Sequenciamento de proteoformas de inibidores de protease do quiabo e avaliação de citotoxicidade em células de Leucemia Mieloide Crônica

• Ano de defesa: 2019
• Autor(a) principal: Lacerda, Jose Thalles Jocelino Gomes De [UNIFESP]
• Tipo de documento: Tese
• Tipo de acesso: Acesso aberto
• Idioma: por
• Instituição de defesa: Universidade Federal de São Paulo (UNIFESP)
• Palavras-chave em Português: Okra; Protease Inhibitor; Proteoforms; De Novo Sequencing; Leukemia; Quiabo; Inibidor De Protease; Proteoformas; Sequenciamento De Novo; Leucemia
• Link de acesso: https://sucupira.capes.gov.br/sucupira/public/consultas/coleta/trabalhoConclusao/viewTrabalhoConclusao.jsf?popup=true&id_trabalho=7741934; https://repositorio.unifesp.br/handle/11600/59823

Objective: To perform the sequencing of new proteoforms of Kunitz-type protease inhibitors with lectin-like domain extracted from okra (Abelmoschus esculentus L. Moench) seeds, besides evaluating the cytotoxicity of these proteins against chronic myeloid leukemia (CML) K562 and Lucena cell lines. Methods: Proteoforms were sequenced by an integrative analysis with liquid chromatography coupled to mass spectrometry in-tandem (LC-MS/MS), de novo peptide sequencing, manual assembly of coding sequences from the transcripts (CDS), proteogenomic analysis and assessment of intact mass by LC-MS analysis. The cytotoxic effect of the inhibitors was evaluated by measuring mitochondrial dehydrogenase activity (MTT assay). Results: From the peptides produced by 6 proteolytic enzymes, the overlap of 193 peptides was obtained by de novo sequencing analysis, resulting in a primary structure of 193 amino acids. However, given the complexity resulting from a mixture of peptides belonging to more than one proteoform, the complete sequencing could not be done only by de novo sequencing analysis. Thus, the peptide sequences of this previously assembled structure were submitted to alignment against the raw okra transcriptome data to build the nucleotide sequences encoding the proteoforms and by an iterative process, the protein sequences of 7 proteoforms were assembled, as well as their respective CDSs. Then, the MS/MS spectra of the de novo peptides were submitted to search against a personalizated database containing the 7 proteoforms sequences, resulting in coverage of 91-95%. The intact mass analysis indicated 11 average masses of 20.4-20.9 kDa, of which 7 corresponded to the mature proteoforms previously reported by the CDS assembly; 2 belonging to the mutated forms of AelKI-1 (M162V) and AelKI-4 (E146K); and 2 belonging to truncated forms of AelKI-3. In this analysis, the differences between experimentally calculated and theoretical masses were equivalent to 3 disulfide bonds, as well as in the prediction of the resulting tertiary structure. The AelKI proteoforms were able to reduce cell viability of Lucena (IC50 = 17.4 ±4 μg/mL) and K562 (IC50 = 30.7 ±9 μg/mL) cell lines and had low cytotoxicity on the fibroblast cell line (control) in which the IC50 was undetermined at the highest dosage (100 μg/mL). Conclusion: The sequencing of AelKI proteoforms shows the utility of using these methods in a complementary way to reveal the primary structures and their characteristics, especially when the genome is lacking. In addition, the antitumor activities make the AelKI proteoforms promising antileukemic agent candidates.