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

Cividini, Federico and Tozzi, Maria Grazia and Galli, Alvaro and Pesi, Rossana and Camici, Marcella and Dumontet, Charles and Jordheim, Lars Petter and Allegrini, Simone (2015) Cytosolic 5’-nucleotidase II interacts with the leucin rich repeat of NLR family member Ipaf. PLoS One, Vol. 10 (3), e0121525. ISSN 1932-6203. Article.

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DOI: 10.1371/journal.pone.0121525

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.

Item Type:Article
ID Code:11781
Status:Published
Refereed:Yes
Uncontrolled Keywords:Protein interactions, recombinant proteins, two-hybrid screening, cloning, Saccharomyces cerevisiae, apoptosis, plasmid construction, transfection
Subjects:Area 05 - Scienze biologiche > BIO/10 Biochimica
Divisions:001 Università di Sassari > 01-a Nuovi Dipartimenti dal 2012 > Chimica e Farmacia
Publisher:Public Library of Science
ISSN:1932-6203
Copyright Holders:© 2015 Cividini et al.
Deposited On:20 Jun 2017 11:44

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