Orientador: Ana Carolina de Mattos Zeri / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-21T00:57:37Z (GMT). No. of bitstreams: 1 Nogueira_MariaLuizaCaldas_M.pdf: 8261820 bytes, checksum: 7c9ed2193fd77623b3a8beea794eb743 (MD5) Previous issue date: 2012 / Resumo: A mTOR é uma via de sinalização muito conservada que controla o crescimento celular em resposta à presença de nutrientes e fatores de crescimento. A desregulação dessa via em humanos está relacionada a doenças como câncer e diabetes. A quinase TOR é ativada na presença de aminoácidos e recentemente descobriu-se que as pequenas GTPases da família Rag são mediadoras da sinalização por Leucina. Essas GTPases são ancoradas na superfície do lisossomo por meio da interação com um complexo de três proteínas denominado Ragulator. Esse complexo também ancora um braço da via das MAPKs (MEK-ERK) aos lisossomos. O entendimento deste complexo pode nos ajudar a elucidar doenças em que a via da mTOR se encontra desregulada. Neste trabalho obtivemos o complexo Ragulator, através da expressão da proteína p18 em corpos de inclusão e sua renaturação através da adição de suas parceiras Mp1/p14 à diálise. Foram realizados estudos biofísicos com a intenção de caracterização do complexo, entretanto o alto grau de dissociação do mesmo resultou em certa dificuldade para caracterizá-lo completamente. Neste trabalho caracterizamos os agregados formados pela p18 e conseguimos reduzir sua formação através de diálise contendo agente redutor e suas proteínas parceiras. A renaturação da p18 na presença de MP1/p14 favoreceu seu rendimento, indicando a interação entre estas proteínas, porém não foi possível estabilizar o complexo Ragulator O estudo da divisão bacteriana é centralmente dependente de FtsZ, um homólogo procariótico das tubulinas. FtsZ desencadeia a divisão ao formar o "anel Z", uma estrutura supramolecular constituída por polímeros de FtsZ que circunda o interior da célula e funciona como arcabouço do aparato de divisão. A formação do anel Z é regulada por moduladores, proteínas que afetam tanto negativamente como positivamente a capacidade de FtsZ polimerizar-se. A proteína MinC é um inibidor da polimerização de FtsZ, recrutada por MinD para a face interna da membrana plasmática, onde o complexo MinCD exerce sua função. MinCD representa um inibidor sítio-específico da polimerização da FtsZ, previnindo a formação do anel Z nos pólos das células mas permitindo que isto aconteça na região central. A elucidação deste processo seria de grande valia para o desenho racional de inibidores da divisão bacteriana. Neste trabalho, comprovamos a interação entre MinC e FtsZ por Ressonância Magnética Nuclear. Estes proteínas não se encontravam em sua forma monomérica e o alto peso molecular do complexo impossibilitou a identificação dos aminoácidos envolvidos nesta interação, devido a limites da técnica 15NHSQC. No momento, a proteína MinC está sendo expressa em presença de deutério, o que aumenta significativamente o limite da técnica de 15NHSQC. Foram realizados ainda estudos biofísicos com intuito de caracterização da interação / Abstract: The mTOR signaling pathway is a very well conserved pathway that controls cell growth in response to the presence of nutrients and growth factors. Deregulation of this pathway in humans is related to diseases like cancer and diabetes. The TOR kinase is activated in the presence of amino acids and it was recently discovered that the Rag small GTPases family are mediators of signaling by Leucine. These GTPases are anchored on the surface of the lysosome through interactions with a complex of three proteins called Ragulator. This complex also anchors an arm of the pathway of MAPKs (MEK-ERK) to lysosomes. Understanding this can help us to elucidate complex diseases in which the mTOR pathway is upregulated. In this work, the Ragulator complex was obtained through the expression of p18 protein in inclusion bodies and their refolding by adding their partners MP1/p14 to dialysis. Biophysical studies were conducted with the intention of characterizing the complex, however its high degree of dissociation resulted in some difficulty to characterize it completely. In this work we characterized the aggregates formed by p18 and managed reduce its formation by dialysis containing reducing agent and its partner proteins. The p18 renatuation with MP1/p14 improve its yield, indicating interaction among these proteins, however the Ragulator complex wasn't stabilized. The study of bacterial division is centrally dependent on FtsZ, a prokaryotic homologue of tubulin. FtsZ triggers the division to form the "Z ring", a supramolecular structure consisting in FtsZ polymers that surrounds the cell and acts as a frame of the division apparatus. The formation of the Z ring is regulated by modulators, proteins that affect both negatively and positively the ability of FtsZ to polymerize. The MinC protein is an inhibitor of FtsZ polymerization, recruits MinD to the inner surface of the plasma membrane, where the complex MinCD exerts its function. MinCD is an inhibitor of site-specific polymerization of FtsZ, preventing the formation of the Z ring at the poles of the cells but allowing this to happen in the central region. The elucidation of this process would be invaluable for the rational design of bacterial division inhibitors. In this work, we confirmed the interaction between MinC and FtsZ by Nuclear Magnetic Resonance. These proteins were not in their monomeric form and the high molecular weight of the complex prevented the identification of the amino acids involved in this interaction, due to limitations of the 15NHSQC technology. At present, the MinC protein is being expressed in the presence of deuterium, which significantly increases the limit of this technique 15NHSQC. Biophysical studies were also performed with the aim of characterizing the interaction / Mestrado / Bioquimica / Mestre em Biologia Funcional e Molecular

Mycobacterium tuberculosis, the principal causative agent of tuberculosis (TB) in humans, is considered to be a successful pathogen owing to the elicitation of multidrug resistance, ability to survive inside macrophage phagosomes by taking nutrients from host cell cytoplasm, and the capacity to alternate between proliferating and dormant (nonproliferating) conditions of growth. Thus, whether one looks at tubercle bacillus from the standpoint of regulation of cell division in the host system, or uptake of nutrients from the host cell cytoplasm or elicitation of drug resistance, the requirement for ATP Binding Cassette (ABC) transporter type protein complexes, which might be involved in the transport of drugs, nutrients or proteins, could be of critical importance to the pathogen. Therefore the present study was initiated to characterize ABC transporter type proteins, FtsE and FtsX of M. tuberculosis (MtFtsE and MtFtsX), and their interaction with FtsZ and FtsQ, which are the septation proteins that are recruited respectively before and after the localization of FtsE and FtsX proteins. The study was carried out in 3 parts. 1. Cloning, overexpression and purification of MtFtsE and MtFtsX proteins and elucidation of ATP binding activity of MtFtsE There exists considerable extent of homology between the FtsE and FtsX proteins of M. tuberculosis and E. coli. Therefore, in order to verify whether the structural homology is reflected in functional homology, complementation of growth defect of E. coli ftsE (Ts) by MtFtsE and MtFtsX was carried out. The MtFtsE protein could partially complement growth defect of E. coli ftsE temperature sensitive strain MFT1181, whereas co-expression of MtFtsE and MtFtsX efficiently complemented growth defect, indicating that the MtFtsE and MtFtsX proteins functionally complement E. coli FtsE and FtsX and that the two proteins together might be performing an associated function. Subsequently, in order to biochemically characterize MtFtsE and MtFtsX proteins of M. tuberculosis, MtftsE gene was cloned in pQE30, overexpressed, purified by Ni2+-NTA agarose affinity chromatography under denaturing conditions and refolded. MtFtsX protein, being toxic to E. coli cells, could not be expressed to sufficient amounts. Western blotting with anti-MtFtsE antibody showed that the recombinant 6xHis-MtFtsE protein and the native MtFtsE protein were localized to the membrane of E. coli and M. tuberculosis cells respectively. 6xHis-MtFtsE protein showed ATP binding in vitro, whereas K42R mutation abolished ATP binding. Thus, like in the case of E. coli FtsE, the K42 residue, which is positionally equivalent to K41 in EcFtsE in Walker A motif, was found to be essential for ATP binding. At 1.3 nM concentration of [α32P] ATP,70 molar excess of ATP, ADP, AMP, and GTP competed out respectively 97%, 87%, 73% and 57% of the [α32P] ATP bound to 6xHis-MtFtsE. 2. Biochemical characterization of MtFtsE protein The functional architecture of an ABC transporter consists of two each of nucleotide binding domain (NBD) and transmembrane domain (TMD), which are either part of a single polypeptide chain or individual subunits. The functional NBD is a ‘nucleotide-sandwich dimer’ with ATP flanked by the Walker A and B motifs of one NBD and the signature motif and D-loop of the other. NBD, through ATPase activity, is involved in energizing the transport of substrates namely drugs, proteins, ions, and solutes across the membrane. Since MtFtsE possesses Walker A and Walker B motifs that constitute NBD, and MtFtsX possesses TMD (four transmembrane segments), the two proteins together might constitute an ABC transporter type complex. Therefore, we wanted to know whether MtFtsE could hydrolyze ATP. MtFtsE not only could bind ATP with high affinity but could hydrolyse it also (Km, 1.5 µM; Vmax, 0.87 nmole/mg/min). It could bind and hydrolyse GTP as well, but not CTP, albeit with lower affinity and rate (Km, 25 µM; Vmax, 0.54 nmole/mg/min). The ATPase activity is strongly dependent on Mg2+ or Mn2+, with a pH optimum of 6.5 – 8.0 and temperature range of 27oC - 40oC. Kinetic analysis of ATPase and GTPase activities indicated nucleotide- dependent cooperativity (Hill coefficient for ATP is 1.7 and for GTP, 2.1). Inhibition of ATPase activity, to almost similar extent, in the presence of 10-fold excess of ATPγS, ADP, AMP, GTP, and CTP, but not TTP, indicated that nucleotide binding is through nitrogenous base of the nucleotide. Inhibition of MtFtsE by orthovanadate classified the enzyme as a P-type ATPase. Partially purified MtFtsE in soluble fraction also showed ATPase activity. The ATPase-active form of MtFtsE is a dimer with the sole cysteine (C84) at the dimer interface. Homology modeling of MtFtsE, using MalK (the NBD component of an ABC transporter for maltose) as the template, supported this observation. Stabilization of the dimer through cys-cys disulphide bond increased ATPase activity by 3.7-fold, although C84 does not have any role in ATPase activity. 3. Identification and elucidation of interaction among cell division proteins FtsE, FtsX, FtsQ and FtsZ of Mycobacterium tuberculosis Septum synthesis in E. coli is mediated by a dozen of proteins, among which the bacterial cytoskeletal protein FtsZ is the first molecule to localise to the mid-cell site, where it forms a scaffold for the localization of downstream cell division proteins namely, FtsA /ZipA < FtsE / FtsX < FtsK < FtsQ < FtsL < FtsB < FtsW < FtsI < FtsN and AmiC. If the above order of recruitment of proteins holds true for M. tuberculosis as well, the immediate proteins recruited to the mid-cell site after MtFtsZ in M. tuberculosis would be MtFtsE and MtFtsX, followed with MtFtsK and MtFtsQ. Thus it is possible that MtFtsE and MtFtsX could be interacting with MtFtsZ and MtFtsQ. Therefore attempts were made to delineate the interaction network among MtFtsE, MtFtsX, MtFtsQ and MtFtsZ of M. tuberculosis. Ni2+-NTA agarose pulldown, co-immunoprecipitation and bacterial two-hybrid assays using wild type and deletion mutants of the proteins showed that MtFtsE interacts with MtFtsQ and MtFtsX through its C-terminus. In addition, MtFtsX could interact with MtFtsZ and MtFtsQ. MtFtsX was found to homodimerise and interact with MtFtsQ in vivo. The ATPase-active of MtFtsE in vivo being a dimer, a hypothetical model for the translocation of MtFtsQ into the membrane at mid-cell site was proposed. According to this model, MtFtsQ might be inserted into the membrane at the mid-cell site by (MtFtsX)2 functioning as the membrane channel for the transport, which could be energized by the ATPase subunit (MtFtsE)2 of the (MtFtsE)2(MtFtsX)2 complex. MtFtsX might have a role in tethering the FtsZ-ring with the membrane at the mid-cell site. An altogether different possibility could be that the (FtsE)2(FtsX)2 complex might have a role in the stabilization or constriction of FtsZ-ring during the inward growth of septum.