Estrutura oligomérica e dinâmica de Major Royal Jelly Protein 1 (MRJP1)/apisimina analisadas por espectrometria de massas e técnicas complementares

Tese (doutorado)—Universidade de Brasília, Instituto de Ciências Biológicas, Departamento de Biologia Celular, Pós-Graduação em Biologia Molecular, 2017.Geleia Real (GR) dispara o desenvolvimento de larvas de abelhas fêmeas até rainhas. Este efeito tem sido atribuído à presença de Major Royal Jelly Protein 1 (MRJP1) presente na geleia real. MRJP1 isolada de GR está intimamente associada a apisimina, um peptídeo helicoidal com 54 resíduos de aminoácidos que promove uma associação não covalente de MRJP1 em oligômeros de diferentes tamanhos. Não existem dados de alta resolução disponíveis para essas estruturas e até mesmo sua estequiometria ainda não é clara. Nesta tese, examinamos a relação MRJP1/apisimina usando um arsenal de técnicas biofísicas. Também, investigamos o comportamento de MRJP1/apisimina em amostras após remoção de seus carboidratos e de apisimina associada. Nossos dados de espectrometria de massas (MS) nativos demonstraram que os complexos existem predominantemente numa estequiometria de MRJP14/apisimina4. Blue native PAGE demonstrou a prevalência de estruturas tetraméricas e monoméricas. Microscopia de força atômica demonstrou a presença de populações que puderam ser agrupadas em dois grandes grupos. Troca do hidrogênio por deutério (HDX) seguida de análises por espectrometria de massas revelaram que MRJP1, nesses complexos, é desordenada na extensão dos resíduos 20-265. Estruturas secundárias (provavelmente folhas beta antiparalelas) estáveis são encontradas marginalmente ao redor dos resíduos 266-432. Estas são regiões fracamente estruturadas com conformações que variam entre estruturada e desestruturada, gerando uma distribuição isotópica bimodal (EX1). Nós propomos que os complexos nativos (tetrâmeros) têm uma estrutura quaternária formada por “dímero de dímero”, onde as cadeias de MRJP1 são ligadas por apisimina. Especificamente, nossos dados sugerem que apisimina age como um ligante que forma contatos hidrofóbicos envolvendo o segmento 316VLFFGLV322 de MRJP1. Esta proteína tem dois sítios de glicosilação localizados nos resíduos de aminoácidos 144 e 177. Por 2DE podemos ver 9 proteoformas de MRJP1, mesmo após a remoção dos carboidratos. Deglicosilação produz grandes agregados solúveis, enfatizando o papel dos glicanos como inibidores de agregação. Amostras com apisimina parcialmente removida formam complexos diméricos com estequiometria (MRJP12/apisimina1). As informações produzidas neste trabalho podem contribuir para uma melhor compreensão da relação estrutura/função de MRJP1, que possui papéis únicos na biologia da abelha.Royal jelly (RJ) triggers the development of female honeybee larvae into queens. This effect has been attributed to the presence of major royal jelly protein 1 (MRJP1) in RJ. MRJP1 isolated from royal jelly is tightly associated with apisimin, a 54-residue -helical peptide that promotes the noncovalent assembly of MRJP1 into multimers. No high resolution structural data are available for these complexes, and their binding stoichiometry remains uncertain. We examined MRJP1/apisimin using a range of biophysical techniques. In addition, we investigated the behavior of deglycosylated samples, as well as samples with reduced apisimin content. Our mass spectrometry (MS) data demonstrated that the native complexes predominantly exist in a (MRJP14 apisimin4) stoichiometry. Blue native and showed the prevalence of tetrameric and monomeric structures in native conditions. Atomic force microscopy also showed two populations. Hydrogen/deuterium exchange (HDX) MS revealed that MRJP1 within these complexes is extensively disordered in the range of the residues 20-265. Marginally stable secondary structure (likely antiparallel -sheet) exists around residues 266-432. These weakly structured regions interchange with conformations that are extensively unfolded, giving rise to bimodal (EX1) isotope distributions. We propose that the native complexes have a “dimer of dimers” quaternary structure in which MRJP1 chains are bridged by apisimin. Specifically, our data suggest that apisimin acts as a linker that forms hydrophobic contacts involving the MRJP1 segment 316VLFFGLV322. MRJP1 has 2 glycosites located at amino acids 144 and 177. By using 2-DE, we observed 9 MRJP1 proteoforms, even after carbohydrate removal. Deglycosylation produces large soluble aggregates, highlighting the role of glycans as aggregation inhibitors. Samples with reduced apisimin content form dimeric complexes with a (MRJP12 apisimin1) stoichiometry. Therefore, the information uncovered in this work should help pave the way towards a better understanding of the structure/function relationship for MRJP1, which possesses unique roles in the honey bee biology

Análise de proteínas nucleares reguladas na amastigogênese e de complexos proteicos de Trypanosoma Cruzi

Dissertação (mestrado)—Universidade de Brasília, Instituto de Ciências Biológicas, Departamento de Biologia Celular, 2013.Uma característica do Trypanosoma cruzi são as mudanças morfológicas que ocorrem durante seu ciclo de vida. A amastigogênese é um importante processo no ciclo de vida do parasita onde as formas tripomastigotas infectivas se diferenciam em amastigotas replicativas. A duplicação celular é um processo complexo, onde vários eventos estão envolvidos, dentre eles, a correta segregação das cromátides irmãs após a replicação do DNA, função exercida por um complexo denominado coesina. Neste trabalho, foi proposta caracterização de complexos proteicos de T.cruzi assim como a identificação de proteínas de complexos nucleares, cuja expressão se altera durante a amastigogênese. Por meio da técnica Blue Native PAGE (BN-PAGE) foi possível realizar a separação de complexos proteicos de lisado total das formas epimastigotas de Trypanosoma cruzi. Os componentes individuais dos complexos puderam ser visualizados em segunda dimensão após o gel ter sido corado com nitrato de prata. Experimentos de detecção imunológica por western blotting demonstraram a ausência da subunidade SCC1 do complexo coesina em amastigotas gerados por diferenciação in vitro de formas tripomastigotas em cultura axênica em pH 5. Porém, a subunidade SCC1 pode ser detectada em extratos proteicos de formas amastigotas que foram reincubadas por 10 h em meio de cultura com pH ajustado para 7,5, sugerindo que essas formas são capazes de manter suas propriedades replicativas. Por espectrometria de massas do tipo LC-MS/MS, 1.339 proteínas de T. cruzi foram identificadas de amostras provenientes dos ensaios de amastigogênese. Destas, 560 proteínas foram anotadas em bancos de dados como proteínas reguladas, 65 possuíam localização nuclear e 66 foram preditas como pertencentes a complexos proteicos. Na classificação das proteínas identificadas os grupos que correspondem àqueles envolvidos em metabolismo de ácidos nucleicos aparecem como predominantes. Proteínas que se ligam a DNA (9,23%), proteínas de ligação a RNA (13,85%) e proteínas que se ligam a nucleotídeos (18,46%) correspondem juntas a 41,54% do total de proteínas nucleares reguladas, corroborando o fato de que intensa atividade de transcrição e processamento de DNA e RNA ocorre em todas as etapas de diferenciação, além de outros principais grupos como as peptidases (15,38%), proteínas estruturais (13,85%) e atividade de hidrolases (12,31%). Para proteínas reguladas e envolvidas com complexos proteicos durante a amastigogênese os maiores grupos estão envolvidos em processos de transporte (25,58%), processos metabólicos (19,77%), processos metabólicos de compostos contendo nucleotídeos (16,28%), geração de precursores metabólicos e de energia (8,14%). ______________________________________________________________________________ ABSTRACTA feature of Trypanosoma cruzi is the morphological changes that occur during their life cycle. The amastigogenesisis an important process in the life cycle of the parasite where the infective trypomastigotes differentiate into replicative amastigotes. A cell replication is a complex process, where several events are involved, among them the correct segregation of sister chromatids after DNA replication, function performed by a complex called cohesin. In this work we proposed the characterization of T. cruzi protein complexes and the identification of proteins from nuclear complexes, whose expression is altered during amastigogenesis. Through the technique of Blue Native PAGE (BN-PAGE) it was possible the separation of protein complexes from total lysate of T. cruzi epimastigotes. The individual components of the complexes could be visualized after the second dimensional gel after stainingwith silver nitrate. Immunological detection experiments by western blotting demonstrated the absence of SCC1 subunit of cohesin complex in amastigotes generated by in vitro differentiation of trypomastigotes in axenic culture at pH 5.0. However, the subunit SCC1 was detected in protein extracts of amastigotes that were reincubated for 10 hours in a culture medium adjusted at pH 7.5, suggesting that these forms are able to maintain their replicative properties. By LC-MS/MS mass spectrometry, 1,339 proteins ofT.cruzi were identified in samples from mastigogenesis. Of these, 560 proteins were annotated in databases as regulated proteins, 65 have presented nuclear localization and 66 were predicted as belonging to protein complexes. In the classification of proteins involved in nucleic acid metabolism, this group appears to be predominant. Proteins which bind to DNA (9.23%), RNA-binding proteins (13.85%) and proteins that bind nucleotides (18.46%) correspond together to 41.54% of the total regulated nuclear proteins, confirming that strong transcription activity and DNA and RNA processing occur in all stages of differentiation, and other major groups such as peptidases (15.38%), structural proteins (13.85%) and activity of hydrolases (12.31%). For regulated proteins and protein complexes involved in amastigogenesis the largest groups are involved in transport processes (25.58%), metabolic processes (19.77%), metabolic processes of compounds containing nucleotides (16.28%), generation of metabolic precursors and energy (8.14%)

Dynamic proteomic analysis of Aedes aegypti Aag-2 cells infected with Mayaro virus

Background Mayaro virus (MAYV) is responsible for a mosquito-borne tropical disease with clinical symptoms similar to dengue or chikungunya virus fevers. In addition to the recent territorial expansion of MAYV, this virus may be responsible for an increasing number of outbreaks. Currently, no vaccine is available. Aedes aegypti is promiscuous in its viral transmission and thus an interesting model to understand MAYV-vector interactions. While the life-cycle of MAYV is known, the mechanisms by which this arbovirus affects mosquito host cells are not clearly understood. Methods After defining the best conditions for cell culture harvesting using the highest virus titer, Ae. aegypti Aag-2 cells were infected with a Brazilian MAYV isolate at a MOI of 1 in order to perform a comparative proteomic analysis of MAYV-infected Aag-2 cells by using a label-free semi-quantitative bottom-up proteomic analysis. Time-course analyses were performed at 12 and 48 h post-infection (hpi). After spectrum alignment between the triplicates of each time point and changes of the relative abundance level calculation, the identified proteins were annotated and using Gene Ontology database and protein pathways were annotated using the Kyoto Encyclopedia of Genes and Genomes. Results After three reproducible biological replicates, the total proteome analysis allowed for the identification of 5330 peptides and the mapping of 459, 376 and 251 protein groups, at time 0, 12 hpi and 48 hpi, respectively. A total of 161 mosquito proteins were found to be differentially abundant during the time-course, mostly related to host cell processes, including redox metabolism, translation, energy metabolism, and host cell defense. MAYV infection also increased host protein expression implicated in viral replication. Conclusions To our knowledge, this first proteomic time-course analysis of MAYV-infected mosquito cells sheds light on the molecular basis of the viral infection process and host cell response during the first 48 hpi. Our data highlight several mosquito proteins modulated by the virus, revealing that MAYV manipulates mosquito cell metabolism for its propagation

Proteomic mapping of multifunctional complexes within Triatomine saliva

Triatomines are hematophagous insects that transmit Trypanosoma cruzi, the etiological agent of Chagas disease. This neglected tropical disease represents a global health issue as it is spreading worldwide. The saliva of Triatominae contains miscellaneous proteins crucial for blood feeding acquisition, counteracting host's hemostasis while performing vasodilatory, anti-platelet and anti-coagulant activities, besides modulating inflammation and immune responses. Since a set of biological processes are mediated by protein complexes, here, the sialocomplexomes (salivary protein complexes) of five species of Triatominae were studied to explore the protein-protein interaction networks. Salivary multiprotein complexes from Triatoma infestans, Triatoma dimidiata, Dipetalogaster maxima, Rhodnius prolixus, and Rhodnius neglectus were investigated by Blue-Native- polyacrylamide gel electrophoresis coupled with liquid chromatography tandem mass spectrometry. More than 70 protein groups, uncovering the landscape of the Triatominae salivary interactome, were revealed. Triabin, actin, thioredoxin peroxidase and an uncharacterized protein were identified in sialocomplexes of the five species, while hexamerin, heat shock protein and histone were identified in sialocomplexes of four species. Salivary proteins related to triatomine immunity as well as those required during blood feeding process such as apyrases, antigen 5, procalins, and nitrophorins compose different complexes. Furthermore, unique proteins for each triatomine species were revealed. This study represents the first Triatominae sialocomplexome reference to date and shows that the approach used is a reliable tool for the analysis of Triatominae salivary proteins assembled into complexes

An integrative sialomic analysis reveals molecules from Triatoma sordida (Hemiptera: Reduviidae)

Triatomines have evolved salivary glands that produce versatile molecules with various biological functions, including those leading their interactions with vertebrate hosts’ hemostatic and immunological systems. Here, using high-throughput transcriptomics and proteomics, we report the first sialome study on the synanthropic triatomine Triatoma sordida. As a result, 57,645,372 reads were assembled into 26,670 coding sequences (CDS). From these, a total of 16,683 were successfully annotated. The sialotranscriptomic profile shows Lipocalin as the most abundant protein family within putative secreted transcripts. Trialysins and Kazal-type protease inhibitors have high transcript levels followed by ubiquitous protein families and enzyme classes. Interestingly, abundant trialysin and Kazal-type members are highlighted in this triatomine sialotranscriptome. Furthermore, we identified 132 proteins in T. sordida salivary gland soluble extract through LC-MS/MS spectrometry. Lipocalins, Hemiptera specific families, CRISP/Antigen-5 and Kazal-type protein inhibitors proteins were identified. Our study provides a comprehensive description of the transcript and protein compositions of the salivary glands of T. sordida. It significantly enhances the information in the Triatominae sialome databanks reported so far, improving the understanding of the vector’s biology, the hematophagous behaviour, and the Triatominae subfamily’s evolution