bluuebunny/crossref_metadata_2025 huggingface dataset:
Inhibition of HDAC1 and 2 Modulates the Expression and Signaling of Natriuretic Peptide Receptor A in Male and Female Gene‐targeted Mutant Mice
Authors: Kumar Prerna , Nguyen Christian , Samivel Ramachandran , Bloodworth Meaghan , Pandey Kailash N.
Published: June 2021

Guanylyl cyclase/natriuretic peptide receptor‐A (GC‐A/NPRA) signaling activates natriuresis, diuresis, and vasodilatation, and inhibits the cell proliferation and fibrosis. Mice lacking functional Npr1 gene (coding for GC‐A/NPRA) exhibit hypertension and kidney disorders, however, the mechanisms regulating Npr1 expression and function are not well understood. The objective of the present study was to elucidate the mechanisms of Npr1 expression and receptor function involving epigenetic regulatory mechanisms activated by class I histone deacetylase (HDAC) inhibitor, Mocetinostat (MGCD0103). Male and female (16–20 weeks old) Npr1 gene‐disrupted heterozygous (1‐copy; Npr1+/−), wild‐type (2‐copy; Npr1+/+) and gene‐duplicated heterozygous (3‐copy; Npr1++/+) mice were injected intraperitoneally with MGCD0103 (2 mg/kg) at alternate days for 2‐weeks. The Western blot analysis showed that MGCD0103 significantly increased the renal NPRA protein levels in Npr1+/−, Npr1+/+, and Npr1++/+ mice groups compared with vehicle‐treated controls. Female mice showed increased levels of plasma cGMP compared with male mice and treatment with MGCD0103 significantly induced cGMP levels in the drug‐treated mice groups. The renal HDAC activity and HDAC1 and 2 protein levels in male mice were higher than the corresponding female mice in all the three genotypes. MGCD0103 treatment attenuated HDAC activity by 30%‐40% and HDAC1/2 protein levels in the treated mice groups. Male and female mice showed differential acetylation levels of H3‐K9 and H3‐K27 in the renal tissues and treatment with MGCD0103 significantly enhanced the acetylation levels in the treated mice groups. Both male and female Npr1+/− mice exhibited higher systolic blood pressure (SBP) compared with the corresponding wild type Npr1+/+ mice, whereas female Npr1+/− mice exhibited significantly lower SBP than male Npr1+/− mice. A significant decrease in SBP was observed in MGCD0103‐treated male and female Npr1+/− mice. The present results suggest that MGCD0103 epigenetically regulates NPRA expression in vivo in male and female Npr1+/− mice, via inhibition of HDAC1 and 2 protein expression and histone modifications, which will have important implications in the hypertension and renal disease states.

Support or Funding Information

This work was supported by NIH grants (HL057531 and HL062147) and by an Institutional Development Award (IDeA) from the NIGMS.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
An Investigation on Collagen Production by Cells on Ligament Scaffolds
Authors: Johnson Jordan Leigh , Guardia Angelica , Alismail Kathm , Li Yawen , Zwiesler‐Vollick Julie
Published: June 2021

Ligament tears are a major injury that is common place among sports, car accidents, and falls. Each year hundreds of thousands of people receive treatment for a ligament tears. Current repair methods include biological and synthetic grafts that often result with complications such as swelling, rejection, and failure of the graft. When these complications arise further treatment is necessary, resulting in more medical bills and a longer recovery time. There is a need for a more effective repair method. Another approach for ligament repair is to use tissue engineering. Effective tissue engineering requires a scaffold which can recruit cells and promote growth and differentiation. Previous work with our collaborators has shown that a scaffold made from extracted collagen, which is electrospun into fibers, can effectively recruit cells. One of the hallmarks of ligament differentiation is collagen production by the recruited cells. However, we cannot use collagen protein detection to characterize differentiation because we cannot discriminate between newly synthesized collagen (translated from Col1a mRNA) from cells and collagen present in the scaffold. We are developing a reverse‐transcriptase quantitative polymerase chain reaction (RT‐qPCR) assay to determine the relative amounts of Col1a mRNA present in cells. This will allow us to determine if cells on collagen scaffolds are actively synthesizing collagen. We are currently refining our procedure by analyzing the 3T3 stable line of Mus musculus ovarian stem cells maintained in culture. We have successfully extracted RNA, synthesized cDNA, and measured Col1a expression. Glycolic acid has been shown to increase collagen production in cell culture. We will be comparing cells treated with glycolic acid to control cells to determine if we can detect changes in Col1a mRNA levels. Once these procedures have been established we will examine cells from collagen scaffolds as well. The results from this study will be presented.

Support or Funding Information

Funding for this experiment is provided by Lawrence Technological University.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
High throughput discovery of novel regulators of human ribosome biogenesis
Authors: Baserga Susan J. , Farley‐Barnes Kathleen I. , McCann Kathleen , Ogawa Lisa , Merkel Janie , Surovtseva Yulia
Published: June 2021

It is well known that in cancer an increased size and number of nucleoli correlates with increased malignant potential. Nucleoli make ribosomes, the essential protein synthesizers of the cell. Previously we have shown (PLoS Genetics 2012) that disruption of nucleolar function in mammalian cells by depletion of the ribosome biogenesis factors UTP4 or NOL11 causes a change in the number of nucleoli per cell from 2–3 to only 1. Taking advantage of this relationship between number and function, we exploited this assay to discover new regulators of nucleolar function in human cells via a high‐content, high‐throughput, genome‐wide siRNA screen. This screen successfully identified approximately 200 proteins whose depletion caused a change in the number of nucleoli per cell from 2–3 to only 1. The function of a subset of these hits in ribosome biogenesis has been validated by oligonucleotide deconvolution. Of these high‐confidence, validated hits, 20 were chosen for further study, including both nucleolar and non‐nucleolar proteins. While the nucleolar proteins are directly involved in ribosome biogenesis, the non‐nucleolar proteins likely reflect new pathways of nucleolar regulation in mammalian cells. Out of the 20 tested hits, we have identified new roles for 17 hits in regulating either the RNA polymerase I transcription of the ribosomal DNA (7/20), or in the processing of the pre‐ribosomal RNA (16/22). These hits increase the complexity of ribosome biogenesis, extending it beyond the nucleolus to extra‐nucleolar protein regulators of nucleolar function.

Support or Funding Information

Supported by NIH R01GM0115710 and by the Yale Cancer Center.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Translation termination on mRNAs lacking a stop codon
Authors: Zeng Fuxing , Jing Hong
Published: June 2021

Translation of mRNAs often terminates at the end of the coding sequence when a stop codon is encountered. However, in nonstop mRNAs that lack a stop codon, the ribosome stalls at the 3′ end of the mRNA and needs to be rescued. In bacteria, stalled ribosomes are rescued by the transfer‐messenger (tmRNA)‐SmpB, the alternative rescue factor A (ArfA)‐RF2 or ArfB proteins (1–3). Although crystal structures of ribosomes complexed with tmRNA‐SmpB and ArfB were solved, the mechanism and molecular interactions involved in the ArfA‐RF2 pathway remained unclear. Recently, an induced‐fit mechanism has been reported for this pathway (4). Here we present a cryoEM structure of the Escherichia coli 70S ribosome on a nonstop mRNA in the presence of ArfA and methylated RF2 (5). Molecular interactions revealed by the structure show how ArfA recognizes a truncated mRNA and recruits the canonical termination factor RF2 to rescue the stalled ribosome (Figure 1). The positively charged C terminus of ArfA occupies the empty mRNA entry channel on the 30S subunit. Consistent with our kinetic and biochemical data, specific molecular interactions show how ArfA recruits RF2, not RF1, to the stalled ribosome. The N‐terminal domain of ArfA interacts with the switch loop of RF2, directing the universally conserved methylated Gly‐Gly‐Glnm (GGQm) motif of RF2 into the peptidyl‐transferase center (PTC) of the ribosome. Furthermore, the conformation of the methylated GGQm motif was determined, which shows that the post‐translational modification stabilizes the side chain of glutamine and thus helps orient the carbonyl oxygen of the glutamine towards the catalytic water molecule. Taken together, we determined the molecular mechanism and interactions important for the ArfA/RF2‐mediated ribosome rescue on nonstop mRNAs and provide insights into the function of the methyl group on the GGQ motif in translation termination on the ribosome.

Support or Funding Information

This study was supported from the National Institute of General Medical Sciences of the NIH (R01‐GM120552). The computational part of the study was supported by the National Institute of General Medical Sciences (P41‐GM104601 to J.C.P. and E.T.).

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Rps10 Protein Contribution to Ribosomal mRNA Selectivity
Authors: Bush Jessica A. , Ferretti Max , Karbstein Katrin
Published: June 2021

Previous work from the Karbstein Lab on ribosomal biogenesis has shown that interactions between the ribosomal protein Rps26 and the mRNA promotes mRNA selectivity, thereby shaping protein homeostasis. Importantly, cells lacking sufficient amounts of Rps26 follow a distinct translational program, which might explain in part the pathogenesis of Diamond‐Blackfan Anemia (DBA), a disease caused by lack of ribosomal proteins, including both Rps26 and Rps10. Both Rps26 and Rps10 are located near the translated mRNA within the entry channel and can be used to predict contact with mRNA downstream of the start and stop codon during translation initiation and termination, respectively. Initial tests were conducted to observe the effects from mutation of the putative Rps10‐contact region on translation. Five mutated plasmids were studied for their affect on Rps10 binding. Results from dual luciferase assays have shown a significant decrease in protein production as a result of mutations in the mRNA plasmid. This decrease appears be dependent on Rps10 levels when compared to the traditional Kozak sequence. To test this hypothesis three different yeast strains were used, WT, TEF and Dox yeast strains. Wild type yeast cells contain their own endogenous copies of the Rps10 genes. TEF and Dox are both genetically modified yeast strains in which the endogenous copies of Rps10 were removed from the genome and replaced by a newly introduced copy in a plasmid under control of either a TEF or Dox promoter. These promoters allow for controlled Rps10 production levels. Doxycycline can be added to cells to further repress their Rps10 production. Using western analysis, we observed that yeast cells with the Dox promoter and treated with doxycycline had significantly reduced levels of Rps10 in their ribosomes. Cells that were successfully depleted of Rps10 had significantly lower protein production then their Rps10 replete counterparts. This translational difference is believed to be a result of a decreased ability of the ribosome to bind mRNA as a result of diminished Rps10 levels.

Support or Funding Information

K. Karbstein is an HHMI Faculty Scholar

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 and characterization of serpin genes in Manduca sexta
Authors: Li Miao , Christen Jayne , Dittmer Neal , Cao Xiaolong , Zhang Xiufeng , Jiang Haobo , Kanost Michael
Published: June 2021

Insects adopt a variety of immune mechanisms to defend against invading pathogens, such as phagocytosis, encapsulation, melanization and antimicrobial peptide synthesis. Serine protease cascades play an important role in some of these immune responses, and serpins (serine protease inhibitors) regulate the proteases after zymogen activation. Serpins are a superfamily of proteins, which share a conserved tertiary structure composed of three β‐sheets, 8–9 α‐helices, and an exposed reactive center loop (RCL). Once encountering its specific serpin, the protease cleaves the serpin RCL at the scissile bond, resulting in the insertion of RCL into β‐sheet A as a new strand and the formation of an irreversible inhibitory complex. Our group uses Manduca sexta a model insect to study the insect immune system. By searching the whole genome of M. sexta, 32 serpin genes were identified. MsSerpin1, 15 and 28 are capable of encoding more than one polypeptide with different RCL sequences via alternative splicing. Besides 12 isoforms of MsSerpin1 previously known, two additional isoforms were identified, increasing the total number of splicing isoforms of MsSerpin1 to 14. Phylogenetic analysis of serpins in M. sexta and the silk worm Bombyx mori indicate evolutionary conservation of some serpins, with 17 orthologous pairs identified. These include 7 characterized M. sexta serpins that are involved in protease cascades activating the prophenoloxidase or Toll immune pathways. The majority of M. sexta and B. mori serpin genes contain 6–10 exons except for a clade including 10 M. sexta serpin genes and 7 B. mori serpin genes with only 1–3 exons. The analysis of RCL regions indicates that about three quarters of M. sexta serpin proteins are likely to be inhibitory. Transcription level of MsSerpin‐1–7, 11–12, 14–15, 17–18 and 22 displayed an up‐regulation in fat body or hemocytes upon immune challenge. Transcript profiles give hints to the temporal and spatial expression patterns of each serpin, which lead to suggestions for their functional study in the future.

Support or Funding Information

NIH grants GM041247, GM58634 and AI11266

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
“Effect of inhibiting protein conformational changes by introducing disulfide bonds in the transmembrane domains of the Hendra virus fusion protein”
Authors: Slaughter Kerri Beth , Dutch Rebecca
Published: June 2021

Hendra virus (HeV) is a zoonotic pathogen that causes severe respiratory illness and encephalitis. However, no human vaccine or antiviral treatment is currently available. As a member of the Paramyxoviridae family, HeV consists of a non‐segmented negative‐sense RNA genome and is enclosed within a lipid bilayer which contains the surface glycoproteins known as the attachment protein (G) and the fusion protein (F). The G protein plays a role in target cell receptor binding, and the F protein promotes membrane fusion between the viral envelope and the target cell membrane to allow for viral entry and infection. Previous studies have shown that viral fusion proteins undergo conformational changes to drive fusion. For HeV, the F protein associates as a homotrimer, and research from our group suggests that the transmembrane domains (TMDs) stabilize the protein in its pre‐fusion state.1 Based on this research, we hypothesized that modulation of TMD interactions is critical for initiation and completion of the conformational changes that drive membrane fusion. To test this, three HeV F constructs (T483C/V484C, V484C/N485C, N485C/P486C) were generated with double cysteine mutations near the N‐terminal region of the TMD to study the effect of altered conformational flexibility in this region. Initially, the mutants were analyzed for intermolecular disulfide bond formation. Results showed that the mutants primarily associated in a trimeric form under non‐reducing conditions, in contrast to the wild type (WT) protein which was primarily monomeric, suggesting that the mutant TMDs were linked by disulfide bonds. Further experiments showed that all three mutants expressed in cells at levels similar to WT HeV F. However, cleavage of HeV F to its fusion‐active form was reduced for the three mutants, suggesting that the mutants are trafficked more slowly or undergo less proteolytic processing by the endosomal protease cathepsin L compared to WT HeV F. The mutants were then assessed for their ability to promote membrane fusion. Preliminary fusion studies showed that fusion was dramatically reduced for the mutants, suggesting that TMD dissociation is critical for initial conformational changes in HeV F. Future studies will further examine the conformational changes that can occur when dissociation of the TMDs is blocked by the introduction of disulfide bonds.

Support or Funding Information

The authors gratefully acknowledge funding from the NIH/NIAID, grant R01 AI051517.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Crystallographic studies of human acetylcholinesterase inhibition by organophosphates and reactivation by oximes
Authors: Gerlits Oksana , Fajer Mikolai , Cheng Xiaolin , Blumenthal Donald , Taylor Palmer , Radic Zoran , Kovalevsky Andrey
Published: June 2021

Human acetylcholinesterase (hAChE; EC 3.1.1.7) is responsible for degrading neurotransmitter acetylcholine at synapses of the nervous system. Organophosphate (OP) nerve agents and pesticides inactivate hAChE through chemical modifications of the enzyme's active center leading to a life‐threatening increase of acetylcholine (ACh) concentration. Exposure to OPs may be fatal if not treated. The current generation of oxime antidotes is not highly efficient; the rates of nucleophilic reactivation are far slower than the catalytic rate of ACh hydrolysis. Insights into the molecular structures of Torpedo and mammalian AChEs reveal the underlying limitations in enhancing reactivation rates, but limited in their ability to resolve positions of hydrogens important for understanding proton transfer in reactivation of OP‐inhibited AChEs. The only experimental method that can provide this information is macromolecular neutron crystallography.

In preparation for the neutron diffraction studies we have obtained several new X‐ray structures of native and phosphonylated (with VX and/or POX) hAChE in complex with oxime reactivators, RS2‐170B and RS‐194B, at 2.15–2.45 Å resolution range. In these complexes the enzyme forms crystals in previously unseen unit cell (a=b=124.3, c=129.1 Å; space group P31) amenable to neutron crystallography. Ternary structures with RS2‐170B reveal that the oxime binds in two conformations to non‐modified and OP‐conjugated active center suggesting that conformational changes can happen inside the active center cavity for this reactivator. In one conformation the oxime moiety extends towards the catalytic Ser203 and could represent reactive orientation of the oxime. Conversely, in our structures RS‐194B binds with the oximate pointing away from Ser203 and towards the entrance to the gorge in the non‐modified active center and the entire molecule is pushed out of the active center to bind in the peripheral site in the VX‐modified structure. POX binding induces significant changes in the acyl pocket loop conformation expelling the weakly binding RS‐194B from the active site gorge completely. This alternation may hinder oxime binding for reactivation.

In addition, we have determined the first room temperature (RT) structure of hAChE in complex with the oxime reactivator RS2‐170B at 2.8 Å. Detailed analysis of interactions between the reactivator and the enzyme in both 100K and room‐temperature structures will be presented.

Support or Funding Information

Supported by Grant 1U01NS083451 by the COUNTERACT program from NINDS

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Dynamics of L‐Kynureninase Orthologs during Catalysis
Authors: D'Arcy Sheena , Karamitros Christos S. , Stone Everett , Georgiou George , Murray Kyle
Published: June 2021

The breakdown of tryptophan in eukaryotes and some bacteria can proceed via a L‐Kynurenine (Kyn) intermediate. This intermediate has three potential fates; it can be converted to kynurenic acid, hydroxylated to 3‐hydroxyl‐kynurenine (3OH‐Kyn), or hydrolyzed to L‐anthranillic acid and alanine. L‐Kynureninase (Kynase) is the aminotransferase enzyme responsible for hydrolysis of either Kyn or 3OH‐Kyn. Kynase functions as a homodimer, with one chain contributing an essential pyridoxal‐5′‐phosphate cofactor, and the other chain contributing three catalytic residues. High‐resolution structural studies show that while human and bacterial Kynase orthologs are structurally similar, they have distinct catalytic residues. Enzyme studies also identify different substrate specificities, with the human ortholog preferring 3OH‐Kyn and the bacterial ortholog preferring Kyn. The basis for this preference is beyond the catalytic residues, as simply switching active sites between orthologs fails to switch substrate preference. The goal of this study was to characterize the dynamics of Kynase enzymes during catalysis. To do this, we have used hydrogen/deuterium exchange coupled to mass spectrometry. Through a substantial series of experiments, we have compared the dynamics of several Kynase orthologs and engineered Kynase mutants. Data sets were collected for enzymes without substrate, as well as with Kyn and 3OH‐Kyn. The results identify the determinants of substrate specificity and provide insight into mechanisms of catalysis. This information is invaluable in the design of Kynase‐based enzyme therapeutics, as well as our general understanding of enzyme dynamics.

Support or Funding Information

Startup funds to S. D'Arcy from The University of Texas at Dallas.

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 control of metabolite flux through a bacterial heme biosynthetic pathway
Authors: Celis Arianna Ixcheel , Choby Jacob , Skaar Eric , DuBois Jennifer
Published: June 2021

Heme is an essential Fe‐containing cofactor. The final two reactions of the heme biosynthesis pathway in Gram‐positive bacteria, including several important pathogens, are unique to these organisms. The substrates for these reactions, including coproporphyrin III, Fe(II), and H2O2, are highly reactive and toxic to the cell. We hypothesized that metabolite flux via a transient protein‐protein interaction between the final two pathway enzymes, designated HemH and HemQ, might therefore be essential in these species. In this study, we showed that the UV/Vis spectral features of coproporphyrin III serve as an indicator for an in vitro protein‐protein interaction between HemH and HemQ from Staphylococcus aureus. HemH and HemQ interacted in a 5:1 HemH:Q monomer ratio specifically at high concentrations of both proteins. Interestingly, the rates of reaction for these enzymes were not affected by the presence of the other, suggesting that the role of this interaction solely supports trafficking of pathway intermediates. The ΔhemQ knock out strain of S. aureus was complemented with single amino acid mutants of HemQ, in which either the enzymatic function or putative sites of protein‐protein interaction were lost. The molecular and growth phenotypes of these mutants were examined, with the goal of confirming possible sites of protein‐protein interaction that were essential to function. Understanding the nature of interactions between these enzymes and those further upstream in the pathway could provide new means to specifically target pathogenic Gram‐positive bacteria like S. aureus.

Support or Funding Information

NIH ROI GM‐090260

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Arachidonic Acid and Oxidation in the Myosin II Motor Domain
Authors: Wong Kellie , Thomas David , Moen Rebecca
Published: June 2021

Myosin is the motor protein responsible for movement and muscle contraction in all eukaryotic cells. The catalytic motor domain of myosin II contains three functionally important subdomains: the actin‐binding cleft, the force‐generating domain, and the nucleotide binding pocket. The chemomechanical coupling in the myosin catalytic domain allows for hydrolysis of adenosine triphosphate (ATP) into adenosine diphosphate (ADP) and inorganic phosphate (Pi), producing the energy needed for the contraction of muscle. This chemical reaction of ATP hydrolysis in the nucleotide binding cleft must be accompanied by a crucial interaction with actin to allow for the proper production of mechanical force. Various physiological and pharmacological effector molecules interact with myosin, specifically in its actinbinding cleft, and affect the actomyosin functional interaction. For example, oxidation of a methionine amino acid residue in the actin‐binding cleft, M394, decreases myosin's actin‐activated ATPase activity and changes the structural dynamics of the actin‐binding cleft of myosin (1). Conversely, arachidonic acid, a bioactive lipid produced naturally in eukaryotic cells, acts as a positive allosteric effector of myosin and is postulated to bind near the actin‐binding cleft of myosin, increasing the rate at which myosin hydrolyzes ATP (2). We have investigated the combined functional and structural effects of oxidation and arachidonic acid on the myosin motor domain in a Dictyostelium myosin II model system. Changes in actomyosin functional interaction in the presence of arachidonic acid after in vitro oxidation was assessed using a myosin ATPase activity assay and an actomyosin cosedimentation assay while changes in structural dynamics of the actin‐binding cleft was investigated using electron paramagnetic resonance (EPR). The results illustrate the importance of myosin's actin‐binding cleft structural dynamics and how allosteric effectors effect actomyosin functional interaction.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Dissecting the Function of IAP (Inhibitor of Apoptosis) Protein Domains in Inhibiting an Apoptotic Caspase
Authors: Storm Mackenzie Bryan , Junker Matthew
Published: June 2021

Apoptosis (programmed cell death) is an essential process in all animals for normal development and tissue regeneration. Apoptosis also eliminates cells at risk for becoming cancerous or spreading a viral infection. Apoptosis requires the activity of intracellular caspase enzymes that are normally kept inhibited in living cells by inhibitor of apoptosis proteins (IAPs). Certain apoptosis‐promoting proteins bind IAPs to relieve caspase inhibition. IAPs contain 2–3 BIR (baculovirus IAP repeat) domains with intervening linkers. In many IAPs, residues in a linker bind and block the caspase active site but the BIR domains also influence caspase inhibition. To determine how the BIRs contribute to caspase inhibition, we compared the ability of different fragments of the Drosophila IAP DIAP1 to inhibit a Drosophila caspase using purified recombinant proteins and an in vitro caspase assay. 2 μM of a DIAP1 fragment containing the BIR1 and BIR2 domains with intervening linker inhibited the caspase by 72 ± 1%. Removing either BIR domain reduced the inhibition to 39–45 ± 10%. Neither BIR domain alone inhibited the caspase even at 6 μM. However, 6 μM added BIR1 domain interfered with the 2 μM BIR1‐linker, reducing its caspase inhibition from 56 ± 1% to 30 ± 2%. An affinity chromatography binding assay detected direct BIR1 domain binding to the caspase. This suggests that the BIR1 interfered with the BIR1‐linker by binding to and blocking a critical BIR1‐binding site on the caspase. Thus, the BIR1 likely functions within DIAP1 by helping anchor the linker to the caspase active site. The BIR2 domain did not interfere with the linker‐BIR2 nor show direct binding to the caspase, indicating that its mechanism for enhancing caspase inhibition by DIAP1 must differ from that of the BIR1 domain. The role of the BIR2 as well as of other linker residues is being further investigated.

Support or Funding Information

This research was funded by a Kutztown University Research Grant to M. Junker and a Kutztown University BEARS grant and a Kutztown University Undergraduate Research Committee Grant to M. Storm.

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 Protein‐Protein Interactions and Allosteric Regulation in the Corticosteroid Synthesis Pathway in Vertebrates
Authors: Kreutzmann Sydney Nikole , Olson‐Manning Carrie Nikole
Published: June 2021

The regulation of biochemical pathways is controlled at a variety of levels, from transcriptional timing to protein localization. Here we study the corticosteroid synthesis pathway in order to understand a transition from a biochemical pathway controlled by allosteric regulation to a phenotypically equivalent pathway controlled by differential expression of paralogs and enzyme specificity.

The corticosteroids aldosterone and cortisol are produced in different layers of the adrenal cortex – a phenomenon known as zonation. Most mammals have a single enzyme, CYP11B, which produces aldosterone in the exterior layer of the adrenal cortex and cortisol in the interior layers. In mammals with a single‐CYP11B, the specific production of aldosterone and cortisol in different tissues is thought to be accomplished by allosteric regulation by another enzyme, CYP11A, which is only present in the interior layer of the cortex. It is thought that the presence of CYP11A changes the function of CYP11B so that it preferentially produces cortisol instead of aldosterone.

However, throughout mammalian evolution CYP11B has duplicated independently in several lineages, giving rise to two CYP11B paralogs. These species still experience zonation and produce aldosterone and cortisol in different layers of the adrenal cortex. It has been demonstrated that this zonation in species with two CYP11B enzymes is accomplished by differential expression of the paralogs in the different tissues along with the evolution of enzymatic preference for their respective products. However, it is unknown whether the interaction between CYP11B and CYP11A has been lost and whether the zonation of hormones produced is regulated solely by the two differential expression and enzymatic activity of the CYP11B enzymes.

Here we used Forster Resonance Energy Transfer (FRET) High Performance Liquid Chromatography (HPLC) to determine whether CYP11B and CYP11A physically interact and whether or not that interaction would contribute to the allosteric regulation of this pathway. Our preliminary analyses demonstrate a physical interaction between CYP11B and CYP11A in a single‐CYP11B species. Further experiments are aimed to test the interaction of duplicated CYP11Bs with CYP11A. We will monitor in vivo enzymatic assays by HPLC to determine what hormones are produced in the presence of different enzyme combinations. These results will shed light on the various ways metabolic pathways are regulated.

Support or Funding Information

SD BRIN P20GM103443

SD EPSCOR IIA‐1355423

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Effect of Genotype on the Ergogenic Effects of Caffeine in Collegiate Nordic Skiers
Authors: Zhang Yun , Millard Julie T.
Published: June 2021

Despite its widespread use, caffeine's effects vary widely between individuals. Two factors that are likely to modulate response to caffeine are the rate of its metabolism and the variant of the receptor to which it binds. The activity of the liver enzyme CYP1A2, which is responsible for metabolizing caffeine, is reportedly associated with a single nucleotide polymorphism (A/C) that leads to higher activity in AA individuals than in C‐allele carriers. CYP1A2 genotype appears to affect caffeine outcomes, as evidenced by reports that slow metabolizers have increased risk of heart attack and reduced benefits of caffeine on athletic performance. The adenosine A2A receptor (ADORA2A) is the principal CNS target of caffeine, and a single nucleotide polymorphism (T/C) of ADORA2A is reported to modulate caffeine consumption habits, caffeine‐induced anxiety, and the ergogenic effects of caffeine. The goal of this project was to determine whether genotypes for caffeine metabolism and/or receptor modulate exercise performance in collegiate athletes following caffeine ingestion. Nordic skiers provided DNA samples, which were genotyped for CYP1A2 (rs762551) and ADORA2A (rs5751876). Subjects were tested for lactate threshold under three conditions: baseline, after ingestion of placebo, and after ingestion of 2 mg/kg caffeine. This work may contribute to a better understanding of the variable effects of caffeine consumption on athletic performance.

Support or Funding Information

Supported by an Institutional Development Award (IDeA) from the National Institute of General Medical Sciences of the National Institutes of Health under grant number P20GM0103423.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Development of a Microscopic Method to Diagnose Hemoglobin C Conditions for Use in Developing Countries
Authors: Schmidt Kayla L. , Randolph Tim R.
Published: June 2021

Hemoglobin C is one of the second most prevalent hemoglobinopathies worldwide behind sickle cell anemia (HbS). HbC disease (HbCC) produces mild symptoms, but is a life‐threatening disorder if inherited with HbS (HbSC). HbS and HbC are most prevalent in countries underequipped to diagnose its presence through gold standard methods like electrophoresis, HPLC or isoelectric focusing. This study aims to create a simple, inexpensive method to identify the presence of Hemoglobin C using limited resources. The hypothesis is that when blood is incubated in a hypertonic salt solution, Hemoglobin C will crystalize intracellularly and become visible microscopically when stained with New Methylene Blue. The method was optimized by modifying the salt type, salt concentration, incubation time, and incubation temperature. The optimized method incubates RBCs in a 5x Dulbecco's phosphate buffered saline (DPBS) at 37°C for 4 hours. Blood samples with the HbSC genotype yielded between 600–750 HbC crystals for every 1000 RBCs counted. Future studies will include blood samples of AC, CC, SC, and AA genotypes to determine if this method will differentiate genotype.

Support or Funding Information

KS is supported by DeNardo Education and Research Foundation 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.
Extraction and Separation of Collagen on SDS PAGE to Determine Effect of Whitening Strips on Teeth
Authors: Mariche‐Banos David , Rotsides Photis , Keenan Kelly
Published: June 2021

In the United States, billions of dollars are spent each year of tooth whitening products including treatments in dentist's offices as well as whitening strips which are sold over the counter. Both of these products use hydrogen peroxide as the active ingredient which has long been known to damage proteins. The outermost part of teeth is the enamel which contains several soluble proteins and the dentin underneath contains nearly 90 to 95 % collagen. The goal of this project was to extract collagen from both untreated teeth as well as those treated with whitening strips and examine the collagen on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS PAGE). Teeth were placed into artificial saliva and either treated with whitening strips for one or three rounds according to manufacturer's instructions or were untreated. Following a treatment, teeth were placed in artificial saliva for 30 minutes. Following treatments, teeth were dialyzed against 10% EDTA, pH 9.0 for two weeks with several changes of EDTA solution. The teeth were then dialyzed against water for two weeks with several changes. The tooth remnants were placed in liquid nitrogen, hammered into shards and ground into a powder using a blender. The powder was suspended in 0.5 M acetic acid and is the collagen fraction (COL); it was treated with pepsin for four hours, neutralized with TRIS, centrifuged and the supernatant was the pepsin soluble collagen fraction (PSC). Gels of varying concentrations of acrylamide were prepared and COL and PSC fractions were loaded. Two modified procedures for the Coomassie Blue staining protocol were used. In one, the washing solution did not contain methanol and this caused the COL proteins to turn a pink color. In addition, the gels were stained with the Oriole fluorescent stain. In order to determine the type of collagen present, standards made from collagen I and III were also used. Results will be presented that shows the level of COL in treated and untreated teeth.

Support or Funding Information

Funding was provided by the Research and Professional Development Grant from Stockton University.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Measurement of protein and collagen in teeth before and after whitening strips
Authors: Tadros Jonathan , Giunta Nick , Keenan Kelly
Published: June 2021

Americans spend billions of dollars each year on whitening products for the teeth. Whether it is performed in a dentist's office or with over‐the‐counter whitening strips, the active ingredient, hydrogen peroxide, is the same and has long been known to damage proteins. The goal was to measure the level of protein extracted from teeth untreated as well as those treated with one or three rounds of whitening strips. Teeth were purchased and treated according to manufacturer's instructions. In one round, teeth were dry when treated and washed with water after for 30 minutes. In another round, teeth were suspended in artificial saliva during treatment and for 30 minutes afterwards. Afterwards, the teeth were dialyzed against 10% EDTA pH 9.0 for 2 weeks. The fluid contained the soluble non collagen proteins and total protein was measured using the Lowry assay. The tooth remnants were dialyzed for two weeks against water and placed in liquid nitrogen, hammered in shards and then ground into a powder in a blender. The powder was suspended in an acetic acid, sodium chloride solution and contained collagen. The collagen was treated with pepsin for four hours to collect a pepsin soluble fraction (PSC). The amount of protein in the collagen and PSC fraction was tested using a modified Lowry assay procedure. Based on the Lowry assay, there was a significant decrease in protein content after treatment with whitening strips and the loss increased with more treatments. Based on the modified Lowry procedure, there was also a significant loss of collagen as measured by the modified Lowry procedure for both the collagen and PSC fraction. Similar losses were observed with or without the artificial saliva.

Support or Funding Information

Authors thank Stockton University for the Research and Professional Development grant to support this project.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Optimization of Breast Cancer Resistance Protein (BCRP) Expression in the Yeast Pichia pastoris
Authors: Oliveira Maisa C. , Lavigne Collette A. , Tran Brandon M. , Fowler Michael E. , Okwuone Dakota D. D. , Farokhnia Roxana , Wise John G. , Vogel Pia D.
Published: June 2021

Multidrug resistance (MDR) has been linked to the failure of chemotherapy, and poor prognoses and reduced survival rates in patients in several cancer types. MDR cancers often become resistant to pharmacologically and structurally diverse drugs by overexpressing members of the family of ATP Binding Cassette (ABC) transporters. The breast cancer resistance protein (BCRP) is expressed in many tissues, including those with barrier or detoxifying functions such as blood‐brain barrier, placenta, and is responsible for the active export of a wide variety of toxins across the plasma membrane. Given its role in MDR, inhibitors of BCRP have been studied for years. To search for BCRP inhibitors as potential leads for the development of co‐therapeutics to combat MDR, we used high‐throughput in silico ligand docking similar to studies performed previously in the search for inhibitors of the P‐glycoprotein (Brewer et al., Mol. Pharmacol. 86, 716–726, 2014). A number of drug like molecules were identified that reversed multidrug resistance of a BCRP‐overexpressing breast cancer cell line generated in our lab (Nanayakkara, unpublished). To understand the mechanism of inhibition of BCRP by these compounds, biochemical assays assessing ATP hydrolysis activity as well as other biochemical properties of BCRP are required. To obtain protein amounts that are large enough for biochemical and biophysical evaluation of inhibitor action, a gene for human BCRP was designed with optimized codon usage for translation and expression in P. pastoris. Maximum expression of BCRP in the PichiaPink™ system was observed after 9 hours of growth on a methanol based medium. Protein expression was confirmed by Western blot analysis using both BCRP and anti‐His antibodies. ATP hydrolysis assays indicated that the protein expressed was catalytically active and that the ATP hydrolysis activity could be stimulated by the BCRP transport substrate, Hoechst 33342. Here we also report our attempts to purify BCRP from PichiaPink™ membranes and characterize the protein both as solubilized in mixed micelles as well as upon reconstitution into membrane nanodiscs

Support or Funding Information

This work is supported by NIH NIGMS [R15GM094771‐02] to John G. Wise, SMU University Research Council, the SMU Center for Drug Discovery, Design and Delivery, Ms. Lynn McBee and New England Biolabs, the Communities Foundation of Texas, a private gift from Ms. Suzy Ruff of Dallas, Texas, as well as the Hamilton Undergraduate Research Scholarship the SMU Undergraduate Research Assistantship Programs.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Expression of ABC Transporters in Multidrug Resistant Cancer Cell Lines
Authors: Holcomb‐Webb Kasi L. , Nanayakkara Amila K. , Aljowni Maha , Lippert Alexander R. , Vogel Pia D. , Wise John G.
Published: June 2021

ATP‐binding cassette (ABC) transporters are transmembrane proteins that act as drug efflux pumps and are associated with multi‐drug resistance (MDR) in cancers. Two specific proteins that belong to this protein superfamily are P‐glycoprotein (P‐gp) and breast cancer resistant protein (BCRP). These two proteins are known to pump chemotherapeutics out of cells, rendering treatments of some cancers less efficient. Certain cancer cell lines overexpress these proteins, leading to MDR. If a compound can be identified that inhibits these efflux pumps, then sensitivity to chemotherapeutics can be restored. The levels of expression of P‐gp and BCRP were quantitatively measured in four cancer cell lines that are used by us to screen for inhibitors of P‐gp or BCRP. The expression of P‐gp, BCRP, and MRP1 (multidrug resistance‐associated protein 1) was measured using quantitative One‐Step qPCR in a prostate cancer cell line, DU145, and compared to an MDR derivative line that overexpresses P‐gp, DU145 TXR. We found that DU145 TXR expresses P‐gp, BCRP, and MRP1 by about 14,000‐fold, 4‐fold, and 0.75‐fold, respectively, when compared to the parental cell line, DU145. A breast cancer cell line, MCF‐7, and its mitoxantrone‐resistant derivative, MCF‐7 M100, were also analyzed. The MCF‐7 M100 cell line was found to express BCRP, P‐gp, and MRP1 by about 600‐fold, 0.3‐fold, and 1.6‐fold, respectively, compared to the parental cell line, MCF‐7. The results were qualitatively confirmed at the protein level using Western Blot analyses. In other experiments, we demonstrated that P‐gp and BCRP inhibitors identified by us that reversed multidrug resistances in the MDR prostate and breast cancer cell lines, DU145 TXR and MCF‐7 M100, did not down‐regulate expression levels of P‐gp or BCRP, suggesting that reversal of MDR was accomplished by inhibiting transport processes and not transporter expression levels.

Support or Funding Information

This work is supported by NIH NIGMS [R15GM09477102] to John G. Wise, SMU University Research Council, the SMU Center for Drug Discovery, Design and Delivery, the Communities Foundation of Texas, and a private gift from Ms. Suzy Ruff of Dallas, Texas, as well as the Hamilton Undergraduate Research Scholars Program and the SMU Summer Research Assistantship and Undergraduate Research Assistantship Programs.

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