<p>The glutathione transferases (GSTs) represent a superfamily of dimeric proteins involved in cellular detoxication by catalyzing the nucleophilic addition of the reduced glutathione (GSH) to the hydrophobic electrophiles. The present work focuses on the functional role of the conserved structures of GSTP1-1. The lock-and-key motif is a highly conserved hydrophobic interaction in the subunit interface of Pi, Mu, and Alpha class GSTs. The key residue (Tyr⁵⁰ in hGSTP1-1) of one subunit is wedged into a hydrophobic pocket of the neighboring subunit. The heterodimer GSTP1/Y50A was constructed from the fully active wild-type GSTP1-1 and the nearly inactive Y50A in order to study how an essentially inactive subunit influences the activity of the neighboring subunit. The results illuminate the vital role of the lock-and-key motif in modulating the GSH binding and the rate of catalysis. Additionally, the two active sites of the dimeric enzyme work synergistically. An observed water network, in hGSTP1-1 structures, connects the two active sites, thereby offering a mechanism for communication between the two active sites.</p><p>Cys⁴⁸ and Tyr⁵⁰ were targeted by mutations and chemical modifications for understanding how the α2 loop residues modulate GSH binding and catalysis. The replacement of Tyr⁵⁰ with different unnatural amino acids showed that the nature of the key residue side-chain influences the interaction with the lock structure and, consequently, the catalytic activity. The K_M^GSH, GSH affinity and protein stability can be modulated by fitting key residue into the lock cavity of the neighbor subunit and, consequently, restriction of the flexibility of the α2 loop. Optimization of the interaction between the key residue and the lock-cavity increases k_cat. Also, the crystal structure of the Cys-free variant was determined. The result indicated that Cys⁴⁸ restricts the flexibility of the α2 loop by interacting with surrounding residues and, consequently, contributes to GSH binding and protein stability.</p>

This thesis describes stereoselective syntheses of four amino acids, three of which are C-glycosidic analogues of glycosylated amino acids. The overall goal of the project was to probe the interactions between MHC molecules, glycopeptide antigens and T cell receptors, that are essential for development of collagen induced arthritis. Collagen induced arthritis is a frequently used mouse model for rheumatoid arthritis, an autoimmune disease that attacks joint cartilage and leads to a painful and eventually crippling condition. The thesis is based on four studies. The first study describes the synthesis of hydroxylysine, an amino acid that is found in collagen and is an important constituent of the glycopeptide proposed as an antigen in collagen induced arthritis. During the synthesis of hydroxylysine some new insight into the mechanism of the reductive opening of p-methoxybenzylidene acetals was obtained. The remaining three studies deals with the synthesis of C-glycosidic analogues of glycosylated amino acids, hydroxy norvaline, threonine and hydroxylysine.The synthesis of each amino acid required control of several stereogenic centra and utilizes a variety of approaches such as use of stereoselective reactions, chiral auxilaries, chiral templates and asymmetric catalysis. The C-glycosidic analogues of galactosylated hydroxynorvaline and hydroxylysine were incorporated in glycopeptides from type II collagen and evaluated in T cell response assays. It was found that the T cells were stimulated by the C-glycopeptides, but that higher concentrations were required than for the native O-glycopeptide

This thesis describes studies directed towards development of novel antiadhesive agents, with particular emphasis on compounds that prevent attachment of bacteria to a host-cell. Three different proteins involved in the assembly or function of adhesive pili in uropathogenic Escherichia coli have been targeted either by rational structure based design or statistical molecular methods. A library of substituted galabiose (Galα1-4Gal) derivatives was screened for binding to the E. coli adhesin PapG in an assay based on surface plasmon resonance, and for inhibition of Streptococcus suis adhesins PN and PO in a hemagglutination assay. The results were used to generate QSAR models which had good predictive powers and provided further insight in the structural requirements needed for high affinity binding. 2-pyridones and amino acid derivatives were modelled into the binding site of chaperones involved in pilus assembly in E. coli and a heuristic method, VALIDATE, was used for affinity prediction. The affinity of the compounds for the chaperones PapD and FimC were assessed in assays based on surface plasmon resonance and relaxation-edited NMR spectroscopy. Their ability to disrupt chaperone/subunit complexes was investigated in vitro through a FPLC assay and their capacity to inhibit pilus formation in vivo was determined via hemagglutination and confirmed with atomic force microscopy. Statistical molecular design was used to design a diverse peptide library targeting pili subunits, and an ELISA was developed to investigate the ability of the peptides to inhibit chaperone/subunit complexation. The resulting QSAR model provided extensive information regarding binding of the peptides to the subunits. Because the peptides were suggested to bind in an extended β-strand formation, β-strand mimetics consisting of oligomeric enaminones were designed. Finally, new methods to synthesize enaminone building blocks were developed using microwave assisted chemistry. The projects described have generated compounds that besides their value as leads for developing novel antibacterial agents, also constitute new chemical tools to study the mechanisms underlying bacterial virulence.

Organ-specific autoimmunity constitutes a large health problem, where both the clinical management and our understanding of the pathogenetic mechanisms need to improve. Women with Addison's disease have abnormally low levels of dehydroepiandrosterone (DHEA), its sulphate ester (DHEA-S) and androgens relative to age, and many patients complain of physical and mental fatigue and low stress tolerance. To define a suitable dose, the effect of oral DHEA replacement was evaluated in women with Addison's disease. DHEA was administered for three months to nine women with Addison's disease in either of two doses, 50 mg (n=5) or 200 mg (n=4). A dose of 50 mg restored the DHEA(S) and androgen levels to normal without altering the insulin sensitivity, body composition or serum lipid profile. Autoimmune polyendocrine syndrome type I (APS I) is a rare but useful model disorder of autoimmunity, characterised by multiple organ-specific autoimmune manifestations and high-titre autoantibodies and with adrenocortical insufficiency, Addison's disease, as one of its cardinal manifestations. Approximately 10-20% of APS I patients suffer from autoimmune hepatitis, which carries a high mortality, if untreated. The presence of putative antigenic targets in the liver was investigated. Cytochrome P4501A2 (CYP1A2) and aromatic L-amino acid decarboxylase (AADC) were identified as hepatic autoantigens with the use of APS I sera for immunofluorescent staining of normal human liver, Western blot of microsomal and cytosol fractions of human liver homogenate, and immunoprecipitation of in vitro transcribed and translated radioactively labelled proteins. The presence of CYP1A2- and AADC-antibodies was significantly correlated to AIH, and CYP1A2 antibodies inhibited enzyme activity in vitro. In conclusion, a daily replacement dose of 50 mg of DHEA sufficiently restores levels of DHEA, DHEA(S) and androgens in women with Addison's disease, without severe side-effects. We have further identified CYP1A2 and AADC as hepatic autoantigens associated with autoimmune hepatitis in APS I.

Lichens are fascinating symbiotic systems, where a fungus and a unicellular alga, most often green (bipartite green algal lichens; 90% of all lichens), or a fi lamentous cyanobacterium (bipartite cyanobacterial lichens; 10% of all lichens) form a new entity (a thallus) appearing as a new and integrated organism: in about 500 lichens the fungus is associated with both a cyanobacterium and an alga (tripartite lichens). In the thallus, the lichen bionts function both as individual organisms, and as a symbiont partner. Hence, in lichens, the participating partners must both be able to receive and acquire resources from the other partner(s) in a controlled way. Lichens are particularly successful in harsh terrestrial environments. In part this is related to their poikilohydric nature and subsequent ability to repeatedly become desiccated and hydrated. Metabolic activity, i.e. photosynthesis, respiration, and for cyanobacterial lichens N2-fixation, is limited to periods when the thallus is suffi ciently hydrated. Mineral nutrients are mainly acquired from dry or wet deposition directly on the thallus. Taken together it then appears that lichens are to a large extent passively controlled by their environment, making their control over resource allocation and acquisition particularly challenging. The aim of this thesis was to investigate resource acquisition and allocation processes in different lichens, and to see how these respond to changes in resource availability. This was done by following lichen growth in the fi eld during manipulation of water, light, and nutrient supply, and by assessing the responses of both the integrated thallus as well as the individual bionts. As a fi rst step, resource allocation and acquisition was investigated for a broad range of lichens aiming to determine the magnitude of metabolic variation across lichens. Seventy-fi ve lichen species were selected to cover as broad a spectrum as possible regarding taxonomy, morphology, habitat, and nitrogen requirements. The lichens had invested their nitrogen resources so that photosynthetic capacity matched respiratory carbon demand around a similar equilibrium across the contrasting species. Regulation of lichen growth was investigated in another study, using the two tripartite species Nephroma arcticum and Peltigera aphthosa, emphasizing the contribution of both internal and external factors. The empirical growth models for the two lichens were similar, showing that weight gain is to a higher extent dependent on those external factors that regulate their photosynthesis, whilst area gain is more controlled by internal factors, such as their nitrogen metabolism. This might be inferred from another study of the same species, where nitrogen manipulations resulted in an undisturbed weight gain, a similar resource allocation pattern between the bionts, but a distorted area gain. Aiming to investigate lichen nitrogen relations even further, lichens’ capacities to assimilate combined nitrogen in the form of ammonium, nitrate and amino acids were assessed using 14 contrasting boreal species. All these had the capacity to assimilate all the three nitrogen forms, with ammonium absorption being more passive, and nitrate uptake being low in bipartite cyanobacterial lichens. Differences in uptake capacities between species were more correlated to photobiont than to morphology or substrate preferences. Finally, to investigate intra-specifi c plasticity in relation to altered nutrient supply, resource investments between photo- and mycobiont were investigated in the two bipartite green algal lichens Hypogymnia physodes and and Platismatia glauca in a low and a high nutrient environ- in a low and a high nutrient environ- ment. In both species, more of the resources had been directed to the photobiont in the high nutrient environment also increasing their overall carbon status. Taken together, my studies indicate that in spite of the apparent passive environmental control on lichen metabolism, these symbiotic organisms are able to both optimize and control their resource acquisition and allocation processes.

The glutathione transferases (GSTs) represent a superfamily of dimeric proteins involved in cellular detoxication by catalyzing the nucleophilic addition of the reduced glutathione (GSH) to the hydrophobic electrophiles. The present work focuses on the functional role of the conserved structures of GSTP1-1. The lock-and-key motif is a highly conserved hydrophobic interaction in the subunit interface of Pi, Mu, and Alpha class GSTs. The key residue (Tyr50 in hGSTP1-1) of one subunit is wedged into a hydrophobic pocket of the neighboring subunit. The heterodimer GSTP1/Y50A was constructed from the fully active wild-type GSTP1-1 and the nearly inactive Y50A in order to study how an essentially inactive subunit influences the activity of the neighboring subunit. The results illuminate the vital role of the lock-and-key motif in modulating the GSH binding and the rate of catalysis. Additionally, the two active sites of the dimeric enzyme work synergistically. An observed water network, in hGSTP1-1 structures, connects the two active sites, thereby offering a mechanism for communication between the two active sites. Cys48 and Tyr50 were targeted by mutations and chemical modifications for understanding how the α2 loop residues modulate GSH binding and catalysis. The replacement of Tyr50 with different unnatural amino acids showed that the nature of the key residue side-chain influences the interaction with the lock structure and, consequently, the catalytic activity. The KMGSH, GSH affinity and protein stability can be modulated by fitting key residue into the lock cavity of the neighbor subunit and, consequently, restriction of the flexibility of the α2 loop. Optimization of the interaction between the key residue and the lock-cavity increases kcat. Also, the crystal structure of the Cys-free variant was determined. The result indicated that Cys48 restricts the flexibility of the α2 loop by interacting with surrounding residues and, consequently, contributes to GSH binding and protein stability.

名古屋大学年代測定総合研究センターシンポジウム報告

Chemical biology encompasses the study and manipulation of biological system using chemistry, often by virtue of small molecules or unnatural amino acids. Much insight has been gained into the mechanisms of biological processes with regards to protein structure and function, metabolic processes and changes between healthy and diseased states. As an ever expanding field, developing new tools to interact with and impact biological systems is an extremely valuable goal. Herein, work is described towards the synthesis of a small library of heterocyclic-containing small molecules and the mechanistic details regarding the interesting and unexpected chemical compounds that arose; an alternative set of non-toxic copper catalyzed azide-alkyne click conditions for in vivo metabolic labelling; and the synthesis of an unnatural amino acid for further chemical modification via [3+2] cycloadditions with nitrones upon incorporation into a peptide of interest. Altogether, these projects strive to supplement pre-existing methodology for the synthesis of small molecule libraries and tools for metabolic labelling, and thus provide further small molecules for understanding biological systems.

Traditional multiple hypothesis testing procedures, such as that of Benjamini and Hochberg, fix an error rate and determine the corresponding rejection region. In 2002 Storey proposed a fixed rejection region procedure and showed numerically that it can gain more power than the fixed error rate procedure of Benjamini and Hochberg while controlling the same false discovery rate (FDR). In this thesis it is proved that when the number of alternatives is small compared to the total number of hypotheses, Storeys method can be less powerful than that of Benjamini and Hochberg. Moreover, the two procedures are compared by setting them to produce the same FDR. The difference in power between Storeys procedure and that of Benjamini and Hochberg is near zero when the distance between the null and alternative distributions is large, but Benjamini and Hochbergs procedure becomes more powerful as the distance decreases. It is shown that modifying the Benjamini and Hochberg procedure to incorporate an estimate of the proportion of true null hypotheses as proposed by Black gives a procedure with superior power.<p> Multiple hypothesis testing can also be applied to regression diagnostics. In this thesis, a Bayesian method is proposed to test multiple hypotheses, of which the i-th null and alternative hypotheses are that the i-th observation is not an outlier versus it is, for i=1,...,m. In the proposed Bayesian model, it is assumed that outliers have a mean shift, where the proportion of outliers and the mean shift respectively follow a Beta prior distribution and a normal prior distribution. It is proved in the thesis that for the proposed model, when there exists more than one outlier, the marginal distributions of the deletion residual of the i-th observation under both null and alternative hypotheses are doubly noncentral t distributions. The outlyingness of the i-th observation is measured by the marginal posterior probability that the i-th observation is an outlier given its deletion residual. An importance sampling method is proposed to calculate this probability. This method requires the computation of the density of the doubly noncentral F distribution and this is approximated using Patnaiks approximation. An algorithm is proposed in this thesis to examine the accuracy of Patnaiks approximation. The comparison of this algorithms output with Patnaiks approximation shows that the latter can save massive computation time without losing much accuracy.<p> The proposed Bayesian multiple outlier identification procedure is applied to some simulated data sets. Various simulation and prior parameters are used to study the sensitivity of the posteriors to the priors. The area under the ROC curves (AUC) is calculated for each combination of parameters. A factorial design analysis on AUC is carried out by choosing various simulation and prior parameters as factors. The resulting AUC values are high for various selected parameters, indicating that the proposed method can identify the majority of outliers within tolerable errors. The results of the factorial design show that the priors do not have much effect on the marginal posterior probability as long as the sample size is not too small.<p> In this thesis, the proposed Bayesian procedure is also applied to a real data set obtained by Kanduc et al. in 2008. The proteomes of thirty viruses examined by Kanduc et al. are found to share a high number of pentapeptide overlaps to the human proteome. In a linear regression analysis of the level of viral overlaps to the human proteome and the length of viral proteome, it is reported by Kanduc et al. that among the thirty viruses, human T-lymphotropic virus 1, Rubella virus, and hepatitis C virus, present relatively higher levels of overlaps with the human proteome than the predicted level of overlaps. The results obtained using the proposed procedure indicate that the four viruses with extremely large sizes (Human herpesvirus 4, Human herpesvirus 6, Variola virus, and Human herpesvirus 5) are more likely to be the outliers than the three reported viruses. The results with thefour extreme viruses deleted confirm the claim of Kanduc et al.

The chiral vicinal diamine moiety is “privileged” and is widely found in catalysts and bio-active compounds. A series of seven chiral vicinal diamines with heterocyclic substituents have been synthesized with great enantiospecificity via the resonance assisted hydrogen bond driven diaza-Cope rearrangement reaction using 1,2-bis(2-hydroxyphenyl)-1,2-diaminoethane and heterocyclic aldehydes as starting materials. This thesis will also discuss the development of a new guanidine-based chiral shift rea-gent for determining the enantiopurity and the absolute configuration of α-amino acids by proton nuclear magnetic resonance (1H NMR) spectroscopy. The chiral shift reagent is easily synthesized from the commercially-available 1,2-diphenyl-1,2-diaminoethane. This method is advantageous over many previously described procedures for determining amino acid enantiopurity as it does not require prior derivatization of the analyte.