Análise de associações de major royal jelly protein 1 por cromatografia de exclusão molecular

Dissertação (mestrado)—Universidade de Brasília, Instituto de Ciências Biológicas, Departamento de Biologia Celular, Pós-Graduação em Biologia Molecular, 2012.Polifenismo é a habilidade de um genoma expressar múltiplos fenótipos morfológica e comportamentalmente distintos. Em abelhas Apis mellifera¸ o polifenismo é essencial, uma vez que gera divisão de tarefas na colméia, vital para sua viabilidade. A plasticidade fenotípica das abelhas é mediada nutricionalmente pela geleia real (GR). A major royal jelly protein 1 (MRJP1) é a mais abundante proteína da GR, e é descrita como o principal fator ativo na diferenciação de castas de Apis mellifera. A MRJP1 é uma glicoproteína que se liga fortemente a um peptídeo chamado apisimina (5,5 kDa) e se auto-associa, formando complexos de diferentes tamanhos, podendo se apresentar em diversas formas moleculares numa única solução. O objetivo deste trabalho foi caracterizar associações de MRPJ1 sob diversas condições. MRJP1 foi purificada em um único passo de cromatografia de troca iônica. Por cromatografia de exclusão molecular foi possível observar que a MRJP1 tende a se oligomerizar ou até mesmo a se agregar em tampões ácidos, em amostras aquecidas a 60ºC ou em tampões com alta molaridade de sal (2 M de NaCl) ou de glicina (2 M),. Em tampão PBS pH neutro e tampão carbonato/bicarbonato pH 10,0, a MRJP1 está presente em três formas -pentâmero (290 kDa), dímero (120 kDa) e monômero (55 kDa). Ao se adicionar 0,5 M de glicina em tampão carbonato/bicarbonato pH 10,0, a MRJP1 se dissocia, apresentando-se mais abundante nas formas dimérica e monomérica. A presença dos surfactantes dodecil sulfato de sódio, Tween® 20 e Brij 35 induz um aumento na massa dos complexos de MRJP1, causado provavelmente pela ligação de moléculas do detergente com a proteína, podendo também induzir oligomerização ou agregação. Logo, conclui-se que a liofilização e aquecimento das amostras de MRJP1 induzem oligomerização e agregação da amostra. Não liofilizar o monômero e mantê-lo em baixas temperaturas mantém a amostra nesta forma molcular. O tampão carbonato/bicarbonato, pH 10,0, contendo glicina 0,5 M foi o melhor para se obter menor quantidade de formas de MRJP1 em solução com grande quantidade de monômero. O processo de ultrafiltração impede a purificação de MRJP1 em cromatografia de troca-iônica. ______________________________________________________________________________ ABSTRACTPolyphenism is the hability of a genome to express multiple phenotypes with different morphologies and behaviors. In Apis mellifera bees, the polyphenism is essential, once it generates labor division in the hive, vital to its viability. The bee s phenotypic plasticity is nutritionally mediated by royal jelly (GR). The major royal jelly protein 1 (MRJP1) is the most abundant protein in GR, and it is described as the main active factor in differentiating Apis mellifera castes. The MRJP1 is a glycoprotein that binds strongly to a peptide named apsimin (5,5 kDa) and self-associates, forming complexes of various sizes, and may present itself in different molecular forms in one solution. The purpose of this study was to characterize MRJP1 associations under different conditions. MRJP1 was purified in a single step of ionic exchange chromatography. By size exclusion chromatography, it was possible to observe the MRJP1 oligomerization or even aggregation in acid buffers, in samples heated to 60ºC or in buffers with high salt molarity (2 M of NaCl) or glycine (2 M). In PBS buffer at neutral pH and carbonate/bicarbonate pH 10,0 buffer, MRJP1 was present in three forms - pentamer (290 kDa), dimer (120 kDa) or monomer (55 kDa). By adding 0.5 M of glycine in carbonate/bicarbonate pH 10,0 buffer, MRJP1 dissociates, leading to monomer and dimmer as the most abundant forms. The presence of the surfactants sodium dodecyl sulfate, Tween® 20 and Brij 35 induces a mass increase in the MRJP1 complex, probably caused by the binding of detergent molecules to the protein. Detergents may also induce oligomerization or aggregation. Therefore, it is concluded that lyophilization and heating the MRJP1 samples induce oligomerization and aggregation of the sample. To not lyophilize the monomer and keep it at low temperatures maintains the sample in this molecular form. The carbonate/bicarbonate buffer, pH 10.0, containing 0.5 M of glycine was the best for obtaining fewer MRJP1 forms in solution with high amounts of monomer. The ultrafiltration process prevents MRJP1 purification by ion exchange chromatography

FYCO1 Increase and Effect of Arimoclomol–Treatment in Human VCP–Pathology

Dominant VCP–mutations cause a variety of neurological manifestations including inclusion body myopathy with early–onset Paget disease and frontotemporal dementia 1 (IBMPFD). VCP encodes a ubiquitously expressed multifunctional protein that is a member of the AAA+ protein family, implicated in multiple cellular functions ranging from organelle biogenesis to ubiquitin–dependent protein degradation. The latter function accords with the presence of protein aggregates in muscle biopsy specimens derived from VCP–patients. Studying the proteomic signature of VCP–mutant fibroblasts, we identified a (pathophysiological) increase of FYCO1, a protein involved in autophagosome transport. We confirmed this finding applying immunostaining also in muscle biopsies derived from VCP–patients. Treatment of fibroblasts with arimoclomol, an orphan drug thought to restore physiologic cellular protein repair pathways, ameliorated cellular cytotoxicity in VCP–patient derived cells. This finding was accompanied by increased abundance of proteins involved in immune response with a direct impact on protein clearaqnce as well as by elevation of pro–survival proteins as unravelled by untargeted proteomic profiling. Hence, the combined results of our study reveal a dysregulation of FYCO1 in the context of VCP–etiopathology, highlight arimoclomol as a potential drug and introduce proteins targeted by the pre–clinical testing of this drug in fibroblasts

Quantifying Missing (Phospho)Proteome Regions with the Broad-Specificity Protease Subtilisin

Despite huge efforts to map the human proteome using mass spectrometry the overall sequence coverage achieved to date is still below 50%. Reasons for missing areas of the proteome comprise protease-resistant domains including the lack/excess of enzymatic cleavage sites, nonunique peptide sequences, impaired peptide ionization/separation and low expression levels. To access novel areas of the proteome the beneficial use of enzymes complementary to trypsin, such as Glu-C, Asp-N, Lys-N, Arg-C, LysargiNase has been reported. Here, we present how the broad-specificity protease subtilisin enables mapping of previously hidden areas of the proteome. We systematically evaluated its digestion efficiency and reproducibility and compared it to the gold standard in the field, trypsin. Notably, subtilisin allows reproducible near-complete digestion of cells lysates in 1–5 min. As expected from its broad specificity the generation of overlapping peptide sequences reduces the number of identified proteins compared to trypsin (8363 vs 6807; 1% protein FDR). However, subtilisin considerably improved the coverage of missing and particularly proline-rich areas of the proteome. Along 14 628 high confidence phosphorylation sites identified in total, only 33% were shared between both enzymes, while 37% were exclusive to subtilisin. Notably, 926 of these were not even accessible by additional in silico digestion with either Asp-N, Arg-C, Glu-C, Lys-C, or Lys-N. Thus, subtilisin might be particularly beneficial for system-wide profiling of post-translational modification sites. Finally, we demonstrate that subtilisin can be used for reporter-ion based in-depth quantification, providing a precision comparable to trypsindespite broad specificity and fast digestion that may increase technical variance

Mutations in glycyl-tRNA synthetase impair mitochondrial metabolism in neurons.

The nuclear-encoded glycyl-tRNA synthetase gene (GARS) is essential for protein translation in both cytoplasm and mitochondria. In contrast, different genes encode the mitochondrial and cytosolic forms of most other tRNA synthetases. Dominant GARS mutations were described in inherited neuropathies, while recessive mutations cause severe childhood-onset disorders affecting skeletal muscle and heart. The downstream events explaining tissue-specific phenotype-genotype relations remained unclear. We investigated the mitochondrial function of GARS in human cell lines and in the GarsC210R mouse model. Human-induced neuronal progenitor cells (iNPCs) carrying dominant and recessive GARS mutations showed alterations of mitochondrial proteins, which were more prominent in iNPCs with dominant, neuropathy-causing mutations. Although comparative proteomic analysis of iNPCs showed significant changes in mitochondrial respiratory chain complex subunits, assembly genes, Krebs cycle enzymes and transport proteins in both recessive and dominant mutations, proteins involved in fatty acid oxidation were only altered by recessive mutations causing mitochondrial cardiomyopathy. In contrast, significant alterations of the vesicle-associated membrane protein-associated protein B (VAPB) and its downstream pathways such as mitochondrial calcium uptake and autophagy were detected in dominant GARS mutations. The role of VAPB has been supported by similar results in the GarsC210R mice. Our data suggest that altered mitochondria-associated endoplasmic reticulum (ER) membranes (MAM) may be important disease mechanisms leading to neuropathy in this condition