Cytosolic 5'-nucleotidase II interacts with the leucin rich repeat of NLR family member Ipaf

PLoS One. 2015 Mar 26;10(3):e0121525. doi: 10.1371/journal.pone.0121525. eCollection 2015.

Abstract

IMP/GMP preferring cytosolic 5'-nucleotidase II (cN-II) is a bifunctional enzyme whose activities and expression play crucial roles in nucleotide pool maintenance, nucleotide-dependent pathways and programmed cell death. Alignment of primary amino acid sequences of cN-II from human and other organisms show a strong conservation throughout the entire vertebrata taxon suggesting a fundamental role in eukaryotic cells. With the aim to investigate the potential role of this homology in protein-protein interactions, a two hybrid system screening of cN-II interactors was performed in S. cerevisiae. Among the X positive hits, the Leucin Rich Repeat (LRR) domain of Ipaf was found to interact with cN-II. Recombinant Ipaf isoform B (lacking the Nucleotide Binding Domain) was used in an in vitro affinity chromatography assay confirming the interaction obtained in the screening. Moreover, co-immunoprecipitation with proteins from wild type Human Embryonic Kidney 293 T cells demonstrated that endogenous cN-II co-immunoprecipitated both with wild type Ipaf and its LRR domain after transfection with corresponding expression vectors, but not with Ipaf lacking the LRR domain. These results suggest that the interaction takes place through the LRR domain of Ipaf. In addition, a proximity ligation assay was performed in A549 lung carcinoma cells and in MDA-MB-231 breast cancer cells and showed a positive cytosolic signal, confirming that this interaction occurs in human cells. This is the first report of a protein-protein interaction involving cN-II, suggesting either novel functions or an additional level of regulation of this complex enzyme.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • 5'-Nucleotidase / metabolism*
  • Animals
  • CARD Signaling Adaptor Proteins / metabolism*
  • Calcium-Binding Proteins / metabolism*
  • Cattle
  • Cell Extracts
  • Cell Line, Tumor
  • HEK293 Cells
  • Humans
  • Immunoprecipitation
  • Protein Binding
  • Proteins / metabolism*
  • Recombinant Proteins / metabolism
  • Structural Homology, Protein
  • Transfection

Substances

  • CARD Signaling Adaptor Proteins
  • Calcium-Binding Proteins
  • Cell Extracts
  • NLRC4 protein, human
  • Proteins
  • Recombinant Proteins
  • leucine-rich repeat proteins
  • 5'-Nucleotidase
  • NT5C2 protein, human

Grant support

CD’s laboratory receives funding through ANR grant 11-BS07-032-01 “cN-II Focus”. MGT’s laboratory was financially supported by a grant from the REGIONE AUTONOMA DELLA SARDEGNA, L.R. 07/08/2007, CRP 3360 (TITLE: Modulation of expression by inducible silencing; heterologous expression in S. cerevisiae; two-hybrid system. Three models to make clear the physiological role of cytosolic 5′nucleotidase II). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.