Structural studies with importin-± and nuclear localization sequences (NLS) of proteins related with fungi metabolism

• Grant number: 14/10289-0
• Support Opportunities: Scholarships in Brazil - Doctorate
• Start date: September 01, 2014
• End date: May 26, 2018
• Field of knowledge: Biological Sciences - Biochemistry - Molecular Biology
• Agreement: Coordination of Improvement of Higher Education Personnel (CAPES)
• Principal Investigator: Marcos Roberto de Mattos Fontes
• Grantee: Natália Elisa Bernardes
• Host Institution: Instituto de Biociências (IBB). Universidade Estadual Paulista (UNESP). Campus de Botucatu. Botucatu , SP, Brazil
• Associated research grant: 13/24705-3 - The filamentous fungus Neurospora crassa as a model organism for the functional characterization of proteins/transcription factors that regulate the carbohydrate metabolism, AP.TEM
• Associated scholarship(s): 15/17228-0 - Nuclear import assays with potential fungal NLS peptides, BE.EP.DR

Abstract

Communication between the cell nucleus and the cytoplasm occurs through the transport mechanisms that allow the passage of molecules in pores present in the nuclear envelope. Among the known transport pathways that enable the transport of macromolecules into or out of the nucleus through recognition of specific signaling sequences, the so-called Nuclear Import Classical Pathway is the best characterized. In this pathway, importin - ± (Imp±) identifies the proteins to be transported to the nucleus through recognition of nuclear localization sequences (NLS). The filamentous fungus Neurospora crassa is widely used in biochemical and molecular biological studies from the early twentieth century. Early studies of structural characterization of Imp± from Neurospora crassa (Imp±Nc) and its crystal structure revealed the presence of regions that can be related to specific protein recognition of NLSs. NLSs were recognized in proteins related to glycogen metabolism and response to light variations in fungi. The goal of this project is to identify the specifics of Imp±Nc by characterizing the interaction of the protein with specific NLSs of fungi and comparison of the functionality of these NLSs in nuclear import.Initially will be analyzed the affinities of Imp±Nc interactions with potential NLSs identified. Then the complex Imp±Nc / NLS peptides will be submitted to crystallization experiments and the best crystals subjected to X-ray diffraction, data collection and data processing. After steps of modeling and refinement, the structures will be elucidated and analyzed. In parallel, nuclear import assays in vitro will be realized to verify the functionality of NLSs interaction with Imp±Nc and Imp± from other organisms. The laboratory of Prof. Marcos R M Fontes, in the Institute of Biosciences of Botucatu, has the necessary infrastructure to conduct all the experiments described in the project, except the functional assays that should be conducted in collaboration with an international group. The candidate was a scientific initiation scholarship by FAPESP (Proc.: 2011/22043-8) and currently have a Masters scholarship (Proc.: 2012/16371-5). This project is linked to the Thematic Project recently approved (Proc. 2013/24705-3). (AU)

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)

BERNARDES, NATALIA E.; TAKEDA, AGNES A. S.; DREYER, THIAGO R.; CUPERTINO, FERNANDA B.; VIRGILIO, STELA; PANTE, NELLY; BERTOLINI, MARIA CELIA; FONTES, MARCOS R. M.. Nuclear transport of the Neurospora crassa NIT-2 transcription factor is mediated by importin-alpha. Biochemical Journal, v. 474, p. 14-pg., 2017-12-15. (14/10289-0, 13/24705-3)

Academic Publications

(References retrieved automatically from State of São Paulo Research Institutions)

BERNARDES, Natália Elisa. Structural studies with importin-α from Neurospora crassa and nuclear localization sequences (NLS) of proteins related with fungi metabolism. 2018. Doctoral Thesis - Universidade Estadual Paulista (Unesp). Instituto de Biociências. Botucatu Botucatu.