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https://www.readbyqxmd.com/read/28340408/ongoing-evolution-of-pseudomonas-aeruginosa-pao1-sublines-complicates-studies-of-dna-damage-repair-and-tolerance
#1
Julia Sidorenko, Tatjana Jatsenko, Maia Kivisaar
Sublines of the major P. aeruginosa reference strain PAO1 are derivatives of the original PAO1 isolate, which are maintained in laboratories worldwide. These sublines display substantial genomic and phenotypic variation due to ongoing microevolution. Here, we examined four sublines, MPAO1, PAO1-L, PAO1-DSM and PAO1-UT, originated from different laboratories, and six DNA polymerase-deficient mutants from the P. aeruginosa MPAO1 transposon library for their employment in elucidation of DNA damage repair and tolerance mechanisms in P...
March 16, 2017: Mutation Research
https://www.readbyqxmd.com/read/28340354/mechanism-of-substrate-translocation-in-an-alternating-access-transporter
#2
Naomi R Latorraca, Nathan M Fastman, A J Venkatakrishnan, Wolf B Frommer, Ron O Dror, Liang Feng
Transporters shuttle molecules across cell membranes by alternating among distinct conformational states. Fundamental questions remain about how transporters transition between states and how such structural rearrangements regulate substrate translocation. Here, we capture the translocation process by crystallography and unguided molecular dynamics simulations, providing an atomic-level description of alternating access transport. Simulations of a SWEET-family transporter initiated from an outward-open, glucose-bound structure reported here spontaneously adopt occluded and inward-open conformations...
March 23, 2017: Cell
https://www.readbyqxmd.com/read/28340294/a-well-defined-osmium-cupin-complex-hyperstable-artificial-osmium-peroxygenase
#3
Nobutaka Fujieda, Takumi Nakano, Yuki Taniguchi, Haruna Ichihashi, Hideki Sugimoto, Yuma Morimoto, Yosuke Nishikawa, Genji Kurisu, Shinobu Itoh
Thermally stable TM1459 cupin superfamily protein from Thermotoga maritima was repurposed as an osmium (Os) peroxygenase by metal-substitution strategy employing the metal-binding promiscuity. This novel artificial metalloenzyme bears a datively bound Os ion supported by the 4-histidine motif. The well-defined Os center is responsible for not only the catalytic activity but also the thermodynamic stability of the protein folding, leading to the robust biocatalyst (Tm ≈ 120 °C). The spectroscopic analysis and atomic resolution X-ray crystal structures of Os-bound TM1459 revealed two types of donor sets to Os center with octahedral coordination geometry...
March 24, 2017: Journal of the American Chemical Society
https://www.readbyqxmd.com/read/28340291/-head-to-head-prenyl-synthases-in-some-pathogenic-bacteria
#4
Christopher J Schwalen, Xinxin Feng, Weidong Liu, Bing O-Dowd, Tzu-Ping Ko, Christopher J Shin, Rey-Ting Guo, Douglas A Mitchell, Eric Oldfield
Many organisms contain "head-to-head" isoprenoid synthases and here, we investigate three types of such enzymes from the pathogens Neisseria meningitidis, N. gonorrhoeae, and Enterococcus hirae. The E. hirae enzyme was found to produce dehydrosqualene and we solved an inhibitor-bound structure which revealed a fold similar to that of CrtM from Staphylococcus aureus. In contrast, the homologous proteins from Neisseria spp. carried out only the "first half" reaction, yielding presqualene diphosphate (PSPP). Based on product analyses, bioinformatics, and mutagenesis we conclude that the Neisseria proteins are HpnDs (PSPP synthases)...
March 24, 2017: Chembiochem: a European Journal of Chemical Biology
https://www.readbyqxmd.com/read/28339688/an-activity-staining-method-on-filtration-paper-enables-high-throughput-screening-of-temperature-sensitive-and-inactive-mutations-of-rice-%C3%AE-amylase-for-improvement-of-rice-grain-quality
#5
Hiromoto Yamakawa, Rieko Hirai-Kimura, Yuriko Nakata, Masaru Nakata, Masaharu Kuroda, Takeshi Yamaguchi
α-Amylase is a starch-hydrolyzing enzyme (EC 3.2.1.1) indispensable for germination of cereal seeds, but it is also expressed during the ripening stage. Previous studies demonstrated that the enzyme is activated in developing rice seeds under extremely hot weather and triggers a loss of grain quality by hindering the accumulation of storage starch in the endosperm. Since inactive or, preferably, heat-labile α-amylases are preferable for breeding premium rice, we developed a method for rapid screening of inactive and temperature-sensitive mutants of the enzyme by combining the random mutagenesis by error-prone PCR and an on-filter activity test of the recombinant enzyme expressed by Escherichia coli...
February 21, 2017: Plant & Cell Physiology
https://www.readbyqxmd.com/read/28339613/a-nonsense-mutation-in-ccdc62-gene-is-responsible-for-spermiogenesis-defects-and-male-infertility-in-repro29-repro29-mice%C3%A2
#6
Yuchi Li, Cailing Li, Shouren Lin, Bo Yang, Weiren Huang, Hanwei Wu, Yuanbin Chen, Lihua Yang, Manling Luo, Huan Guo, Jianbo Chen, Tiantian Wang, Qian Ma, Yanli Gu, Lisha Mou, Zhimao Jiang, Jun Xia, Yaoting Gui
Phenotype-driven mutagenesis is an unbiased method to identify novel genes involved in spermatogenesis and other reproductive processes. Male repro29/repro29 mice generated by the Reproductive Genomics Program at the Jackson Laboratory were infertile with deformed sperm and poor motility. Using selected exonic capture and massively parallel sequencing technologies, we identified a nonsense mutation in the exon 6 of coiled-coil domain-containing 62 gene (Ccdc62), which results in a formation of a premature stop codon and a truncated protein...
January 28, 2017: Biology of Reproduction
https://www.readbyqxmd.com/read/28339459/functional-classification-of-dna-variants-by-hybrid-minigenes-identification-of-30-spliceogenic-variants-of-brca2-exons-17-and-18
#7
Eugenia Fraile-Bethencourt, Beatriz Díez-Gómez, Valeria Velásquez-Zapata, Alberto Acedo, David J Sanz, Eladio A Velasco
Mutation screening of the breast cancer genes BRCA1 and BRCA2 identifies a large fraction of variants of uncertain clinical significance (VUS) whose functional and clinical interpretations pose a challenge for genomic medicine. Likewise, an increasing amount of evidence indicates that genetic variants can have deleterious effects on pre-mRNA splicing. Our goal was to investigate the impact on splicing of a set of reported variants of BRCA2 exons 17 and 18 to assess their role in hereditary breast cancer and to identify critical regulatory elements that may constitute hotspots for spliceogenic variants...
March 24, 2017: PLoS Genetics
https://www.readbyqxmd.com/read/28338942/structure-and-mechanism-of-benzaldehyde-dehydrogenase-from-pseudomonas-putida-atcc-12633-a-member-of-the-class-3-aldehyde-dehydrogenase-superfamily
#8
Megan P D Zahniser, Shreenath Prasad, Malea M Kneen, Cheryl A Kreinbring, Gregory A Petsko, Dagmar Ringe, Michael J McLeish
Benzaldehyde dehydrogenase from Pseudomonas putida (PpBADH) belongs to the Class 3 aldehyde dehydrogenase (ALDH) family. The Class 3 ALDHs are unusual in that they are generally dimeric (rather than tetrameric), relatively non-specific and utilize both NAD+ and NADP+. To date, X-ray structures of three Class 3 ALDHs have been determined, of which only two have cofactor bound, both in the NAD+ form. Here we report the crystal structure of PpBADH in complex with NADP+ and a thioacyl intermediate adduct. The overall architecture of PpBADH resembles that of most other members of the ALDH superfamily, and the cofactor binding residues are well conserved...
March 9, 2017: Protein Engineering, Design & Selection: PEDS
https://www.readbyqxmd.com/read/28338893/site-directed-mutant-libraries-for-isolating-minimal-mutations-yielding-functional-changes
#9
Dong Hee Chung, Sarah C Potter, Ammon C Tanomrat, Krishnakumar M Ravikumar, Michael D Toney
Powerful, facile new ways to create libraries of site-directed mutants are demonstrated. These include: (1) one-pot-PCR, (2) multi-pot-PCR, and (3) split-mix-PCR. One-pot-PCR uses mutant oligonucleotides to generate megaprimers in situ, and it was used to randomly incorporate 28 mutations in a gabT gene in a single reaction. In more difficult cases, multi-pot-PCR can be employed: mutant megaprimers are synthesized individually, then combined in a single mutagenesis PCR. This method was used to incorporate 14 out of 15 mutations in a pabB gene...
March 9, 2017: Protein Engineering, Design & Selection: PEDS
https://www.readbyqxmd.com/read/28338789/phosphorylation-of-the-phytosulfokine-peptide-receptor-pskr1-controls-receptor-activity
#10
Christine Kaufmann, Michael Motzkus, Margret Sauter
The phytosulfokine peptide receptor PSKR1 is modified by phosphorylation of its cytoplasmic kinase domain. We analyzed defined phosphorylation sites by site-directed mutagenesis with regard to kinase activity in vitro and receptor activity in planta. S696 and S698 in the juxtamembrane (JM) domain are phosphorylated in planta. The phosphomimetic S696D/S698D replacements resulted in reduced transphosphorylation activity of PSKR1 kinase in vitro but did not reduce autophosphorylation activity. Growth-promoting activity of the PSKR1(S696D/S698D) receptor isoform was impaired in the shoot but not in the root...
February 23, 2017: Journal of Experimental Botany
https://www.readbyqxmd.com/read/28337968/crispr-cas9-mutagenesis-invalidates-a-putative-cancer-dependency-targeted-in-on-going-clinical-trials
#11
Ann Lin, Christopher J Giuliano, Nicole M Sayles, Jason M Sheltzer
The Maternal Embryonic Leucine Zipper Kinase (MELK) has been reported to be a genetic dependency in several cancer types. MELK RNAi and small-molecule inhibitors of MELK block the proliferation of various cancer cell lines, and MELK knockdown has been described as particularly effective against the highly-aggressive basal/triple-negative subtype of breast cancer. Based on these preclinical results, the MELK inhibitor OTS167 is currently being tested as a novel chemotherapy agent in several clinical trials. Here, we report that mutagenizing MELK with CRISPR/Cas9 has no effect on the fitness of basal breast cancer cell lines or cell lines from six other cancer types...
March 24, 2017: ELife
https://www.readbyqxmd.com/read/28336574/switching-head-group-selectivity-in-mammalian-sphingolipid-biosynthesis-by-active-site-engineering-of-sphingomyelin-synthases
#12
Matthijs Kol, Radhakrishnan Panatala, Mirjana Nordmann, Leoni Swart, Leonie Van Suijlekom, Birol Cabukusta, Angelika Hilderink, Tanja Gabrietz, John G M Mina, Pentti Somerharju, Sergei Korneev, Fikadu G Tafesse, Joost C M Holthuis
Sphingomyelin (SM) is a fundamental component of mammalian cell membranes that contributes to mechanical stability, signaling and sorting. Its production involves the transfer of phosphocholine from phosphatidylcholine onto ceramide, a reaction catalyzed by SM synthase SMS1 in the Golgi and SMS2 at the plasma membrane. Mammalian cells also synthesize trace amounts of the SM analog ceramide phosphoethanolamine (CPE), but the physiological relevance of CPE production is unclear. Previous work revealed that SMS2 is a bifunctional enzyme producing both SM and CPE whereas a closely related enzyme, SMSr/SAMD8, acts as a monofunctional CPE synthase in the ER...
March 23, 2017: Journal of Lipid Research
https://www.readbyqxmd.com/read/28336556/human-microrna-548p-decreases-hepatic-apolipoprotein-b-secretion-and-lipid-synthesis
#13
Liye Zhou, M Mahmood Hussain
OBJECTIVE: MicroRNAs (miRs) play important regulatory roles in lipid metabolism. Apolipoprotein B (ApoB), as the only essential scaffolding protein in the assembly of very-low-density lipoproteins, is a target to treat hyperlipidemia and atherosclerosis. We aimed to find out miRs that reduce apoB expression. APPROACH AND RESULTS: Bioinformatic analyses predicted that hsa-miR-548p can interact with apoB mRNA. MiR-548p or control miR was transfected in human and mouse liver cells to test its role in regulating apoB secretion and mRNA expression levels...
March 23, 2017: Arteriosclerosis, Thrombosis, and Vascular Biology
https://www.readbyqxmd.com/read/28336547/glycans-in-the-intestinal-peptide-transporter-pept1-contribute-to-function-and-protect-from-proteolysis
#14
Tamara Stelzl, Kerstin Elisabeth Geillinger-Kästle, Jürgen Stolz, Hannelore Daniel
Despite the fact that many membrane proteins carry extracellular glycans, little is known about whether the glycan chains also affect protein function. We recently demonstrated that the proton-coupled oligopeptide transporter 1 (PEPT1) in the intestine is glycosylated at six asparagine residues (N50, N406, N439, N510, N515, N532). Mutagenesis-induced disruption of the individual N-glycosylation site N50, which is highly conserved among mammals, was detected to significantly enhance the PEPT1 mediated inward transport of peptides...
March 23, 2017: American Journal of Physiology. Gastrointestinal and Liver Physiology
https://www.readbyqxmd.com/read/28336404/mapping-region-of-human-restriction-factor-apobec3h-critical-for-interaction-with-hiv-1-vif
#15
Masaaki Nakashima, Shinya Tsuzuki, Hiroaki Awazu, Akiko Hamano, Ayaka Okada, Hirotaka Ode, Masami Maejima, Atsuko Hachiya, Yoshiyuki Yokomaku, Nobuhisa Watanabe, Hirofumi Akari, Yasumasa Iwatani
The APOBEC3 (A3) family of cellular cytidine deaminases comprises seven members (A, B, C, D, F, G, and H) that potently inhibit retroviral replication. Human immunodeficiency virus type 1 (HIV-1) Vif is a small pleiotropic protein that specifically inactivates these enzymes, targeting them for ubiquitin-mediated proteasomal degradation. A3 Vif-interaction sites are presumed to fall into three distinct types: A3C/D/F, A3G, and A3H. To date, two types of A3G and A3C/D/F sites have been well characterized, whereas the A3H Vif-binding site remains poorly defined...
March 20, 2017: Journal of Molecular Biology
https://www.readbyqxmd.com/read/28335548/study-of-different-variants-of-mo-enzyme-crarc-and-the-interaction-with-its-partners-crcytb5-r-and-crcytb5-1
#16
Alejandro Chamizo-Ampudia, Aurora Galvan, Emilio Fernandez, Angel Llamas
The mARC (mitochondrial Amidoxime Reducing Component) proteins are recently discovered molybdenum (Mo) Cofactor containing enzymes. They are involved in the reduction of several N-hydroxylated compounds (NHC) and nitrite. Some NHC are prodrugs containing an amidoxime structure or mutagens such as 6-hydroxylaminopurine (HAP). We have studied this protein in the green alga Chlamydomonas reinhardtii (crARC). Interestingly, all the ARC proteins need the reducing power supplied by other proteins. It is known that crARC requires a cytochrome b₅ (crCytb5-1) and a cytochrome b₅ reductase (crCytb5-R) that form an electron transport chain from NADH to the substrates...
March 21, 2017: International Journal of Molecular Sciences
https://www.readbyqxmd.com/read/28335409/the-formyl-peptide-receptors-diversity-of-ligands-and-mechanism-for-recognition
#17
REVIEW
Hui-Qiong He, Richard D Ye
The formyl peptide receptors (FPRs) are G protein-coupled receptors that transduce chemotactic signals in phagocytes and mediate host-defense as well as inflammatory responses including cell adhesion, directed migration, granule release and superoxide production. In recent years, the cellular distribution and biological functions of FPRs have expanded to include additional roles in homeostasis of organ functions and modulation of inflammation. In a prototype, FPRs recognize peptides containing N-formylated methionine such as those produced in bacteria and mitochondria, thereby serving as pattern recognition receptors...
March 13, 2017: Molecules: a Journal of Synthetic Chemistry and Natural Product Chemistry
https://www.readbyqxmd.com/read/28334874/efficient-cnv-breakpoint-analysis-reveals-unexpected-structural-complexity-and-correlation-of-dosage-sensitive-genes-with-clinical-severity-in-genomic-disorders
#18
Ling Zhang, Jingmin Wang, Cheng Zhang, Dongxiao Li, Claudia M B Carvalho, Haoran Ji, Jianqiu Xiao, Ye Wu, Weichen Zhou, Hongyan Wang, Li Jin, Yang Luo, Xiru Wu, James R Lupski, Feng Zhang, Yuwu Jiang
Genomic disorders are the clinical conditions manifested by submicroscopic genomic rearrangements including copy number variants (CNVs). The CNVs can be identified by array-based comparative genomic hybridization (aCGH), the most commonly used technology for molecular diagnostics of genomic disorders. However, clinical aCGH only informs CNVs in the probe interrogated regions. Neither orientational information nor the resulting genomic rearrangement structure is provided, which is a key to uncovering mutational and pathogenic mechanisms underlying genomic disorders...
March 14, 2017: Human Molecular Genetics
https://www.readbyqxmd.com/read/28333944/mechanisms-of-glycosylase-induced-genomic-instability
#19
Daniel E Eyler, Kylie A Burnham, Thomas E Wilson, Patrick J O'Brien
Human alkyladenine DNA glycosylase (AAG) initiates base excision repair (BER) to guard against mutations by excising alkylated and deaminated purines. Counterintuitively, increased expression of AAG has been implicated in increased rates of spontaneous mutation in microsatellite repeats. This microsatellite mutator phenotype is consistent with a model in which AAG excises bulged (unpaired) bases, altering repeat length. To directly test the role of base excision in AAG-induced mutagenesis, we conducted mutation accumulation experiments in yeast overexpressing different variants of AAG and detected mutations via high-depth genome resequencing...
2017: PloS One
https://www.readbyqxmd.com/read/28333442/photoaffinity-ligand-for-the-inhalational-anesthetic-sevoflurane-allows-mechanistic-insight-into-potassium-channel-modulation
#20
Kellie A Woll, Wesley Peng, Qiansheng Liang, Lianteng Zhi, Jack A Jacobs, Lina Maciunas, Natarajan Bhanu, Benjamin A Garcia, Manuel Covarrubias, Patrick J Loll, William P Dailey, Roderic G Eckenhoff
Sevoflurane is a commonly used inhaled general anesthetic. Despite this, its mechanism of action remains largely elusive. Compared to other anesthetics, sevoflurane exhibits distinct functional activity. In particular, sevoflurane is a positive modulator of voltage-gated Shaker-related potassium channels (Kv1.x), which are key regulators of action potentials. Here, we report the synthesis and validation of azisevoflurane, a photoaffinity ligand for the direct identification of sevoflurane binding sites in the Kv1...
March 23, 2017: ACS Chemical Biology
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