title: Cytosolic 5'-nucleotidase II interacts with the leucin rich repeat of NLR family member Ipaf.
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.
authors: University of Pisa, Department of Biology, Biochemistry Unit, Pisa, Italy.
authors: Federico
authors: F
authors: Cividini
authors: University of Pisa, Department of Biology, Biochemistry Unit, Pisa, Italy.
authors: Maria Grazia
authors: MG
authors: Tozzi
authors: National Research Counsil (CNR), Institute of Clinical Physiology, Pisa, Italy.
authors: Alvaro
authors: A
authors: Galli
authors: University of Pisa, Department of Biology, Biochemistry Unit, Pisa, Italy.
authors: Rossana
authors: R
authors: Pesi
authors: University of Pisa, Department of Biology, Biochemistry Unit, Pisa, Italy.
authors: Marcella
authors: M
authors: Camici
authors: Université de Lyon, Lyon, France; Université de Lyon 1, Lyon, France; INSERM U1052, Centre de Recherche en Cancérologie de Lyon, Lyon, France; CNRS UMR 5286, Centre de Recherche en Cancérologie de Lyon, Lyon, France.
authors: Charles
authors: C
authors: Dumontet
authors: Université de Lyon, Lyon, France; Université de Lyon 1, Lyon, France; INSERM U1052, Centre de Recherche en Cancérologie de Lyon, Lyon, France; CNRS UMR 5286, Centre de Recherche en Cancérologie de Lyon, Lyon, France.
authors: Lars Petter
authors: LP
authors: Jordheim
authors: University of Sassari, Department of Chemistry and Pharmacology, Sassari, Italy.
authors: Simone
authors: S
authors: Allegrini
mesh_terms: D015720:5'-Nucleotidase / Q000378:metabolism*; D000818:Animals; D053468:CARD Signaling Adaptor Proteins / Q000378:metabolism*; D002135:Calcium-Binding Proteins / Q000378:metabolism*; D002417:Cattle; D002457:Cell Extracts; D045744:Cell Line, Tumor; D057809:HEK293 Cells; D006801:Humans; D047468:Immunoprecipitation; D000090263:Leucine-Rich Repeat Proteins; D011485:Protein Binding; D011506:Proteins / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D040681:Structural Homology, Protein; D014162:Transfection
chemical_list: D053468:CARD Signaling Adaptor Proteins; D002135:Calcium-Binding Proteins; D002457:Cell Extracts; D000090263:Leucine-Rich Repeat Proteins; C435692:NLRC4 protein, human; D011506:Proteins; D011994:Recombinant Proteins; D015720:5'-Nucleotidase; C508048:NT5C2 protein, human