bluuebunny/crossref_metadata_2025 huggingface dataset:
Discovery of a novel Complex I biogenesis factor regulating mitochondrial gene expression (571.1)
Authors: Subrahmanian Nitya , Hamel Patrice
Published: June 2021

Mitochondrial Complex I (CI), a proton pumping NADH:ubiquinone oxidoreductase, is the most complicated respiratory enzyme. The assembly of this highly conserved enzyme, consisting of more than 40 subunits of dual genetic origin, is an intricate process that is poorly understood. The objective of this study was to identify novel factors involved in CI biogenesis using Chlamydomonas reinhardtii, a unicellular photosynthetic alga, as an ideal model system. From a mutant screen, the amc11 mutant with a CI deficiency was isolated. In‐gel activity shows that amc11 accumulates a sub‐complex diagnostic of an assembly defect in the membrane arm of CI. The mutation in amc11 was mapped to a novel gene encoding a unique low complexity protein with no conserved domains. The role of this AMC11 locus in CI assembly was confirmed by complementation studies. In order to understand the basis for the CI deficiency due to loss of AMC11, the transcript levels of the five mitochondrial encoded CI subunits was determined. Interestingly, the accumulation of only the nd4 transcript, encoding a subunit in CI membrane arm was partially reduced, the levels of which are restored upon complementation with the WT nuclear gene. Also, substitution of mitochondrial nd4 gene with a reporter in the amc11 mutant results in loss of reporter protein levels. This suggests that loss of AMC11 function yields a specific defect in the translation of the nd4 transcript. This is in accord with previous studies showing that loss of function mutations in the nd4 gene yields a CI‐deficiency with accumulation of a subcomplex, similar to the observed phenotype in the amc11. Thus, our forward genetic screen has led to the discovery of AMC11, a novel nuclear‐encoded protein required for the translation of the mitochondrial nd4 transcript.

Grant Funding Source: The Ohio State University
Insulin‐dependent mitochondrial Ca2+ uptake in skeletal muscle is quickly disrupted in high‐fat diet fed mice (572.3)
Authors: Contreras‐Ferrat Ariel , Llanos Paola , Jaimovich Enrique
Published: June 2021

The mitochondrial function quickly responds to energy‐dense food supply in order to deal with the demand but the role of mitochondrial Ca2+ in these processes has not been understood.

Male C57BL/6J mice were fed a normal chow diet (NCD) or a high fat diet (HFD) for 1 week. The insulin resistance was evaluated by IPGTT, fasting glucose and insulin, and HOMA‐IR. GLUT4myc‐eGFP was electroporated in the Flexor digitorum brevis muscle (FDB).

Insulin induces an increase in cytoplasmic and mitochondrial Ca2+ signal in adult fibers. The uncoupler FCCP, releases Ca2+ from mitochondria to cytoplasm. Insulin‐dependent mitochondrial Ca2+ uptake is decreased in fibers from short‐term HFD fed mice while insulin‐dependent cytoplasmic Ca2+ increase appear to be faster and stronger. In fibers from NCD fed mice, insulin‐dependent mitochondrial Ca2+ uptake was inhibited by xestospongin B, a specific IP3R inhibitor. Using TMRE+ in the non‐quenching mode (5 nM), we found that the ΔΨm was larger in fibers from short‐term HFD fed mice v/s NCD derived fibers. Using TMRE+ in quenching mode (100 nM), we found that the TMRE+ released after uncoupler stimuli was higher in fibers from short‐term HFD fed mice v/s NCD fed mice. The glucose analogue (2‐NBDG) uptake and the redistribution of GLUT4myceGFP induced by insulin were decreased in presence of xestospongin or ruthenium red (MCU inhibitor). This effect suggest a retrograde regulation of insulin signaling by mitochondrial Ca2+ uptake.

Grant Funding Source: Supported by FONDECYT 11130267 and ACT1111
Examination of enzyme and metabolic activity in yeast expressing mutant alleles of triose phosphate isomerase (578.1)
Authors: Koziara Kortnee , Barnes Carol , Squires Kevin , Fisher Isaac , Hrizo Stacy
Published: June 2021

Glycolysis is a highly conserved metabolic pathway by which the enzymatic breakdown of glucose occurs to yield energy in the form of ATP. Our research focuses on a mutation in an enzyme involved in glycolysis called triose phosphate isomerase (TPI). This enzyme converts DHAP to G3P in glycolysis. Importantly, mutations in TPI are associated with a human neurodegenerative disease called TPI deficiency and the pathology of TPI deficiency is still poorly understood. In order to study the effects of the TPI deficiency causing mutations on cellular processes we will use the yeast, Saccharomyces cerevisiae, as a biochemical model system for glycolysis, which is highly conserved between eukaryotes. S cerevisiae, when cultured without shaking, will rely heavily on the fermentation for energy production. We hypothesized that if the mutations that cause TPI deficiency resulted in reduced pyruvate production through glycolysis, then we would see a corresponding drop in carbon dioxide and ethanol production through fermentation. In addition, we examined redox activity due to cellular respiration with an alamar blue assay with shaking cultures. In addition, the activity of each TPI mutant allele was assessed by a spectrophotometric assay. Overall, we found that indeed some, but not all of the TPI mutants exhibited altered isomerase activity which affected metabolic output.
Structural insights into the thermal stability of endo‐mannanase belonging to family 26 from Bacillus sp. CFR1601 (580.2)
Authors: Srivastava Praveen , Appu Rao Appu Rao , Kapoor Mukesh
Published: June 2021

Structural insights into the thermal stability of endo‐mannanase belonging to family 26 from Bacillus sp. CFR1601 Praveen K. Srivastava, Appu Rao G. Appu Rao, Mukesh Kapoor: Department of Protein Chemistry and Technology, CSIR‐Central Food Technological Research Institute, Cheluvamba Mansion, Mysore‐570020.

Endo‐β‐mannanase (endo‐1,4‐β‐mannosidase; EC 3.2.1.78) is the key enzyme that catalyzes random hydrolysis of β‐mannosidic linkages in mannans and heteromannans. Mannanases play an important role in modification of gums, oil drilling industry and in the preparation of pre‐biotics for food industry. An endo‐mannanase obtained from soil bacterium Bacillus sp. CFR1601 was purified to apparent homogeneity and characterized. Partial biochemical and molecular characterization of purified enzyme reveals that it belongs to the glycosyl hydrolase family 26 with highest specific activity (10,400 ± 100 IU/mg) in its class. The enzyme retains its structure and activity up to 700C with activation energy of 10.8 ± 1.0 kcalmol‐1. Based on kinetics of thermal inactivation, enthalpy, free energy and entropy were found to be 86.7± 0.0 kcalmol‐1, 24.192± 0.7 kcalmol‐1 and 186.3± 0.21cal mol‐1 K‐1, respectively. Thermal stability measurements in presence of additives suggested the role of surface charges in thermal inactivation. Modification of surface charges could lead to development of highly stable and active endo‐mannanase for food and oil drilling applications.

Source of research support: Council of Scientific and Industrial Research (CSIR), New Delhi, India
Engineering a delta protein kinase C inhibitor that selectively inhibits interaction and phosphorylation of one substrate of the multi‐substrate kinase (582.3)
Authors: Qvit Nir , Disatnik Marie‐Helene , Mochly‐Rosen Daria
Published: June 2021

Rationale: Many kinases phosphorylate multiple substrates. To assess the role of an individual phosphorylation event without using laborious mutagenesis studies, selective pharmacological tools that inhibit phosphorylation of specific one substrate are needed. We focused on delta protein kinase C (δPKC) because δPKC activation after heart attack leads to cardiac damage. We reasoned that since pyruvate dehydrogenase kinase (PDK) phosphorylation by δPKC leads to inhibition of ATP production, it might be critical in the injurious effect δPKC after heart attack. To determine if of the many substrates of δPKC, PDK is the substrate that mediates this injurious effect in the heart, we designed a selective inhibitor of δPKC binding and phosphorylation of PDK.

Methods: We designed a short peptide inhibitor that mimics a unique interaction between PDK and δPKC to selectively inhibit δPKC phosphorylation of PDK. This protein‐protein interaction inhibitory peptide (PPIIP) was used in vitro, in cells and in intact heart, in models of heart attack.

Results: PDK/δPKC interaction was inhibited by PPIIP in vitro and in vivo. PDK/δPKC PPIIP selectively inhibited phosphorylation of PDK without affecting phosphorylation of several other δPKC substrates. Treatment with PDK/δPKC PPIIP inhibited myocardial infarction‐induced PDK phosphorylation without inhibiting the phosphorylation of other δPKC substrates. Further, PDK/δPKC PPIIP treatment led to a 50% reduction in infarct size, in release of cardiac enzyme (a measure of cell lysis) and in JNK phosphorylation, all markers of cardiac injury.

Conclusion: PDK/δPKC PPIIP is a selective inhibitor of PDK phosphorylation by δPKC. Of the several δPKC‐mediated phosphorylation events following heart attack, δPKC‐induced PDK phosphorylation is sufficient to cause cardiac injury as a selective inhibitor of this phosphorylation event alone was sufficient to provide great reduction in δPKC‐mediated cardiac injury after heart attack.
Determining the kinetics of DnaE, an enzyme for DNA replication in S. aureus (583.8)
Authors: Kosoy Gabrielle , Lahiri Indrajit , Pata Janice
Published: June 2021

DnaE is an DNA polymerase of the C family enzymes. It is used in DNA replication in S.aureus bacterium where this enzyme has been found to be crucial for lagging strand replication. We conducted pre‐steady state and steady state assays to look at the kinetic nature of DnaE in the replicative pathway. The pathway begins with DnaE binding to DNA substrate. Pre‐steady state condition assays showed that the affinity of the enzyme to bind to DNA was kDDNA=7±2nM. Single‐turn over condition assays gave a value of kDdNTP=30±2μM for the affinity of a nucleotide to be added to the duplex with enzyme complex. After this step the replicative polymerase may bind to other DNA to continue replication. A koff=0.1145sec‐1 rate was found for the disassociation of the enzyme from the binary structure. Replication with DnaE continues at a steady state rate of kcat=0.1 sec‐1.These values helped to determine that the rate at which the enzyme dissociates from the DNA duplex must be the rate limiting step in DnaE replication. Comparing these values to PolC, the enzyme that replicates the leading strand in S.aureus, and other C family polymerases may help answer why this enzyme must begin lagging strand replication at the primer.
Mechanistic studies of polysaccharide monooxygenases (584.8)
Authors: Vu Van , Beeson William , Phillips Christopher , Cate Jamie , Marletta Michael
Published: June 2021

Polysaccharide monooxygenases (PMOs) from filamentous fungi have recently been shown to play a central role in oxidative degradation of cellulose. These enzymes contain a mononuclear copper active site on a flat surface of the protein that would allow for direct interaction with the substrate. The active site copper coordination consists of two histidine ligands. One of these ligands is the unusual N‐terminal Ne‐methyl‐histidine residue that binds in a bidentate mode using its imidazole and amino groups. PMOs activate dioxygen leading to hydroxylation of either C1 or C4 position of the glycosidic linkage, which subsequently results in the cleavage of this bond. Newly developed expression methods have enabled the characterizeation and classification of the PMO family into three major groups based on their selectivity toward C1 and C4. Factors that control the regioselectivity of PMOs are underway, including residues that contribute to orientation of the copper site on the cellulose surface. Several PMOs have been identified that hydroxylate soluble cellodextrins to form similar products as observed for cellulose. Oxygen activation by these PMOs is also underway.
Mass spectrometry characterization of trypanothione and novel peptides of medical importance isolated from Acanthamoeba polyphaga (585.1)
Authors: Ondarza Raul
Published: June 2021

This paper presents unequivocal results about the presence of trypanothione and its precursor glutathione‐spermidine from the opportunistic human pathogen Acanthamoeba polyphaga.

They were isolated by RP‐HPLC as thiol‐bimane derivatives and characterized using matrix‐assisted laser desorption ionization‐time‐of‐flight (MALDI‐TOF/TOF).

Besides trypanothione, we want to report four new peptides in trophozoites, a tetrapeptide, a hexapeptide, a heptapeptide and a nonapeptide. Trypanothione and two of the thiol peptides, the hexapeptide and heptapeptide, are oxidized since the reduced forms increase in amount when the normal extract is treated by DTT or by electrolytic reduction that convert the oxidized forms to reduced ones. On the other hand, they disappear when the amoeba extract is treated with NEM or when the amoeba culture is treated with various inhibitors of NADPH‐dependent disulfide reducing enzymes.

Comparison of the thiol peptides, including trypanothione from A. polyphaga with extracts from human lymphocytes showed that they are not present in the latter. Therefore, some of the peptides here reported could be used as antigens for rapid detection of these parasites.

In regard to the presence of the enzymes that synthesize and reduce trypanothione in A. polyphaga we suggest that they can be used as drug targets.

Grant Funding Source: no grant funding
Expression and characterisation of asparagine‐tRNA synthetase: the key enzyme that mediates angiogenesis in lymphatic filariasis (585.10)
Authors: Dhanasekaran Jeeva Jothi , Solaiyappan Shanmugam , D Dhanraj Muthu , Chatterjee Suvro , Kron Michael , Dhanasekaran Anuradha
Published: June 2021

Asparagine‐tRNA synthetase is an enzyme which belongs to the class‐IIb aminoacyl‐tRNA synthetases(AARSs) an heterogeneous family of enzymes,that play a role in protein biosynthesis. Brugi Malayi parasites secrete this 63kDa high molecular weight enzyme. It has not been documented clearly,how humans infected with filariasis have lymphatic dysfunction. We have cloned and purified the AsnRs enzyme to evaluate the role played by this enzyme in angiogenesis in Filarial patients.The recombinant plasmid of pET‐15B encoding Brugia malayi Asparagine‐tRNA synthetase was transformed into the competent cells of E.coli BL21(DE3),induced to express by IPTG,purified by Immobilized metal ion affinity chromatography and identified by western blotting. Purified Recombinant Brugia malayi AsnRS was used to perform cellular based assays using endothelial cells‐ MTT assay,Chemotactic assay,ring formation assay and tube formation assay in EAhy926 cells.We also carried out angiogenesis assays in chick chorioallantoic membrane (CAM) models. Recombinant AsnRS induces proliferation in MTT assay,migration in chemotaxis assay,formation of endothelial ring structure in ring formation assay and tubular structures in matric gel assay. Additionally,BmAsnRs promotes the formation and widening of blood vessel mediated angiogenesis in CAM model. Our results showed the enzyme BmAsnRs to be a potent pro‐angiogenic mediator in vitro and CAM models. These results throw light on the possibility of AsnRs as a drug target for the control of Filariasis and thus this enzyme can be targeted for the control of angiogenesis‐associated pathology in Filariasis.
Computational fluid dynamic modeling to predict wall shear stress in the cephalic vein of human subjects (588.1)
Authors: Hammes Mary , Boghosian Michael , Kevin Cassel , Hines Jane
Published: June 2021

Background Cephalic arch stenosis (CAS) leads to access failure in hemodialysis patients with brachiocephalic fistula. The objective of this study was to use computational fluid dynamic modeling (CFD) to predict wall shear stress (WSS) in fistula access to determine the effects of increased arterial flow in the cephalic arch. Methods A venogram, Doppler and whole blood viscosity were performed pre‐fistula and at 3 months. Geometric measurements of arch angle and vein diameter were made. CFD of the cephalic arch was created to predict wall shear stress throughout the vessel. Results Eleven subjects receiving hemodialysis were studied. The arch angle decreased with a mean average of 133° at baseline to 116° at three months (p = 0.002). The venous diameter increased from 0.26 cm to 0.44 cm (p = 0.002). The blood flow increased from 3.95 cm/sec to 47.14 cm/sec (p < 0.001). A comparison of the CFD streamline plots between baseline and three months showed angle reduction, coupled with high flows leading to flow separation in most cases at three months. Flow separation was associated with a change in flow direction and low WSS. Conclusion Fistula creation results in increased arterial pressure and flow in the native vein. The vein dilates over three months with an observed decrease in [LOCAL] WSS made worse by the acute angulation of the arch, both contributing to intimal hyperplasia and CAS.

Grant Funding Source: Supported By: NIH‐NDK 1 RO1 DK090769
Imaging enzymes at work: metabolic mapping by enzyme histochemistry to study functional mechanisms of the isocitrate dehydrogenase IDH1 mutation in glioblastoma (59.3)
Authors: Van Noorden Cornelis
Published: June 2021

Activity localization of NADP+‐ dependent isocitrate dehydrogenases (IDH) was studied quantitatively using metabolic mapping and image analysis to study the metabolic consequences of the IDH1 mutation in glioblastoma, the most aggressive form of brain tumors. The mutation is found in 70‐80% of secondary glioblastoma patients. Metabolic rewiring of cells due to the mutation causes gliomagenesis by the production of 2‐hydroxyglutarate. On the other hand, glioblastoma patients with the IDH1 mutation have a significant survival benefit of approx. one year. We found that the NADPH production capacity in glioblastoma is reduced by 38% when IDH1 is mutated (Bleeker et al. 2010). We hypothesized that this reduced production capacity of NADPH, a major metabolic involved in detoxification processes, renders glioblastoma cells less resistant against irradiation and chemotherapy and thus prolong patient survival (Baldewpersad Tewarie et al. 2013). We also demonstrated with metabolic mapping that cellular metabolism is rewired in IDH1‐mutated glioblastoma cells by elevated mitochondrial activity (Navis et al. 2013). Further metabolic mapping of human and rodent tissues showed that IDH activity is responsible for 65% of the NADPH production capacity in human brain and only for 15% in rodent brain whereas the pentose phosphate pathway was responsible for 85% of the NADPH production capacity in rodent tissues (Atai et al. 2011). It explains why the pentose phosphate pathway was always linked with cancer and not IDH because most studies on metabolism and cancer have been performed in rodents. This study clearly shows the importance of metabolic mapping as a histochemical tool to understand the consequences of a mutation in the gene of an enzyme such as IDH1.

Grant Funding Source: none
A novel NUT translocation partner binds to BRD4 and is necessary for the blockade of differentiation in NUT midline carcinoma (59.8)
Authors: French Christopher , Walsh Erica , Kuhnle Simone , Rahman Shaila , Grayson Adlai , Lemieux Madeleine , Howley Peter
Published: June 2021

NUT midline carcinoma (NMC) is an aggressive type of squamous cell carcinoma that typically harbors BRD4/3‐NUT fusion oncogenes which encode chimeric proteins that block differentiation and maintain tumor growth. However, in 30% of cases NUT is fused to yet to be identified non‐BRD gene(s). Using RNA sequencing, we identified a novel gene fused to NUT in a NMC cell line (1221), fusing a novel 5’ coding sequence to the NUT gene. Like Brd4/3, this gene encodes a protein that is also involved in epigenetic regulation, and we find that siRNA knockdown of this protein leads to differentiation of the 1221 cells as well as of three BRD4‐NUT‐positive NMC cell lines. We have established that the novel NUT fusion protein encoded by this translocated gene binds to BRD4. We have mapped the regions of Brd4 and of the novel fusion protein involved in complex formation and are exploring whether association of the novel protein with Brd4 is required for the blockade of differentiation in NMC. Differentiation of TC‐797 cells induced by knockdown of BRD4‐NUT is abrogated by enforced expression of the novel NUT‐fusion gene. Together, these findings identify a novel BRD4‐NUT‐interacting protein whose expression is required for the maintenance of the undifferentiated state in NMC, and when fused to NUT, is oncogenic and can recapitulate the function of BRD4‐NUT to block differentiation.

Grant Funding Source: NCI
Changes of proteins induced by anticoagulants can be more sensitively detected in urine than in plasma (591.2)
Authors: Li Menglin , Zhao Mindi , Gao Youhe
Published: June 2021

Biomarker studies commonly monitor the composition of plasma, which is under strict homeostatic control, while urine is not. Urine is a partial filtrate of blood; and its composition reflects systemic physiology. Here we show that changes in the coagulation status of blood can be more sensitively detected in urine than in plasma. Plasma and urine protein composition was analysed by LC‐MS/MS and Western blot in six SD female rats before and after treatment with heparin or argatroban. LC‐MS/MS and Western blot analyses demonstrated drug‐induced increases in transferrin and hemopexin levels in urine but not in plasma. Our data indicates that urine may serve as a source for more sensitive detection of protein biomarkers than plasma.

Grant Funding Source: Supported by the National Basic Research Program of China
Identifying prostate cancer biomarkers by profiling glycoproteins in human prostate tissue (591.6)
Authors: Spiciarich David , Maund Sophia , Peehl Donna , Bertozzi Carolyn
Published: June 2021

Prostate‐specific antigen (PSA), the current clinical biomarker for prostate cancer, suffers from high false positive and high false negative rates that leave many men without a conclusive diagnosis. Virtually all clinically approved cancer biomarkers are cell‐surface or secreted glycoproteins. We seek to identify more specific clinical biomarkers of prostate cancer by developing an efficient method for specifically labeling and enriching glycoproteins, followed by MS‐based analysis of normal and cancerous human prostate tissue. We present a novel biomarker identification strategy where human prostate tissue derived from radical prostatectomies, at varying states of prostatic disease is chemically tagged. To identify cell‐surface or secreted glycoproteins from prostate tissue, we employ metabolic labeling, a technique in which unnatural azide or alkyne‐functionalized monosaccharides are incorporated, into cell‐surface glycoproteins via the cells’ own metabolic machinery. Tissue slices incubated with these labeled sugars are subsequently reacted with a biotin‐functionalized probe for the capture of sialylated glycoproteins. Analysis of labeled tissue by Western blot confirmed azide or alkyne‐dependent labeling. Proteins were then digested with trypsin and the resultant peptides were analyzed on a linear ion trap mass spectrometer with collision‐induced dissociation (CID). Greater than 60% of the proteins identified by LC‐MS/MS were cell‐surface or secreted proteins, indicating that this strategy achieves enrichment of low abundance proteins. Subsequently, we performed a comparative proteomics experiment between diseased and healthy tissue and identified proteins unique to each tissue type, which are undergoing further validation as potential biomarkers. This work extends the application of existing metabolic labeling techniques, while developing new methodologies for cell surface labeling to discover biomarkers associated with prostate cancer.
Analysis of the oligomerization propensity of prohibitin1 (603.1)
Authors: Padilla‐Meier Gay Pauline , Meier Markus , Stetefeld Jorg , Mishra Suresh
Published: June 2021

Prohibitins (PHB) are highly conserved and ubiquitously expressed proteins in eukaryotic cells. The prohibitin family exist in two isoforms PHB1 (also known as BAP32) and PHB2 ( also known as BAP37 and REA). The PHB1 and PHB2 together form large ring shaped complexes in the inner‐mitochondrial membrane, although PHB has also been reported to be localized in the nucleus and the cell membrane. PHB proteins have been implicated in mitochondrial biogenesis as well as scaffold for mitochondrial proteins. Details on the structure and function of PHB1 are still unclear. To better understand the functional mechanisms of PHB1 , we conducted biophysical analysis on recombinant human PHB1 and a truncated form of PHB1 (aa 52‐197). Our results show that PHB1 variants can form oligomers, and that varying salt concentration can modulate the polymerization of PHB1. Furthermore, addition of iron to PHB1 in solution affects the stability of the PHB1 oligomers. These data suggest that PHB1 could have diverse oligomeric forms depending on its local environment

Grant Funding Source: Supported by: Manitoba Health Research Council,
Role of RKTG, a novel Golgi localized receptor, in Gβγ regulated transport of secretory proteins from the Golgi to the plasma membrane (604.4)
Authors: Hewavitharana Thamara , Wedegaertner Philip
Published: June 2021

Recent studies have revealed a role for G protein betagamma signaling at the Golgi via protein kinase D (PKD) regulating fission of plasma membrane (PM) destined secretory vesicles from the trans Golgi network (TGN). The mechanism by which the Gbetagamma subunits are recruited to and activated at the Golgi in this process remains elusive. Recent studies reveal that a newly characterized Golgi localized, membrane protein belonging to the Progestin and AdipoQ Receptor (PAQR) family, RKTG (Raf Kinase Trapping to the Golgi), interacts with the Gbeta subunit and sequesters Gbetagamma to the Golgi thereby regulating the functions of Gbetagamma. Here we show that over expression of RKTG causes vesiculation of the Golgi, while a Gbeta binding mutant of RKTG does not cause vesiculation. Also, the C‐terminal fragment of GRK2 (GRK2ct), which interacts with, and inhibits Gbetagamma signaling, and Gallein, a small molecule inhibitor of Gbetagamma, were both able to inhibit Golgi vesiculation in the presence of RKTG. Furthermore, a dominant negative form of PKD (PKD‐DN) and a pharmacological inhibitor of PKD, Go6976, also inhibited RKTG mediated vesiculation of the Golgi. Collectively, these results reveal a novel role for the newly characterized, Golgi localized RKTG receptor, in regulating Gbetagamma at the Golgi, thus controlling Golgi to PM protein transport via the Gbetagamma‐PKD signaling pathway.

Grant Funding Source: NIH GM056444
Identification of novel genes for arachidonic acid metabolism in Saccharomyces cerevisiae (605.11)
Authors: Batugedara Hashini , Chikhalya Aniska , Haas‐Stapleton Eric
Published: June 2021

Saccharomyces cerevisiae produces prostaglandin E2 (PGE2) in the presence of arachidonic acid (AA). S. cerevisiae and its metabolites may be consumed in products manufactured using the yeast (e.g. beer) and yeast PGE2 can potentially modify human immune cell functions to exacerbate pre‐existing inflammation. The biochemical pathway utilized by S. cerevisiae to produce PGE2 is unknown. The identification of genes involved in AA metabolism may direct approaches to reduce off‐target effects of yeast PGE2 on immune cells. As S. cerevisiae does not have genes homologous to those involved in mammalian AA metabolism, identification of such genes will reveal a novel pathway for lipid biosynthesis. RNAseq transcriptome sequencing was conducted and we observed 2044 genes upregulated in yeast cultured with AA relative to the control yeast cultured without AA. Notably, genes encoding mitochondrial proteins that direct fatty acid beta‐oxidation (CRC1, YAT1 and CEM1) were upregulated 1.1‐3.0‐fold and genes encoding peroxisomal proteins that mediate beta‐oxidation of fatty acids (POT1, POX1, FAA1 and FAA2) were upregulated 1‐2.3‐fold. Upregulation of fatty acid metabolizing genes points to enzymes that may metabolize AA to produce PGE2 in S. cerevisiae. Currently, we are testing knock‐out strains of S. cerevisiae that lack the genes identified in the RNA‐seq studies for reduced PGE2 production.

Grant Funding Source: Supported by a grant from the National Institutes of Health to E.J.H‐S (SC3GM092298).
S. cerevisiae coq5 mutants require stabilization of the CoQ‐synthome for rescue by COQ5 homologues (605.13)
Authors: Nguyen Theresa , Clarke Catherine , Salviati Leonardo , Casarin Alberto
Published: June 2021

Coenzyme Q (Ubiquinone or Q) is an essential lipid electron carrier in the respiratory electron chain. In Saccharomyces cerevisiae, eleven genes (COQ1‐9, ARH1, YAH1) are required for coenzyme Q biosynthesis, several of which encode proteins involved in a multi‐subunit complex termed the CoQ‐synthome. One of these genes, COQ5, encodes a C‐methyltransferase responsible for making the late stage intermediate demethoxy Q6 (DMQ6). COQ5 has three conserved methyltransferase motifs, and yeast point mutants within or adjacent to these motifs (coq5‐2, coq5‐5) lack Coq5 activity but retain steady state levels of the Coq5 polypeptide. We show that the E. coli ubiE and human homologues for COQ5 are able to rescue coq5‐2 and coq5‐5 point mutants, but are unable able to rescue the yeast coq5Δ null mutant. However when putative kinase Coq8 is over‐expressed to stabilize the CoQ‐synthome, non‐yeast Coq5 homologues are able to rescue the yeast COQ5 knockout. We demonstrate that co‐expression of COQ8 and COQ5 homologues in a coq5Δ null mutant restores Q6 levels and supports growth on non‐fermentable carbon sources. By observing the accumulation of Q6‐intermediates in coq5 mutants, we have been able to elucidate steps within a novel Q6 biosynthetic pathway.
Quantitative analysis of the green microalga Coccomyxa subellipsoidea C169 reveals homeostasis of complex lipid synthesis retained during nitrogen stress (605.14)
Authors: Allen James , DiRusso Concetta , Black Paul
Published: June 2021

The inevitable decline of fossil fuel availability and the destructive escalation of atmospheric carbon dioxide concentration are pressing problems that scientific innovation can potentially address. Algae accumulate large amounts of oil when exposed to abiotic stress, enough to possibility use their cultivation to produce carbon‐neutral replacements of conventional transportation fuels. Unraveling the intricacies of triglyceride (TG) synthesis during abiotic stress is a means to generate genetic intervention strategies for improving lipid productivity. Our research is focused on understanding TG accumulation in the context of lipid biosynthesis for Coccomyxa subellipsoidea C169, a sequenced relative of Chlorella and a sensible proxy for biofuel production species. A thorough analysis of lipid synthesis was conducted including GC‐MS fatty acid profiles of isolated lipid pools and LC‐MS/MS quantification of membrane lipid (ML) and TG pools through a time course of N starvation. Stable isotopic labeling studies were done with 13CO2 and D2O to analyze the roles of photosynthesis and de novo synthesis from previously reduced carbon in TG synthesis. The data quantify an initial redistribution of membrane lipids into the TG pool followed by synthesis and incorporation of fatty acids into all complex lipid pools. Reproductive growth is inhibited by N stress, however lipid synthesis continues normally.

Grant Funding Source: Supported by NSF‐EPSCoR, Nebraska Center for Algal Biology and Biotechnology
Characterization of proteins associated with a coenzyme Q biosynthetic complex in yeast mitochondria (605.19)
Authors: Allan Christopher , Johnson Jarrett , Shirasaki Dyna , Loo Joseph , Clarke Catherine
Published: June 2021

Coenzyme Q (Q) is a fully substituted benzoquinone lipid crucial to the respiratory electron transport chain where it transfers electrons from NADH and succinate to cytochrome c. In the yeast Saccharomyces cerevisiae Q synthesis requires eleven known mitochondrial proteins, Coq1p‐Coq9p, Yah1p, and Arh1p. Several of these proteins have been shown to exist in a high molecular weight complex localized to the matrix side of the inner mitochondrial membrane. The function of some of these proteins in Q biosynthesis is not well characterized and it is not known whether additional proteins are required. Additionally, the organization of the biosynthetic complex as well as the role of protein phosphorylation in Q biosynthesis is not fully understood. To better characterize the Q biosynthetic complex as well as to identify potentially novel associated proteins we generated chromosomally‐integrated tandem affinity tags for Coq3p and Coq9p which were analyzed through co‐precipitation of the dual‐tagged Coq proteins followed by proteomic analysis. This tag, termed the CNAP tag, allows for purification of the tagged proteins under native conditions, preserving any noncovalent protein‐protein interactions that may exist in a macromolecular complex. The presence of the tag on either Coq3p or Coq9p was shown not to disrupt steady‐state levels of the Coq proteins and not to impair Q biosynthesis. Using this tag we demonstrated that Coq3p and Coq9p co‐precipitate each other as well as Coq4p, Coq5p, Coq6p, and Coq7p. Proteomic analysis also identified potentially phosphorylated peptides in Coq4p and Coq6p, which have not previously been shown to be phosphorylated. Three additional uncharacterized candidate proteins were found in this analysis, two of which were shown to have impaired de novo Q biosynthesis.
Contrasting the incorporation of glycerol into lipids caused by the presence of two isoforms of diacylglycerol kinase (605.20)
Authors: Epand Richard , Jalan Divyanshi , Topham Matthew , Hatch Grant
Published: June 2021

The mammalian isoforms of diacylglycerol kinase (DGK) include the epsilon isoform (DGKε) and the delta isoform (DGKδ). DGKε has specificity for substrates with an arachidonoyl group, while DGKδ shows no acyl chain specificity for the substrate, but it promotes fatty acid synthesis resulting in an increased fraction of lipids with shorter and less unsaturated acyl chains. We have compared the effects of knocking out each of these isoforms individually using SV40 transformed mouse embryo fibroblasts (MEFs). We compared wild‐type (WT) MEFs with those from knock‐out (KO) mice. We find that DGKδ‐KO MEFs have lower incorporation of 3H‐glycerol into lipids compared with their WT counterparts. This is consistent with our recent demonstration (Biochemistry (2013) 52, 7766) that the depletion of this DGK isoform results in less fatty acid synthesis. This change is not caused by a decreased expression of glycerol kinase in the KO cells. In contrast, the DGKε‐KO MEFs show greater incorporation of 3H‐glycerol into lipids compared with their WT counterparts, with no change in the relative amounts of various lipids between the DGKε‐KO and WT MEFs. This result is explained by our observation that glycerol kinase is more highly expressed in the DGKε‐KO cells than in their WT counterparts.
Effects of temperature and pressure level on lipid oxidation and physicochemical properties during chafing dish fried process (606.1)
Authors: Wang Qiang , Wang Rui , Wang Cun
Published: June 2021

Chafing Dish is very important and common condiment for Chinese customers. The effects of different temperatures and pressure levels on Acid value, TBARS and physicochemical properties of the chafing dish products were investigated during fried process. The chemical parameters, such as Acid Value, TBARS, NaNO3 and NaNO2, slightly increased as temperature, but Aw decreased from 0.45 to 0.38 when temperature was increased from 80 °C to 160 °C. In addition, L* value (66.05) at 100 °C were highest in all of groups, while the lowest of a* value (18.66) was observed at 160 °C. For the chemical parameters of the chafing dish products, they slightly increased as pressure except for Aw. For 400 MPa, chemical parameters were significantly different (p < 0.05) from other treated samples. Of importance, the lower pressure restrained the increase of NaNO3 and NaNO2 which were harmful for health. At 100 MPa, the treatment increased significantly the L* and a* value. However, the higher pressure (>100Mpa) decreased the L* and a* value of samples. Moreover, compared with temperature and pressure individually, combination of temperature and pressure effectively (p < 0.05) prolong the oxidation induction period of samples. Therefore, the study suggests that the lipid oxidation and physicochemical properties of chafing dish product can be regulated effectively by combination of temperature and pressure.

Grant Funding Source: This research was supported by Application and Development Plan of CQ CSTC (No. CSTC2014yykfA0094)
Structure‐function studies of the sterol transfer protein, STARD4 (606.2)
Authors: Iaea David , Maxfield Frederick
Published: June 2021

Significant differences in lipid distribution are maintained between intracellular organelles. Cholesterol comprises ~30% of the lipid molecules in the plasma membrane and is also enriched in the endocytic recycling compartment (ERC). However, in the endoplasmic reticulum (ER) cholesterol accounts for 5% of the lipid molecules. Sterols are transported as components of lipid bilayers in vesicular transport and are transferred by non‐vesicular processes involving carrier proteins. The steroidogenic acute regulator‐related lipid‐transfer (START) domain containing proteins are involved in several pathways of non‐vesicular trafficking of sterols. Among the soluble START proteins, StARD4 has been shown to increase cholesteryl ester accumulation in lipid droplets, in an acyl‐CoA:cholesterol acyl‐transferase (ACAT) dependent manner, and is controlled at the transcriptional level by sterols. Our laboratory has previously shown that StARD4 is efficient in transporting sterols between membranes in vivo and in vitro. However, the precise molecular mechanisms that mediate StARD4 membrane targeting, interaction and sterol extraction are unknown. Here we show that STARD4 has a marked preference for negatively charged membranes, for membranes rich in unsaturated lipids and that specific phosphatidylinositol phosphates (PIPs) modulate the sterol transfer activity of STARD4. Additionally, mutations in either the basic patch or Ω1 loop greatly alter the sterol transfer activity of STARD4, likely by perturbing membrane interaction or sterol absorption/release. Overall, these findings provide insight into the biophysical and cellular mechanisms that facilitate StARD4 activity and distribution required to maintain cholesterol homeostasis.
Amelioration of the foam cell formation by simvastatin due to increased expression of Niemann‐Pick disease type C1 protein and enhanced lysosomal lipids transport (606.4)
Authors: Zhang Qiufang , Li Ningjun , Dewey William , Li Pin‐Lan , Zhang Fan
Published: June 2021

It is well accepted that statins, hypercholesterol‐lowing agents, have direct therapeutic effects on the atherosclerotic lesions. However, the underlying mechanism remains unknown. Given the pivotal role of macrophages in atherosclerosis, the present study was designed to investigate the effects of simvastatin, a prototype of the most statins used clinically, on the foam cell formation by examining lysosomal lipids in macrophages. Using fluorescent microplate reader and confocal microscopy, we found that simvastatin dose‐dependently (in nM: 0, 10, 50, 100, 150 and 200) decreased lysosomal lipid deposition in macrophages treated with oxLDL ( 40 µg/ml ) as stained by lysosomal lipid specific dye of Bodipy 493/503. Oil red O staining and electron microscopy showed that the foam cell formation was substantially attenuated. In simvastatin‐ treated macrophages, the cholesterol level in the purified lysosome fractions was markedly decreased. RT‐PCR analysis revealed that the expression of NPC1, a lysosomal cholesterol transporter, was significantly enhanced. Silencing of NPC1 markedly decreased simvastatin‐induced lysosomal lipids transport. These results indicate that simvastatin‐induced cholesterol transport out of lysosome in macrophages leads to foam cell formation attenuation via enhancing NPC1 expression, which may be a therapeutic mechanism of statins to ameliorate atherosclerotic lesions.

Grant Funding Source: NIH R01HL115068
Role of membrane contact sites in PM ‐ ER sterol transport (606.7)
Authors: Sere Yves , Johansen Jesper , Beh Christopher , Menon Anant
Published: June 2021

Intracellular trafficking of sterols occurs largely by non‐vesicular processes that are poorly understood. Cytoplasmic sterol transport proteins (STPs) are presumed to ferry sterols between membranes and this may be enhanced in regions where the participating membranes are closely apposed (membrane contact sites or MCSs). The major sterol trafficking pathway in the yeast Saccharomyces cerevisiae involves direct transfer between the endoplasmic reticulum (ER) and plasma membrane (PM). Yeast STPs have not been identified, although the cytoplasmic protein Kes1/Osh4 has been implicated in the phosphoinositide‐driven cycling of ergosterol between the ER and TGN. We considered whether MCSs between the ER and PM might play a role in sterol transport between the two organelles. We tested this hypothesis by analyzing sterol trafﬁc in Saccharomyces cerevisiae cells that were largely devoid of ER ‐ PM contact sites (∆tether cells). We used ﬂuorescence microscopy to track the retrograde movement of exogenously supplied dehydroergosterol (DHE) from the PM to the ER and lipid droplets, and high performance liquid chromatography to quantify, in parallel, the transport‐coupled formation of DHE esters. We found that sterol transport from PM to ER is unaffected in ∆tether cells. We are currently generating strains based on ∆tether cells in which all residual ER‐PM MCSs are eliminated, and also testing whether vesicular pathways might play a redundant role in the absence of MCSs.
Role of heparan sulfate in intestinal normal and cancer stem cells (607.10)
Authors: Nonaka Motohiro , Nakayama Jun , Fukuda Minoru
Published: June 2021

The epithelial linage in small and large intestines develops from stem cells residing bottom of the crypt domain. Lgr5 (leucine‐rich‐repeat‐containing G‐protein‐coupled receptor 5) is currently a leading candidate for an intestinal normal stem cell (NSC) and cancer stem cell (CSC) marker. Hepran sulfate, which is ubiquitously expressed in cells and extracellular matrices, interacts with various factors such as growth factors, cytokines and signaling molecules. While heparan sulfate is involved in many aspects of inflammation, its significance in carcinogenesis is poorly understood.

To determine the role of heparan sulfate in NSCs and CSCs, we utilized Lar5‐driven inducible gene knockout, which was generated by Hans Clevers (Nature; 459: 262‐265, 2009). In this system, Cre enzyme activation which is induced by tamoxifen intraperitoneal injection allows deletion of floxed EXT‐1 gene in the Lgr5+ stem cells. The Lgr5‐EXT‐1 KO mice exhibit shorter villus at jejunum, but not at proximal or distal colon. When the intestinal tissues were stained with proliferation maker Ki‐67, the number of Ki‐67+ cells in colon crypts as well as in small intestinal cryps was dramatically decreased in Lgr5‐EXT‐1 KO mice. Surprisingly, we observed that undifferentiated Lgr5+ cells were accumulated in villi in Lgr5‐EXT‐1 KO mice, suggesting that function of NSCs was disordered. Moreover, Lgr5‐driven elimination of APC tumor suppressor gene inactivation resulted in the formation of adenocarcinoma cell; however formation of adenocarcinoma was abrogated when EXT1 gene was also inactivated. The results strongly suggest that CSCs also require heparan sulfate to produce cancer progenies. Further work is critical to determine the role of heparan sulfate in cancer formation, possibly by utilizing organoid culture.

Grant Funding Source: Supported by NCI U01CA168925
O‐GlcNAc modulates β‐catenin localization during M‐phase in HeLa cells (607.18)
Authors: Lucena Miguel , Hu Ping , Todeschini Adriane , Han Guanghui Han , Dias Wagner , Hart Gerald
Published: June 2021

β ‐catenin is already known to be O‐GlcNAcylated. However, the function of this PTM is not well established. Here, we show that increased O‐GlcNAc levels induced the interaction with OGT, providing the accumulation of glycosylated form of β ‐catenin in cytoplasm. In addition, the WNT treatment rapidly decreased the O‐GlcNAc levels indicating an interplay between this PTM and the canonical pathway. Further, we showed that β‐catenin co‐localized with OGT and OGA during the mitosis. However, a strong co‐localization with OGA was observed during the metaphase specially with centrossome. Surprisingly, the β‐catenin strongly co‐localized with OGT at anaphase within the central spindle. In addition, the co‐localization of β‐catenin with OGT seems to be stronger at the midbody during cytokinesis when compared with OGA. Taken together, our date indicates that O‐GlcNAcylation of β‐catenin modulates its sub‐cellular localization during cell cycle.

Grant Funding Source: CNPq, CAPES, FAPERJ
5‐AZA‐2'‐deoxycytidine induced demethylation influences N‐glycosylation of secreted glycoproteins in ovarian cancer (608.1)
Authors: Saldova Radka , Dempsey Eugene , Perez‐Garay Marta , Marino Karina , Watson Jenny , Blanco‐Fernández Alfonso , Struwe Weston , Harvey David , Madden Stephen , Peracaula Rosa , McCann Amanda , Rudd Pauline
Published: June 2021

Glycosylation is the most common posttranslational modification of proteins and is highly reflective of changes in the environment of a cell. Epigenetic modifications to the genome are stably transmitted to daughter cells without the requirement for genetic sequence alterations. Aberrant regulation of both epigenetic programming and glycosylation are integral aspects of carcinogenesis. The objective of this study was to determine the interplay between these two complex cellular processes. We demonstrate that global DNA methylation changes in ovarian cancer epithelial cells OVCAR3 resulted in significant alterations in the glycosylation of secreted glycoproteins. These changes included a reduction in core fucosylation, increased branching and increased sialylation. We further show that the change in core fucose levels was mirrored by altered expression of key enzymes in fucose biosynthesis. Alterations in the expression of key glycosyltransferase enzymes reflect the changes seen in the branching and sialylation of secreted glycans. Our results highlight that modifications to the epigenetic machinery have a profound effect on the glycan structures generated by cells, which may be a key step in understanding metastasis and drug resistance during cancer progression.