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Quantitative targeted proteomics by target LC-MS (SRM) to determine the ligands and interaction interface of cystatin B complex, a prognostic marker for oral cancer

Grant number: 19/14828-7
Support type:Scholarships in Brazil - Master
Effective date (Start): October 01, 2019
Effective date (End): July 31, 2021
Field of knowledge:Health Sciences - Medicine
Principal Investigator:Adriana Franco Paes Leme
Grantee:Guilherme Araújo Câmara
Home Institution: Centro Nacional de Pesquisa em Energia e Materiais (CNPEM). Ministério da Ciência, Tecnologia, Inovações e Comunicações (Brasil). Campinas , SP, Brazil

Abstract

Cystatin B (CSTB) has emerged as an important mediator of carcinogenesis, as well as angiogenesis and tumor growth. In our previous study (Carnielli et al., 2018), we have demonstrated that low level of CSTB, in front of tumor region, is correlated to poor prognostic in patients with oral cancer squamous cells carcinoma (OSCC). Additionally, we found out CSTB levels are correlated to local recurrence of OSCC patients. Although some studies have shown the diagnostic and prognostic value of CSTB, its role in cancer is not well understood. Thus, the aim of this study is i) to identify interaction partners of CSTB to understand the signaling pathways of this protein; ii) to select CSTB partners to perform analysis of protein-protein interaction interface; and iii) to determine stoichiometric of this interaction complex. For this, we will use quantitative strategy based on discovery (DDA, dependent-data acquisition) and target proteomics, SRM mode (Selected Reaction Monitoring). The CSTB partners will be evaluated in at least four cell lines. For that, two approaches will be utilized: i) immunoprecipitation of the endogenous protein with cross-linker in vivo and ii) protein-protein interaction of selected recombinant proteins and CSTB expressed in E.coli with cross-linker to stabilize the complex bonds, followed by mass spectrometry proteomics (XL-MS), using DDA and SRM, respectively. Therefore, the combination of both approaches will provide the identification of interaction partners of CSTB, the interaction interface of these partners, as well as the stoichiometric of the complex protein content, by SRM quantitative analysis. This project is pioneered to be conducted in oral cancer studies, since besides CSTB has been revealed as an independent prognostic marker and a potential therapeutic target in this disease, we will also study strategies to modulate this protein signalization through the analysis of the interaction between CSTB and its partners. (AU)