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146 results on '"chemical chaperones"'

101. Role of the Extremolytes Ectoine and Hydroxyectoine as Stress Protectants and Nutrients: Genetics, Phylogenomics, Biochemistry, and Structural Analysis

Author: Laura Czech, Alexandra A. Richter, Erhard Bremer, Nadine Stöveken, Lucas Hermann, Johann Heider, Sander H. J. Smits, and Astrid Höppner
Subjects: 0301 basic medicine, Osmotic shock, enzymes, 030106 microbiology, high salinity, Review, Ectoine, crystal structures, 03 medical and health sciences, chemistry.chemical_compound, Phylogenomics, genomics, Genetics, Genetics (clinical), chemical chaperones, Osmotic concentration, biology, Chemistry, biology.organism_classification, Biochemistry, Osmolyte, gene expression, Osmoprotectant, osmotic stress, Chemical chaperone, growth temperature extremes, biotechnology, Archaea
Abstract: Fluctuations in environmental osmolarity are ubiquitous stress factors in many natural habitats of microorganisms, as they inevitably trigger osmotically instigated fluxes of water across the semi-permeable cytoplasmic membrane. Under hyperosmotic conditions, many microorganisms fend off the detrimental effects of water efflux and the ensuing dehydration of the cytoplasm and drop in turgor through the accumulation of a restricted class of organic osmolytes, the compatible solutes. Ectoine and its derivative 5-hydroxyectoine are prominent members of these compounds and are synthesized widely by members of the Bacteria and a few Archaea and Eukarya in response to high salinity/osmolarity and/or growth temperature extremes. Ectoines have excellent function-preserving properties, attributes that have led to their description as chemical chaperones and fostered the development of an industrial-scale biotechnological production process for their exploitation in biotechnology, skin care, and medicine. We review, here, the current knowledge on the biochemistry of the ectoine/hydroxyectoine biosynthetic enzymes and the available crystal structures of some of them, explore the genetics of the underlying biosynthetic genes and their transcriptional regulation, and present an extensive phylogenomic analysis of the ectoine/hydroxyectoine biosynthetic genes. In addition, we address the biochemistry, phylogenomics, and genetic regulation for the alternative use of ectoines as nutrients.
Published: 2018

102. The Protein Translocation Defect of MCT8 L291R Is Rescued by Sodium Phenylbutyrate.

Author: Braun D and Schweizer U
Abstract: Introduction: The monocarboxylate transporter 8 (MCT8; SLC16A2) is a specific transporter for thyroid hormones. MCT8 deficiency, formerly known as the Allan-Herndon-Dudley syndrome, is a rare genetic disease that leads to neurological impairments and muscle weakness. Current experimental treatment options rely on thyromimetic agonists that do not depend on MCT8 for cellular uptake. Another approach comes from studies with the chemical chaperone sodium phenylbutyrate (NaPB), which was able to stabilize MCT8 mutants having protein folding defects in vitro. In addition, NaPB is known as a compound that assists with plasma membrane translocation., Objective: The pathogenic MCT8 L291R leads to the same severe neurological impairments found for other MCT8-deficient patients but, unexpectedly, lacks alterations in plasma 3,3',5-triiodothyronine (T 3 ) levels. Here we tried to unravel the underlying mechanism of MCT8 deficiency and tested whether the pathogenic MCT8 L291R mutant responds to NaPB treatment. Therefore, we overexpressed the mutant in Madin-Darby canine kidney cells in the human choriocarcinoma cell line JEG1 and in COS7 cells of African green monkey origin., Results: In our recent study we describe that the MCT8 L291R mutation most likely leads to a translocation defect. The pathogenic mutant is not located at the plasma membrane, but shows overlapping expression with a marker protein of the lysosome. Mutation of the corresponding amino acid in murine Mct8 (Mct8 L223R ) displays a similar effect on cell surface expression and transport function as seen before for MCT8 L291R . NaPB was able to correct the translocation defect of MCT8 L291R /Mct8 L223R and restored protein function by increasing T 3 transport activity. Furthermore, we detected enhanced mRNA levels of wild-type and mutant MCT8/Mct8 after NaPB treatment. The increase in mRNA levels could be an explanation for the positive effect on protein expression and function detected for wild-type MCT8., Conclusion: NaPB is not only suitable for the treatment of mutations leading to misfolding and protein degradation, but also for a mutant wrongly sorted inside a cell which is otherwise functional., Competing Interests: No competing financial interests exist., (Copyright © 2020 by S. Karger AG, Basel.)
Published: 2020
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103. Getting CD19 Into Shape: Expression of Natively Folded "Difficult-to- Express" CD19 for Staining and Stimulation of CAR-T Cells.

Author: Lobner E, Wachernig A, Gudipati V, Mayrhofer P, Salzer B, Lehner M, Huppa JB, and Kunert R
Abstract: The transmembrane protein CD19 is exclusively expressed on normal and malignant B cells and therefore constitutes the target of approved CAR-T cell-based cancer immunotherapies. Current efforts to assess CAR-T cell functionality in a quantitative fashion both in vitro and in vivo are hampered by the limited availability of the properly folded recombinant extracellular domain of CD19 (CD19-ECD) considered as "difficult-to-express" (DTE) protein. Here, we successfully expressed a novel fusion construct consisting of the full-length extracellular domain of CD19 and domain 2 of human serum albumin (CD19-AD2), which was integrated into the Rosa26 bacterial artificial chromosome vector backbone for generation of a recombinant CHO-K1 production cell line. Product titers could be further boosted using valproic acid as a chemical chaperone. Purified monomeric CD19-AD2 proved stable as shown by non-reduced SDS-PAGE and SEC-MALS measurements. Moreover, flow cytometric analysis revealed specific binding of CD19-AD2 to CD19-CAR-T cells. Finally, we demonstrate biological activity of our CD19-AD2 fusion construct as we succeeded in stimulating CD19-CAR-T cells effectively with the use of CD19-AD2-decorated planar supported lipid bilayers., (Copyright © 2020 Lobner, Wachernig, Gudipati, Mayrhofer, Salzer, Lehner, Huppa and Kunert.)
Published: 2020
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104. Chaperones Promote Remarkable Solubilization of Salmonella enterica serovar Enteritidis Flagellin Expressed in Escherichia coli.

Author: Bakhtiarvand B, Sadeghi Z, Tarahomjoo S, and Yaghmaie S
Subjects: Electrophoresis, Polyacrylamide Gel, Escherichia coli genetics, Escherichia coli Proteins metabolism, Flagellin genetics, Flagellin metabolism, Inclusion Bodies chemistry, Inclusion Bodies genetics, Inclusion Bodies metabolism, Models, Molecular, Protein Conformation, Protein Folding, Recombinant Proteins chemistry, Recombinant Proteins metabolism, Salmonella enteritidis chemistry, Solubility, Sorbitol pharmacology, Escherichia coli growth & development, Flagellin chemistry, Molecular Chaperones metabolism, Salmonella enteritidis metabolism
Abstract: Background: Flagellin of Salmonella enterica serovar Enteritidis (SEF) stimulates immune responses to both itself and coapplied antigens. It is therefore used in vaccine development and immunotherapy. Removal of pathogenic S. enterica ser. Enteritidis from SEF production process is advantageous due to the process safety improvement. The protein solubility analysis using SDS-PAGE indicated that 53.49% of SEF expressed in Escherichia coli formed inclusion bodies. However, the protein recovery from inclusion bodies requires a complex process with a low yield., Objective: We thus aim to study possibility of enhancing SEF expression in E. coli in soluble form using chemical and molecular chaperones., Methods: Chemical chaperones including arginine, sorbitol, trehalose, sodium chloride and benzyl alcohol were used as cultivation medium additives during SEF expression. SEF solubilization by coexpression of molecular chaperones DnaK, DnaJ, and GrpE was also investigated., Results: All of the chemical chaperones were effective in improving SEF solubility. However, sorbitol showed the most profound effect. SEF solubilization by molecular chaperones was slightly better than that using sorbitol and this approach enhanced noticeably SEF soluble concentration and SEF solubility percentage to almost two folds and 96.37% respectively. Results of limited proteolysis assay and native PAGE indicated similar conformational states and proper folding for SEF obtained without using chaperones and for those obtained using sorbitol and the molecular chaperones. However, the molecular chaperones based system was less costly than the sorbitol based system., Conclusion: The coexpression of molecular chaperones was then considered as the most appropriate approach for soluble SEF production. Therefore, SEF production for medical purposes is expected to be facilitated., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)
Published: 2020
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105. Revealing Protein Aggregates under Thapsigargin-Induced ER Stress Using an ER-Targeted Thioflavin.

Author: Verwilst P, Kim K, Sunwoo K, Kim HR, Kang C, and Kim JS
Subjects: Benzothiazoles analysis, Endoplasmic Reticulum chemistry, Endoplasmic Reticulum metabolism, HeLa Cells, Humans, Molecular Structure, Protein Aggregates drug effects, Spectrometry, Fluorescence, Thapsigargin chemistry, Benzothiazoles metabolism, Biosensing Techniques, Endoplasmic Reticulum drug effects, Endoplasmic Reticulum Stress drug effects, Thapsigargin pharmacology
Abstract: Endoplasmic reticulum-thioflavin T (ER-ThT ), a thioflavin T-based fluorescent chemosensor, was developed to detect protein aggregates in the endoplasmic reticulum (ER) and was applied to live cells under various forms of ER stress. Upon dithiothreitol (DTT)-induced reductive denaturation of lysozyme and albumin, the intensity was increased in a protein concentration-dependent way, following a nonfluorescent lag phase. ER-ThT detects protein aggregates rather than unfolded proteins in solution, and the protein aggregation can be visualized in the presence of lipid membranes or native proteins. Within live HeLa cells, ER-ThT is localized in the ER and its fluorescence was dramatically increased upon ER stress induction by DTT, Thapsigargin, or Brefeldin A. Moreover, in the presence of ER stress modulators (tauroursodeoxycholic acid, trimethylamine N -oxide, or 4-phenylbutyric acid), also known as chemical chaperones, the fluorescence under Thapsigargin treatment was suppressed to the level of the control group. Thus, ER-ThT is capable of detecting the accumulation of protein aggregates under ER stress in living cells and acts as an in vitro screening tool for ER stress modulators, putative prodrugs against ER-related proteopathy. Overall, the results strongly suggest that protein aggregation is intricately involved in the activation of the unfolded protein response following ER stress.
Published: 2019
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106. The unfolded protein response in hereditary haemochromatosis

Author: M. de Sousa and S. F. de Almeida
Subjects: Protein Folding, hereditary haemochromatosis (HH), Reviews, Poison control, Disease, major histocompatibility complex class-I (MHC-I), Endoplasmic Reticulum, Models, Biological, Stress, Physiological, Homeostasis, Humans, Medicine, unfolded protein response (UPR), Hemochromatosis Protein, Hemochromatosis, chemical chaperones, Antigen Presentation, business.industry, Endoplasmic reticulum, Histocompatibility Antigens Class I, Membrane Proteins, Cell Biology, medicine.disease, Iron Metabolism Disorders, Penetrance, Cell biology, Membrane protein, Mutation, Immunology, Unfolded protein response, Molecular Medicine, HFE, Signal transduction, business, Forecasting, Molecular Chaperones, Signal Transduction
Abstract: To cope with the accumulation of unfolded or misfolded proteins the endoplasmic reticulum (ER) has evolved specific signalling pathways collectively called the unfolded protein response (UPR). Elucidation of the mechanisms governing ER stress signallinghas linked this response to the regulation of diverse physiologic processes as well as to the progression of a number of diseases. Interest in hereditary haemochromatosis (HH) has focused on the study of proteins implicated in iron homeostasis and on the identification of new alleles related with the disease. HFE has been amongst the preferred targets of interest, since the discovery that its C282Y mutation was associated with HH. However, the discrepancies between the disease penetrance and the frequency of this mutation have raised the possibility that its contribution to disease progression might go beyond the mere involvement in regulation of cellular iron uptake. Recent findings revealed that activation of the UPR is a feature of HH and that this stress response may be involved in the genesis of immunological anomalies associated with the disease. This review addresses the connection of the UPR with HH, including its role in MHC-I antigen presentation pathway and possible implications for new clinical approaches to HH.
Published: 2008

107. The chemical chaperone sodium 4-phenylbutyrate improves the secretion of the protein CA267T mutant in CHO-K1 cells trough the GRASP55 pathway

Author: Chollet, Maria Eugenia, Skarpen, Ellen, Iversen, Nina, Sandset, Per Morten, and Skretting, Grethe
Subjects: Misfolding, Chemical chaperones, Mutant, Letter to the Editor, GRASP55, Protein C
Abstract: Some inherited coagulation factor deficiencies are caused by intracellular retention or degradation of misfolded proteins, and chemical chaperones have been shown to reverse protein misfolding. The purpose of the present study was to investigate whether chemical chaperones may improve secretion of the protein CA267T (PCA267T) mutant in a cellular model. Using stably transfected Chinese hamster ovary cells (CHO-K1) expressing PCA267T we demonstrate that sodium 4-phenylbutyrate (PBA) increased the secretion of PCA267T by approximately 4-fold in comparison with untreated cells, and that this secretion seemed to follow an unconventional pathway via the Golgi reassembly stacking protein (GRASP55).
Published: 2015

108. An unfolded protein response is the initial cellular response to the expression of mutant matrilin-3 in a mouse model of multiple epiphyseal dysplasia

Author: Nundlall, Seema, Rajpar, M. Helen, Bell, Peter A., Clowes, Christopher, Zeeff, Leo A. H., Gardner, Benjamin, Thornton, David J., Boot-Handford, Raymond P., and Briggs, Michael D.
Published: 2010
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109. Modulation of the Activity of Newly Synthesized Human Phenylalanine Hydroxylase Mutant Proteins by Low-Molecular-Weight Compounds

Author: Nascimento, Cátia, Leandro, João, Tavares de Almeida, Isabel, and Leandro, Paula
Published: 2008
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110. Bacterial cryoprotectants

Author: Chattopadhyay, M K
Published: 2002
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111. (Bio)chemical Strategies To Modulate Amyloid-β Self-Assembly.

Author: Ayala S, Genevaux P, Hureau C, and Faller P
Subjects: Alzheimer Disease metabolism, Alzheimer Disease pathology, Amyloid beta-Peptides metabolism, Animals, Humans, Plaque, Amyloid metabolism, Plaque, Amyloid pathology, Amyloid beta-Peptides chemistry, Protein Aggregation, Pathological metabolism, Protein Aggregation, Pathological pathology
Abstract: Amyloid plaques are one of the two hallmarks of Alzheimer's disease (AD). They consist mainly of fibrils made of self-assembled amyloid-β (Aβ) peptides. Aβ is produced in healthy brains from proteolytic cleavage of the amyloid precursor protein. Aβ aggregates, in particular smaller, soluble aggregates, are toxic to cells. Hence, modulating the self-assembly of Aβ became a very active field of research, with the aim to reduce the amount of the toxic aggregates of Aβ or to block their toxic action. A great variety of molecules, chemical and biological, are able to modify the aggregation of Aβ. Here we give an overview of the different mechanistic ways to modulate Aβ aggregation and on which step in the self-assembly molecules can interfere. We discuss the aggregation modulators according to different important parameters, including the type of interaction (weak interaction, coordination or covalent bonds), the importance of kinetics and thermodynamics, the size of the modulating molecules, and binding specificity.
Published: 2019
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112. Deciphering the role of trehalose in hindering antithrombin polymerization.

Author: Naseem A, Khan MS, Ali H, Ahmad I, and Jairajpuri MA
Subjects: Anilino Naphthalenesulfonates chemistry, Circular Dichroism, Guanidine chemistry, Humans, Hydrophobic and Hydrophilic Interactions, Protein Binding physiology, Protein Conformation, Protein Folding, Serpins metabolism, Anticoagulants chemistry, Antithrombins metabolism, Blood Coagulation physiology, Drug Design, Trehalose metabolism
Abstract: Serine protease inhibitors (serpins) family have a complex mechanism of inhibition that requires a large scale conformational change. Antithrombin (AT), a member of serpin superfamily serves as a key regulator of the blood coagulation cascade, deficiency of which leads to thrombosis. In recent years, a handful of studies have identified small compounds that retard serpin polymerization but abrogated the normal activity. Here, we screened small molecules to find potential leads that can reduce AT polymer formation. We identified simple sugar molecules that successfully blocked polymer formation without a significant loss of normal activity of AT under specific buffer and temperature conditions. Of these, trehalose proved to be most promising as it showed a marked decrease in the bead like polymeric structures of AT shown by electron microscopic analysis. A circular dichroism (CD) analysis indicated alteration in the secondary structure profile and an increased thermal stability of AT in the presence of trehalose. Guanidine hydrochloride (GdnHCl)-based unfolding studies of AT show the formation of a different intermediate in the presence of trehalose. A time-dependent fluorescence study using 1,1'-bi(4-anilino)naphthalene-5,5'-disulfonic acid (Bis-ANS) shows that trehalose affects the initial conformational change step in transition from native to polymer state through its binding to exposed hydrophobic residues on AT thus making AT less polymerogenic. In conclusion, trehalose holds promise by acting as an initial scaffold that can be modified to design similar compounds with polymer retarding propensity., (© 2019 The Author(s).)
Published: 2019
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113. Protein misfolding in endoplasmic reticulum stress with applications to renal diseases.

Author: Nademi S and Dickhout JG
Subjects: Humans, Kidney Diseases genetics, Kidney Diseases therapy, Mutation, Proteins chemistry, Proteins genetics, Proteins metabolism, Thermodynamics, Unfolded Protein Response, Endoplasmic Reticulum Stress, Kidney Diseases metabolism, Protein Folding
Abstract: Protein misfolding may be the result of a variety of different processes that disrupt the ability of a protein to form a thermodynamically stable tertiary structure that allows it to perform its proper function. In this chapter, we explore the nature of a protein's form that allows it to have a stable tertiary structure, and examine specific mutation that are known to occur in the coding regions of DNA that disrupt a protein's ability to be folded into a thermodynamically stable tertiary structure. We examine the consequences of these protein misfoldings in terms of the endoplasmic reticulum stress response and resulting unfolded protein response. These conditions are specifically related to renal diseases. Further, we explore novel therapeutics, pharmacological chaperones, that are being developed to alleviate the disease burden associated with protein misfolding caused by mutations. These interventions aim to stabilize protein folding intermediates and allow proper folding to occur as well as prevent protein aggregation and the resulting pathophysiological consequences., (Copyright © 2020 Elsevier Inc. All rights reserved.)
Published: 2019
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114. Thermal Shift and Stability Assays of Disease-Related Misfolded Proteins Using Differential Scanning Fluorimetry.

Author: Lucas TG, Gomes CM, and Henriques BJ
Subjects: Fluorescent Dyes, Humans, Molecular Chaperones chemistry, Molecular Chaperones metabolism, Protein Stability, Proteins metabolism, Proteostasis Deficiencies, Transition Temperature, Fluorometry methods, Protein Folding, Proteins chemistry, Thermodynamics
Abstract: Systematic identification of buffer formulations and small molecule chaperones that improve the expression, stability, and storage of proteins with therapeutic interest has gained enormous importance in biochemical research as well as in biotechnology and biomedical applications. In particular, the biochemical characterization of disease-related proteins and their genetic variants that result in misfolding requires systematic determination of protein stability, screening of optimal buffer conditions for biophysical and structural studies, and in some cases, the identification of small molecule chaperones with the potential to ameliorate folding defects. Among the several techniques available, differential scanning fluorimetry (DSF) is currently an extensively employed screening and analysis method for thermal shift and protein stability assays. Here we describe a step-by-step generic protocol for fast characterization of protein thermal stability and analysis of stabilization in thermal-shift assays by additives, ligands and chemical chaperones using β-oxidation mitochondrial dehydrogenases as model. These enzymes are associated to inborn errors of metabolism caused by mutant variants with folding and stability defects for which we previously established folding correction afforded by their cognate cofactors and substrates. With this example we thus illustrate the potential applications of the method in screening small molecule folding correctors among metabolites, ligands, cofactors or candidate drugs with therapeutic potential in protein folding diseases.
Published: 2019
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115. Role of the Extremolytes Ectoine and Hydroxyectoine as Stress Protectants and Nutrients: Genetics, Phylogenomics, Biochemistry, and Structural Analysis.

Author: Czech, Laura, Hermann, Lucas, Stöveken, Nadine, Richter, Alexandra A., Höppner, Astrid, Smits, Sander H. J., Heider, Johann, and Bremer, Erhard
Subjects: *CYTOPLASM, *OSMOLAR concentration, *PATHOGENIC microorganisms, *POLYSACCHARIDES, *MICROORGANISMS
Abstract: Fluctuations in environmental osmolarity are ubiquitous stress factors in many natural habitats of microorganisms, as they inevitably trigger osmotically instigated fluxes of water across the semi-permeable cytoplasmic membrane. Under hyperosmotic conditions, many microorganisms fend off the detrimental effects of water efflux and the ensuing dehydration of the cytoplasm and drop in turgor through the accumulation of a restricted class of organic osmolytes, the compatible solutes. Ectoine and its derivative 5-hydroxyectoine are prominent members of these compounds and are synthesized widely by members of the Bacteria and a few Archaea and Eukarya in response to high salinity/osmolarity and/or growth temperature extremes. Ectoines have excellent function-preserving properties, attributes that have led to their description as chemical chaperones and fostered the development of an industrial-scale biotechnological production process for their exploitation in biotechnology, skin care, and medicine. We review, here, the current knowledge on the biochemistry of the ectoine/hydroxyectoine biosynthetic enzymes and the available crystal structures of some of them, explore the genetics of the underlying biosynthetic genes and their transcriptional regulation, and present an extensive phylogenomic analysis of the ectoine/hydroxyectoine biosynthetic genes. In addition, we address the biochemistry, phylogenomics, and genetic regulation for the alternative use of ectoines as nutrients. [ABSTRACT FROM AUTHOR]
Published: 2018
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116. Systems metabolic engineering of Corynebacterium glutamicum for production of the chemical chaperone ectoine

Author: Rudolf Schäfer, Nadine Stöveken, Judith Becker, Erhard Bremer, Michael Kohlstedt, Christoph Wittmann, Björn J Harder, and Nicole S Borchert
Subjects: Industrial fermentation, Bioengineering, Biology, Ectoine, Applied Microbiology and Biotechnology, Corynebacterium glutamicum, Metabolic engineering, Synthetic biology, chemistry.chemical_compound, Gene cluster, Amino Acids, Heterologous production, Aspartokinase, Research, Compatible solutes, Amino Acids, Diamino, Codon optimization, Biochemistry, chemistry, Metabolic Engineering, Chemical chaperones, Fermentation, Osmoprotectant, Biotechnology
Abstract: Background The stabilizing and function-preserving effects of ectoines have attracted considerable biotechnological interest up to industrial scale processes for their production. These rely on the release of ectoines from high-salinity-cultivated microbial producer cells upon an osmotic down-shock in rather complex processor configurations. There is growing interest in uncoupling the production of ectoines from the typical conditions required for their synthesis, and instead design strains that naturally release ectoines into the medium without the need for osmotic changes, since the use of high-salinity media in the fermentation process imposes notable constraints on the costs, design, and durability of fermenter systems. Results Here, we used a Corynebacterium glutamicum strain as a cellular chassis to establish a microbial cell factory for the biotechnological production of ectoines. The implementation of a mutant aspartokinase enzyme ensured efficient supply of L-aspartate-beta-semialdehyde, the precursor for ectoine biosynthesis. We further engineered the genome of the basic C. glutamicum strain by integrating a codon-optimized synthetic ectABCD gene cluster under expressional control of the strong and constitutive C. glutamicum tuf promoter. The resulting recombinant strain produced ectoine and excreted it into the medium; however, lysine was still found as a by-product. Subsequent inactivation of the L-lysine exporter prevented the undesired excretion of lysine while ectoine was still exported. Using the streamlined cell factory, a fed-batch process was established that allowed the production of ectoine with an overall productivity of 6.7 g L-1 day-1 under growth conditions that did not rely on the use of high-salinity media. Conclusions The present study describes the construction of a stable microbial cell factory for recombinant production of ectoine. We successfully applied metabolic engineering strategies to optimize its synthetic production in the industrial workhorse C. glutamicum and thereby paved the way for further improvements in ectoine yield and biotechnological process optimization.
Published: 2013

117. Therapeutic Targeting of Endoplasmic Reticulum Stress in Inflammatory Bowel Disease

Author: Das, Indrajit
Subjects: Anti-inflammatory agents, Chemical chaperones, Endoplasmic reticulum stress, Inflammatory bowel diseases
Abstract: Endoplasmic reticulum (ER) stress occurs when proteins misfold during biosynthesis in the ER. ER stress in intestinal secretory cells has been implicated in the aetiology of inflammatory bowel diseases (IBD) and intestinal inflammation in mice. Intestinal secretory cells are susceptible to ER stress due to high rates of protein synthesis, and ER stress in these cells results in reduced production of cell surface and secreted proteins leading to thinner mucus with a lower anti-microbial content, allowing penetration by luminal microbes, leading to inflammation. Cells experiencing ER stress attempt to restore homeostasis via the unfolded protein response (UPR), which enables the cells to increase the protein folding capacity of the ER. The primary focus of this thesis is to examine whether the drugs that are efficacious in IBD treatment modulate goblet cell function and ER homeostasis, and whether drugs that modulate ER stress can suppress intestinal inflammation and restore intestinal homeostasis. In order to explore the ER stress-inflammation nexus I utilized well established IBD anti-inflammatory agents, 5-Aminosalicylate (5-ASA), 6-thioguanine (6-TG), the anti-TNF antibody, infliximab, and the glucocorticoid dexamethasone (DEX). Chemical chaperones (TUDCA and sodium 4-PBA) and UPR modulators (guanabenz, salubrinal and 4μ8C) were used to investigate how modulation of ER stress affects goblet cell function and intestinal inflammation. In vivo studies were carried out in Winnie mice, which have ER stress in goblet cells due to a Muc2 misfolding mutation resulting in colitis involving both innate and adaptive immunity. To understand the direct effect of these drugs on ER stress in goblet cells in the absence of confounding inflammatory factors, in vitro experiments were performed using the human colonic adenocarcinoma LS174T cell line, a cell line containing cells with goblet cell differentiation, where ER stress was induced either by inhibition of N-glycosylation by tunicamycin or by depletion of Ca2+ by thapsigargin.
Published: 2012
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118. Pharmacological chaperone therapy for Gaucher disease: A patent review

Author: Juan M. Benito, Carmen Ortiz Mellet, José M. García Fernández, Universidad de Sevilla. Departamento de Química orgánica, Ministerio de Ciencia e Innovación (MICIN). España, and Junta de Andalucía
Subjects: Glucocerebrosidase, Protein Folding, Pharmacological chaperone therapy, Iminosugar, Drug Evaluation, Preclinical, Gaucher disease, Pharmacology, Biology, Iminosugars, lysosomal storage disorders, Patents as Topic, Miglustat, Drug Discovery, medicine, Humans, Substrate reduction therapy, Enzyme Replacement Therapy, Gaucher Disease, Nojirimycin, Glycosidase inhibitors, Folding diseases, General Medicine, Enzyme replacement therapy, Isofagomine, Pharmacological chaperone, Ambroxol, Chemical chaperones, Blood-Brain Barrier, Drug Design, Immunology, Chemical chaperone, medicine.drug, Imino Pyranoses, Molecular Chaperones
Abstract: Introduction: Mutations in the gene encoding for acid β-glucosidase (β-glucocerebrosidase, GlcCerase) are seen in Gaucher disease (GD), which give rise to significant protein misfolding effects and result in progressive accumulation of glucosyl ceramide. The main treatment for GD is enzyme replacement therapy (ERT). The iminosugar glycosidase inhibitor N-(n-butyl)-1-deoxynojirimycin (miglustat, Zavesca™) is used in a second treatment modality known as substrate reduction therapy. At the beginning of the 21st century, a third therapeutic paradigm was launched, namely, pharmacological chaperone therapy (PCT). This therapeutic strategy relies on the capability of such inhibitors to promote the correct folding and stabilize mutant forms of lysosomal enzymes, such as GlcCerase, as they pass through the secretory pathway. Areas covered: This review summarizes the different approaches used to implement the concept of PCT for GD. It discusses the relevant research, patents and patent applications filed in the last decade. Expert opinion: While the significance of PCT remains a matter of debate, the great interest gathered regarding it in a relatively few years reflects its broad potential scope, well beyond GD. The fact that pharmacological chaperones can be designed to cross the blood brain barrier (BBB) make them candidates for the treatment of neuronopathic forms of GD that are not responsive to ERT. Combined therapies offer even broader possibilities that deserve to be fully explored. © 2011 Informa UK, Ltd., The authors thank the the Spanish Ministerio de Ciencia e Innovación (contract numbers CTQ2007-61180/PPQ and SAF2010-84015670), the Fundación Ramón Areces, the Junta de Andalucía (Project P08-FQM-03711) and the European Union for sustained financial support in the field covered by this review.
Published: 2011

119. Synthesis of lipophilic 1-deoxygalactonojirimycin derivatives as D-galactosidase inhibitors

Author: Elisabeth Scheucher, Andreas Steiner, Don J. Mahuran, Georg Schitter, Stephen G. Withers, Arnold E. Stütz, Jacqueline Wicki, Eduard Paschke, Katrin Fantur, Martin Thonhofer, Brigitte Rigat, Michael B. Tropak, Tanja M. Wrodnigg, and Chris A. Tarling
Subjects: chemistry.chemical_classification, Stereochemistry, Chemistry, Organic Chemistry, Mutant, Ring (chemistry), Agrobacterium sp, lipophilic galactosidase inhibitor, Full Research Paper, 1-deoxygalactonojirimycin, lcsh:QD241-441, lcsh:Organic chemistry, N-modified iminosugars, iminosugars, lcsh:Q, 1-deoxy-D-galactonojirimycin, Chemical chaperone, lcsh:Science, Green coffee, Alkyl, chemical chaperones
Abstract: N-Alkylation at the ring nitrogen of the D-galactosidase inhibitor 1-deoxygalactonojirimycin with a functionalised C6 alkyl chain followed by modification with different aromatic substituents provided lipophilic 1-deoxygalactonojirimycin derivatives which exhibit inhibitory properties against β-glycosidases from E. coli and Agrobacterium sp. as well as green coffee bean α-galactosidase. In preliminary studies, these compounds also showed potential as chemical chaperones for GM1-gangliosidosis related β-galactosidase mutants.
Published: 2010

120. An unfolded protein response is the initial cellular response to the expression of mutant matrilin-3 in a mouse model of multiple epiphyseal dysplasia

Author: Raymond P. Boot-Handford, M. Helen Rajpar, Christopher Clowes, Michael D. Briggs, David J. Thornton, Peter A. Bell, Leo A. H. Zeeff, Seema Nundlall, and Benjamin Gardner
Subjects: Time Factors, Mutant, Apoptosis, Endoplasmic Reticulum, Biochemistry, Unfolded protein response, Mice, 0302 clinical medicine, CRELD2, 0303 health sciences, Extracellular Matrix Proteins, ARMET, Phenylbutyrates, Schmid metaphyseal chondrodysplasia, 3. Good health, Cell biology, Up-Regulation, medicine.anatomical_structure, Phenotype, Chondrodysplasia, medicine.medical_specialty, Ribs, Biology, Osteochondrodysplasias, Phenylbutyrate, Chondrocyte, Multiple epiphyseal dysplasia, Mouse model, 03 medical and health sciences, Chondrocytes, Internal medicine, medicine, Animals, Matrilin Proteins, Nerve Growth Factors, 030304 developmental biology, Original Paper, Cartilage, Endoplasmic reticulum, Gene Expression Profiling, Cell Biology, medicine.disease, Disease Models, Animal, Endocrinology, Chemical chaperones, Mutation, Matrilin-3, 030217 neurology & neurosurgery
Abstract: Multiple epiphyseal dysplasia (MED) can result from mutations in matrilin-3, a structural protein of the cartilage extracellular matrix. We have previously shown that in a mouse model of MED the tibia growth plates were normal at birth but developed a progressive dysplasia characterised by the intracellular retention of mutant matrilin-3 and abnormal chondrocyte morphology. By 3 weeks of age, mutant mice displayed a significant decrease in chondrocyte proliferation and dysregulated apoptosis. The aim of this current study was to identify the initial post-natal stages of the disease. We confirmed that the disease phenotype is seen in rib and xiphoid cartilage and, like tibia growth plate cartilage is characterised by the intracellular retention of mutant matrilin-3. Gene expression profiling showed a significant activation of classical unfolded protein response (UPR) genes in mutant chondrocytes at 5 days of age, which was still maintained by 21 days of age. Interestingly, we also noted the upregulation of arginine-rich, mutated in early stage of tumours (ARMET) and cysteine-rich with EGF-like domain protein 2 (CRELD2) are two genes that have only recently been implicated in the UPR. This endoplasmic reticulum (ER) stress and UPR did not lead to increased chondrocyte apoptosis in mutant cartilage by 5 days of age. In an attempt to alleviate ER stress, mutant mice were fed with a chemical chaperone, 4-sodium phenylbutyrate (SPB). SPB at the dosage used had no effect on chaperone expression at 5 days of age but modestly decreased levels of chaperone proteins at 3 weeks. However, this did not lead to increased secretion of mutant matrilin-3 and in the long term did not improve the disease phenotype. We performed similar studies with a mouse model of Schmid metaphyseal chondrodysplasia, but again this treatment did not improve the phenotype. Electronic supplementary material The online version of this article (doi:10.1007/s12192-010-0193-y) contains supplementary material, which is available to authorized users.
Published: 2010

121. Protein quality control: the who’s who, the where’s and therapeutic escapes

Author: Roger Santimaria, Valérie Le Fourn, Katarina Gaplovska-Kysela, Christian Zuber, Tania Torossi, Jing-Yu Fan, Jürgen Roth, Kiyoko Hirano, Bruno Guhl, Martin Ziak, Gary Hin-Fai Yam, University of Zurich, and Roth, Jürgen
Subjects: Protein Folding, Histology, 610 Medicine & health, Review, Endoplasmic-reticulum-associated protein degradation, Endoplasmic Reticulum, 142-005 142-005, 2722 Histology, Chaperonin, 1307 Cell Biology, JUNQ and IPOD, Glucosyltransferase, 1312 Molecular Biology, Humans, Molecular Biology, Secretory pathway, biology, 3607 Medical Laboratory Technology, Membrane Proteins, Proteins, Cell Biology, ERAD, Glucosidase II, Cell biology, Co-chaperone, Medical Laboratory Technology, Biochemistry, Membrane protein, Chemical chaperones, Chaperone (protein), Drug Design, biology.protein, 570 Life sciences, Protein folding, Endomannosidase, Protein folding disease, EDEM1, Metabolism, Inborn Errors, Molecular Chaperones
Abstract: In cells the quality of newly synthesized proteins is monitored in regard to proper folding and correct assembly in the early secretory pathway, the cytosol and the nucleoplasm. Proteins recognized as non-native in the ER will be removed and degraded by a process termed ERAD. ERAD of aberrant proteins is accompanied by various changes of cellular organelles and results in protein folding diseases. This review focuses on how the immunocytochemical labeling and electron microscopic analyses have helped to disclose the in situ subcellular distribution pattern of some of the key machinery proteins of the cellular protein quality control, the organelle changes due to the presence of misfolded proteins, and the efficiency of synthetic chaperones to rescue disease-causing trafficking defects of aberrant proteins.
Published: 2008

122. Therapeutic Targeting of Endoplasmic Reticulum Stress in Inflammatory Bowel Disease

Author: Crane, Denis, McGuckin, Michael, Florin, Timothy, Eri, Rajaraman, Das, Indrajit, Crane, Denis, McGuckin, Michael, Florin, Timothy, Eri, Rajaraman, and Das, Indrajit
Abstract: Endoplasmic reticulum (ER) stress occurs when proteins misfold during biosynthesis in the ER. ER stress in intestinal secretory cells has been implicated in the aetiology of inflammatory bowel diseases (IBD) and intestinal inflammation in mice. Intestinal secretory cells are susceptible to ER stress due to high rates of protein synthesis, and ER stress in these cells results in reduced production of cell surface and secreted proteins leading to thinner mucus with a lower anti-microbial content, allowing penetration by luminal microbes, leading to inflammation. Cells experiencing ER stress attempt to restore homeostasis via the unfolded protein response (UPR), which enables the cells to increase the protein folding capacity of the ER. The primary focus of this thesis is to examine whether the drugs that are efficacious in IBD treatment modulate goblet cell function and ER homeostasis, and whether drugs that modulate ER stress can suppress intestinal inflammation and restore intestinal homeostasis., Thesis (PhD Doctorate), Doctor of Philosophy (PhD), School of Biomolecular and Physical Sciences, Science, Environment, Engineering and Technology, Full Text
Published: 2012

123. Epidermolysis bullosa simplex due to KRT5 mutations : mutation-related differences in cellular fragility and the protective effects of trimethylamine N-oxide in cultured primary keratinocytes

Author: Chamcheu, Jean Christopher, Virtanen, Marie, Navsaria, Harshad, Bowden, Paul E., Vahlquist, Anders, Törmä, Hans, Chamcheu, Jean Christopher, Virtanen, Marie, Navsaria, Harshad, Bowden, Paul E., Vahlquist, Anders, and Törmä, Hans
Abstract: Summary Background Epidermolysis bullosa simplex (EBS) is a mechanobullous skin fragility disease characterized by cytolysis of basal keratinocytes and intraepidermal blistering often caused by mutations in keratin genes (KRT5 or KRT14). No remedies exist for these disorders presenting a need for development of novel therapies. Objectives To identify new genotype-phenotype relationships in vivo and in cultured primary EBS keratinocytes in vitro, and to study the cytoskeletal stabilizing effects of trimethylamine N-oxide (TMAO) in heat-stressed EBS cells. Methods Genomic DNA and cDNA samples from three Swedish patients with EBS were analysed for keratin mutations. Primary EBS keratinocyte cultures were established, heat stressed with and without added TMAO, followed by evaluation of cellular fragility. Results In addition to the previously reported KRT5 mutation (V186L) in one patient, two patients were found to have a novel I183M and recurrent E475G replacements in KRT5. Cultured EBS keratinocytes did not exhibit keratin aggregates or cell loss, except in the patient with the p.I183M mutation who showed 3% aggregates and 2% cell loss. Upon transient heat stress the number of aggregate-containing cells increased to 21%, 27% and 13%, respectively, in the p.I183M, p.E475G and p.V186L mutant cells. Interestingly, pretreatment with TMAO prior to heat stress, dose dependently reduced the number of aggregate-containing cells and cell loss. Conclusion These results revealed a genotype-phenotype correlation in EBS keratinocytes upon heat stress and suggest protein stabilization as a new therapeutic strategy.
Published: 2010
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124. A chemical chaperone-based drug candidate is effective in a mouse model of amyotrophic lateral sclerosis (ALS).

Author: Getter T, Zaks I, Barhum Y, Ben-Zur T, Böselt S, Gregoire S, Viskind O, Shani T, Gottlieb H, Green O, Shubely M, Senderowitz H, Israelson A, Kwon I, Petri S, Offen D, and Gruzman A
Subjects: Amides chemical synthesis, Amides chemistry, Animals, Cells, Cultured, Disease Models, Animal, Dose-Response Relationship, Drug, Humans, Mice, Mice, Transgenic, Molecular Structure, Amides therapeutic use, Amyotrophic Lateral Sclerosis drug therapy
Abstract: Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by the selective death of motor neurons and skeletal muscle atrophy. The majority of ALS cases are acquired spontaneously, with inherited disease accounting for only 10 % of all cases. Recent studies provide compelling evidence that aggregates of misfolded proteins underlie both types of ALS. Small molecules such as artificial chaperones can prevent or even reverse the aggregation of proteins associated with various human diseases. However, their very high active concentration (micromolar range) severely limits their utility as drugs. We synthesized several ester and amide derivatives of chemical chaperones. The lead compound 14, 3-((5-((4,6-dimethylpyridin-2-yl)methoxy)-5-oxopentanoyl)oxy)-N,N-dimethylpropan-1-amine oxide shows, in the micromolar concentration range, both neuronal and astrocyte protective effects in vitro; at daily doses of 10 mg kg(-1) 14 improved the neurological functions and delayed body weight loss in ALS mice. Members of this new chemical chaperone derivative class are strong candidates for the development of new drugs for ALS patients., (© 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)
Published: 2015
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125. Differential functional rescue of Lys(513) and Lys(516) processing mutants of MRP1 (ABCC1) by chemical chaperones reveals different domain-domain interactions of the transporter.

Author: Iram SH and Cole SP
Subjects: Biological Transport, GABA Agents pharmacology, Glycerol pharmacology, HEK293 Cells, Humans, Lysine genetics, Multidrug Resistance-Associated Proteins chemistry, Multidrug Resistance-Associated Proteins genetics, Mutation genetics, Substrate Specificity, Surface-Active Agents pharmacology, Cell Membrane metabolism, Lysine metabolism, Multidrug Resistance-Associated Proteins metabolism, Polyethylene Glycols pharmacology, Protein Conformation drug effects, Protein Folding, gamma-Aminobutyric Acid pharmacology
Abstract: Multidrug resistance protein 1 (MRP1) extrudes drugs as well as pharmacologically and physiologically important organic anions across the plasma membrane in an ATP-dependent manner. We previously showed that Ala substitutions of Lys(513) and Lys(516) in the cytoplasmic loop (CL5) connecting transmembrane helix 9 (TM9) to TM10 cause misfolding of MRP1, abrogating its expression at the plasma membrane in transfected human embryonic kidney (HEK) cells. Exposure of HEK cells to the chemical chaperones glycerol, DMSO, polyethylene glycol (PEG) and 4-aminobutyric acid (4-PBA) improved levels of K513A to wild-type MRP1 levels but transport activity was only fully restored by 4-PBA or DMSO treatments. Tryptic fragmentation patterns and conformation-dependent antibody immunoreactivity of the transport-deficient PEG- and glycerol-rescued K513A proteins indicated that the second nucleotide binding domain (NBD2) had adopted a more open conformation than in wild-type MRP1. This structural change was accompanied by differences in ATP binding and hydrolysis but no changes in substrate Km. In contrast to K513A, K516A levels in HEK cells were not significantly enhanced by chemical chaperones. In more permissive insect cells, however, K516A levels were comparable to wild-type MRP1. Nevertheless, organic anion transport by K516A in insect cell membranes was reduced by >80% due to reduced substrate Km. Tryptic fragmentation patterns indicated a more open conformation of the third membrane spanning domain of MRP1. Thus, despite their close proximity to one another in CL5, Lys(513) and Lys(516) participate in different interdomain interactions crucial for the proper folding and assembly of MRP1., (Copyright © 2013 Elsevier B.V. All rights reserved.)
Published: 2014
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126. The therapeutic potential of chemical chaperones in protein folding diseases.

Author: Cortez L and Sim V
Subjects: Clinical Trials as Topic, Humans, Molecular Chaperones chemistry, Neurodegenerative Diseases metabolism, Translational Research, Biomedical, Molecular Chaperones therapeutic use, Neurodegenerative Diseases drug therapy, Protein Folding
Abstract: Several neurodegenerative diseases are caused by defects in protein folding, including Alzheimer, Parkinson, Huntington, and prion diseases. Once a disease-specific protein misfolds, it can then form toxic aggregates which accumulate in the brain, leading to neuronal dysfunction, cell death, and clinical symptoms. Although significant advances have been made toward understanding the mechanisms of protein aggregation, there are no curative treatments for any of these diseases. Since protein misfolding and the accumulation of aggregates are the most upstream events in the pathological cascade, rescuing or stabilizing the native conformations of proteins is an obvious therapeutic strategy. In recent years, small molecules known as chaperones have been shown to be effective in reducing levels of misfolded proteins, thus minimizing the accumulation of aggregates and their downstream pathological consequences. Chaperones are classified as molecular, pharmacological, or chemical. In this mini-review we summarize the modes of action of different chemical chaperones and discuss evidence for their efficacy in the treatment of protein folding diseases in vitro and in vivo.
Published: 2014
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127. Mutations in intracellular loops 1 and 3 lead to misfolding of human P-glycoprotein (ABCB1) that can be rescued by cyclosporine A, which reduces its association with chaperone Hsp70.

Author: Kapoor K, Bhatnagar J, Chufan EE, and Ambudkar SV
Subjects: ATP Binding Cassette Transporter, Subfamily B, ATP Binding Cassette Transporter, Subfamily B, Member 1 genetics, Aspartic Acid genetics, Aspartic Acid metabolism, Biological Transport, Active drug effects, Biological Transport, Active genetics, Endoplasmic Reticulum genetics, Endoplasmic Reticulum metabolism, HSP70 Heat-Shock Proteins genetics, HeLa Cells, Humans, Immunosuppressive Agents pharmacology, Protein Structure, Secondary, Protein Structure, Tertiary, Tacrolimus pharmacology, ATP Binding Cassette Transporter, Subfamily B, Member 1 metabolism, Cyclosporine pharmacology, HSP70 Heat-Shock Proteins metabolism, Mutation, Protein Folding drug effects
Abstract: P-glycoprotein (P-gp) is an ATP binding cassette transporter that effluxes a variety of structurally diverse compounds including anticancer drugs. Computational models of human P-gp in the apo- and nucleotide-bound conformation show that the adenine group of ATP forms hydrogen bonds with the conserved Asp-164 and Asp-805 in intracellular loops 1 and 3, respectively, which are located at the interface between the nucleotide binding domains and transmembrane domains. We investigated the role of Asp-164 and Asp-805 residues by substituting them with cysteine in a cysteine-less background. It was observed that the D164C/D805C mutant, when expressed in HeLa cells, led to misprocessing of P-gp, which thus failed to transport the drug substrates. The misfolded protein could be rescued to the cell surface by growing the cells at a lower temperature (27 °C) or by treatment with substrates (cyclosporine A, FK506), modulators (tariquidar), or small corrector molecules. We also show that short term (4-6 h) treatment with 15 μM cyclosporine A or FK506 rescues the pre-formed immature protein trapped in the endoplasmic reticulum in an immunophilin-independent pathway. The intracellularly trapped misprocessed protein associates more with chaperone Hsp70, and the treatment with cyclosporine A reduces the association of mutant P-gp, thus allowing it to be trafficked to the cell surface. The function of rescued cell surface mutant P-gp is similar to that of wild-type protein. These data demonstrate that the Asp-164 and Asp-805 residues are not important for ATP binding, as proposed earlier, but are critical for proper folding and maturation of a functional transporter.
Published: 2013
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128. Dipeptides promote folding and peptide binding of MHC class I molecules.

Author: Saini SK, Ostermeir K, Ramnarayan VR, Schuster H, Zacharias M, and Springer S
Subjects: Dipeptides genetics, Endoplasmic Reticulum metabolism, Escherichia coli, Flow Cytometry, Fluorescence Polarization, Humans, Hydrophobic and Hydrophilic Interactions, Microscopy, Fluorescence, Molecular Dynamics Simulation, Protein Binding, Protein Folding, Amino Acids metabolism, Dipeptides metabolism, HLA-A2 Antigen metabolism, Protein Conformation
Abstract: MHC class I molecules bind only those peptides with high affinity that conform to stringent length and sequence requirements. We have now investigated which peptides can aid the in vitro folding of class I molecules, and we find that the dipeptide glycyl-leucine efficiently supports the folding of HLA-A*02:01 and H-2K(b) into a peptide-receptive conformation that rapidly binds high-affinity peptides. Treatment of cells with glycyl-leucine induces accumulation of peptide-receptive H-2K(b) and HLA-A*02:01 at the surface of cells. Other dipeptides with a hydrophobic second amino acid show similar enhancement effects. Our data suggest that the dipeptides bind into the F pocket like the C-terminal amino acids of a high-affinity peptide.
Published: 2013
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129. A microbial sensor for discovering structural probes of protein misfolding and aggregation.

Author: Waraho-Zhmayev D, Gkogka L, Yu TY, and DeLisa MP
Subjects: Amyloid metabolism, Antibodies metabolism, Biotechnology, Escherichia coli metabolism, Escherichia coli Proteins chemistry, Escherichia coli Proteins metabolism, High-Throughput Screening Assays, Intracellular Space chemistry, Intracellular Space metabolism, Membrane Transport Proteins chemistry, Membrane Transport Proteins metabolism, Models, Molecular, Molecular Probe Techniques, Protein Binding, Protein Folding, Amyloid analysis, Biosensing Techniques methods
Abstract: In all cell types, protein homeostasis, or "proteostasis," is maintained by sophisticated quality control networks that regulate protein synthesis, folding, trafficking, aggregation, disaggregation, and degradation. In one notable example, Escherichia coli employ a proteostasis system that determines whether substrates of the twin-arginine translocation (Tat) pathway are correctly folded and thus suitable for transport across the tightly sealed cytoplasmic membrane. Herein, we review growing evidence that the Tat translocase itself discriminates folded proteins from those that are misfolded and/or aggregated, preferentially exporting only the former. Genetic suppressors that inactivate this mechanism have recently been isolated and provide direct evidence for the participation of the Tat translocase in structural proofreading of its protein substrates. We also discuss how this discriminatory "folding sensor" has been exploited for the discovery of structural probes (e.g., sequence mutations, pharmacologic chaperones, intracellular antibodies) that modulate the folding and solubility of virtually any protein-of-interest, including those associated with aggregation diseases (e.g., α-synuclein, amyloid-β protein). Taken together, these studies highlight the utility of engineered bacteria for rapidly and inexpensively uncovering potent anti-aggregation factors.
Published: 2013
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130. Additive Effects of Alcohols and Polyols on Thermostability of Pepper Leaf Extracts.

Author: Anderson, Jeffrey A.
Subjects: *CULTIVARS, *POLYOLS, *MOLECULAR chaperones, *PLANT hormones, *PLANT physiology, *PLANT proteins
Abstract: Chemical chaperones (CC) are plant stress-related compounds that can stabilize protein structure in adverse environments. Modes of action are thought to involve hydrogen bonding, primarily with the solvent, and hydrophobic stabilization of the protein core. The objective of this study was to determine structure—function relationships between CC (and structurally related compounds) and thermal stability of pepper (Capsicum annuum L.) leaf proteins. Both polarity [based on log Kow (the oil—water partition coefficient)] and capacity for hydrogen bonding (based on the number of OH groups) contributed to whether low-molecular-weight alcohols and polyols stabilized or destabilized proteins at elevated temperatures. Thermal stability increased with increasing number of OH groups at a fixed number of carbon atoms per molecule. Conversely, thermal stability decreased with increasing number of carbon atoms with a fixed number of OH groups. When CC solution concentrations were adjusted to the same concentration of OH groups (1.51 × 1022 OH groups per milliliter), protein thermal stability increased with increasing CC polarity. Mixtures of different CC had additive effects on increasing protein thermostability, but mixtures of stabilizing (mannitol) and destabilizing (methanol) compounds negated each other. As a strategy for increasing plant thermotolerance, identification and removal of destabilizing compounds should be equally effective as increasing levels of stabilizers in protecting protein conformation at elevated temperatures. [ABSTRACT FROM AUTHOR]
Published: 2007
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131. Synthesis of lipophilic 1-deoxygalactonojirimycin derivatives as D-galactosidase inhibitors.

Author: Schitter G, Scheucher E, Steiner AJ, Stütz AE, Thonhofer M, Tarling CA, Withers SG, Wicki J, Fantur K, Paschke E, Mahuran DJ, Rigat BA, Tropak M, and Wrodnigg TM
Abstract: N-Alkylation at the ring nitrogen of the D-galactosidase inhibitor 1-deoxygalactonojirimycin with a functionalised C ₆alkyl chain followed by modification with different aromatic substituents provided lipophilic 1-deoxygalactonojirimycin derivatives which exhibit inhibitory properties against β-glycosidases from E. coli and Agrobacterium sp. as well as green coffee bean α-galactosidase. In preliminary studies, these compounds also showed potential as chemical chaperones for GM1-gangliosidosis related β-galactosidase mutants.
Published: 2010
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132. Differential functional rescue of Lys513 and Lys516 processing mutants of MRP1 (ABCC1) by chemical chaperones reveals different domain–domain interactions of the transporter

Author: Surtaj H. Iram and Susan P.C. Cole
Subjects: Glycerol, Protein Folding, GABA Agents, Protein Conformation, Biophysics, ATP-binding cassette transporter, Biology, Biochemistry, Polyethylene Glycols, Substrate Specificity, Surface-Active Agents, Multidrug resistance protein 1, Humans, gamma-Aminobutyric Acid, ATP-binding cassette, Lysine, Cell Membrane, HEK 293 cells, Biological Transport, Cell Biology, Transmembrane protein, Transmembrane domain, HEK293 Cells, Chemical chaperones, Cyclic nucleotide-binding domain, Mutation, Organic anion transport, Protein folding, Plasma membrane trafficking, Multidrug Resistance-Associated Proteins, Chemical chaperone, Organic anion transporter, Protein misfolding
Abstract: Multidrug resistance protein 1 (MRP1) extrudes drugs as well as pharmacologically and physiologically important organic anions across the plasma membrane in an ATP-dependent manner. We previously showed that Ala substitutions of Lys(513) and Lys(516) in the cytoplasmic loop (CL5) connecting transmembrane helix 9 (TM9) to TM10 cause misfolding of MRP1, abrogating its expression at the plasma membrane in transfected human embryonic kidney (HEK) cells. Exposure of HEK cells to the chemical chaperones glycerol, DMSO, polyethylene glycol (PEG) and 4-aminobutyric acid (4-PBA) improved levels of K513A to wild-type MRP1 levels but transport activity was only fully restored by 4-PBA or DMSO treatments. Tryptic fragmentation patterns and conformation-dependent antibody immunoreactivity of the transport-deficient PEG- and glycerol-rescued K513A proteins indicated that the second nucleotide binding domain (NBD2) had adopted a more open conformation than in wild-type MRP1. This structural change was accompanied by differences in ATP binding and hydrolysis but no changes in substrate Km. In contrast to K513A, K516A levels in HEK cells were not significantly enhanced by chemical chaperones. In more permissive insect cells, however, K516A levels were comparable to wild-type MRP1. Nevertheless, organic anion transport by K516A in insect cell membranes was reduced by >80% due to reduced substrate Km. Tryptic fragmentation patterns indicated a more open conformation of the third membrane spanning domain of MRP1. Thus, despite their close proximity to one another in CL5, Lys(513) and Lys(516) participate in different interdomain interactions crucial for the proper folding and assembly of MRP1.
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133. Využití mikrokalorimetrických metod při studiu protektivních účinků chemických chaperonů

Author: Krouská, Jitka, Pekař, Miloslav, Krouská, Jitka, and Pekař, Miloslav
Abstract: Tato bakalářská práce se zabývá studiem ochranných látek před denaturačními procesy, označovanými jako chemické chaperony. V teoretické části byla popsána obecná charakteristika proteinů, popis vybraných chaperonů a metody diferenční kompenzační kalorimetrie. V experimentální části byly zkoumány protektivní účinky čtyř potenciálních protektivních látek – trehalóza, guanidine hydrochloride, 3-hydroxybutyrát a hydroxyectoine – na modelovém proteinu lysozymu. Protektivní účinky jednotlivých látek byly zkoumány pomocí diferenciální kompenzační kalorimetrie (DSC), kterou byla stanovena denaturační teplota lysozymu v přítomnosti ochranných látek. Ze všech zkoumaných chemických chaperonů byl nejvyšší protektivní efekt pozorován u 3-hydroxybutyrátu, který teplotu denaturace posunoval k vyšším hodnotám, a guanidine hydrochloride, který naopak denaturační teplotu snižoval. Zároveň byl u některých látek zjištěn reverzibilní denaturační proces, který byl u GuHCl nejintenzivnější., This bachelor thesis deals with the study of protective substances against denaturation processes, called chemical chaperones. The theoretical part describes the general characteristics of proteins, description of selected chaperones and methods of differential scanning calorimetry. In the experimental part, the protective effects of four potential protective agents - trehalose, guanidine hydrochloride, 3-hydroxybutyrate and hydroxyectoine - were investigated on the lysozyme model protein. The protective effects of the individual substances were examined by differential scanning calorimetry (DSC), which determined the denaturation temperature of lysozyme in the presence of preservatives. Of all the chemical chaperones examined, the highest protective effect was observed with 3-hydroxybutyrate, which shifted the denaturation temperature to higher levels, and guanidine hydrochloride, which on the other hand lowered the denaturation temperature. At the same time, a reversible denaturation process was found in some substances, which was the most intense in GuHCl.

134. Fyzikálně-chemická charakterizace 3-hydroxybutyrátu ve vztahu k jeho bioprotektivním účinkům

Author: Sedláček, Petr, Smilek, Jiří, Sedláček, Petr, and Smilek, Jiří
Abstract: Hlavným cieľom práce bolo overiť bioprotektívne vlastnosti 3-hydroxybutyrátu (3HB) z hľadiska jeho vplyvu na denaturáciu proteínu (konkrétne lipázy) a stanoviť relevantné fyzikálno-chemické charakteristiky 3HB, ktoré by mohli viesť k vysvetleniu mechanizmu tohto ochranného efektu. Rešeršná časť práce sa zamerala predovšetkým na mechanizmus ochranného účinku tzv. kompatibilných solutov, medzi ktoré patrí 3HB. Ochranný účinok 3HB bol stanovený metódou dynamického rozptylu svetla (DLS). Ako ďalšie parametre vodného roztoku 3HB boli stanovené: koncentračná závislosť povrchového napätia 3HB, vplyv rôznych koncentrácií 3HB na zeta potenciál lipázy. Hydrofílny/hydrofóbny charakter 3HB bol charakterizovaný parametrom log Kow. Pre kvantitatívne stanovenie 3HB vo vodnom roztoku bola optimalizovaná jednoduchá metóda. Touto metódou je nepriama spektroskopia. Je založená na konverzii 3HB na krotonovú kyselinu v prítomnosti kyseliny sírovej za zvýšenej teploty., The main aim of the work was to verify bio-protective properties of 3-hydroxybutyrate (3HB) in terms of its effect on protein denaturation (especially lipase) and determine the physicochemical characteristics of 3HB that could lead to the explanation of the mechanism of its protective effect. The literature search focused primarily on the mechanism of the protective effect of the compatible solutes. The 3HB belongs to the group of compatible solutes. The protective effect of the 3HB was monitored by Dynamic Light Scattering (DLS). As the other parameters of the aqueous solution 3HB were determined: the dependence of the surface tension on the concentration of the 3HB, the effect of different 3HB concentrations on the zeta potential of the lipase. Hydrophobic and hydrophilic character of the 3HB was characterized with the log Kow parameter. A simple method for quantity determination of the 3HB aqueous solution was optimized. The method is based on conversion of the 3HB into crotonic acid by reaction with sulphuric acid at higher temperatures and on subsequent psectrophotometric determination of the amount of crotonic acid.

135. Studium protektivního účinku 3-hydroxybutyrátu a jeho derivátů

Author: Obruča, Stanislav, Smilek, Jiří, Obruča, Stanislav, and Smilek, Jiří
Abstract: Cílem této bakalářské práce je studium chemických chaperonů, jakožto ochranných látek enzymů před denaturačními procesy. V experimentální části práce byly zkoumány protektivní účinky pěti vybraných potenciálních protektivních látek – derivátů 3-hydroxybutyrátu – na dvou modelových enzymech: lipáze a lysozymu. Protektivní účinky potenciálních chemických chaperonů byly zkoumány metodou dynamického rozptylu světla (DLS), kterou byla stanovována distribuce velikosti vznikajících agregátů lipázy v závislosti na teplotě při tepelně indukované denaturaci. Dále byla použita diferenciální kompenzační kalorimetrie (DSC), kterou byla stanovena teplota denaturace lysozymu v přítomnosti nebo nepřítomnosti potenciálně protektivních látek. Posledním parametrem bylo stanovení vlivu potenciálně protektivních látek při různých koncentracích na katalytickou aktivitu lipázy a stanovení residuální aktivity lipázy po aplikaci dvou denaturačních faktorů – zvýšené teploty a mražení. Ze všech testovaných strukturních analogů 3-hydroxybutyrátu byl nejvyšší protektivní efekt (stabilizace molekuly enzymu) pozorován u jantaranu, který disponuje dvěma karboxylovými skupinami. Naopak 1,3-butandiol nevykázal prakticky žádnou protektivní účinnost, což dokazuje, že přítomnost karboxylové skupiny je pro protektivní účinek zcela zásadní. Nízká protektivní účinnost butyrátu dále naznačuje, že je nezbytné, aby efektivní protektant kromě karboxylové skupiny obsahoval i další funkční skupinu – ať už karboxylovou nebo hydroxylovou. Výraznější protektivní účinek byl pozorován u 3-hydroxybutyrátu, než-li u 2-hydroxybutyrátu. Je zajímavé, že efektivní protektanty zároveň při vyšších koncentracích výrazně inhibují enzymovou aktivitu modelového enzymu, což pravděpodobně souvisí se solvatací a konformací proteinu v přítomnosti protektantů a dostupností aktivního místa pro substrát., The aim of this thesis is the study of chemical chaperones, as a preservative enzymes before denaturing processes. In the experimental part of the study we investigated the protective effects of five selected potential protective compounds – derivatives of 3-hydroxybutyrate – two model enzymes: lipase and lysozyme. Protective effects of potential chemical chaperones were investigated by dynamic light scattering (DLS), which was determined by the size distribution of the aggregates lipase produced depending on the temperature at the heat-induced denaturation. Further we have been used differential scanning calorimetry (DSC), which was determined by the temperature of the denaturation of lysozyme in the presence or absence of potentially protective substances. The last parameter was to determine the effect of potentially protective agents at different concentrations on the catalytic activity of lipase and determining the residual lipase activity after application of denaturing two factors – the high temperature and freezing. Of all tested structural analogues of 3-hydroxybutyrate was the highest protective effect observed (stabilization of enzyme molecules) with the succinate, which has two carboxyl groups. Conversely, 1,3-butanediol showed virtually no protective activity, indicating that the presence of carboxyl groups on the protective effect of essential. The low protective efficacy butyrate further indicates that it is essential that an effective protectant in addition to the carboxyl group contained as a further functional group – either carboxyl or hydroxyl. More significant protective effect was observed in 3-hydroxybutyrate than in 2-hydroxybutyrate. Interestingly, the effective protectant while at higher concentrations strongly inhibit the enzyme activity of the model enzyme, which is probably related to the solvation and conformations of the protein in the presence of protectant and the availability of the active site for the substrate.

136. Fyzikálně-chemická charakterizace 3-hydroxybutyrátu ve vztahu k jeho bioprotektivním účinkům

Author: Sedláček, Petr, Smilek, Jiří, Sedláček, Petr, and Smilek, Jiří
Abstract: Hlavným cieľom práce bolo overiť bioprotektívne vlastnosti 3-hydroxybutyrátu (3HB) z hľadiska jeho vplyvu na denaturáciu proteínu (konkrétne lipázy) a stanoviť relevantné fyzikálno-chemické charakteristiky 3HB, ktoré by mohli viesť k vysvetleniu mechanizmu tohto ochranného efektu. Rešeršná časť práce sa zamerala predovšetkým na mechanizmus ochranného účinku tzv. kompatibilných solutov, medzi ktoré patrí 3HB. Ochranný účinok 3HB bol stanovený metódou dynamického rozptylu svetla (DLS). Ako ďalšie parametre vodného roztoku 3HB boli stanovené: koncentračná závislosť povrchového napätia 3HB, vplyv rôznych koncentrácií 3HB na zeta potenciál lipázy. Hydrofílny/hydrofóbny charakter 3HB bol charakterizovaný parametrom log Kow. Pre kvantitatívne stanovenie 3HB vo vodnom roztoku bola optimalizovaná jednoduchá metóda. Touto metódou je nepriama spektroskopia. Je založená na konverzii 3HB na krotonovú kyselinu v prítomnosti kyseliny sírovej za zvýšenej teploty., The main aim of the work was to verify bio-protective properties of 3-hydroxybutyrate (3HB) in terms of its effect on protein denaturation (especially lipase) and determine the physicochemical characteristics of 3HB that could lead to the explanation of the mechanism of its protective effect. The literature search focused primarily on the mechanism of the protective effect of the compatible solutes. The 3HB belongs to the group of compatible solutes. The protective effect of the 3HB was monitored by Dynamic Light Scattering (DLS). As the other parameters of the aqueous solution 3HB were determined: the dependence of the surface tension on the concentration of the 3HB, the effect of different 3HB concentrations on the zeta potential of the lipase. Hydrophobic and hydrophilic character of the 3HB was characterized with the log Kow parameter. A simple method for quantity determination of the 3HB aqueous solution was optimized. The method is based on conversion of the 3HB into crotonic acid by reaction with sulphuric acid at higher temperatures and on subsequent psectrophotometric determination of the amount of crotonic acid.

137. Využití mikrokalorimetrických metod při studiu protektivních účinků chemických chaperonů

Author: Krouská, Jitka, Pekař, Miloslav, Krouská, Jitka, and Pekař, Miloslav
Abstract: Tato bakalářská práce se zabývá studiem ochranných látek před denaturačními procesy, označovanými jako chemické chaperony. V teoretické části byla popsána obecná charakteristika proteinů, popis vybraných chaperonů a metody diferenční kompenzační kalorimetrie. V experimentální části byly zkoumány protektivní účinky čtyř potenciálních protektivních látek – trehalóza, guanidine hydrochloride, 3-hydroxybutyrát a hydroxyectoine – na modelovém proteinu lysozymu. Protektivní účinky jednotlivých látek byly zkoumány pomocí diferenciální kompenzační kalorimetrie (DSC), kterou byla stanovena denaturační teplota lysozymu v přítomnosti ochranných látek. Ze všech zkoumaných chemických chaperonů byl nejvyšší protektivní efekt pozorován u 3-hydroxybutyrátu, který teplotu denaturace posunoval k vyšším hodnotám, a guanidine hydrochloride, který naopak denaturační teplotu snižoval. Zároveň byl u některých látek zjištěn reverzibilní denaturační proces, který byl u GuHCl nejintenzivnější., This bachelor thesis deals with the study of protective substances against denaturation processes, called chemical chaperones. The theoretical part describes the general characteristics of proteins, description of selected chaperones and methods of differential scanning calorimetry. In the experimental part, the protective effects of four potential protective agents - trehalose, guanidine hydrochloride, 3-hydroxybutyrate and hydroxyectoine - were investigated on the lysozyme model protein. The protective effects of the individual substances were examined by differential scanning calorimetry (DSC), which determined the denaturation temperature of lysozyme in the presence of preservatives. Of all the chemical chaperones examined, the highest protective effect was observed with 3-hydroxybutyrate, which shifted the denaturation temperature to higher levels, and guanidine hydrochloride, which on the other hand lowered the denaturation temperature. At the same time, a reversible denaturation process was found in some substances, which was the most intense in GuHCl.

138. Studium protektivního účinku 3-hydroxybutyrátu a jeho derivátů

Author: Obruča, Stanislav, Smilek, Jiří, Obruča, Stanislav, and Smilek, Jiří
Abstract: Cílem této bakalářské práce je studium chemických chaperonů, jakožto ochranných látek enzymů před denaturačními procesy. V experimentální části práce byly zkoumány protektivní účinky pěti vybraných potenciálních protektivních látek – derivátů 3-hydroxybutyrátu – na dvou modelových enzymech: lipáze a lysozymu. Protektivní účinky potenciálních chemických chaperonů byly zkoumány metodou dynamického rozptylu světla (DLS), kterou byla stanovována distribuce velikosti vznikajících agregátů lipázy v závislosti na teplotě při tepelně indukované denaturaci. Dále byla použita diferenciální kompenzační kalorimetrie (DSC), kterou byla stanovena teplota denaturace lysozymu v přítomnosti nebo nepřítomnosti potenciálně protektivních látek. Posledním parametrem bylo stanovení vlivu potenciálně protektivních látek při různých koncentracích na katalytickou aktivitu lipázy a stanovení residuální aktivity lipázy po aplikaci dvou denaturačních faktorů – zvýšené teploty a mražení. Ze všech testovaných strukturních analogů 3-hydroxybutyrátu byl nejvyšší protektivní efekt (stabilizace molekuly enzymu) pozorován u jantaranu, který disponuje dvěma karboxylovými skupinami. Naopak 1,3-butandiol nevykázal prakticky žádnou protektivní účinnost, což dokazuje, že přítomnost karboxylové skupiny je pro protektivní účinek zcela zásadní. Nízká protektivní účinnost butyrátu dále naznačuje, že je nezbytné, aby efektivní protektant kromě karboxylové skupiny obsahoval i další funkční skupinu – ať už karboxylovou nebo hydroxylovou. Výraznější protektivní účinek byl pozorován u 3-hydroxybutyrátu, než-li u 2-hydroxybutyrátu. Je zajímavé, že efektivní protektanty zároveň při vyšších koncentracích výrazně inhibují enzymovou aktivitu modelového enzymu, což pravděpodobně souvisí se solvatací a konformací proteinu v přítomnosti protektantů a dostupností aktivního místa pro substrát., The aim of this thesis is the study of chemical chaperones, as a preservative enzymes before denaturing processes. In the experimental part of the study we investigated the protective effects of five selected potential protective compounds – derivatives of 3-hydroxybutyrate – two model enzymes: lipase and lysozyme. Protective effects of potential chemical chaperones were investigated by dynamic light scattering (DLS), which was determined by the size distribution of the aggregates lipase produced depending on the temperature at the heat-induced denaturation. Further we have been used differential scanning calorimetry (DSC), which was determined by the temperature of the denaturation of lysozyme in the presence or absence of potentially protective substances. The last parameter was to determine the effect of potentially protective agents at different concentrations on the catalytic activity of lipase and determining the residual lipase activity after application of denaturing two factors – the high temperature and freezing. Of all tested structural analogues of 3-hydroxybutyrate was the highest protective effect observed (stabilization of enzyme molecules) with the succinate, which has two carboxyl groups. Conversely, 1,3-butanediol showed virtually no protective activity, indicating that the presence of carboxyl groups on the protective effect of essential. The low protective efficacy butyrate further indicates that it is essential that an effective protectant in addition to the carboxyl group contained as a further functional group – either carboxyl or hydroxyl. More significant protective effect was observed in 3-hydroxybutyrate than in 2-hydroxybutyrate. Interestingly, the effective protectant while at higher concentrations strongly inhibit the enzyme activity of the model enzyme, which is probably related to the solvation and conformations of the protein in the presence of protectant and the availability of the active site for the substrate.

139. Využití mikrokalorimetrických metod při studiu protektivních účinků chemických chaperonů

Author: Krouská, Jitka, Pekař, Miloslav, Krouská, Jitka, and Pekař, Miloslav
Abstract: Tato bakalářská práce se zabývá studiem ochranných látek před denaturačními procesy, označovanými jako chemické chaperony. V teoretické části byla popsána obecná charakteristika proteinů, popis vybraných chaperonů a metody diferenční kompenzační kalorimetrie. V experimentální části byly zkoumány protektivní účinky čtyř potenciálních protektivních látek – trehalóza, guanidine hydrochloride, 3-hydroxybutyrát a hydroxyectoine – na modelovém proteinu lysozymu. Protektivní účinky jednotlivých látek byly zkoumány pomocí diferenciální kompenzační kalorimetrie (DSC), kterou byla stanovena denaturační teplota lysozymu v přítomnosti ochranných látek. Ze všech zkoumaných chemických chaperonů byl nejvyšší protektivní efekt pozorován u 3-hydroxybutyrátu, který teplotu denaturace posunoval k vyšším hodnotám, a guanidine hydrochloride, který naopak denaturační teplotu snižoval. Zároveň byl u některých látek zjištěn reverzibilní denaturační proces, který byl u GuHCl nejintenzivnější., This bachelor thesis deals with the study of protective substances against denaturation processes, called chemical chaperones. The theoretical part describes the general characteristics of proteins, description of selected chaperones and methods of differential scanning calorimetry. In the experimental part, the protective effects of four potential protective agents - trehalose, guanidine hydrochloride, 3-hydroxybutyrate and hydroxyectoine - were investigated on the lysozyme model protein. The protective effects of the individual substances were examined by differential scanning calorimetry (DSC), which determined the denaturation temperature of lysozyme in the presence of preservatives. Of all the chemical chaperones examined, the highest protective effect was observed with 3-hydroxybutyrate, which shifted the denaturation temperature to higher levels, and guanidine hydrochloride, which on the other hand lowered the denaturation temperature. At the same time, a reversible denaturation process was found in some substances, which was the most intense in GuHCl.

140. Studium protektivního účinku 3-hydroxybutyrátu a jeho derivátů

Author: Obruča, Stanislav, Smilek, Jiří, Obruča, Stanislav, and Smilek, Jiří
Abstract: Cílem této bakalářské práce je studium chemických chaperonů, jakožto ochranných látek enzymů před denaturačními procesy. V experimentální části práce byly zkoumány protektivní účinky pěti vybraných potenciálních protektivních látek – derivátů 3-hydroxybutyrátu – na dvou modelových enzymech: lipáze a lysozymu. Protektivní účinky potenciálních chemických chaperonů byly zkoumány metodou dynamického rozptylu světla (DLS), kterou byla stanovována distribuce velikosti vznikajících agregátů lipázy v závislosti na teplotě při tepelně indukované denaturaci. Dále byla použita diferenciální kompenzační kalorimetrie (DSC), kterou byla stanovena teplota denaturace lysozymu v přítomnosti nebo nepřítomnosti potenciálně protektivních látek. Posledním parametrem bylo stanovení vlivu potenciálně protektivních látek při různých koncentracích na katalytickou aktivitu lipázy a stanovení residuální aktivity lipázy po aplikaci dvou denaturačních faktorů – zvýšené teploty a mražení. Ze všech testovaných strukturních analogů 3-hydroxybutyrátu byl nejvyšší protektivní efekt (stabilizace molekuly enzymu) pozorován u jantaranu, který disponuje dvěma karboxylovými skupinami. Naopak 1,3-butandiol nevykázal prakticky žádnou protektivní účinnost, což dokazuje, že přítomnost karboxylové skupiny je pro protektivní účinek zcela zásadní. Nízká protektivní účinnost butyrátu dále naznačuje, že je nezbytné, aby efektivní protektant kromě karboxylové skupiny obsahoval i další funkční skupinu – ať už karboxylovou nebo hydroxylovou. Výraznější protektivní účinek byl pozorován u 3-hydroxybutyrátu, než-li u 2-hydroxybutyrátu. Je zajímavé, že efektivní protektanty zároveň při vyšších koncentracích výrazně inhibují enzymovou aktivitu modelového enzymu, což pravděpodobně souvisí se solvatací a konformací proteinu v přítomnosti protektantů a dostupností aktivního místa pro substrát., The aim of this thesis is the study of chemical chaperones, as a preservative enzymes before denaturing processes. In the experimental part of the study we investigated the protective effects of five selected potential protective compounds – derivatives of 3-hydroxybutyrate – two model enzymes: lipase and lysozyme. Protective effects of potential chemical chaperones were investigated by dynamic light scattering (DLS), which was determined by the size distribution of the aggregates lipase produced depending on the temperature at the heat-induced denaturation. Further we have been used differential scanning calorimetry (DSC), which was determined by the temperature of the denaturation of lysozyme in the presence or absence of potentially protective substances. The last parameter was to determine the effect of potentially protective agents at different concentrations on the catalytic activity of lipase and determining the residual lipase activity after application of denaturing two factors – the high temperature and freezing. Of all tested structural analogues of 3-hydroxybutyrate was the highest protective effect observed (stabilization of enzyme molecules) with the succinate, which has two carboxyl groups. Conversely, 1,3-butanediol showed virtually no protective activity, indicating that the presence of carboxyl groups on the protective effect of essential. The low protective efficacy butyrate further indicates that it is essential that an effective protectant in addition to the carboxyl group contained as a further functional group – either carboxyl or hydroxyl. More significant protective effect was observed in 3-hydroxybutyrate than in 2-hydroxybutyrate. Interestingly, the effective protectant while at higher concentrations strongly inhibit the enzyme activity of the model enzyme, which is probably related to the solvation and conformations of the protein in the presence of protectant and the availability of the active site for the substrate.

141. Studium protektivního účinku 3-hydroxybutyrátu a jeho derivátů

Author: Obruča, Stanislav, Smilek, Jiří, Obruča, Stanislav, and Smilek, Jiří
Abstract: Cílem této bakalářské práce je studium chemických chaperonů, jakožto ochranných látek enzymů před denaturačními procesy. V experimentální části práce byly zkoumány protektivní účinky pěti vybraných potenciálních protektivních látek – derivátů 3-hydroxybutyrátu – na dvou modelových enzymech: lipáze a lysozymu. Protektivní účinky potenciálních chemických chaperonů byly zkoumány metodou dynamického rozptylu světla (DLS), kterou byla stanovována distribuce velikosti vznikajících agregátů lipázy v závislosti na teplotě při tepelně indukované denaturaci. Dále byla použita diferenciální kompenzační kalorimetrie (DSC), kterou byla stanovena teplota denaturace lysozymu v přítomnosti nebo nepřítomnosti potenciálně protektivních látek. Posledním parametrem bylo stanovení vlivu potenciálně protektivních látek při různých koncentracích na katalytickou aktivitu lipázy a stanovení residuální aktivity lipázy po aplikaci dvou denaturačních faktorů – zvýšené teploty a mražení. Ze všech testovaných strukturních analogů 3-hydroxybutyrátu byl nejvyšší protektivní efekt (stabilizace molekuly enzymu) pozorován u jantaranu, který disponuje dvěma karboxylovými skupinami. Naopak 1,3-butandiol nevykázal prakticky žádnou protektivní účinnost, což dokazuje, že přítomnost karboxylové skupiny je pro protektivní účinek zcela zásadní. Nízká protektivní účinnost butyrátu dále naznačuje, že je nezbytné, aby efektivní protektant kromě karboxylové skupiny obsahoval i další funkční skupinu – ať už karboxylovou nebo hydroxylovou. Výraznější protektivní účinek byl pozorován u 3-hydroxybutyrátu, než-li u 2-hydroxybutyrátu. Je zajímavé, že efektivní protektanty zároveň při vyšších koncentracích výrazně inhibují enzymovou aktivitu modelového enzymu, což pravděpodobně souvisí se solvatací a konformací proteinu v přítomnosti protektantů a dostupností aktivního místa pro substrát., The aim of this thesis is the study of chemical chaperones, as a preservative enzymes before denaturing processes. In the experimental part of the study we investigated the protective effects of five selected potential protective compounds – derivatives of 3-hydroxybutyrate – two model enzymes: lipase and lysozyme. Protective effects of potential chemical chaperones were investigated by dynamic light scattering (DLS), which was determined by the size distribution of the aggregates lipase produced depending on the temperature at the heat-induced denaturation. Further we have been used differential scanning calorimetry (DSC), which was determined by the temperature of the denaturation of lysozyme in the presence or absence of potentially protective substances. The last parameter was to determine the effect of potentially protective agents at different concentrations on the catalytic activity of lipase and determining the residual lipase activity after application of denaturing two factors – the high temperature and freezing. Of all tested structural analogues of 3-hydroxybutyrate was the highest protective effect observed (stabilization of enzyme molecules) with the succinate, which has two carboxyl groups. Conversely, 1,3-butanediol showed virtually no protective activity, indicating that the presence of carboxyl groups on the protective effect of essential. The low protective efficacy butyrate further indicates that it is essential that an effective protectant in addition to the carboxyl group contained as a further functional group – either carboxyl or hydroxyl. More significant protective effect was observed in 3-hydroxybutyrate than in 2-hydroxybutyrate. Interestingly, the effective protectant while at higher concentrations strongly inhibit the enzyme activity of the model enzyme, which is probably related to the solvation and conformations of the protein in the presence of protectant and the availability of the active site for the substrate.

142. Fyzikálně-chemická charakterizace 3-hydroxybutyrátu ve vztahu k jeho bioprotektivním účinkům

Author: Sedláček, Petr, Smilek, Jiří, Sedláček, Petr, and Smilek, Jiří
Abstract: Hlavným cieľom práce bolo overiť bioprotektívne vlastnosti 3-hydroxybutyrátu (3HB) z hľadiska jeho vplyvu na denaturáciu proteínu (konkrétne lipázy) a stanoviť relevantné fyzikálno-chemické charakteristiky 3HB, ktoré by mohli viesť k vysvetleniu mechanizmu tohto ochranného efektu. Rešeršná časť práce sa zamerala predovšetkým na mechanizmus ochranného účinku tzv. kompatibilných solutov, medzi ktoré patrí 3HB. Ochranný účinok 3HB bol stanovený metódou dynamického rozptylu svetla (DLS). Ako ďalšie parametre vodného roztoku 3HB boli stanovené: koncentračná závislosť povrchového napätia 3HB, vplyv rôznych koncentrácií 3HB na zeta potenciál lipázy. Hydrofílny/hydrofóbny charakter 3HB bol charakterizovaný parametrom log Kow. Pre kvantitatívne stanovenie 3HB vo vodnom roztoku bola optimalizovaná jednoduchá metóda. Touto metódou je nepriama spektroskopia. Je založená na konverzii 3HB na krotonovú kyselinu v prítomnosti kyseliny sírovej za zvýšenej teploty., The main aim of the work was to verify bio-protective properties of 3-hydroxybutyrate (3HB) in terms of its effect on protein denaturation (especially lipase) and determine the physicochemical characteristics of 3HB that could lead to the explanation of the mechanism of its protective effect. The literature search focused primarily on the mechanism of the protective effect of the compatible solutes. The 3HB belongs to the group of compatible solutes. The protective effect of the 3HB was monitored by Dynamic Light Scattering (DLS). As the other parameters of the aqueous solution 3HB were determined: the dependence of the surface tension on the concentration of the 3HB, the effect of different 3HB concentrations on the zeta potential of the lipase. Hydrophobic and hydrophilic character of the 3HB was characterized with the log Kow parameter. A simple method for quantity determination of the 3HB aqueous solution was optimized. The method is based on conversion of the 3HB into crotonic acid by reaction with sulphuric acid at higher temperatures and on subsequent psectrophotometric determination of the amount of crotonic acid.

143. Fyzikálně-chemická charakterizace 3-hydroxybutyrátu ve vztahu k jeho bioprotektivním účinkům

Author: Sedláček, Petr, Smilek, Jiří, Sedláček, Petr, and Smilek, Jiří
Abstract: Hlavným cieľom práce bolo overiť bioprotektívne vlastnosti 3-hydroxybutyrátu (3HB) z hľadiska jeho vplyvu na denaturáciu proteínu (konkrétne lipázy) a stanoviť relevantné fyzikálno-chemické charakteristiky 3HB, ktoré by mohli viesť k vysvetleniu mechanizmu tohto ochranného efektu. Rešeršná časť práce sa zamerala predovšetkým na mechanizmus ochranného účinku tzv. kompatibilných solutov, medzi ktoré patrí 3HB. Ochranný účinok 3HB bol stanovený metódou dynamického rozptylu svetla (DLS). Ako ďalšie parametre vodného roztoku 3HB boli stanovené: koncentračná závislosť povrchového napätia 3HB, vplyv rôznych koncentrácií 3HB na zeta potenciál lipázy. Hydrofílny/hydrofóbny charakter 3HB bol charakterizovaný parametrom log Kow. Pre kvantitatívne stanovenie 3HB vo vodnom roztoku bola optimalizovaná jednoduchá metóda. Touto metódou je nepriama spektroskopia. Je založená na konverzii 3HB na krotonovú kyselinu v prítomnosti kyseliny sírovej za zvýšenej teploty., The main aim of the work was to verify bio-protective properties of 3-hydroxybutyrate (3HB) in terms of its effect on protein denaturation (especially lipase) and determine the physicochemical characteristics of 3HB that could lead to the explanation of the mechanism of its protective effect. The literature search focused primarily on the mechanism of the protective effect of the compatible solutes. The 3HB belongs to the group of compatible solutes. The protective effect of the 3HB was monitored by Dynamic Light Scattering (DLS). As the other parameters of the aqueous solution 3HB were determined: the dependence of the surface tension on the concentration of the 3HB, the effect of different 3HB concentrations on the zeta potential of the lipase. Hydrophobic and hydrophilic character of the 3HB was characterized with the log Kow parameter. A simple method for quantity determination of the 3HB aqueous solution was optimized. The method is based on conversion of the 3HB into crotonic acid by reaction with sulphuric acid at higher temperatures and on subsequent psectrophotometric determination of the amount of crotonic acid.

144. Studium protektivního účinku 3-hydroxybutyrátu a jeho derivátů

Author: Obruča, Stanislav, Smilek, Jiří, Černá, Klára, Obruča, Stanislav, Smilek, Jiří, and Černá, Klára
Abstract: Cílem této bakalářské práce je studium chemických chaperonů, jakožto ochranných látek enzymů před denaturačními procesy. V experimentální části práce byly zkoumány protektivní účinky pěti vybraných potenciálních protektivních látek – derivátů 3-hydroxybutyrátu – na dvou modelových enzymech: lipáze a lysozymu. Protektivní účinky potenciálních chemických chaperonů byly zkoumány metodou dynamického rozptylu světla (DLS), kterou byla stanovována distribuce velikosti vznikajících agregátů lipázy v závislosti na teplotě při tepelně indukované denaturaci. Dále byla použita diferenciální kompenzační kalorimetrie (DSC), kterou byla stanovena teplota denaturace lysozymu v přítomnosti nebo nepřítomnosti potenciálně protektivních látek. Posledním parametrem bylo stanovení vlivu potenciálně protektivních látek při různých koncentracích na katalytickou aktivitu lipázy a stanovení residuální aktivity lipázy po aplikaci dvou denaturačních faktorů – zvýšené teploty a mražení. Ze všech testovaných strukturních analogů 3-hydroxybutyrátu byl nejvyšší protektivní efekt (stabilizace molekuly enzymu) pozorován u jantaranu, který disponuje dvěma karboxylovými skupinami. Naopak 1,3-butandiol nevykázal prakticky žádnou protektivní účinnost, což dokazuje, že přítomnost karboxylové skupiny je pro protektivní účinek zcela zásadní. Nízká protektivní účinnost butyrátu dále naznačuje, že je nezbytné, aby efektivní protektant kromě karboxylové skupiny obsahoval i další funkční skupinu – ať už karboxylovou nebo hydroxylovou. Výraznější protektivní účinek byl pozorován u 3-hydroxybutyrátu, než-li u 2-hydroxybutyrátu. Je zajímavé, že efektivní protektanty zároveň při vyšších koncentracích výrazně inhibují enzymovou aktivitu modelového enzymu, což pravděpodobně souvisí se solvatací a konformací proteinu v přítomnosti protektantů a dostupností aktivního místa pro substrát., The aim of this thesis is the study of chemical chaperones, as a preservative enzymes before denaturing processes. In the experimental part of the study we investigated the protective effects of five selected potential protective compounds – derivatives of 3-hydroxybutyrate – two model enzymes: lipase and lysozyme. Protective effects of potential chemical chaperones were investigated by dynamic light scattering (DLS), which was determined by the size distribution of the aggregates lipase produced depending on the temperature at the heat-induced denaturation. Further we have been used differential scanning calorimetry (DSC), which was determined by the temperature of the denaturation of lysozyme in the presence or absence of potentially protective substances. The last parameter was to determine the effect of potentially protective agents at different concentrations on the catalytic activity of lipase and determining the residual lipase activity after application of denaturing two factors – the high temperature and freezing. Of all tested structural analogues of 3-hydroxybutyrate was the highest protective effect observed (stabilization of enzyme molecules) with the succinate, which has two carboxyl groups. Conversely, 1,3-butanediol showed virtually no protective activity, indicating that the presence of carboxyl groups on the protective effect of essential. The low protective efficacy butyrate further indicates that it is essential that an effective protectant in addition to the carboxyl group contained as a further functional group – either carboxyl or hydroxyl. More significant protective effect was observed in 3-hydroxybutyrate than in 2-hydroxybutyrate. Interestingly, the effective protectant while at higher concentrations strongly inhibit the enzyme activity of the model enzyme, which is probably related to the solvation and conformations of the protein in the presence of protectant and the availability of the active site for the substrate.

145. Využití mikrokalorimetrických metod při studiu protektivních účinků chemických chaperonů

Author: Krouská, Jitka, Pekař, Miloslav, Bohunská, Miroslava, Krouská, Jitka, Pekař, Miloslav, and Bohunská, Miroslava
Abstract: Tato bakalářská práce se zabývá studiem ochranných látek před denaturačními procesy, označovanými jako chemické chaperony. V teoretické části byla popsána obecná charakteristika proteinů, popis vybraných chaperonů a metody diferenční kompenzační kalorimetrie. V experimentální části byly zkoumány protektivní účinky čtyř potenciálních protektivních látek – trehalóza, guanidine hydrochloride, 3-hydroxybutyrát a hydroxyectoine – na modelovém proteinu lysozymu. Protektivní účinky jednotlivých látek byly zkoumány pomocí diferenciální kompenzační kalorimetrie (DSC), kterou byla stanovena denaturační teplota lysozymu v přítomnosti ochranných látek. Ze všech zkoumaných chemických chaperonů byl nejvyšší protektivní efekt pozorován u 3-hydroxybutyrátu, který teplotu denaturace posunoval k vyšším hodnotám, a guanidine hydrochloride, který naopak denaturační teplotu snižoval. Zároveň byl u některých látek zjištěn reverzibilní denaturační proces, který byl u GuHCl nejintenzivnější., This bachelor thesis deals with the study of protective substances against denaturation processes, called chemical chaperones. The theoretical part describes the general characteristics of proteins, description of selected chaperones and methods of differential scanning calorimetry. In the experimental part, the protective effects of four potential protective agents - trehalose, guanidine hydrochloride, 3-hydroxybutyrate and hydroxyectoine - were investigated on the lysozyme model protein. The protective effects of the individual substances were examined by differential scanning calorimetry (DSC), which determined the denaturation temperature of lysozyme in the presence of preservatives. Of all the chemical chaperones examined, the highest protective effect was observed with 3-hydroxybutyrate, which shifted the denaturation temperature to higher levels, and guanidine hydrochloride, which on the other hand lowered the denaturation temperature. At the same time, a reversible denaturation process was found in some substances, which was the most intense in GuHCl.

146. Fyzikálně-chemická charakterizace 3-hydroxybutyrátu ve vztahu k jeho bioprotektivním účinkům

Author: Sedláček, Petr, Smilek, Jiří, Kahanovská, Kristína, Sedláček, Petr, Smilek, Jiří, and Kahanovská, Kristína
Abstract: Hlavným cieľom práce bolo overiť bioprotektívne vlastnosti 3-hydroxybutyrátu (3HB) z hľadiska jeho vplyvu na denaturáciu proteínu (konkrétne lipázy) a stanoviť relevantné fyzikálno-chemické charakteristiky 3HB, ktoré by mohli viesť k vysvetleniu mechanizmu tohto ochranného efektu. Rešeršná časť práce sa zamerala predovšetkým na mechanizmus ochranného účinku tzv. kompatibilných solutov, medzi ktoré patrí 3HB. Ochranný účinok 3HB bol stanovený metódou dynamického rozptylu svetla (DLS). Ako ďalšie parametre vodného roztoku 3HB boli stanovené: koncentračná závislosť povrchového napätia 3HB, vplyv rôznych koncentrácií 3HB na zeta potenciál lipázy. Hydrofílny/hydrofóbny charakter 3HB bol charakterizovaný parametrom log Kow. Pre kvantitatívne stanovenie 3HB vo vodnom roztoku bola optimalizovaná jednoduchá metóda. Touto metódou je nepriama spektroskopia. Je založená na konverzii 3HB na krotonovú kyselinu v prítomnosti kyseliny sírovej za zvýšenej teploty., The main aim of the work was to verify bio-protective properties of 3-hydroxybutyrate (3HB) in terms of its effect on protein denaturation (especially lipase) and determine the physicochemical characteristics of 3HB that could lead to the explanation of the mechanism of its protective effect. The literature search focused primarily on the mechanism of the protective effect of the compatible solutes. The 3HB belongs to the group of compatible solutes. The protective effect of the 3HB was monitored by Dynamic Light Scattering (DLS). As the other parameters of the aqueous solution 3HB were determined: the dependence of the surface tension on the concentration of the 3HB, the effect of different 3HB concentrations on the zeta potential of the lipase. Hydrophobic and hydrophilic character of the 3HB was characterized with the log Kow parameter. A simple method for quantity determination of the 3HB aqueous solution was optimized. The method is based on conversion of the 3HB into crotonic acid by reaction with sulphuric acid at higher temperatures and on subsequent psectrophotometric determination of the amount of crotonic acid.

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