Abstract
Nucleophosmin (NPM1) is a multifunctional nucleolar protein implicated in ribogenesis, centrosome duplication, cell cycle control, regulation of DNA repair and apoptotic response to stress stimuli. The majority of these functions are played through the interactions with a variety of protein partners. NPM1 is frequently overexpressed in solid tumors of different histological origin. Furthermore NPM1 is the most frequently mutated protein in acute myeloid leukemia (AML) patients. Mutations map to the C-terminal domain and lead to the aberrant and stable localization of the protein in the cytoplasm of leukemic blasts. Among NPM1 protein partners, a pivotal role is played by the tumor suppressor Fbw7γ, an E3-ubiquitin ligase that degrades oncoproteins like c-MYC, cyclin E, Notch and c-jun. In AML with NPM1 mutations, Fbw7γ is degraded following its abnormal cytosolic delocalization by mutated NPM1. This mechanism also applies to other tumor suppressors and it has been suggested that it may play a key role in leukemogenesis. Here we analyse the interaction between NPM1 and Fbw7γ, by identifying the protein surfaces implicated in recognition and key aminoacids involved. Based on the results of computational methods, we propose a structural model for the interaction, which is substantiated by experimental findings on several site-directed mutants. We also extend the analysis to two other NPM1 partners (HIV Tat and CENP-W) and conclude that NPM1 uses the same molecular surface as a platform for recognizing different protein partners. We suggest that this region of NPM1 may be targeted for cancer treatment.
Highlights
The pleiotropic behavior of NPM1 is due to its modular structure consisting of: (i) an N-terminal oligomerization domain involved in protein–protein interactions and containing two nuclear export signals (NES);[1,4] (ii) an intrinsically unstructured central region which contains a bipartite nuclear localization signal (NLS) and (iii) a C-terminal nucleic acid binding domain where the nucleolar localization signal (NoLS) is located.[6]
In an effort to understand the molecular mechanism whereby Fbw7γ localizes in nucleoli, we carried out a bioinformatic analysis of Fbw[7] isoforms using the NoD algorithm (Nucleolar Localization Signal Detector; http://www.compbio.dundee.ac.uk/www-nod/ index.jsp) to identify putative nucleolar localization signals (NoLS) in these proteins (Figures 1a–c)
This analysis, which relies on sequence only, is based on the observation that the NoLS of many proteins consists of a short motif rich in lysines and arginines positioned in variably spaced clusters.[20]
Summary
Nucleophosmin (NPM1) is an abundant and ubiquitous protein[1] mainly localized in nucleoli, where it contributes to their structure and organization,[2,3] and shuttles between nucleolus and cytoplasm to perform its functions.[4,5,6] NPM1 has a primary role in ribosome biogenesis and transport[7,8] and contributes to the maintenance of genomic stability and DNA repair,[9,10] histones assembly,[11,12] centrosome duplication,[13,14] cell cycle regulation and response to stress stimuli.[5]. WD40 domain, which recognizes phosphorylated substrates.[22] The Fbw[7] gene codes for three protein isoforms (namely α, β and γ) differing in their N-terminal region and displaying distinct cellular localization: Fbw7α is nucleoplasmic, Fbw7β is cytoplasmic and Fbw7γ is nucleolar.[23] Many of Fbw[7] targets are oncoproteins, including c-MYC, Notch, Cyclin E and c-Jun[22] and isoforms localization may be instrumental in their regulation through the compartmentalization of substrates recognition and degradation. We provide and validate a structural model for the interaction through protein-peptide docking and molecular dynamics simulations We extend this analysis to two other NPM1 interacting proteins, namely Tat[26] and CENP-W,27,28 demonstrating that the same region of NPM1 recognizes all these proteins, substantiating the proposed role of NPM1 as a ‘nucleolar hub’. We suggest that this protein region may be targeted for the treatment of AML with NPM1 mutations
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