bluuebunny/crossref_metadata_2025_split huggingface dataset:
Genome‐Wide Analysis of Bromodomain Histone Di‐Acetyllysine Ligands
Authors: Signorelli Sylvia , Gerrard Diana , Glass Karen , Frietze Seth 
Published: June 2021

Combinatorial patterns of epigenetic modifications on core histone proteins serve in the marking of genomic elements for the recruitment of regulatory proteins to effect critical biological functions. The recognition of acetylated histone lysine residues by bromodomain chromatin readers appears to be an essential cellular mechanism that regulates protein‐protein interactions, chromatin remodeling, and gene expression. However, the functional relevance of the association of bromodomain chromatin readers and their genome‐wide association with various combinations of histone acetylations remains poorly understood. ATAD2 is a bromodomain‐containing protein highly overexpressed in solid tumors, and has been correlated to an increased risk of cancer metastasis and recurrence. ATAD2B (KIAA1240) is a poorly studied paralog of the ATAD2 gene, and although ATAD2 and ATAD2B are highly conserved, there is little known about the function of ATAD2B. In vitro binding assays demonstrate ATAD2 and ATAD2B bromodomains selectively recognize H4K5/H4K12 and H4K5/H4K8 di‐acetyllysine marks, respectively. To study the genome‐wide patterns of ATAD2/2B histone ligands, we used a Chromatin Immunoprecipitation followed by high‐throughput sequencing (ChIP‐seq) assay to characterize the genomic distribution of H4 tail lysine acetylation patterns in MCF7 breast cancer cells. We performed replicate H4K5, H4K8, H4K12 ChIP‐seq assays as well as H4K5/K8 or H4K5/K12 sequential ChIP‐seq assays. We provide a high‐resolution map of combinations of histone tail acetylation in MCF7 cells. Our results reveal unique patterns of chromatin states, genes and putative transcription factor binding sites associated with different combinations of diacetyllysine residues. Ongoing work seeks to define the recruitment of ATAD2/B, as well as other chromatin effector proteins, and to understand the functional relevance of histone ligand recognition on breast cancer cell gene expression 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.
Salt Bridge Formation within the RKS Motif of Histone H3 Detected by NMR Spectroscopy
Authors: Estrada‐Tobar Zoila M. , Fuentes Daniel , Zurita‐Lopez Cecilia I. 
Published: June 2021

Histone N‐terminal tails are subject to covalent post‐translational modifications (PTMs) including methylation and phosphorylation which can help signal gene activation. More recently, PTMs are studied for their impact on each other. In many cases, one modification may prevent that of another. We set out to physically characterize the formation of a salt bridge between arginine‐8 and phosphoserine‐10 in histone H3 by nuclear magnetic resonance (NMR) spectroscopy. We hypothesize that both methylated arginine and phosphorylated serine do not co‐exist when in close proximity; phosphorylation of serine‐10 forms a salt bridge with neighboring unmodified arginine‐8, preventing arginine‐8 methylation. To investigate the contributions of these two modifications, 31P NMR was used for salt bridge detection because the natural isotopic abundance of phosphorus (100 %) is high enough to observe without the need for isotopic labeling. To calculate the relative free energy of the salt bridge, we carried out a 31P NMR titration using four modified peptides that correspond to histone H3. Our NMR data confirm the formation of a salt bridge. The detection of a salt bridge provides evidence that this interaction changes the conformation of the histone H3 tail and also prevents subsequent methylation of arginine‐8. This work can lead to mechanistic insights into the modification of histone H3 tails in both healthy and diseased states.Support or Funding InformationNational Science Foundation Louis Stokes Alliance for Minority Participation HDR‐1602210This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Stoichiometry of Multi‐protein Complexes containing RTT109, Vps75, and Histone H3–H4
Authors: D'Arcy Sheena , Akhavantabib Noushin , Krzizike Daniel 
Published: June 2021

RTT109 is a fungal‐specific acetyltransferase that acetylates lysine residues in histone H3–H4. These acetylations play a critical role in nucleosome dynamics as required for DNA transcription, replication and repair. RTT109 acetylation of specific histone H3 lysine residues requires Vps75. Vps75 is a histone chaperone belonging to the Nap‐family; it directly binds the RTT109 enzyme and the histone H3–H4 substrate. The goal of this study was to determine the absolute stoichiometry of the active complex containing RTT109, Vps75, and histone H3–H4. The stoichiometry was unclear as a Vps75 homodimer can crystalize with either one or two copies of RTT109. A high‐resolution structure of the complex between Vps75 and histone H3–H4 is not available, and both Vps75 and histone H3–H4 are known to form oligomers in solution. To measure absolute stoichiometry, we have used an elegant series of analytical ultracentrifugation experiments. We characterize all possible combinations of RTT109, Vps75, and histone H3–H4. These centrifugation experiments are complemented by other biophysical approaches. Our final stoichiometry, combined with targeted mutagenesis, reveals the organization of the active complex containing RTT109, Vps75, and histone H3–H4. Our results pave the way for high‐resolution structural studies and have repercussions for the mechanism and preferred substrate of RTT109 bound by Vps75.Support or Funding InformationStartup 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.
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 InformationThis 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 InformationFunding 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 InformationSupported 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 InformationThis 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 InformationK. Karbstein is an HHMI Faculty ScholarThis 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 InformationNIH grants GM041247, GM58634 and AI11266This 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 InformationThe 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.