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Pull down assay

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https://www.readbyqxmd.com/read/29140695/rna-g-quadruplexes-in-kirsten-ras-kras-oncogene-as-targets-for-small-molecules-inhibiting-translation
#1
Giulia Miglietta, Susanna Cogoi, Jessica Marinello, Giovanni Capranico, Alexander S Tikhomirov, Andrey E Shchekotikhin, Luigi E Xodo
The human KRAS transcript contains a G-rich 5'-UTR sequence (77 % GC) harbouring several G4 motifs capable to form stable RNA G-quadruplex (RG4) structures that can serve as targets for small molecules. A biotin-streptavidin pull-down assay showed that 4,11-bis(2-aminoethylamino)anthra[2,3-b]furan-5,10-dione (2a) binds to RG4s in the KRAS transcript under low-abundance cellular conditions. Dual-luciferase assays demonstrated that 2a and its analogue 4,11-bis(2-aminoethylamino)anthra[2,3-b]thiophene-5,10-dione 2b) repress translation in a dose-dependent manner...
November 15, 2017: Journal of Medicinal Chemistry
https://www.readbyqxmd.com/read/29129743/comprehensive-identification-of-proteins-binding-to-rna-g-quadruplex-motifs-in-the-5-utr-of-tumor-associated-mrnas
#2
Tatsuo Serikawa, Christos Spanos, Annekathrin von Hacht, Nediljko Budisa, Juri Rappsilber, Jens Kurreck
G-quadruplex structures in the 5' UTR of mRNAs are widely considered to suppress translation without affecting transcription. The current study describes the comprehensive analysis of proteins binding to four different G-quadruplex motifs located in mRNAs of the cancer-related genes Bcl-2, NRAS, MMP16, and ARPC2. Following metabolic labeling (Stable Isotope Labeling with Amino acids in Cell culture, SILAC) of proteins in the human cell line HEK293, G-quadruplex binding proteins were enriched by pull-down assays and identified by LC-orbitrap mass spectrometry...
November 9, 2017: Biochimie
https://www.readbyqxmd.com/read/29128594/soft-interactions-with-model-crowders-and-non-canonical-interactions-with-cellular-proteins-stabilize-rna-folding
#3
May Daher, Julia R Widom, Wendy Tay, Nils G Walter
Living cells contain diverse biopolymers, creating a heterogeneous crowding environment, the impact of which on RNA folding is poorly understood. Here, we have used single-molecule fluorescence resonance energy transfer to monitor tertiary structure formation of the hairpin ribozyme as a model to probe the effects of polyethylene glycol (PEG) and yeast cell extract as crowding agents. As expected, PEG stabilizes the docked, catalytically active state of the ribozyme, in part through excluded volume effects; unexpectedly, we found evidence that it additionally displays soft, non-specific interactions with the ribozyme...
November 8, 2017: Journal of Molecular Biology
https://www.readbyqxmd.com/read/29126425/hdac7-promotes-lung-tumorigenesis-by-inhibiting-stat3-activation
#4
Yubin Lei, Lingling Liu, Shujing Zhang, Shicheng Guo, Xiaoqing Li, Jiucun Wang, Bo Su, Yuchao Fang, Xiaofeng Chen, Hengning Ke, Wufan Tao
BACKGROUND: Lung cancer is the leading cause of cancer death worldwide. However, the molecular mechanisms underlying lung cancer development have not been fully understood. The functions of histone deacetylases (HDACs), a class of total eighteen proteins (HDAC1-11 and SIRT1-7 in mammals) that deacetylate histones and non-histone proteins, in cancers are largely unknown. METHODS: Hdac7 (+/-)/K-Ras mice and HDAC7-depleted human lung cancer cell lines were used as models for studying the function of Hdac7 gene in lung cancer...
November 10, 2017: Molecular Cancer
https://www.readbyqxmd.com/read/29121672/beyond-genome-wide-scan-association-of-a-cis-regulatory-ncr3-variant-with-mild-malaria-in-a-population-living-in-the-republic-of-congo
#5
Sabrina Baaklini, Sarwat Afridi, Thy Ngoc Nguyen, Felix Koukouikila-Koussounda, Mathieu Ndounga, Jean Imbert, Magali Torres, Lydie Pradel, Francine Ntoumi, Pascal Rihet
Linkage studies have revealed a linkage of mild malaria to chromosome 6p21 that contains the NCR3 gene encoding a natural killer cell receptor, whereas NCR3-412G>C (rs2736191) located in its promoter region was found to be associated with malaria in Burkina Faso. Here we confirmed the association of rs2736191 with mild malaria in a Congolese cohort and investigated its potential cis-regulatory effect. Luciferase assay results indicated that rs2736191-G allele had a significantly increased promoter activity compared to rs2736191-C allele...
2017: PloS One
https://www.readbyqxmd.com/read/29115671/fatty-acid-elongase-7-is-regulated-via-sp1-and-is-involved-in-lipid-accumulation-in-bovine-mammary-epithelial-cells
#6
Si Chen, Zhigang Hu, Hua He, Xiaolin Liu
Fatty Acid Elongase 7 (ELOVL7) is the newly discovered protein on human that catalyzes the rate-limiting step towards the synthesis of very long-chain fatty acids and exhibits the highest activity toward C18:3 (n-3) acyl-CoAs, which is the precursor of eicosapentaenoic acid (EPA, 20:5n-3). However, in ruminants, an overall understanding of ELOVLs gene family and the transcriptional regulation of ELOVL7 remain unknown. The purpose of this study is to investigate the transcriptional regulation and the influence of bovine ELOVL7 in bovine mammary epithelial cells (bMECs)...
November 8, 2017: Journal of Cellular Physiology
https://www.readbyqxmd.com/read/29113909/the-chaperone-dynein-ll1-mediates-cytoplasmic-transport-of-empty-and-mature-hepatitis-b-virus-capsids
#7
Quentin Osseman, Lara Gallucci, Shelly Au, Christian Cazenave, Elodie Berdance, Marie-Lise Blondot, Aurélia Cassany, Dominique Bégu, Jessica Ragues, Cindy Aknin, Irina Sominskaya, Andris Dishlers, Birgit Rabe, Fenja Anderson, Nelly Panté, Michael Kann
BACKGROUND & AIMS: Hepatitis B virus (HBV) has a DNA genome but replicates within nucleus by transcription of an RNA pregenome, which is converted to DNA in cytoplasmic capsids. Capsids in this compartment are correlated with inflammation and epitopes of the capsid protein core (Cp) are the main target for T cell-mediated immune response. We investigated the mechanism of cytoplasmic capsid transport, which is important for infection but also for cytosolic capsid removal. METHODS: We used virion-derived capsids with a mature rcDNA (matC), empty capsids (empC), and RNA containing capsids (rnaC) as control...
November 4, 2017: Journal of Hepatology
https://www.readbyqxmd.com/read/29113749/long-non-coding-rna-atb-promotes-emt-during-silica-induced-pulmonary-fibrosis-by-competitively-binding-mir-200c
#8
Yi Liu, Yan Li, Qi Xu, Wenxi Yao, Qiuyun Wu, Jiali Yuan, Weiwen Yan, Tiantian Xu, Xiaoming Ji, Chunhui Ni
Long non-coding RNAs (lncRNAs) are important signal transduction regulators that act by various patterns. However, little is known about the molecular mechanisms of lncRNA related pathways in occupational lung fibrosis. Our previous study found that epithelial-mesenchymal transition (EMT) was one of the key events in silica-induced pulmonary fibrosis. This study showed that the lncRNA-ATB promoted EMT by acting as a miR-200c sponge. miR-200c was identified by miRNA array as a potential target of lncRNA-ATB and verified by dual luciferase reporter gene together with RNA pull-down assays...
November 4, 2017: Biochimica et Biophysica Acta
https://www.readbyqxmd.com/read/29112934/long-non-coding-rna-gas5-antagonizes-the-chemoresistance-of-pancreatic-cancer-cells-through-down-regulation-of-mir-181c-5p
#9
Zhi-Qiang Gao, Jun-Feng Wang, De-Hua Chen, Xue-Song Ma, Wu Yang, Tang Zhe, Xiao-Wei Dang
OBJECTIVE: To explore the core mechanism of long non-coding RNA (lncRNA) growth arrest-specific transcript 5 (GAS5) in the regulation of multidrug resistance of pancreatic cancer cells. METHODS: mRNA levels of GAS5, miR-181c-5p and Hippo pathway related genes were detected by quantitative real-time PCR (qRT-PCR). Protein levels of MDR-1, MST1, YAP and TAZ were measured by western blot. Cell viability was detected by MTT assay. The combination between GAS5 and miR-181c-5p was confirmed by RNA pull-down and RNA immunoprecipitation (RIP) assay...
November 4, 2017: Biomedicine & Pharmacotherapy, Biomédecine & Pharmacothérapie
https://www.readbyqxmd.com/read/29110463/8-nitro-cgmp-attenuates-the-interaction-between-snare-complex-and-complexin-through-s-guanylation-of-snap-25
#10
Yusuke Kishimoto, Kohei Kunieda, Atsushi Kitamura, Yuki Kakihana, Takaaki Akaike, Hideshi Ihara
8-Nitroguanosine 3',5'-cyclic monophosphate (8-nitro-cGMP) is the second messenger in nitric oxide/reactive oxygen species redox signaling. This molecule covalently binds to protein thiol groups, called S-guanylation, and exerts various biological functions. Recently, we have identified synaptosomal-associated protein 25 (SNAP-25) as a target of S-guanylation, and demonstrated that S-guanylation of SNAP25 enhanced SNARE complex formation. In this study, we have examined the effects of S-guanylation of SNAP-25 on the interaction between the soluble SNARE complex and complexin (cplx), which binds to the SNARE complex with a high affinity...
November 7, 2017: ACS Chemical Neuroscience
https://www.readbyqxmd.com/read/29106533/ywhae-is-a-novel-interaction-partner-of-helicobacter-pylori-caga
#11
Xiaoyan Zhang, Bangwei Zeng, Chunyan Wen, Shurong Zheng, Hao Chen, Feifei She
CagA, an important virulence factor of Helicobacter pylori, targets and interacts with a series of host proteins to activate signaling factors involved in many functions, such as development, cytoskeleton rearrangement, and inflammatory molecule release. Despite extensive efforts, the relationship between CagA and gastric cancer is far from completely understood. Here, the GAL4 yeast two-hybrid system was used to screen cellular proteins for binding to CagA, and five cellular proteins, including tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein, epsilon (YWHAE), were identified...
November 2, 2017: FEMS Microbiology Letters
https://www.readbyqxmd.com/read/29102705/a-laminin-receptor-like-protein-regulates-white-spot-syndrome-virus-infection-by-binding-to-the-viral-envelope-protein-vp28-in-red-claw-crayfish-cherax-quadricarinatus
#12
Ling-Ke Liu, Wei-Dong Li, Yan Gao, Rong-Yuan Chen, Xiao-Lu Xie, Heng Hong, Ke-Jian Wang, Hai-Peng Liu
White spot syndrome virus (WSSV) is a lethal pathogen of shrimp and many other crustaceans, which has been causing huge economic losses in global aquaculture. Laminin receptor (LR) is a cell surface receptor which participates in the interactions between cells as well as cells and extracellular matrix. Previously, we found that a CqLR-like gene was responsive to WSSV infection in the hematopoietic tissue (Hpt) cells from red claw crayfish Cherax quadricarinatus. To further reveal the role of CqLR-like gene involved in WSSV infection, the full-length cDNA of CqLR-like gene was cloned with 1000 bp, and the open reading frame encoded 308 amino acids with a conserved laminin-binding domain...
November 1, 2017: Developmental and Comparative Immunology
https://www.readbyqxmd.com/read/29100983/acetyl-coa-carboxylase-in-escherichia-coli-exhibits-a-pronounced-hysteresis-when-inhibited-by-palmitoyl-acyl-carrier-protein
#13
Alexandra Evans, Wendy Ribble, Erin Schexnaydre, Grover L Waldrop
Acetyl-CoA carboxylase (ACC) in bacteria is composed of three components: biotin carboxylase, biotin carboxyl carrier protein, and carboxyltransferase. ACC catalyzes the first committed step in fatty acid synthesis: the carboxylation of acetyl-CoA to form malonyl-CoA via a two-step reaction. In the first half-reaction, biotin carboxylase catalyzes the ATP-dependent carboxylation of the vitamin biotin covalently linked to biotin carboxyl carrier protein. In the second half-reaction, the carboxyl group is transferred from biotin to acetyl-CoA by the enzyme carboxyltransferase...
October 31, 2017: Archives of Biochemistry and Biophysics
https://www.readbyqxmd.com/read/29097358/lncrna-miat-promoted-proliferation-and-invasion-of-hcc-cells-via-sponging-mir-214
#14
Xinli Huang, Yun Gao, Jianjie Qin, Sen Lu
BACKGROUND: The aberrant expression of lncRNAs has been involved in various human tumors including hepatocellular carcinoma (HCC). Our study aimed to investigate the potential molecular mechanism of lncRNA-MIAT in HCC. METHODS: The expression of MIAT and miR-214 in HCC tissues and cells was examined by qRT-PCR, and the levels of EZH2 and β-catenin were detected by western blot assay. The immunoprecipitation analysis was used to detect the level of H3/H4 histone acetylation...
November 2, 2017: American Journal of Physiology. Gastrointestinal and Liver Physiology
https://www.readbyqxmd.com/read/29093731/twin-sister-of-ft-tsf-interacts-with-fructokinase6-and-inhibits-its-kinase-activity-in-arabidopsis
#15
Suhyun Jin, Sun Young Kim, Ji Hoon Ahn
In flowering plants, the developmental switch to the reproductive phase is tightly regulated and involves the integration of internal and external signals. FLOWERING LOCUS T (FT) and TWIN SISTER OF FT (TSF) integrate signals from multiple pathways. FT and TSF function as florigenic substances, and share high sequence similarity with mammalian Raf kinase inhibitor protein (RKIP). Despite their strong similarity to RKIP, the kinase inhibitory activity of FT and TSF remains to be investigated. We performed a yeast two-hybrid screen and found that TSF interacted with FRUCTOKINASE6 (FRK6), which phosphorylates fructose for various metabolic pathways...
2017: Frontiers in Plant Science
https://www.readbyqxmd.com/read/29093508/vp2-of-chicken-anaemia-virus-interacts-with-apoptin-for-down-regulation-of-apoptosis-through-de-phosphorylated-threonine-108-on-apoptin
#16
Guan-Hua Lai, Yi-Yang Lien, Ming-Kuem Lin, Jai-Hong Cheng, Jason Tc Tzen, Fang-Chun Sun, Meng-Shiunn Lee, Hsi-Jien Chen, Meng-Shiou Lee
Chicken anaemia virus (CAV) is an important contagious agent that causes immunosuppressive disease in chickens. CAV Apoptin is a nucleoplasmic shuffling protein that induces apoptosis in chicken lymphoblastoid cells. In the present study, confocal microscopy revealed co-localisation of expressed CAV non-structural protein VP2 with Apoptin in the nucleus of MDCC-MSB1 cells and the nucleoplasmic compartment of CHO-K1 cells. In vitro pull-down and ex vivo biomolecular fluorescent complementation (BiFC) assays further showed that the VP2 protein directly interacts with Apoptin...
November 1, 2017: Scientific Reports
https://www.readbyqxmd.com/read/29091771/multiple-cis-acting-rdnas-contribute-to-nucleoid-separation-and-recruit-the-bacterial-condensin-smc-scpab
#17
Koichi Yano, Hironori Niki
Condensins load onto DNA to organize chromosomes. Smc-ScpAB clearly loads onto the parS sites bound by Spo0J, but other loading site(s) must operate independently of parS. In this study, we asked where and how Smc-ScpAB normally selects its loading site. Our results suggest that rDNA is also a loading site. A pull-down assay revealed that Smc-ScpAB preferentially loads onto rDNA in the wild-type cell and even in a Δspo0J mutant but not in a Δsmc mutant. Moreover, we showed that deletion mutants of rDNAs cause a defect in nucleoid separation, and at least two rDNAs near oriC are essential for separation...
October 31, 2017: Cell Reports
https://www.readbyqxmd.com/read/29081444/ca-2-calmodulin-dependent-protein-kinase-ii-%C3%AE-dependent-serine727-phosphorylation-is-required-for-tmem16a-ca-2-activated-cl-channel-regulation-in-cerebrovascular-cells
#18
Cai-Xia Lin, Xiao-Fei Lv, Feng Yuan, Xiang-Yu Li, Ming-Ming Ma, Can-Zhao Liu, Jia-Guo Zhou, Guan-Lei Wang, Yong-Yuan Guan
BACKGROUND: TMEM16A is a critical component of Ca(2+)-activated chloride channels (CaCCs) and mediates basilar arterial smooth muscle cell (BASMC) proliferation in hypertensive cerebrovascular remodeling. CaMKII is a negative regulator of CaCC, and four CaMKII isoforms (α, β, γ and δ) are expressed in vasculature; however, it is unknown which and how CaMKII isoforms affect TMEM16A-associated CaCC and BASMC proliferation.Methods and Results:Patch clamp and small interfering RNA (siRNA) knockdown of different CaMKII isoforms revealed that only CaMKIIγ inhibited native Ca(2+)-activated chloride currents (ICl...
October 28, 2017: Circulation Journal: Official Journal of the Japanese Circulation Society
https://www.readbyqxmd.com/read/29081216/long-noncoding-rna-heih-promotes-colorectal-cancer-tumorigenesis-via-counteracting-mir-939%C3%A2-mediated-transcriptional-repression-of-bcl-xl
#19
Chunhui Cui, Duanyang Zhai, Lianxu Cai, Qiaobin Duan, Lang Xie, Jinlong Yu
Purpose: Studies have found that long noncoding RNA HEIH (lncRNA-HEIH) is upregulated and facilitates hepatocellular carcinoma tumor growth. However, its clinical significances, roles and action mechanism in colorectal cancer (CRC) remains unidentified. Materials and Methods: lncRNA-HEIH expression in CRC tissues and cell lines was measured by quantitative real-time polymerase chain reaction. Cell Counting Kit-8, ethynyl deoxyuridine incorporation assay, terminal deoxynucleotidyl transferase dUTP nick end labeling staining, and nude mice xenografts assays were performed to investigate the roles of lncRNA-HEIH...
October 30, 2017: Cancer Research and Treatment: Official Journal of Korean Cancer Association
https://www.readbyqxmd.com/read/29075625/chemical-proteomics-for-target-discovery-of-head-to-tail-cyclized-mini-proteins
#20
Roland Hellinger, Kathrin Thell, Mina Vasileva, Taj Muhammad, Sunithi Gunasekera, Daniel Kümmel, Ulf Göransson, Christian W Becker, Christian W Gruber
Target deconvolution is one of the most challenging tasks in drug discovery, but a key step in drug development. In contrast to small molecules, there is a lack of validated and robust methodologies for target elucidation of peptides. In particular, it is difficult to apply these methods to cyclic and cysteine-stabilized peptides since they exhibit reduced amenability to chemical modification and affinity capture; however, such ribosomally synthesized and post-translationally modified peptide natural products are rich sources of promising drug candidates...
2017: Frontiers in Chemistry
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