bluuebunny/crossref_metadata_2025_split huggingface dataset:
Detecting Released Peptidoglycan Associated Lipoprotein (Pal) from Escherichia coli
Authors: Snyder Emma , Zavorin Mark , Farquaharson Kara , Panullo Nicole , Pichichero Michael , Hellman Judith , Michel Lea Vacca 
Published: June 2021

Gram‐negative sepsis can result from a bacterial infection, such as Escherichia coli (E. coli), which leads to an over‐exuberant inflammatory response in humans. During E. coli sepsis, a bacterial lipoprotein, Pal (peptidoglycan associated lipoprotein), has been shown to be released from the outer membrane of E. coli and is thought to contribute to an inflammatory response in human cells. In E. coli, Pal has two subpopulations: periplasmic Pal, which is bound to peptidoglycan, and surface‐exposed Pal. A biotinylation technique was used to roughly quantify the amount of each Pal subpopulation that was released from E. coli in vitro. Several different antibiotics were tested for their effect on Pal release; ultracentrifugation was also employed to quantify the amount of Pal released in outer membrane vesicles. Preliminary results suggest that both subpopulations of Pal are capable of release from E. coli, and that the majority of Pal is released from E. coli in outer membrane vesicles.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
CONFORMATIONAL FLUCTUATIONS RELATED TO CATALYSIS IN HUMAN RIBONUCLEASE SUPERFAMILY
Authors: Bafna Khushboo , Narayanan Chitra , Bernard David , Doucet Nicolas , Agarwal Pratul 
Published: June 2021

The high catalytic efficiency of enzymes is a result of structural and dynamical effects. The role of enzyme structure in catalysis has been understood for some time. The enzymes of the ribonuclease A (RNase) family possess identical or similar active site residues and conserved fold architecture. However, their catalytic efficiency differs by 105–106 fold. Moreover, the rate of dynamics among the RNases ranges from microsecond to second time‐scale, a factor 106 difference. Recent investigations provide insights into the role of conformational fluctuations (dynamics at multiple time‐scales) in enzyme catalysis. The fluctuations allow enzymes to sample inter‐converting conformations (called as conformational sub‐states). These conformational sub‐states are proposed to contain features that can promote the function of an enzyme. Therefore, correlating the role of time dependent dynamics in sampling functionally relevant conformational sub‐states would enable a better understanding of the catalytic mechanism of the various RNases. NMR relaxation dispersion experiments show distinct patterns of dynamical variations among the RNases clustered into sub‐families having different biological functions within the same fold. A combination of theoretical modeling, computer simulations and higher order statistics has enabled the identification of conformational sub‐states that regulate the mechanism substrate binding, catalysis and product removal in representative members of each sub‐family found in the human genome. The representative members of each sub‐family have diverse conformational sub‐states associated with each step of the catalytic cycle suggesting a possible correlation between dynamics and function.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Phe36 Plays a Key Role in the Fluorine Recognition of Fluoroacetyl‐CoA Thioesterase FlK
Authors: Wang Ningkun , Pelton Jeffrey G. , Chang Michelle C. 
Published: June 2021

Fluorinated small molecules play an important role in the design of bioactive compounds for a broad range of applications. Hence there is great interest in developing a deeper understanding of how proteins recognize fluorine moieties in ligands. The fluoroacetyl‐CoA thioesterase FlK from Streptomyces cattleya is one of the few enzymes that display a native selectivity towards organofluorine substrates, providing us a rare opportunity to study this enzyme‐ligand pair and the molecular interactions that lead to the 106‐fold selectivity for a single fluorine moiety. In this study, we have used protein NMR to dissect the molecular details of the interaction between FlK and substrate analogs. Previous studies have found that Phe36, a residue at the binding pocket of FlK, is important to the substrate specificity of the enzyme. Our work here shows that Phe36 displays a change in chemical shift specific to the binding of the fluoroacetyl‐CoA analog, and has determined that this discrimination between fluoroacetyl‐CoA and acetyl‐CoA analogs can be contributed to a decrease in exchange rate between the bound and free form of FlK as conveyed through Phe36.Support or Funding InformationThis work was funded by the generous support of the National Institutes of Health (R01 GM123181).The funds for the 900 MHz NMR spectrometer housed in the College of Chemistry NMR Facility at U.C. Berkeley were kindly provided by the NIH (GM68933).This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Evidence for Distinct CoA Binding Sites in Nudt19 and Nudt7, Two Mammalian Nudix Hydrolases that Degrade Coenzyme A
Authors: Shumar Stephanie , Geldenhuys Werner , Leonardi Roberta 
Published: June 2021

Coenzyme (CoA) is an essential cofactor required for hundreds of metabolic processes as well as protein acetylation, a post‐translational modification that regulates gene expression and activity of thousands of cellular proteins. CoA levels are tightly regulated. In diabetic mice, loss of regulation and an abnormally high concentration of CoA drive excess glucose production and contribute to hyperglycemia. This phenotype is associated with constitutively low CoA degradation, a process that is emerging as an important mechanism for CoA regulation. Nudt7 and Nudt19 are two peroxisomal enzymes with CoA‐degrading activity, which are highly expressed in the liver and kidney, respectively. Two members of the Nudix hydrolase superfamily, Nud7 and Nudt19 both catalyze the Mg2+‐dependent hydrolysis of free and acyl‐CoAs at the phosphodiester bond. Nudt19 (42 kDa) is larger than Nudt7 (27 kDa) and contains a unique 46–50‐residue insertion in the putative Mg2+‐binding box of unknown function. Nudt7 accepts a wider range of CoA substrates compared to Nudt19. Limited information is available on the biochemical and regulatory properties of Nudt7 and Nudt19. Additionally, the involvement of these enzymes in regulation CoA levels in vivo has not been proven. We identified residues on both Nudt7 and Nudt19 that play an essential role in CoA and Mg2+ binding. By screening selected metabolites, we discovered that chenodeoxycholic acid (CDCA) is a specific inhibitor of Nudt19 but not Nudt7. A Nudt19 mutant lacking the Nudix box insertion, as well as four chimeric Nudt7/Nudt19 proteins, were generated to investigate the contribution of the CoA binding motif and the unique Nudix box of Nudt19 to its sensitivity to CDCA inhibition and substrate specificity. Finally, we show that mice lacking Nudt7 exhibit elevated liver CoA in the fed state and mice lacking Nudt19 exhibit higher CoA levels in the kidneys. These data establish the importance of Nudt7‐ and Nudt19‐mediated degradation in the regulation of tissue CoA levels.Support or Funding InformationThis work was supported by West Virginia University's School of Medicine, (10020452.15.F2R226W) startup foundation funding, and National Institutes of Health Grant GM119528This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
The Cytochrome P450 24A1 Interaction with Adrenodoxin Modulates Substrate Binding and Relies on Species‐variable Recognition Sites
Authors: Estrada D. Fernando 
Published: June 2021

Redox partner and accessory protein interactions are known to modulate function in mammalian cytochrome P450 enzymes by exerting a level of allosteric control, most notably demonstrated by the interaction of cytochrome b5 with P450 enzymes in the endoplasmic reticulum. However, in the inner mitochondrial membrane, in which P450 electron transfer occurs solely via interaction with the ferredoxin protein adrenodoxin (Adx), the events that modulate P450 function are not clear. In this study, we report the use of combined spectroscopic techniques to demonstrate that Adx serves to modulate substrate binding in the mitochondrial vitamin D metabolizing enzyme CYP24A1. The presence of equal molar amounts of Adx potentiates substrate binding for 25(OH)D3 and 1,24,25(OH)3D3, but not for the primary substrate 1,25(OH)2D3. We follow up on the CYP24A1‐Adx interaction by utilizing solution nuclear magnetic resonance (NMR) spectroscopy to monitor the spectra of uniformly labeled 15N Adx while titrating with unlabeled CYP24A1 bound to the P450 inhibitor clotrimazole. The pattern of NMR line broadening, combined with single and double mutants of Adx, suggest the presence of multiple recognition sites between the proteins. In order to evaluate redox partner complexes for CYP24A1 isoforms displaying distinct regioselectivity for vitamin D, all of the binding data were acquired in parallel for a carbon 23 hydroxylase, a carbon 24 hydroxylase, and a combined carbon 23/24 hydroxylase. Interestingly, disruption of the CYP24A1‐Adx complex in response to Adx mutagenesis was variable between CYP24A1 isoforms, suggesting a correlation between redox partner binding and regioselectivity and consistent with the ability of Adx to modulate substrate binding. Therefore, this work provides structural evidence in support of Adx modulation of function in a mitochondrial P450 enzyme.Support or Funding InformationThis work was supported by R00GM112862 and the State University of New YorkThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Fluorogenic structure activity library pinpoints molecular variations in the substrate specificity of structurally homologous esterases
Authors: Johnson R. Jeremy , White Alex , Koelper Andrew , Russell Arielle , Larsen Erik , Hoops Geoffrey 
Published: June 2021

Ubiquitous cellular esterases catalyze many essential biological functions by performing hydrolysis reactions on diverse substrates. These diverse substrates and functions however complicate a priori prediction of their substrate preferences and biological functions. Analogous to substrate activity screening for serine protease activity, we designed a 36‐member structure activity relationship (SAR) library of fluorogenic esterase substrates with low background hydrolysis, high sensitivity, and modular, straightforward synthesis. In three parallel substrate series containing alkyl, ether, and thioether substituents, the SAR library systemically interrogates esterase preference for chain length, branching patterns, polarity, and hydrogen bonding to differentiate common classes of esterase substrates. Applying this library against two structurally homologous bacterial esterases, previously broad overlapping substrate specificity was refined to preferences for γ‐position thioethers and ethers for ybfF from Vibrio cholerae and branched substrates with and without thioethers for Rv0045c from Mycobacterium tuberculosis. Structural control over this substrate differentiation was then assigned to individual substrate selectivity residues of Tyr116 in ybfF and His187 in Rv0045c whose reciprocal substitution inverted each esterase's substrate preference. This SAR esterase library could have multi‐faceted future applications including in vivo imaging, biocatalyst screening, molecular fingerprinting, and inhibitor design.Support or Funding InformationThis work was supported by a grant from the National Institutes of Health (NIH 1 R15 GM110641‐01A1).This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Setting all‐carbon quaternary stereocenters with stereospecific, nickel‐catalyzed cross couplings
Authors: Duke Alana D. , Pound Sarah M. , Xu Jianyu , Basch Corey H. , Hoerrner Megan E. , Bampo Earl M. , Watson Mary P. 
Published: June 2021

Formation of quaternary stereocenters is an essential goal for organic chemistry due to these centers' presence in numerous drugs and their importance in biological activity. Transition metal catalysis offers a unique approach to this challenge, but previous attempts have been limited in either scope or enantiomeric enrichment. We are introducing a stereospecific Suzuki cross coupling of enantioenriched benzyl alcohol derivatives via C‐O bond activation that yields quaternary stereocenters with both aryl and vinyl substituents. Using benzylic carboxylates available in high enantiopurity, an air‐stable, inexpensive nickel source, and pinacol boronates, the reaction yields products in high yield and enantioenrichment and can tolerate a wide range of functional groups, including heteroaromatics. The optimization and scope of these reactions will be presented.Support or Funding InformationThe National Institutes of Health (R01 GM111820) is gratefully acknowledged.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
CYTOTOXIC AND GENOTOXIC ASSESSMENT OF SOME ANTI‐TUMOUR PLANTS FROM SOUTHWEST NIGERIA IN CERVICAL CANCER (HeLa) CELL LINE.
Authors: Adebesin Olumide , James Babatunde , Magbagbeola Olubunmi , Omilabu Sunday 
Published: June 2021

Natural products have contributed immensely to the development of anti‐tumour agents in which genotoxic and cytotoxic activities are consistent with these agents. Hence, this study is aimed at evaluating the cytotoxic and genotoxic effects of hit fractions from the leaves of Catharanthus roseus, Amaranthus viridis, Piper guineense, Zanthoxylum zanthoxyloides, Costus afer, and Annona muricata. Neutral red (NR) cytotoxicity assay was carried out to determine the IC50 values of each hit fraction using HeLa Cell lines. Alkaline comet assay was used to assess both double‐ and single‐stranded DNA breaks and detect alkali‐labile sites, which are expressed as strand breaks in DNA. Apoptosis was evaluated using DNA Fragmentation assay. Results shows that tannin and ethylacetate fractions of A. viridis, A. muricata (tannin fraction), C. roseus chloroform fractions had LC50 values< 100 μg/ml. Significant (P<0.05) percentage DNA comet tail was observed with C. roseus (chloroform), Piper guineense (hexane), and A. muricata (tannin fraction). Apoptotic DNA fragmentation was also observed in Piper guineense (hexane), A. viridis (ethylacetate), Z. zanthoxyloides (ethylacetete) and A. muricata (tannin fraction).In conclusion, this study provides experimental evidence that supports the cytotoxic and genotoxic effects of Piper guineense (hexane), A. viridis (ethylacetate), Z. zanthoxyloides (ethylacetete) and A. muricata (tannin) fractions as potentials for anti‐tumour drug development sources.Support or Funding InformationSelf FundedThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Nickel Catalyzed Cross Couplings of Amino Acids and Peptide Derivatives via C‐N Bond Activation
Authors: Bampo Earl Michel Osei , Watson Mary , Hoerrner Megan , Baker Kristen , Basch Corey 
Published: June 2021

Primary alkyl amines are well appreciated as building blocks for the synthesis of nitrogen‐containing molecules. However, their use as alkylating agents via cleavage of the carbon–nitrogen (C–N) bond is largely undeveloped. Recognizing this opportunity to expand the utility of alkyl amines in synthesis, the M. Watson group is developing strategies to convert primary alkyl amines into alkyl arenes via nickel‐ catalyzed cross‐couplings of Katrizky pyridinium salt intermediates. We are now developing this chemistry to enable cross‐couplings of alpha‐amino acids and peptides via cleavage of the alpha‐C–N bond. Pyridinium salts were formed from the N‐termini of a number of α‐amino acids and a tripeptide, prepared via solid phase peptide synthesis. Once formed, the pyridinium salts were allowed to undergo nickel‐catalyzed cross‐coupling. Our preliminary scope, as well as future work, will be presented.Support or Funding InformationThe National Institutes of Health (R01 GM111820) University of Delaware David A. Plastino FellowshipThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Systems Genetics Analysis of Arrhythmogenic Cardiomyopathy Induced by p.S368L Mutation in Transmembrane Protein 43
Authors: Munkhsaikhsan Undral , Hiltenbrand Ryan , Khuchua Zaza , Lu Lu , Towbin Jeffrey A. , Purevjav Enkhsaikhan 
Published: June 2021

BackgroundA missense mutation, p.S358L, in the transmembrane protein 43 (TMEM43/LUMA) gene causes arrhythmogenic right ventricular cardiomyopathy type 5 (ARVC5). ARVC5 is characterized by sudden cardiac death and fibro‐fatty replacement of the myocardium, particularly, in the right ventricle (RV). It has been hypothesized that the TMEM43 mutation impacts function via Ppar signaling, major regulators of adipogenesis. Little is known regarding the mechanistic basis of the S358L mutation‐induced effects.ObjectivesThe aim of this study is to discover novel pathways and gene networks associated with the clinical phenotype elicited by the S358L mutation in the Tmem43 gene using a systems genetics approach.MethodsThe mutant Tmem43S358L mouse was created using the conventional knock‐in approach. BXD strains were derived from a cross between C57BL/6J (B6) and DBA/2J (D2) mice. Total RNAs were extracted from heart of mutant Tmem43S358L mice and 40 BXD strains, and hybridized to Affymetrix Mouse Gene 2.0ST arrays. Differential expression of gene was analyzed using two‐way ANOVA and t‐test. Genetic correlation, functional enrichment, and co‐expression network were performed using GeneNetwork (http://genenetwork.org) and Webgestalt (http://www.webgestalt.org) online tools, respectively.ResultsWe found highly significant expression of Tmem43 hearts among the BXD strains with broad variability in expression levels suggesting an important role for Tmem43 in the heart. A co‐expression Tmem43‐gene network consisting of 25 highly associated genes was created and includes many cardiomyopathy‐related genes, including Tmem43, Ppargc1a and Jup, that are highly significantly connected with Tmem43 and each other (r>0.5, p< 0.00075). A significant negative correlation (p<0.01) between Tmem43 expression and heart mass and heart rate was also noted. Levels of high‐density lipoprotein (HDL) in plasma in BXD mice was significantly correlated with Tmem43 on a normal or high‐fat diet. Microarray analysis of the myocardium revealed significant downregulation of Ppargc1a and Pparg (p<0.0001) in Tmem43S358L mice hearts compared to WT littermates. Decrease in Ppargc1a was significantly related with the Tmem43 decrease in RV, suggesting direct inhibition of Ppargc1a by S358L‐Tmem43 mutation.ConclusionsWe conclude that Tmem43 is essential for normal cardiac function. The Tmem43‐S358L mutation causes alterations in gene expression of numerous cardiomyopathy‐associated genes, including Jup, Ppargc1a and Pparg, resulting in the development of ARVC5.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
C‐reactive Protein Binds to Integrin α2 and Fcγ receptor I, Leading to Breast Cell Adhesion and Breast Cancer Progression
Authors: Moon Aree , Kim Eun‐Sook , Kim Sun Young , Koh Minsoo , Lee Hye‐Min , Kim Kyoungmee , Jung Joohee , Kim Hoe Suk , Moon Woo Kyung , Hwang Sejin 
Published: June 2021

C‐reactive protein (CRP) is an acute phase protein synthesized upon the inflammatory responses, associated with breast cancer. The process of tumor cell invasion and metastasis involves the adherence of cells to the extracellular matrix via integrin as a receptor for matrix molecules. The present study investigated the role of CRP in the adhesive phenotype of breast cells and the underlying mechanisms. Here, we first showed that CRP induces adhesion of MCF10A human breast epithelial cells through the activation of integrin α2 signaling. Expression of integrin α2 was induced by CRP in which transcription factors c‐fos and SP1 may be involved. Binding of CRP with integrin α2 leads to the activation of focal adhesion kinase (FAK), paxillin and ERKs. CRP also binds to an Fcγ receptor I (FcγRI), and induces activation of paxillin, FAK and ERKs. Integrin α2 and FAK have crucial roles in the adhesive and invasive phenotypes as well as MMP‐9 upregulation induced by CRP in MCF10A cells. Treatment with an inflammatory lipid sphingosine‐1‐phosphate induced CRP, which may be secreted and exert an autocrine effect by binding to FcγRI and integrin α2. Involvement of CRP in adhesion, invasion, anchorage‐independent growth and upregulation of integrin α2, paxillin and FAK was observed in MDA‐MB‐231 triple‐negative human breast cancer (TNBC) cells. Using an in vivo invasion model and an orthotopic mouse tumor model with MDA‐MB‐231 cells, we showed that CRP has an important role in intravasation and tumor growth in vivo, demonstrating the in vivo relevance of our in vitro results. The present study elucidates a critical molecular basis between CRP, integrin α2 and FcγRI pathways in MCF10A breast cells and MDA‐MB‐231 TNBC cells, thereby providing useful information on CRP‐induced aggressiveness of breast cells in the inflammatory microenvironment.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.