bluuebunny/crossref_metadata_2025_split huggingface dataset:
Variations in Nuclear Localization Strategies Among Pol X Family Enzymes
Authors: KIRBY THOMAS , PEDERSEN LARS C. , GABEL SCOTT A. , GASSMAN NATALIE R. , LONDON ROBERT E. 
Published: June 2021

The pol X family enzymes DNA polymerase β (pol β), DNA polymerase λ (pol λ), DNA polymerase μ (pol μ), and terminal deoxynucleotidyl transferase (TdT) have critical roles in DNA repair. Nevertheless, information about the structural basis of their nuclear import is limited. Recent studies revealed the unexpected presence of a functional NLS in DNA polymerase β, indicating the importance of active nuclear targeting, even for enzymes likely to leak in and out of the nucleus. Here we further explore the active nuclear transport of the three remaining human pol X enzymes by identifying and structurally characterizing their functional NLS sequences. We used X‐ray crystallography and fluorescence polarization binding assays to characterized the three pol X NLS sequences and their interactions with Importin α that lacks the Importin β binding domain (ImpαΔIBB). All three polymerases use NLS sequences located near their N‐terminus; the pol μ and TdT NLS sequences are monopartite, while pol λ utilizes a bipartite sequence, unique among the pol X family members. We confirmed the results by fluorescence‐based cellular localization studies, which also verified the bipartite nature of the pol λ NLS. This bipartite sequence, was somewhat unexpected, since NLS prediction programs either identified very low probability possibilities or completely failed to identify a potential NLS. Further validation of the identified NLS sequences was conducted by fluorescence polarization peptide‐binding to ImpαΔIBB, where TdT and pol λ demonstrated submicromolar dissociation constants, while pol μ had a low μM Kd value. Nuclear targeting is unique to each pol X family enzyme with variations in sequence context and affinity likely regulating the import and retention of these enzymes in the nuclear compartment.Support or Funding InformationThis work was supported by the Intramural Research Program of the NIH, National Institute of Environmental Health Sciences, project number ZIA ES050111 to REL.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Structure and mechanism of DNMT3A‐mediated DNA methylation
Authors: Song Jikui , Zhang Zhi‐Min , Lu Rui , Wang Gang Greg 
Published: June 2021

Mammalian DNA methylation is an important epigenetic mechanism crucial for gene silencing and imprinting, X‐inactivation, genome stability, and cell fate determination. It is established mainly at CpG dinucleotides by de novo methyltransferases DNMT3A and DNMT3B, and subsequently maintained by DNA methyltransferase 1 (DNMT1) in a replication‐dependent manner. Deregulation of this process is implicated in various diseases, notably cancer. However, the mechanisms underlying DNMT3 substrate recognition and enzymatic specificity remain elusive. We determined the crystal structure of the DNMT3A – DNMT3L tetramer in complex with a DNA substrate containing two target sites separated by fourteen base pairs. Structural analysis of the DNMT3A – DNMT3L – DNA complex reveals that the two DNMT3A monomers simultaneously attack the two target sites, supporting the notion that DNMT3A homodimer can co‐methylate two adjacent CpG dinucleotides in one DNA‐binding event. Importantly, we identified that a residue from the target recognition domain makes crucial contacts with CpG, ensuring DNMT3A enzymatic preference towards CpG sites in cells. Mutational, enzymatic and cellular analyses further demonstrate that the hematological cancer‐associated somatic mutations of the substrate‐binding residues decrease DNMT3A activity, induce CpG hypomethylation, and promote transformation of hematopoietic cells. Together, our study reveals the mechanistic basis for DNMT3A‐mediated DNA methylation.Support or Funding InformationSidney Kimmel Foundation for Cancer Research;March of Dimes Foundation (1‐FY15‐345);National Institute of Health (1R35GM119721)This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Screening and identification of four serum miRNAs as novel potential biomarkers for cured pulmonary tuberculosis
Authors: Wang Chong , Yang Su , Liu Chang‐Ming , Jiang Ting‐Ting , Chen Zhong‐Liang , Tu Hui‐Hui , Li Ji‐Cheng 
Published: June 2021

Rapid and efficient methods for the determination of cured pulmonary tuberculosis (TB) are lacking. We screened serum miRNAs using the Solexa sequencing method among untreated TB patients, two‐month treated TB patients, cured TB patients, and healthy controls. A total of 100 differentially expressed miRNAs were identified in cured TB patients, including 37 up‐regulated (fold change >1.50, P<0.05) and 63 down‐regulated (fold change <0.60, P<0.05) miRNAs. Gene ontology (GO) enrichment analysis revealed that most of the predicted genes were present in the nucleus with a strong protein binding function. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis strongly suggested alterations in the metabolic pathways. Following quantitative real time chain reaction (qRT‐PCR), significantly reduced expression levels of miR‐21‐5p (0.59, P=0.039), miR‐92a‐3p (0.46, P=0.038), and miR‐148b‐3p (0.96, P=0.049) were found in the cured TB patients compared with the untreated TB patients. Also, significantly increased expression levels of miR‐92a‐3p (2.82, P<0.001), miR‐125a‐5p (5.09, P<0.001), and miR‐148b‐3p (4.25, P<0.001) were found in the untreated TB patients compared with the healthy controls. We established a cured TB model with 73.08% accuracy by four miRNAs (miR‐21‐5p, miR‐92a‐3p, miR‐148b‐3p, and miR‐125a‐5p), and also established a diagnostic model with 71.43% accuracy. Our study provides experimental data for establishing objective indicators of cured TB, and also provides a new experimental basis to understand the pathogenesis and prognosis of TB.Support or Funding InformationThis work was supported by grants from the National Natural Science Foundation of China (No. 81573709), the Zhejiang Provincial Natural Science Foundation of China (No. LQ18H040009), and Zhejiang Provincial Medical Science and Technology Foundation of China (2017KY550).This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Selenium Regulation of Selenoprotein Enzyme Activity and Transcripts in a Pilot Study with Founder Mouse Strains from the Collaborative Cross
Authors: Sunde Roger A. 
Published: June 2021

Rodents and humans have 24–25 selenoproteins, and these proteins contain the 21st amino acid, selenocysteine, incorporated co‐translationally into the peptide backbone in a series of reactions dependent on at least 6 unique gene products. In selenium (Se) deficiency, there is differential regulation of selenoprotein expression, whereby levels of some selenoproteins and their transcripts decrease dramatically in Se deficiency, but other selenoprotein transcripts are spared this decrease; the underlying mechanism, however, is not fully understood. To begin explore the genetic basis for this variation in regulation by Se status in a pilot study, we fed Se‐deficient or Se‐adequate diets (0.005 or 0.2 μg Se/g, respectively) for eight weeks to the eight Founder strains of the Collaborative Cross. We found rather uniform expression of selenoenzyme activity for glutathione peroxidase (Gpx) 3 in plasma, Gpx1 in red blood cells, and Gpx1, Gpx4, and thioredoxin reductase in liver. In Founder mice, Se deficiency decreased each of these activities to a similar extent. Regulation of selenoprotein transcript expression by Se status was also globally retained intact, with dramatic down‐regulation of Gpx1, Selenow, and Selenoh transcripts in all 8 strains of Founder mice. These results indicate that differential regulation of selenoprotein expression by Se status is an essential aspect of Se metabolism and selenoprotein function. A few lone differences in Se regulation were observed for individual selenoproteins in this pilot study, but these differences did not single‐out one strain or one selenoprotein that consistently had unique Se regulation of selenoprotein expression. These differences should be affirmed in larger studies; use of the Diversity Outbred and Collaborative Cross strains may help to better define the functions of these selenoproteins.Support or Funding InformationSupported by the Wisconsin Alumni Foundation Selenium Nutrition Research Fund (No. 12046295)This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
How Cell Stress and 3′ End Alterations Control the Metabolism of a Cellular Non‐Coding RNA
Authors: Rivas Thomas , Kugel Jennifer F. , Goodrich James 
Published: June 2021

RNA Polymerase II (Pol II) canonically acts as a DNA‐dependent RNA polymerase (DdRP), using double stranded DNA to synthesize protein‐encoding mRNAs and some non‐coding RNAs. Pol II also has RNA‐dependent RNA polymerase activity (RdRP), which uses an RNA as a template to synthesize RNA. An example of Pol II RdRP activity is the 3′ end extension of the non‐coding B2 RNA to generate extended B2 (eB2) RNA. B2 RNA binds to Pol II and is a substrate for a Pol II‐dependent 18 nucleotide RdRP‐catalyzed extension to form eB2 RNA. eB2 RNA can be detected in cells and has a reduced half‐life as compared to B2 RNA.B2 RNA is transcribed by Pol III from Short Interspersed Elements (SINEs), which exist in over 350,000 copies in the mouse genome due to retrotransposition. Upon cellular stress, transcription of non‐coding B2 RNAs from B2 SINEs is greatly increased, thereby increasing the likelihood of retrotransposition of B2 SINEs. The RdRP activity of Pol II could control the levels of B2 RNA post‐transcriptionally by generating eB2 RNA to promote its degradation, thereby impacting the frequency of retrotransposition.To study the effects of 3′ end extension and cellular stress on the lifecycle and metabolism of B2 RNA we are developing a heterologous expression system. Because the B2 SINE sequence is embedded in many mouse mRNA transcripts (e.g. UTRs and introns), it is impossible to perform hybridization and PCR‐based experiments to distinguish between a Pol III‐derived B2 RNA and those sequences within mRNA transcripts. To overcome this, we express B2 and eB2 RNA in human cells, which do not contain B2 SINEs, using a human Pol III promoter. The expressed RNAs are the correct size, have the correct 5′ ends, have the correct 3′ secondary structure, and exogenous eB2 has decreased stability compared to B2 RNA. Therefore, the heterologous system mimics characteristics of B2 RNA and eB2 RNA in mouse cells.Using this system, ongoing experiments are investigating how extension alters the cellular localization of B2 RNA and retrotransposition efficiency in unstressed and stressed cells. Altogether, these experiments will determine the interplay between B2 RNA expression, Pol II RdRP extension, cellular localization, and cellular stress on the life cycle of this ncRNA.Support or Funding InformationR01 GM68414 T32GM008759This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Enzymatic Site‐Specific Labeling of RNA for Affinity Isolation of RNA‐Protein Complexes
Authors: Busby Kayla N. , Devaraj Neal K. 
Published: June 2021

The role of RNA in regulating cellular processes has generated tremendous interest in methods to image RNAs and profile their interactions within cells. Building on our recently developed enzymatic technique, RNA‐TAG (Transglycosylation At Guanosine), we demonstrate the site‐specific labeling and affinity purification of RNA transcripts. This technology takes advantage of a bacterial tRNA guanine transglycosylase (TGT) for the incorporation of nucleobase derivatives bearing functional probes, such as biotin, into an encodable RNA stem loop recognition motif. Affinity purification enables high levels of enrichment of target RNA complexes and analysis of bound proteins. Using this methodology, we have demonstrated that an RNA of interest can be selectively biotinylated in live mammalian cells or cell lysate. Through the development of RNA‐TAG, we aim to create a robust, convenient methodology for the elucidation of RNA function via affinity purification of cellular RNA‐protein complexes.Support or Funding InformationThis work is supported by the NIH under grant R01 GM123285. K.N.B. is supported by the Chemical Biology Interfaces Training Grant, NIH Grant T32 GM112584.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Thermal Stability of Type II Polyketide Acyl Carrier Proteins
Authors: Rivas Marco , Courouble Valentine ,   2016 Haverford College Biochemistry Superlab, Sisto Ashley , Beld Joris , Kokona Bashkim , Charkoudian Louise K. 
Published: June 2021

Engineering biosynthetic pathways in microorganisms is a compelling route to gain access to novel molecules with drug‐like properties. Polyketide synthases (PKSs) utilize acyl carrier proteins (ACPs) to convert simple acetate building blocks into structurally complex molecules, many of which are antibiotics and anticancer agents. In a process known as combinatorial synthesis, biochemists have attempted to mix‐and‐match enzymes from different PKSs to create new hybrid synthases capable of synthesizing novel polyketide natural products. However, the ACP has proven to be a roadblock in these studies, as little is known about how the ACP functions and interacts with other enzymes due to its small and dynamic nature. To further our knowledge about the structure and function of ACPs, we cloned, expressed, and characterized a subset of 14 type II PKS ACPs. Temperature‐dependent circular dichroism (CD) experiments were conducted to determine the thermostability of the ACPs in the presence or absence of divalent cations. Our results indicate that despite strong sequence similarity, ACPs display a range in thermal stability. Further, the presence of metal ions confers additional stability to the ACP, presumably by decreasing electrostatic repulsive forces. Analytical ultracentrifugation‐sedimentation velocity experiments revealed that all ACPs sediment as monomers, and that despite sharing a common ancestor with fatty acid synthases, only one of the PKS ACPs studied interacted with the E. coli fatty acid ketosynthase, FabF.Support or Funding InformationWe would like to acknowledge an NSF CAREER Award #R15GM120704 and an NIH Award #CHE‐1652424 to Louise K. Charkoudian, as well as the Beckman Scholars Program, as sources of funding for 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.
Site‐selective alterations within the hemolysin A non‐polar core
Authors: Grosskopf Julian David , Weaver Todd Michael , Grilley Daniel Patrick 
Published: June 2021

The secretion of virulence factors often aids bacterial pathogenesis. The two‐partner secretion (TPS) pathway, harboring both A (TpsA) and B‐components (TpsB), is the most commonly used gram‐negative virulence factor secretion system. In fact, whooping cough, meningitis, and certain food‐borne illnesses have been attributed to TPS pathway containing gram‐negative bacterial species. Systematically, TpsA members are activated concomitant with Tps‐dependent secretion across the outer membrane. Upon secretion, TpsA members elicit a variety of functions including cytolysis, adhesion, contact‐growth inhibition, and iron sequestration. Collectively, these TpsA functions provide advantageous invasion and proliferation strategies within host cells. Structurally, TpsA members can be divided into a two‐partner secretion (TPS) domain and a functional domain. All TPS domains are constructed from a 300‐residue right‐handed, parallel β‐helix structure, and recognize their cognate TpsB partner. In order to further understand the relationship between TPS domains and TpsA structure and function, we have implemented a truncated form of hemolysin A (HpmA265), a TpsA member from Proteus mirabilis. In previous studies, HpmA265 was structurally separated into three sequential folding subdomains: polar core, non‐polar core, and carboxy‐terminus. Specifically, these research investigations targeted valine 158 (V158) and phenylalanine 215 (F215) located within the first and last parallel β‐strands of the non‐polar core subdomain. A series of site‐selective variations were established at both V158 and F215. These variant forms of HpmA265 were characterized structurally via protease sensitivity and protein folding techniques, while functionality was ascertained within a template‐assisted hemolysis assay. Structurally, the V158 variants have destabilized the unfolding transitions associated with both the polar and non‐polar core subdomains, while leaving functionality unaffected. Site‐selective variants at F215 have selectively destabilized the non‐polar core subdomain, while leaving the unfolding transition attributed to the polar core subdomain unaffected. Additionally, the F215 variants do not affect template‐assisted hemolysis. Therefore, our results have been able to dissect the structural stability within the non‐polar core subdomain from template‐assisted function. These results have expanded the understanding for the implementation of TPS domains within gram‐negative bacteria.Support or Funding InformationFunding for this research was provided by: University Wisconsin – La Crosse Faculty Research Grant Program (TMW) and University Wisconsin – La Crosse Undergraduate Research and Creativity Grant Program (JDG).This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
How and why internal cavities destabilize proteins
Authors: Mulder Frans AA. , Xue Mengjun , Yoshimura Yuichi , Kitahara Ryo 
Published: June 2021

Proteins are marginally stable as a consequence of the large and opposing contributions from enthalpy and entropy to the folded state. The microscopic contributions to the total system ΔS and ΔH are still highly enigmatic. Here, we use hydrostatic pressure ‐– coupled to NMR spectroscopy detection – to explore the protein energy landscape in residue‐specific detail. As the conjugate of pressure is volume, this approach offers a mild and clearly defined perturbation of the system, as opposed to mutagenesis, temperature or denaturants. We could thus directly examine the Van der Waals contribution to stability. Pressure‐variation also allowed us to detect several partially unfolded states that consist of cooperative modules, a result that argues for a rather discrete route that connects the folded and unfolded forms of a protein.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
LDLR Processing Dysregulation by Statin Treatment of Class II Transport Mutant Cells
Authors: Omer Linda , Boyd Nolan L. 
Published: June 2021

The familial hypercholesterolemia (FH) Class II transport mutation is believed to be the most common defect in the low‐density lipoprotein receptor (LDLR). Class II mutations cause mis‐folding of the LDLR precursor preventing its transport from the ER for maturation in the Golgi and transport to the plasma membrane. The homozygous 3‐nucleotide deletion, c.654_656delTGG (FH‐Piscataway), in LDLR exon 4, caused a Class II mutation resulting in serum LDL over 800mg/dl in a 9yo female (GM03040, Coriell). Induced pluripotent stem cells (iPSC) derived from this patient's fibroblasts showed restored LDL‐receptor mediated endocytosis in homozygous CRISPR corrected clones. When statin treated, LDLR in non‐corrected FH‐iPSC (FH‐iPSC‐NC) persisted in the precursor (120kDa) form, while corrected cells (FH‐iPSC‐C) processed the LDLR to the mature form (160kDa). Quantification of total LDLR (immature + mature) showed 2.2‐fold more protein in NC cells. LDLR transcript quantification of statin treated NC, C and LDLR normal cells (H1 human embryonic stem cells, hESC) showed no difference, indicating LDLR accumulation is a dysregulation of post‐translational protein processing. This data and evidence in the literature led to our hypothesis that statin‐treatment of Class II LDLR mutant cells causes ER accumulation of mis‐folded LDLR and ER stress, leading to the unfolded protein response activation, which is reversed by LDLR correction. Since LDL cholesterol is primarily regulated by hepatocytes, we differentiated FH‐NC, C and H1 to hepatocyte‐like cells (HLC) using a modified version of Hay's protocol (PMID: 25081564) where stage 3 HLC were formed into spheroids of ~50/aggregate using AggreWell 400 plates. The HLC spheroids were treated with 5 mM Lovastatin or ethanol control in 5% lipoprotein‐deficient serum media overnight after which protein or RNA lysate was collected for Western blotting or qPCR, respectively. LDLR transcript levels were unchanged between groups, indicating that dysregulation is occurring at the post‐translational level in HLC. This work will provide new information as to whether statin treatment is implicated in inducing ER stress in Class II LDLR mutations and contribute to understanding the downstream effects and significance of statin treatment in patients with Class II LDLR FH.Support or Funding InformationJewish Heritage Fund for Excellence (NB)This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
The Molecular Mechanism of Intermolecular Signal Transduction in Cystathionine‐β‐Synthase (CBS)
Authors: Chandrasekaran Sita S. , Ruiz Donovan George , Kennedy Dana Renae , Esquerra Raymond M. 
Published: June 2021

Human cystathionine‐β‐synthase (hCBS) is a pyridoxal phosphate‐dependent (PLP) enzyme that contains heme and catalyzes the β‐replacement reactions of serine (L‐Ser) by homocysteine (L‐Hcy) to produce cystathionine. Mutation in this key mammalian enzyme causes hyperhomocysteinemia, which leads to aggressive arterial diseases, a clinical phenotype observed in patients. Although the role of PLP is similar to other PLP dependent enzymes that catalyze the exchange of the hydroxyl group of serine to thiolate in homocysteine, little is known about the role that heme plays in regulating the catalytic process. My project seeks to develop a continuous, kinetic assay to measure CBS activity. Two approaches are being explored: 1) A coupled kinetic assay, in which the cystathionine produced by CBS is used by Cystathionine‐β‐lyase (hCBL) to generate pyruvate, and pyruvate production is measured phototrically; 2) A novel nuclear magnetic resonance assay, in which we will monitor the direct production of cystathionine over time. These methods will be compared to the standard, but discontinuous, radiometric assay. These assays will be used to understand the effect of small ligand binding to the heme and point mutations at the heme cofactor in relation to the enzyme activity. The long‐term goal of this work is to use time‐resolved absorption and magnetic circular dichroism spectroscopy to understand how small ligand binding to the heme induces conformational changes at the PLP active site in CBS and to measure activity of CBS with various mutated active site residues.Support or Funding InformationArnold and Mabel Beckman Foundation; NIH MARC: T34‐GM008574This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.