Proteomics of human osteosarcoma cells after treatment with lectins from okra seeds

Lectinas são proteínas capazes de reconhecer e se ligar a carboidratos presentes na membrana celular com alta especificidade. Células tumorais frequentemente possuem alterações no padrão de glicosilação da superfície celular, que podem ser seletivamente reconhecidos por lectinas de plantas. Aelki representa um conjunto de isolectinas da semente do quiabo (Abelmoschus esculentus L. Moench) que apresentou em estudos anteriores ação antitumoral seletiva contra células de câncer de mama humanos (MCF7) e baixa toxicidade contra uma linhagem não tumorigênica de células de fibroblastos (CCK-1059 sk). A investigação da atividade dar AelKI poderia ser ampliada a tumores de outras origens histológicas, incluindo tipos raros como o osteosarcoma. Este trabalho investigou a atividade antitumoral in vitro da AelKI, contra células de osteosarcoma humano (U-2 OS). O proteoma das células tratadas com AelKI foi comparado com o das células não tratadas, visando estabelecer alvos moleculares e vias bioquímicas afetados pelo tratamento. O extrato de AelKI foi preparado a partir das sementes do quiabo e purificado por cromatografia de troca iônica. A citotoxicidade de AelKI sobre as células U-2 OS foi avaliada para a determinação das concentrações para incubação, e dois níveis equivalentes a IC25 e IC40 foram selecionadas para o tratamento. Foi realizada a análise proteômica quantitativa de células U-2 OS, pelo método sem marcação. Ensaios de immunoblotting foram utilizados para verificação de marcadores das vias apoptótica e autofágica. AelKI reduziu a viabilidade da população celular em cerca de 90% nas concentrações a partir de 2 micromolar, apresentando IC50=0,26 micromolar. A análise proteômica revelou em torno de 2.200 proteínas quantificadas em todos os grupos, das quais 41 foram diferencialmente abundantes (PDA). Em termos de vias bioquímicas alteradas, as PDAs foram principalmente relacionadas à apoptose, autofagia, metabolismo energético, vesiculação intracelular e replicação de DNA. O ensaio de immunoblotting revelou que marcadores de apoptose (Caspase-3, Caspase-9, Bax e Bcl-2) tiveram seus níveis alterados pelo tratamento, indicando ativação da via intrínseca. Em relação à via autofágica, não houve alteração significativa no nível de expressão dos marcadores testados. Conjuntamente, os resultados apresentados indicam que AelKI induz a morte celular de células de osteosarcoma humano U-2 OS pela ativação da via apoptótica intrínseca, sugerindo o seu potencial como modelo para o desenvolvimento de novos agentes terapêuticos antitumorais.Lectins are proteins with ability to recognize and attach to carbohydrates on cell membrane with high specificity. Tumoral cells often present altered glycosylation pattern on cell surface, that can be selectively recognized by plant lectins. AelKI represents a group of isolectins extracted from okra seeds (Abelmoschus esculentus L. Moench) that presented antitumoral activity against human breast cancer cells (MCF7) and low toxicity in non-tumorigenic fibroblastic cells (CCK-1059 sk). The investigation of this activity could be expanded to cells from different histological origins, including rare cancer types such as osteosarcoma. In this study the in vitro antitumoral activity of AelKI was investigated against a human osteosarcoma cell line (U-2 OS). The proteome of AelKI- treated cells was compared with that of untreated cells, aiming to unveil the molecular targets and biochemical pathways affected by the treatment. AelKI extract was prepared from okra seeds and purified by ion-exchange chromatography. AelKI cytotoxicity against U-2 OS cells was evaluated to determine the incubation concentrations, and two levels equivalent to IC25 and IC40 were selected for treatment. After treatment, quantitative proteomics analysis of U-2 OS cells was performed by label-free method. Immunoblotting assays were used to check apoptotic and autophagy markers. The results showed a 90% reduction in cell growth at 2 micromolar of AelKI, and the IC50 was 0.26 micromolar. Proteomics analysis revealed around 2,200 proteins quantified in all groups, from which 41 were differentially abundant proteins (DAPs). In terms of affected biochemical pathways, the main DAPs were related to apoptosis, autophagy, energy metabolism, intracellular vesicles, and DNA replication pathways. Immunoblotting assays revealed modulation of apoptotic markers (cleaved caspase-3, caspase-9, Bax, and Bcl-2) evidencing the intrinsic pathway activation. In contrast, no significative stimulation of autophagy was evidenced. Overall, the data indicates that AelKI induces cell death in human osteosarcoma U-2 OS by activating the intrinsic apoptosis pathway. This suggests that AelKI has the potential to serve as a scaffold for the development of novel antitumoral therapies.Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)CNPq [142126/2018-7]CAPES-PrInt (88887.372047/2019-00)FAPESP [2017/20106-9

Análise Do Dimorfismo Sexual Em Venenos Da Aranha Acanthoscurria Juruenicola Por Peptidômica Quantitativa

Arachnida Class Is Considered One Of The Most Successful Evolutionary Group Among Venomous Animals. This Is In Part Due To The Production Of A Pharmacologically Complex Venom Consisting In A Collection Of Biologically Active And Highly Specific Proteins And Peptides, Which May Be Biotechnologically Applied In Pharmacological And Biopesticidal Fields. However, Peptidomics Studies Are Still Scarce, So That Their Identities And Biological Activities Remain Unknown. The Objective Of This Study Was To Compare And Characterize The Peptidic Profile Of The Venom Of Male And Female Specimens Of The Brazilian Spider Specie Acanthoscurria Juruenicola. The Material Was Extracted From The Animals By Electrostimulation Of Venom Glands. Native Peptides Were Obtained By Sep-Pak C-18 Cartridge Purification, Followed By Mass Spectrometry Analysis (Lc-Ms/Ms), As Well As The Fragments Generated By The Digestion Isolated By 5 Different Enzymes (Trypsin, Chymotrypsin, Thermolysin, Glu-C And Asp-N). Peptidic Fractions Obtained By GOs Aracnídeos Estão Entre Os Grupos De Maior Sucesso Evolutivo Da História Do Planeta, E Um Dos Fatores De Maior Importância Para Isso É A Produção De Um Veneno Farmacologicamente Complexo, Constituído Por Uma Coleção De Proteínas E Peptídeos Biologicamente Ativos E Altamente Específicos, Com Potencial Aplicação Biotecnológica Nas Áreas Farmacológica E De Bioinseticidas. Entretanto, Estudos Peptidômicos Ainda São Escassos, Consequentemente As Identidades E Atividades Biológicas Desses Peptídeos Permanecem Desconhecidas. O Objetivo Deste Trabalho Foi Caracterizar E Comparar O Perfil Peptídico Do Veneno De Espécimes Machos E Fêmeas Da Aranha Acanthoscurria Juruenicola. O Material Foi Obtido Por Eletroestimulação Das Glândulas De Veneno. Peptídeos Brutos Foram Fracionados Por Extração Em Fase Sólida Em Cartuchos Sep-Pak" C-18, E Analisados Por Espectrometria De Massas, Assim Como Seus Fragmentos Gerados Pela Digestão Isolada De 5 Enzimas Distintas (Tripsina, Quimotripsina, Termolisina, Glu-C E Asp-N). Frações PeDados abertos - Sucupira - Teses e dissertações (2018

Glucose metabolism is upregulated in the mononuclear cell proteome during sepsis and supports endotoxin-tolerant cell function

Metabolic adaptations shape immune cell function. In the acute response, a metabolic switch towards glycolysis is necessary for mounting a proinflammatory response. During the clinical course of sepsis, both suppression and activation of immune responses take place simultaneously. Leukocytes from septic patients present inhibition of cytokine production while other functions such as phagocytosis and production of reactive oxygen species (ROS) are preserved, similarly to the in vitro endotoxin tolerance model, where a first stimulation with lipopolysaccharide (LPS) affects the response to a second stimulus. Here, we sought to investigate how cellular metabolism is related to the modulation of immune responses in sepsis and endotoxin tolerance. Proteomic analysis in peripheral blood mononuclear cells (PBMCs) from septic patients obtained at intensive care unit admission showed an upregulation of proteins related to glycolysis, the pentose phosphate pathway (PPP), production of ROS and nitric oxide, and downregulation of proteins in the tricarboxylic acid cycle and oxidative phosphorylation compared to healthy volunteers. Using the endotoxin-tolerance model in PBMCs from healthy subjects, we observed increased lactate production in control cells upon LPS stimulation, while endotoxin-tolerant cells presented inhibited tumor necrosis factor-α and lactate production along with preserved phagocytic capacity. Inhibition of glycolysis and PPP led to impairment of phagocytosis and cytokine production both in control and in endotoxin-tolerant cells. These data indicate that glucose metabolism supports leukocyte functions even in a condition of endotoxin tolerance

Comparative compositional and functional analyses of Bothrops moojeni specimens reveal several individual variations.

Snake venoms are complex protein mixtures with different biological activities that can act in both their preys and human victims. Many of these proteins play a role in prey capture and in the digestive process of these animals. It is known that some snakes are resistant to the toxicity of their own venom by mechanisms not yet fully elucidated. However, it was observed in the Laboratory of Herpetology of Instituto Butantan that some Bothrops moojeni individuals injured by the same snake species showed mortalities caused by envenoming effects. This study analyzed the biochemical composition of 13 venom and plasma samples from Bothrops moojeni specimens to assess differences in their protein composition. Application of sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) showed distinct venom protein profiles, but very homogeneous plasma profiles. Western Blotting (WB) was performed with plasma samples, which were submitted to incubation with the respective venom. Some individuals showed an immunorecognized band zone around 25 kDa, indicating interaction between the same individual plasma and venom proteins. Crossed-WB assay using non-self-plasma and venom showed that this variability is due to venom protein composition instead of plasma composition. These venoms presented higher caseinolytic, collagenolytic and coagulant activities than the venoms without these regions recognized by WB. Mass spectrometry analyses performed on two individuals revealed that these individuals present, in addition to higher protein concentrations, other exclusive proteins in their composition. When these same two samples were tested in vivo, the results also showed higher lethality in these venoms, but lower hemorrhagic activity than in the venoms without these regions recognized by WB. In conclusion, some Bothrops moojeni specimens differ in venom composition, which may have implications in envenomation. Moreover, the high individual venom variability found in this species demonstrates the importance to work with individual analyses in studies involving intraspecific venom variability and venom evolution

Combined Transcriptome and Proteome Leukocyte’s Profiling Reveals Up-Regulated Module of Genes/Proteins Related to Low Density Neutrophils and Impaired Transcription and Translation Processes in Clinical Sepsis

Sepsis is a global health emergency, which is caused by various sources of infection that lead to changes in gene expression, protein-coding, and metabolism. Advancements in “omics” technologies have provided valuable tools to unravel the mechanisms involved in the pathogenesis of this disease. In this study, we performed shotgun mass spectrometry in peripheral blood mononuclear cells (PBMC) from septic patients (N=24) and healthy controls (N=9) and combined these results with two public microarray leukocytes datasets. Through combination of transcriptome and proteome profiling, we identified 170 co‐differentially expressed genes/proteins. Among these, 122 genes/proteins displayed the same expression trend. Ingenuity Pathway Analysis revealed pathways related to lymphocyte functions with decreased status, and defense processes that were predicted to be strongly increased. Protein-protein interaction network analyses revealed two densely connected regions, which mainly included down‐regulated genes/proteins that were related to the transcription of RNA, translation of proteins, and mitochondrial translation. Additionally, we identified one module comprising of up‐regulated genes/proteins, which were mainly related to low-density neutrophils (LDNs). LDNs were reported in sepsis and in COVID-19. Changes in gene expression level were validated using quantitative real-time PCR in PBMCs from patients with sepsis. To further support that the source of the upregulated module of genes/proteins found in our results were derived from LDNs, we identified an increase of this population by flow cytometry in PBMC samples obtained from the same cohort of septic patients included in the proteomic analysis. This study provides new insights into a reprioritization of biological functions in response to sepsis that involved a transcriptional and translational shutdown of genes/proteins, with exception of a set of genes/proteins related to LDNs and host‐defense system