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Site directed mutations

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https://www.readbyqxmd.com/read/28649619/variable-frequency-of-lrrk2-variants-in-the-latin-american-research-consortium-on-the-genetics-of-parkinson-s-disease-large-pd-a-case-of-ancestry
#1
Mario Cornejo-Olivas, Luis Torres, Mario R Velit-Salazar, Miguel Inca-Martinez, Pilar Mazzetti, Carlos Cosentino, Federico Micheli, Claudia Perandones, Elena Dieguez, Victor Raggio, Vitor Tumas, Vanderci Borges, Henrique B Ferraz, Carlos R M Rieder, Artur Shumacher-Schuh, Carlos Velez-Pardo, Marlene Jimenez-Del-Rio, Francisco Lopera, Jorge Chang-Castello, Brennie Andreé-Munoz, Sarah Waldherr, Dora Yearout, Cyrus P Zabetian, Ignacio F Mata
Mutations in Leucine-Rich Repeat Kinase 2 (LRRK2), primarily located in codons G2019 and R1441, represent the most common genetic cause of Parkinson's disease in European-derived populations. However, little is known about the frequency of these mutations in Latin American populations. In addition, a prior study suggested that a LRRK2 polymorphism (p.Q1111H) specific to Latino and Amerindian populations might be a risk factor for Parkinson's disease, but this finding requires replication. We screened 1734 Parkinson's disease patients and 1097 controls enrolled in the Latin American Research Consortium on the Genetics of Parkinson's disease (LARGE-PD), which includes sites in Argentina, Brazil, Colombia, Ecuador, Peru, and Uruguay...
2017: NPJ Parkinson's Disease
https://www.readbyqxmd.com/read/28647474/antiviral-profiling-of-the-capsid-assembly-modulator-bay41-4109-on-full-length-hbv-genotype-a-h-clinical-isolates-and-core-site-directed-mutants-in-vitro
#2
Jan Martin Berke, Ying Tan, Thierry Verbinnen, Pascale Dehertogh, Karen Vergauwen, Ann Vos, Oliver Lenz, Frederik Pauwels
The HBV core protein represents an attractive target for new antiviral therapies due to its multiple functions within the viral life-cycle. Here, we report the antiviral activity of the capsid assembly modulator (CAM) BAY41-4109 and two nucleos(t)ide analogues (NAs) on a diverse panel of 54 HBV clinical isolates from genotype (GT) A-H and assessed the impact of core amino acid (aa) substitutions using site-directed mutants (SDMs). The median EC50 values of BAY41-4109 across genotypes ranged from 26 nM in GT G to 215 nM in GT F irrespective of the presence of NA resistance mutations compared to 43 nM for the GT D reference construct...
June 21, 2017: Antiviral Research
https://www.readbyqxmd.com/read/28646206/crispr-cas9-mediated-genome-editing-via-postnatal-administration-of-aav-vector-cures-haemophilia-b-mice
#3
Tsukasa Ohmori, Yasumitsu Nagao, Hiroaki Mizukami, Asuka Sakata, Shin-Ichi Muramatsu, Keiya Ozawa, Shin-Ichi Tominaga, Yutaka Hanazono, Satoshi Nishimura, Osamu Nureki, Yoichi Sakata
Haemophilia B, a congenital haemorrhagic disease caused by mutations in coagulation factor IX gene (F9), is considered an appropriate target for genome editing technology. Here, we describe treatment strategies for haemophilia B mice using the clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 system. Administration of adeno-associated virus (AAV) 8 vector harbouring Staphylococcus aureus Cas9 (SaCas9) and single guide RNA (sgRNA) to wild-type adult mice induced a double-strand break (DSB) at the target site of F9 in hepatocytes, sufficiently developing haemophilia B...
June 23, 2017: Scientific Reports
https://www.readbyqxmd.com/read/28645318/detecting-consistent-patterns-of-directional-adaptation-using-differential-selection-codon-models
#4
Sahar Parto, Nicolas Lartillot
BACKGROUND: Phylogenetic codon models are often used to characterize the selective regimes acting on protein-coding sequences. Recent methodological developments have led to models explicitly accounting for the interplay between mutation and selection, by modeling the amino acid fitness landscape along the sequence. However, thus far, most of these models have assumed that the fitness landscape is constant over time. Fluctuations of the fitness landscape may often be random or depend on complex and unknown factors...
June 23, 2017: BMC Evolutionary Biology
https://www.readbyqxmd.com/read/28645089/drug-resistance-mechanism-of-l10f-l10f-n88s-and-l90m-mutations-in-crf01_ae-hiv-1-protease-molecular-dynamics-simulations-and-binding-free-energy-calculations
#5
C S Vasavi, Ramasamy Tamizhselvi, Punnagai Munusami
HIV-1 protease plays a crucial role in viral replication and maturation, which makes it one of the most attractive targets for anti-retroviral therapy. The majority of HIV infections in developing countries are due to non-B subtype. Subtype AE is spreading rapidly and infecting huge population worldwide. The mutations in the active site of subtype AE directly impair the interactions with the inhibitor. The non-active site mutations influence the binding of the inhibitor indirectly and their resistance mechanism is not well understood...
June 8, 2017: Journal of Molecular Graphics & Modelling
https://www.readbyqxmd.com/read/28643343/alpha-beta-hydrolases-a-unique-structural-motif-coordinates-catalytic-acid-residue-in-40-protein-fold-families
#6
Polytimi S Dimitriou, Alexander Denesyuk, Seiji Takahashi, Satoshi Yamashita, Mark S Johnson, Toru Nakayama, Konstantin Denessiouk
The alpha/beta-hydrolases (ABH) are a family of acid-base-nucleophile catalytic triad enzymes with a common fold, but using a wide variety of substrates, having different pH optima, catalyzing unique catalytic reactions and often showing improved chemical and thermo stability. The ABH enzymes are prime targets for protein engineering. Here, we have classified active sites from 51 representative members of 40 structural ABH fold families into eight distinct conserved geometries. We demonstrate the occurrence of a common structural motif, the catalytic acid zone, at the catalytic triad acid turn...
June 23, 2017: Proteins
https://www.readbyqxmd.com/read/28642232/a-single-amino-acid-residue-at-transmembrane-domain-4-of-the-alpha-subunit-influences-carisoprodol-direct-gating-efficacy-at-gabaa-receptors
#7
Manoj Kumar, Manish Kumar, John Freund, Glenn H Dillon
The muscle relaxant carisoprodol (CSP, trade name Soma) has recently been controlled at the federal level as a Schedule IV drug due to its high abuse potential and consequences of misuse, such as withdrawal syndrome, delusions, seizures and even death. Recent work has shown that carisoprodol can directly gate and allosterically modulate the GABAA receptor. These actions are subunit-dependent; compared to other GABAA receptors, carisoprodol has nominal direct gating effects in α3β2γ2receptors. Here, using site-directed-mutagenesis and whole cell patch clamp electrophysiology in transiently transfected HEK293 cells, we examined the role of GABAA receptor α subunit transmembrane domain 4 (TM4) amino acids in direct gating and allosteric modulatory actions of carisoprodol...
June 22, 2017: Journal of Pharmacology and Experimental Therapeutics
https://www.readbyqxmd.com/read/28641563/pentapeptide-prosequence-enhances-expression-and-structure-folding-of-recombinant-thermomyces-lanuginosus-lipase-in-pichia-pastoris
#8
Haiying Cai, Minjie Zhao, Yang Li, Jianwei Mao, Chenggang Cai, Fengqin Feng
Propeptides have been demonstrated to influence many properties of their mature proteins as intramolecular chaperones. In our study, the role of pentapeptide prosequence of Thermomyces lanuginosus lipase (TLL) was determined through its effect on the recombinant expression of TLL in P. pastoris. The result showed that the average lipase activity of recombinant strains GS-pTL reached 434.32 U/mL, higher than that of GS-TL 377.71 U/mL, indicating that the propeptide "SPIRR-" sequence is beneficial for enhancing TLL expression...
June 21, 2017: Protein and Peptide Letters
https://www.readbyqxmd.com/read/28640983/multiplexed-crispr-cas9-mediated-metabolic-engineering-of-%C3%AE-aminobutyric-acid-levels-in-solanum-lycopersicum
#9
Rui Li, Ran Li, Xindi Li, Daqi Fu, Benzhong Zhu, Huiqin Tian, Yunbo Luo, Hongliang Zhu
In recent years, the type II CRISPR system has become a widely used and robust technique to implement site-directed mutagenesis in a variety of species including model and crop plants. However, few studies manipulated metabolic pathways in plants using the CRISPR system. Here, we introduced the pYLCRISPR/Cas9 system with one or two single-site guide RNAs to target the tomato phytoene desaturase gene. An obvious albino phenotype was observed in T0 regenerated plants, and more than 61% of the desired target sites were edited...
June 22, 2017: Plant Biotechnology Journal
https://www.readbyqxmd.com/read/28639260/improving-thermostability-of-r-selective-amine-transaminase-from-aspergillus-terreus-through-introduction-of-disulfide-bonds
#10
Dong-Fang Xie, Hui Fang, Jia-Qi Mei, Jin-Yan Gong, Hong-Peng Wang, Xiu-Ying Shen, Jun Huang, Le-He Mei
To improve the thermostability of (R)-selective amine transaminase from Aspergillus terreus (AT-ATA), we used computer software Disulfide by Design (DbD) and Modelling of Disulfide Bonds in Proteins (MODIP) to identify mutation sites where the disulfide bonds were most likely to form. We obtained three stabilised mutants (N25C-A28C, R131C-D134C, M150C-M280C) from seven candidates by site-directed mutagenesis. Compared to the wild-type, the best two mutants N25C-A28C and M150C-M280C showed improved thermal stability with a 3...
June 21, 2017: Biotechnology and Applied Biochemistry
https://www.readbyqxmd.com/read/28637869/heat-shock-proteins-stimulate-apobec-3-mediated-cytidine-deamination-in-the-hepatitis-b-virus
#11
Zhigang Chen, Thomas L Eggerman, Alexander V Bocharov, Irina N Baranova, Tatyana G Vishnyakova, Roger Kurlander, Amy P Patterson
Apolipoprotein B mRNA-editing enzyme catalytic subunit 3 (APOBEC-3) enzymes are cytidine deaminases that are broadly and constitutively expressed. They are often upregulated during carcinogenesis and candidate genes for causing the major single-base substitution in cancer-associated DNA mutations. Moreover, APOBEC-3s are involved in host innate immunity against many viruses. However, how APOBEC-3 mutational activity is regulated in normal and pathological conditions remains largely unknown. Heat shock protein levels are often elevated in both carcinogenesis and viral infection and are associated with DNA mutations...
June 21, 2017: Journal of Biological Chemistry
https://www.readbyqxmd.com/read/28637270/in-planta-recapitulation-of-isoprene-synthases-evolution-from-ocimene-synthases
#12
Mingai Li, Jia Xu, Alberto Algarra Alarcon, Silvia Carlin, Enrico Barbaro, Luca Cappellin, Violeta Velikova, Urska Vrhovsek, Francesco Loreto, Claudio Varotto
Isoprene is the most abundant biogenic volatile hydrocarbon compound naturally emitted by plants and plays a major role in atmospheric chemistry. It has been proposed that isoprene synthases (IspS) may readily evolve from other terpene synthases, but this hypothesis has not been experimentally investigated.We isolated and functionally validated in Arabidopsis the first isoprene synthase gene, AdoIspS, from a monocotyledonous species (Arundo donax L., Poaceae). Phylogenetic reconstruction indicates that AdoIspS and dicots isoprene synthases most likely originated by parallel evolution from TPS-b monoterpene synthases...
June 16, 2017: Molecular Biology and Evolution
https://www.readbyqxmd.com/read/28637173/crystallographic-analyses-illustrate-significant-plasticity-and-efficient-recoding-of-meganuclease-target-specificity
#13
Rachel Werther, Jazmine P Hallinan, Abigail R Lambert, Kyle Havens, Mark Pogson, Jordan Jarjour, Roberto Galizi, Nikolai Windbichler, Andrea Crisanti, Tony Nolan, Barry L Stoddard
The retargeting of protein-DNA specificity, outside of extremely modular DNA binding proteins such as TAL effectors, has generally proved to be quite challenging. Here, we describe structural analyses of five different extensively retargeted variants of a single homing endonuclease, that have been shown to function efficiently in ex vivo and in vivo applications. The redesigned proteins harbor mutations at up to 53 residues (18%) of their amino acid sequence, primarily distributed across the DNA binding surface, making them among the most significantly reengineered ligand-binding proteins to date...
June 20, 2017: Nucleic Acids Research
https://www.readbyqxmd.com/read/28636382/development-of-efficient-chemistry-to-generate-site-specific-disulfide-linked-protein-and-peptide-payload-conjugates-application-to-thiomab%C3%A2-antibody-drug-conjugates
#14
Jack David Sadowsky, Thomas H Pillow, Jinhua Chen, Fang Fan, Changrong He, Yanli Wang, Gang Yan, Hui Yao, Zijin Xu, Shanique Martin, Donglu Zhang, Phillip Chu, Josefa Dela Cruz-Chuh, Aimee O'Donohue, Guangmin Li, Geoffrey Del Rosario, Jintang He, Luna Liu, Carl K Ng, Dian Su, Gail D Lewis Phillips, Katherine Ruth Kozak, Shang-Fan Yu, Keyang Xu, Douglas Leipold, John S Wai
Conjugation of small molecule payloads to specific cysteine residues on proteins via a disulfide bond represents an attractive strategy to generate redox-sensitive bioconjugates, which have value as potential diagnostic reagents or therapeutics. Advancement of such "direct-disulfide" bioconjugates to the clinic necessitates chemical methods to form disulfide connections efficiently, without byproducts. The disulfide connection must also be resistant to premature cleavage by thiols prior to arrival at the targeted tissue...
June 21, 2017: Bioconjugate Chemistry
https://www.readbyqxmd.com/read/28634712/site-directed-mutagenesis-under-the-direction-of-in-silico-protein-docking-modeling-reveals-the-active-site-residues-of-3-ketosteroid-%C3%AE-1-dehydrogenase-from-mycobacterium-neoaurum
#15
Ning Qin, Yanbing Shen, Xu Yang, Liqiu Su, Rui Tang, Wei Li, Min Wang
3-Ketosteroid-Δ(1)-dehydrogenases (KsdD) from Mycobacterium neoaurum could transform androst-4-ene-3,17-dione (AD) to androst-1,4-diene-3,17-dione. This reaction has a significant effect on the product of pharmaceutical steroid. The crystal structure and active site residues information of KsdD from Mycobacterium is not yet available, which result in the engineering of KsdD is tedious. In this study, by the way of protein modeling and site-directed mutagenesis, we find that, Y122, Y125, S138, E140 and Y541 from the FAD-binding domain and Y365 from the catalytic domain play a key role in this transformation...
July 2017: World Journal of Microbiology & Biotechnology
https://www.readbyqxmd.com/read/28634062/site-directed-mutagenesis-of-gh10-xylanase-a-from-penicillium-canescens-for-determining-factors-affecting-the-enzyme-thermostability
#16
Yury A Denisenko, Alexander V Gusakov, Aleksandra M Rozhkova, Dmitry O Osipov, Ivan N Zorov, Veronika Yu Matys, Igor V Uporov, Arkady P Sinitsyn
In order to investigate factors affecting the thermostability of GH10 xylanase A from Penicillium canescens (PcXylA) and to obtain its more stable variant, the wild-type (wt) enzyme and its mutant forms, carrying single amino acid substitutions, were cloned and expressed in Penicillium verruculosum B1-537 (niaD-) auxotrophic strain under the control of the cbh1 gene promoter. The recombinant PcXylA-wt and I6V, I6L, L18F, N77D, Y125R, H191R, S246P, A293P mutants were successfully expressed and purified for characterization...
June 17, 2017: International Journal of Biological Macromolecules
https://www.readbyqxmd.com/read/28631420/expression-of-pten-long-mediated-by-crispr-cas9-can-repress-u87-cell-proliferation
#17
Na Fang, Tingxuan Gu, Yahui Wang, Shuzhen Wang, Fengling Wang, Yang An, Wenqiang Wei, Weijuan Zhang, Xiangqian Guo, Adil J Nazarali, Shaoping Ji
PTEN is a tumour suppressor that is frequently mutated in a variety of cancers. Hence, PTEN has significant potential as a therapeutic molecule. PTEN-long is an alternative translation variant, with an additional 173 amino acids added to the N-terminal of the canonical PTEN when CUG of the mRNA is utilized as the start codon. PTEN-long is secreted into serum and can re-enter cells throughout the body. One of the major barriers for gene therapy is to efficiently and specifically deliver DNA or RNA material to target cells...
June 19, 2017: Journal of Cellular and Molecular Medicine
https://www.readbyqxmd.com/read/28628526/determinants-of-hiv-1-cd4-independent-brain-adaptation
#18
Madina Shakirzyanova, Xiang-Peng Kong, Cecilia Cheng-Mayer
BACKGROUND: HIV-1 is known to adapt to the local environment in its usage of receptors and it can become CD4 independent in the brain where the receptor is scarce. This adaptation is through amino acid variations, but the patterns of such variation are not yet well understood. Given that infection of long-lived CD4-low and CD4-negative cells in anatomic compartments such as the brain expand cell tropism in vivo and may serve as potential viral reservoirs that pose challenge for HIV eradication, understanding the evolution to CD4-independence and envelope conformation associated with infection in the absence of CD4 will not only broaden our insights into HIV pathogenesis, but may guide functional cure strategies as well...
June 15, 2017: Journal of Acquired Immune Deficiency Syndromes: JAIDS
https://www.readbyqxmd.com/read/28627884/hdx-ms-reveals-calcium-binding-properties-and-allosteric-regulation-of-downstream-regulatory-element-antagonist-modulator-dream
#19
Jun Zhang, Jing Li, Theodore A Craig, Rajiv Kumar, Michael L Gross
Downstream regulatory element antagonist modulator (DREAM) is an EF-hand Ca2+-binding protein that also binds to specific DNA sequence, downstream regulatory elements (DRE), and thereby regulates transcription in a calcium-dependent fashion. DREAM binds to DRE in the absence of Ca2+, but detaches from DRE under Ca2+ stimulation, allowing gene expression. The Ca2+-binding properties of DREAM and the consequences of the binding on protein structure are key to understanding the function of DREAM. Here we describe the application of hydrogen deuterium exchange mass spectrometry (HDX-MS) and site-directed mutagenesis to investigate the Ca2+ binding properties and the subsequent conformational changes of full length DREAM...
June 19, 2017: Biochemistry
https://www.readbyqxmd.com/read/28624275/nuclear-receptor-subfamily-1-group-h-member-2-lxrb-is-the-predominant-liver-x-receptor-subtype-regulating-transcription-of-2-major-lipogenic-genes-in-goat-primary-mammary-epithelial-cells
#20
H B Shi, C H Zhang, Z A Xu, X F Xu, Z B Lv, J Luo, J J Loor
In ruminants, recent research has identified a crucial role for liver X receptors (LXR) in regulating lipid metabolism in mammary cells. However, the differences between LXR subtypes in regulating ruminant lipid metabolism are unknown. We used overexpression and knockdown of LXRA and LXRB in goat primary mammary epithelial cells to distinguish subtype-specific regulation of sterol regulatory element binding protein 1c (SREBP1c) and fatty acid synthase (FASN) mRNA expression and their promoter activity. Incubation with siRNA targeting LXR decreased expression of LXRA and LXRB...
June 14, 2017: Journal of Dairy Science
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