scholarly journals Trehalose: is it a potential inhibitor of antithrombin polymerization?

2019 ◽  
Vol 39 (6) ◽  
Author(s):  
Irene Martínez-Martínez
Keyword(s):  
Three Dimensional ◽  
Research Topic ◽  
Site Directed Mutagenesis ◽  
Coagulation Cascade ◽  
Dimensional Structure ◽  
Native Conformation ◽  
Thrombotic Risk

Abstract SERine Protease INhibitorS (Serpins) are a superfamily of proteins that are characterized by having a similar three-dimensional structure. The native conformation is not most thermodynamically stable, so polymerization is the main consequence when its stability is altered as a result of certain mutations. The polymerization of serpins has been a research topic for many years. Different mechanisms have been proposed and in the same way different compounds or strategies have been studied to prevent polymerization. A recent paper published in Bioscience Reports by Naseem et al. [Biosci. Rep. (2019) 5, 39] studies the role of trehalose in the prevention of the polymerization of antithrombin, which belongs to the serpin superfamily. The main consequence of the antithrombin polymerization is the increased thrombotic risk, since antithrombin is the main inhibitor of the coagulation cascade. The authors demonstrate that trehalose is able to prevent the in vitro polymerization of antithrombin, under conditions in which it usually tends to polymerize, and demonstrate it by using different techniques. However, the binding site of trehalose in antithrombin should be defined by site-directed mutagenesis. On the other hand, it is not clear if all serpins polymerize in vivo through the same mechanism and it is also not clear if the same serpin can even polymerize through different mechanisms. Therefore, there are still doubts about the potential of trehalose or its derivatives to prevent in vivo antithrombin polymerization and, therefore, reduce thrombotic risk, as well as whether trehalose would be able to reduce polymerization in other serpins.

Understanding the highly efficient catalysis of prokaryotic peptide deformylases by shedding light on the determinants specifying the low activity of the human counterpart

2014 ◽  
Vol 70 (2) ◽  
pp. 242-252 ◽  
Author(s):  
Sonia Fieulaine ◽  
Michel Desmadril ◽  
Thierry Meinnel ◽  
Carmela Giglione
Keyword(s):  
Sequence Similarity ◽  
Three Dimensional ◽  
Binding Pocket ◽  
Dimensional Structure ◽  
Structural Basis ◽  
Human Counterpart ◽  
Low Activity

Peptide deformylases (PDFs), which are essential and ubiquitous enzymes involved in the removal of theN-formyl group from nascent chains, are classified into four subtypes based on the structural and sequence similarity of specific conserved domains. All PDFs share a similar three-dimensional structure, are functionally interchangeablein vivoand display similar propertiesin vitro, indicating that their molecular mechanism has been conserved during evolution. The human mitochondrial PDF is the only exception as despite its conserved fold it reveals a unique substrate-binding pocket together with an unusual kinetic behaviour. Unlike human PDF, the closely related mitochondrial PDF1As from plants have catalytic efficiencies and enzymatic parameters that are similar to those of other classes of PDFs. Here, the aim was to identify the structural basis underlying the properties of human PDF compared with all other PDFs by focusing on plant mitochondrial PDF1A. The construction of a chimaera composed of plant PDF1A with the nonrandom substitutions found in a conserved motif of its human homologue converted it into an enzyme with properties similar to the human enzyme, indicating the crucial role of these positions. The crystal structure of this human-like plant PDF revealed that substitution of two residues leads to a reduction in the volume of the ligand-binding site together with the introduction of negative charges, unravelling the origin of the weak affinity of human PDF for its substrate. In addition, the substitution of the two residues of human PDF modifies the transition state of the reaction through alteration of the network of interactions between the catalytic residues and the substrate, leading to an overall reduced reaction rate.

scholarly journals In Vivo and In Vitro Studies of Bacillus subtilis Ferrochelatase Mutants Suggest Substrate Channeling in the Heme Biosynthesis Pathway

2002 ◽  
Vol 184 (14) ◽  
pp. 4018-4024 ◽  
Author(s):  
Ulf Olsson ◽  
Annika Billberg ◽  
Sara Sjövall ◽  
Salam Al-Karadaghi ◽  
Mats Hansson
Keyword(s):  
Bacillus Subtilis ◽  
Amino Acid ◽  
Three Dimensional ◽  
Site Directed Mutagenesis ◽  
Dimensional Structure ◽  
Amino Acid Residues ◽  
Three Dimensional Structure ◽  
Activity Measurements

ABSTRACT Ferrochelatase (EC 4.99.1.1) catalyzes the last reaction in the heme biosynthetic pathway. The enzyme was studied in the bacterium Bacillus subtilis, for which the ferrochelatase three-dimensional structure is known. Two conserved amino acid residues, S54 and Q63, were changed to alanine by site-directed mutagenesis in order to detect any function they might have. The effects of these changes were studied in vivo and in vitro. S54 and Q63 are both located at helix α3. The functional group of S54 points out from the enzyme, while Q63 is located in the interior of the structure. None of these residues interact with any other amino acid residues in the ferrochelatase and their function is not understood from the three-dimensional structure. The exchange S54A, but not Q63A, reduced the growth rate of B. subtilis and resulted in the accumulation of coproporphyrin III in the growth medium. This was in contrast to the in vitro activity measurements with the purified enzymes. The ferrochelatase with the exchange S54A was as active as wild-type ferrochelatase, whereas the exchange Q63A caused a 16-fold reduction in V max. The function of Q63 remains unclear, but it is suggested that S54 is involved in substrate reception or delivery of the enzymatic product.

scholarly journals The Role of Pre-Clinical 3-Dimensional Models of Osteosarcoma

2020 ◽  
Vol 21 (15) ◽  
pp. 5499
Author(s):  
Hannah L. Smith ◽  
Stephen A. Beers ◽  
Juliet C. Gray ◽  
Janos M. Kanczler
Keyword(s):  
Three Dimensional ◽  
Chorioallantoic Membrane ◽  
3D Models ◽  
In Ovo ◽  
Future Directions ◽  
3 Dimensional ◽  
In Vivo Animal Models

Treatment for osteosarcoma (OS) has been largely unchanged for several decades, with typical therapies being a mixture of chemotherapy and surgery. Although therapeutic targets and products against cancer are being continually developed, only a limited number have proved therapeutically active in OS. Thus, the understanding of the OS microenvironment and its interactions are becoming more important in developing new therapies. Three-dimensional (3D) models are important tools in increasing our understanding of complex mechanisms and interactions, such as in OS. In this review, in vivo animal models, in vitro 3D models and in ovo chorioallantoic membrane (CAM) models, are evaluated and discussed as to their contribution in understanding the progressive nature of OS, and cancer research. We aim to provide insight and prospective future directions into the potential translation of 3D models in OS.

scholarly journals Protein Folding: Search for Basic Physical Models

2003 ◽  
Vol 3 ◽  
pp. 623-635 ◽  
Author(s):  
Ivan Y. Torshin ◽  
Robert W. Harrison
Keyword(s):  
Protein Folding ◽  
Tertiary Structure ◽  
Three Dimensional ◽  
Physical Models ◽  
Dimensional Structure ◽  
Computational Techniques ◽  
Linear Sequence ◽  
Physical Forces

How a unique three-dimensional structure is rapidly formed from the linear sequence of a polypeptide is one of the important questions in contemporary science. Apart from biological context ofin vivoprotein folding (which has been studied only for a few proteins), the roles of the fundamental physical forces in thein vitrofolding remain largely unstudied. Despite a degree of success in using descriptions based on statistical and/or thermodynamic approaches, few of the current models explicitly include more basic physical forces (such as electrostatics and Van Der Waals forces). Moreover, the present-day models rarely take into account that the protein folding is, essentially, a rapid process that produces a highly specific architecture. This review considers several physical models that may provide more direct links between sequence and tertiary structure in terms of the physical forces. In particular, elaboration of such simple models is likely to produce extremely effective computational techniques with value for modern genomics.

scholarly journals NADPH Oxidases Are Required for Full Platelet Activation In Vitro and Thrombosis In Vivo but Dispensable for Plasma Coagulation and Hemostasis

2020 ◽  
Author(s):  
Dina Vara ◽  
Reiner K. Mailer ◽  
Anuradha Tarafdar ◽  
Nina Wolska ◽  
Marco Heestermans ◽  
...  
Keyword(s):  
Thrombus Formation ◽  
Single Gene ◽  
Coagulation Cascade ◽  
Intracellular Cgmp ◽  
Antithrombotic Effects ◽  
Tail Tip ◽  
Synthase Inhibitor

Objective: Using 3KO (triple NOX [NADPH oxidase] knockout) mice (ie, NOX1 −/− /NOX2 −/− /NOX4 −/− ), we aimed to clarify the role of this family of enzymes in the regulation of platelets in vitro and hemostasis in vivo. Approach and Results: 3KO mice displayed significantly reduced platelet superoxide radical generation, which was associated with impaired platelet aggregation, adhesion, and thrombus formation in response to the key agonists collagen and thrombin. A comparison with single-gene knockouts suggested that the phenotype of 3KO platelets is the combination of the effects of the genetic deletion of NOX1 and NOX2, while NOX4 does not show any significant function in platelet regulation. 3KO platelets displayed significantly higher levels of cGMP—a negative platelet regulator that activates PKG (protein kinase G). The inhibition of PKG substantially but only partially rescued the defective phenotype of 3KO platelets, which are responsive to both collagen and thrombin in the presence of the PKG inhibitors KT5823 or Rp-8-pCPT-cGMPs, but not in the presence of the NOS (NO synthase) inhibitor L-NG-monomethyl arginine. In vivo, triple NOX deficiency protected against ferric chloride–driven carotid artery thrombosis and experimental pulmonary embolism, while hemostasis tested in a tail-tip transection assay was not affected. Procoagulatory activity of platelets (ie, phosphatidylserine surface exposure) and the coagulation cascade in platelet-free plasma were normal. Conclusions: This study indicates that inhibiting NOXs has strong antithrombotic effects partially caused by increased intracellular cGMP but spares hemostasis. NOXs are, therefore, pharmacotherapeutic targets to develop new antithrombotic drugs without bleeding side effects.

scholarly journals Role of YidC in folding of polytopic membrane proteins

2004 ◽  
Vol 165 (1) ◽  
pp. 53-62 ◽  
Author(s):  
Shushi Nagamori ◽  
Irina N. Smirnova ◽  
H. Ronald Kaback
Keyword(s):  
Membrane Proteins ◽  
Three Dimensional ◽  
In Vitro Transcription ◽  
Dimensional Structure ◽  
Lipid Phase ◽  
Lactose Permease ◽  
Primary Role ◽  
Protein Insertion

YidC of Echerichia coli, a member of the conserved Alb3/Oxa1/YidC family, is postulated to be important for biogenesis of membrane proteins. Here, we use as a model the lactose permease (LacY), a membrane transport protein with a known three-dimensional structure, to determine whether YidC plays a role in polytopic membrane protein insertion and/or folding. Experiments in vivo and with an in vitro transcription/translation/insertion system demonstrate that YidC is not necessary for insertion per se, but plays an important role in folding of LacY. By using the in vitro system and two monoclonal antibodies directed against conformational epitopes, LacY is shown to bind the antibodies poorly in YidC-depleted membranes. Moreover, LacY also folds improperly in proteoliposomes prepared without YidC. However, when the proteoliposomes are supplemented with purified YidC, LacY folds correctly. The results indicate that YidC plays a primary role in folding of LacY into its final tertiary conformation via an interaction that likely occurs transiently during insertion into the lipid phase of the membrane.

scholarly journals Analysis of the cross-reactivity and of the 1.5 Å crystal structure of the Malassezia sympodialis Mala s 6 allergen, a member of the cyclophilin pan-allergen family

2006 ◽  
Vol 396 (1) ◽  
pp. 41-49 ◽  
Author(s):  
Andreas G. Glaser ◽  
Andreas Limacher ◽  
Sabine Flückiger ◽  
Annika Scheynius ◽  
Leonardo Scapozza ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Three Dimensional ◽  
Cross Reactivity ◽  
Dimensional Structure ◽  
Cellular Functions ◽  
Human Proteins ◽  
Ige Mediated ◽  
Unit Cell Dimensions

Cyclophilins constitute a family of proteins involved in many essential cellular functions. They have also been identified as a panallergen family able to elicit IgE-mediated hypersensitivity reactions. Moreover, it has been shown that human cyclophilins are recognized by serum IgE from patients sensitized to environmental cyclophilins. IgE-mediated autoreactivity to self-antigens that have similarity to environmental allergens is often observed in atopic disorders. Therefore comparison of the crystal structure of human proteins with similarity to allergens should allow the identification of structural similarities to rationally explain autoreactivity. A new cyclophilin from Aspergillus fumigatus (Asp f 27) has been cloned, expressed and showed to exhibit cross-reactivity in vitro and in vivo. The three-dimensional structure of cyclophilin from the yeast Malassezia sympodialis (Mala s 6) has been determined at 1.5 Å (1 Å=0.1 nm) by X-ray diffraction. Crystals belong to space group P41212 with unit cell dimensions of a=b=71.99 Å and c=106.18 Å. The structure was solved by molecular replacement using the structure of human cyclophilin A as the search model. The refined structure includes all 162 amino acids of Mala s 6, an active-site-bound Ala-Pro dipeptide and 173 water molecules, with a crystallographic R- and free R-factor of 14.3% and 14.9% respectively. The overall structure consists of an eight-stranded antiparallel β-barrel and two α-helices covering the top and bottom of the barrel, typical for cyclophilins. We identified conserved solvent-exposed residues in the fungal and human structures that are potentially involved in the IgE-mediated cross-reactivity.

scholarly journals Three-Dimensional In Vitro Lymphangiogenesis Model in Tumor Microenvironment

2021 ◽  
Vol 9 ◽  
Author(s):  
Youngkyu Cho ◽  
Kyuhwan Na ◽  
Yesl Jun ◽  
Jihee Won ◽  
Ji Hun Yang ◽  
...  
Keyword(s):  
Tumor Microenvironment ◽  
Tumor Metastasis ◽  
Lymphatic Vessel ◽  
Disease Model ◽  
Three Dimensional ◽  
Lymphatic Vessels ◽  
Dimensional Structure ◽  
Biomechanical Stress

Lymphangiogenesis is a stage of new lymphatic vessel formation in development and pathology, such as inflammation and tumor metastasis. Physiologically relevant models of lymphatic vessels have been in demand because studies on lymphatic vessels are required for understanding the mechanism of tumor metastasis. In this study, a new three-dimensional lymphangiogenesis model in a tumor microenvironment is proposed, using a newly designed macrofluidic platform. It is verified that controllable biochemical and biomechanical cues, which contribute to lymphangiogenesis, can be applied in this platform. In particular, this model demonstrates that a reconstituted lymphatic vessel has an in vivo–like lymphatic vessel in both physical and biochemical aspects. Since biomechanical stress with a biochemical factor influences robust directional lymphatic sprouting, whether our model closely approximates in vivo, the initial lymphatics in terms of the morphological and genetic signatures is investigated. Furthermore, attempting an incorporation with a tumor spheroid, this study successfully develops a complex tumor microenvironment model for use in lymphangiogenesis and reveals the microenvironment factors that contribute to tumor metastasis. As a first attempt at a coculture model, this reconstituted model is a novel system with a fully three-dimensional structure and can be a powerful tool for pathological drug screening or disease model.

scholarly journals Functional Analysis of the Carbohydrate-Binding Domains of Erwinia chrysanthemi Cel5 (Endoglucanase Z) and an Escherichia coli Putative Chitinase

1999 ◽  
Vol 181 (15) ◽  
pp. 4611-4616 ◽  
Author(s):  
Helen D. Simpson ◽  
Frederic Barras
Keyword(s):  
Escherichia Coli ◽  
Catalytic Domain ◽  
Three Dimensional ◽  
Site Directed Mutagenesis ◽  
Erwinia Chrysanthemi ◽  
Dimensional Structure ◽  
Carbohydrate Binding ◽  
Binding Domains ◽  
Cellulose Binding

ABSTRACT The Cel5 cellulase (formerly known as endoglucanase Z) fromErwinia chrysanthemi is a multidomain enzyme consisting of a catalytic domain, a linker region, and a cellulose binding domain (CBD). A three-dimensional structure of the CBDCel5 has previously been obtained by nuclear magnetic resonance. In order to define the role of individual residues in cellulose binding, site-directed mutagenesis was performed. The role of three aromatic residues (Trp18, Trp43, and Tyr44) in cellulose binding was demonstrated. The exposed potential hydrogen bond donors, residues Gln22 and Glu27, appeared not to play a role in cellulose binding, whereas residue Asp17 was found to be important for the stability of Cel5. A deletion mutant lacking the residues Asp17 to Pro23 bound only weakly to cellulose. The sequence of CBDCel5 exhibits homology to a series of five repeating domains of a putative large protein, referred to as Yheb, from Escherichia coli. One of the repeating domains (Yheb1), consisting of 67 amino acids, was cloned from the E. coli chromosome and purified by metal chelating chromatography. While CBDCel5 bound to both cellulose and chitin, Yheb1 bound well to chitin, but only very poorly to cellulose. The Yheb protein contains a region that exhibits sequence homology with the catalytic domain of a chitinase, which is consistent with the hypothesis that the Yheb protein is a chitinase.

Three-dimensional imaging of nucleosomes from transcriptionally active genes using electron spectroscopic imaging

1996 ◽  
Vol 54 ◽  
pp. 54-55
Author(s):  
G. J. Czarnota ◽  
D. P. Bazett-Jones ◽  
F. P. Ottensmeyer
Keyword(s):  
Gene Expression ◽  
Three Dimensional ◽  
Spectroscopic Imaging ◽  
Dimensional Structure ◽  
Crystallographic Structure ◽  
Electron Spectroscopic Imaging ◽  
Nucleosome Structure ◽  
Active Genes

The three-dimensional structure of the nucleosome was determined using particles purified from transcriptionally active genes in conjunction with electron spectroscopic imaging, and quaternion-assisted angular reconstitution procedures. The results reveal a configuration which is very different from the canonical compact crystallographic structure for this fundamental chromosome subunit, implying a structural disruption of the nucleosome with the activation of gene expression in accord with numerous physico-chemical observations.Previous analyses of nucleosomes purified from transcriptionally quiescent genes have indicated numerous structural states dependent on factors in vitro which modify charge based interactions in nucleoprotein complexes. Nucleosomes from transcriptionally active genes undergo chemical alterations in vivo which similarly modify charge based interactions. In order to investigate the effects of the gene expression associated chemical alterations on nucleosome structure, particles were purified from transcriptionally active genes using mercury affinity chromatography. These nucleosome particles are hyperacetylated with respect to particles from transcriptionally quiescent genes. Here additionally, sulphydryls normally buried within the protein core of the transcriptionally inactive particle are exposed to chemical modifying agents thus facilitating purification as described.

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