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MicroScale Thermophoresis

Flavia A Mercurio, Daniela Marasco, Concetta Di Natale, Luciano Pirone, Susan Costantini, Emilia M Pedone, Marilisa Leone
The EphA2 receptor controls diverse physiological and pathological conditions and its levels are often upregulated in cancer. Targeting receptor overexpression, through modulation of endocytosis and consequent degradation, appears to be an appealing strategy for attacking tumor malignancy. In this scenario, the Sam domain of EphA2 plays a pivotal role because it is the site where protein regulators of endocytosis and stability are recruited by means of heterotypic Sam-Sam interactions. Because EphA2-Sam heterotypic complexes are largely based on electrostatic contacts, we have investigated the possibility of attacking these interactions with helical peptides enriched in charged residues...
October 20, 2016: Chembiochem: a European Journal of Chemical Biology
Weiying Zhang, Xiangyang Li, Guoping Zhang, Yan Ding, Longlu Ran, Liangzhi Luo, Jian Wu, Deyu Hu, Baoan Song
Tobacco mosaic virus (TMV) is an important plant virus that can cause considerable crop loss. Our group synthesized a series of enantiomeric α-aminophosphonate derivatives with high anti-TMV activities. The activity of (R)-diphenyl-1-(4-methylbenzothiazole-2-amino)-1-(thiphene-2-yl)-methylphosphonate (Q-R) was found to be superior to that of (S)-diphenyl-1-(4-methyl benzothiazole-2-amino)-1-(thiphene-2-yl)-methylphosphonate (Q-S). However, the mechanism for inhibition of the R-isomer (Q-R) of infection activity is not clear...
October 13, 2016: International Journal of Biological Macromolecules
T Dau, E V Edeleva, S A I Seidel, R A Stockley, D Braun, D E Jenne
High abundance proteins like protease inhibitors of plasma display a multitude of interactions in natural environments. Quantitative analysis of such interactions in vivo is essential to study diseases, but have not been forthcoming, as most methods cannot be directly applied in a complex biological environment. Here, we report a quantitative microscale thermophoresis assay capable of deciphering functional deviations from in vitro inhibition data by combining concentration and affinity measurements. We obtained stable measurement signals for the substrate-like interaction of the disease relevant inhibitor α-1-antitrypsin (AAT) Z-variant with catalytically inactive elastase...
October 14, 2016: Scientific Reports
Achim Löf, Jochen P Müller, Martin Benoit, Maria A Brehm
The large multimeric plasma glycoprotein von Willebrand factor (VWF) is essential for primary hemostasis by recruiting platelets to sites of vascular injury. VWF multimers respond to elevated hydrodynamic forces by elongation, thereby increasing their adhesiveness to platelets. Thus, the activation of VWF is force-induced, as is its inactivation. Due to these attributes, VWF is a highly interesting system from a biophysical point of view, and is well suited for investigation using biophysical approaches. Here, we give an overview on recent studies that predominantly employed biophysical methods to gain novel insights into multiple aspects of VWF: Electron microscopy was used to shed light on the domain structure of VWF and the mechanism of VWF secretion...
September 28, 2016: Advances in Biological Regulation
Mindy I Davis, Rajan Pragani, Jennifer T Fox, Min Shen, Kalindi Parmar, Emily F Gaudiano, Li Liu, Cordelle Tanega, Lauren McGee, Matthew David Hall, Crystal McKnight, Paul Shinn, Henrike Nelson, Debasish Chattopadhyay, Alan D D'Andrea, Douglas S Auld, Larry J DeLucas, Zhuyin Li, Matthew B Boxer, Anton Simeonov
Deubiquitinases are important components of the protein degradation regulatory network. We report the discovery of ML364, a small molecule inhibitor of the deubiquitinase USP2 and its use to interrogate the biology of USP2 and its putative substrate Cyclin D1. ML364 has an IC50 of 1.1 uM in a biochemical assay using an internally quenched fluorescent di-ubiquitin substrate. Direct binding of ML364 to USP2 was demonstrated using microscale thermophoresis. ML364 induced an increase in cellular Cyclin D1 degradation and caused cell-cycle arrest as shown in western blots and flow cytometry assays utilizing both mino and HCT116 cancer cell lines...
September 28, 2016: Journal of Biological Chemistry
Markéta Svobodová, Vasso Skouridou, Mary Luz Botero, Miriam Jauset-Rubio, Thomas Schubert, Abdulaziz S Bashammakh, Mohammad S El-Shahawi, Abdulrahman O Alyoubi, Ciara K O'Sullivan
The rapid and sensitive detection of small molecules is garnering increasing importance, and aptamers show great promise in replacing expensive, elaborate detection platforms exploiting chromatographic separation or antibody-based assays. The characterization of aptamer interaction with small molecule targets is not facile, and there is a mature need for a rapid, high-throughput technique for the analysis of aptamer-small molecule kinetics and affinity. In this work we present methodologies for the evaluation of aptamer-small molecule interactions, using the aptamers reported against the steroid 17β-estradiol as a model system...
September 23, 2016: Journal of Steroid Biochemistry and Molecular Biology
Petra Zadravec, Lucie Marečková, Hana Petroková, Vesna Hodnik, Milica Perišić Nanut, Gregor Anderluh, Borut Štrukelj, Petr Malý, Aleš Berlec
Infections with shiga toxin-producing bacteria, like enterohemorrhagic Escherichia coli and Shigella dysenteriae, represent a serious medical problem. No specific and effective treatment is available for patients with these infections, creating a need for the development of new therapies. Recombinant lactic acid bacterium Lactococcus lactis was engineered to bind Shiga toxin by displaying novel designed albumin binding domains (ABD) against Shiga toxin 1 B subunit (Stx1B) on their surface. Functional recombinant Stx1B was produced in Escherichia coli and used as a target for selection of 17 different ABD variants (named S1B) from the ABD scaffold-derived high-complex combinatorial library in combination with a five-round ribosome display...
2016: PloS One
Kristina Szameit, Katharina Berg, Sven Kruspe, Erica Valentini, Eileen Magbanua, Marcel Kwiatkowski, Isaure Chauvot de Beauchêne, Boris Krichel, Kira Schamoni, Charlotte Uetrecht, Dmitri I Svergun, Hartmut Schlüter, Martin Zacharias, Ulrich Hahn
G-quadruplexes have recently moved into focus of research in nucleic acids, thereby evolving in scientific significance from exceptional secondary structure motifs to complex modulators of gene regulation. Aptamers (nucleic acid based ligands with recognition properties for a specific target) that form G-quadruplexes may have particular potential for therapeutic applications as they combine the characteristics of specific targeting and G-quadruplex mediated stability and regulation. We have investigated the structure and target interaction properties of one such aptamer: AIR-3 and its truncated form AIR-3A...
July 29, 2016: RNA Biology
Tatiana P Soares da Costa, Sebastien Desbois, Con Dogovski, Michael A Gorman, Natalia E Ketaren, Jason J Paxman, Tanzeela Siddiqui, Leanne M Zammit, Belinda M Abbott, Roy M Robins-Browne, Michael W Parker, Geoffrey B Jameson, Nathan E Hall, Santosh Panjikar, Matthew A Perugini
Dihydrodipicolinate synthase (DHDPS) catalyzes the first committed step in the lysine biosynthesis pathway of bacteria. The pathway can be regulated by feedback inhibition of DHDPS through the allosteric binding of the end product, lysine. The current dogma states that DHDPS from Gram-negative bacteria are inhibited by lysine but orthologs from Gram-positive species are not. The 1.65-Å resolution structure of the Gram-negative Legionella pneumophila DHDPS and the 1.88-Å resolution structure of the Gram-positive Streptococcus pneumoniae DHDPS bound to lysine, together with comprehensive functional analyses, show that this dogma is incorrect...
August 2, 2016: Structure
Tan Qin, Fangjin Chen, Xiaolong Zhuo, Xiao Guo, Taikangxiang Yun, Ying Liu, Chuanmao Zhang, Luhua Lai
Polo-like kinase 1(Plk1) is vital for cell mitosis and has been identified as anticancer target. Its polo-box domain (PBD) mediates substrate binding, blocking of which may offer selective Plk1 inhibition compared to kinase domain inhibitors. Although several PBD inhibitors were reported, most of them suffer from low cell activity. Here, we report the discovery of novel inhibitors to induce mitotic arrest in HeLa cells by virtual screening with Plk1 PBD and cellular activity testing. Of the 81 compounds tested in the cell assay, 10 molecules with diverse chemical scaffolds are potent to induce mitotic arrest of HeLa at low micromolar concentrations...
August 11, 2016: Journal of Medicinal Chemistry
Yuhua Zhan, Yongliang Yan, Zhiping Deng, Ming Chen, Wei Lu, Chao Lu, Liguo Shang, Zhimin Yang, Wei Zhang, Wei Wang, Yun Li, Qi Ke, Jiasi Lu, Yuquan Xu, Liwen Zhang, Zhihong Xie, Qi Cheng, Claudine Elmerich, Min Lin
Unlike most Pseudomonas, the root-associated bacterium Pseudomonas stutzeri A1501 fixes nitrogen after the horizontal acquisition of a nitrogen-fixing (nif) island. A genome-wide search for small noncoding RNAs (ncRNAs) in P. stutzeri A1501 identified the novel P. stutzeri-specific ncRNA NfiS in the core genome, whose synthesis was significantly induced under nitrogen fixation or sorbitol stress conditions. The expression of NfiS was RNA chaperone Hfq-dependent and activated by the sigma factor RpoN/global nitrogen activator NtrC/nif-specific activator NifA regulatory cascade...
July 26, 2016: Proceedings of the National Academy of Sciences of the United States of America
Brett Thurlow, Joseph H Davis, Vivian Leong, Trevor F Moraes, James R Williamson, Joaquin Ortega
Our understanding regarding the function of YjeQ (also called RsgA), RbfA, RimM and Era in ribosome biogenesis has been derived in part from the study of immature 30S particles that accumulate in null strains lacking one of these factors. However, their mechanistic details are still unknown. Here, we demonstrate that these immature particles are not dead-end products of assembly, but progress into mature 30S subunits. Mass spectrometry analysis revealed that in vivo the occupancy level of these factors in these immature 30S particles is below 10% and that the concentration of factors does not increase when immature particles accumulate in cells...
July 5, 2016: Nucleic Acids Research
Sarah Ameziane-Le Hir, Gilles Paboeuf, Christophe Tascon, Jean-François Hubert, Elisabeth Le Rumeur, Véronique Vié, Céline Raguénès-Nicol
Dystrophin (DYS) is a membrane skeleton protein whose mutations lead to lethal Duchenne muscular dystrophy or to the milder Becker muscular dystrophy (BMD). One third of BMD "in-frame" exon deletions are located in the region that codes for spectrin-like repeats R16 to R21. We focused on four prevalent mutated proteins deleted in this area (called RΔ45-47, RΔ45-48, RΔ45-49, and RΔ45-51 according to the deleted exon numbers), analyzing protein/membrane interactions. Two of the mutants, RΔ45-48 and RΔ45-51, led to mild pathologies and displayed a similar triple coiled-coil structure as the full-length DYS R16-21, whereas the two others, RΔ45-47 and RΔ45-49, induced more severe pathologies and showed "fractional" structures unrelated to the normal one...
July 26, 2016: Biochemistry
Xingqi Wang, Xuefeng Wu, Aihua Zhang, Shiyu Wang, Chunhui Hu, Wei Chen, Yan Shen, Renxiang Tan, Yang Sun, Qiang Xu
PDGF-BB/PDGFR-ββ signaling plays very crucial roles in the process of many diseases such as liver fibrosis. However, drug candidates with selective affinities for PDGF-B/PDGFR-β remain deficient. Here, we identified a natural cyclopeptide termed destruxin A5 that effectively inhibits PDGF-BB-induced PDGFR-β signaling. Interestingly and importantly, the inhibitory mechanism is distinct from the mechanism of tyrosine kinase inhibitors because destruxin A5 does not have the ability to bind to the ATP-binding pocket of PDGFR-β...
May 2016: EBioMedicine
Luise Apelt, Kevin E Knockenhauer, Nina C Leksa, Nouhad Benlasfer, Thomas U Schwartz, Ulrich Stelzl
The nuclear pore complex (NPC) enables transport across the nuclear envelope. It is one of the largest multiprotein assemblies in the cell, built from about 30 proteins called nucleoporins (Nups), organized into distinct subcomplexes. Structure determination of the NPC is a major research goal. The assembled ∼40-112 MDa NPC can be visualized by cryoelectron tomography (cryo-ET), while Nup subcomplexes are studied crystallographically. Docking the crystal structures into the cryo-ET maps is difficult because of limited resolution...
August 2016: Molecular & Cellular Proteomics: MCP
Nathan B P Adams, Cvetelin Vasilev, Amanda A Brindley, C Neil Hunter
In chlorophyll biosynthesis, the magnesium chelatase enzyme complex catalyzes the insertion of a Mg(2+) ion into protoporphyrin IX. Prior to this event, two of the three subunits, the AAA(+) proteins ChlI and ChlD, form a ChlID-MgATP complex. We used microscale thermophoresis to directly determine dissociation constants for the I-D subunits from Synechocystis, and to show that the formation of a ChlID-MgADP complex, mediated by the arginine finger and the sensor II domain on ChlD, is necessary for the assembly of the catalytically active ChlHID-MgATP complex...
May 25, 2016: Journal of the American Chemical Society
Veronica Esposito, Luciano Pirone, Luciano Mayol, Emilia Pedone, Antonella Virgilio, Aldo Galeone
The aptamer d(GGGT)4 (T30923 or T30695) forms a 5'-5' dimer of two stacked parallel G-quadruplexes, each characterized by three G-tetrads and three single-thymidine reversed-chain loops. This aptamer has been reported to exhibit anti-HIV activity by targeting the HIV integrase, a viral enzyme responsible for the integration of viral DNA into the host-cell genome. However, information concerning the aptamer/target interaction is still rather limited. In this communication we report microscale thermophoresis investigations on the interaction between the HIV-1 integrase and d(GGGT)4 aptamer analogues containing abasic sites singly replacing thymidines in the original sequence...
August 2016: Biochimie
Stefania Valenzano, Annalisa De Girolamo, Maria C DeRosa, Maureen McKeague, Roberto Schena, Lucia Catucci, Michelangelo Pascale
Aptamers are synthetic single-stranded DNA or RNA sequences that can fold into tertiary structures allowing them to interact with and bind to targets with high affinity and specificity. This paper describes the first selection and identification of DNA aptamers able to recognize the biogenic amine tyramine. To successfully isolate aptamers to this challenging small molecule target, the SELEX methodology was adapted by combining a systematic strategy to increase the selection stringency and monitor enrichment success...
June 13, 2016: ACS Combinatorial Science
Friederike M Möller, Michael Kieß, Dieter Braun
Intricate spatiotemporal patterns emerge when chemical reactions couple to physical transport. We induce electrophoretic transport by a confined photochemical reaction and use it to infer the binding strength of a second, biomolecular binding reaction under physiological conditions. To this end, we use the photoactive compound 2-nitrobenzaldehyde, which releases a proton upon 375 nm irradiation. The charged photoproducts locally perturb electroneutrality due to differential diffusion, giving rise to an electric potential Φ in the 100 μV range on the micrometer scale...
April 27, 2016: Journal of the American Chemical Society
Avi Harazi, Michal Becker-Cohen, Hagit Zer, Ofra Moshel, Stephan Hinderlich, Stella Mitrani-Rosenbaum
UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase (GNE) is the gene mutated in GNE myopathy. In an attempt to elucidate GNE functions that could account for the muscle pathophysiology of this disorder, the interaction of GNE with α-actinins has been investigated. Surface plasmon resonance and microscale thermophoresis analysis revealed, that in vitro, GNE interacts with α-actinin 2, and that this interaction has a 10-fold higher affinity compared to the GNE-α-actinin 1 interaction. Further, GNE carrying the M743T mutation, the most frequent mutation in GNE myopathy, has a 10-fold lower binding affinity to α-actinin 2 than intact GNE...
March 29, 2016: Molecular Neurobiology
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