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Author: Kevin Vaughn Kepple Publisher: ISBN: Category : Genetic recombination Languages : en Pages : 390
Book Description
Bacteriophage lambda uses the recombination protein Integrase (Int) to incorporate and remove its genome from the chromosomal DNA of E. coli . Intermediates of the recombination reaction are difficult to study because of the high efficiency of the reaction and lack of high-energy cofactor requirements. Previously, peptide libraries were screened in order to isolate inhibitors of the recombination reaction. These peptides function by targeting specific recombination intermediates, but also inhibit the growth of bacterial cells. The exact mechanism by which the peptides function both in vitro and in vivo is not known with certainty. In this work, I am interested in how peptide inhibitors interact with their target structure and if this can be applied to explain their antibiotic activity. The specific questions I set out to answer include: what is the site of interaction between the peptide inhibitors and recombination intermediates? What role does substrate conformation play during recombination and inhibition of recombination? What types of biochemical interactions are important for peptide binding? Are potential in vivo protein targets inhibited by the peptides? If inhibition of these potential targets is observed, what is the mechanism of inhibition? I show that peptide inhibitors bind to the center of the protein-bound Holliday junction complex, in agreement with crystal structure data. The data indicate that contacts are mediated by interactions that mimic base stacking. In addition, peptides bind Holliday junctions in the square-planar conformation even in the absence of Int. This opens up the possibility that inhibitors of lambda recombination that trap Holliday junctions may also inhibit many other proteins that process DNA junctions. Indeed, the peptides prevent unwinding of branched DNA substrates by the RecG helicase of E. coli and interfere with the resolution of Holliday junction substrates by the RuvABC complex. The results indicate that inhibition of lambda Integrase, the RecG helicase, and the RuvABC complex all occur by a similar mechanism and imply that there are a number of potential targets for these peptides in vivo. Finally, the results emphasize the importance of Holliday junction conformation for peptide activity and as a determinant for the directionality of catalysis by lambda Int.
Author: Kevin Vaughn Kepple Publisher: ISBN: Category : Genetic recombination Languages : en Pages : 390
Book Description
Bacteriophage lambda uses the recombination protein Integrase (Int) to incorporate and remove its genome from the chromosomal DNA of E. coli . Intermediates of the recombination reaction are difficult to study because of the high efficiency of the reaction and lack of high-energy cofactor requirements. Previously, peptide libraries were screened in order to isolate inhibitors of the recombination reaction. These peptides function by targeting specific recombination intermediates, but also inhibit the growth of bacterial cells. The exact mechanism by which the peptides function both in vitro and in vivo is not known with certainty. In this work, I am interested in how peptide inhibitors interact with their target structure and if this can be applied to explain their antibiotic activity. The specific questions I set out to answer include: what is the site of interaction between the peptide inhibitors and recombination intermediates? What role does substrate conformation play during recombination and inhibition of recombination? What types of biochemical interactions are important for peptide binding? Are potential in vivo protein targets inhibited by the peptides? If inhibition of these potential targets is observed, what is the mechanism of inhibition? I show that peptide inhibitors bind to the center of the protein-bound Holliday junction complex, in agreement with crystal structure data. The data indicate that contacts are mediated by interactions that mimic base stacking. In addition, peptides bind Holliday junctions in the square-planar conformation even in the absence of Int. This opens up the possibility that inhibitors of lambda recombination that trap Holliday junctions may also inhibit many other proteins that process DNA junctions. Indeed, the peptides prevent unwinding of branched DNA substrates by the RecG helicase of E. coli and interfere with the resolution of Holliday junction substrates by the RuvABC complex. The results indicate that inhibition of lambda Integrase, the RecG helicase, and the RuvABC complex all occur by a similar mechanism and imply that there are a number of potential targets for these peptides in vivo. Finally, the results emphasize the importance of Holliday junction conformation for peptide activity and as a determinant for the directionality of catalysis by lambda Int.
Author: Jasdeep Kaur Mann Publisher: ISBN: Category : Languages : en Pages : 165
Book Description
The main focus of this dissertation is to develop the technology that allows targeting of key cellular pathways such as cell signaling and inflammatory pathways, where dysregulations leads to a disease state. As part of the work, it provides an overview of protein therapeutics and highlights some of the progresses that have been made toward achieving the goal. Comparison is drawn among small molecules, antibodies and non-antibody protein scaffolds to offer a rationale for developing alternative strategies. The dissertation is divided into three sections: The first part describes the engineering of fibronectin type III (FN3) monobodies to target the ERK-2 signaling pathway. ERK-2, along with other mitogen activated protein kinases (MAPKs) forms evolutionarily conserved pathways in eukaryotes, and regulate changes in protein function and gene expression in response to stimuli at the cell surface. Several MAPK pathways exist in humans, which collectively control cell proliferation, apoptosis, and other adaptive behaviors of the cell. Correct activation of different MAPK pathways is crucial to generate appropriate biological responses, whereas overactive signaling results in a number of human diseases, including cancer and neurodegenerative diseases. MAPKs use modular binding interactions outside the catalytic site to achieve specificity of interaction. In particular, all MAPKs contain a common docking domain that selectively binds a linear peptide motif within their respective substrates and regulators. Using engineered monobody inhibitors, we disrupted the docking interaction and thus selectively inhibit the ERK-2 pathway. Monobodies are compact (MW = 11 kDa) and resemble an immunoglobulin domain yet fold without an intradomain disulfide bond. We show that the engineered monobodies inhibit the activity of ERK-2 in vitro by competitively inhibiting the binding of a D-peptide. They also inhibit ERK-2 dependent signaling in transfected mammalian cells and homologous signaling pathways in yeast and worms. The design strategy used in our study is general and a similar approach may be useful to engineer epitope-specific inhibitors against other molecules. The second part discusses the use of rationally designed peptides to target a signaling pathway. Peptides can provide effective and innovative solutions to treat diseases by delivering high biological activity with low toxicity. We demonstrated their relevance by engineering a helical peptide to potentiate NOD2 protein activity associated with Crohn's disease (CD). NOD2 is an intracellular sensor that recognizes the bacterial cell wall component, muramyl dipeptide (MDP), and is critical for the innate immune response in the GI tract. However, the molecular mechanisms by which mutations in NOD2 contribute to the disease are not well understood. Modeling CD associated mutations at a molecular level is important to identify new therapeutic targets and develop potential treatment options. We used computational and structural analysis to model the mechanism of NOD2 activation. The similarity of domain organization in NOD2 to other related proteins for which structural and biochemical data exist suggests an activation mechanism that includes ligand induced removal of auto-inhibition by the carboxy terminal ligand binding domain. Based on the analysis, we designed a peptide predicted to potentiate NOD2 response to MDP by inducing conformational change of NOD2. The designed peptide shows an increase in NOD2 activity for both wild type and CD risk mutants in the presence of MDP. In vitro biochemical assays show that the peptide binds directly to the LRR domain of NOD2 and has an allosteric effect on MDP binding. The peptide may contribute to the elucidation of the activation mechanism of NOD2 and help develop a novel therapeutic agent against CD. The last section describes the novel methods developed in our lab for use in high throughput assays. Protein complexes are common in nature but biochemical characterization of protein-protein interaction is laborious and expensive. We describe two methods that may help characterize protein-protein interaction. First, we show that a combination of yeast surface display and disulfide trapping can detect the formation of a broad spectrum of protein complexes on the yeast surface. This technique is referred to as stabilization of transient and unstable complexes by engineered disulfide (STUCKED). The technique uses co-expression of two potentially interacting proteins in the same yeast cell, one of which is designed to be anchored to the cell wall and the other is designed to be in a soluble form. A structure-based disulfide is then introduced between the subunits to covalently crosslink them so that the bound complex can be efficiently detected irrespective of the stability of the complex. We demonstrated that the technique can be applied to trap a diverse group of complexes, including a protein-peptide complex (MDM2-p53) and an oligomeric protein (streptavidin heterodimer). The method may be used to assist in the testing of rationally designed interactions as well as to develop a directed evolution study to identify novel protein-protein interactions. To study dynamic protein interactions in vivo, we developed a technique which involves the use of monomeric streptavidin (mSA) to induce selective biotinylation of protein molecules based on distance criteria. Selective biotinylation is achieved when mSA forms a noncovalent complex with a target protein and is used to recruit a photoactivatable biotin crosslinker to the target molecule. Photoactivation with UV leads to chemical crosslinking of nearby proteins and results in selective biotinylation, so that the modified proteins can be purified using immobilized streptavidin. By targeting a post translational modified enzyme, mSA induced biotinylation (MIB) can selectively biotinylate enzyme substrates for downstream analysis. The proposed technique is generic and can be easily modified to study different transient interactions.
Author: Claude Malvy Publisher: Springer Science & Business Media ISBN: 9780792384182 Category : Science Languages : en Pages : 332
Book Description
Sequence-specific DNA binding ligands, amongst which triple helix forming oligonucleotides are the most efficient as yet, represent promising tools in a number of fields. One of their most promising applications is as antiviral tools: they can specifically target a viral gene, even if it is integrated into the host genome, and be used to specifically inactivate the viral gene or even destroy the cells harboring this gene. However, from science fiction to science there remains a gap; and we are at the moment on the threshold of this fascinating field. Triple Helix Forming Oligonucleotides considers the different aspects of the design and improvement, current or future, of these molecules and their structural analysis, as well as their applications, with special emphasis on the attempts to obtain biological effects of these potentially important tools. What emerges is that the current state of the research is encouraging, and that these molecules are already useful in some biotechnology applications.
Author: Nouri Neamati Publisher: John Wiley & Sons ISBN: 1118015363 Category : Science Languages : en Pages : 710
Book Description
This book comprehensively covers the mechanisms of action and inhibitor design for HIV-1 integrase. It serves as a resource for scientists facing challenging drug design issues and researchers in antiviral drug discovery. Despite numerous review articles and isolated book chapters dealing with HIV-1 integrase, there has not been a single source for those working to devise anti-AIDS drugs against this promising target. But this book fills that gap and offers a valuable introduction to the field for the interdisciplinary scientists who will need to work together to design drugs that target HIV-1 integrase.
Author: Ellen G. Strauss Publisher: Elsevier ISBN: 0080553168 Category : Medical Languages : en Pages : 476
Book Description
Completely revised and updated, the new edition of this groundbreaking text integrates basic virology with pathophysiological conditions to examine the connection between virology and human disease. Most virology textbooks focus on the molecular biology involved without adequate reference to physiology. This text focuses on viruses that infect humans, domestic animals and vertebrates and is based on extensive course notes from James Strauss’ virology class at the California Institute of Technology taught for over 30 years. Expertly depicting in color the molecular structure and replication of each virus, it provides an excellent overview for students and professionals interested in viruses as agents of human disease. Includes over 30% new material - virtually all of the figures and tables have been redrawn to include the latest information and the text has been extensively rewritten to include the most up-to-date information Includes a new chapter on emerging and reemerging viral diseases such as avian flu, SARS, the spread of West Nile virus across America, and the continuing spread of Nipah virus in Southeast Asia Further reading sections at the end of each chapter make it easy find key references World maps depicting the current distribution of existing and newly emerging viruses are also incorporated into the text
Author: Kevin Vaughn Kepple
Author: Kevin Vaughn Kepple
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Author: Jasdeep Kaur Mann
Author: Bruce Alberts
Author: Peter Matthew Flanagan
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Author: Claude Malvy
Author: Nouri Neamati
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Author: Ellen G. Strauss