Aspergillus flavusis a filamentous fungusthat causes an ear and kernel rot in maize (Zea mays L.). It produces a toxic secondary metabolite, aflatoxin, on the colonized maize kernels. Aflatoxin is a carcinogen to humans and animals. The toxin is also an immunosuppressant and causes aspergillosis in immune compromised individuals. Therefore, the presence of aflatoxin in food is strictly regulated by governmental agencies. Contaminated food leads to severe loss in profit and in marketable yield. There has been extensive research to investigate resistance of maize toA. flavus. Certain lines of maize exhibit increased resistance to A. flavus and aflatoxin accumulation compared to others and correlated with that are proteins and metabolites that differ in abundance in those lines. Among them are members of the cupin superfamily of proteins and products of special nitrogen metabolism (derived from glutamate). The goal here was to identify networks underlying disease resistance indifferent maize genotypes through the identification of protein-protein interactions and the analysis of transcript abundance profiles realting to cupins and glutamate. The outcome will be an understanding of host resistance to A. flavussufficient to develop methods to prevent pre-harvest contamination by aflatoxin. A protein abundant in resistant maize was identified as a cupin and named ZmCUP1. The cDNA isolation, expression in E. coliand characterization of the protein encoded by the mRNA, Zmcup1, lead to the discovery that the ZmCUP1 protein had anti fungal properties and oxalate decarboxylase activity (EC 4.1.1.2). Another part of the project aimed at understanding the involvement of a transgene that encoded bacterial NADPH-glutamate dehydrogenase (GDHA; EC 4.2.3.1) that reduced aflatoxin accumulation by half. A maize partial predicted protein to protein interactome was built and used to identify potential interactions between proteins expressed differentially in lines of maize resistant to A. flavus. These interactions were characterized in-silico and one specific interaction, between Zmcup1 and a maize zinc finger protein was characterized in vitro.

The biophysical properties, biological activity and function of macromolecular systems are highly dependent on their structure. Structure-activity relationships of proteins and their binding partners are critical for drug discovery, biochemical and medical research. While the gas-phase environment might present as an unusual venue from which to explore protein structure, for over the past two decades, nano-electrospray ionization (nESI) coupled to mass spectrometry (MS) has been recognized as having great potential for analysis of protein structure and protein non-covalent complexes. In conjunction with related technique of ion mobility (IM), mass spectrometry (IM-MS) provides insights into protein native-like conformations and any structural changes in may undergo upon ligand binding or alternations induced via physical parameters such as temperature, pressure or solution conditions. As most proteins tend to exist as multiple domains; from the distribution of oligomeric states in the Protein Data Base (PDB) 86% of proteins exist as oligomers; the work presented in this thesis focuses on application of MS techniques to probe the tertiary and quaternary structure of various large and multimeric protein complexes, their dynamics and/or conformational changes. Wherever relevant, the gas-phase studies reported here are complemented by other techniques, such as hydrogen deuterium exchange MS (HDX), molecular modelling (MD) and analytical ultracentrifugation (AUC). Firstly, the dynamics of intact monoclonal antibodies (mAbs) and their fragments are explored with IM-MS. Variations observed in conformational landscapes occupied by two mAb isotypes are rationalized by differences in disulfide linkages and non-covalent interactions between the antibody peptide chains. Moreover, mAb intrinsic flexibility is compared to other multimeric protein complexes in terms of collision cross section distribution span. Secondly, variable temperature MS (VT-MS) and variable temperature IM-MS (IM-MS) are used to probe unfolding and dissociation of four standard multimeric protein complexes (TTR, avidin, conA and SAP) as a function of the of analysis environment temperature. VT-MS is found to allow for decoupling of their melting temperature (Tm) from the protein complex dissociation temperature (TGPD). Whereas, VT-IM-MS is used to investigate structural changes of these protein complexes at elevated temperatures and provide insights into the thermally induced dissociation (TID) mechanism, as well as strength of the non-covalent interactions between subunits. Thirdly, VT-(IM)-MS methodology is applied to study behaviour of three mAbs: IgG1, IgG4 and an engineered IgG4 of increased thermal stability. Such analysis shows to be promising for comparative thermal stability studies for proteins of therapeutic interest. Lastly, the structure of ATP-phosphoribosyltransferase (MtATPPRT), an enzyme catalysing the first step of the biosynthesis of L-histidine in Mycobacterium tuberculosis, is explored. Conformational changes occurring upon feedback allosteric inhibition by L-histidine are probed with MS, IM-MS, HDX-MS and AUC. Reported results serve as the basis for IM-MS/HDX-MS based screening method to be used for screening of a library of novel and promising anti-tuberculosis agents.

Septinas pertencem a uma família de proteínas de ligação a GTP e são encontradas em diversos eucariontes, participando de diferentes processos celulares citoesqueléticos. As septinas apresentam um domínio central de ligação a GTP (domínio G) flanqueado por uma região amino-terminal e outra carboxi-terminal. As septinas se caracterizam por interagirem entre si formando heterocomplexos que se polimerizam, constituindo filamentos. A única estrutura resolvida de um complexo de septinas é de um hexâmero, composto por duas subunidades de três septinas humanas diferentes: SEPT7-SEPT6-SEPT2-SEPT2-SEPT6-SEPT7. Esta estrutura revelou que a formação do filamento envolve interações conservadas entre os domínios G, estando o restante da estrutura desordenado no cristal. Além disso, mostrou que dois tipos de interface se alternam ao longo do filamento, as chamadas interfaces G (que incluem a região de ligação do nucleotídeo de duas subunidades) e interfaces NC (que incluem as regiões N e C-terminais do domínio G). Várias evidências sugerem que as regiões C-terminais da proteína sejam as principais responsáveis pela seleção do parceiro correto de interação para montagem dos heterocomplexos. Nesse contexto, buscou-se avaliar a importância das regiões C-terminais na seleção das parceiras SEPT6 e SEPT7 para formar a interface NC, frente ao domínio G. Inicialmente, uma septina quimérica foi produzida de forma a conter o domínio G de SEPT2 e o C-terminal de SEPT6, gerando SEPT2G6C. As proteínas SEPT7GC, SEPT6GC, SEPT2GC e SEPT2G6C foram expressas e purificadas separadamente. Análises de estabilidade térmica e de afinidade proteína-proteína dos pares indicou que a quimera foi capaz de interagir com SEPT7GC, gerando o heterodímero SEPT7GC-SEPT2G6C, mas este não se mostrou tão estável quanto o heterodímero fisiológico. Foi também avaliada a importância da ligação do nucleotídeo para formação da interface G e, para isso, foram construídos os mutantes SEPT2GT78M e SEPT2GD185N, cujos resíduos importantes para hidrólise e ligação do nucleotídeo, respectivamente, foram alterados. A análise de oligomerização por cromatografia de exclusão molecular mostrou deslocamento no volume de eluição das proteínas expressas sozinhas e coexpressas com SEPT6G, indicando que a formação do dímero via interface G depende da ligação do nucleotídeo, mas não da sua hidrólise. Finalmente, foi avaliada a estabilidade térmica e estrutural e a propensão à formação de amilóides do heterodímero SEPT6G-SEPT2G, o qual apresentou maior estabilidade estrutural quando comparado ao homodímero de SEPT2G, mas ainda exibiu alteração de sua estrutura para um estado capaz de ligar Thioflavina-T, sugerindo a formação de amilóides. Entretanto, isso foi observado em temperaturas cerca de 30 ºC acima daquela observada para o homodímero, confirmando a maior estabilidade do heterodímero e sugerindo que a formação da interface G com o parceiro correto pode ser um fator importante na prevenção da formação de estruturas amilóides à temperaturas fisiológicas. / Septins belong to a family of GTP binding proteins and are found in several eucaryotes, participating in different cytoskeletal cell processes. The septins have a central GTP binding domain (G domain) flanked by an amino-terminal and a carboxy-terminal regions. The septins are characterized by the ability to interact with each other forming heterocomplexes which polymerize themselves, forming filaments. The only solved structure of a septin complex is a hexamer, formed by two subunits of three different human septins: SEPT7-SEPT6-SEPT2- SEPT2-SEPT6-SEPT7. This structure revealed that the filament arrangement involves conserved interaction between G domains, being the remainder of the structure disordered in the crystal. Moreover, two types of interface alternate along the filament were shown, socalled G interfaces (which include the nucleotide binding region of the two subunits) and NC interfaces (which include the N- and C- terminal regions of G domain). Plenty of evidences suggest that C-terminal regions of the protein are the main responsible for the selection of the correct interaction partner to assembly of heterocomplexes. In this context, it was sought to evaluate the importance of the C-terminal regions in the selection of the partnerships SEPT6 and SEPT7 to form the NC interface, against the G domain. For this, a chimerical septin was designed so that contains the G-domain of SEPT2 and the C-terminal of SEPT6, creating SEPT2G6C. The SEPT7GC, SEPT6GC, SEPT2GC and SEPT2G6C proteins were expressed and purified individually. Thermal stability and protein-protein affinity analysis of the pairs indicated that the chimera was able to interact with SEPT7GC, forming the heterodimer SEPT7GC-SEPT2G6C, which, however, did not show as stable as the physiological heterodimer. The importance of nucleotide binding to the interaction through G interface was also evaluated and, for that, SEPT2 mutants on GTP-domain were constructed, SEPT2T78M and SEPT2D185N, whose important residues in the hydrolysis and linking of nucleotide, respectively, were changed. Oligomerization analysis by size exclusion chromatography showed a shift in the elution volume of proteins expressed alone and coexpressed with SEPT6, indicating that the complexation of proteins to form G interface depends on the nucleotide binding, but not on its hydrolysis. Finally, the thermal and structural stability and the propensity to amyloid formation of heterodimer SEPT6G-SEPT2G were evaluated, which showed greater structural stability when compared to SEPT2 homodimers, but still exhibited alteration of its structure to a state that was able to bind Thioflavin-T, suggesting amyloid formation. However, this was observed at temperatures around 30 ºC above that observed for the homodimer, confirming the greater conformational stability of the heterodimer and suggesting that the formation of G interface with the right partner can be an important factor of the amyloid filament prevention at physiological temperatures.

Kyoto University (京都大学) / 0048 / 新制・課程博士 / 博士(工学) / 甲第17599号 / 工博第3758号 / 新制||工||1573(附属図書館) / 30365 / 京都大学大学院工学研究科合成・生物化学専攻 / (主査)教授 濵地 格, 教授 梅田 眞郷, 教授 杉野目 道紀 / 学位規則第4条第1項該当

>Magister Scientiae - MSc / Retinoblastoma Binding Protein 6 (RBBP6) is a 200 kDa human protein known to play an essential role in mRNA 3’-end processing, as well as functioning as an E3 ligase to catalyze ubiquitination and suppression of p53 and other cancer-associated proteins. A RBBP6 knockout mouse model previously suggested that RBBP6 cooperates with MDM2 in polyubiquitinating p53, but is not able to ubiquitinate p53 without the assistance of MDM2. However, unpublished studies from our laboratory suggest that the N-terminal 335 residues of RBBP6, known as R3, are able to ubiquitinate p53 in full in vitro assays, and that the isolated RING finger of RBBP6 is able to catalyse ubiquitination of itself, a phenomenon known as auto-ubiquitination. It is, however, possible that other domains within RBBP6, in particular the ubiquitin-like DWNN domain situated near to the RING finger, may modulate the autoubiquitination and substrate-ubiquitination potentials of the complete protein. / 2022

2-ω-carboxyethylpyrrole (CEP) is a product of docosahexaenoic acid (DHA) oxidation, which forms covalent adducts with different proteins. CEP-modified proteins can interact with macrophage receptor, integrin αDβ2. This study aims to compare αDβ2 binding to its physiological ligand, fibrinogen, and CEP-modified fibrinogen, which is formed during inflammation. We hypothesize that modification of fibrinogen changes its ligand-binding properties to integrin αDβ2 which can affect macrophage migration and retention. Recombinant αD I-domain and αDβ2-transfected HEK293 cells were used for the experiments. Using biolayer interferometry, we found that the affinity of αD I-domain binding to fibrinogen-CEP was higher than fibrinogen and inhibited by the anti-CEP antibody. In agreement, αDβ2-transfected cells demonstrated stronger adhesion to fibrinogen-CEP and this adhesion was significantly inhibited by polyglutamic acid that mimics CEP-mediated binding. These findings suggest that αDβ2's interaction with DHA-modified extracellular matrix (ECM) proteins significantly increases macrophage adhesion and may serve for macrophage retention during chronic inflammation.

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