bluuebunny/crossref_metadata_2025_split huggingface dataset:
Characterizing the Lone Acyltransferase Homolog in Saccharomyces cerevisiae
Authors: Ullrich Alexander , Hardin Blake , Khanafer Adam , Oelkers Peter 
Published: June 2021

Cell membranes are largely composed of glycerophospholipids (PL) that create a barrier and separate intracellular aqueous environments. PL are heterogeneous, amphipathic lipids containing a glycerol backbone. In Saccharomyces cerevisiae, our previous work found that 108 different PL structural species were maintained at consistent relative abundance. Our aim is to understand the molecular mechanisms that maintain and regulate PL species abundance. The distribution of acyl chains in PL may be controlled by the activity and substrate specificity of acyltransferases which use an acyl‐CoA substrate to attach a hydrocarbon chain to an acceptor's hydroxyl group via an ester bond. In S. cerevisiae, at least 11 characterized acyltransferases contribute to PL metabolism. One, PSI1p, which esterifies 1‐hydroxy‐2‐acyl phosphatidylinositol with stearoyl‐CoA, has an uncharacterized paralog, YDR018c. Determining the acyl acceptor for YDR018c will likely clarify mechanisms for PL homeostasis in S. cerevisiae and provide a functional precedent for its most similar human homologs, 1‐acyl‐3‐glycerophosphate acyltransferase (AGPAT) 3 and 4. Since Psi1p has been shown to utilize an acyl acceptor with a large head group, inositol, it is possible that YDR018c utilizes a similar acyl acceptor.Since work by others and our lab did not identify a PL phenotype for haploid S. cerevisiae harboring a targeted deletion of YDR018c, loss of its function may be masked by the activity of other acyltransferases. To address this, we have made compound, targeted gene deletions of YDR018c, PSI1 and combinations of glycerol‐3‐phosphate O‐acyltransferases (GPATs; GAT1 or GAT2) and AGPATs (SLC1 or LPT1). Growth phenotype assays, including temperature sensitivity and providing various metabolic stresses, are ongoing to identify physiological changes in the compound deletion strains. Phospholipidome analysis, after culturing the strains in conditions shown to elicit a growth phenotype, will follow so to identify the absence or loss of specific PL species in conjunction with YDR018c deletion.Support or Funding InformationThis project was supported by the 2018 Summer Undergraduate Research Experience (SURE) Program from the University of Michigan‐Dearborn's Office of Research and Sponsored Programs (ORSP).This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Regulation of genes responsible for cholesterol uptake and efflux by fatty acids in THP‐1 macrophages
Authors: Moctar Khadija , Rasiah Angela , Medh Jheem D. 
Published: June 2021

Cholesterol is a very hydrophobic molecule that is transported through the blood in complexes containing apolipoproteins. Lipoprotein lipase (LPL) is a secreted enzyme that localizes to the luminal side of blood vessels. LPL is proatherogenic and facilitates occlusion of arterial tissue. When there is an abundance of lipids in the body, cytokines stimulate the release of vascular cell adhesion molecules (VCAM) by endothelial cells lining the arteries. This response induces the adhesion of monocytes to endothelial cells allowing monocytes to pass through gaps in the endothelium into the bloodstream. The monocytes take up oxidized lipids and differentiate to macrophages. If these macrophages are unsuccessful at eradicating lipid particles, they become foam cells and build up as plaques lining arteries.There are certain receptors and transporters that take part in cholesterol homeostasis. Scavenger receptors (CD36, SR‐AI, SR‐BI) mediate the cellular uptake of cholesterol whereas the ATP‐binding cassette transporter A1 (ABCA1) mediates the unidirectional efflux of excess cholesterol. SR‐BI also mediates bidirectional cholesterol transport.It has been reported in the literature that unsaturated fatty acids inhibit ABCA1 expression. This led us to ask, do the products of normal LPL function affect ABCA1 gene expression levels? To investigate this, we differentiate THP‐1 macrophages and stimulated them with various saturated and unsaturated fatty acids, namely, oleic acid a monounsaturated omega‐9 fatty acid, linoleic acid a polyunsaturated omega‐6 essential fatty acid and stearic acid, a common saturated fatty acid. The expression levels of CD36/SRA1 and SRB1 protein levels were compared in treated and untreated cells by Western blot analysis.Support or Funding InformationThis work was supported by National Institutes of Health Awards R15HL083946, SC3GM095413, and GM063787.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Lipidomic and proteomic evaluation of extracellular membrane vesicles from Streptococcus mutans wild‐type, ΔsrtA and Δsfp strains
Authors: Aparicio Joyce Corina Morales , Berdon Ana Lilia Barran , Green Kari B. , Wen Zezhang , Brady L. Jeannine 
Published: June 2021

BackgroundExtracellular membrane vesicles (EMVs) have long been studied in Gram‐negative bacteria. It is more recently established that Gram‐positive bacteria are capable of producing EMVs despite their thick cell wall. Our group showed that the cariogenic bacterium Streptococcus mutans produces EMVs that carry protein and eDNA. EMV protein content is altered in a deletion mutant lacking the transpeptidase, Sortase A. In Bacillus subtilis, EMVs are disrupted by the lipoprotein surfactin and accumulate in a Δsfp mutant. Our current objective is to isolate EMVs from wild‐type, ΔsrtA and Δsfp strains in order to compare their respective lipidomic and proteomic profiles, as well as to compare each strain's EMVs with corresponding cytoplasmic membrane preparations.MethodsEMVs were isolated from four separate replicates of each strain grown in Terleckyj's defined media (TDM). Filtered culture supernatants were concentrated ~50 fold, and EMVs were harvested by high‐speed centrifugation and density gradient isolation. Cytoplasmic membranes were isolated from protoplasts derived from bacterial cells from the same cultures. Lipids were analyzed by capillary‐liquid chromatography mass spectrometry (Cap‐LC‐MS). Proteins were analyzed by Nano‐Liquid chromatography tandem mass spectrometry (Nano‐LC/MS/MS). MV size, concentration, and polydisperity were assessed by transmission electron microscopy, nanoparticle tracking analysis, and dynamic light scattering.ResultsEMVs from the ΔsrtA mutant were enlarged compared to the other strains. Lipid composition of EMV membranes consisted primarily of glycerophospholipids, with some glycerolipids and sphingolipids. Glycerophosphoinsitol (PI) was underrepresented in both mutant strains compared to the WT. Glycerophosphocholine (PC) was overrepresented in the Δsfp mutant compared to the other two strains. Cardiolipin (CL) was overrepresented in EMVs from the WT strain compared to its corresponding cytoplasmic membrane preparations. Proteomics analyses are underway.ConclusionsLipidomic and proteomic analyses will provide information regarding S. mutans vesiculogenesis and potential functional differences in EMVs produced by WT and mutant strains.Support or Funding InformationNIH R21DE025348 NIH R01DE008007This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Mutations in the Human Follicle Stimulating Hormone Receptor Caveolin Binding Motif Cause Increased Basal Activation
Authors: Altman Elizabeth K , Cohen Brian D 
Published: June 2021

Over twelve percent of women aged fifteen to forty‐five in America suffer from infertility and over seven million women have used infertility services, such as intrauterine insemination and in vitro fertilization. Some cases of infertility may be due to dysfunctional human follicle stimulating hormone (hFSH) signaling. hFSH plays a role in spermatogenesis in males, as well as follicle maturation and estrogen production in females. Problems with either hFSH or the hFSH receptor (hFSHR) decrease fertility in males and cause complete infertility in females. As part of the hypothalamic‐pituitary‐gonadal axis, hFSH is released from the pituitary gland and binds to hFSHR in the ovaries and testes. It has been shown that hFSHR interacts with lipid rafts, stiffer regions of the plasma membrane with higher sphingolipid and cholesterol concentrations, and with caveolin, a protein found in certain lipid rafts. This interaction may occur through a specific sequence of amino acids in the 4th transmembrane domain of the hFSHR consistent with a caveolin binding motif (CBM). Previous work from our lab also suggested that hFSHR signaling is regulated through residency in lipid rafts, such that hFSHR signaling is inhibited as a result of recruitment into lipid rafts. The current study investigated the effect of mutations of the CBM on hFSHR signaling. It was hypothesized that complex mutation of the CBM would cause increased hFSHR signaling by disrupting interactions with caveolin, preventing inhibition via residency in lipid rafts. Mutants were created using site‐directed mutagenesis and expression vectors were transiently transfected into HEK293 cells and hFSHR signaling was qualitatively compared between mutants via western blot. Mutation of two sites in the CBM resulted in increased basal and induced hFSHR signaling, yet mutation of three sites decreased signaling below that of the wild type receptor. Mutation of two sites supports the hypothesis, while the decreased signaling from the triple mutant may suggest too large of a conformational change to allow for any receptor function. Further understanding of hFSHR signaling regulation would allow for development of new fertility treatments for men and women.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Direct Imaging of the Cholesterol and Sphingolipid Abundance at the Site of Influenza Virus Assembly with High‐Resolution SIMS
Authors: Kraft Mary L , Yeager Ashley N , Weber Peter K , Joshua Joshua 
Published: June 2021

The influenza virus is hypothesized to assemble and bud from plasma membrane domains that are enriched with cholesterol and sphingolipids. This hypothesis is consistent with the reported enrichment of the influenza viral envelope with cholesterol and sphingolipids, the reduction in influenza virus protein clustering in the plasma membrane induced by cholesterol depletion, and the sensitivity of influenza virus formation to cellular cholesterol levels. Nonetheless, the levels of cholesterol and sphingolipids in the plasma membrane at the site of influenza virus budding have never been directly measured. The main challenge has been a lack of analytical methods with the sensitivity and lateral resolution that is required to measure the relative cholesterol and sphingolipid levels in the plasma membrane on the 100‐nm‐length scales that are relevant to influenza virus budding. We recently overcame this challenge by using a secondary ion mass spectrometry (SIMS) technique with high spatial resolution and sensitivity to image the distributions of stable isotope‐labeled cholesterol, sphingolipids, and immunolabeled influenza hemagglutinin in the plasma membranes of virus‐infected MDCK cells. Our results show that the plasma membranes of both influenza‐infected and uninfected MDCK cells contained regions that were enriched with sphingolipids, but cholesterol appeared to be uniformly distributed within the membrane. Examination of the influenza‐infected MDCK cells revealed the hemagglutinin‐rich sites of influenza virus assembly in the plasma membrane were not enriched with cholesterol, and seldom enriched with sphingolipids. These direct imaging results demonstrate that the influenza virus does not assemble within plasma membrane domains that are enriched with cholesterol and sphingolipids.Support or Funding InformationThis research was supported by the U.S. National Science Foundation under CHE 15‐08662.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Structural and biochemical analysis completes the puzzle of chitin hydrolysis in insects
Authors: Yang Qing , Chen Wei 
Published: June 2021

Chitin is a linear homopolymer of β‐N‐acetyl‐D‐glucosamines and a major structural component of insect cuticles. Chitin hydrolysis involves glycoside hydrolase family 18 (GH18) chitinases and GH20 β‐N‐acetyl‐D‐hexosaminidases. In insects, chitin hydrolysis is essential for periodically shedding of the old cuticle ecdysis and proceeds via a pathway different from that in the well‐studied bacterial chitinolytic system. In Lepidopteran insects, the group II chitinase (ChtII) together with two other chitinases (ChtI and Chi‐h) and one GH20 β‐N‐acetyl‐D‐hexosaminidase (Hex1) are essential (Fig.1). Having revealed the acting mode and catalysis of ChtI, Chi‐h and Hex1, our lab recently elucidated the role of ChtII. ChtII is a widespread chitinolytic enzyme in insects and contains the greatest number of catalytic domains and chitin binding domains. We investigated the structure and enzymology of OfChtII, a ChtII representative derived from the insect pest Ostrinia furnacalis. OfChtII exhibit structural characteristics within the substrate‐binding cleft similar to those in OfChi‐h and OfChtI. However, it lacks structural elements favoring substrate binding beyond the active sites, including an extra wall structure present in OfChi‐h. Nevertheless, the numerous domains in OfChtII may compensate for this difference; a truncation containing one catalytic domain and three chitin binding modules (OfChtII‐B4C1) displayed higher activity toward insoluble polymeric substrates than those of OfChi‐h and OfChtI. Our observations complete the puzzle of chitin hydrolysis in insects and suggest a unique system distinguished from the bacterial ones. And based on the structural uniqueness, we have been able to develop highly efficient inhibitors against OfChi‐h, OfChtI, OfChtII and OfHex1.Support or Funding InformationThis work was supported by the Program for National Natural Science Funds for Distinguished Young Scholar (31425021), the National Natural Science Foundation of China (31402015), the National Key Research and Development Project of China (2017YFD0200502, 2017YFD0201207), the Fundamental Research Funds for the Central Universities (DUT17RC(4)13) and China Postdoctoral Science Foundation funded project (2017M611234).This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
The New Buzz about Sugars: Novel N‐Glycans in Bees, Moths and Mosquitoes
Authors: Wilson Iain B. H. , Hykollari Alba , Eckmair Barbara , Malzl Daniel , Vanbeselaere Jorick , Paschinger Katharina 
Published: June 2021

Sugars cover the surfaces of all cells and a major class of these glycoconjugates are the asparagine‐linked oligosaccharides attached to glycoproteins (N‐glycans). The classical view has been that N‐glycans in ‘lower’ are simple. However, recent data shows that, other than plants, the diversity of N‐glycan modifications in non‐vertebrate species is extremely high. Based on isobaric/isomeric separation combined with chemical or enzymatic treatments and MALDI‐TOF MS/MS, our own studies on dipteran, lepidopteran and hymenopteran species (specifically mosquitoes, moths and the honeybee) reveal a variety of ‘charged’ modifications of N‐glycans from insects, such as glucuronic acid, sulphate, phosphoethanolamine and phosphorylcholine. In addition, unusual core mannosylation, variable antennal length as well as up to three branches increase the range of possibilities. In the honeybee, we also have evidence for tissue‐specific N‐glycosylation patterns when comparing larvae, royal jelly and venom, while immunomodulatory or immunogenic epitopes are found on N‐glycans from insect cell lines used as baculovirus expression hosts. It is also clear that each analysed insect species has its own N‐glycosylation profile due to variations in relative abundance or absence of certain glycan structures. Complementary to the MS analyses, binding to antibodies, lectins and pentraxins can be tested with either natural N‐glycan pools in an array format or glycoproteins on blots; thereby, interactions with proteins of the human innate immune system can be demonstrated. Our data not only show that the concept of ‘simple’ organisms possessing only ‘simple’ glycans is not a true reflection of the actually complex biosynthetic glycosylation capacity of insects, but have repercussions for the use of insect cell lines for recombinant protein expression.Support or Funding InformationThis work was supported by the Austrian Science Fund (FWF) by grants to K.P., A.H. and I.B.H.W.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Characterization of O‐GlcNAc Hydrolase with Phosphomimetic Mutations in HeLa Cells
Authors: Hinkle Megan Leigh , Crawford Garland 
Published: June 2021

O‐linked beta N‐acetylglucosamine (O‐GlcNAc) hydrolase, or OGA, is an enzyme involved in a post‐translational modification. This enzyme hydrolyzes a single O‐GlcNAc from serine or threonine residues on target proteins located in the cytosol of metazoan organisms. OGA is the only protein known to carry out this function in the context of cytosolic proteins. This exclusivity of function makes OGA a likely target of post‐translational modifications in order to regulate its activity. Our aim is to generate a phosphomimetic OGA using site‐directed mutagenesis to convert a single serine or threonine to an aspartic acid and to introduce the mutated OGA gene into HeLa cells to study the relationship between OGA phosphorylation and its activity. While multiple serine and threonine residues of OGA have been predicted by bioinformatics and experimental approaches to be phosphorylated, the phosphorylation of OGA at serine 364 is of particular interest. This residue has been identified to be phosphorylated in 45 separate experiments and the introduction of OGA with a mutation from a serine to an aspartic acid residue at this point shuts down bacterial growth. Our aim is to study the effect of the phosphomimetic mutation at serine 364 in a mammalian cell system. Analysis by Western blot will reveal the extent to which the mutants alter OGA expression levels and O‐GlcNAc modification levels in the cell. Additionally, interactions of OGA with known binding partners, such as OGT, the enzyme which adds the O‐GlcNAc sugar to target proteins, can be studied with co‐immunoprecipitation. This work lays the foundation for identifying potential phosphorylation states of OGA and would aid in elucidating the regulatory pathways of this enzyme.Support or Funding InformationSEED grant, Office of the Provost, Mercer UniversityThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Cellulases Specific Activities Study of Enzymes secreted by Fusarium sp. and Phlebia sp. in Aerobic and Anaerobic Conditions
Authors: Mercado Benjamin 
Published: June 2021

Cellulose is the most abundant organic compound on Earth but we can't use it plenty as raw material due to its molecular arrangement and composition. Cellulases are powerful enzymes secreted by some organisms that can be used to degrade cellulose into simple organic molecules like glucose or cellobiose. Although traditional filamentous fungi are known to be excellent cellulose degraders in aerobic conditions, there is little known about cellulose degradation by fungi in anaerobic condition. In this study two facultative anaerobic filamentous fungi isolated and identified using ITS were used to compare cellulase specific activities in aerobic and anaerobic conditions. One Fusarium sp. and Phlebia sp. were grown individually in Potato Dextrose agar for 7 days. Suspensions of spores were prepared for each organism and use it as inoculums. Liquid media culture was prepared and enriched with Carboxylmethylcellulose, Wheat Brand Flour and Whatman no.1 filer paper and inoculated with 1mL of 106 suspension spores of each fungi individually. Cellulase specific activities were determined using Dinitrosalicylic acid method at ph 4, 5, 6, and 7 at 30°C, 40°C, 50°C and 60°C after one month of incubation. Fusarium sp. showed a maximum of cellulase activity at 40°C and pH 5 for both aerobic and anaerobic conditions with values of 0.436 and 0.403 respectively. On the other hand Phlebia sp. showed better results in anaerobic condition with a value of 0.715 at 60°C and pH 6 against a value of 0.632 in aerobic condition at 60°C and ph 5. These results confirm different trending in cellulase specific activities in aerobic and anaerobic conditions and could suggest the presence of different enzymes secreted depending of oxygen concentrations.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Unraveling the activity of glycosyltransferases and other PTM enzymes with bioluminescent biochemical and cell based assays
Authors: Zegzouti Hicham , Engel Laurie , Hwang Brian (Byounghoon) , Alves Juliano , Goueli Said 
Published: June 2021

Post translational modifications (PTMs) are central to all aspects of biological regulation. PTMs amplify the diverse functions of the proteome by covalently adding functional groups to proteins. These modifications include phosphorylation, methylation, glycosylation, and ubiquitination, and influence many aspects of normal cell biology and pathogenesis. Examples of post translational modification enzymes include but are not limited to, kinases/phosphatases, methyltransferases/demethylases, and glycosyltransferases/glucanases. Under normal physiological conditions, the regulation of PTM enzymes is tightly regulated. However, under pathological conditions, these enzyme activities can be dysregulated, and the disruption of the intracellular networks they govern leads to an array of diseases including cancer and inflammation. Consequently, PTM enzymes have become important targets for drug discovery creating a need for development of activity detection assays. Current assays to analyze cellular or biochemical PTM enzyme activities can be tedious, non‐homogeneous, and not easily adaptable to HTS. They can also suffer from generation of false hits due to compound interference or require special instruments for detection. To overcome these shortcomings, we developed an array of biochemical and cellular bioluminescent assays to detect these enzyme activities. This presentation will showcase how cellular mechanisms driven by PTM in general and phosphorylation and glcnacylation in particular can be addressed by analyzing these enzyme's cellular activity using the NanoLuc Binary Technology (NanoBiT), a two‐subunit complementation system based on NanoLuc luciferase. We will also discuss how the bioluminescent nucleotide assays that detect UDP, GDP, UMP and CMP as means to measure glycosyltransferase activities can streamline studies on GT specificity of transfer of different sugars to different acceptors. We will show their use in screening for specific inhibitors as well as in studies of their modes of action. Development of these bioluminescent detection assays will enable the investigation of key signaling pathways and the study of many PTM enzymes in general and GTs in particular, which may have significant impact on diverse areas of glycobiology research.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.