ABSTRACT
The tumor suppressor p14arf interacts, in response to oncogenic signals, with the p53 E3-ubiquitin ligase HDM2, thereby resulting in p53 stabilization and activation. In addition, it also exerts tumor-suppressive functions in p53-independent contexts. The activities of p14arf are regulated by the nucleolar chaperone nucleophosmin (NPM1), which controls its levels and cellular localization. In acute myeloid leukemia with mutations in the NPM1 gene, mutated NPM1 aberrantly translocates in the cytosol carrying with itself p14arf that is subsequently degraded, thus impairing the p14arf-HDM2-p53 axis. In this work we investigated the complex between these two proteins by means of NMR and other techniques. We identified a novel NPM1-interacting motif in the C-terminal region of p14arf, which corresponds to its predicted nucleolar localization signal. This motif recognizes a specific region of the NPM1 N-terminal domain and, upon binding, the two proteins form soluble high molecular weight complexes. By NMR, we identified critical residues on both proteins involved in the interaction. Collectively, our data provide a structural framework to rationalize the overall assembly of the p14arf-NPM1 supramolecular complexes. A number of p14arf cancer-associated mutations cluster in this motif and their effect on the interaction with NPM1 was also analyzed.
Subject(s)
Nuclear Proteins/chemistry , Tumor Suppressor Protein p14ARF/chemistry , Amino Acid Motifs , Amino Acid Sequence , Animals , Humans , Leukemia, Myeloid, Acute/genetics , Leukemia, Myeloid, Acute/metabolism , Mice , Models, Molecular , Molecular Targeted Therapy , Mutation , Neoplasm Proteins/chemistry , Neoplasm Proteins/genetics , Neoplasm Proteins/physiology , Neoplasms/drug therapy , Neoplasms/genetics , Neoplasms/metabolism , Nuclear Magnetic Resonance, Biomolecular , Nuclear Proteins/genetics , Nuclear Proteins/physiology , Nucleophosmin , Protein Aggregates , Protein Conformation , Protein Interaction Domains and Motifs , Protein Interaction Mapping , Recombinant Fusion Proteins/chemistry , Sequence Alignment , Sequence Deletion , Sequence Homology, Amino Acid , Spectrometry, Fluorescence , Tumor Suppressor Protein p14ARF/genetics , Tumor Suppressor Protein p14ARF/physiologyABSTRACT
Nucleophosmin is a highly and ubiquitously expressed protein, mainly localized in nucleoli but able to shuttle between nucleus and cytoplasm. Nucleophosmin plays crucial roles in ribosome maturation and export, centrosome duplication, cell cycle progression, histone assembly and response to a variety of stress stimuli. Much interest in this protein has arisen in the past ten years, since the discovery of heterozygous mutations in the terminal exon of the NPM1 gene, which are the most frequent genetic alteration in acute myeloid leukemia. Nucleophosmin is also frequently overexpressed in solid tumours and, in many cases, its overexpression correlates with mitotic index and metastatization. Therefore it is considered as a promising target for the treatment of both haematologic and solid malignancies. NPM1 targeting molecules may suppress different functions of the protein, interfere with its subcellular localization, with its oligomerization properties or drive its degradation. In the recent years, several such molecules have been described and here we review what is currently known about them, their interaction with nucleophosmin and the mechanistic basis of their toxicity. Collectively, these molecules exemplify a number of different strategies that can be adopted to target nucleophosmin and we summarize them at the end of the review.
Subject(s)
Antineoplastic Agents/therapeutic use , Hematologic Neoplasms/drug therapy , Neoplasms/drug therapy , Nuclear Proteins/antagonists & inhibitors , Amino Acid Sequence , Antineoplastic Agents/chemistry , Hematologic Neoplasms/genetics , Hematologic Neoplasms/metabolism , Humans , Indoles/chemistry , Indoles/therapeutic use , Molecular Structure , Molecular Targeted Therapy/methods , Neoplasms/genetics , Neoplasms/metabolism , Nuclear Proteins/genetics , Nuclear Proteins/metabolism , Nucleophosmin , Sequence Homology, Amino AcidABSTRACT
Nucleophosmin (NPM1) is a nucleocytoplasmic shuttling protein, mainly localized at nucleoli, that plays a number of functions in ribosome biogenesis and export, cell cycle control, and response to stress stimuli. NPM1 is the most frequently mutated gene in acute myeloid leukemia; mutations map to the C-terminal domain of the protein and cause its denaturation and aberrant cytoplasmic translocation. NPM1 C-terminal domain binds G-quadruplex regions at ribosomal DNA and at gene promoters, including the well characterized sequence from the nuclease-hypersensitive element III region of the c-MYC promoter. These activities are lost by the leukemic variant. Here we analyze the NPM1/G-quadruplex interaction, focusing on residues belonging to both the NPM1 terminal three-helix bundle and a lysine-rich unstructured tail, which has been shown to be necessary for high affinity recognition. We performed extended site-directed mutagenesis and measured binding rate constants through surface plasmon resonance analysis. These data, supported by molecular dynamics simulations, suggest that the unstructured tail plays a double role in the reaction mechanism. On the one hand, it facilitates the formation of an encounter complex through long range electrostatic interactions; on the other hand, it directly contacts the G-quadruplex scaffold through multiple and transient electrostatic interactions, significantly enlarging the contact surface.
Subject(s)
G-Quadruplexes , Nuclear Proteins/physiology , Amino Acid Sequence , Base Sequence , DNA Primers , Molecular Dynamics Simulation , Molecular Sequence Data , Nuclear Proteins/chemistry , Nuclear Proteins/metabolism , Nucleophosmin , Protein Binding , Sequence Homology, Amino Acid , Surface Plasmon ResonanceABSTRACT
Nucleophosmin (NPM1) is a nucleolar protein implicated in ribosome biogenesis, centrosome duplication and cell cycle control; the NPM1 gene is the most frequent target for mutations in Acute Myeloid Leukemia. Mutations map to the C-terminal domain of the protein and cause its unfolding, loss of DNA binding properties and aberrant cellular localization. Here we investigate the folding pathway and denatured state properties of a NPM1 C-terminal domain construct encompassing the last 70 residues in the reference sequence. This construct is more stable than the previously characterized domain, which consisted of the last 53 residues. Data reveal that, similarly to what was discovered for the shorter construct, also the 70-residue construct of NPM1 displays a detectable residual structure in its denatured state. The higher stability of the latter domain allows us to conclude that the denatured state is robust to changes in solvent composition and that it consists of a discrete state in equilibrium with the expanded fully unfolded conformation. This observation, which might appear as a technicality, is in fact of general importance for the understanding of the folding of proteins. The implications of our results are discussed in the context of previous works on single domain helical proteins.
Subject(s)
Nuclear Proteins/chemistry , Protein Folding , Protein Structure, Tertiary , Protein Unfolding , Dose-Response Relationship, Drug , Humans , Kinetics , Nuclear Proteins/genetics , Nuclear Proteins/metabolism , Nucleophosmin , Protein Denaturation/drug effects , Protein Stability , Spectrometry, Fluorescence , Urea/chemistry , Urea/pharmacologyABSTRACT
Nucleophosmin (NPM1) is an abundant nucleolar protein implicated in ribosome maturation and export, centrosome duplication and response to stress stimuli. NPM1 is the most frequently mutated gene in acute myeloid leukemia. Mutations at the C-terminal domain led to variant proteins that aberrantly and stably translocate to the cytoplasm. We have previously shown that NPM1 C-terminal domain binds with high affinity G-quadruplex DNA. Here, we investigate the structural determinants of NPM1 nucleolar localization. We show that NPM1 interacts with several G-quadruplex regions found in ribosomal DNA, both in vitro and in vivo. Furthermore, the most common leukemic NPM1 variant completely loses this activity. This is the consequence of G-quadruplex-binding domain destabilization, as mutations aimed at refolding the leukemic variant also result in rescuing the G-quadruplex-binding activity and nucleolar localization. Finally, we show that treatment of cells with a G-quadruplex selective ligand results in wild-type NPM1 dislocation from nucleoli into nucleoplasm. In conclusion, this work establishes a direct correlation between NPM1 G-quadruplex binding at rDNA and its nucleolar localization, which is impaired in the acute myeloid leukemia-associated protein variants.