18 research outputs found
An allosteric redox switch in domain V of β2-glycoprotein I controls membrane binding and anti-domain I autoantibody recognition
No full textβ2-glycoprotein I (β2GPI) is an abundant multi-domain plasma protein that plays various roles in the clotting and complement cascades. It is also the main target of antiphospholipid antibodies (aPL) in the acquired coagulopathy known as Antiphospholipid Syndrome (APS). Previous studies have shown that β2GPI adopts two interconvertible biochemical conformations, oxidized and reduced, depending on the integrity of the disulfide bonds. However, the precise contribution of the disulfide bonds to β2GPI structure and function is unknown. Here, we substituted cysteine residues with serine to investigate how the disulfide bonds C32-C60 in domain I (DI) and C288-C326 in domain V (DV) regulate β2GPI's structure and function. Results of our biophysical and biochemical studies support the hypothesis that the C32-C60 disulfide bond plays a structural role, whereas the disulfide bond C288-C326 is allosteric. We demonstrate that absence of the C288-C326 bond, unlike absence of the C32-C60 bond, diminishes membrane binding without affecting the thermodynamic stability and overall structure of the protein, which remains elongated in solution. We also document that, while absence of the C32-C60 bond directly impairs recognition of β2GPI by pathogenic anti-DI antibodies, absence of the C288-C326 disulfide bond is sufficient to abolish complex formation in the presence of anionic phospholipids. We conclude that the disulfide bond C288-C326 operates as a molecular switch capable of regulating β2GPI's physiological functions in a redox-dependent manner. We propose that in APS patients with anti-DI antibodies, selective rupture of the C288-C326 disulfide bond may be a valid strategy to lower the pathogenic potential of aPL
Mechanistic basis of activation and inhibition of protein disulfide isomerase by allosteric antithrombotic compounds
No full textBackground: Protein disulfide isomerase (PDI) is a promising target for combating thrombosis. Extensive research over the past decade has identified numerous PDI-targeting compounds. However, limited information exists regarding how these compounds control PDI activity, which complicates further development. Objectives: To define the mechanism of action of 2 allosteric antithrombotic compounds of therapeutic interest, quercetin-3-O-rutinoside and bepristat-2a. Methods: A multipronged approach that integrates single-molecule spectroscopy, steady-state kinetics, single-turnover kinetics, and site-specific mutagenesis. Results: PDI is a thiol isomerase consisting of 2 catalytic a domains and 2 inactive b domains arranged in the order a-b-b'-a'. The active sites CGHC are located in the a and a' domains. The binding site of quercetin-3-O-rutinoside and bepristat-2a is in the b' domain. Using a library of 9 Förster resonance energy transfer sensors, we showed that quercetin-3-O-rutinoside and bepristat-2a globally alter PDI structure and dynamics, leading to ligand-specific modifications of its shape and reorientation of the active sites. Combined with enzyme kinetics and mutagenesis of the active sites, Förster resonance energy transfer data reveal that binding of quercetin-3-O-rutinoside results in a twisted enzyme with reduced affinity for the substrate. In contrast, bepristat-2a promotes a more compact conformation of PDI, in which a greater enzymatic activity is achieved by accelerating the nucleophilic step of the a domain, leading to faster formation of the covalent enzyme–substrate complex. Conclusion: This work reveals the mechanistic basis underlying PDI regulation by antithrombotic compounds quercetin-3-O-rutinoside and bepristat-2a and points to novel strategies for furthering the development of PDI-targeting compounds into drugs
Mapping out molecular locations in biological liposomes by fluorescence nanotomography
No full textEThOS - Electronic Theses Online ServiceGBUnited Kingdo
Structure of Coagulation Factor II: Molecular Mechanism of Thrombin Generation and Development of Next-Generation Anticoagulants
No full textCoagulation factor II, or prothrombin, is a multi-domain glycoprotein that is essential for life and a key target of anticoagulant therapy. In plasma, prothrombin circulates in two forms at equilibrium, “closed” (~80%) and “open” (~20%), brokered by the flexibility of the linker regions. Its structure remained elusive until recently when our laboratory solved the first X-ray crystal structure of the zymogen locked in the predominant closed form. Because of this technical breakthrough, fascinating aspects of the biology of prothrombin have started to become apparent, and with this, novel and important questions arise. Here, we examine the significance of the “closed”/“open” equilibrium in the context of the mechanism of thrombin generation. Further, we discuss the potential translational opportunities for the development of next-generation anticoagulants that arise from this discovery. By providing a structural overview of each alternative conformation, this minireview also offers a relevant example of modern structural biology and establishes a practical workflow to elucidate the structural features of analogous clotting and complement factors
Video_1_Structure of Coagulation Factor II: Molecular Mechanism of Thrombin Generation and Development of Next-Generation Anticoagulants.MPG
No full textCoagulation factor II, or prothrombin, is a multi-domain glycoprotein that is essential for life and a key target of anticoagulant therapy. In plasma, prothrombin circulates in two forms at equilibrium, “closed” (~80%) and “open” (~20%), brokered by the flexibility of the linker regions. Its structure remained elusive until recently when our laboratory solved the first X-ray crystal structure of the zymogen locked in the predominant closed form. Because of this technical breakthrough, fascinating aspects of the biology of prothrombin have started to become apparent, and with this, novel and important questions arise. Here, we examine the significance of the “closed”/“open” equilibrium in the context of the mechanism of thrombin generation. Further, we discuss the potential translational opportunities for the development of next-generation anticoagulants that arise from this discovery. By providing a structural overview of each alternative conformation, this minireview also offers a relevant example of modern structural biology and establishes a practical workflow to elucidate the structural features of analogous clotting and complement factors.</p
Structural information on nanomolecular systems revealed by FRET
No full textOur newly developed fluorescence resonance energy transfer (FRET) based technique, fluorescence nanotomography (FN), is used to determine the morphology and dynamics of some soft materials and bio-molecules by attaching donor (D) fluorophores and acceptors (A) to the investigated structure and using fluorescence lifetime measurements to reveal the D-A distance distribution function ρDA(r).We report the effect of the limited sizes of the donor and acceptor, effect of porous polymer, and molecular structure and phase transition in phospholipid bilayers
Flexibility in Transcription Start-Site Selection by RNA Polymerase Involves Transcription-Bubble Expansion (“Scrunching”) or Contraction (“Unscrunching”)
No full textCo-authorship pattern and Collaboration in Colorectal Cancer Research
No full textThe study focused on authorship pattern and collaboration in colorectal cancer research output as reflected in the web of science database for the period 2010-2017. Using various scientometrics approaches, the study presents co-authorship and collaborative patterns for different countries, institutions, and authors. We find multi and mega-author contributions which are increasing and dominate the CRC research. In the case of collaborative patterns, we found domestic collaboration which dominates the CRC research compared to international collaborations. Institution wise we find mostly domestic inter-institutional collaboration. Country pair-wise collaboration pattern shows that the US is the most preferred country for collaborations and the author-wise collaborative pattern in CRC research shows that the collaboration of domestic or local inter-institutional collaboration between the authors and highest possible combinations
An analysis of Research publication on Colorectal Cancer in Asian Countries
No full textThis paper discusses colorectal cancer research output in Asian during 2000- 2017. The data has been downloaded from PubMed databases. A total of 2726 articles were found. Language distribution shows a majority of the papers are published in the English language 2661 (97.61%) irrespective of the native language of the country and those publications were in the form of journal articles i.e. 2225 (81.62%). The authorship pattern indicates the maximum number of papers was published by collaborative work of more than ten authors for which the mean value of Degree of Collaboration is 0.84 indicating the high ratio of collaborative research work undertaken by researchers. The Relative Growth Rate and Doubling Time of total publications show decreasing and increasing trend. The mean relative growth rate is 0.28. It is also found that the average exponential growth rate is 11.55% during the sample periods. The Geographical distribution of productivity of top 20 countries shows that Thailand has contributed 838 (30.74%) publications and ranked top among the countries in terms of publications. Among the authors engaged in CRC research, Wang J is the most productive author with 31 (1.13%) of total contributions. “Wang J’ had collaborated with 82 researchers in colorectal cancer research. The most common keyword used by researcher is “human”. Cluster Density view has identified 932 items with five different clusters, in which studies were grouped right from how alteration or changes take place in the cell or DNA to surgery or therapy or remedy related studies for CRC related cases
