Falini B, Bolli N, Shan J, Martelli MP, Liso A, Pucciarini A et al.. Both carboxy-terminus NES motif and mutated tryptophan(s) are crucial for aberrant nuclear export of nucleophosmin leukemic mutants in NPMc+ AML. Blood 107: 4514-4523
We recently identified aberrant cytoplasmic expression of nucleophosmin (NPM) as the immunohistochemical marker of a large subgroup of acute myeloid leukemia (AML) (about one-third of adult AML) that is characterized by normal karyotype and mutations occurring at the exon-12 of the NPM gene. In this paper, we have elucidated the molecular mechanism underlying the abnormal cytoplasmic localization of NPM. All 29 AML-associated mutated NPM alleles so far identified encode abnormal proteins which have acquired at the C-terminus a nuclear export signal (NES) motif and lost both tryptophan residues 288 and 290 (or only the residue 290) which determine nucleolar localization. We show for the first time that both alterations are crucial for NPM mutant export from nucleus to cytoplasm. In fact, the cytoplasmic accumulation of NPM is blocked by leptomycin-B and ratjadones, specific exportin-1/Crm1-inhibitors, and by reinsertion of tryptophan residues 288 and 290, which respectively relocate NPM mutants in the nucleoplasm and nucleoli. NPM leukemic mutants in turn recruit the wild-type NPM from nucleoli to nucleoplasm and cytoplasm. These findings indicate that potential therapeutic strategies aimed to retarget NPM to its physiological sites will have to overcome 2 obstacles, the new NES motif and the mutated tryptophan(s) at the NPM mutant C-terminus.
"This protein has been found over-expressed in tumors of different histological origins, including gastric, ovarian, bladder and prostate carcinomas and in various hematological malignancies    . Notably, NPM1/ B23 has been identified as the most frequently mutated gene in acute myeloid leukemia (AML) patients, accounting for approximately 30% of cases     . NPM1/B23 belongs to the nucleophosmin/nucleoplasmin family of proteins . "
[Show abstract][Hide abstract] ABSTRACT: Background
Nucleophosmin (NPM1, B23) is a multifunctional protein that is involved in a variety of fundamental biological processes. NPM1/B23 deregulation is implicated in the pathogenesis of several human malignancies. This protein exerts its functions through the interaction with a multiplicity of biological partners. Very recently it is has been shown that NPM1/B23 specifically recognizes DNA G-quadruplexes through its C-terminal region.
Through a rational dissection approach of protein here we show that the intrinsically unfolded regions of NPM1/B23 significantly contribute to the binding of c-MYC G-quadruplex motif. Interestingly, the analysis of the ability of distinct NPM1/B23 fragments to bind this quadruplex led to the identifications of distinct NPM1/B23-based peptides that individually present a high affinity for this motif.
These results suggest that the tight binding of NPM1/B23 to the G-quadruplex is achieved through the cooperation of both folded and unfolded regions that are individually able to bind it. The dissection of NPM1/B23 also unveils that its H1 helix is intrinsically endowed with an unusual thermal stability.
These findings have implications for the unfolding mechanism of NPM1/B23, for the G-quadruplex affinity of the different NPM1/B23 isoforms and for the design of peptide-based molecules able to interact with this DNA motif.
Biochimica et Biophysica Acta (BBA) - General Subjects 06/2014; 1840(6). DOI:10.1016/j.bbagen.2014.02.017 · 4.38 Impact Factor
"We further investigated this issue by re-introducing the Trp residues in their native topological positions but in the context of the mutant A sequence, and we showed that (i) by introducing Trp288 or Trp290 separately, only a partial rescue of folding is observed; (ii) when both Trp288 and Trp290 are inserted in their topological position, the complete refolding of the domain is observed in the context of the variant sequence; and (iii) the NPM1-Cter-MutA refolded variant binds to G-quadruplex regions at rDNA with an affinity similar to the native construct. Importantly, previous work already demonstrated that such variant re-acquire nucleolar localization (41). Taken together, these data demonstrate that a direct correlation exists between NPM1 G-quadruplex binding at rDNA and its nucleolar localization. "
[Show abstract][Hide abstract] ABSTRACT: 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.
Nucleic Acids Research 01/2013; 41(5). DOI:10.1093/nar/gkt001 · 9.11 Impact Factor
"However, these novel mutations show a tryptophan loss at codon 290 only. NPM mutant proteins that retained tryptophan 288 accounted for 31% of all mutants in AML patients, while tryptophan 290 was mutated in all the NPM1 mutations identified . We infer that tryptophan 290 may be more critical to nucleolar localization; thus, a single mutation at tryptophan 290 may be sufficiently relevant to AML pathogenesis. "
[Show abstract][Hide abstract] ABSTRACT: Genetic abnormalities in adult AML are caused most frequently by somatic mutations in exon 12 of the NPM1 gene, which is observed in approximately 35% of AML patients and up to 60% of patients with cytogenetically normal AML (CN-AML).
We performed mutational analysis, including fragment analysis and direct sequencing of exon 12 of the NPM1 gene, on 83 AML patients to characterize the NPM1 mutations completely.
In this study, NPM1 mutations were identified in 19 (22.9%) of the 83 AML patients and in 12 (42.9%) of the 28 CN-AML patients. Among the 19 patients with NPM1 mutations, type A NPM1 mutations were identified in 16 (84.2%) patients, whereas non-A type NPM1 mutations were observed in 3 (15.8%) patients. Two of the 3 non-A type NPM1 mutations were novel: c.867_868insAAAC and c.869_873indelCTTTAGCCC. These 2 novel mutant proteins display a nuclear export signal motif (L-xxx-L-xx-V-x-L) less frequently and exhibit a mutation at tryptophan 290 that disrupts the nucleolar localization signal.
This study suggests that novel NPM1 mutations may be non-rare and that supplementary sequence analysis is needed along with conventional targeted mutational analysis to detect non-A types of NPM1 mutations.
Annals of Laboratory Medicine 01/2013; 33(1):60-4. DOI:10.3343/alm.2013.33.1.60 · 1.48 Impact Factor
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