Cloning of Fbxl17 domains in bacterial expression vectors

• Grant number: 19/03943-0
• Support Opportunities: Scholarships in Brazil - Scientific Initiation
• Start date: May 01, 2019
• End date: December 31, 2021
• Field of knowledge: Biological Sciences - Biochemistry - Molecular Biology
• Principal Investigator: Felipe Roberti Teixeira
• Grantee: Camila Rolemberg Santana Travaglini Berti de Correia
• Host Institution: Centro de Ciências Biológicas e da Saúde (CCBS). Universidade Federal de São Carlos (UFSCAR). São Carlos , SP, Brazil

Abstract

The Fbxl17 protein is one of 69 F-box proteins that interact, through their F-box domain, with the SKP1, Cullin 1 and RBX1 proteins to form the SCF complex, which is the largest class of human E3 ubiquitin ligases. These enzymes are responsible for their substrates ubiquitination for degradation by proteasome or for their cellular function modulation. In addition of F-box domain, FBXL proteins present the LRR domain (Leucine Rich Repeat) for interaction with their substrates. Analysis of 1992 patient samples of the METABRIC project (Molecular Taxonomy of Breast Cancer International Consortium) showed that mutations on the FBXL17 gene in 135 samples led to truncations in its LRR domain. In addition to this, by means of CGH-array (Comparative Genomic Hybridization Array), it was shown that 746 cancer cell lines presented breaks in the FBXL17 gene, and that 3 breast cancer cell lines (BT-474, HCC38 e HCC1395) and 1 esophageal/gastric cancer cell line (OE-19) had FBXL17 variants with truncations in the LRR domain. However, there are no researches correlating the Fbxl17 protein with cancer development or the consequences of its truncations in the functions of SCF(Fbxl17) complexes yet. We have an ongoing FAPESP Regular Project- 2016/25798-3 which has an objective to identify protein partners or substrates of Fbxl17 and their mutants (Fbxl17-D3-LRR and Fbxl17-D10-LRR) founded in breast cancer patient samples. There is no commercial good antibodies for Fbxl17 to be used in western blot or immunofluorescence assays, which are essential in the ongoing project. So, the aim of this proposal is to cloning Fbxl17 or their mutants N terminus (1-228), central (210-422) e C terminus (412-711) in bacterial expression vectors in order to express and purify these fragments. We intend be able to get antibodies for each fragment to be used in our research. In addition, these fragments will be useful for in vitro protein protein interaction assays with the Fbxl17 substrates and partners. Thus, the results of this proposal will contribute with our investigation of the Fbxl17 function in cellular proliferation.

Scientific publications

(References retrieved automatically from Web of Science and SciELO through information on FAPESP grants and their corresponding numbers as mentioned in the publications by the authors)

SPAGNOL, VALENTINE; OLIVEIRA, CAIO A. B.; RANDLE, SUZANNE J.; PASSOS, PATRICIA M. S.; CORREIA, CAMILA R. S. T. B.; SIMAROLI, NATALIA B.; OLIVEIRA, JOICE S.; MEVISSEN, TYCHO E. T.; MEDEIROS, ANA CARLA; GOMES, MARCELO D.; et al. The E3 ubiquitin ligase SCF(Fbxo7) mediates proteasomal degradation of UXT isoform 2 (UXT-V2) to inhibit the NF-kappa B signaling pathway. BIOCHIMICA ET BIOPHYSICA ACTA-GENERAL SUBJECTS, v. 1865, n. 1, JAN 2021. (16/25798-3, 16/21310-6, 16/00792-2, 17/22153-4, 17/07879-9, 18/09204-1, 19/03943-0, 18/01308-2)

NASCIMENTO, EVAIR D.; FONSECA, WILSON T.; DE OLIVEIRA, TASSIA R.; DE CORREIA, CAMILA R. S. T. B.; FACA, VITOR M.; DE MORAIS, BEATRIZ P.; SILVESTRINI, VIRGINIA C.; POTT-JUNIOR, HENRIQUE; TEIXEIRA, FELIPE R.; FARIA, RONALDO C.. COVID-19 diagnosis by SARS-CoV-2 Spike protein detection in saliva using an ultrasensitive magneto-assay based on disposable electrochemical sensor. SENSORS AND ACTUATORS B-CHEMICAL, v. 353, p. 9-pg., 2022-02-15. (19/03943-0, 16/25798-3, 20/12920-0, 20/04635-4, 20/07539-6)