Protein kinases are important regulatory enzymes in signal transduction and in cell regulation. Understanding inhibition mechanisms of kinases is important for the further development of new therapies for cancer and inflammatory diseases. I have developed a statistical approach based on the Mantel test to find the relationship between the shapes of ATP binding sites and their affinities for inhibitors. My shape-based dendrogram shows clustering of the kinases based on similarity in shape. I investigate the pocket in terms of conservation of surrounding amino acids and atoms in order to identify the key determinants of ligand binding. I find that the most conserved regions are the main chain atoms in the hinge region and I show that the tetrahydropyran ring of staurosporine causes induced-fit of the glycine rich loop. I apply multiple linear regression to select distances measured between the distinctive parts of residues which correlate with the binding constants. This method allows me to understand the importance of the size of the gatekeeper residue and the closure between the first glycine of the GXGXXG motif and the aspartate of the DFG loop, which act together to promote tight binding to staurosporine. I also find that the greater the number of hydrogen bonds made by the kinase around the methylamine group of staurosporine, the tighter the binding to staurosporine. The website I have developed allows a better understanding of cross reactivity and may be useful for narrowing down the options for a synthetic strategy to design kinase inhibitors.

京都大学 / 0048 / 新制・課程博士 / 博士(理学) / 甲第21946号 / 理博第4524号 / 新制||理||1650(附属図書館) / 京都大学大学院理学研究科生物科学専攻 / (主査)准教授 佐藤 ゆたか, 教授 高橋 淑子, 教授 中務 真人 / 学位規則第4条第1項該当 / Doctor of Science / Kyoto University / DGAM

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English Abstract Shortly after the identification of HIV as a causative agent of AIDS, an aspartic protease was identified in the viral genetic information. The very same time protease has become one of the dominant therapeutical targets in AIDS therapy. The introduction of protease inhibitors into the antiretroviral therapy has led to a significant improvement in the quality and length of life of HIV patients. However, the virus is still able to effectively prevent the impact of an inhibitor via generating inhibitor-resistant mutated protease variants. Thus, there is a constant need for novel types of inhibitors that would be capable of effectively blocking these resistant variants and simultaneously not supporting the development of novel resistant viral strains. One way to identify such inhibitors could be searching for compounds interacting with the enzyme at different sites than the active cavity, via the mechanisms of noncompetitive or uncompetitive inhibition. The group of compounds called metallacarboranes - inorganic compounds consisting of carbon, boron, hydrogen and metall ion - were shown to exhibit such an activity against HIV-1 protease. However, for further optimization of these inhibitors, detailed biophysical investigation of the enzyme-inhibitor complex is needed. This work focuses on the...

Background Promoters are key players in gene regulation. They receive signals from various sources (e.g. cell surface receptors) and control the level of transcription initiation, which largely determines gene expression. In vertebrates, transcription start sites and surrounding regulatory elements are often poorly defined. To support promoter analysis, we present CORG http://corg.molgen.mpg.de, a framework for studying upstream regions including untranslated exons (5' UTR). Description The automated annotation of promoter regions integrates information of two kinds. First, statistically significant cross-species conservation within upstream regions of orthologous genes is detected. Pairwise as well as multiple sequence comparisons are computed. Second, binding site descriptions (position-weight matrices) are employed to predict conserved regulatory elements with a novel approach. Assembled EST sequences and verified transcription start sites are incorporated to distinguish exonic from other sequences. As of now, we have included 5 species in our analysis pipeline (man, mouse, rat, fugu and zebrafish). We characterized promoter regions of 16,127 groups of orthologous genes. All data are presented in an intuitive way via our web site. Users are free to export data for single genes or access larger data sets via our DAS server http://tomcat.molgen.mpg.de:8080/das. The benefits of our framework are exemplarily shown in the context of phylogenetic profiling of transcription factor binding sites and detection of microRNAs close to transcription start sites of our gene set. Conclusion The CORG platform is a versatile tool to support analyses of gene regulation in vertebrate promoter regions. Applications for CORG cover a broad range from studying evolution of DNA binding sites and promoter constitution to the discovery of new regulatory sequence elements (e.g. microRNAs and binding sites).

The localization of substance P (SP) binding sites in guinea-pig heart was studied by in vitro autoradiography, and pharmacological effects of SP were examined with isolated heart preparations. Specific binding of [125I]SP was found in association with cardiac parasympathetic ganglia and some coronary arteries. No specific SP binding sites were associated with coronary veins, atria, ventricles, ascending aorta or pulmonary trunk. Local bolus injections of SP (2.5 and 25 nmol) caused a bradycardia which, in some preparations, was followed by a slight tachycardia. SP produced a prominent coronary vasodilator effect after basal perfusion pressure had been elevated by 1 μM vasopressin. The vasodilator response was probably mediated by the SP binding sites associated with the coronary arteries. Bradycardia might be elicited by binding of SP to the receptors present in the parasympathetic ganglia and subsequent release of acetylcholine. It is suggested that these effects of SP on the isolated heart could be of physiological significance.

Zinc is an essential metal ion for the activity of multiple enzymes and transcription factors. Among many other transporting proteins human serum albumin (HSA) is the main carrier of Zn(II) in the blood plasma. HSA displays multiple ligand binding sites with extraordinary binding capacity for a wide range of ions and molecules including fatty acids. Hence, HSA controls the availability and distribution of those molecules throughout the body. Previous studies have established that the existence of one zinc site with high affinity (MBS-A) that is modulated by the presence of fatty acids. Therefore, the fatty acid concentration in the blood influences zinc distribution which may result in a significant effect on both normal physiological processes and a range of diseases. Based on the current knowledge of HSA's structure and its coordination chemistry with zinc ion, here, we attempted to investigate zinc interactions and coordination with HSA and the effect of different fatty acids on the protein structure, stability and on Zn(II) binding. By NMR titration, we examine the Zn(II) binding to HSA and the spectra show distinct movements of some resonances showing a conformational change has occurred as a result of Zn(II) binding. Isothermal calorimetry titrations study was performed to evaluate zinc binding affinity to HSA in the absence and presence of fatty acids. Free HSA results indicates the existence of one high affinity site and multiple low affinity sites. Upon the binding of fatty acids to HSA, three distinct behaviors of Zn(II) affinity was observed ranging from no effect to moderate to significant depending on the FAs. By the use of circular dichroism, we investigate secondary and tertiary structure of HSA in the presence and absence of FAs and Zn(II). We found albumin is predominately α-helical and the overall conformation of the protein remains unchanged even after interacting with FAs and Zn(II) with some exception. The structural stability of HSA was evaluated by obtaining the denaturation temperature in the presence and absence of fatty acid and we found the thermal denaturation of HSA increases with the increase of amount of fatty acids.

Influenza A viruses (IAVs) cause occasional pandemics and seasonal epidemics, thus presenting continuous challenges to public health. Vaccination is the primary strategy for the prevention and control of influenza outbreaks. The antigenicity matched high-yield seed strain is critical for the success of influenza vaccine. Currently, there are several limitations for the influenza vaccine manufacture: 1) the conventional methods for generating such strains are time consuming; 2) egg-based vaccines, the predominant production platform, have several disadvantages including the emergence of viral antigenic variants that can be induced during egg passage; 3) vaccine seed viruses often do not grow efficiently in mammalian cell lines. Previous studies suggested that mutations in the receptor binding site (RBS) that locates at the globular head of the HA1 can change IAVs’ binding specificity, antigenicity, and yield and thus RBS would be an potential target for engineering vaccine seed strain. However, systematic analysis of the mutations on RBS affecting those viral phenotypes is lacking. Specifically, this dissertation has following aims: Firstly, we developed a novel method to rapidly generate high-yield candidate vaccine strains by integrating error-prone PCR, site-directed mutagenesis strategies, and reverse genetics. The error-prone PCR- based reverse genetic system could also be applied to gain-ofunction studies for influenza virus and other pathogens; Secondly, in this dissertation, we identified an Y161F mutation in the hemagglutinin (HA) that enhanced the infectivity and thermostability of virus without changing its original antigenic properties which would prompted the development of cell-based vaccines; Thirdly, the molecular mechanisms underlying host adaption of equine-origin influenza A(H3N8) virus from horses to dogs are unknown. This dissertation identified that a substitution of W222L in the HA of the equine-origin A(H3N8) virus facilitated its host adaption to dogs. This mutation increased binding avidity of the virus specifically to sialyl Lewis X motifs, which were found abundantly in the submucosal glands of dog trachea but not in equine trachea. To summary, this dissertation investigated the role of RBS in IAVs biology and expanded the current knowledge toward IAV vaccine strain engineering, IAV host adaption and evolution.

The canonical model of transcriptional regulation in prokaryotes restricted binding site locations to promoter regions and suggested that the binding sequences serve as the main determinants of binding. In this dissertation, I challenge these assumptions. As a member of the TB Systems Biology Consortium, I analyzed and validated ChIP-Seq and microarray experiments for over 100 transcription factors (TFs). In order to study the transcriptional functions of predicted binding sites, I integrated binding and expression data and assigned potential regulatory roles to 20% of the binding sites. Stronger binding sites were more often associated with regulation than weaker sites, suggesting a correlation between binding strength and regulatory impact. Seventy-six percent of the sites fell into annotated coding regions and a significant proportion was assigned to regulatory functions. To study the importance of binding sequences, I compared experimental sites with computational motif predictions. Although a conservative binding motif was found for most TFs, only a fraction of the observed motifs appeared bound in the experiment. Some low-affinity binding sites appeared occupied by the corresponding TF while many high-affinity binding sites were not. Interestingly, I found exactly the same nucleotide sequences (up to 15 residues long) bound in one area of the genome but not bound in another area, pointing to DNA accessibility as an important factor for in vivo binding. To investigate the evolutionary conservation of binding-site occupancy, sequence, and transcriptional impact, I analyzed ChIP-Seq and expression experiments for five conserved TFs for two-to-four Mycobacterial relatives. The regulon composition showed significantly less conservation than expected from the overall gene conservation level across Mycobacteria. Despite expectations, sequence conservation did not serve as a good indicator of whether or not a computationally predicted motif was bound experimentally; and in some cases, a fully conserved motif was bound in one relative but not in the other. Conservation of genic binding sites was higher than expected from the random model, adding to the evidence that at least some genic sites are functional. Understanding the evolutionary story of binding sites allowed me to explain unusual site configurations, some of which indicated a role for DNA looping.

Subsurface microbes are extremophiles adapted to thrive in deep, resource-limited environments, performing crucial roles in a myriad of biogeochemical processes. The extremozymes they produce might play a pivotal role in catalyzing these processes. Identifying and characterizing those enzymes could contribute to the advancements in industrially important biocatalytic reactions. Among various enzymes, sorbitol dehydrogenases are enzymes that catalyze the reversible conversion of sorbitol into fructose in the presence of NAD+. In this study, we focus on the exploration of a sorbitol dehydrogenase (SDHSIUC1) derived from the novel strictly anaerobic, thermophilic, subsurface bacterium, Caldiatribacterium inferamans SIUC1, which is one of the first cultured members from the candidate phylum Atribacteria OP9. As SDHSIUC1 originated from a subsurface microbe, we hypothesized that the enzyme has industrially beneficial characteristics such as higher thermostability and can be used for bioindustry applications such as synthesis of rare sugars and chiral alcohols. We successfully cloned, expressed, and purified the functional SDHSIUC1 enzyme aerobically using E. coli BL21(DE3) and did biochemical assays to characterize its properties. Additionally, in combination with the findings of biochemical characterization, we applied in silico approaches such as molecular modeling and molecular docking to describe the functional mechanism of the enzyme. Initial phylogenetic tree analysis using a pool of 24 amino acid sequences showed that the closest relative for SDHSIUC1 is a Candidatus Caldiatribacterium californiense, which is an uncultured member of the Atribacteria phylum. Size exclusion chromatography and Native-PAGE suggested that SDHSIUC1 is a hexamer with a size of 225 kDa. Kinetic characterization of the SDHSIUC1 showed that the enzyme has a higher affinity for sorbitol and fructose in the presence of NAD+ and NADH, respectively. Furthermore, SDHSIUC1 enzyme is promiscuous as it could utilize other polyols (i.e., glycerol, xylitol, inositol), diols (i.e., butanediol), aldehydes (i.e., glycolaldehyde), and ketoses (i.e., sorbose) in the presence of NAD+/NADH cofactors. We observed a significant increase in enzyme activity in the presence of Zn2+, where other metal ions such as Mn2+ and Mg2+ also resulted in rate improvements. The enzyme is an alkaline dehydrogenase that prefers a higher pH above 8. The effect of temperature on SDHSIUC1 activity showed that it’s a thermophilic enzyme with activity at 85 ℃. The thermal denaturation points of the enzyme at 85 ℃ was increased when the enzyme was preincubated at 85 ℃ in the presence of Zn2+. Notably, the enzyme preincubated 25 min at 85 ℃ in the presence of Zn2+ prefers fructose conversion and ceased the sorbitol conversion. We identified the presence of a structural Zn2+ binding site in SDHSIUC1 in addition to a catalytic Zn2+ binding site. We speculated that the structural Zn2+ involves thermal stability of the enzyme. Hence, we mutated the cysteine with serine of potential structural Zn2+ binding site (Cys96, Cys99, Cys102, and Cys110). Indeed, the Inductively coupled plasma mass spectrometry (ICP-MS) analysis revealed the mutated enzyme contains a lower amount of Zn2+ relative to the native enzyme. The data revealed that the mutated enzyme has low melting temperature (78 ℃) relative to the native enzyme (92 ℃), suggesting that structural Zn2+ is key to enhance the thermal stability of the SDHSIUC1. Surprisingly, we observed that the mutant enzyme completely lost its activity. The data suggests the role of structural Zn2+ binding site on both the structural and functional stability of SDHSIUC1. In consistence with the in-vitro data, the preliminary computational modeling data suggest that the losing structural Zn2+ unstable the enzyme and we are conducting in depth in-silico study to unveil the mechanism(s). We envisioned that the mechanisms behind the thermal stability of SDHSIUC1 could be used as basic model to enhance thermostable protein for the industrial application (e.g., design thermostable plastic hydrolyzing enzymes). To further demonstrate the potential applications of the SDHSIUC1, we genome-integrated it into the industrially important microorganism Pseudomonas putida KT2440. The resulting strain exhibited significantly increased growth in the presence of sorbitol compared to the wild-type P. putida KT2440, highlighting the potential of this enzyme for industrial applications such as enabling sorbitol catabolism or establishing xylose reductase pathway in P. putida KT2440 (i.e., leverage xylitol dehydrogenase activity of SDHSIUC1). In summary, this study has uncovered a novel thermostable sorbitol dehydrogenase from a subsurface microbe, which could have potential applications in the bioindustry where thermostable sorbitol dehydrogenases are required for the application in food and beverage industry, pharmaceutical industry, biofuel production etc. as it would be advantageous for the industrial processes.