Nuclear import assays with potential fungal NLS peptides

• Grant number: 15/17228-0
• Support Opportunities: Scholarships abroad - Research Internship - Doctorate
• Start date: February 01, 2016
• End date: October 26, 2016
• Field of knowledge: Biological Sciences - Biochemistry - Molecular Biology
• Principal Investigator: Marcos Roberto de Mattos Fontes
• Grantee: Natália Elisa Bernardes
• Supervisor: Nelly Ofelia Pante
• Host Institution: Instituto de Biociências (IBB). Universidade Estadual Paulista (UNESP). Campus de Botucatu. Botucatu , SP, Brazil
• Institution abroad: University of British Columbia, Vancouver (UBC), Canada
• Associated to the scholarship: 14/10289-0 - Structural studies with importin-± and nuclear localization sequences (NLS) of proteins related with fungi metabolism, BP.DR

Abstract

Importin-alpha (Impa) is a protein that mediates nuclear import via recognition of nuclear localization sequences (NLSs) in the macromolecule that will be transported. The concave surface of Impa contains conserved residues that form two NLS binding sites known as major and minor NLS binding sites. To better understand NLS recognition and gain information about the fungal nuclear import process, the first crystal structure of Impa from a filamentous fungus (Neurospora crassa; NcImpa), complexed with a classical NLS, was elucidated by our group. Now, the focus is to study the interactions between NcImpa with potential fungal NLSs, by crystallographic, thermodynamic and functional experiments. N. crassa is widely used as a model organism to understand the regulation of basic cellular processes, such as glycogen metabolism. Many proteins and transcription factors have been identified that likely play a role in this process. Some of these proteins and transcription factors possess putative nuclear localization signals (NLSs), suggesting that they can be transported into the nucleus via the classical nuclear import pathway, which is mediated by Importins. The analysis of these potentials NLSs by isothermal titration calorimetry showed a high interaction affinity with NcImp±. The aim of this project is to verify the functionality of these NLSs from filamentous fungus to be transported into the nucleus by Imp± and to identify changes in their transport by adding mutated NLS or a NLS competitor. For this, these promising NLSs will be submitted to in vivo studies with digitonin-permeabilized HeLa cells to observe the nuclear import of these peptides, under biological conditions. (AU)