Sonia Bañuelos

Phosphorylation of Both Nucleoplasmin Domains Is Required for Activation of Its Chromatin Decondensation Activity

Nucleoplasmin (NP) is a histone chaperone involved in nucleosome assembly, chromatin decondensation at fertilization, and apoptosis. To carry out these activities NP has to interact with different types of histones, an interaction that is regulated by phosphorylation. Here we have identified a number of phosphorylated residues by mass spectrometry and generated mutants in which these amino acids are replaced by Asp to mimic the effect of phosphorylation. Our results show that, among the eight phosphoryl groups experimentally detected, four are located at the flexible N terminus, and the rest are found at the tail domain, flanking the nuclear localization signal. Phosphorylation-mimicking mutations render a recombinant protein as active in chromatin decondensation as hyperphosphorylated NP isolated from Xenopus laevis eggs. Comparison of mutants in which the core and tail domains of the protein were independently or simultaneously “activated” indicates that activation or phosphorylation of both protein domains is required for NP to efficiently extract linker-type histones from chromatin.

A global survey of CRM1-dependent nuclear export sequences in the human deubiquitinase family.

The mechanisms that regulate the nucleocytoplasmic localization of human deubiquitinases remain largely unknown. The nuclear export receptor CRM1 binds to specific amino acid motifs termed NESs (nuclear export sequences). By using in silico prediction and experimental validation of candidate sequences, we identified 32 active NESs and 78 inactive NES-like motifs in human deubiquitinases. These results allowed us to evaluate the performance of three programs widely used for NES prediction, and to add novel information to the recently redefined NES consensus. The novel NESs identified in the present study reveal a subset of 22 deubiquitinases bearing motifs that might mediate their binding to CRM1. We tested the effect of the CRM1 inhibitor LMB (leptomycin B) on the localization of YFP (yellow fluorescent protein)- or GFP (green fluorescent protein)-tagged versions of six NES-bearing deubiquitinases [USP (ubiquitin-specific peptidase) 1, USP3, USP7, USP21, CYLD (cylindromatosis) and OTUD7B (OTU-domain-containing 7B)]. YFP-USP21 and, to a lesser extent, GFP-OTUD7B relocated from the cytoplasm to the nucleus in the presence of LMB, revealing their nucleocytoplasmic shuttling capability. Two sequence motifs in USP21 had been identified during our survey as active NESs in the export assay. Using site-directed mutagenesis, we show that one of these motifs mediates USP21 nuclear export, whereas the second motif is not functional in the context of full-length USP21.

Interaction of native and partially folded conformations of α-lactalbumin with lipid bilayers: characterization of two membrane-bound states

α‐Lactalbumin (αLA) can adopt two different membrane‐bound states depending on the physical properties of the lipid bilayer, namely adsorbed and inserted. The latter, but not the adsorbed state, is able to disrupt the permeability barrier of the bilayer. The structure of both states is strongly affected by the conformational properties of the αLA conformer considered: as protein flexibility increases the helical content of the membrane‐bound conformation decreases, especially in the adsorbed form. Moreover, the adsorbed and the inserted states of those conformers containing 3 or 4 disulfides can interconvert in response to changes in the physical properties of the host membrane.

Recognition of nucleoplasmin by its nuclear transport receptor importin α/β: insights into a complete import complex.

Nuclear import of the pentameric histone chaperone nucleoplasmin (NP) is mediated by importin α, which recognizes its nuclear localization sequence (NLS), and importin β, which interacts with α and is in charge of the translocation of the NP/α/β complex through the nuclear pore. Herein, we characterize the assembly of a functional transport complex formed by full-length NP with importin α/β. Isothermal titration calorimetry (ITC) was used to analyze the thermodynamics of the interactions of importin α with β, α with NP, and the α/β heterodimer with NP. Our data show that binding of both importin α and α/β to NP is governed by a favorable enthalpic contribution and that NP can accommodate up to five importin molecules per NP pentamer. Phosphomimicking mutations of NP, which render the protein active in histone chaperoning, do not modulate the interaction with importin. Using small-angle X-ray scattering, we model the α/β heterodimer, NP/α, and NP/α/β solution structures, which reveal a glimpse of a complete nuclear import complex with an oligomeric cargo protein. The set of alternative models, equally well fitting the scattering data, yields asymmetric elongated particles that might represent consecutive geometries the complex can adopt when stepping through the nuclear pore.

Activation of nucleoplasmin, an oligomeric histone chaperone, challenges its stability.

Nucleoplasmin (NP) is a pentameric, ring-shaped histone chaperone involved in chromatin remodeling processes such as sperm decondensation at fertilization. Monomers are formed by a core domain, responsible for oligomerization, that confers the protein a high stability and compactness and a flexible tail domain, that harbors a polyglutamic tract and the nuclear localization signal. Fully activated NP presents multiple phosphorylated residues in the tail and in flexible regions of the core domain. In this work, we analyze the effect of activation on the structure and stability of the full-length protein and the isolated core domain through phosphorylation mimicking mutations. We have solved the crystal structure of an activated NP core domain that, however, is not significantly different from that of the wild-type,inactive, NP core. Nevertheless, we find that NP activation results in a strong destabilization of the pentamer probably due to electrostatic repulsion. Moreover, characterization of the hydrodynamic properties of both full-length and core domain proteins indicates that activating mutations lead to an expansion of the NP pentamer in solution. These findings suggest that NP needs a compact and stable structure to afford the accumulation of negative charges that weakens its quaternary interactions but is required for its biological function.

Recognition of intermolecular G-quadruplexes by full length nucleophosmin. Effect of a leukaemia-associated mutation.

Nucleophosmin (NPM) is a nucleolar protein involved in ribosome biogenesis. NPM1 gene is frequently mutated in acute myeloid leukaemia (AML), correlating with aberrant cytoplasmic localization of the protein. NPM attachment to the nucleolus in physiological conditions probably depends on binding to nucleic acids, and this recognition could be altered in AML. NPM associates to guanine‐rich DNA sequences, able to fold as “G‐quadruplexes”. We have analyzed the interaction of pentameric, full length NPM with G‐rich oligonucleotides, finding that the protein binds preferentially high‐order G‐quadruplexes. AML‐associated mutation significantly hampers DNA binding, pointing to a possible mechanism contributing to pathological mislocalization of NPM.

Thermodynamic characterization of nucleoplasmin unfolding: interplay between function and stability.

The unfolding equilibrium of recombinant (rNP) and natural variants of nucleoplasmin (NP) from Xenopus laevis has been analyzed using biochemical and spectroscopic techniques. In the presence of denaturing concentrations of guanidinium salts (GuHCl and GuSCN), both domains, core and tail, of the rNP pentamer unfold as proven using single-carrying tryptophan mutants, whereas urea is remarkably unable to fully unfold rNP. Chemical unfolding is reversible and can be described well as a two-state transition in which the folded pentamer is directly converted to unfolded monomers, with no evidence of (partially) folded monomers. Therefore, rNP dissociates and fully unfolds simultaneously (N 5 <--> 5U). Activation of the protein by hyperphosphorylation is accompanied by a destabilization of the protein oligomer. A comparison of natural NP forms isolated from eggs and oocytes of X. laevis and recombinant NP reveals that natural variants can be fully unfolded by urea and exhibit D 50 (denaturant concentration at the transition midpoint) values lower than that of the nonphosphorylated protein. Progressive phosphorylation of NP correlates with a gradual loss of stability of 6 kcal/mol (oNP) and 10 kcal/mol (eNP), as compared with the nonphosphorylated protein pentamer. These results suggest that the remarkable stability of the recombinant protein is required to cope with the destabilization brought about by its phosphorylation-induced activation.

Leukemia-Associated Mutations in Nucleophosmin Alter Recognition by CRM1: Molecular Basis of Aberrant Transport

Nucleophosmin (NPM) is a nucleocytoplasmic shuttling protein, normally enriched in nucleoli, that performs several activities related to cell growth. NPM mutations are characteristic of a subtype of acute myeloid leukemia (AML), where mutant NPM seems to play an oncogenic role. AML-associated NPM mutants exhibit altered subcellular traffic, being aberrantly located in the cytoplasm of leukoblasts. Exacerbated export of AML variants of NPM is mediated by the nuclear export receptor CRM1, and due, in part, to a mutationally acquired novel nuclear export signal (NES). To gain insight on the molecular basis of NPM transport in physiological and pathological conditions, we have evaluated the export efficiency of NPM in cells, and present new data indicating that, in normal conditions, wild type NPM is weakly exported by CRM1. On the other hand, we have found that AML-associated NPM mutants efficiently form complexes with CRM1HA (a mutant CRM1 with higher affinity for NESs), and we have quantitatively analyzed CRM1HA interaction with the NES motifs of these mutants, using fluorescence anisotropy and isothermal titration calorimetry. We have observed that the affinity of CRM1HA for these NESs is similar, which may help to explain the transport properties of the mutants. We also describe NPM recognition by the import machinery. Our combined cellular and biophysical studies shed further light on the determinants of NPM traffic, and how it is dramatically altered by AML-related mutations.

The Nuclear Transport Machinery Recognizes Nucleoplasmin–Histone Complexes

The nuclear transport of the chromatin remodeling protein nucleoplasmin and chromatin building histones is mediated by importins. Nucleoplasmin (NP) contains a classical bipartite nuclear localization signal (NLS) that is recognized by the importin α/β heterodimer, while histones present multiple NLS-like motifs that are recognized by importin β family members for nuclear targeting. To explore the possibility of a cotransport of histones and their chaperone NP to the nucleus, we have analyzed the assembly of complexes of NP/histones with importins by means of fluorescence anisotropy, centrifugation in sucrose gradients, and isothermal titration calorimetry. Data show that importin α ΔIBB (a truncated form of importin α lacking the autoinhibitory N-terminal domain) and histones (linker, H5, and nucleosomal core, H2AH2B) can simultaneously bind to NP. Analysis of the binding energetics reveals an enthalpy-driven formation of high affinity ternary, NP/Δα/H5 and NP/Δα/H2AH2B, complexes. We find that different amount of importin α molecules can be loaded on NP/histone complexes dependent on the histone type, linker or core, and the amount of bound histones. We further demonstrate that NP/H5 complexes can also incorporate importin α/β, thus forming quaternary NP/histones/α/β complexes that might represent a putative coimport pathway for nuclear import of histones and their chaperone protein NP, enhancing the histone import efficiency.

A cellular reporter to evaluate CRM1 nuclear export activity: functional analysis of the cancer-related mutant E571K

The exportin CRM1 binds nuclear export signals (NESs), and mediates active transport of NES-bearing proteins from the nucleus to the cytoplasm. Structural and biochemical analyses have uncovered the molecular mechanisms underlying CRM1/NES interaction. CRM1 binds NESs through a hydrophobic cleft, whose open or closed conformation facilitates NES binding and release. Several cofactors allosterically modulate the conformation of the NES-binding cleft through intramolecular interactions involving an acidic loop and a C-terminal helix in CRM1. This current model of CRM1-mediated nuclear export has not yet been evaluated in a cellular setting. Here, we describe SRV100, a cellular reporter to interrogate CRM1 nuclear export activity. Using this novel tool, we provide evidence further validating the model of NES binding and release by CRM1. Furthermore, using both SRV100-based cellular assays and in vitro biochemical analyses, we investigate the functional consequences of a recurrent cancer-related mutation, which targets a residue near CRM1 NES-binding cleft. Our data indicate that this mutation does not necessarily abrogate the nuclear export activity of CRM1, but may increase its affinity for NES sequences bearing a more negatively charged C-terminal end.