bluuebunny/crossref_metadata_2025_split huggingface dataset:
Short‐range and long‐range transcriptional repressors exhibit distinct and highly context‐specific chromatin signatures (741.2)
Authors: Arnosti David , Kok Kurtulus , Morsdorf David , Payankaulam Sandhya 
Published: June 2021

Transcriptional repressors employ a variety of mechanisms to regulate eukaryotic gene expression, and research from the Drosophila embryo has highlighted two distinct functional classes of repressor. Short‐range repressors such as Knirps work locally to interfere with proximal transcription factors, and others, such as the HES family protein Hairy, dominantly repress transcription of an entire locus with multiple regulatory elements. Both classes of repressor interact with common corepressors such as the evolutionarily conserved C‐terminal binding protein CtBP and WD‐40 domain protein Groucho. The basis and importance of such complexity is unknown, however it is possible that these types of repressors may exert unique effects in a gene‐specific manner. Our previous studies have highlighted the very localized chromatin modifications induced by the Knirps short‐range repressor, contrasted with kbp‐wide alterations observed with Hairy, however these studies were limited to individual target genes, leaving open the question about gene specificity, and whether repressors exert the same effect on all loci, regardless of gene activity or activator inputs. We have now analyzed the impact of Knirps and Hairy repression on a genome‐wide basis. We observe strong indications of correlated chromatin modifications on a wide set of targets, for instance, both H3K4 and H3K27 acetylation are coordinately reduced at Hairy targets, showing changes over a range an average of ~2kbp (often up to 10 kbp). A subset of targeted genes lacks significant deacetylation marks, but reveals strong upregulation of H3K9 trimethylation, as well as increased occupancy of histone H1 on the gene body. Our initial analysis indicates that complex and diverse repression pathways may be tailored to specific gene contexts, permitting a uniform control of heterogeneously regulated genes.Grant Funding Source: Supported by NIH GMS 056976
Mechanism of strong inhibition of HNF4α1 protein expression by its 5' UTR (749.7)
Authors: Guo Shangdong , Lu Hong 
Published: June 2021

HNF4α is a liver‐enriched master regulator down‐regulated in liver cancer and cirrhosis. Restoration of HNF4α expression can treat both liver cancer and liver fibrosis. However, lack of known activating ligand makes HNF4α “undruggable”. The purpose of this study was to elucidate the mechanism of translational regulation of HNF4α1, the adult‐predominant HNF4α isoform, so as to develop a novel approach to restore HNF4α1 expression for cancer therapy. The 5' UTR of both human and mouse HNF4α1 have potential G‐quadruplex (G4), a stable secondary structure known to inhibit transcription and translation. FRET assay of dual‐labeled oligo from human HNF4α1 5' UTR indicated the formation of G4. Luciferase reporter assay showed the wild‐type, but not G4‐mutated, 5' UTR of HNF4α1 strongly inhibited protein translation without affecting mRNA expression, which is further confirmed by Western blot analysis of protein expression of HNF4α1 in the presence/absence of its 5' UTR. In HEK 293 cells, protein expression of mouse Hnf4α1 was completely inhibited by its 5' UTR. Luciferase assays of the deletion/mutation reporter constructs of Hnf4α1 5' UTR suggest G4 motif and its adjacent stem loop may play the key role in suppressing Hnf4α1 protein expression. In summary, the 5' UTR of human and mouse HNF4α1 markedly inhibit HNF4α1 expression at translational levels, most likely due to interaction of G4 motif with stem loop within 5' UTR.Grant Funding Source: NIH Grant CA143656
A role for the ribosome in deciding the fate of damaged RNA (752.10)
Authors: Zaher Hani 
Published: June 2021

Fast and faithful translation of the cellular messenger RNAs is a defining feature of the ribosome and the translation factors. High‐accuracy protein synthesis ensures that errant proteins, which are more prone to misfold, are not made. On aberrant mRNAs, such as truncated ones and those either containing premature or lacking stop codons, a number of ribosome‐based quality control processes ensure that these RNAs are not translated and instead are targeted for degradation. While these mRNA‐surveillance mechanisms have received much attention during the past decade, curiously a different class of aberrant mRNAs has received little study. In particular, chemically‐damaged RNAs pose a significant hurdle to translational fidelity and efficiency. Of particular interest to us is oxidized RNA in the form of 8‐oxoguanine, which has been linked to a number of neurodegenerative diseases. The exact mechanism by which the cell recognizes the aberrant mRNAs and targets them for degradation remains elusive. To this end, we have begun to investigate the effects of oxidation on the decoding process using a high‐resolution‐in‐vitro translation system. Preliminary data suggests that 8‐oxoguanine is detrimental to the decoding process, for which we measure rates of peptide‐bond formation that are 4 to 5 orders of magnitude lower relative to undamaged RNA. We are currently investigating tRNA‐selection steps preceding the final step of peptidyl transfer in an attempt to understand the mechanism by which such a minor change to the nucleobase could have dramatic effects on the decoding process. We are also interested in studying the fate of the oxidized RNAs and the role of the ribosome in this process. Preliminary data is promising and suggests that the ribosome takes advantage of existent quality control processes to target these mRNAs for degradation.Grant Funding Source: NIH
Fis1 and Drp1 cooperate in mitochondrial homeostasis (756.8)
Authors: Hill Blake , Manlandro Cara Marie , Cleland Megan , Kennedy Nolan , Bakkum Amber 
Published: June 2021

Defects in mitochondrial fission and fusion cause numerous human disorders yet how these defects affect mitochondrial function and contribute to disease is unknown. Mitochondrial fission requires the dynamin‐related GTPase Drp1 and a lethal mutation (A395D) was reported in a neonate with microcephaly, abnormal brain development, optic atrophy, and lactic acidemia (Waterham et al., 2007, N. Engl. J. Med. 356, 1736‐1741). Another Drp1 mutation (C446F) was reported to cause severe cardiomyopathy in mice (Ashrafian et al., 2010, PLoS Genet 6, e1001000). We are determining how these mutants affect mechanoenzyme activity and function. For A395D, we found impaired localization, recruitment, assembly, and GTP hydrolysis. For C446F, we found impaired GTP hydrolysis and assembly. Thus, these mutants impair higher order assembly of Drp1 at mitochondria, leading to decreased fission, elongated mitochondria, and altered cellular distribution of mitochondria. Curiously, these mutants show enhanced in vitro interactions with the mitochondrial protein Fis1, a possible recruiter of Drp1. To determine the consequences of disrupting these interactions, we devised an unbiased and general method to rapidly identify residues critical to protein interfaces and applied this technology to yeast Fis1 interactions. Of the >3000 Fis1 alleles screened, ~9% selectively disrupted interactions with one of the three protein partners including yeast Drp1. To test the functional consequences, each allele was parsed into its corresponding point mutation and tested for mitochondrial fission. Of 211 yeast Fis1 mutants tested to date, 97 resulted in nonfunctional fission indicating that our method identifies residues essential for mitochondrial fission. Orthologous mutations were introduced into human Fis1 and also found to impair interactions with Drp1 and mitochondrial morphology consistent with a role for Fis1 in mitochondrial homeostasis.Grant Funding Source: RO1‐GM067180
Activation of ceramide‐metabolizing pathway in mitochondria from diabetic heart (758.3)
Authors: Novgorodov Sergei , Riley Christopher , Gudz Tatyana 
Published: June 2021

Mitochondrial dysfunction is an important contributor to the development of cardiac dysfunction in diabetic cardiomyopathy. Sphingolipids are key mediators of cell‐stress responses and effectors of mitochondrial function. The objective of the study was to investigate mechanisms underlying sphingolipid‐mediated mitochondrial dysfunction. The alterations of mitochondrial sphingolipid metabolism in cardiac mitochondria from mice with streptozotocin‐ induced type‐1 diabetes were examined. Sphingolipid analysis revealed profound increases in dihydroceramides in both subsarcolemmal (SSM) and interfibrillar (IFM) mitochondria. However, ceramide levels have not changed. Analysis of sphingolipid‐metabolizing enzyme gene expression indicated that, of the 42 genes investigated, only four genes exhibited an up‐regulation of > 2 fold: acid ceramidase, dihydroceramide synthase 2, desaturase 1 and galactosidase. Increased dihydroceramide levels along with activation of desaturase1 and dihydroceramide synthase 2 are indicative of an activation of de novo ceramide biosynthesis in diabetic heart. However, the lack of an increase in ceramide suggests concomitant up‐regulation of ceramide‐metabolizing pathways. Indeed, knocking down mitochondria‐resident neutral ceramidase (NCDase) drastically increased ceramide level in both SSM and IFM mitochondria from diabetic hearts. Ceramide increase in mitochondria from diabetic NCDase KO mouse hearts correlated with concomitant decrease in mitochondrial calcium retention capacity and suppression of mitochondrial respiration. These data attest that diabetes increases ceramide flux in cardiac mitochondria, which is controlled at least in part by mitochondrial NCDase.Grant Funding Source: These studies were supported by grant from The American Diabetes Association (#7‐12‐IN‐28) to SN.
The regulation of apoptosis by RNA processing factors in Saccharomyces cerevisiae (762.4)
Authors: Chirigos Allison , Schwotzer Erica , Luo Yi , Whitworth Gregg 
Published: June 2021

We have identified a novel link between mRNA processing and the regulation of apoptosis in the yeast Saccharomyces cerevisiae: the mRNA export factor Npl3, and its cytoplasmic kinase Sky1, are both necessary for the induction of apoptosis in response to environmental stress conditions. In the normal nuclear export cycle, Sky1 phoshorylation of serine 411 in the RS domain of Npl3 is associated with cytoplasmic release of the mRNA cargo. Provocatively, deletion of either factor (sky1Δ, npl3Δ), or mutation of the phosphorylation site (npl3‐S411A), abrogates induction of apoptosis in response to cation toxicity (CuSO4, LiCl). We are currently using HPLC‐coupled mass spectroscopy to quantify Npl3 phosphorylation levels at both the canonical site and adjacent residues which have been suggested as possible phosphorylation targets by high‐throughput mass spectrometry sequencing. Additionally, we are using high‐throughput epistatic‐analysis of double mutants to construct a genetic interaction network which probes the role of NPL3 and SKY1 in apoptotic stress conditions in the context of well described regulators of both the metacaspase‐mediated (YCA1) and mitochondrial (AIF1) pathways. In total, our data suggest a role for the Npl3/Sky1 export machinery in the mitochondrial pathway.Grant Funding Source: EJS, AJC, YL & GW supported by the HHMI Fellows Program & Grant Initiative; GW by Lenfest Grants.
Structural mechanisms of starch dephosphorylation (769.2)
Authors: Gentry Matthew , Meekins David , Guo Hou‐Fu , Husodo Satrio , Vander Kooi Craig 
Published: June 2021

Starch is comprised of glucose chains long enough to interact and self‐assemble into tightly packed helices that make starch water‐insoluble. Plants utilize reversible phosphorylation of glucose residues within starch to disrupt the helices and render the granule surface accessible to hydrolyzing enzymes.Glucan dikinases solubilize starch outer glucan chains by phosphorylating different positions on glucose units and allow starch degradation to initiate. However, β‐amylase activity is inhibited when a phosphate group is reached and glucan phosphatases must then act. We initially discovered glucan phosphatase activity and now define the structural basis of dephosphorylation by glucan phosphatases.We determined the structure of two Arabidopsis starch glucan phosphatases Starch Excess 4 (SEX4) and Like Sex Four2 (LSF2) both with phospho‐glucan bound and without glucan. SEX4 utilizes a carbohydrate binding module to position the oxygen of the C6 carbon into its catalytic cleft. LSF2 binds maltohexaose‐phosphate using an aromatic channel within an extended phosphatase active site and positions the C3 carbon into its active site.These structures are the first of glucan‐bound glucan phosphatases, and they provide insights into the molecular basis of this agriculturally‐ and industry‐relevant enzyme family, and their unique mechanisms of catalysis, substrate specificity, and interaction with starch.Grant Funding Source: NSF CAREER MCB1252345, NIH R01NS070899, KSEF 2268‐RDE‐014
Communication between the zinc and tetrahydrobiopterin binding sites in nitric oxide synthase (774.1)
Authors: Chreifi Georges , Li Huiying , McInnes Craig , Suckling Colin , Poulos Thomas 
Published: June 2021

The nitric oxide synthase (NOS) dimer is stabilized by a Zn2+ ion coordinated to symmetry related Cys residues exactly on the dimer axis. Each of the two essential tetrahydrobiopterin (H4B) molecules in the dimer interacts directly with the heme, and each H4B molecule is about 12 Å from the Zn2+. We have solved the crystal structures of the bovine endothelial NOS (eNOS) dimer oxygenase domain bound to three different pterin compounds and they reveal an intimate structural communication between the H4B and Zn2+ sites. The structure of one of these compounds, WSG1002, bound to eNOS with varying levels of Zn2+, showed that WSG1002 and Zn2+ cannot bind to eNOS simultaneously. WSG1002 both directly and indirectly disrupts hydrogen bonding between key residues in the zinc binding motif, resulting in destabilization of the dimer. Addition of excess Zn2+ stabilizes the Zn2+ site which decreases binding of WSG1002. These results have directed our attention to differences between eNOS and the other two mammalian NOS isoforms, neuronal NOS (nNOS) and inducible NOS (iNOS), that provide a structural basis for why the order of dimer stability is eNOS>nNOS>iNOS.Grant Funding Source: Supported by NIH
Identification of missing genetic variants in primary congenital glaucoma (776.8)
Authors: Ganguly Sriparna , Mukherjee Ankur , Chakrabarti Subhabrata , Kaur Inderjeet , Mandal Anil , Senthil Sirisha , Kabra Meha , Majumder Partha 
Published: June 2021

Background and Objective: Variations in the cytochrome P450, family 1, subfamily B, polypeptide 1 (CYP1B1) gene and mutations in the latent transforming growth factor beta binding protein 2 (LTBP2) gene cause autosomal recessive Primary Congenital Glaucoma (PCG). However, in India, only 45% of PCG patients harbor alterations in the CYP1B1 gene. The present study aimed to identify novel genetic variations contributing to unexplained PCG cases in India.Methods: 112 unrelated Indian PCG patients in whom neither chromosome carried a CYP1B1 or an LTBP2 mutation, and 106 matched controls, were recruited for this study. DNA isolated from each case and control was screened for a total of 906,600 single nucleotide polymorphism (SNP) loci using an Affymetrix 6.0 whole genome genotyping array. The marker data were first used for an association analysis followed by multiple test corrections, after an initial adjustment for population stratification. A second approach undertaken in this study was detection of regions of homozygosity (ROH) across all autosomes that are significantly found more often in cases than in normal controls.Results: Eleven SNPs in 5 genes and intergenic regions showed statistically significant association with PCG. These genes are neuronal growth regulator 1 (NEGR1) [1p31.1], glutamate receptor, ionotropic, kainate 4 (GRIK4) [11q22.3], plexin domain containing 2 (PLXDC2) [10p12.31], YY1 associated factor 2 (YAF2) [12q12] and copine IV (CPNE4) [3q22.1]. The results of the ROH analysis have indicated the presence of two regions of homozygosity that cover two important genes, namely kelch‐like family member 26 (KLHL26) [19p13.11] and teashirt zinc finger homeobox 2 (TSHZ2) [20q13.2].Conclusions: The present study has identified likely locations of new putative genes underlying PCG. These genes have previously not been associated with any type of glaucoma, but the products of these genes appear to be involved in pathways related to eye development and physiology.Grant Funding Source: Department of Biotechnology
Metallomic study of mercury in fish muscle (Semaprochilodus sp.) from Amazon ‐ Brazil (778.3)
Authors: Moraes Paula , Queiroz João , Braga Camila , Leite Aline , Buzalaf Marília , Zara Luiz , Okoshi Marina , Padilha Pedro 
Published: June 2021

The aim of this study is to present the metallomics results obtained for the protein fractionation in muscle samples of “jaraqui” (Semaprochilodus sp) from JIRAU Hydroelectric Power Plant ‐ Madeira River/Amazon, Brazil. The protein spots obtained after the fractionation (2D‐PAGE) were cut from the gel and analyzed by graphite furnace atomic absorption spectrometry (GFAAS) and the protein spots in which were detected mercury were characterized by mass spectrometry (ESI‐MS/MS). The GFAAS determinations indicated that 65% of the mercury linked to protein fraction with molar mass (Mm) lower than 50 kDa. The mercury concentration present in five selected spots was between 12 and 40 mg kg‐1. Based on these concentrations was possible to estimate approximately one to three mercury atoms per molecule of protein spots. The ESI‐MS/MS analysis allowed to characterize the follow proteins: Hemoglobin subunit beta (Mm= 16.60, pI= 6.00); Hemoglobin subunit beta‐A/B (Mm= 16.95, pI= 8.90); Parvalbumin alpha (Mm= 12.40, pI= 3.20); Parvalbumin beta (Mm= 13.10, pI=3.60) and Parvalbumin 2 (Mm= 11.82, pI= 3.40).Grant Funding Source: Financial Support: Fapesp 2009/54856‐8 and ESBR PD‐6631‐0001/2012
Expression, purification and functional characterization of influenza hemagglutinin II (HA2) protein containing transmembrane (TM) domain and ectodomain (781.1)
Authors: Ratnayake Punsisi Upeka , Weliky David 
Published: June 2021

Developing better vaccines against influenza requires investigating the viral infection process. Influenza hemagglutinin protein consists of two polypeptide chains HA1 and HA2. Conformational changes occurring in HA2 subunit initiate the fusion of viral membrane to endosomal membrane. The objective of this project was to assess the structure and function of the HA2 transmembrane domain while it is embedded in the lipid bilayer, during viral fusion process. Hydrophobicity and limited membrane space are two factors that limit membrane protein expression in cells. Therefore, expression of nonnative proteins in E‐coli causes formation of inclusion bodies. HA2 consisting of ectodomain and different lengths of TM without fusion peptide (FP) were expressed. Next, the proteins were purified from inclusion bodies followed by refolding. Also, HA2 ectodomain with and without FP were expressed and purified. Folding of all proteins was verified using circular dichroism spectroscopy. The activities of various proteins were analyzed using a lipid mixing assays at pH 5.0 and pH 7.0. No significance difference in the fusion activity was observed at pH 5.0 between the constructs with different lengths of TM domain. However, in the presence of FP, a significant increase of fusion activity was observed leading to conclude that fusion peptide is the main contributor in the fusion process.Grant Funding Source: Supported by National Institute Health (NIH) research grant 5R01AI047153‐13
A chemical genetic approach to understand inositol polyphosphate signaling (794.6)
Authors: Dul Barbara , Bratz Jeffrey , Fiedler Dorothea 
Published: June 2021

Cells regulate their growth in response to nutritional and mitogenic signals through signaling pathways that are well defined and tightly controlled by second messengers. It is critical that these messengers respond quickly and balance cellular responses. Inositol polyphosphates (IPs) have been found to play an important role in signaling and highly conserved from yeast to higher organisms. Genetic studies in yeast ascribe important functions for IPs in many cellular processes, including telomere maintenance and nutrient sensing. IP deficient mice exhibit several defects in insulin secretion and are resistant to age and diet induced obesity. We are interested in understanding the mechanisms by which the various IPs function within cells and have begun to study this pathway with a chemical genetic approach. This approach relies on designing orthogonal protein/ligand pairs ‐ by introducing a mutation in the kinase active site and using a size complementary ATP analog ‐ and has successfully been applied to numerous protein kinases. We found that this approach works with yeast inositol kinases Arg82, Ipk1, and Kcs1. Because yeast screens remain the most versatile tool for assessing functional genomic interactions at an organismal level, we exploit the yeast system to investigate IP signaling in transcriptional regulation. We anticipate that a number of these fundamental signaling roles will be maintained in higher organisms.Grant Funding Source: NIH
Role of G‐protein coupled estrogen receptor in estrogen‐mediated protection from cardiomyocyte hypertrophy (795.6)
Authors: Burton Tyler , Waikel Rebekah , Malone Payton 
Published: June 2021

Independent, preceding studies have reported decreased risk and progression of cardiac disease in pre‐menopausal women as compared to men. 17‐β estradiol has been implicated as the molecular variable underlying this sex‐specific observation and is primarily thought to signal through nuclear receptor ER‐β. Our study seeks to understand the regulatory role of the extracellular G‐Protein Coupled Estrogen Receptor (GPER) in estrogen‐mediated protection from cardiac hypertrophy. Primary, neonatal cardiomyocytes were cultured in a sex‐dependent manner. Cellular response to the hypertrophic agent, phenylephrine, in the presence and absence of GPER activity, was assessed. Cell surface area reduction was observed in response to GPER activation, independent of nuclear estrogen receptor activity. Related studies have predominantly made use mixed cultures. Interestingly, sex‐specific cardiomyocyte cultures differ in cycling and ligand responses. The significance of sex‐specific cellular characteristics is currently being assessed. Differential expression levels identified specific miRNAs as downstream mediators of GPER, which are consistent with our previous sex‐specific Next Generation Sequencing data from hypertrophic and wild‐type hearts.NIH 5P20RR016481‐12, 8P20GM103436‐12, R15HL108192‐02Grant Funding Source: NIH 5P20RR016481‐12, 8P20GM103436‐12, R15HL108192‐02