Conformational Rearrangements Regulating the DNA Repair Protein APE1.

Apurinic apyrimidinic endonuclease 1 (APE1) is a key enzyme of the Base Excision Repair (BER) pathway, which primarily manages oxidative lesions of DNA. Once the damaged base is removed, APE1 recognises the resulting abasic site and cleaves the phosphodiester backbone to allow for the correction by subsequent enzymes of the BER machinery. In spite of a wealth of information on APE1 structure and activity, its regulation mechanism still remains to be understood. Human APE1 consists of a globular catalytic domain preceded by a flexible N-terminal extension, which might be involved in the interaction with DNA. Moreover, the binding of the nuclear chaperone nucleophosmin (NPM1) to this region has been reported to impact APE1 catalysis. To evaluate intra- and inter-molecular conformational rearrangements upon DNA binding, incision, and interaction with NPM1, we used Förster resonance energy transfer (FRET), a fluorescence spectroscopy technique sensitive to molecular distances. Our results suggest that the N-terminus approaches the DNA at the downstream side of the abasic site and enables the building of a predictive model of the full-length APE1/DNA complex. Furthermore, the spatial configuration of the N-terminal tail is sensitive to NPM1, which could be related to the regulation of APE1.

Molecular Mechanisms Regulating the DNA Repair Protein APE1: A Focus on Its Flexible N-Terminal Tail Domain.

APE1 (DNA (apurinic/apyrimidinic site) endonuclease 1) is a key enzyme of one of the major DNA repair routes, the BER (base excision repair) pathway. APE1 fulfils additional functions, acting as a redox regulator of transcription factors and taking part in RNA metabolism. The mechanisms regulating APE1 are still being deciphered. Structurally, human APE1 consists of a well-characterized globular catalytic domain responsible for its endonuclease activity, preceded by a conformationally flexible N-terminal extension, acquired along evolution. This N-terminal tail appears to play a prominent role in the modulation of APE1 and probably in BER coordination. Thus, it is primarily involved in mediating APE1 localization, post-translational modifications, and protein-protein interactions, with all three factors jointly contributing to regulate the enzyme. In this review, recent insights on the regulatory role of the N-terminal region in several aspects of APE1 function are covered. In particular, interaction of this region with nucleophosmin (NPM1) might modulate certain APE1 activities, representing a paradigmatic example of the interconnection between various regulatory factors.

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.

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.

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/ß heterodimer, while histones present multiple NLS-like motifs that are recognized by importin ß 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/ß, thus forming quaternary NP/histones/α/ß complexes that might represent a putative coimport pathway for nuclear import of histones and their chaperone protein NP, enhancing the histone import efficiency.

Recognition of nucleoplasmin by its nuclear transport receptor importin α/ß: 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 ß, which interacts with α and is in charge of the translocation of the NP/α/ß complex through the nuclear pore. Herein, we characterize the assembly of a functional transport complex formed by full-length NP with importin α/ß. Isothermal titration calorimetry (ITC) was used to analyze the thermodynamics of the interactions of importin α with ß, α with NP, and the α/ß heterodimer with NP. Our data show that binding of both importin α and α/ß 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/ß heterodimer, NP/α, and NP/α/ß 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.

Nucleophosmin, a multifunctional nucleolar organizer with a role in DNA repair.

Nucleophosmin (NPM1) is a mostly nucleolar protein with crucial functions in cell growth and homeostasis, including regulation of ribosome biogenesis and stress response. Such multiple activities rely on its ability to interact with nucleic acids and with hundreds of proteins, as well as on a dynamic subcellular distribution. NPM1 is thus regulated by a complex interplay between localization and interactions, further modulated by post-translational modifications. NPM1 is a homopentamer, with globular domains connected by long, intrinsically disordered linkers. This configuration allows NPM1 to engage in liquid-liquid phase separation phenomena, which could underlie a key role in nucleolar organization. Here, we will discuss NPM1 conformational and functional versatility, emphasizing its emerging, and still largely unexplored, role in DNA damage repair. Since NPM1 is altered in a subtype of acute myeloid leukaemia (AML), we will also present ongoing research on the molecular mechanisms underlying its pathogenic role and potential NPM1-targeting therapeutic strategies.

Nucleophosmin interaction with APE1: Insights into DNA repair regulation.

Nucleophosmin (NPM1), an abundant, nucleolar protein with multiple functions affecting cell homeostasis, has also been recently involved in DNA damage repair. The roles of NPM1 in different repair pathways remain however to be elucidated. NPM1 has been described to interact with APE1 (apurinic apyrimidinic endonuclease 1), a key enzyme of the base excision repair (BER) pathway, which could reflect a direct participation of NPM1 in this route. To gain insight into the possible role(s) of NPM1 in BER, we have explored the interplay between the subnuclear localization of both APE1 and NPM1, the in vitro interaction they establish, the effect of binding to abasic DNA on APE1 conformation, and the modulation by NPM1 of APE1 binding and catalysis on DNA. We have found that, upon oxidative damage, NPM1 is released from nucleoli and locates on patches throughout the chromatin, perhaps co-localizing with APE1, and that this traffic could be mediated by phosphorylation of NPM1 on T199. NPM1 and APE1 form a complex in vitro, involving, apart from the core domain, at least part of the linker region of NPM1, whereas the C-terminal domain is dispensable for binding, which explains that an AML leukemia-related NPM1 mutant with an unfolded C-terminal domain can bind APE1. APE1 interaction with abasic DNA stabilizes APE1 structure, as based on thermal unfolding. Moreover, our data suggest that NPM1, maybe by keeping APE1 in an "open" conformation, favours specific recognition of abasic sites on DNA, competing with off-target associations. Therefore, NPM1 might participate in BER favouring APE1 target selection as well as turnover from incised abasic DNA.

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.

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.

Conformational stabilization as a strategy to prevent nucleophosmin mislocalization in leukemia.

Nucleophosmin (NPM) is a nucleolar protein involved in ribosome assembly and cell homeostasis. Mutations in the C-terminal domain of NPM that impair native folding and localization are associated with acute myeloid leukemia (AML). We have performed a high-throughput screening searching for compounds that stabilize the C-terminal domain. We identified three hit compounds which show the ability to increase the thermal stability of both the C-terminal domain as well as full-length NPM. The best hit also seemed to favor folding of an AML-like mutant. Computational pocket identification and molecular docking support a stabilization mechanism based on binding of the phenyl/benzene group of the compounds to a particular hydrophobic pocket and additional polar interactions with solvent-accessible residues. Since these results indicate a chaperoning potential of our candidate hits, we tested their effect on the subcellular localization of AML-like mutants. Two compounds partially alleviated the aggregation and restored nucleolar localization of misfolded mutants. The identified hits appear promising as pharmacological chaperones aimed at therapies for AML based on conformational stabilization of NPM.

Actin binding of a minispectrin.

A "minispectrin" has been constructed from the tail end of the alpha/beta heterodimer, and its actin-binding properties have been characterised. It is a complex of the N-terminal fragment of the beta-subunit consisting of the actin-binding domain plus the two first triple-helical repeats beta 1 and beta 2, and the C-terminal fragment of the alpha-subunit containing the repeats alpha 19 and alpha 20 plus the calmodulin-like domain. This minispectrin exists in a dimeric form that contains one copy of each polypeptide and binds to actin in a cooperative manner with an apparent K(d) of 2.5 microM. Calcium seems not to have any effect on its binding to actin. Electron microscopic analysis shows that the minispectrin decorates actin filaments as clusters, and induces formation of actin bundles. This study shows that the actin-binding region of the spectrin alpha/beta heterodimer retains its functional properties in a truncated form and establishes basis for further research on spectrin's structure and function.

Activation mechanism of the nuclear chaperone nucleoplasmin: role of the core domain.

Nucleoplasmin (NP) mediates nucleosome assembly by removing basic proteins from sperm chromatin and exchanging them with histones. This function is modulated by phosphorylation of NP at multiple sites. NP is pentameric, each monomer consisting of two domains: a core, which forms a stable ring-like pentamer, and a tail, that holds a polyglutamic tract and the nuclear localization signal. In the present study, we have explored the role of the core domain in the functionality of NP. Despite lacking the poly-Glu region, a putative binding site for basic proteins, the isolated core domain of the hyperphosphorylated protein isolated from eggs of Xenopus laevis is able to bind sperm basic proteins and decondense chromatin, in contrast to the inactive, non-phosphorylated recombinant core. This activity can be reproduced artificially in the recombinant core domain through mutation of putative phosphorylation sites to aspartate, thus mimicking the charge effect of phosphorylation. The mutated residues locate in flexible or loop regions exposed on the "distal face" of the core pentamer, where a short acidic region is also found, indicating that phosphorylation might activate the core domain of NP by generating a strong localized negative potential. Our results show that the phosphorylated core domain of NP is active in chromatin decondensation, thus it could contribute together with the poly-Glu containing tail in displaying a binding surface for sperm basic proteins on the NP pentamer.

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.

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.

A mechanism for histone chaperoning activity of nucleoplasmin: thermodynamic and structural models.

Nucleoplasmin (NP), a histone chaperone, acts as a reservoir for histones H2A-H2B in Xenopus laevis eggs and can displace sperm nuclear basic proteins and linker histones from the chromatin fiber of sperm and quiescent somatic nuclei. NP has been proposed to mediate the dynamic exchange of histones during the expression of certain genes and assists the assembly of nucleosomes by modulating the interaction between histones and DNA. Here, solution structural models of full-length NP and NP complexes with the functionally distinct nucleosomal core and linker histones are presented for the first time, providing a picture of the physical interactions between the nucleosomal and linker histones with NP core and tail domains. Small-angle X-ray scattering and isothermal titration calorimetry reveal that NP pentamer can accommodate five histones, either H2A-H2B dimers or H5, and that NP core and tail domains are intimately involved in the association with histones. The analysis of the binding events, employing a site-specific cooperative model, reveals a negative cooperativity-based regulatory mechanism for the linker histone/nucleosomal histone exchange. The two histone types bind with drastically different intrinsic affinity, and the strongest affinity is observed for the NP variant that mimicks the hyperphosphorylated active protein. The different "affinity windows" for H5 and H2A-H2B might allow NP to fulfill its histone chaperone role, simultaneously acting as a reservoir for the core histones and a chromatin decondensing factor. Our data are compatible with the previously proposed model where NP facilitates nucleosome assembly by removing the linker histones and depositing H2A-H2B dimers onto DNA.

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.

GroEL stability and function. Contribution of the ionic interactions at the inter-ring contact sites.

The chaperonin GroEL consists of a double ring structure made of identical subunits that display different modes of allosteric communication. The protein folding cycle requires the simultaneous positive intra-ring and negative inter-ring cooperativities of ATP binding. This ensures GroES binding to one ring and release of the ligands from the opposite one. To better characterize inter-ring allosterism, the thermal stability as well as the temperature dependence of the functional and conformational properties of wild type GroEL, a single ring mutant (SR1) and two single point mutants suppressing one interring salt bridge (E434K and E461K) were studied. The results indicate that ionic interactions at the two interring contact sites are essential to maintain the negative cooperativity for protein substrate binding and to set the protein thermostat at 39 degrees C. These electrostatic interactions contribute distinctly to the stability of the inter-ring interface and the overall protein stability, e.g. the E434K thermal inactivation curve is shifted to lower temperatures, and its unfolding temperature and activation energy are also lowered. An analysis of the ionic interactions at the inter-ring contact sites reveals that at the so called "left site" a network of electrostatic interactions involving three charged residues might be established, in contrast to what is found at the "right site" where only two oppositely charged residues interact. Our data suggest that electrostatic interactions stabilize protein-protein interfaces depending on both the number of ionic interactions and the number of residues engaged in each of these interactions. In the case of GroEL, this combination sets the thermostat of the protein so that the chaperonin distinguishes physiological from stress temperatures.

Electrostatic interactions at the C-terminal domain of nucleoplasmin modulate its chromatin decondensation activity.

The chromatin decondensation activity, thermal stability, and secondary structure of recombinant nucleoplasmin, of two deletion mutants, and of the protein isolated from Xenopus oocytes have been characterized. As previously reported, the chromatin decondensation activity of recombinant, unphosphorylated nucleoplasmin is almost negligible. Our data show that deletion of 50 residues at the C-terminal domain of the protein, containing the positively charged nuclear localization sequence, activates its chromatin decondensation ability and decreases its stability. Interestingly, both the decondensation activity and thermal stability of this deletion mutant resemble those of the phosphorylated protein isolated from Xenopus oocytes. Deletion of 80 residues at the C-terminal domain, containing the above-mentioned positively charged region and a poly(Glu) tract, inactivates the protein and increases its thermal stability. These findings, along with the effect of salt on the thermal stability of these proteins, suggest that electrostatic interactions between the positive nuclear localization sequence and the poly(Glu) tract, at the C-terminal domain, modulate protein activity and stability.