Orientador: Mário Tyago Murakami / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-19T22:14:14Z (GMT). No. of bitstreams: 1 Pinto_AlineSampaio_M.pdf: 3074349 bytes, checksum: 0fdb140ae2fd1c1975ba6cde0cf00bca (MD5) Previous issue date: 2012 / Resumo: As miosinas formam uma superfamília de proteínas de alto peso molecular com atividade mecanoquímica capaz de hidrolisar a molécula de ATP e de interagir com os filamentos de actina. A estrutura das miosinas pode ser divida de modo geral em cabeça motora, pescoço e cauda. São conhecidas 35 classes de miosinas em eucariotos sendo a classe II de miosinas denominada miosinas convencionais e as demais chamadas de não convencionais. Em plantas são somente encontradas as miosinas não convencionais de classe VIII e XI. A classe VIII caracteriza-se por sua alta processividade sobre os filamentos de actina e a classe XI é a maior classe em número de genes, tendo uma estrutura muito semelhante às miosinas de classe V. Uma terceira classe, a classe XIII, foi posteriormente descoberta e somente foi encontrada no gênero Acetabularia apresentando dois genes, porém essa classificação é controversa havendo aqueles que dizem que as miosinas da classe XIII possuem tanta afinidade filogenética com as da classe XI que elas deveriam compor uma única classe. As miosinas VIII e XI desempenham papéis chave no transporte direcional de componentes intracelulares em plantas, principalmente devido às grandes dimensões das células de plantas que não sobrevivem utilizando somente a difusão como mecanismo de transporte intracelular. Neste trabalho, buscamos selecionar os melhores representantes de cada classe a partir de análises in silico para desenvolvermos os testes de expressão, purificação e análises biofísicas. Os domínios selecionados, cauda globular (GT), dilute e SH3, foram clonados em pET28a e pET28aSUMO, os testes de expressão com diversas cepas de E coli, mostraram que o domínio dilute expressa em grande quantidade na fração solúvel, porém forma agregados impedindo as análises biofísicas e os ensaios de cristalização. O domínio SH3 também foi obtido na forma solúvel, porém em pouca quantidade e apresentou migração anômala no gel, sendo identificado a partir de análises de espectrometria de massas. Foram realizados testes iniciais de cristalização para o domínio SH3, mas não resultou na formação de cristais adequados a difração de raios X. A cauda globular foi obtida apenas na fração insolúvel e submetida a procedimentos de refolding para sua solubilização. Mesmo conseguindo o reenovelamento da construção, a mesma se manteve agregada inviabilizando os testes de cristalização. Por outro lado foram analisadas as interações da cauda globular da miosina XIh com presas identificadas por duplo-híbrido realizado pelo nosso grupo com a miosina humana Va. Três das proteínas que interagiram com a miosina Va humana também mostraram sinais de interação com a miosina XIh de Arabidopsis, indicando que mesmo havendo diferenças nas sequências polipeptídicas entre as classes de miosina, a estrutura terciária se mantém permitindo que ambas apresentem interações com algumas proteínas em comum / Abstract: Myosins belong to a superfamily of high molecular weight proteins, presenting mechanochemical activity by hydrolyzing ATP molecule and ability to interact with actin filaments. The myosin structure can be divided into motor head, neck and tail. 35 myosin classes are known in eukaryotic cells, where class II is known as conventional myosins and all others, as unconventional myosins. Plants possess only unconventional myosins including classes VIII and XI. The class VIII, is characterized by its high processivity on actin filaments while class XI possesses multiple genes and its protein structure is very similar to class V myosins. A third class, XIII, was later discovered and only found in Acetabularia genome presenting two genes. However this classification is controversial, because some studies shown that class XIII myosins share such high phylogenetic similarity with class XI that they should form the same class. Myosins VIII and XI play key roles on directional intracellular components transport in plants, mainly due to the large plant cell size which would not survive only by the diffusion mechanism for intracellular transport. In this work, we have selected the best representative targets of each class from in silico analyses to develop the protein expression, purification and biophysical characterization. The selected domains (globular tail (GT), dilute and SH3) were cloned into pET28a and pET28aSUMO expression vectors. Results of expression tests with several E coli strains, showed that dilute domain is expressed in large amounts in the soluble fraction; however, it aggregates preventing biophysical analysis and crystallization trials. The SH3 domain, expressed in low soluble concentration, presented an abnormal migration in SDS-PAGE and its identity was confirmed by mass spectrometry analysis. Initial crystallization tests were conducted with SH3 domain, but owing to the low protein concentration only clear drops have appeared, and no crystals or aggregates were observed. The globular tail domain, obtained only in insoluble fraction, was subjected to refolding procedures/ techniques in order to recover its native-like state; however, even the refolded protein displayed secondary structure, the protein remained aggregated. Furthermore, we analyzed the interactions between of myosin XIh globular tail with pre-identified proteins by yeast two-hybrid conducted by our group with human myosin Va. Three proteins that interacted with human myosin Va also showed interaction with Arabidopsis myosin XIh, indicating that despite of differences in polypeptide sequences between the classes XI and V, the tertiary structure may be maintained allowing both of them to have interactions with common proteins / Mestrado / Bioquimica / Mestre em Biologia Funcional e Molecular

Myosin 10 is a vertebrate specific actin-based motor protein that is expressed in a variety of cell types. Cell biological evidences suggest that myosin 10 plays a role in cargo transport and filopodia extension. In order to fully appreciate these physiological processes, it is crucial to understand the motor property of myosin 10. However, little is known about its mechanoenzymatic characteristics. In vitro biochemical characterization of myosin 10 has been hindered by the low expression level of the protein in most tissues. In this study, we succeeded in obtaining sufficient amount of recombinant mammalian myosin 10 using the baculovirus expression system. The movement directionality of the heterologously expressed myosin 10 was determined to be plus end-directed by the in vitro motility assay with polarity-marked actin filament we developed. The result is consistent with the proposed physiological function of myosin 10 as a plus end-directed transporter inside filopodia. The duty ratio of myosin 10 was determined to be 0.6~0.7 by the enzyme kinetic analysis, suggesting that myosin 10 is a processive motor. Unexpectedly, we were unable to confirm the processive movement of dimeric myosin 10 along actin filaments in a single molecule study. The result does not support the proposed function of myosin 10 as a transporter. One possible explanation for this discrepancy is that the apparent nonprocessive nature of myosin 10 is important for generating sufficient force required for the intrafilopodial transport by working in concert with numbers of other myosin 10 molecules while not interfering with each other. Altogether, the present study provided qualitative and quantitative biochemical evidences for the better understanding of the motor property of myosin 10 and of the biological processes in which it is involved. Finally, a general molecular mechanism of myosin motors behind the movement directionality and the processivity is discussed based on our results together with the currently available experimental evidences. The validity of the widely accepted ‘leverarm hypothesis’ is reexamined.

Intraflagellar transport (IFT), the bi-directional movement of particles along the length of flagella, is required for flagellar assembly. The IFT particles are moved by kinesin II from the base to the tip of the flagellum, where flagellar assembly occurs. The IFT particles are then moved in the retrograde direction by cytoplasmic dynein 1b/2 to the base of the flagellum. The IFT particles of Chlamydomonas are composed of ~16 proteins, organized into complexes A and B. Alhough IFT is believed to transport cargoes into flagella, few cargoes have been identified and little is known about how the cargos are transported. To study the mechanism of IFT and how IFT is involved in flagellar assembly, this thesis focuses on two questions. 1) In addition to a heavy chain, DHC1b, and a light chain, LC8, what other proteins are responsible for the retrograde movement of IFT particles? 2) What is the specific function of an individual IFT-particle protein? To address these two questions, I screened for Chlamydomonas mutants either defective in retrograde IFT by immunofluorescence microscopy, or defective in IFT-particle proteins and D1bLIC, a dynein light intermediate chain possibly involved in retrograde IFT, by Southern blotting. I identified several mutants defective in retrograde IFT and one of them is defective in the D1bLIC gene. I also identified several mutants defective in several IFT-particle protein genes. I then focused on the mutant defective in D1bLIC and the one defective in IFT46, which was briefly reported as an IFT complex B protein. My results show that as a subunit of the retrograde IFT motor, D1bLIC is required for the stability of DHC1b and is involved in the attachment of IFT particles to the retrograde motor. The P-loop in D1bLIC is not necessary for the function of D1bLIC in retrograde IFT. My results also show that as a complex B protein, IFT46 is necessary for complex B stability and is required for the transport of outer dynein arms into flagella. IFT46 is phosphorylated in vivo and the phosphorylation is not critical for IFT46’s function in flagellar assembly.