions in Plasma Membranes
Authors: Endapally Shreya , Radhakrishnan Arun 
Published: November 2023

Sphingomyelin (SM) and cholesterol are lipids that are critical for the function of animal cells. Interplay between these two lipids is important for maintaining integrity of the myelin sheath that encases axonal cells and for maintaining functionally distinct pools of cholesterol in the plasma membranes of fibroblast cells. The nature of the interaction between SM and cholesterol has been long debated, with proposals ranging from complexes of the two lipids to no specific interaction between these two lipids.Here, we have developed a tool to examine the interaction between SM and cholesterol in animal cell membranes. Our tool is derived from a fungal toxin, ostreolysin A (OlyA). We find that OlyA binds to membranes of CHO‐K1 hamster cells when they contain both SM and cholesterol, but not when levels of either lipid are diminished by treatment with sphingomyelinase (which degrades SM) or cyclodextrin (which depletes cholesterol). In model membranes, OlyA shows no binding to 1, 2‐dioleoyl‐sn‐glycero‐3‐phosphocholine (DOPC) – cholesterol membranes even when the cholesterol concentration exceeds 50 mole%. Also, OlyA shows no binding to SM‐containing membranes when epicholesterol (a diasteromer of cholesterol) is present at concentration exceeding 50 mole%. These observations combined with direct binding experiments have given us insights into OlyA's dual lipid specificity and into how SM and cholesterol are organized in membranes.Support or Funding InformationThis work was supported by the Welch Foundation (I‐1793) and the American Heart Association (12SDG12040267).
Investigating Phospholipid Binding Residues in the C Terminus of Ebola Virus Matrix Protein, VP40
Authors: Budicini Melissa , Johnson Kristen , Stahelin Robert V. 
Published: November 2023

IntroductionEbola virus (EBOV) is a lipid‐enveloped virus that causes hemorrhagic fever and a fatality rate of 50–90%. Though EBOV only has 7 genes in its genome, one, VP40 is the main driver of viral egress. VP40 localizes to the plasma membrane where it forms virus like particles (VLPs). While the mechanism of the viral egress is not completely understood, previous studies have found that phospholipids phosphatidylserine (PS) and phosphatidylinositol 4,5‐bisphosphate (PIP2) are required for plasma membrane localization, self‐oligomerization, VLP formation and VLP budding from the cell. VP40 transforms through self‐oligomerization from a dimer to a hexamer and eventually a longer filament. PS likely induces the dimer to hexamer transition while PIP2 binding is required for stability of larger oligomers (n=12 or more). In an effort to understand the process of VP40 oligomerization, we have identified a likely PIP2 binding pocket on the C terminus of VP40; the C terminus of VP40 is hypothesized to interact with the PM. This pocket shares structural similarity to the PIP2 binding pocket of viral matrix protein HIV‐GAG from HIV‐1.ObjectiveWe are mutating potential binding residues and control residues to alanine on the C terminus of VP40 in order to understand the interactions between VP40, PIP2, and localization to the plasma membrane.MethodsSite Directed Mutagenesis, Confocal Laser Scanning Microscopy (CLSM), Liposome Pelleting Assays (LPA), and Transmission Electron Microscopy (TEM).ResultsVP40 PM localization can be blocked by elimination of phospholipid substrates through various assays in live COS‐7 cells. We are now investigating how VP40 binds to phospholipids. We have made several mutations with site directed mutagenesis and expressed or purified each in live cells for CLSM, or for lipid binding assays (LPA, TEM). We have identified several amino acids in the proposed binding pocket that are required for PI(4,5)P2 binding and oligomerization.ConclusionsPhospholipid‐focused assays in live cells and in vitro assays reveal residues in the C‐terminal domain of VP40 that are required to bind to PI(4,5)P2. We aim to better understand this interaction and it's role in oligomerization through future assays.Support or Funding InformationNIH AI081077
Binding Interaction of Lipid‐bound ApoA–I with Lipopolysaccharides and Phosphatidylglycerol
Authors: Haeri Bahareh , Weers Paul M.M. 
Published: November 2023

Apolipoprotein A‐I (apoA‐I) is a major component of high‐density lipoprotein (HDL), an anti‐antherogenic complex responsible for reverse cholesterol transport from peripheral tissues to liver, and mediates several essential metabolic functions related to heart disease. Human apoA‐I is 28 kDa protein found in lipid‐free and lipid‐bound forms. Previous studies have shown that lipid‐free apoA‐I binds and neutralizes lipopolysaccharides (LPS), which are major constituents of the outer bacterial membrane of gram‐negative bacteria, therefore reducing endotoxic effects. In addition, the protein destabilizes bilayers of negatively charged phosphatidylglycerol (PG), a phospholipid found in the inner bacterial membrane, which may result in cell lysis. However, the majority of apoA‐I is found in the lipid‐bound form as HDL; therefore binding interactions with LPS and PG were studied. To this end reconstituted HDL (rHDL) was produced with 1‐palmitoyl‐2‐oleoyl‐sn‐glycero‐3‐phosphocholine (POPC) and apoA‐I, which was isolated by KBr density ultracentrifugation. rHDL was analyzed by native PAGE and showed three distinct lipid‐protein complexes of 246, 203, and 149 kDa. To compare the binding of lipid‐free and lipid bound apoA‐I to LPS, non‐denaturing PAGE was employed. This showed that LPS binding was decreased for rHDL in comparison to lipid‐free apoA‐I. Both forms of apoA‐I were able to disaggregate LPS micelles, as shown by LPS carbohydrate staining of native PAGE. To investigate binding to PG, unilamellar PG vesicles with encapsulated calcein were prepared by extrusion. Calcein is a fluorescent dye and used as an indicator of lipid vesicle leakage, and is self‐quenched at a concentration above 70 mM. Binding to the vesicles was measured by the increase in calcein fluorescence, which was released upon protein‐vesicle binding. Lipid‐free apoA‐I induced the release of 60 to 70% calcein from PG vesicles; however, calcein release was reduced to 20 to 30% for rHDL. These results show that when apoA‐I is in the lipid‐bound state, binding interaction with LPS and PG bilayer vesicles was decreased significantly. Thus in order to be most effective as an antimicrobial protein, apoA‐I needs to be in the lipid‐free conformation.Support or Funding InformationThis research was supported by a grant from the National Institutes of Health (NIGMS #GM089564).