Дисертації з теми "Protein mechanism"

Thesis (Ph. D.)--University of Oregon, 2006.
Typescript. Includes vita and abstract. Includes bibliographical references (leaves 115-121). Also available for download via the World Wide Web; free to University of Oregon users.

G-protein coupled receptor signaling (GPCR) is essential for regulating a large variety of hormonal, sensory and neuronal processes in eukaryotic cells. Because the regulation of these physiological responses is critical, GPCR signaling pathways are carefully controlled at different levels within the cascade. Phosducin-like protein 1 (PhLP1) can bind the G protein βγ dimer and participate in GPCR signaling. Recent evidence has supported the concept that PhLP1 can serve as a co-chaperone of the eukaryotic cytosolic chaperonin complex CCT/TRiC to mediate G βγ assembly. Although a general mechanism of PhLP1-mediated G βγ assembly has been postulated, many of the details about this process are still missing. Structural analysis of key complexes that are important intermediates in the G βγ assembly process can generate snapshots that provide molecular details of the mechanism beyond current understanding. We have isolated two important intermediates in the assembly process, the Gβ1-CCT and PhLP1-Gβ1-CCT complexes assembled in vivo in insect cells, and have determined their structures by cryo-electron microscopy (cryo-EM). Structural analysis reveals that Gβ1, representing the WD40 repeat proteins which are a major class of CCT substrates, interacts specifically with the apical domain of CCTβ. Gβ1 binding experiments with several chimeric CCT subunits confirm a strong interaction of Gβ1 with CCTβ and map Gβ1 binding to α-Helix 9 and the loop between β-strands 6 and 7. These regions are part of a hydrophobic surface of the CCTβ apical domain facing the chaperonin cavity. Docking the Gβ molecule into the two 3D reconstructions (Gβ1-CCT and PhLP1-Gβ1-CCT) reveals that upon PhLP1 binding to Gβ1-CCT, the quasi-folded Gβ molecule is constricted to a more native state and shifted to an angle that can lead to the release of folded Gβ1 from CCT. Moreover, mutagenesis of the CCTβ subunit suggests that PhLP1 can interact with the tip of the apical domain of CCTβ subunit at residue S260, which is a downstream phosphorylation target site of RSK and S6K kinases from the Ras-MAPK and mTOR pathways. These results reveal a novel mechanism of PhLP1-mediated Gβ folding and its release from CCT. The next important step in testing the PhLP1-mediated Gβγ assembly hypothesis is to investigate the function of PhLP1 in vivo. We have prepared a rod-specific PhLP1 conditional knockout mouse in which the physiological consequences of the loss of PhLP1 functions have been characterized. The loss of PhLP1 has led to profound consequences on the ability of these rods to detect light as a result of a significant reduction in the expression of transducin (Gt) subunits. Expression of other G protein subunits as well as Gβ5-RGS9-1 complexes was also greatly decreased, yet all of this occurs without resulting in rapid degeneration of the photoreceptor cells. These results show for the first time the essential nature of PhLP1 for Gβγ and Gβ5-RGS dimer assembly in vivo, confirming results from cell culture and structural studies.

Thesis (M.S.)--University of Alabama at Birmingham, 2009.
Title from PDF t.p. (viewed June 30, 2010). Additional advisors: Lori McMahon, Stephen Watts. Includes bibliographical references (p. 31-35).

It is well known that folded proteins in water are destabilized by the addition of urea. When a protein loses its ability to perform its biological activity due to a change in its structure, it is said to denaturate. The mechanism by which urea denatures proteins has been thoroughly studied in the past but no proposed mechanism has yet been widely accepted. The topic of this thesis is the study of the mechanism of urea-induced protein denaturation, by means of Molecular Dynamics (MD) computer simulations and Nuclear Magnetic Resonance (NMR) spectroscopy. Paper I takes a thermodynamic approach to the analysis of protein – urea solution MD simulations. It is shown that the protein – solvent interaction energies decrease significantly upon the addition of urea. This is the result of a decrease in the Lennard-Jones energies, which is the MD simulation equivalent to van der Waals interactions. This effect will favor the unfolded protein state due to its higher number of protein - solvent contacts. In Paper II, we show that a combination of NMR spin relaxation experiments and MD simulations can successfully be used to study urea in the protein solvation shell. The urea molecule was found to be dynamic, which indicates that no specific binding sites exist. In contrast to the thermodynamic approach in Paper I, in Paper III we utilize MD simulations to analyze the affect of urea on the kinetics of local processes in proteins. Urea is found to passively unfold proteins by decreasing the refolding rate of local parts of the protein that have unfolded by thermal fluctuations. Based upon the results of Paper I – III and previous studies in the field, I propose a mechanism in which urea denatures proteins mainly by an enthalpic driving force due to attractive van der Waals interactions. Urea interacts favorably with all the different parts of the protein. The greater solvent accessibility of the unfolded protein is ultimately the factor that causes unfolded protein structures to be favored in concentrated urea solutions.

A protein-mobilising factor of estimated molecular weight 24 KDa (p24) was purified both from the cachexia-inducing MAC 16 tumour and the urine of cachectic cancer patients by a combination of ammonium sulphate precipitation and affinity chromatography using a monoclonal antibody developed against the murine material. Administration of p24 to non tumour-bearing mice caused a decrease in body weight 24 h after the first injection, which was attenuated by prior treatment with the monoclonal antibody. Loss of body weight was accompanied by an accelerated loss of skeletal muscle protein, as determined by the release of tyrosine from this tissue. This was associated with an increased release of PGE2 and both protein degradation and PGE2 release were attenuated by the monoclonal antibody. Loss of protein mass arose from both a decrease in the rate of protein synthesis and an elevation of protein breakdown; the latter due to an activation of the ubiquitin-proteasome proteolytic system. In isolated muscle, p24 was capable of promoting protein breakdown and this was also associated with increased PGE2 levels. Both tyrosine and PGE2 release, were inhibited by PGE2 inhibitors and a specific inhibitor of cPLA2. When added to muscle cells in culture, p24 caused an elevation in the rates of total and myofibrillar protein breakdown and a depression in the rate of protein synthesis which was inhabitable by short-term incubation in insulin, suggesting that p24 may inhibit protein synthesis by causing an arrest in the translational process.

Thesis (Ph. D.)--Case Western Reserve University, 2006.
[School of Medicine] Department of Molecular Biology and Microbiology. Includes bibliographical references. Available online via OhioLINK's ETD Center.

京都大学
0048
新制・課程博士
博士(農学)
甲第20008号
農博第2192号
新制||農||1045(附属図書館)
学位論文||H28||N5017(農学部図書室)
33104
京都大学大学院農学研究科農学専攻
(主査)教授 裏出 令子, 教授 松村 康生, 教授 三上 文三
学位規則第4条第1項該当
Doctor of Agricultural Science
Kyoto University
DFAM

Kyoto University (京都大学)
0048
新制・課程博士
博士(農学)
甲第20008号
農博第2192号
新制||農||1045(附属図書館)
学位論文||H28||N5017(農学部図書室)
33104
京都大学大学院農学研究科農学専攻
(主査)教授 裏出 令子, 教授 松村 康生, 教授 三上 文三
学位規則第4条第1項該当

Thesis (PhD)--Stellenbosch University, 2014.
ENGLISH ABSTRACT: Left ventricular hypertrophy (LVH) is a major risk factor for cardiovascular morbidity and mortality, and is a feature of common diseases, such as hypertension and diabetes. It is therefore vital to understand the underlying mechanisms influencing its development. However, investigating the mechanisms underlying LVH in such complex disorders can be challenging. For this reason, many researchers have focused their attention on the autosomal dominant cardiac muscle disorder, hypertrophic cardiomyopathy (HCM), since it is considered a model disease in which to study the causal molecular factors underlying isolated cardiac hypertrophy. HCM is a heterogeneous disease that manifests with various phenotypes and clinical symptoms, even in families with the same genetic defects, suggesting that additional factors contribute to the disease phenotype. Despite the identification of several HCM-causing genes, the genetic factors that modify the extent of hypertrophy in HCM patients remain relatively unknown. The gene encoding the sarcomeric protein, cardiac myosin binding protein C, cMyBPC (MyBPC3) is one of the most frequently implicated genes in HCM. Identification of proteins that interact with cMyBPC has led to improved insights into the function of this protein and its role in cardiac hypertrophy. However, very little is known about another member of the myosin binding protein family, myosin binding protein H (MyBPH). Given the sequence homology and similarity in structure between cMyBPC and MyBPH, we propose that MyBPH, like cMyBPC, may play a critical role in the structure and functionality of the cardiac sarcomere and could therefore be involved in HCM pathogenesis. The present study aimed to identify MyBPH-interacting proteins by using yeast two-hybrid (Y2H) analysis and to verify these interactions using three-dimensional (3D) co-localisation and co-immunoprecipitation (Co-IP) analyses. We further hypothesized that both MyBPH and cMyBPC may be involved in autophagy. To test this hypothesis, both MyBPH and cMyBPC were analysed for co-localisation with a marker for autophagy, LC3b-II. The role of MyBPH and cMyBPC in cardiac cell contractility were analysed by measuring the planar cell surface area of differentiated H9c2 rat cardiomyocytes in response to β-adrenergic stress after individual and concurrent siRNA-mediated knockdown of MyBPH and cMyBPC. In the present study we employed a family-based genetic association analysis approach to investigate the contribution of genes encoding the novel MyBPH-interacting proteins in modifying the hypertrophy phenotype. This study investigated the hypertrophy modifying effects of 38 SNPs and haplotypes in four candidate HCM modifier genes, in 388 individuals from 27 HCM families, in which three unique South African HCM-causing founder mutations segregate. Yeast two-hybrid analysis identified three putative MyBPH-interacting proteins namely, cardiac β-myosin heavy chain (MYH7), cardiac α-actin (ACTC1) and the SUMO-conjugating enzyme UBC9 (UBC9). These interactions were verified using both 3D co-localisation and Co-IP analyses. Furthermore, MyBPH and cMyBPC were implicated in autophagy, since both these proteins were being recruited to the membrane of autophagosomes. In addition, a cardiac contractility assay demonstrated that the concurrent siRNA-mediated knockdown of MyBPH and cMyBPC resulted in a significant reduction in cardiomyocyte contractility, compared to individual protein and control knockdowns under conditions of β-adrenergic stress. These results indicated that MyBPH could compensate for cMyBPC, and vice versa, further confirming that both these proteins are required for efficient sarcomere contraction. Results from genetic association analyses found a number of SNPs and haplotypes that had a significant effect on HCM hypertrophy. Single SNP and haplotype analyses identified SNPs and haplotypes within genes encoding MyBPH, MYH7, ACTC1 and UBC9, which contribute to the extent of hypertrophy in HCM. In addition, we found that several variants and haplotypes had markedly different statistical significant effects in the presence of each of the three HCM founder mutations. The results of this study ascribe novel functions to MyBPH. Cardiac MyBPC and MyBPH play a critical role in sarcomere contraction and have been implicated in autophagy. This has further implications for understanding the patho-etiology of HCM-causing mutations in the gene encoding MyBPH and its interacting proteins. This is to our knowledge the first genetic association analysis to investigate the modifying effect of interactors of MyBPH, as indication of the risk for developing LVH in the context of HCM. Our findings suggest that the hypertrophic phenotype of HCM is modulated by the compound effect of a number of variants and haplotypes in MyBPH, and genes encoding protein interactors of MyBPH. These results provide a basis for future studies to investigate the risk profile of hypertrophy development in the context of HCM, which could consequently lead to improved risk stratification and patient management.
AFRIKAANSE OPSOMMING: Linker ventrikulêre hipertrofie (LVH) is 'n primêre risikofaktor vir kardiovaskulêre morbiditeit en mortaliteit asook 'n kenmerk van algemene siektes soos hipertensie en diabetes. Daarom is dit van kardinale belang om te verstaan wat die onderliggende meganismes is wat die ontwikkeling van LVH beïnvloed. Die ondersoek na die onderliggende meganismes wat lei tot LVH in sulke komplekse siektes is ‟n uitdaging. Om hierdie rede fokus baie navorsers hul aandag op die autosomaal dominante hartspier siekte, hipertrofiese kardiomiopatie (HKM), wat beskou word as 'n model siekte om die molekulêre oorsake onderliggend tot geïsoleerde kardiovaskulêre hipertrofie te ondersoek. HKM is 'n heterogene siekte wat manifesteer met verskeie fenotipes en kliniese simptome, selfs in families met dieselfde genetiese defekte, wat impliseer dat addisionele faktore bydra tot die modifisering van die siekte fenotipe. Ten spyte van die identifisering van verskeie HKM-versoorsakende gene, bly die genetiese faktore wat die mate van hipertrofie in HKM pasiente modifiseer relatief onbekend. Die geen wat kodeer vir die sarkomeriese proteïen, kardiale miosien-bindingsproteïen C (kMyBPC) is die algemeenste betrokke in HKM. Die identifisering van proteïene wat bind met kMyBPC het gelei tot verbeterde insigte tot die funksie van hierdie proteïen en die rol wat hierdie proteïen in hipertrofie speel. Ten spyte hiervan, is daar baie min inligting beskikbaar oor 'n ander lid van die miosien-bindingsproteïen families, miosien-bindingsproteïen H (MyBPH). Gegewe die ooreenstemming tussen die DNA basispaar-volgorde en struktuur tussen hierdie twee proteïene, stel ons voor dat MyBPH, net soos kMyBPC, 'n kritiese rol in die struktuur en funksie van die kardiale sarkomeer speel en kan daarom betrokke wees in die patogenese van HKM. Die huidige studie het beoog om proteïene wat met MyBPH bind te identifiseer deur die gebruik van gis-twee-hibried (G2H) kardiale biblioteek sifting en om hierdie interaksies te verifieer met behulp van drie-dimensionele (3D) ko-lokalisering en ko-immunopresipitasie eksperimente. Ons het verder gehipotiseer dat beide MyBPH and kMyBPC betrokke kan wees in outofagie. Om hierdie hipotese te toets is beide MyBPH en kMyBPC geanaliseer vir ko-lokalisering met 'n merker vir outofagie, LC3b-II. Verder het ons beplan om die rol van MyBPH en kMyBPC in kardiale spiersel-sametrekking te ondersoek deur die oppervlak van gedifferensieerde H9c2 rot kardiomiosiete in reaksie op β-adrenergiese stres te meet, na individuele en gesamentlike siRNA-bemiddelde uitklopping van MyBPH en kMyBPC. In hierdie studie het ons 'n familie-gebaseerde genetiese assosiasie analise benadering gevolg om vas te stel of MyBPH en gene wat kodeer vir die geverifieerde bindingsgenote van MyBPH bydra tot die modifisering van die hipertrofiese fenotipe. Die doel van hierdie studie was om die hipertrofiese effek van 38 enkel nukleotied polimorfismes (SNPs) en haplotipes in vier kandidaat HKM modifiserende gene in 388 individue van 27 HCM families te toets, waarin drie unieke Suid-Afrikaanse HKM-stigters mutasies segregeer. G2H analise het drie verneemde MyBPH bindingsgenote geidentifiseer, naamlik miosien (MYH7), alfa kardiale aktien (ACTC1) en die SUMO-konjugerende ensiem UBC9 (UBC9). Hierdie interaksies is geverifieer deur middel van 3D ko-lokalisering en ko-immunopresipitasie analises. Verder is bewys dat MyBPH en kMyBPC betrokke is in outofagie, siende dat beide proteïene gewerf is tot die membraan van die outofagosoom. 'n Kardiale sametrekkings eksperiment het gevind dat die gesamentlike siRNA-bemiddelde uitklopping van MyBPH en kMyBPC 'n merkwaardige vermindering in die kardiomiosiet sametrekking veroorsaak het in reaksie op β-adrenergiese stres kondisies, in vergelyking met die individuele proteïen en kontrole uitkloppings eksperimente. Hierdie resultate bevestig dat MyBPH vir kMyBPC kan instaan en ook andersom, wat verder bevestig dat beide proteïene benodig word vir effektiewe sarkomeer sametrekking. Resultate van die genetiese assosiasie studie het gevind dat 'n aantal SNPs en haplotipes 'n beduidende effek of HKM hipertrofie het. Enkel SNP en haplotipe analises in gene wat kodeer vir MyBPH, MYH7, ACTC1 en UBC9 het SNPs en haplotipes geidentifiseer wat bydra tot die omvang van hipertrofie in HKM. Verder het ons gevind dat sekere SNPs en haplotipes kenmerkend verskillende statisties beduidende effekte in die teenwoordigheid van elk van die drie HKM-stigter mutasies gehad het. Die resultate van hierdie studie skryf twee nuwe funksies aan MyBPH toe. Kardiale MyBPC en MyBPH speel 'n kritiese rol in sarkomeer sametrekking en is betrokke in outofagie. Hierdie resultate het verdere implikasies vir die verstaan van die pato-etiologie van die HKM-veroorsakende mutasies in die MyBPH, MYH7, ACTC1 en UBC9 gene. So vêr dit ons kennis strek is dit die eerste genetiese assosiasie studie wat die modifiserende effek van bindingsgenote van MyBPH ondersoek as risiko aanduiding vir die ontwikkeling van LVH in die konteks van HKM. Ons bevindinge bewys dat die hipertrofiese fenotipe van HKM gemoduleer word deur die komplekse effekte van SNPs en haplotipes in die MyBPH geen en gene wat MyBPH proteïen-bindingsgenote enkodeer. Hierdie resultate verskaf dus 'n basis vir toekomstige studies om die risiko profiel van hipertrofie ontwikkeling met betrekking tot HKM te ondersoek, wat gevolglik kan bydra tot die verbeterde risiko stratifikasie en pasiënte bestuur.

Factor H (FH) is a 155-kDa plasma protein that regulates the alternative pathway of the complement system. Its 20 CCP modules, of 51-62 amino acid residues each, are linked by short stretches (“linkers’) of three to eight residues. We set out to test the hypothesis that long linkers towards the middle of FH play a role in ensuring that its architecture allows binding sites near its N- and C-termini to engage cooperatively with the main target, C3b, which is the key complement pathway-triggering product of C3 cleavage. In initial work, site-directed mutagenesis was used to test whether two mutations, R53H and R78G, located within CCP 1 and linked to the kidney disease atypical hemolytic uremic syndrome, are functionally deficient. Mutant versions and a native-sequence version of CCPs 1-4 of FH (i.e. FH 1-4) were tested for their ability to act as a cofactor for the FI-mediated cleavage of C3b, and accelerate the decay of the C3 convertase. It was shown that FH 1-4 R53H binds normally to C3b but has no regulatory activity while FH 1-4 R78G binds very poorly and is also deficient in cofactor and decay-accelerating activities. In subsequent work, mutagenesis was used to make the eight-residue CCPs 12-13 linker shorter (SL), or more flexible through introduction of glycine residues (3xGLY), within recombinant (r) module pair FH 12-13, and in rFH 10-15 and rFH 8-15 as well as full-length rFH. NMR showed CCPs 12 and 13 remain intact following mutation of the linker but (in FH 12-13) are more flexibly mutually disposed, as expected. SAXS indicated that both FH 10-15 SL and FH 10-15 3xGLY nonetheless have similar compact structures to native sequence (WT) FH 10-15. On the other hand, FH linker mutants interact with C3b (according to surface plasmon resonance) somewhat less well than WT FH and in the case of FH SL, affinity is similar to that of FH 19-20, i.e. there is no evidence that both C3b-binding sites in this mutant bind to the target simultaneously. Nonetheless, the bacterial protein PspCN boosts binding of linker mutants to C3b by a similar factor (three-to-fivefold) to that observed for FH WT. Thus, while interactions between non-sequential CCPs are important for FH architecture, a bend at the 12-13 linker is needed for full-length FH to adopt a fully biological activity confirmation. The use of EPR for structural studies of rFH and its mutants was explored. Free cysteines were engineered in so they could have spin labels site-specifically attached. Alternatively, a recognition site for transglutaminase was introduced so a spin label could be incorporated. These strategies were applied to rFH 12-13 and rFH 10-15 as a prelude to studies of full-length FH. Several suitably engineered proteins were prepared but only one paramagnetically labeled sample (of FH 12-13) made it for EPR; this yielded results commensurate with the NMR-derived structure. Taken together, these promising data lay the groundwork for a future, potentially very insightful, combined mutagenesis and EPR study of FH architecture and its role in complement activation.

Two of the central building blocks of life are proteins and carbohydrates. The interactions between these two disparate biomolecules playa key role in numerous biological processes. Carbohydrates are a major component of the plant cell wall, which consists of a complex network of polysaccharides. The complex structure of the plant cell wall is highly inaccessible to enzyme degradation. To overcome their limited accessibilirf to polysaccharides, microbial cellulases and hemicellulases have developed complex molecular architectures generally comprising both catalytic modules and non-catalytic carbohydrate b1...''1ding modules (CBMs). CBMs display considerable variation L.''1 primary structtlte and are grouped into 51 sequence-based families collated in the continuously updated carbohydrate-active enzyme data base CAZy. Family 35 CBMs provide an interesting example of how the fme-tuning of these modules can have a profound influence on polysaccharide recognition. The ligand specificity of fom members of this CBM family was determined. A CBM35 derived from a modular pectate lyase, Pell0A-CBM35, binds specifically to the unsaturated ends of poly galacturonic acid (.6.GaLL\.-GalA), while QAbf62.l\.CBM35 and CtCBM3Sb display affinity for glucuronic acid and .6.GalA-GalA. A fomth CBM35, CtCBM3Sa, is the only one of these proteins to recognise galactose. The crystal structures of these four CBM35 have been solved, while the structure of QAbf62A-CBM35 was also determined in complex with glucmonic acid. Structure comparison of the four CBM35s, and directed-mutagenesis studies of Pe110A-CBM35 and QAbf62A-CBM35, revealed that the binding sites of the four protein modules exhibit slight differences in the positioning of the key residues, allowing the CBMs to accommodate different ligands. Finally, as demonstrated in a previous study on QAbf62A-CBM35, ligand recognition by Pell0A-CBM35 and CtCBM35a is calcium dependent. This thesis also presents the characterisation of two novel families of CBM L.''lat display new ligand specificities. A member of a Clostridium thermol'ellum family 52 CBM, CtCBM52, was shown to bind selectively to galactopyranose and arabinofuranose, with preference for the galactopyranose sidechain of xyloglucan. This was supported by the crystal structure of CtCBM52 in complex \.viL.''l xyloglucan oligosaccharide, 61-tX-D-galactosyl mannotriose and arabinobiose. The putative role of CtCBM52 in the formation of a polysaccharide lattice that engulfs the host bacterium is discussed. The X14 modules present in several xylanases, including QXynl1A, represent a family of non-catalytic modules whose function was previously unknown. It is shown in this thesis that a member of this family, X14-BD7340, binds to a range of different polysaccharides that include galactans, glucomannans, ~-glucans and AJlans, \.v1.th similar affinities. The crystal structure of X14-BD7340 and site-directed mutagenesis provided insight how this module displays such plasticirf in ligand recogrutlon. The complete degradation of the plant cell wall requires enzymes that are able to release decorations from the backbones of many polysaccharides, such as acetyl groups. This thesis reveals that four family 2 carbohydrate esterases, CtCE2, CjCE2A, CjCE2B and CjCE2C, displayed activity against 4-nitrophenyl acetate, glucomannan, galactoglucomannan and acetylated xylan. Intriguingly, CtCE2, the only CE2 member to be linked to a second catalytic module, which displays cellulase activity, is inhibited by cellulosic polymers. The crystal structure of CtCE2, in complex with cellohexaose, and the apo forms of CjCE2A and QCE2B revealed the structural basis for the observed non-competitive inhibition of CtCE2 by cellulose. The enzyme contains three planar aromatic residues lirJng the deep active site cleft that stack against the glucopyranose rings of cellulose. The biological significance for the dual CBM-esterase activity 1...''1 relation to the appended cellulase, and in the wider context of plant cell wall degradation, is discussed. The crystal structures of two of the CE2 enzymes showed that these serine esterases did not display the canonical catalytic triad; the position of the histidine that activates the serine nucleophile of these enzymes is stabilized through hydrogen bonds with polar backbone carbonyl and amine moieties.

Viral myocarditis, the inflammation of myocardium initiated by viral infection, is an important cause of mortality in neonates and children. In addition, it is a precursor to dilated cardiomyopathy (DCM). To date, no effective therapy is available for viral myocarditis/DCM. Coxsackievirus B3 (CVB3) is an important human pathogen of viral myocarditis. Extensive research efforts on CVB3 have broadened our understanding of the virus-host protein interactions. However, the pathogenesis of coxsackievirus-induced myocarditis is not fully understood. The objective of this dissertation is to explore the role of host protein manipulation in coxsackieviral replication and pathogenicity. My hypotheses are that (1) coxsackievirus hijacks host’s cellular autophagy mechanism to facilitate its own replication; and (2) the serum response factor (SRF) is cleaved by viral protease 2A during coxsackievirus infection and contributes to impaired myocardial function and progression to DCM. For project 1, I demonstrated that CVB3 manipulates the host autophagy pathway to supplement viral replication. Autophagy is an evolutionary conserved homeostatic mechanism in eukaryotes that degrades and recycles long-lived cytoplasmic proteins, as well as damaged organelles. The hallmark of autophagy is the formation of double-membrane vesicles known as autophagosomes. I provided the initial evidence that CVB3 infection induces the formation of autophagosomes. Up-regulation of autophagosome formation enhances CVB3 replication, whereas downregulation of autophagy pathway reduces CVB3 replication. My results help clarify the nature of the intracellular membranes previously shown to be required for viral replication. For project 2, I demonstrated that CVB3 manipulates SRF expression via protein cleavage. SRF is a transcription factor vital for the expression of cardiac contractile/regulator genes, as well as gene silencing microRNAs. Cardiac-specific knockout of SRF in adult transgenic mice results in disruption of cardiac gene expression and development of severe DCM. I showed that SRF is cleaved in CVB3-infected mouse hearts and cardiomyocytes. Further studies revealed that SRF is cleaved at the 327 amino acids position by CVB3-encoded protease 2A. I demonstrated that SRF cleavage contributes to DCM by abolishing the transactivation property of SRF and generating dominant-negative SRF-truncates. Taken together, these novel viral strategies bridged existing knowledge and may serve as therapeutic targets for viral myocarditis/DCM.

The structure and mechanism of the Protein Tyrosine Phosphatase-like Phytases (PTPLPs) from Selenomonas ruminantium (PhyAsr) and Mitsuokella multacida (PhyAmm) were investigated using a combination of enzyme kinetics, site-directed mutagenesis, and X-ray crystallography. I show that PTPLPs use a classical protein tyrosine phosphatase catalytic mechanism and adopt a core PTP fold. Several unique structural features of PTPLPs confer specificity for inositol phosphates. The effect of ionic strength and oxidation on the kinetics and structure of PTPLPs was investigated. The structural consequences of reversible and irreversible oxidation on PTPLPs and PTPs are compared and discussed. We determine the structural basis of substrate specificity in PTPLPs and propose a novel reaction mechanism for the hydrolysis of inositol polyphosphates by PTPLPs. Finally, the structure and function of a unique tandemly repeated phytase has been determined. We show that the active sites of the tandem repeat possess significantly different specificities for inositol polyphosphate.
xix, 148 leaves : ill. (some col.) ; 29 cm

本文データは平成22年度国立国会図書館の学位論文(博士)のデジタル化実施により作成された画像ファイルを基にpdf変換したものである
Kyoto University (京都大学)
0048
新制・課程博士
博士(農学)
甲第7067号
農博第954号
新制||農||754(附属図書館)
学位論文||H10||N3109(農学部図書室)
UT51-98-C180
京都大学大学院農学研究科農芸化学専攻
(主査)教授 小田 順一, 教授 清水 昌, 教授 江﨑 信芳
学位規則第4条第1項該当

La cellule est un système chimique complexe qui a bénéficié de milliards d’années d’évolution pour se perfectionner, et représente une machinerie très bien organisée ne laissant rien au hasard. Pour assurer son rôle, elle contrôle un ensemble de processus d’auto-assemblage où des composants isolés interagissent spontanément entre eux pour conduire à la formation de structures organisées et fonctionnelles telles que les microtubules, le collagène ou les fibres d’actine. En m’inspirant de l’organisation cellulaire, mon projet doctoral consiste en la conception de systèmes chimiques artificiels basés sur l’auto-assemblage de peptides originaux. Ces édifices donnent naissance à des hydrogels supramoléculaires d’intérêts dans le domaine des biomatériaux. Je m’intéresse à la fois à des aspect fondamentaux concernant la compréhension de l’initiation des processus d’auto-assemblage en présence de biomacromolécules, mais aussi à des problématiques plus appliquées consistant à élaborer des stratégies pour contrôler le lieu mais aussi le moment où ces édifices moléculaires auto-assemblés prennent naissance. Enfin, je m’intéresse à l’émergence des différentes propriétés apparaissant lors de la formation de certains auto-assemblages comme la catalyse ou l’auto-catalyse
The cell is a complex chemical system that has benefited from billions of years of evolution to perfect itself, and represents a very well organized machinery leaving nothing to chance. To ensure its role, it controls a set of self-assembly processes where isolated components interact spontaneously with each other to lead to the formation of organized and functional structures such as microtubules, collagen or actin fibers. Inspired by cellular organization, my doctoral project involves the design of artificial chemical systems based on the self-assembly of original peptides. These buildings give rise to supramolecular hydrogels of interest in the field of biomaterials. I am interested at the same time in fundamental aspects concerning the comprehension of the initiation of the processes of self-assembly in the presence of biomacromolecules, but also with more applied problems of elaborating strategies to control the place but also the moment where these self-assembled molecular structures originate. Finally, I am interested in the emergence of the different properties appearing during the formation of certain self-assemblies such as catalysis and auto-catalysis

The aim of this work was to understand the molecular mechanism of translation and the mechanism of translation termination, in particular. Cleavage of peptidyl-tRNA and peptide release terminates translation of mRNA on the ribosome. In prokaryotes, three release factors (RFs) are involved in this process. RF1 and RF2 recognise the three stop codons on mRNA and induce hydrolysis of the ester bond in peptidyl-tRNA. RF3 accelerates the rate of RF1 and RF2 recycling between ribosome in a GTP-dependent manner. We have clarified the mechanism of action of peptide release factor RF3. In the cell, free RF3 is in the GDP conformation. When RF3∙GDP binds to ribosome in complex with RF1 or RF2, these ribosome complexes act as guanine exchange factors for RF3 by inducing rapid dissociation of GDP. If, and only if, the peptide has been removed from tRNA, GDP is quickly replaced by GTP. Binding of GTP to RF3 induces a conformation of the factor with high affinity for the ribosome, which forces RF1 or RF2 to rapidly dissociate. Subsequent hydrolysis of GTP on RF3 induces a factor conformation with low affinity for the ribosome and rapid release of RF3∙GDP. It was further shown how the position of peptidyl-tRNA on the ribosome and the presence or absence of its peptide regulates the binding and GTPase activity of translation factors IF2, EF-G and EF-Tu. The result explains how idling GTPase hydrolysis and negative interference between different translation factors are minimized in living cells. The present biochemical observations, in conjunction with cryo-EM results, lead to new proposals for the role of hybrid sites in translocation of tRNAs, recycling of RF1 and RF2 by RF3 and recycling of post-termination ribosomes back to a new round of initiation.

Protein arginine methyltransferase 1 (PRMT1) is a key posttranslational modification enzyme that catalyzes the methylation of specific arginine residues in histone and nonhistone protein substrates, regulating diverse cellular processes such as transcriptional initiation, RNA splicing, DNA repair, and signal transduction. Recently the essential roles of PRMT1 in cancer and cardiovascular complications have intrigued much attention. Developing effective PRMT inhibitors therefore is of significant therapeutic value. The research on PRMT inhibitor development however is greatly hindered by poor understanding of the biochemical basis of protein arginine methylation and lack of effective assays for PRMT1 inhibitor screening. Herein, we report our effort in the kinetic mechanism study as well as the fluorescent probe and inhibitor development for PRMT1. New fluorescent reporters were designed and applied to perform single-step analysis of substrate binding and methylation of PRMT1. Using these reporters, we performed transient-state fluorescence measurements to dissect the rate constants along the PRMT1 catalytic coordinate. The data give evidence that the chemistry of methyl transfer is the major rate-limiting step, and that binding of the cofactor SAM or SAH affects the association and dissociation of H4 with PRMT1. Importantly, we identified a critical kinetic step suggesting a precatalytic conformational transition induced by substrate binding. On the other hand, we discovered a type of naphthyl-sulfo (NS) compounds that block PRMT1- mediated arginine methylation at micromolar potency through a unique mechanism: they directly target the substrates but not PRMT enzymes for the observed inhibition. We also found that suramin, an anti-parasite and anti-cancer drug bearing similar functional groups, effectively inhibited PRMT1 mediated methylation. These findings about novel PRMT inhibitors and their unique inhibition mechanism provide a new way for chemical regulation of protein arginine methylation. Addionally, to dissect the interplaying relationship between different histone modification marks, we investigated how individual lysine acetylations and their different combinations at the H4 tail affect Arg-3 methylation in cis. Our data reveal that the effect of lysine acetylation on arginine methylation depends on the site of acetylation and the type of methylation. While certain acetylations present a repressive impact on PRMT-1 mediated methylation (type I methylation), lysine acetylation generally is correlated with enhanced methylation by PRMT5 (type II dimethylation). In particular, Lys-5 acetylation decreases activity of PRMT1 but increases that of PRMT5. Furthermore, hyperacetylation increases the content of ordered secondary structures of H4 tail. These findings provide new insights into the regulatory mechanism of Arg-3 methylation by H4 acetylation, and unravel that complex intercommunications exist between different posttranslational marks in cis.

Mutations in the gene for peripheral myelin protein 22 (PMP22) are associated with peripheral neuropathy in mice and humans. PMP22 is produced mainly in Schwann cells in the peripheral nervous system where it is localised to compact myelin. The function of PMP22 is unclear but its low abundance makes it unlikely to be a structural myelin protein. I have studied the peripheral nerves of two different mouse models with alterations in the pmp22 gene. (1) The Trembler-J (Tr^J) mouse which has a point mutation [L16P] in the first transmembrane domain of PMP22. (2) PMPP22 overexpressing transgenic mice which have 7 (C22), 4 (C61) and 2 (C2) copies of the human PMP22 gene in addition to the mouse pmp22 gene. In the nerves of adult Tr^J mice there was considerable evidence of abnormal Schwann cell-axon interactions. Abnormal features were reproduced in the early stages of regeneration following crush injury. This demonstrates that the abnormalities are a result of an intrinsic abnormality of Tr^J Schwann cells and not secondary changes related to demyelination. In the initial stages of postnatal development the number of axons that were singly ensheathed was the same in all the mutants examined, indicating that PMP22 does not function in the initial enclosure of groups of axons and subsequent separation of single axons. All strains examined had an increased proportion of fibres that were incompletely surrounded by Schwann cell cytoplasm indicating that this step is disrupted in PMP22 mutants. Increasing the number of copies of PMP22 resulted in an increasing severity of phenotype. In C22 (7 copy) animals myelin formation was delayed or non-existent in many fibres whereas in C61 animals myelination initially appeared normal with abnormality appearing later in a small population of fibres. The C2 strain appeared relatively unaffected. It is concluded that PMP22 functions in the initiation of myelination and most probably involves the ensheathment of the axon by the Schwann cell, and the extension of this cell along the axon. Abnormalities are most likely to result from defective interactions between the axon and the Schwann cell.

Dissertation presented to obtain the Ph.D degree in Biology
Human cytomegalovirus (HCMV) is a -herpesvirus that infects healthy individuals, usually asymptomatically, but can cause severe or fatal disease in immunocompromised individuals such as transplant recipients or AIDS patients. Primary HCMV infection, as with other herpesviruses, is followed by establishment of lifelong latency and periodic reactivation. To ensure its survival and propagation within the host, HCMV has evolved many strategies to subvert both innate and adaptive host immunity. It is known that HCMV infection induces production of interleukin-8 (IL-8), a proinflammatory chemokine with neutrophil chemotatic activity. Significantly, neutrophils are a major carrier of HCMV during viremia and they are able to transmit infectious virus to other cells, playing a key role in virus dissemination through their contact with endothelial cells. In addition, IL-8 enhances HCMV virion production. This work has identified an HCMV gene (UL76), with the relevant property of inducing IL-8 expression at both transcriptional and protein levels. Interestingly, the murine homologue, MHV-68 ORF20, has no significant effect in the expression of IL-8. The main objective of this work was to characterize the mechanism of IL-8 induction by UL76 and the impact of its expression during the viral infection. The UL76-mediated enhancement of luciferase activity was abolished when the NF-kB binding element was mutated in the IL-8 promoter luciferase reporter, thereby demonstrating that activation of NF-kB is essential for the UL76-mediated induction of IL-8. Consistent with the requirement for NF-kB pathway activation, IKK- and degradation of Ik were essential for IL-8 induction by UL76. In addition, and as might be predicted, expression of UL76 resulted in the translocation of p65 to the nucleus.(...)

Cardiovascular disease is the most common cause of morbidity and mortality worldwide, of which cardiomyopathies account for a proportion. One of the hallmarks of progressive heart disease is diminished energy metabolism associated with cardiac mitochondrial dysfunction. Recent evidence directly implicates malfunctioning mitochondria and altered mitochondrial dynamics in the development of heart disease. Yet, little is known about mitochondrial remodelling changes that might contribute to the development of heart failure. A recently identified mouse mutant in the Dnm1l gene, Python, leads to the development of dilated cardiomyopathy at specific ages. The work reported herein focussed on understanding the mechanisms responsible for the development of cardiomyopathy in this model. Evidence was obtained of alteration in mitochondria, peroxisome and endoplasmic reticulum (ER) morphology in various cell types. There was a suggestion of altered physical interaction between the mitochondria and the ER. Increased cytosolic calcium levels and reduced mitochondrial uptake of calcium were also observed in Python fibroblasts. Mitochondrial membranes were also depolarised. These changes resulted in reduced oxidative phosphorylation activity in the hearts of Python mice, which showed a progressive reduction with age. Ultimately a decrease in ATP levels occurred, which was unsustainable with normal heart function. DNM1L expression was found to increase with age in hearts of wild type mice, but not other tissues. This may be suggestive of an increase in the importance for the DNM1L protein in the ageing heart, though the exact reasons for this remain elusive. Fragmented mitochondria have been postulated to contribute to the development of Huntington’s disease. However, a reduction in fragmentation through introduction of the Python mutation did not alleviate the progressive development of symptoms in these animals. We hypothesize that the Python mutation impairs the ability of mitochondrial-ER tethering. Subsequent dysfunctional mitochondrial calcium uptake and altered mitochondrial membrane potential, leads to a progressive decline in oxidative phosphorylation activity and ATP production.

La rémorine du groupe 1 isoforme 3 de Solanum tuberosum (StREM1.3) est une protéine membranaire de la famille multigénique de protéines de plante appelée rémorines (REMs), impliquées dans l’immunité des plantes, la symbiose, la résistance aux stress abiotiques et la signalisation hormonale. La caractéristique la plus connue des REMs est leur capacité à se ségréger en nanodomaines au feuillet interne de la membrane plasmique (MP). Pour StREM1.3, ceci se fait via une interaction entre deux lysines de l’ancre C-terminale de la rémorine (RemCA) et le phosphatidylinositol 4-phosphate (PI4P) négativement chargé. Ainsi, RemCA modifie sa conformation et s’enfonce partiellement dans la MP, résultant en un accrochage membranaire intrinsèque. Capitalisant sur les données structurales déjà disponibles concernant cet isoforme, nous investiguons StREM1.3 davantage quant à ses propriétés d’interaction membranaire, en utilisant un large éventail de techniques, allant de la microscopie de fluorescence et de la RMN à l’état solide (ssNMR) à la microscopie de force atomique (AFM), la cryo-microscopie électronique (cryoEM) et la modélisation informatique. Nous souhaitons découvrir l’impact de l’oligomérisation et de la phosphorylation de StREM1.3 sur ses interactions membranaires et son activité biologique, ainsi que d’examiner son influence sur la dynamique des lipides et les lipides requis pour l’accrochage à la membrane et le regroupement en nanodomaines. Enfin, forts de toutes les données structurales disponibles, nous entreprendrons la reconstruction in vitro et la caractérisation de nanodomaines minimaux de StREM1.3
Group 1 isoform 3 remorin from Solanum tuberosum (StREM1.3) is a membrane protein belonging to the multigenic family of plant proteins called remorins (REMs), involved in plant immunity, symbiosis, abiotic stress resistance and hormone signalling. REMs’ most well known feature is their ability to segregate into nanodomains at the plasma membrane’s (PM) inner leaflet. For StREM1.3, this is achieved by an interaction between two lysines of the remorin C-terminal anchor (RemCA) and negatively charged phosphatidylinositol 4-phosphate (PI4P). Thus, RemCA undergoes conformational changes and partially buries itself in the PM, resulting in an intrinsic membrane anchoring. Capitalising on pre-existing structural data about this isoform, we investigate StREM1.3’s membrane-interacting properties further, using a wide array of techniques, ranging from fluorescence microscopy and solid-state nuclear magnetic resonance (ssNMR) to atomic force microscopy (AFM), cryo-electron microscopy (cryoEM) and computational modelling. We aim to discover the impact of StREM1.3’s oligomerisation and phosphorylation on its membrane interactions and biological activity, and to assess its influence on lipid dynamics as well as its lipid requirements for membrane binding and nanoclustering. Finally, based on all available structural data, we will undertake the in vitro reconstruction and characterisation of minimal nanodomains of StREM1.3

Isotope partitioning experiments were carried out with the adenosine 3',5'-monophosphate-dependent protein kinase catalytic subunit (cAPK) from bovine hearts to obtain information on the order of addition of reactants and the relative rates of reactant release from enzyme compared to the catalytic step(s). A value of 100% trapping for both ErMgATP-[γ-32P] and E:3H-Serpeptide at low Mgf indicates that MgATP and Serpeptide dissociate slowly from the enzyme compared to the catalytic step(s). The K_Serpeptide for MgATP trapping is 17 μM, while the K_MgATP for Serpeptide trapping is 0.58 mM. The latter data indicate that the off-rate for MgATP from the E:MgATP complex is 14 s^-1 while that for Serpeptide from the E: Serpeptide complex is 64 s^-1. At high Mg^, 100% trapping is obtained for the E:MgATP-[γ-32P] complex but only 40% is obtained for the E:Serpeptide complex. Thus, the off-rate for Serpeptide from the E:MgATP:Serpeptide complex becomes significant at high Mg_f. Data suggest a random mechanism in which MgATP is sticky. The V for the cAPK reaction increases 1.5-1.7 fold in the presence of the R_II in the presence of saturating cAMP at a stoichiometry of R:C of 1:1. No change is obtained with the type-I complex under these conditions. At higher ratio of R:C (up to 100) no further change is observed with the type-II complex but inhibition by the type-I R_2(cAMP)_4 complex competitive vs. Serpeptide is observed. The activiation observed in the presence type-II R_2(cAMP)_4 effects neither the K_m for Serpeptide nor the K_m for MgATP. Both the activating affect of the type-II complex and the inhibitory effect of the type-I complex are dependent on the Mg_f with more type-II activation obtained the higher the Mg_f and more type-I complex required for inhibition the higher the Mg_f. The activation and inhibition are discussed in terms of the mechanism of the protein kinase.

Cancer cachexia is characterised by selective depletion of skeletal muscle protein reserves. The ubiquitin-proteasome proteolytic pathway has been shown to be responsible for muscle wasting in a range of cachectic conditions including cancer cachexia. To establish the importance of this pathway in muscle wasting during cancer (and sepsis), a quantitative competitive RT-PCR (QcRT-PCR) method was developed to measure the mRNA levels of the proteasome sub units C2a and C5ß and the ubiquitin-conjugating enzyme E214k. Western blotting was also used to measure the 20S proteasome and E214k protein expression. In vivo studies in mice bearing a cachexia inducing murine colon adenocarcinoma (MAC16) demonstrated the effect of progressive weight loss on the mRNA and protein expression for 20S proteasome subunits, as well as the ubiquitin-conjugating enzyme, E214k, in gastrocnemius and pectoral muscles. QcRT-PCR measurements showed a good correlation between expression of the proteasome subunits (C2 and CS) and the E214k enzyme mRNA and weight loss in gastrocnemius muscle, where expression increased with increasing weight loss followed by a decrease in expression at higher weight losses (25-27%). Similar results were obtained in pectoral muscles, but with the expression being several fold lower in comparison to that in gastrocnemius muscle, reflecting the different degrees of protein degradation in the two muscles during the process of cancer cachexia. Western blot analysis of 20S and E214k protein expression followed a similar pattern with respect to weight loss as that found with mRNA. In addition, mRNA and protein expression of the 20S proteasome subunits and E214k enzyme was measured in biopsies from cachectic cancer patients, which also showed a good correlation between weight loss and proteasome expression, demonstrating a progressive increase in expression of the proteasome subunits and E214k mRNA and protein in cachectic patients with progressively increasing weight loss. The effect of the cachexia-inducing tumour product PIF (proteolysis inducing factor) and 15-hydroxyeicosatetraenoic acid (15-HETE), the arachidoinic acid metabolite (thought to be the intracellular transducer of PIF action) has also been determined. Using a surrogate model system for skeletal muscle, C2C12 myotubes in vitro, it was shown that both PIF and 15-HETE increased proteasome subunit expression (C2a and C5ß) as well as the E214k enzyme. This increase gene expression was attenuated by preincubation with EPA or the 15-lipoxygenase inhibitor CV-6504; immunoblotting also confirmed these findings. Similarly, in sepsis-induced cachexia in NMRI mice there was increased mRNA and protein expression of the 20S proteasome subunits and the E214k enzyme, which was inhibited by EPA treatment. These results suggest that 15-HETE is the intracellular mediator for PIF induced protein degradation in skeletal muscle, and that elevated muscle catabolism is accomplished through upregulation of the ubiquitin-proteasome-proteolytic pathway. Furthermore, both EPA and CV -6504 have shown anti-cachectic properties, which could be used in the future for the treatment of cancer cachexia and other similar catabolic conditions.