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Polymerase substrate specificity

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https://www.readbyqxmd.com/read/29329981/molecular-cloning-and-characterization-of-leucine-aminopeptidase-gene-from-taenia-pisiformis
#1
Shaohua Zhang, Xuepeng Cai, Xuenong Luo, Shuai Wang, Aijiang Guo, Junling Hou, Run Wu
Leucine aminopeptidase (LAP, EC: 3.4.11.1) is an important metalloexopeptidase that catalyze the hydrolysis of amino-terminal leucine residues from polypeptides and proteins. In this study, a full length of cDNA encoding leucine aminopeptidase of Taenia pisiformis (TpLAP) was cloned by rapid amplification of cDNA-ends using the polymerase chain reaction (RACE-PCR) method. The full-length cDNA of the TpLAP gene is 1823 bp and contains a 1569 bp ORF encoding 533 amino acids with a putative mass of 56.4 kDa...
January 9, 2018: Experimental Parasitology
https://www.readbyqxmd.com/read/29295939/structure-of-hiv-1-reverse-transcriptase-cleaving-rna-in-an-rna-dna-hybrid
#2
Lan Tian, Min-Sung Kim, Hongzhi Li, Jimin Wang, Wei Yang
HIV-1 reverse transcriptase (RT) contains both DNA polymerase and RNase H activities to convert the viral genomic RNA to dsDNA in infected host cells. Here we report the 2.65-Å resolution structure of HIV-1 RT engaging in cleaving RNA in an RNA/DNA hybrid. A preferred substrate sequence is absolutely required to enable the RNA/DNA hybrid to adopt the distorted conformation needed to interact properly with the RNase H active site in RT. Substituting two nucleotides 4 bp upstream from the cleavage site results in scissile-phosphate displacement by 4 Å...
January 2, 2018: Proceedings of the National Academy of Sciences of the United States of America
https://www.readbyqxmd.com/read/29290984/estrogen-receptor-activation-contributes-to-rnf146-expression-and-neuroprotection-in-parkinson-s-disease-models
#3
Hyojung Kim, Sangwoo Ham, Joon Yeop Lee, Areum Jo, Gum Hwa Lee, Yun-Song Lee, MyoungLae Cho, Heung-Mook Shin, Donghoon Kim, Olga Pletnikova, Juan C Troncoso, Joo-Ho Shin, Yun-Il Lee, Yunjong Lee
RNF146 is an E3 ubiquitin ligase that specifically recognizes and polyubiquitinates poly (ADP-ribose) (PAR)-conjugated substrates for proteasomal degradation. RNF146 has been shown to be neuroprotective against PAR polymerase-1 (PARP1)-induced cell death during stroke. Here we report that RNF146 expression and RNF146 inducers can prevent cell death elicited by Parkinson's disease (PD)-associated and PARP1-activating stimuli. In SH-SY5Y cells, RNF146 expression conferred resistance to toxic stimuli that lead to PARP1 activation...
December 5, 2017: Oncotarget
https://www.readbyqxmd.com/read/29278652/parp1-induces-cardiac-fibrosis-by-mediating-mtor-activity
#4
Shuya Sun, Yuehuai Hu, Qiyao Zheng, Zhen Guo, Duanping Sun, Shaorui Chen, Yiqiang Zhang, Peiqing Liu, Jing Lu, Jianmin Jiang
Cardiac fibrosis is involved in nearly all forms of heart diseases, and is characterized by excessive deposition of extracellular matrix (ECM) proteins by cardiac fibroblasts (CFs). We and others have reported the possibility of poly(ADP-ribose) polymerase 1 (PARP1), the founding subtype of the PARPs enzyme family, as a novel therapeutic target of heart diseases. The cardiac fibrotic induction of mTOR (mammalian target of rapamycin) is mainly due to collagen expression, Smad3 and p53/JNK-mediated apoptosis...
December 26, 2017: Journal of Cellular Biochemistry
https://www.readbyqxmd.com/read/29224322/-%C3%AE-1-antitrypsin-pittsburg-mutations-report-of-two-cases-in-the-same-family
#5
L J Cao, X Bai, Z Q Yu, W Zhang, J Su, Z Y Wang, C G Ruan
Objective: To analyze the clinical and laboratory abnormalities of two patients with α1-antitrypsin (α1-AT) Pittsburgh in a family and review the literatures. Methods: Both plasma clotting time and factor activities were performed using clotting or substrate methods. Platelet aggregation was evaluated using an optical aggregometer. The serum protein electrophoresis was performed on Sebia HYDRASYS by using Agarose gel. The exons of α1-AT were amplified by using polymerase chain reaction (PCR) and then sequenced and compared with NCBI GenBank records...
November 14, 2017: Zhonghua Xue Ye Xue za Zhi, Zhonghua Xueyexue Zazhi
https://www.readbyqxmd.com/read/29180510/the-mrna-processing-factor-cstf-50-and-ubiquitin-escort-factor-p97-are-brca1-bard1-cofactors-involved-in-chromatin-remodeling-during-dna-damage-response
#6
Danae Fonseca, Jorge Baquero, Michael R Murphy, Gamage Aruggoda, Sophia Varriano, Carmen Sapienza, Oksana Mashadova, Shadaqur Rahman, Frida E Kleiman
The cellular response to DNA damage is an intricate mechanism that involves the interplay among several pathways. In this study, we provide evidence of the role of the polyadenylation factor CstF-50 and the ubiquitin (Ub)-escort factor p97 as cofactors of BRCA1/BARD1 E3 Ub ligase, facilitating chromatin remodeling during DNA damage response (DDR). CstF-50 and p97 formed (a) complex(es) with BRCA1/BARD1, Ub and some of BRCA1/BARD1 substrates, such as RNA polymerase II (RNAP II) and histones. Besides, CstF-50 and p97 had an additive effect on the activation of the ubiquitination of these BRCA1/BARD1 substrates during DDR...
November 27, 2017: Molecular and Cellular Biology
https://www.readbyqxmd.com/read/29159585/kfoa-the-udp-glucose-4-epimerase-of-escherichia-coli-strain-o5-k4-h4-shows-preference-for-acetylated-substrates
#7
Hao-Miao Zhu, Bin Sun, Yi-Jun Li, Dan-Hua Meng, Shuang Zheng, Ting-Ting Wang, Feng-Shan Wang, Ju-Zheng Sheng
Capsule of Escherichia coli O5:K4:H4 is formed of a chondroitin-repeat disaccharide unit of glucuronic acid (GlcA)-N-acetylgalactosamine (GalNAc). This polysaccharide, commonly referred to as K4CP, is a potentially important source of precursors for chemoenzymatic or bioengineering synthesis of chondroitin sulfate. KfoA, encoded by a gene from region 2 of the K4 capsular gene cluster, shows high homology to the UDP-glucose-4-epimerase (GalE) from E. coli. KfoA is reputed to be responsible for uridine 5'-diphosphate-N-acetylgalactosamine (UDP-GalNAc) supply for K4CP biosynthesis in vivo, but it has not been biochemically characterized...
November 20, 2017: Applied Microbiology and Biotechnology
https://www.readbyqxmd.com/read/29155210/transition-from-androgenic-to-neurosteroidal-action-of-5%C3%AE-androstane-3%C3%AE-17%C3%AE-diol-through-the-type-a-%C3%AE-aminobutyric-acid-receptor-in-prostate-cancer-progression
#8
Ding Xia, Doan V Lai, Weijuan Wu, Zachary D Webb, Qing Yang, Lichao Zhao, Zhongxin Yu, Jessica E Thorpe, Bryan C Disch, Michael A Ihnat, Muralidharan Jayaraman, Danny N Dhanasekaran, Kelly L Stratton, Michael S Cookson, Kar-Ming Fung, Hsueh-Kung Lin
Androgen ablation is the standard of care prescribed to patients with advanced or metastatic prostate cancer (PCa) to slow down disease progression. Unfortunately, a majority of PCa patients under androgen ablation progress to castration-resistant prostate cancer (CRPC). Several mechanisms including alternative intra-prostatic androgen production and androgen-independent androgen receptor (AR) activation have been proposed for CRPC progression. Aldo-keto reductase family 1 member C3 (AKR1C3), a multi-functional steroid metabolizing enzyme, is specifically expressed in the cytoplasm of PCa cells; and positive immunoreactivity of the type A γ-aminobutyric acid receptor (GABAAR), an ionotropic receptor and ligand-gated ion channel, is detected on the membrane of PCa cells...
November 15, 2017: Journal of Steroid Biochemistry and Molecular Biology
https://www.readbyqxmd.com/read/29153392/polycomb-repressive-complex-2-methylates-elongin-a-to-regulate-transcription
#9
M Behfar Ardehali, Anthony Anselmo, Jesse C Cochrane, Sharmistha Kundu, Ruslan I Sadreyev, Robert E Kingston
Polycomb repressive complex 2 (PRC2-EZH2) methylates histone H3 at lysine 27 (H3K27) and is required to maintain gene repression during development. Misregulation of PRC2 is linked to a range of neoplastic malignancies, which is believed to involve methylation of H3K27. However, the full spectrum of non-histone substrates of PRC2 that might also contribute to PRC2 function is not known. We characterized the target recognition specificity of the PRC2 active site and used the resultant data to screen for uncharacterized potential targets...
December 7, 2017: Molecular Cell
https://www.readbyqxmd.com/read/29148714/evaluating-the-rate-and-substrate-specificity-of-laboratory-evolved-xna-polymerases
#10
Ali Nikoomanzar, Matthew R Dunn, John C Chaput
Engineered polymerases that can copy genetic information between DNA and xeno-nucleic acids (XNA) hold tremendous value as reagents in future biotechnology applications. However, current XNA polymerases function with inferior activity relative to their natural counterparts, indicating that current polymerase engineering efforts would benefit from new benchmarking assays. Here, we describe a highly parallel, low-cost method for measuring the average rate and substrate specificity of XNA polymerases in a standard qPCR instrument...
November 21, 2017: Analytical Chemistry
https://www.readbyqxmd.com/read/29138906/influence-of-polyaluminum-chloride-on-microbial-characteristics-in-anaerobic-membrane-bioreactors-for-sludge-digestion
#11
Wanlin Zheng, Zhiyong Yu, Yu Xia, Xianghua Wen
In this study, two parallel lab-scale anaerobic membrane bioreactors (AnMBRs), one of which was dosed with polyaluminum chloride (PAC) for membrane fouling control, were operated for treating excess activated sludge collected from a wastewater treatment plant (WWTP). The AnMBRs were inoculated with anaerobic digested sludge collected from an anaerobic digester of another WWTP. The microbial community of digested sludge and cake layer in AnMBRs, as well as that of excess sludge, was analyzed through polymerase chain reaction coupled with denaturing gradient gel electrophoresis (PCR-DGGE) and Illumina MiSeq...
November 14, 2017: Applied Microbiology and Biotechnology
https://www.readbyqxmd.com/read/29130210/imaging-newly-transcribed-rna-in-cells-by-using-a-clickable-azide-modified-utp-analog
#12
Anupam A Sawant, Sanjeev Galande, Seergazhi G Srivatsan
Robust RNA labeling and imaging methods that enable the understanding of cellular RNA biogenesis and function are highly desired. In this context, we describe a practical chemical labeling method based on a bioorthogonal reaction, namely, azide-alkyne cycloaddition reaction, which facilitates the fluorescence imaging of newly transcribed RNA in both fixed and live cells. This strategy involves the transfection of an azide-modified UTP analog (AMUTP) into mammalian cells, which gets specifically incorporated into RNA transcripts by RNA polymerases present inside the cells...
2018: Methods in Molecular Biology
https://www.readbyqxmd.com/read/29118367/impaired-oxidative-stress-response-characterizes-huwe1-promoted-x-linked-intellectual-disability
#13
Matthias Bosshard, Rossana Aprigliano, Cristina Gattiker, Vuk Palibrk, Enni Markkanen, Paul Hoff Backe, Stefania Pellegrino, F Lucy Raymond, Guy Froyen, Matthias Altmeyer, Magnar Bjørås, Grigory L Dianov, Barbara van Loon
Mutations in the HECT, UBA and WWE domain-containing 1 (HUWE1) E3 ubiquitin ligase cause neurodevelopmental disorder X-linked intellectual disability (XLID). HUWE1 regulates essential processes such as genome integrity maintenance. Alterations in the genome integrity and accumulation of mutations have been tightly associated with the onset of neurodevelopmental disorders. Though HUWE1 mutations are clearly implicated in XLID and HUWE1 regulatory functions well explored, currently much is unknown about the molecular basis of HUWE1-promoted XLID...
November 8, 2017: Scientific Reports
https://www.readbyqxmd.com/read/29115461/histone-deacetylase-inhibition-of-cardiac-autophagy-in-rats-on-a-high%C3%A2-fat-diet-with-low%C3%A2-dose-streptozotocin-induced-type-2-diabetes-mellitus
#14
Ting-I Lee, Kuan-Jen Bai, Yao-Chang Chen, Ting-Wei Lee, Cheng-Chih Chung, Wen-Chih Tsai, Shin-Yi Tsao, Yu-Hsun Kao
Autophagy serves a role in preserving cellular homeostasis. Diabetes mellitus (DM) impairs cardiac autophagy and is associated with an accumulation of cytotoxic proteins that may provoke apoptosis and damage cardiomyocytes. Histone deacetylase (HDAC) inhibitors attenuate cardiac fibrosis and inflammation, and improve cardiomyopathy resulting from DM. However, the effect of HDAC inhibition on autophagy in DM cardiomyopathy has not been investigated. The purpose of the present study was to evaluate whether HDAC inhibition modulates cardiac autophagy and to investigate the potential mechanisms in type 2 DM (T2DM) hearts...
October 26, 2017: Molecular Medicine Reports
https://www.readbyqxmd.com/read/29091409/cucurbit-7-uril-driven-host-guest-chemistry-for-reversible-intervention-of-5-formylcytosine-targeted-biochemical-reactions
#15
Shao-Ru Wang, Yan-Yan Song, Lai Wei, Chao-Xing Liu, Bo-Shi Fu, Jia-Qi Wang, Xi-Ran Yang, Yi-Nong Liu, Si-Min Liu, Tian Tian, Xiang Zhou
5-Formylcytosine (5fC) is identified as one of the key players in active DNA demethylation and also as an epigenetic mark in mammals, thus representing a novel attractive target to chemical intervention. The current study represents an attempt to develop a reversible 5fC-targeted intervention tool. A supramolecular aldehyde reactive probe was therefore introduced for selective conversion of the 5fC to 5fC-AD nucleotide. Using various methods, we demonstrate that cucurbit[7]uril (CB7) selectively targets the 5fC-AD nucleotide in DNA, however, the binding of CB7 to 5fC-AD does not affect the hydrogen bonding properties of natural nucleobases in duplex DNA...
November 9, 2017: Journal of the American Chemical Society
https://www.readbyqxmd.com/read/29061787/-epigallocatechin-3-gallate-down-regulates-doxorubicin-induced-overexpression-of-p-glycoprotein-through-the-coordinate-inhibition-of-pi3k-akt-and-mek-erk-signaling-pathways
#16
Hana Satonaka, Kumiki Ishida, Miho Takai, Ryoji Koide, Ryota Shigemasa, Jun Ueyama, Tetsuya Ishikawa, Kazuhiko Hayashi, Hidemi Goto, Shinya Wakusawa
BACKGROUND/AIM: (-)-Epigallocatechin-3-gallate (EGCG) has been indicated to regulate the function of P-glycoprotein (P-gp), which is a drug transporter encoded by the MDR1 (ABCB1) gene. P-gp expression is induced by doxorubicin (DOX). We aimed to clarify the mechanisms and inhibitory effects of EGCG on DOX-induced P-gp expression in HepG2 cells. MATERIALS AND METHODS: Rhodamine 123 (Rho123) was used for P-gp substrate. Western blotting and polymerase chain reactions (PCRs) were conducted using specific antibodies and primer sets...
November 2017: Anticancer Research
https://www.readbyqxmd.com/read/28994814/real-time-quaking-induced-conversion-assay-for-detection-of-cwd-prions-in-fecal-material
#17
Yo Ching Cheng, Samia Hannaoui, Theodore Ralph John, Sandor Dudas, Stefanie Czub, Sabine Gilch
The RT-QuIC technique is a sensitive in vitro cell-free prion amplification assay based mainly on the seeded misfolding and aggregation of recombinant prion protein (PrP) substrate using prion seeds as a template for the conversion. RT-QuIC is a novel high-throughput technique which is analogous to real-time polymerase chain reaction (PCR). Detection of amyloid fibril growth is based on the dye Thioflavin T, which fluoresces upon specific interaction with ᵦ-sheet rich proteins. Thus, amyloid formation can be detected in real time...
September 29, 2017: Journal of Visualized Experiments: JoVE
https://www.readbyqxmd.com/read/28992937/substrate-specificity-of-the-kinase-p-tefb-towards-the-rna-polymerase-ii-c-terminal-domain
#18
Eric B Gibbs, Tatiana N Laremore, Grace A Usher, Bede Portz, Erik C Cook, Scott A Showalter
The positive transcription elongation factor b (P-TEFb) promotes transcription elongation through phosphorylation of the RNA polymerase II C-terminal domain. This process is not well understood, partly due to difficulties in determining the specificity of P-TEFb toward the various heptad repeat motifs within the C-terminal domain. A simple assay using mass spectrometry was developed to identify the substrate specificity of the Drosophila melanogaster P-TEFb (DmP-TEFb) in vitro. This assay demonstrated that DmP-TEFb preferentially phosphorylates Ser5 and, surprisingly, that pre-phosphorylation or conserved amino acid variation at the 7-position in the heptad can alter DmP-TEFb specificity, leading to the creation of distinct double-phosphorylation marks...
November 7, 2017: Biophysical Journal
https://www.readbyqxmd.com/read/28984438/engineering-the-genetic-code-in-cells-and-animals-biological-considerations-and-impacts
#19
Lei Wang
Expansion of the genetic code allows unnatural amino acids (Uaas) to be site-specifically incorporated into proteins in live biological systems, thus enabling novel properties selectively introduced into target proteins in vivo for basic biological studies and for engineering of novel biological functions. Orthogonal components including tRNA and aminoacyl-tRNA synthetase (aaRS) are expressed in live cells to decode a unique codon (often the amber stop codon UAG) as the desired Uaa. Initially developed in E...
November 21, 2017: Accounts of Chemical Research
https://www.readbyqxmd.com/read/28977649/the-rna-chaperone-la-promotes-pre-trna-maturation-via-indiscriminate-binding-of-both-native-and-misfolded-targets
#20
Ana Vakiloroayaei, Neha S Shah, Marlene Oeffinger, Mark A Bayfield
Non-coding RNAs have critical roles in biological processes, and RNA chaperones can promote their folding into the native shape required for their function. La proteins are a class of highly abundant RNA chaperones that contact pre-tRNAs and other RNA polymerase III transcripts via their common UUU-3'OH ends, as well as through less specific contacts associated with RNA chaperone activity. However, whether La proteins preferentially bind misfolded pre-tRNAs or instead engage all pre-tRNA substrates irrespective of their folding status is not known...
August 31, 2017: Nucleic Acids Research
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