Structural and calorimetric studies with importin alpha of Neorospora crassa and nuclear localization sequences of transciption factores

• Grant number: 16/09314-6
• Support Opportunities: Scholarships in Brazil - Scientific Initiation
• Start date: August 01, 2016
• End date: December 31, 2018
• Field of knowledge: Biological Sciences - Biophysics - Molecular Biophysics
• Principal Investigator: Marcos Roberto de Mattos Fontes
• Grantee: Hamine Cristina de Oliveira
• 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

Abstract

The communication between the cell nucleus and cytoplasm occurs through transport mechanisms that allow the passage of molecules through pores present in the nuclear envelope. In the group of the mechanisms responsible for nuclear transport of macromolecules into or out of the nucleus through recognition of specific signal sequences, the classical pathway composed by Importin-alfa/beta heterodimer is one of the major mechanisms of transport. Importin-beta (ImpB) acts as the carrier while the importin-alfa (ImpA) acts as an adapter recognizing the nuclear localization sequence (NLS) present in proteins that have function into the nucleus. Since the beginning of XX century, the filamentous fungus Neurospora crassa is widely used in biochemical studies and molecular biology. The first studies of characterization and the crystallographic structure of Neurospora crassa ImpA (ImpANc) revealed the presence of regions that may be related to the nuclear localization sequence (NLSs). In fungi, NLSs were recognized in proteins related to glycogen metabolism and in response to light variations. The mechanisms responsible for the regulation of nuclear import, the classical pathway constituted by the heterodimer ImpA/ImpB is a major shift mechanism. In this process, the structural study of ImpA is the key for your understanding. This protein was recently crystallized and its structure was elucidated by our research group. Biophysics experiments with ImpANc protein have also been performed by us and showed that the protein retains some features present in Mus Musculus and Saccharomyces cerevisiae isoforms that already elucidate your crystallographic structure. However, the ImpANc shows amino acids not conserved near the IBB domain, which may be related to the specificity of this protein. So we are proposing in this project: a) The expression and purification of Neurospora crassa Imp± truncated at its N-terminal; b) To perform experiments that quantify the ImpANc interaction with NLS peptides of the hypothetical protein NCU01629 (VPRPKRQQRRL) using calorimetric techniques; c) To co-crystallize and elucidate the structure of ImpANc with peptide (VPRPKRQQRRL) of the protein involved in the regulation of glycogen metabolism.