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Molecular cloning of human glycogen synthase kinase-3α promoter and expression study of the protein.January 1998 (has links)
by Chan Ying Chi, Jessica. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1998. / Includes bibliographical references (leaves 113-127). / Abstract also in Chinese. / Acknowledgments --- p.i / Abstract in English --- p.ii / Abstract in Chinese --- p.iv / Contents --- p.vi / Abbreviations --- p.xi / Single Letter Amino Acid Code --- p.xvi / Chapter Chapter 1 --- Introduction / Chapter 1.1 --- Glycogen Synthase (EC 2.4.1.11) --- p.1 / Chapter 1.2 --- Glycogen Synthase Kinase-3 --- p.4 / Chapter 1.3 --- Structure of Glycogen Synthase Kinase-3 --- p.5 / Chapter 1.4 --- Functions of Glycogen Synthase Kinase-3 --- p.8 / Chapter 1.4.1 --- Substrate Recognition --- p.8 / Chapter 1.4.2 --- Glycogen Synthase Kinase-3 Homologs --- p.10 / Chapter 1.4.2.1 --- Drosophila --- p.10 / Chapter 1.4.2.2 --- Xenopus --- p.11 / Chapter 1.4.2.3 --- Dictyostelium and Others --- p.12 / Chapter 1.4.3 --- Regulation of Glycogen Synthase-3 in Mammalian Systems --- p.13 / Chapter 1.4.4 --- The role of Glycogen Synthase Kinase-3in Mammalian Brain --- p.16 / Chapter 1.4.4.1 --- Glycogen Synthase Kinase-3β --- p.18 / Chapter 1.4.4.2 --- Glycogen Synthase Kinase-3α --- p.21 / Chapter 1.4.5 --- Glycogen Synthase Kinase-3α in Certain Tumor Cells --- p.23 / Chapter 1.5 --- Objectives --- p.25 / Chapter Chapter 2 --- Materials and Methods / Chapter 2.1 --- General Techniques / Chapter 2.1.1 --- Plasmid Minipreparation --- p.26 / Chapter 2.1.2 --- Large Scale of Plasmid DNA Purification Using QIAGEN-tip500 --- p.28 / Chapter 2.1.3 --- Extraction of Human Blood Genomic DNA --- p.30 / Chapter 2.1.4 --- UV Spectroscopy for determining DNA/RNA Concentration --- p.31 / Chapter 2.1.5 --- Agarose Gel Electrophoresis of DNA --- p.31 / Chapter 2.1.6 --- Purification of DNA Fragment from Agarose Gel using GeneClean III ® (BIO 101 Inc.) Kit --- p.32 / Chapter 2.1.7 --- Restriction Digestion of DNA --- p.32 / Chapter 2.1.8 --- Southern Blot --- p.33 / Chapter 2.1.9 --- Probe Labelling --- p.33 / Chapter 2.1.10 --- Hybridization by Radio-labelling --- p.34 / Chapter 2.1.11 --- DNA Sequencing Reaction --- p.35 / Chapter 2.1.12 --- "Preparation of 6% Polyacrylamide, 8M Urea Denaturing Gel for DNA Sequencing Analysis" --- p.37 / Chapter 2.1.13 --- Preparation of Escherichia coli DH5α Competent Cells --- p.38 / Chapter 2.1.14 --- Modification of 5'Protruding end with T4DNA Polymerase --- p.39 / Chapter 2.1.15 --- Ligation and Transformation of Foregin DNA --- p.39 / Chapter 2.1.16 --- Rapid Screening for the Presence of Plasmid --- p.40 / Chapter 2.2 --- Expression of Glycogen Synthase Kinase-3 / Chapter 2.2.1 --- Preparation of Mammalian cells in Culture --- p.41 / Chapter 2.2.2 --- SDS-Polyacrylamide Gel Electrophoresis (SDS-PAGE) --- p.42 / Chapter 2.2.3 --- Western Blot Detection of Glycogen Synthase Kianse-3 --- p.43 / Chapter 2.3 --- Assay of Glycogen Synthase Kinase Promoter Activity / Chapter 2.3.1 --- Preparation of SHSY5Y in Culture --- p.45 / Chapter 2.3.2 --- Trypsinization for Removing Adherent Cells --- p.45 / Chapter 2.3.3 --- Transfection of Mammalian Cells by Calcium Phosphate Precipitation --- p.46 / Chapter 2.3.4 --- Stimulation of Transfection Cells by different Chemicals and Preparation of Cell Extract --- p.47 / Chapter 2.3.5 --- CAT-ELISA and β-Gal ELISA Assay --- p.47 / Chapter 2.4 --- Isolation of Glycogen Synthase Kinase-3α 5,Promoter Region / Chapter 2.4.1 --- 5'Rapid Amplification of cDNA End (5'RACE) --- p.48 / Chapter 2.4.2 --- PromoterFinder DNA Walking --- p.49 / Chapter 2.4.3 --- YAC Clone Genomic Construction --- p.50 / Chapter 2.5 --- Construction of Plasmid for Assay of Glycogen Synthase Kinase-3α Promoter Activity --- p.53 / Chapter 2.6 --- Genomic Organization of Glycogen Synthase Kinase-3α --- p.53 / Chapter 2.7 --- Primer Extension Assay / Chapter 2.7.1 --- Isolation of Total RNA by TRIZOL Reagent --- p.57 / Chapter 2.7.2 --- Primer Extension by SuperScript II --- p.57 / Chapter 2.8 --- Reagents and Buffers / Chapter 2.8.1 --- Nucleic Acid Electrophoresis Buffers --- p.59 / Chapter 2.8.2 --- Reagents for Preparation of Plasmid DNA --- p.59 / Chapter 2.8.3 --- Media for Bacterial Culture --- p.60 / Chapter 2.8.4 --- Reagents for Southern Blot --- p.60 / Chapter 2.8.5 --- Reagents for SDS-PAGE --- p.61 / Chapter 2.8.6 --- Reagents for Western Blot --- p.62 / Chapter 2.8.7 --- Reagents for DNA Sequencing --- p.62 / Chapter Chapter 3 --- Isolation of 5´ة Glycogen Synthase Kinase-3α Promoter Region / Chapter 3.1 --- Introduction --- p.63 / Chapter 3.2 --- Results --- p.66 / Chapter 3.2.1 --- 5' Rapid Amplification of cDNA End (5'RACE) --- p.66 / Chapter 3.2.2 --- PromoterFinder DNA Walking --- p.68 / Chapter 3.2.3 --- YAC Clone Library Construction --- p.71 / Chapter 3.2.3.1 --- Southern Blotting --- p.71 / Chapter 3.2.3.2 --- Isolation of Sequence Upstream of Glycogen Synthase Kinase-3α region from YAC Clone Using PromoterFider DNA Walking --- p.71 / Chapter 3.2.3.3 --- Sequences of 5,Glycogen Synthase Kinase -3α Promoter --- p.73 / Chapter 3.2.4 --- Primer Extension Assay --- p.78 / Chapter 3.2.5 --- Assay of Glycogen Synthase Kinase-3α Promoter Activity using CAT-ELISA --- p.78 / Chapter 3.2.6 --- Genomic Structure of Glycogen Synthase Kinase-3α --- p.84 / Chapter 3.3 --- Discussion --- p.90 / Chapter 3.3.1 --- Glycogen Synthase Kinase-3a Promoter --- p.90 / Chapter 3.3.2 --- Glycogen Synthase Kianse-3a Promoter Activity --- p.92 / Chapter 3.3.3 --- Prospective and Future Studies --- p.94 / Chapter Chapter 4 --- Expression of Glycogen Synthase Kinase-3 / Chapter 4.1 --- Introduction --- p.96 / Chapter 4.2 --- Results Expression of GSK-3 under Stresses --- p.97 / Chapter 4.3 --- Discussion --- p.105 / Chapter 4.3.1 --- Post-translation regulation of Glycogen Synthase Kinase-3 --- p.105 / Chapter 4.3.2 --- Prospective and Future Studies --- p.107 / Chapter Chapter 5 --- Conclusion --- p.109 / Chapter 5.1 --- Promoter study --- p.110 / Chapter 5.2 --- Genomic organization study --- p.111 / Chapter 5.3 --- Expression study --- p.112 / Reference --- p.113 / Appendices / Appendix I G/C contents of GSK-3α Promoter Region --- p.128 / Appendix II Restriction sites of GSK-3α Promoter Region --- p.134 / Appendix III Primers designed on GSK-3α Promoter Region --- p.139 / Appendix IV Restriction sites of GSK-3α cDNA --- p.142 / Appendix V Vectors --- p.150 / Appendix VI Adaptors Sequences --- p.152 / Appendix VII Anti-GSK-3 Antibody --- p.153 / Appendix VIII Raw data of GSK-3α promoter activity assay --- p.154
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Multi-omics network analysis to discover novel type 2 diabetes related genes. / CUHK electronic theses & dissertations collectionJanuary 2013 (has links)
Gao, Zhibo. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2013. / Includes bibliographical references (leaves 147-157). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese.
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The allelic features revealed by whole genome, methylome and transcriptome sequencing analysis of a type 2 diabetes trio. / CUHK electronic theses & dissertations collectionJanuary 2013 (has links)
Liu, Xin. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2013. / Includes bibliographical references (leaves 157-164). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese.
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Investigation of Physical Characteristics Impacting Fate and Transport of Viral Surrogates in Water SystemsCharest, Abigail J. 29 January 2015 (has links)
A multi-scale approach was used to investigate the occurrence and physical characteristics of viral surrogates in water systems. This approach resulted in a methodology to quantify the dynamics and physical parameters of viral surrogates, including bacteriophages and nanoparticles. Physical parameters impacting the occurrence and survival of viruses can be incorporated into models that predict the levels of viral contamination in specific types of water. Multiple full-scale water systems (U.S., Italy and Australia) were tested including surface water, drinking water, stormwater and wastewater systems. Water quality parameters assessed included viral markers (TTV, polyomavirus, microviridae and adenovirus), bacteriophages (MS2 and ΦX-174), and coliforms (total coliforms and E. coli). In this study, the lack of correlations between adenovirus and that of bacterial indicators suggests that these bacterial indicators are not suitable as indicators of viral contamination. In the wastewater samples, microviridae were correlated to the adenovirus, polyomavirus, and TTV. While TTV may have some qualities which are consistent with an indicator such as physical similarity to enteric viruses and occurrence in populations worldwide, the use of TTV as an indicator may be limited as a result of the detection occurrence. The limitations of TTV may impede further analysis and other makers such as coliphages, and microviridae may be easier to study in the near future. Batch scale adsorption tests were conducted. Protein-coated latex nanospheres were used to model bacteriophages (MS2 and ΦX-174) and includes a comparison of the zeta potentials in lab water, and two artificial groundwaters with monovalent and divalent electrolytes. This research shows that protein-coated particles have higher average log10 removals than uncoated particles. Although, the method of fluorescently labeling nanoparticles may not provide consistent data at the nanoscale. The results show both that research on viruses at any scale can be difficult and that new methodologies are needed to analyze virus characteristics in water systems. A new dynamic light scattering methodology, area recorded generalized optical scattering (ARGOS) method, was developed for observing the dynamics of nanoparticles, including bacteriophages MS2 and ΦX-174. This method should be further utilized to predict virus fate and transport in environmental systems and through treatment processes. While the concentration of MS2 is higher than ΦX-174 as demonstrated by relative total intensity, the RMSD shows that the dynamics are greater and have more variation in ΦX-174 than MS2 and this may be a result of the hydrophobic nature of ΦX-174. Relationships such as these should be further explored, and may reflect relationships such as particle bonds or hydrophobicity.
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Týr : a dependent type based code transformation for spatial memory safety in LLVM / Týr : uma transformação de código baseada em tipos dependentes para segurança especial de memória em LLVMAraújo, Vítor Bujés Ubatuba de January 2018 (has links)
A linguagem C não provê segurança espacial de memória: não garante que a memória acessada através de um ponteiro para um objeto, tal como um vetor, de fato pertence ao objeto em questão. Em vez disso, o programador é responsável por gerenciar informações de alocações e limites, e garantir que apenas acessos válidos à memória são realizados pelo programa. Por um lado, isso provê flexibilidade: o programador tem controle total sobre o layout dos dados em memória, e sobre o momento em que verificações são realizadas. Por outro lado, essa é uma fonte frequente de erros e vulnerabilidades de segurança em programas C. Diversas técnicas já foram propostas para prover segurança de memória em C. Tipicamente tais sistemas mantêm suas próprias informações de limites e instrumentam o programa para garantir que a segurança de memória não seja violada. Isso causa uma série de inconvenientes, tais como mudanças no layout de memória de estruturas de dados, quebrando assim a compatibilidade binária com bibliotecas externas, e/ou um aumento no consumo de memória. Uma abordagem diferente consiste em usar tipos dependentes para descrever a informação de limites já latente em programas C e assim permitir que o compilador use essa informação para garantir a segurança espacial de memória. Embora tais sistemas tenham sido propostos no passado, eles estão atrelados especificamente à linguagem C. Outras linguagens, como C++, sofrem de problemas similares de segurança de memória, e portanto poderiam se beneficiar de uma abordagem mais independente de linguagem. Este trabalho propõe Týr, uma transformação de código baseada em tipos dependentes para garantir a segurança espacial de memória de programas C ao nível LLVM IR. O sistema permite que o programador descreva no nível dos tipos as relações entre pointeiros e informação de limites já presente em programas C. Dessa maneira, Týr provê segurança espacial de memória verificando o uso consistente desses metadados pré-existentes, através de verificações em tempo de execução inseridas no programa guiadas pela informação de tipos dependentes. Ao trabalhar no nível mais baixo do LLVM IR, Týr tem por objetivo ser usável como uma fundação para segurança espacial de memória que possa ser facilmente estendida no futuro para outras linguagens compiláveis para LLVM IR, tais como C++ e Objective C. Demonstramos que Týr é eficaz na proteção contra violações de segurança espacial de memória, com um overhead de tempo de execução relativamente baixo e de consumo de memória próximo de zero, atingindo assim um desempenho competitivo com outros sistemas para segurança espacial de memória de uma maneira mais independente de linguagem. / The C programming language does not enforce spatial memory safety: it does not ensure that memory accessed through a pointer to an object, such as an array, actually belongs to that object. Rather, the programmer is responsible for keeping track of allocations and bounds information and ensuring that only valid memory accesses are performed by the program. On the one hand, this provides flexibility: the programmer has full control over the layout of data in memory, and when checks are performed. On the other hand, this is a frequent source of bugs and security vulnerabilities in C programs. A number of techniques have been proposed to provide memory safety in C. Typically such systems keep their own bounds information and instrument the program to ensure that memory safety is not violated. This has a number of drawbacks, such as changing the memory layout of data structures and thus breaking binary compatibility with external libraries and/or increased memory usage. A different approach is to use dependent types to describe the bounds information already latent in C programs and thus allow the compiler to use that information to enforce spatial memory safety. Although such systems have been proposed before, they are tied specifically to the C programming language. Other languages such as C++ suffer from similar memory safety problems, and thus could benefit from a more language-agnostic approach. This work proposes Týr, a program transformation based on dependent types for ensuring spatial memory safety of C programs at the LLVM IR level. It allows programmers to describe at the type level the relationships between pointers and bounds information already present in C programs. In this way, Týr ensures spatial memory safety by checking the consistent usage of this pre-existing metadata, through run-time checks inserted in the program guided by the dependent type information. By targeting the lower LLVM IR level, Týr aims to be usable as a foundation for spatial memory which could be easily extended in the future to other languages that can be compiled to LLVM IR, such as C++ and Objective C. We show that Týr is effective at protecting against spatial memory safety violations, with a reasonably low execution time overhead and nearly zero memory consumption overhead, thus achieving performance competitive with other systems for spatial memory safety, in a more language-agnostic way.
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Expression analysis of glycogen synthase kinase-3 in human tissues and cloning of the beta-isoform promoter. / Expression analysis of glycogen synthase kinase-3 in human tissues and cloning of the b-isoform promoter / CUHK electronic theses & dissertations collectionJanuary 1999 (has links)
"November 1999." / Thesis (Ph.D.)--Chinese University of Hong Kong, 1999. / Includes bibliographical references (p. 131-152). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Mode of access: World Wide Web. / Abstracts in English and Chinese.
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Role of cyclin-dependent kinase 9 in the resolution of innate inflammation in a zebrafish tailfin injury modelHoodless, Laura Jane January 2016 (has links)
Neutrophils are an important cell in host defence and migrate rapidly to sites of inflammation when the host is compromised (e.g., in infection or wounding). There, they produce and/or release inflammatory mediators (e.g., LTB4, TNF, IL-8) and ingest and degrade pathogens (e.g., by release of granule proteins and reactive oxygen species). Neutrophils then undergo apoptosis and are cleared by phagocytes such as macrophages, to allow efficient resolution of inflammation. Inducing neutrophil apoptosis by pharmacological means could be a therapeutic strategy to dampen inflammation in diseases where neutrophils are prevalent, e.g., acute respiratory distress syndrome (ARDS) and rheumatoid arthritis (RA). Inhibition of cyclin-dependent kinases (CDKs) using CDK inhibitor (CDKi) compounds induces mammalian neutrophil apoptosis in vitro, and can drive resolution of inflammation in vivo in mouse models. Evidence indicated that this is due to inhibition of CDK9 and CDK7-mediated transcription of the anti-apoptotic protein Mcl-1. The hypothesis of this project was that CDK9, CDK7 and Mcl-1 are pivotal regulators of resolution of inflammation in vivo. The model selected to test this hypothesis was tailfin injury of embryonic zebrafish (Danio rerio). Zebrafish are optically transparent and reporter transgenic lines with neutrophils labelled by enhanced GFP (EGFP - Tg[mpx:EGFP]i114) and macrophages (Tg[MPEG1:mCherry]) have been created, permitting the imaging of the behaviour of these cells in vivo. The model of tailfin transection was chosen to cause an inflammatory response in these animals, with neutrophil and macrophage recruitment to the tailfin. This response was manipulated using CDKi compounds and specific gene knockdowns (using morpholino and CRISPR/cas9 technologies). It was shown that CDKi compounds could reduce neutrophil numbers at 24 h post-injury at the transected tailfin, but did not affect macrophage numbers. The CDKi AT7519 increased neutrophil apoptosis at 12 h post-injury. Specific CDK9 knockdown using morpholinos or CRISPR/cas9 also reduced neutrophilic inflammation at the tailfin 24 h after transection, accompanied by increased apoptosis levels at 8 h in the morpholino-treated group. Inhibition of an endogenous CDK9 inhibitor, LaRP7, had the opposite effect and increased neutrophil numbers; and could oppose the neutrophil- reducing effect of AT7519 and CDK9 morpholino knockdown. Preliminary genetic knockdown studies into the roles of CDK7 and Mcl-1 have been carried out. Taken together, the results demonstrate CDK9 is important in the resolution of neutrophilic inflammation, indicating that manipulation of CDK9 activity could be a good target for therapeutic intervention in inflammatory disease.
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model for the risk of complications in Hong Kong type 2 diabetic patients. / 香港二型糖尿病併發症風險評估模型 / A model for the risk of complications in Hong Kong type 2 diabetic patients. / Xianggang er xing tang niao bing bing fa zheng feng xian ping gu mo xingJanuary 2011 (has links)
Fok, Tsz Nam = 香港二型糖尿病併發症風險評估模型 / 霍梓楠. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2011. / Includes bibliographical references (p. 71-72). / Abstracts in English and Chinese. / Fok, Tsz Nam = Xianggang er xing tang niao bing bing fa zheng feng xian ping gu mo xing / Huo Zinan. / Abstract --- p.i / 概要 --- p.iii / Acknowledgements --- p.iv / Chapter 1 --- Introduction --- p.1 / Chapter 2 --- Dataset Information --- p.3 / Chapter 3 --- Background and Literature Review --- p.9 / Chapter 3.1 --- The Idea of Risk Model --- p.9 / Chapter 3.2 --- Discrimination Problem --- p.10 / Chapter 3.3 --- Receiver Operating Characteristic (ROC) Curve --- p.11 / Chapter 3.4 --- Summary Indices of the ROC Curve --- p.13 / Chapter 3.4.1 --- Area Under ROC Curve (AROC) --- p.14 / Chapter 3.4.2 --- Maximum Vertical Distance --- p.16 / Chapter 3.5 --- Discrimination Performance in Prognostic Model --- p.18 / Chapter 3.5.1 --- Survival Data --- p.18 / Chapter 3.5.2 --- Survival Function --- p.20 / Chapter 3.5.3 --- Time-dependent ROC Curve for Censored Data --- p.21 / Chapter 3.6 --- Earlier Work on Diabetic Complications Risk Models --- p.22 / Chapter 3.6.1 --- Maximization of the AROC --- p.28 / Chapter 4 --- Model Development --- p.29 / Chapter 4.1 --- Overview --- p.29 / Chapter 4.2 --- Estimating the ROC curve and the AROC --- p.29 / Chapter 4.3 --- Choosing Suitable Risk Factors --- p.30 / Chapter 4.4 --- Mixing Risk Factors and Optimizing Coefficients --- p.31 / Chapter 4.5 --- Validation of Risk Equations Using Test Set --- p.33 / Chapter 5 --- Results and Validation --- p.34 / Chapter 5.1 --- Performance of the Risk Factor Candidates --- p.34 / Chapter 5.2 --- Estimation of the Coefficients --- p.37 / Chapter 5.3 --- Checking the Uniqueness of the Solution --- p.41 / Chapter 5.4 --- Validation Using Test Set --- p.46 / Chapter 5.5 --- Comparison of AROC-optimized and MVD-optimized Risk Equations --- p.56 / Chapter 6 --- Comparison of our Results with Earlier Work --- p.58 / Chapter 7 --- "Discussion, Outstanding Issues and Future Works" --- p.66 / Chapter 7.1 --- Comparison Between the AROC and the MVD --- p.66 / Chapter 7.2 --- Applications of Risk Models --- p.68 / Chapter 7.3 --- Limitations of the study --- p.69 / Chapter 7.4 --- Outstanding Issues and Future Works --- p.69 / Chapter 7.4.1 --- The Estimation of Error Due to Sampling Variance --- p.69 / Chapter 7.4.2 --- Time-dependent Coefficients --- p.69 / Chapter 7.4.3 --- Extending the Idea to other Datasets --- p.70 / Chapter 7.5 --- Conclusion --- p.70 / Bibliography --- p.71
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Thermodynamic and structural determinants of calcium-independent interactions of CalmodulinFeldkamp, Michael Dennis 01 July 2010 (has links)
Calmodulin (CaM) is an essential protein found in all eukaryotes ranging from vertebrates to unicellular organisms such as Paramecia. CaM is a calcium sensor protein composed of two domains (N and C) responsible for the regulation of numerous calcium-mediated signaling pathways. Four calcium ions bind to CaM, changing its conformation and determining how it recognizes and regulates its cellular targets. Since the discovery of CaM, most studies have focused on the role of its calcium-saturated form.
However, an increasing number of target proteins have been discovered that preferentially bind apo (calcium-depleted) CaM. My study focused on understanding how apo CaM recognizes drugs and protein sequences, and how those interactions differ from those of calcium-saturated CaM. I have used spectroscopic methods to explore CaM binding the drug Trifluoperazine (TFP) and the IQ-motif of the type 2 Voltage-Dependent Sodium Channel (Nav1.2IQp). These studies have shown that both TFP and Nav1.2IQp preferentially bind to the "semi-open" conformation of apo CaM.
TFP was shown to be an unusual allosteric effector of calcium binding to CaM. Using 15N-HSQC NMR spectroscopy, I determined the stoichiometry of TFP binding to apo Cam to be 2:1 and to (Ca2+)4-CaM to be 4:1 TFP:CaM. That difference in stoichiometry determined whether TFP decreased or increased the affinity of CaM for calcium. Analysis of residue-specific chemical shift differences indicated that TFP binding to apo and (Ca2+)4-CaM perturbed the C-domain more than the N-domain, prompting high-resolution structural studies of the isolated C-domain of CaM.
Crystallographic studies of TFP bound to a calcium-saturated C-domain fragment of CaM (CaM76-148) revealed that CaM adopted an "open" tertiary conformation. The unit cell contained two protein and 4 drug molecules. The orientation of TFP revealed that its trifluoromethyl group was found in two alternative positions (one in each protein in the unit cell), and that Met 144 acted as a gatekeeper to select the orientation of TFP.
In contrast to TFP binding to the "open" conformation of calcium-saturated CaM76-148, my NMR studies showed that TFP bound the "semi-open" conformation of apo CaM76-148. TFP interacted with CaM residues near the perimeter of the hydrophobic pocket, but did not contact residues that are solvent-accessible only in the "open" form. Allosteric effects due to TFP binding were observed in the calcium-binding loops of apo CaM76-148. These properties suggest that TFP may antagonize interactions between apo CaM and target proteins such as ion channels that preferentially bind apo CaM.
Nav1.2, is responsible for the passage of Na+ ion across cellular membranes. Apo binding of CaM to Nav1.2 poises it for action upon calcium release in the cell. My NMR studies of CaM binding to the Nav1.2 IQ-motif sequence (Nav1.2IQp) showed that the C-domain of apo CaM was necessary and sufficient for binding. My high-resolution structure of the isolated C-domain of CaM bound to Nav1.2IQp revealed that the domain adopted a "semi-open" conformation. At the interface between the IQ-motif and CaM, the highly conserved I and two Y residues of Nav1.2IQp interacted with hydrophobic residues of CaM, while the invariant Q residue interacted with residues in the loop between helices F and G of CaM. This is the first CaM-IQ complex to be determined by NMR; the only other available structure of apo CaM bound to an IQ-motif was determined crystallographically.
To accomplish its regulatory roles in response to cellular Ca2+ fluxes, CaM has evolved multiple binding interfaces that are allosterically linked to its Ca2+-ligation state. My studies of CaM binding to TFP and NaV1.2 demonstrate the versatility of CaM functioning as a regulatory protein comprised of domains having separable functions.
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Mitochondrial Ca2+/Calmodulin-dependent kinase ii (CaMKII) regulates smooth muscle cell migration and neointimal formation via mitochondrial Ca2+ uptake and mobilityNguyen, Emily Kim 01 May 2019 (has links)
No description available.
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