bluuebunny/crossref_metadata_2025 huggingface dataset:
LPS/TLR4‐NF‐[kappa]B axis signaling amplifies STIM1 expression to augment PAR‐1‐induced Calcium entry and permeability response in lung microvessels
Authors: DEBROY AUDITI , Sundivakkam Premanand , Singh Vandana , Malik Asrar B , Tiruppathi Chinnaswamy
Published: June 2021

Rationale

Human sepsis is characterized by lung microvascular leakage. Store‐operated Calcium Entry (SOCE) in endothelial cells (ECs) mediates increase in lung vascular permeability. Here we investigated the role of the transcription factor NF‐κB, activated downstream of TLR4 in mediating the expression of the crucial activator of SOCE, Stromal Interacting Molecule 1 (STIM1), and its consequences on PAR‐1‐mediated Ca2+ entry in ECs and lung vascular leak.

Results

LPS induced STIM1 expression in lung ECs. Increased STIM1 expression was associated with augmented PAR‐1‐mediated SOCE and permeability increase. These results were recapitulated in vivo mouse model LPS‐induced sepsis and the response was abrogated in TLR4 knockout mice. To characterize the transcriptional mechanism of LPS induction of STIM1, we analyzed the 5′‐regulatory region of STIM1 gene and found the consensus binding sites for LPS‐sensitive transcription factors NF‐κB, SP1, and AP1. By electrophoretic mobility shift assay, we observed the binding of NF‐κB and SP1 to the hSTIM1 promoter. We cloned the hSTIM1 promoter and showed LPS induction of promoter activity in ECs. Silencing of p65/RelA markedly reduced LPS‐induced STIM1 expression and subsequent PAR‐1 responses in ECs.

Conclusion

These results collectively suggest the novel and critical role of STIM1 in mediating lung vascular hyper‐permeability during gram‐negative sepsis.
Store‐operated calcium entry regulate mesenchymal stem cell proliferation
Authors: Maliske Joshua Keith , Pani Biswaranjan , Bollimuntha Sunitha , Ohm Joyce E. , Singh Brij B.
Published: June 2021

Mesenchymal stem cells (MSC) are a unique class of stromal cells which like other stem cells have the special ability to self‐renew and differentiate. MSCs have multi‐lineage differentiation potential, and can be coaxed to differentiate into adipocytes, chondrocytes, osteocytes or neurons in vitro. It is this remarkable plasticity which makes the MSCs important for cell‐based therapy and regenerative applications. Thus, understanding the molecular mechanisms that control growth and differentiation of MSCs is of clinical importance. Calcium (Ca2+) is an essential signaling messenger that modulates a variety of cellular functions ranging from cell growth to differentiation to cell death. One of the prime physiological modalities that regulate cellular Ca2+homeostasis is store‐operated calcium entry (SOCE). SOCE represents a universal phenomenon of Ca2+influx across the plasma membrane which is triggered, following activation of PM‐GPCRs, by IP3‐mediated depletion of endoplasmic reticulum Ca2+stores. Studies from this laboratory and others have suggested the non‐selective cation channel TRPC1 as an integral component of store‐operated Ca2+channel (SOC). Further, TRPC1 forms a complex with the recently identified calcium‐selective channel Orai1 and following store‐depletion is activated by the endoplasmic reticulum‐associated Ca2+sensor STIM1. The objective of this study was to identify the ‘TRPC1‐STIM1‐Orai1’ component of Ca2+signaling and to test the significance of SOCE in MSC proliferation. Using RT‐PCR we identified the expression of TRPC1, STIM1 and Orai1, and co‐immunoprecipitation analysis identified their functional interaction. Further, pharmacological inhibition of SOCE in proliferating MSCs resulted in growth attenuation. In conclusion, our study suggests the ‘TRPC1‐STIM1‐Orai1’ complex as the core SOCE machinery that is critical for MSC proliferation
Rapid and Specific Measurements of Superoxide Using Fluorescence Spectroscopy
Authors: Nazarewicz Rafal , Bikineyeva Alfiya , Harrison David G , Dikalov Sergey
Published: June 2021

Superoxide plays a critical role in number of pathological conditions including cardiovascular diseases. Development of specific and reliable methods for superoxide detection is critical for investigation of these conditions. Cellular reactions of dihydroethydium (DHE) produce both the superoxide specific product 2‐OH‐ethidium (2‐OH‐E) and non‐specific oxidation product ethidium which have overlapping fluorescence spectra. Specific superoxide detection therefore is difficult. HPLC can separate ethidium and 2‐OH‐E but this method is limited by inability to detect site‐specific 2‐OH‐E and duration of the HPLC procedure. In this study, we show optimized fluorescence spectroscopy protocol allowing reliable superoxidedetection in cell free systems, subcellular fractions, intact cells and tissue. Analysis of fluorescence emission spectra showed four times higher contribution of 2‐OH‐E fluorescence comparing to ethidium when excitation was changed from 480 nm to 405 nm. Selection of specific emission enables selective detection of 2‐OH‐E with negligible contribution of ethidium. The best selectivity of fluorescence signal for detection of 2‐OH‐E at excitation at 405 and 480 nm was achieved at emission 570 nm (1:21 ethidium:2‐OH‐E ratio) and 530 nm (1:52 Ethidium:2‐OH‐E ratio), respectively. We applied these optimized conditions for on‐line superoxidedetection generated by xanthine oxidase. Fluorescent signal was inhibited by SOD supplementation confirming specific detection of 2‐OH‐E with xanthine oxidase concentration as low as 10 microunits per ml. Specificity of plate reader‐based superoxide measurements was confirmed by HPLC analysis of 2‐OH‐E and ethidium. Our study shows that limitations of HPLC in superoxide detection in biological samples can be substantially overcome by using optimized fluorescence spectroscopy.

This work has been funded by National Institute of health PO‐1 HL058000.
Rapid method for monitoring muscle protein metabolites by LC‐MS
Authors: Auell Olivia , Hayes Benjamin , Chow Caroline , King Daniel , Hammond D.G.
Published: June 2021

America's growing elderly population has led to an increase in research on the aging process, including the relationship of muscle retention to health. Monitoring muscle development at the molecular level is an excellent complement to traditional whole‐muscle assessment methods. 3‐Methylhistidine (3‐MH) is an amino acid derivative and breakdown product of muscle protein. Urinary 3‐MH levels vary with diet and exercise, meriting its use as a molecular marker of muscle turnover. A method to perform rapid quantitation of 3‐MH in urine was developed using single‐ion monitoring (SIM) LC‐MS. After being spiked with an added internal standard and desalted by solid‐phase extraction, samples were analyzed by SIM LC‐MS, quantified versus the added internal standard and normalized versus a ubiquitous internal standard, creatinine. The use of LC‐MS eliminates the need for derivatization as required in established HPLC fluorescence and GC‐MS techniques. Constructed calibration curves plotting the ratio of 3‐MH to internal standard show a strong linear relationship with 3‐MH concentrations, with R‐squared values as high as 0.9983. The method presented is not only effective for monitoring 3‐MH, but holds potential for simultaneous monitoring of additional urine metabolites with run times of approximately 13 minutes.
Identification of the Differentiation Status of Individual Hematopoietic Cells from Mouse Bone Marrow using Secondary Ion Mass Spectrometry
Authors: Kraft Mary L , Frisz Jessica F. , Choi Ji Sun , Wilson Robert L. , Harley Brendan A.
Published: June 2021

The ability to self‐renew and differentiate into multiple types of blood and immune cells renders hematopoietic stem cells (HSCs) of significant value for tissue engineering applications. To enable engineering stem cell fate within laboratory settings, the combination of chemotactic and micromechanical properties that direct HSC maturation must be elucidated. This task requires determining the differentiation stage of individual cells within ex vivo culture systems. New experimental approaches are required to identify the differentiation stage of individual cells and to correlate this information on maturation stage with cell location within their environment. Here we demonstrate that multivariate analysis of time‐of‐flight secondary ion mass spectrometry (TOF‐SIMS) data can be used to successfully classify individual hematopoietic cells from mouse bone marrow according to their distinct lymphopoiesis phenotype, specifically HSCs, common lymphoid progenitors, or mature B cells.

This research was supported by a Career Award at the Scientific Interface from the Burroughs Wellcome Fund.
Protease digestion of P6: Demonstrating a novel dual orientation of P6 in Nontypable Haemophilus influenzae
Authors: Schmidt Rachel , Milillo Jennifer , Sharma Sharad , Pichichero Michael , Michel Lea Vacca
Published: June 2021

Nontypable Haemophilus influenzae (NTHi) causes otitis media (ear infections) and other respiratory illnesses. Currently, NTHi's outer membrane protein P6 is a leading vaccine candidate for the pathogenic bacteria. We have recently demonstrated that P6 is surface exposed, but not a transmembrane protein. In this study, we performed a protease digestion experiment to show that P6 is also present on the inside of NTHi cells. We exposed whole and lysed NTHi cells to a serine protease called Proteinase K and collected time resolved samples to test for the digestion of P6. P6 present on the inside of whole cells were protected from Proteinase K digestion, while P6 in lysed cells were readily digested by Proteinase K. This experiment, in light of other studies from our lab, has demonstrated that P6 exists in two orientations in the outer membrane of NTHi. Additionally, we have made progress toward quantifying the amount of P6 present on the inside versus the outside of the cell. This study was funded by the Rochester Institute of Technology and by NIH NIDCD RO1 08671 (to MEP).
Phospholipase A2 (PLA2) activity of phosphoPeroxiredoxin6 (pPrdx6) is regulated by the interaction with p67phox
Authors: Krishnaiah Saikumari Y. , Dodia Chandra , Feinstein Sheldon I. , Chatterjee Shampa , Fisher Aron B.
Published: June 2021

Phospholipase A2 activity of pPrdx6 is required for activation of NADPH oxidase (NOX2) (J. Biol. Chem. 286:11696, 2011). Prdx6 has been reported to interact with p67phox PMN (J. Biol. Chem. 277:45181, 2002). We further investigated the interaction of these two proteins and its physiological role. Binding of p67phoxto Prdx6 was demonstrated by several techniques. His‐tagged p67phoxbound to Ni2+chelated beads resulted in pull down Prdx6 or pPrdx6 while His‐tagged Prdx6 pull down p67phox. Using recombinant protein or lysates of endothelial cells, immunoprecipitation of Prdx6 or pPrdx6 co‐precipitated of p67phoxand immunoprecepitation of p67phoxco‐precipitated Prdx6 or pPrdx6. The interactions with p67phox appeared to be stronger with phosphorylated protein. By Isothermal Titration Calorimetry, p67phoxbound by one to one stoichiometry to pPrdx6, but did not bind to Prdx6. PLA2 activity, of pPrdx6, measured at pH 7 in Ca2+‐free buffer, was 1750 nmol/min/mg protein and decreased to 14–28 nmol/min/mg in the presence of p67phox. The dissociation constant of p67phox/phosphoPrdx6 calculated by PLA2 activity, was 60nM. With knock down of p67phox by siRNA in pulmonary microvascular endothelial cells, PLA2 activity following AngII treatment increased from 12.6 (control) to 21.7. These data indicate that p67phox binds to pPrdx6 and inhibits its PLA2 activity and thus could function to terminate the PLA2 ‐mediated NOX2 activation signal.
Cloning of a putative L‐3‐hydroxyacyl CoA dehydrogenase from micrococcus luteus
Authors: Takkalapalli Vamshi , Huang Jenq-Kuen , Wen Lisa
Published: June 2021

A novel secondary alcohol dehydrogenase (2‐ADH) was previously purified from Micrococcus luteus WIUJH20 and it N‐terminal amino acid sequence determined. This 2‐ADH gene has been assigned to NAD+ dependant L‐3‐hydroxyacyl CoA dehydrogenase (an enzyme in fatty acid β‐oxidation) due to its high sequence homology (96%). The 2‐ADH can oxidize many hydroxyl fatty acids to keto fatty acids with hydroxyl groups in either D‐or L‐ form on a number of different carbon positions. Therefore this 2‐ADH has low substrate specificity unlike other L‐3‐hydroxyacyl CoA dehydrogenases that requires hydroxyl group in L‐form and on carbon 3. These two genes are excellent candidates for studying the structure‐function relationship that distinguishes their substrate specificities. The 2‐ADH gene has been cloned previously and the putative residues responsible for substrate specificity studied by site‐directed mutagenesis. In the present study, we report the cloning of a putative L‐3‐hydroxyacyl CoA dehydrogenase from micrococcus luteus WIUJH20. The gene has been amplified by PCR using the micrococcus luteus genomic DNA as template. The candidate gene was cloned into pET28a expression vector and transformed to JM109 host cell. The target protein will be expressed in BL21(DE3)pLysS host cells and the substrate specificity will be studied and compared to that of the 2‐ADH. Supported by Western Illinois University.
Lithocholic acid delays yeast aging by altering mitochondrial dynamics
Authors: Titorenko Vladimir , Beach Adam , Burstein Michelle
Published: June 2021

We found that an exogenously added bile acid called lithocholic acid (LCA) delays yeast aging and accumulates in the inner mitochondrial membrane (IMM). To define the mechanism by which LCA extends yeast longevity, we 1) examined how LCA influences mitochondrial proteome and lipidome; 2) investigated the effect of LCA on the composition and stoichiometry of respiratory complexes and supercomplexes in the IMM; 3) assessed how LCA affects mitochondrial oxygen consumption, membrane potential and reactive oxygen species (ROS); 4) examined how LCA influences mitochondrial morphology and how it affects the chronology of events characteristic of age‐related, mitochondria‐controlled apoptosis; and 5) investigated the effect of LCA on the lifespans of long‐ and short‐lived mutants lacking individual components of the mitochondrial fission and fusion machines. Our findings imply that LCA delays yeast aging by increasing the level of phosphatidylserine (PS) and decreasing the levels of phosphatidylethanolamine (PE) and cardiolipin in the IMM. By altering the abundance of these lipid species, LCA greatly expands mitochondrial membrane cristae. In addition, LCA enhances the positive effect of PS and weakens the negative effect of PE on membrane protein machines whose activity they modulate – thereby 1) stimulating protein machines driving mitochondrial respiration, the maintenance of mitochondrial membrane potential and ROS homeostasis, mitochondrial fusion, and protein synthesis in mitochondria; and 2) inhibiting protein machines promoting mitochondrial fission and mitochondria‐controlled apoptosis. Supported by NSERC of Canada.
Cardiac‐specific deletion of prohibitin‐2 in adult mice destabilizes mitochondrial proteins, nucleoids, and cristae morphology, resulting in lethal cardiomyopathy
Authors: Gawlowski Thomas , Yan Jenny , Merkwirth Carsten , Dillmann Wolfgang , Langer Thomas , Hoshijima Masahiko
Published: June 2021

Evolutionally conserved prohibitins (PHBs) primarily localize in the inner mitochondrial membrane (IMM) and play a significant role in the maintenance of normal mitochondrial biogenesis, metabolism, and micro‐domain structures. In a heart failure mouse model, we found PHB was significantly decreased in the heart. Using a tamoxifen (tmx)‐inducible chimeric Cre‐lox system, the current study selectively removed PHB2 in adult cardiac myocytes in vivo. Five weeks after tmx treatment, PHB2 deletion accompanied nearly complete loss of PHB1, which is hetero‐oligomerized with PHB2, and reduced expression of oxidative phosphorylation complex I‐IV proteins. Remarkably, ~70% of the long isoforms of Opa1 in the IMM disappeared, while outer membrane GTPases Mfn1 and 2 were preserved. Serial echocardiography showed that cardiac contractility started to decline and dilated cardiomyopathy rapidly progressed after 10 weeks: all tmx‐treated mice ultimately died due to heart failure. PicoGreen staining identified disorganized mitochondrial nucleoids in PHB2‐deleted isolated cardiomyocytes. Tomographic electron microscopy found enlarged and aberrantly organized mitochondrial cristae, while outer membrane structure was not overtly affected. These results indicate that PHB is essential to the integrity of IMM micro‐domain structures and associated molecular functions in postmitotic adult mammalian cells.
Effects of Manganese and Copper on Mitochondrial Membrane Potential in the Gill of Crassostrea virginica
Authors: Boisette Beatrix , Dailey Fiona , Catapane Edward J. , Carroll Margaret A.
Published: June 2021

Accumulations of manganese (Mn) or copper (Cu) is characteristic of the neurodegenerative disorders Manganism and Wilsonˈs Disease, respectively. Mitochondria are a source and target of oxidative stress. Previously we found gill mitochondria from the oyster Crassostrea virginica, treated with Mn or Cu had impaired oxygen utilization. Oxidative damage causes a loss of mitochondrial membrane potential (MMP) with associated mitochondrial dysfunction. Here we used two fluorescent dyes, TMRM and JC‐1, to determine effects of Mn and Cu treatments on MMP. Mitochondria from gill of C. virginica were isolated and treated. For JC‐1 we compared fluorescence intensities at 525/590 nm of Mn treated mitochondria to that of control. Mn treated showed dose dependant decreases in fluorescence. For TMRM we compared slopes of the fluorescence intensity ratios. Treating isolated mitochondria with Cu resulted in a dose dependant reversal in the slopes. Cu was more toxic than Mn. Both fluorescent dyes were effective in demonstrating short term treatments with Mn or Cu could de energize gill MMP. This information correlates well with our previous findings on the toxic effects of Mn and Cu on mitochondrial respiration. Identifying the molecular and cellular mechanisms of metal induced oxidative stress will provide a better understanding of the pathophysiological of neurodegenerative disorders associated with metal toxicity.
Structural and energetic changes in mitochondria associated with aging rodent ooctyes may be overcome by mitochondrial microinjection
Authors: Castora Frank J. , Duran Fatma , Li Fang , Ford Wentia , Birdsall Paige , Mezezi Aami , Swanson R. James
Published: June 2021

Growing evidence suggests that age‐related sub‐fertility in mammals is linked to oocyte senescence and the resultant changes in oocyte structure and function that accumulate over time. Recent studies have shown that low quality oocytes have age‐related dysfunctions including reduced mitochondrial membrane potential, increased mitochondrial DNA (mtDNA) damage and changes in mitochondrial gene expression. In this study, we demonstrate a significant reduction in oocyte ATP level and oocyte mtDNA number during mouse and hamster aging. These age‐related deficits are also reflected in changes in the oocyte cytoplasmic lamellae and mitochondrial cristae. We have chosen the hamster as a model system to evaluate the effects of microinjection of purified mitochondria into aged oocytes. We recover mitochondria from platelets from age‐matched surrogates. We posit that mitochondria from “old” animal platelets behave in many ways like those from “young” animals. ATP production increased significantly in the microinjected group compared to a mock‐injected control, however mtDNA number was not significantly altered. Embryo development to blastocyst stage was not improved by microinjection. These results were promising but inconsistent. We are developing an approach using magnetic beads to prepare mitochondria of high purity and quality. Support was from the Jones Institute Foundation and the EVMS Dept. of OBGYN.
Selenoprotein W is required for epidermal growth factor (EGF)‐stimulated proliferation via a pathway involving MKK4 and p53
Authors: Hawkes Wayne Chris , Alkan Zeynep
Published: June 2021

Selenoprotein W (SEPW1) is a highly conserved thioredoxin‐like protein whose depletion causes a p53‐ and p21‐dependent G1‐phase cell cycle arrest in breast and prostate epithelial cells. SEPW1 promotes the ubiquitination, nuclear export, and proteasomal degradation of p53, effects that are associated with decreased phosphorylation of Ser‐33 in p53. We now report that SEPW1 is required for EGF‐stimulated proliferation in breast and prostate cells. SEPW1 depletion blocked EGF‐stimulated G1 to S‐phase progression and altered signaling via downstream MAP kinase pathways. In an siRNA screen of 4 MAP kinase kinases and 6 MAP kinase kinase kinases, only silencing of MKK4 (JNKK1/SEK1) blocked G1 arrest, Ser‐33 phosphorylation, and p53 activation from SEPW1 depletion in EGF‐stimulated cells, suggesting MKK4 is downstream from SEPW1 in a p53‐dependent pathway regulating EGF‐stimulated cell cycle entry. We found that silencing p53 in freely cycling cells accelerated G1 to S‐phase progression, suggesting SEPW1 stimulates proliferation by suppressing the already low basal level of p53 expression. Taken together, these results suggest SEPW1 mediates EGF‐stimulated proliferation, at least in part, by suppression of p53 via MKK4, and presumably, one or more downstream MAP kinases.

This research was supported by USDA CRIS Project 5306‐51530‐018‐00D. USDA is an equal opportunity provider and employer.
Fat storage‐inducing Transmembrane Protein 2 adipose tissue deficiency results in decreased adipose tissue mass
Authors: Miranda Diego A , Silver David L
Published: June 2021

Adipose tissue serves as the main storage depot of neutral lipids in cytosolic lipid droplets. We recently identified a two‐gene family of proteins involved in lipid droplet biogenesis, which we name Fat storage‐Inducing Transmembrane protein (FITM1/FIT1 & FITM2/FIT2. FITM2 is ubiquitously expressed in tissues with high expression in white and brown adipose tissue. Overexpression of either member of the FITM family results in increased accumulation of cytosolic lipid droplets both in vitro and in vivo, not by enhancing triglyceride synthesis but by partitioning newly synthesized triglyceride into lipid droplets. To understand the role of FITM2 in lipid droplet biogenesis in vivo we generated adipose tissue‐specific knockouts (AF2KO) using the aP2‐cre to drive gene deletion of FIT2 in mature adipocytes. AF2KO mice exhibited protection from diet‐induced weight gain, which correlated with a 40% decreased in fat mass relative to wild‐type mice. This was due to an increased in basal and isoproterenol stimulated lipolysis in white fat pads isolated from AF2KO mice. AF2KO mice exhibited massive increased accumulation of adipose tissue macrophage, but this did not result in changes in glucose or insulin tolerance. Taken together our studies indicate that FIT2 plays an important role in triglyceride storage in mature adipocytes in vivo.
CGI‐58 regulates triacylglycerol metabolism and lipid signaling pathways in plant cells
Authors: Park Sunjung , Gidda Satinder , Khuu Nicholas , Horn Patrick , James Chris , Chapman Kent , Mullen Robert , Dyer John
Published: June 2021

CGI‐58 (Comparative Gene Identification‐58) encodes an α/β hydrolase‐type protein that is involved in multiple aspects of lipid metabolism in mammals, including the breakdown of triacylglycerols (TAGs) and formation of the hydrophobic barrier of the skin. Mutations in CGI‐58 result not only in aberrant skin formation, but also in accumulation of lipid droplets (LDs) in non‐lipid‐storing tissues such as skin, liver, and blood cells. The activity of CGI‐58 is determined at least in part by its interaction with other proteins including perilipin, which serves to anchor CGI‐58 to the surface of LDs. Phosphorylation of perilipin releases CGI‐58 and allows it to interact with Adipose Triglyceride Lipase (ATGL), thereby stimulating TAG breakdown. Interestingly, disruption of the CGI‐58 homolog in Arabidopsis results also in the accumulation of LDs in tissues that do not normally store lipids (e.g., leaves and stems), but plants lack any apparent homologs to perilipin or ATGL. In an effort to better understand the function of CGI‐58 in plants, we conducted a yeast 2‐hybrid assay using Arabidopsis CGI‐58 as bait. Although we did not identify any obvious LD‐associated proteins or lipases, we did identify several proteins known to be involved in distinct aspects of plant lipid metabolism. Evidence will be presented showing that CGI‐58 modulates the level of linolenic acid as well as lipid signaling pathways in plant cells.
Characterization of a novel short‐form of perilipin‐5
Authors: DuBreuil Daniel M , Tansey John T
Published: June 2021

The perilipins are a family of five interrelated genes with conserved sequences. Perilpin‐5 is expressed most strongly in oxidative tissues such as heart and fasting liver and has been shown to interact with both the lipase ATGL and the ATGL co‐activator CGI‐58 (ABDH‐5). Perilipin‐5 has recently been shown to localize to the lipid storage droplet and a small lipoprotein‐like structure found in the cytosol. Using western blotting, we have identified a short form of perilipin‐5 we have termed perilipin‐5B. This 35 kDa protein is found in liver, heart, and c2c12 myoblasts. Immunofluorescence of c2c12 myoblasts shows staining in cytosol and lipid droplets, as well as some other membranous structures. Analysis of murine genomic DNA sequences reveals stop codons found in the introns between exons 7 and 8 and 8 and 9, any of which could give rise to a protein of approximately 35 kDa. Interestingly, many of the functions ascribed to perilipin‐5 have been attributed to the carboxy‐terminus of the protein, which would be missing in this proposed truncation. Support for this research provided by the Chemistry Department of Otterbein University and the Bert and Jane Horn endowed student research fund in the sciences.
Regulation of macrophage cholesterol homeostasis by trans fatty acid
Authors: Shao Fei , Ford David A.
Published: June 2021

Trans fatty acid (TFA) consumption causes adverse changes in plasma lipids and lipoprotein profile, which leads to increased incidence of atherosclerosis. Macrophage cholesterol efflux, an atheroprotective process, is mediated by ATP binding cassette transporter A1 (ABCA1). ABCA1 is highly regulated by transcription factors Liver X receptor‐α (LXRα) and Retinoic X Receptor‐α (RXRα). Our studies are focusing on the mechanism by which individual trans fatty acid molecular species regulate ABCA1‐mediated macrophage cholesterol efflux and macrophage cholesterol efflux. Our results show that trans fatty acid species differentially regulate ABCA1 protein expression in mouse macrophage J774. The most abundant trans fatty acid elaidic acid (9t‐18:1) protects ABCA1 protein degradation, whereas oleic acid (9c‐18:1) induce ABCA1 protein degradation. Elaidic acid upregulates LXRα/RXRα activity and sterol regulatory element (SRE)‐driven luciferase activity, which suggest that elaidic acid may increase activities of sterol regulatory element binding proteins (SREBPs) family. These results demonstrated that trans fatty acids regulate ABCA1‐mediated cholesterol efflux and cholesterol homeostasis in a distinctive way compared to cis fatty acids.

This research was supported by NIH grants HL074214 and HL098907 (DAF)
Class A Scavenger receptor (SR‐A) mediated adhesion is regulated by lipid raft localization and cytoplasmic motifs
Authors: Vadali Shanthi , Post Steven R
Published: June 2021

SR‐A is a membrane receptor that mediates cell adhesion and ligand internalization. Previous studies suggest that SR‐A‐mediated macrophage adhesion, but not ligand internalization, activates signaling pathways that promote inflammation. We hypothesized that SR‐A function is regulated by localization of SR‐A in distinct plasma membrane domains; specifically, that SR‐A‐ mediated cell adhesion requires localization in cholesterol‐rich lipid rafts. To test this hypothesis, HEK cells were transfected to express either wild‐type (SR‐Aw/t) or a truncated receptor (SR‐AΔ1‐ 49), which lacks all but the membrane‐proximal 6 cytoplasmic amino acids and mediates only adhesion. A detergent‐free cell fractionation protocol was used to show that SR‐A and SR‐AΔ1–49 localized to a similar extent in lipid rafts. To determine if lipid raft localization was required for cell adhesion, cells were treated with methyl‐beta‐cyclodextrin (MβCD), which depletes cell cholesterol and disrupts lipid rafts. MβCD treatment inhibited the adhesion of cells expressing SR‐Aw/t, but did not affect adhesion mediated by SR‐AΔ1–49. Together, these results demonstrate that SR‐A localizes in lipid rafts, that this localization is required for cell adhesion, and that the membrane proximal amino acids are sufficient to localize the receptor in lipid rafts but not sufficient for functional regulation. Support: NIH‐HLRO1‐076682 and AHA Predoctoral fellowship AHA‐11PRE‐7950020.
Cloning and characterization of novel CTP: phosphoethanolamine cytidylyltransferase (Pcyt2) isoforms made by alternative splicing
Authors: Pavlovic Zvezdan , Bakovic Marica
Published: June 2021

Pcyt2 controls the rate limiting step of de novo biosynthesis of phosphatidylethanolamine. We previously identified the exon‐7 skipping as a mechanism for alternative splicing of two different Pcyt2 isoforms, Pcyt2α and Pcyt2β. Homology searches of genome databases indicated the presence of additional Pcyt2 transcripts. In order to isolate and characterize those alternative transcripts, we used total cDNA prepared from C2C12 and multiple mouse tissues. Methodology included specific PCR amplification, cloning into pGEMt vector and sequencing. We detected two distinctive Pcyt2 isoforms, named γ1 and γ2. The Pcyt2γ1 is a longer form, composed of exons 1–8 and two additional regions from introns 7 and 8. The first intronic segment of 184 bp located at the beginning of intron 7 was named γ‐specific region 1. Next, exon 8 was followed by 258 bp of sequence from intron 8, named γ‐specific region 2. The new isoforms resemble the first half of Pcyt2α in containing the first catalytic site and in maintaining the exon 7 linker peptide region. Pcyt2γ1 contains two additional regions made of partial introns 7 and 8, while Pcyt2γ2 retains only parts of intron 8. This composition made the C‐terminal ends of both γ1 and γ2 completely different from isoform Pcyt2α. Both isororms were expressed in different mouse tissues as well as in mouse embryos at different stages of development. This research was supported by CIHR research grant.
Lipid‐protein interactions as a determinant of the function and topogenesis of membrane proteins
Authors: Vitrac Heidi , Bogdanov Mikhail , Dowhan William
Published: June 2021

LacY (lactose permease) expressed in Escherichia coli lacking PE (phosphatidylethanolamine) and containing only anionic phospholipids exhibits downhill but not uphill transport and is topologically misassembled. Substitution in vivo of PE by foreign lipids (including phosphatidylcholine (PC)) with no net charge supports native LacY topology and uphill transport. Here, we further investigate the effects of phospholipid head group and fatty acid composition on topology and function of LacY reconstituted into proteoliposomes. Anionic phospholipids alone do not support uphill transport or native topology. Addition of E. coli derived PE or PC supports both uphill transport and native topology. Synthetic PE or PC containing only unsaturated fatty acids supports proper topology but not uphill transport. PE or PC with one saturated fatty acid supports uphill transport. These results show that LacY structure and function are sensitive to both the polar head group and acyl group composition of phospholipids. Our results demonstrate that membrane protein organization and function are determined through direct interactions of the protein with its lipid environment. Supported by NIH grant R37‐GM20478.
Phosphatidylcholine Transfer Protein Attenuates High Fat Diet Induced Changes in Mitochondrial Membrane Phosphatidylcholine Composition
Authors: Maner-Smith Kristal , Cohen David E.
Published: June 2021

Phosphatidylcholine transfer protein (PC‐TP) is a phospholipid binding protein that is enriched in liver and catalyzes intermembrane exchange of phosphatidylcholines (PCs) in vitro. Because it associates with the mitochondria, we explored the hypothesis that PC‐TP regulates the distribution of PC molecular species in mitochondrial membranes. Pctp−/− and Pctp+/+ mice were fed chow or high fat diets. Liver mitochondria were purified and membrane lipid extracts were analyzed by tandem mass spectrometry. The distribution of PC species was determined by precursor ion scanning of m/z 184, which corresponds to the headgroup. In chow fed mice, PC‐TP expression did not influence the distribution of PCs. High fat fed Pctp+/+ mice exhibited a modest 21% decrease in mitochondrial 16:0–18:2 PC and a 27% increase in 16:0–20:4 PC. By contrast, high fat feeding of Pctp−/− mice led to more pronounced changes in the same PCs, with a 43% decrease in 16:0–18:2 PC and 34% increase in 16:0–20:4 PC. We conclude that under physiological conditions, PC‐TP expression is not a determinant of the PC composition of mitochondrial membranes. In response to dietary fat, PC‐TP expression mitigates the enrichment of mitochondria with polyunsaturated PCs, which may modulate biophysical properties of the membrane. Supported by NIH.
Functional and structural analysis of transmembrane segment IV of Na+/H+ exchanger of Schizosaccharomyces pombe
Authors: Ullah Asad , Alves Claudia , Lee Brian L , Sykes Brian D , Fliegel Larry
Published: June 2021

Sod2 is the plasma membrane Na+/H+ exchanger of fission yeast S. pombe. It plays an important role in providing salt tolerance to S. pombe by removing excess intracellular sodium (or lithium) in exchange for protons. We examined the role of amino acids residues of TMIV (126FQINFLGSLLIAGCITSTDPVLSALI152) in activity of this protein using alanine scanning mutagenesis, and examining LiCl or NaCl tolerance in Sod2‐deficient S. pombe. We identified two amino acids, T144 and V147, critical for the function of Sod2. The mutations T144A and V147A resulted in a defective sod2 protein that did not confer salt tolerance when reintroduced into to S. pombe. Sod2 protein with other mutations in TMIV, exported either sodium or lithium as efficiently as the wild type Sod2 or had intermediate effects on salt tolerance of S. pombe. When we mutated T144 to aspartic acid, lysine or serine, the mutant proteins were impaired in their ability to allow growth in both lithium and sodium containing media, comparable to the knock out sod2 yeast (sod2::ura4). Western blot analysis showed that mutants T144A and V147A were expressed at comparable levels as the wild type sod2. To determine the structure of TMIV, we expressed and purified TMIV as a fusion protein with maltose binding protein. The TMIV peptide was released by cleaving the fusion protein with tobacco etch virus and re‐purified by HPLC. The structure is being determined by NMR.

Supported by Natural Sciences and Engineering Research Council of Canada
A fibrinogen‐derived peptide induces clathrin‐ and caveolaeindependent endocytosis in endothelial cells
Authors: Serrano Daniel , Garnacho Carmen , Muro Silvia
Published: June 2021

Endocytosis serves key functions in the vascular endothelium, with clathrin‐ and caveolae‐mediated endocytosis as the most prevalent pathways in this tissue. For instance clathrin mediates E‐ and P‐selectin uptake, and turnover of thrombomodulin occurs via caveolae. Clathrin‐ and caveolae‐independent pathways are also found in the endothelium, as is the case of syndecan‐4‐mediated endocytosis of fibroblast growth factor and uptake of antibody‐targeted drug nanocarriers (NCs) by intercellular adhesion molecule 1 (ICAM‐1), a protein involved in inflammation and thrombosis. The latter pathway, cell adhesion molecule (CAM)‐ mediated endocytosis, depends on amiloride‐sensitive sodium proton exchanger 1 (NHE1) and actin stress fibers. To assess whether ICAM‐1 can also mediate endocytosis of a natural binding partner, we coated NCs with a fibrinogen‐derived peptide, gamma 3 (γ3). Radioisotope tracing, fluorescence and electron microscopy revealed that γ3 NCs specifically bound to ICAM‐1, induced actin stress fibers, and elicited amiloride‐sensitive endocytosis in cell culture and mice, followed by trafficking to lysosomes. These results show that clathrin‐ and caveolae‐independent CAM‐mediated endocytosis also operates upon binding of ligands derived from natural binding partners of ICAM‐1, providing a framework for understanding the physiological role of this endocytic pathway.
Characterization of the interaction between Sorting Nexin 27 and β‐Catenin in kidney epithelial cells
Authors: Duchez Brian , Milgram Sharon , Playford Martin Peter
Published: June 2021

Sorting Nexin 27 (SNX27) is a 62‐kDa protein localized to early endosomes and known to regulate the intracellular trafficking of ion channels and receptors. In addition to a PX domain, SNX27 is the only sorting family member that contains a PDZ domain. To identify novel SNX27‐PDZ binding partners, we performed a proteomic screen in mouse principal kidney cortical collecting duct cells (mpkCCD) using a GST‐SNX27 fusion construct as bait. We found that the C‐terminal type I PDZ binding motif (DTDL) of β‐Catenin, an adherens junction scaffolding protein and transcriptional co‐activator, interacts directly with SNX27. Using biochemical and immunofluorescent techniques, β‐Catenin was identified in endosomal compartments where co‐localization with SNX27 was observed. As expected, over‐expressed β‐Catenin wild‐type protein was identified in nuclear regions and increased TCF‐LEF dependent transcriptional activity was observed. In contrast, over‐expressed β‐Catenin ΔDTDL was found at sites of cell‐cell contact with transcriptional activity at control levels. These results imply importance of the C‐terminal PDZ binding motif in localization and transcriptional activity of β‐Catenin.
The Vibrio parahaemolyticus effector VopQ induces autophagosome accumulation in host cells
Authors: Sreelatha Anju , Bennett Terry , Starai Vincent , Orth Kim
Published: June 2021

Vibrio parahaemolyticus, a Gram‐negative bacterium, is the leading cause of seafood‐borne illness in the United States. This pathogen is able to infect and survive in humans by manipulating the host cellular machinery via bacterial Type III effector proteins that are translocated into the host cell, where they disrupt cellular signaling. One of the principal effector proteins, VopQ, has been demonstrated to be necessary and sufficient to induce autophagosome accumulation. I propose to elucidate the precise cellular and molecular mechanism by which VopQ induces autophagosome accumulation. Since autophagosomes are transient vesicles, the accumulation could be due to an increase in formation of autophagosomes or a decrease in lysosomal fusion. Using biochemical assays and yeast genetics we have uncovered a novel target that reveals the role of VopQ in vesicle fusion during autophagy.

With the discovery of several bacterial effectors that function to manipulate host membranes, many eukaryotic vesicle trafficking pathways have been unveiled, and many new questions have surfaced about the mechanisms of novel bacterial effectors. Understanding the mechanism by which VopQ induces autophagosome accumulation could be essential in understanding V. para infection and could give new insights into the mechanisms of eukaryotic vesicle trafficking.
Receptor determinants for the avian coronavirus infectious bronchitis virus: Roles of host cell lectins and glycans
Authors: Zhang Yueting , Whittaker Gary
Published: June 2021

The Coronavirus family consists of a variety of single‐stranded RNA, enveloped viruses that infect a wide range of animals, with many new coronaviruses from unanticipated hosts discovered in recent years. The prototype avian coronavirus, infectious bronchitis virus (IBV) is a continuing economic concern in the poultry industry around the globe and can be used as model for the study of pathogenesis for the Coronavirus family. Even though the putative receptor for IBV has not been identified, sialic acid, a terminal sugar on glycoproteins or glycolipids, has been proposed to be an attachment factor for IBV. Intriguingly, IBV is able to infect many different tissues/organs in the host chicken; yet IBV is unable to infect any cells from other animal species than chicken. The sialic acid binding pattern in multiple chicken organs has also been implicated in the pathogenesis in the host, but the correlation of binding could not explain the limited susceptibility of IBV to non‐chicken cells since sialic acid is ubiquitous in animal tissues. The lack of knowledge in IBV receptor leaves a big gap in our understanding in the relatively wide chicken tissue tropism and strict species tropism of IBV. When we expressed DC‐SIGN, a human C‐type lectin that may resemble a chicken analog lectin, into mammalian cell lines, we were surprised to find that not only IBV infection is rescued in these cell lines but also the infection is not dependent on the level of sialic acid on the cell surface. In addition, we examined whether sialic acid also plays a role in the IBV infection in chicken peripheral blood derived monocytes (chPBMCs), cells that potentially harbor the authentic proteinaceous receptor for IBV, unexpectedly we also found that the established attachment factor sialic acid does not play a critical role in the IBV infection in PBMC. Our data suggest that, sialic acid may be dispensable or may play a lesser role in virus‐receptor complex formation in the presence of a potential high‐affinity receptor molecule.
Global increases in O‐GlcNAc levels lead to differentiation of myoblasts
Authors: Vaidyanathan Krithika , Wells Lance
Published: June 2021

O‐GlcNAc is a posttranslational modification occurring on hundreds of nuclear and cytoplasmic proteins, which is cycled by the joint action of OGT, for its addition, and OGA, for its removal. O‐GlcNAc modification has been demonstrated to regulate transcription, differentiation, disease pathology and cell cycle regulation in various settings. In order to further understand the regulation of OGA, our laboratory performed a yeast two‐hybrid screen with the enzyme as bait and identified IFRD1, a transcriptional co‐activator/co‐repressor, as a candidate interacting protein. We have successfully demonstrated the OGA/IFRD1 interaction through reciprocal immunoprecipation, western blotting experiments. This led to our interest in examining the role of this interaction in skeletal muscle cells where IFRD1 is known to play a role in myoblast differentation. When OGA and IFRD1 are co‐expressed, there is a global increase in O‐GlcNAc levels suggesting that OGA could either be sequestered by IFRD1 thus allowing for increased global O‐GlcNAc levels, or IFRD1 inhibits OGA activity. When C2C12 cells were treated with GlcNAcstatin, an OGA inhibitor for 18 hours at 60% confluency, they spontaneously differentiate into myotubes in a dose‐dependent manner. Current work is recapitulating these results via genetic manipulation of the O‐GlcNAc cycling enzymes and more carefully defining the differentiation process using a variety of markers. We are currently localizing the two proteins as well as determining the activity of IFRD1‐associated OGA using immunofluorescence, biochemical fractionation, and enzyme activity assays. Current work is aimed at exploring the mechanism by which elevated O‐GlcNAc levels lead to myoblast differentiation with the working hypothesis that the IFRD1/OGA interaction positively regulates myogenesis.
Antibacterial Activity of Some Coumarins and their Metal Complexes
Authors: Govori Sevdije R , Buza Aida , Ajvazi Njomza , Haziri Arben
Published: June 2021

A series of some new coumarin derivatives and their Cu(II) complexes have been synthesized and characterized by a variety of physicochemical techniques. The ligands and its metal complexes are screened for their in vitro antibacterial activity against Escherichia coli and Enterochoaco.

The studies demonstrated that metallation can increase the antibacterial activity rather than the free ligand.
Study of a Potent Small‐Molecule Benzosuberene Anti‐Cancer Agent
Authors: Charlton-Sevcik Amanda K. , Strecker Tracy E. , Odutola Samuel O. , Tidmore Justin K. , Tanpure Rajendra P. , George Clinton S. , Sriram Madhavi , Chaplin David J. , Pinney Kevin G. , Trawick Mary Lynn
Published: June 2021

The discovery of a benzosuberene‐based, small‐molecule anti‐cancer agent (KGP18) with remarkable cytotoxicity against selected human cancer cell lines (BMC 2008 16, 8161–8171) provided the inspiration for a collaborative research program to investigate the mechanism of action of this compound and related benzosuberene analogues. The structure of KGP18 and its ability to inhibit tubulin polymerization into microtubules in vitro suggests that it may act as a vascular disrupting agent (VDA). An adequate blood supply is required for tumor growth and metastasis. VDAs represent a promising new class of anti‐cancer agents that function by selectively disrupting the tumor vasculature, thus starving the tumor of nutrients and oxygen via an adequate blood supply. Tubulin‐binding VDAs inhibit microtubule assembly in the activated endothelial cells, lining the tumor vasculature, leading to cytoskeletal reorganization, activation of RhoA and RhoA kinase, increased vascular permeability, cell rounding and detachment, and vessel occlusion. The lead benzosuberene analogue (KGP18) binds to tubulin at the colchicine binding site as determined by a competitive radiometric assay. Flow cytometry indicates that MDA‐MB‐231 cells treated with KGP18 are arrested at the G2/M phase of the cell cycle. (Supported by Oxigene Inc., the NIH, and CPRIT).
Optimization of expression of Schizophyllum commune metacaspase gene scp1 via codon‐optimization, prodomain removal, and autoinduction
Authors: Major Ajay , Fox Kristin M
Published: June 2021

In plants, fungi, and protists, programmed cell death is controlled by a family of proteases known as metacaspases, which appear to be homologous to aspartate‐specific mammalian caspases with high similarity of primary sequence and conservation of the cysteine‐histidine catalytic dyad. As proteases, metacaspases also contain N‐terminal prodomains that are cleaved off during proteolytic activation of the enzyme. However, metacaspases are arginine and lysine‐specific and thus have different self‐cleaving mechanisms and substrate interactions during programmed cell death. Expression and purification of S. commune metacaspase protein Scp1 is important for understanding these apoptotic mechanisms, but previous attempts at expression of the complete scp1 gene in E. coli were unsuccessful. Codon‐optimization of the scp1 gene resulted in no increase in expression. However, removal of the prodomain of codon‐optimized scp1 via PCR resulted in successful expression of Scp1 protein. Preliminary experiments with autoinduction, an expression technique in E. coli that utilizes glucose/lactose‐rich media, have shown increased expression of soluble Scp1 protein compared to IPTG‐induced overexpression.