bluuebunny/crossref_metadata_2025 huggingface dataset:
CRISPR‐Cas9 knock out of gravin variant 1 impairs in vitro angiogenic sprouting
Authors: Ali Ashrifa , Spagnolia Alessandra , Hull Maddysen , Geffre Marcus , Bialk Pawel , Kmiec Eric , Grove Bryon Dennis
Published: June 2021

Angiogenesis is a highly regulated physiological process that is controlled by several signaling pathways including Src dependent and cAMP dependent pathways. Currently, the mechanisms underlying the coordination and integration of these signaling pathways are unknown, but based on several knockdown studies, we postulate that gravin (AKAP12), which is expressed as two variants in endothelial cells, may serve as a scaffold to coordinate Src and cAMP dependent signaling during angiogenesis. To investigate this hypothesis, we are using gene editing and protein expression approaches to identify the role of each gravin variant and the role of the Src and PKA binding domains in gravin in angiogenesis. Here, we report on the effect of knocking out gravin variant 1 using CRISPR‐Cas9 on angiogenic sprouting in a 3D‐collagen gel model. Immortalized human umbilical vein endothelial cells (HUVEC‐TERT2) were transfected with a CRISPR‐Cas9 construct in which Cas9 was targeted to exon 3. An RFLP assay designed to identify changes in an HpyCH4III site at the targeted double stranded break point identified a positive clone which lacked the restriction site. Subsequent sequencing revealed a single base deletion at the targeted site in exon 3 of the AKAP12 gene. Reverse transcriptase PCR showed a partial loss of variant 1 mRNA and sequencing of the RT‐PCR product confirmed the single base deletion. Western blotting demonstrated loss of variant 1 in the CRISPR‐Cas9 modified cells while immunofluorescence microscopy using an anti‐gravin antibody that recognized the C‐terminal region of both variants revealed approximately a 50% decrease in fluorescence intensity in CRISPR‐Cas9 treated cells. This CRISPR modified cell line, expressing only variant 2 of gravin, showed altered cell morphology and behavior compared to wild type cells. When cultured on collagen gels, variant 1 knockout cultures showed significantly fewer sprouts than wild type cultures. Confocal microscopy revealed that fewer variant 1 knockout cells migrated into collagen gels, resulting in shorter sprouts with fewer branches, and that these cells displayed fewer filopodia than wild type cells. These results provide evidence that variant 1 of gravin plays a role in invasive cell migration and angiogenic sprouting, which are essential cellular events that regulate angiogenesis. Future studies will investigate the mechanism by which variant 1 contributes to angiogenic sprouting and the role that each variant of gravin plays in Src and cAMP dependent signaling in endothelial cell migration, adhesion and angiogenic sprouting.

Support or Funding Information

Supported by NIH grants P30GM103329, P20GM103442 and P20GM113123

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Effects of aspirin on melanogenesis in B16F10 cells grown as 3D hanging drop tumor organoids.
Authors: Martin Mike , Lyden Timothy
Published: June 2021

Aspirin (acetylsalicylic acid)has been shown to significantly reduce the melanin content in murine melanoma cells cultured in 2D. While this phenomenon has been attributed to lower tyrosinase expression and ERK‐induced degradation of MITF, the effect of aspirin treatment on the growth of melanoma cells in 3D, their morphology and melanosome secretion have yet to be characterized. Melanin secretion and gene expression data were generated from three and five, respectively, replicates of the following. Twelve hanging drop (HD) plates were seeded with B16F10 cells (5000 cells/well). Cells in six plates were cultured in DMEM+10% FBS+Pen/Strep (CONTROL; 50 ml/well) at 37C in a 5% CO2 in air environment. Cells in the remaining plates were cultured in the same medium supplemented with aspirin. Medium was changed daily (20 ml/well). Cells and supernatants were harvested (1 plate/treatment) at 24 hour intervals up to and including 144 hrs. of culture. Cells were washed in PBS and stored in either lysis buffer at −20C for Western analysis or RNA‐Later at −80C for qPCR. Supernatants were centrifuged in order to remove cells and 405l absorbance was measured. Aspirin visibly reduced the melanin content in B16F10 cells within 48 hrs. of initial exposure. Supernatants above both ASPIRIN and CONTROL cells contained significantly (P<0.05)more melanin at 120 hrs. of HD culture than any previous time point. The melanin content in ASPIRIN cell supernatants was also significantly (P<0.05) higher than that of CONTROL cell supernatants at 120 hrs. of HD culture. Tyrosinaseexpression and tyrosinaseprotein content were lower (P<0.03) in ASPIRIN as compared to CONTROL cells during the last 72 hrs. of HD culture. MITFexpression was also reduced (P<0.05)in ASPIRIN cells during the last 48 hrs. of HD culture. We have demonstrated that aspirin can reducethe melanin content in B16F10 cells cultured in 3D. This phenomenon may be due to aspirin's downregulation of two genes, tyrosinase and MITF, associated with the melanogenic pathway. Aspirin, however, did not affect melanin secretion by B16F10 cells grown in this HD plate format.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Identification of Genes Involved in Low Temperature Growth of Cronobacter sakazakii ATCC 29544
Authors: LIU JING , Hong Seong Tschool
Published: June 2021

Cronobacter sakazakii ATCC 29544 is a gram‐negative opportunistic pathogen, which can cause local necrotizing enterocolitis, bacteremia, and meningitis. Powdered infant formula is considered to be the major transmission route for newborn infections. Even though powdered infant formula is stored at a refrigerator temperature (5~10°C), Cronobacter sakazakii can still cause infection. In order to understand its molecular mechanism for low temperature growth, we first constructed random mutant library and screened for no growth at 8 °C. By using the plasmid recovery technique, soluble pyridine nucleotide transhydrogenase (sth), nusB and two ribosomal RNA genes were identified to be responsible for the reduced growth at low temperature. Overall, we report 11 genes that play an important role in the growth of C. sakazakii ATCC 29544 at low temperature.

Support or Funding Information

This research was financially supported by the “Global accompanied growth R&BD program (N042600010)” through the Ministry of Trade, Industry and Energy (MOTIE, Korea).

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Biochemical Characterization of the Set1 H3K4 Methyltransferase Complexes
Authors: Kim Jaehoon
Published: June 2021

Methylation on lysine 4 of histone H3 (H3K4) is one of the prominent histone modification marks that correlate strongly with active transcription in eukaryotes. Accumulating studies in metazoans have implicated misregulation of H3K4 methylation in the pathogenesis of cancer and in developmental defects, further emphasizing the importance of understanding the regulation of H3K4 methylation. In budding yeast, a single H3K4 methyltransferase (Set1) complex can methylate all H3K4 in an H2B ubiquitylation‐dependent manner. However, presence of at least six H3K4 methyltransferase (SET1A, SET1B, MLL1, MLL2, MLL3 and MLL4) complexes in mammalian cells has complicated functional characterization of each complex because of their possible redundant and non‐redundant roles. To understand the roles of H3K4 methyltransferases in transcriptional regulation in mammalian cells, we have deployed biochemical analyses with purified human H3K4 methyltransferase complexes. By taking advantage of an in vitro histone methyltransferarse assay employing a reconstituted human H3K4 methyltransferase complexes and a recombinant chromatin template containing fully ubiquitylated H2B, we found that a subset of human H3K4 methyltransferase complexes exhibit H2B ubiquitylation‐dependent H3K4 methylation activity. In addition, we demonstrate different subunit compositions and subunit interaction networks in human H3K4 methyltransferase complexes. Our studies establish minimal components of the H3K4 methyltransferase complexes required for H3K4 methylation and provide a mechanistic basis for H3K4 methylation in mammalian cells.

Support or Funding Information

National Research Foundation (NRF) of Korea (2015R1A1A1A05001593)

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
A novel approach to identify regulated long non‐coding RNAs in selected pathways
Authors: Okino Steven T. , Kong Michelle , Flynn James M. , Wang Yan
Published: June 2021

We have developed a highly sensitive RT‐qPCR‐based workflow that quantifies the expression of lncRNAs in a RNA sample. The workflow utilizes a novel reagent that enables streamlined genomic DNA removal, cDNA synthesis and multi‐target pre‐amplification and allows for reliable quantification of as little as 25 pg of RNA (single cell equivalent). This reagent can be used together with pre‐designed qPCR array panels that contain lncRNA targets identified as being involved in specific biological/pathophysiological pathways through perturbation experiments, guilt‐by‐association experiments or literature mining. We demonstrate the utility of this workflow by identifying seven lncRNAs that are regulated in early stem cell differentiation using the NTera2 model system. Two lncRNAs are suppressed and five lncRNAs are induced during the differentiation process and may play a role in the maintenance of the pluripotent state or in stem cell differentiation respectively. Two of the lncRNAs have been previously reported to be involved in stem cell differentiation; the other five lncRNAs appear to be a new finding. We believe that this workflow represents a powerful discovery tool to identify lncRNAs involved in biological/pathophysiological pathways and can promote new insights in lncRNA research.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Micro‐RNA‐205 Regulates Heart Size Through Direct Modulation of the Hippo Pathway
Authors: Weldrick Jonathan James , Kouri Lara , Yi Rui , Burgon Patrick Gary
Published: June 2021

During fetal development, the mammalian myocardium undergoes a period of hyperplastic growth, which establishes the number of cardiomyocytes in the adult heart. After birth, cardiomyocytes proceed through a final round of cell division in the absence cytokinesis that results in binucleation of >95% of adult cardiomyocytes. Nearly all subsequent increase in myocardial mass is due to myocardial hypertrophy, with extremely low number of new cardiomyocytes being produced throughout post‐natal life. Despite the importance of this phenomenon, little is known about the molecular/genetic basis, especially with regard to the role of micro‐RNAs, for the transition from hyperplastic to hypertrophic‐based myocardial growth.

We hypothesize a specific perinatal heart micro‐RNA‐mediated gene program is necessary for the normal transition from a fetal heart to an adult heart gene program.

To identify the molecular mechanisms and genetic pathways involved in cardiac myocyte differentiation, RNA was isolated from E19, and 1, 3, 5, 7, 10 and 35‐day old mouse hearts (n=9 hearts/time point pooled). Cardiomyocyte micro‐RNA profiles (n=3 arrays/time point) were measured and bioinformatic analysis was used to identify genes that are transiently and significantly changing (p<0.05, fold change >1.5) during the perinatal period.

Our analysis identified microRNA‐205 as a candidate for playing a role in the cardiac transitional program. Previous studies have shown a global knockout of miR‐205 to be neonatally lethal. We observe a transient 20‐fold increase in miR‐205 expression between day 1 and day 5 of post‐natal life, with levels returning to baseline by day 10. In‐situ hybridization revealed miR‐205 expression to be restricted to the epicardium of the heart.

Mice harbouring a cardiomyocyte‐specific deletion of miR‐205 using αMHC‐Cre are born healthy with expected Mendelian ratios, and develop through the neonatal period normally. Hearts collected from adult mice show signs of abnormal growth and hypertrophy, up to 50% larger than controls. Previous studies demonstrated that miR‐205 directly targets YAP within the evolutionarily conserved hippo pathway that controls organ size. Hearts lacking miR‐205 exhibit a substantial increase in YAP protein expression. Increased cardiac size has been demonstrated in a constitutively active YAP transgenic mouse model. We conclude that miR‐205 plays a direct role in regulating post‐natal heart size through direct modulation of the Hippo pathway.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
The Effect of 1,4‐dimethylnaphthalene on mir‐166 microRNA
Authors: Alharbi Alhanouf , Campbell Michael
Published: June 2021

The compound 1,4‐dimethylnaphthalene (DMN) is a natural growth inhibitor produced in dormant potatoes. It prevents potato tubers from sprouting by halting cell division. Recent studies have shown DMN prevents the growth of microbiomes present on the surface of stored potato tubers. However, the effect of DMN on small RNAs has not been yet investigated. The molecular miR166 is a highly conserved class of microRNA in many plants. It is found in meristems, and it modulates transcripts of the III homeodomain‐leucine zipper family genes to control plant growth. High expression of miR166 in Arabidopsis thaliana downregulated transcription factors such as Phavulota (PHV) and Phabulosa (PHB), suppressing growth of various parts of the plant. Therefore, we hypothesized that DMN increases miR166 expression to inhibit sprouting. Potatoes were exposed to DMN or nuclease‐free water (control) for three days. Meristems were isolated, frozen in liquid nitrogen, and stored at −80 C. RNAs were isolated using mirVana kit (Thermofisher.com). Total RNA was measured and converted into cDNAs using stem‐loop primers, specifically made for mir166. TaqMan qtPCR was used to quantify the expression of mir166 in control and treated samples. The results obtained from TaqMan qtPCR showed that the expression of miR166 in the treated sample was equivalent to the control sample, suggesting that DMN did not change miR166 expression in dormant potatoes. Further experiments will determine if DMN alters miR166 expression in potatoes after dormancy terminates.

Support or Funding Information

Supported in part by a grant from the 1,4‐Group, Meridian, ID

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Calcineurin Gene Subfunctionalization and Swimming Behavior in Paramecium tetraurelia
Authors: McKenna Strange Madigan , Day Alex , Fraga Dean , Welker Jordan , Gaines Ray , Barrin William
Published: June 2021

The protein phosphatase calcineurin (PP2B) plays a multifaceted role in the motility of the unicellular Paramecium tetraurelia. The Paramecium tetraurelia genome is the product of at least three whole genome duplications resulting in multiple ohnologs pairs for many genes in the Paramecium genome. The most recent duplication resulted in 7 ohnolog pairs that are virtually identical at the amino acid level. Each pair of ohnologs was tested for their role in the swimming behavior of Paramecium using RNAi to silence each ohnolog pair. The behavioral effects were tested in both wildtype and the mutant strain, Dancer, which displays exaggerated backward swimming due to a reduced ability to close the voltage dependent calcium channel after opening. The results suggest that the calcineurin ohnolog pairs are undergoing subfunctionalization. This conclusion is also supported by a real‐time rtPCR analysis of relative gene expression in log‐phase cells in which the different isoforms display different gene expression levels under these growth conditions. These results will be discussed in the context of gene evolution after duplication.

Support or Funding Information

The College of Wooster Sophomore Research Program

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
USP21 and OTUD3 Antagonize Regulatory Ribosomal Ubiquitylation and Ribosome‐associated Quality Control Pathways
Authors: Garshott Danielle M. , Leonard Marilyn , Sundaramoorthy Elayanambi , Bennett Eric J.
Published: June 2021

Ribosomal translation of messenger RNA into properly decoded polypeptides is a tightly‐controlled process that is regulated by a number of cellular quality control pathways. When ribosomes encounter defective mRNA or otherwise experience a terminal stall in translation, the ribosome‐associated quality control pathway is initiated. Conserved ubiquitylation events on specific 40S ribosomal proteins are required for downstream destruction of the mRNA and nascent peptide as well as recycling of ribosomal subunits. We have previously demonstrated that the E3 ubiquitin ligase, ZNF598, is responsible for regulatory ubiquitylation (RRub) at specific sites on the 40S ribosomal subunit during ribosome‐associated quality control events. Here we demonstrate that the deubiquitylating enzymes USP21 and OTUD3 directly antagonize these regulatory ribosomal ubiquitylation events, and catalyze the deubiquitylation of particular 40S ribosomal proteins. For the majority of cell types, the relative RNA expression and protein levels of ZNF598 as compared to USP21 and OTUD3 is substantially higher for the ligase, suggesting a constrained stoichiometric balance that is required to maintain cellular homeostasis. Overexpression of USP21 or OTUD3 results in enhanced read‐through of polyA sequences that would normally induce ribosomal stalling and abort translation elongation. Our results establish USP21 and OTUD3 catalyzed RRub deubiquitylation as a critical checkpoint on ribosome‐associated quality control.

Support or Funding Information

This project described was supported by Grant Number T32 ES007059 from NIH NIEHS and its contents are solely the responsibility of the authors and do not necessarily represent the official views of the NIEHS or NIH.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Plasma metalloproteinase‐9 (MMP9) changes in acute mild traumatic brain injury (mTBI) and correlates with quantitative EEG
Authors: Hubbard Eric , Dawlaty Jessica , Arakaki Xianghong , Cole Soren , Goldweber Robert , Harrington Michael
Published: June 2021

Background and Objectives

We aim to explore blood plasma levels of matrix metalloproteinase‐9 (MMP9) in acute mild traumatic brain injury (mTBI), and test if MMP9 levels correlate with quantitative electroencephalography (qEEG) during working memory (WM) testing.

Methods

Study participants were recruited from the emergency department of Huntington Memorial Hospital in Pasadena, CA, consisting of thirteen acute mTBI civilian patients and seven controls who were trauma patients without head injury ranging between 18–50 years of age. Blood samples were collected from three time points: within 1 week, 14 days, and 30 days after the injury. To study blood‐brain‐barrier (BBB) integrity, we quantified three MMP9 peptides (SLGPALLLLQK, QLSLPETGELDSATLK, and LGLGADVAQVTGALR) using liquid chromatography and mass spectrometry with stable isotope standards. We also employed qEEG at each visit to investigate alpha frequency power during N‐back WM processing.

Results & Discussion

We detected the presence of all three MMP9 peptides in blood plasma. We observed that MMP9 peptide levels in both mTBI and controls were decreasing in abundance in the 2–4 weeks after injury compared to the first week. Importantly, the MMP9 levels of the LGLGADVAQVTGALR peptide but not the peptides from the pre‐protein, were significantly higher in the mTBI group 2–4 weeks after the injury, consistent with known “secondary injury” phenomena. MMP9 levels correlated with alpha power during WM testing, at the first visit for the controls but not for the mTBI patients, at specific brain regions during different WM load. Elevated MMP9 levels indicate the BBB integrity is compromised acutely after mTBI. The correlations between MMP9 and alpha power during WM that we only found in the trauma controls need further investigation.

Support or Funding Information

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
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 Information

This 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 Information

This work was supported by West Virginia University's School of Medicine, (10020452.15.F2R226W) startup foundation funding, and National Institutes of Health Grant GM119528

This 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 Information

This work was supported by R00GM112862 and the State University of New York

This 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 Information

This 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 Information

The 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 Information

Self Funded

This 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 Information

The National Institutes of Health (R01 GM111820) University of Delaware David A. Plastino Fellowship

This 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

Background

A 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.

Objectives

The 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.

Methods

The 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.

Results

We 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.

Conclusions

We 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.
Purinergic Signaling Controls Fibroblast Growth Factor‐21 Expression in Skeletal Muscle through Akt/mTOR Pathway.
Authors: Arias‐Calderón Manuel , Morales Camilo , Vasquez Walter , Hernández Nadia , Jaimovich Enrique , Buvinic Sonja
Published: June 2021

Fibroblast Growth Factor‐21 (FGF‐21) is a myokine which induce fiber type transformation and metabolic adaptations in muscle. However, little is known about the molecular mechanisms involved in the regulation of FGF‐21 expression in skeletal muscle in non‐pathological conditions. We have previously described that purinergic signaling is related with muscle adaptations and IL6‐myokine production through the “excitation‐transcription coupling” (ETC) mechanism, in which extracellular ATP (eATP) promotes gene expression in skeletal muscle. We have also demonstrated that eATP activates the PI3K/Akt signaling pathway in myotubes. Considering that Akt has been proposed as a master regulator for FGF‐21 expression control, the aim of this work is to determine the role of extracellular ATP over the FGF‐21 production in adult mouse skeletal muscle, and their dependence on the Akt/mTOR signaling pathway. Mice FDB‐isolated fibers or whole‐muscle were used in this study. The mRNA levels of FGF‐21 after electrical (20 Hz, 270 pulses, 0.3 msec each) or exogenous ATP (100 μM) stimulation were assessed by qPCR. FGF‐21 protein levels were assessed by immunoblot. We used pharmacological inhibitors of purinergic receptors (100 μM Suramin) and Akt/mTOR pathway inhibitors (50 μM LY294002; 1 μM Akt inhibitor VIII; 100 nM rapamycin), to turn off different interactors of the putative signaling pathway. ATP stimulation (100 μM) evoked an 8‐fold increase in FGF‐21 mRNA levels in isolated FDB fibers at 30 min post stimulation. The increase in FGF‐21 mRNA levels evoked by eATP was inhibited by suramin, a general antagonist of P2Y/P2X nucleotide receptors. Electrical stimulation also evokes an increase in FGF‐21 mRNA levels, which was inhibited by both nifedipine (a DHPR blocker) and suramin. Exogenous ATP increased protein levels of FGF‐21 in whole FDB‐muscles at 120 min, in a concentration‐dependent manner, with a maximal increase of 3.5 folds with 3 μM ATP. The increase in FGF‐21 protein level evoked by ATP, was abolished by LY294002 (a PI3K inhibitor), Akt inhibitor VIII, and rapamycin (mTOR inhibitor). In this work, we demonstrated for the first time that extracellular ATP evokes an increase in the mRNA and protein levels of FGF‐21 in skeletal muscle. The increase on FGF‐21 levels evoked by ATP is dependent on Akt/mTOR pathway activation. These results suggest that ETC mechanism could be involved in regulation of FGF‐21 expression in skeletal muscle.

Support or Funding Information

Funded by CONICYT PCHA 21151035(MA‐C), 63140009(CM) and Fondecyt 1151353(SB), 1151293(EJ)

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Conserved salt‐bridge competition triggered by phosphorylation regulates the protein interactome
Authors: Skinner John James , Wang Sheng , Lee Jiyoung , Ong Colin , Sommese Ruth , Sivaramakrishnan Sivaraj , Koelmel Wolfgang , Hirschbeck Maria , Schindelin Hermann , Kisker Caroline , Lorenz Kristina , Sosnick Tobin , Rosner Marsha Rich
Published: June 2021

Phosphorylation is a major regulator of protein interactions; however, the mechanisms by which regulation occurs are not well understood. Here we identify a salt‐bridge competition or “theft” mechanism that enables a phospho‐triggered swap of protein partners by Raf Kinase Inhibitory Protein (RKIP). RKIP transitions from inhibiting Raf‐1 to inhibiting G‐protein–coupled receptor kinase 2 upon phosphorylation, thereby bridging MAP kinase and G‐Protein–Coupled Receptor signaling. NMR and crystallography indicate that a phosphoserine, but not a phosphomimetic, competes for a lysine from a preexisting salt bridge, initiating a partial unfolding event and promoting new protein interactions. Structural elements underlying the theft occurred early in evolution and are found in 10% of homo‐oligomers and 30% of hetero‐oligomers including Bax, Troponin C, and Early Endosome Antigen 1. In contrast to a direct recognition of phosphorylated residues by binding partners, the salt‐bridge theft mechanism represents a facile strategy for promoting or disrupting peptide interactions using solvent‐accessible residues, and it can provide additional specificity at protein interfaces through local unfolding or conformational change.

Support or Funding Information

GM087630 (to M.R.R.), GM55694 (to T.S.), Deutsche Forschungsgemeinschaft (FZ82: K.L., C.K., and H.S.; SFB688, TPA17: K.L.) and the Ministry for Innovation, Science and Research of the Federal State of North Rhine‐Westphalia (K.L.).

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.