High-Throughput Screening to Identify Inhibitors of Plasmodium falciparum Importin α.

The global burden of malaria and toxoplasmosis has been limited by the use of efficacious anti-parasitic agents, however, emerging resistance in Plasmodium species and Toxoplasma gondii threatens disease control worldwide, implying that new agents/therapeutic targets are urgently needed. Nuclear localization signal (NLS)-dependent transport into the nucleus, mediated by members of the importin (IMP) superfamily of nuclear transporters, has shown potential as a target for intervention to limit viral infection. Here, we show for the first time that IMPα from P. falciparum and T. gondii have promise as targets for small molecule inhibitors. We use high-throughput screening to identify agents able to inhibit P. falciparum IMPα binding to a P. falciparum NLS, identifying a number of compounds that inhibit binding in the µM-nM range, through direct binding to P. falciparum IMPα, as shown in thermostability assays. Of these, BAY 11-7085 is shown to be a specific inhibitor of P. falciparum IMPα-NLS recognition. Importantly, a number of the inhibitors limited growth by both P. falciparum and T. gondii. The results strengthen the hypothesis that apicomplexan IMPα proteins have potential as therapeutic targets to aid in identifying novel agents for two important, yet neglected, parasitic diseases.

Overlapping binding sites for importin ß1 and suppressor of fused (SuFu) on glioma-associated oncogene homologue 1 (Gli1) regulate its nuclear localization.

A key factor in oncogenesis is the transport into the nucleus of oncogenic signalling molecules, such as Gli1 (glioma-associated oncogene homologue 1), the central transcriptional activator in the Hedgehog signalling pathway. Little is known, however, how factors such as Gli are transported into the nucleus and how this may be regulated by interaction with other cellular factors, such as the negative regulator suppressor of fused (SuFu). In the present study we show for the first time that nuclear entry of Gli1 is regulated by a unique mechanism through mutually exclusive binding by its nuclear import factor Impß1 (importin ß1) and SuFu. Using quantitative live mammalian cell imaging, we show that nuclear accumulation of GFP-Gli1 fusion proteins, but not of a control protein, is specifically inhibited by co-expression of SuFu. Using a direct binding assay, we show that Impß1 exhibits a high nanomolar affinity to Gli1, with specific knockdown of Impß1 expression being able to inhibit Gli1 nuclear accumulation, thus implicating Impß1 as the nuclear transporter for Gli1 for the first time. SuFu also binds to Gli1 with a high nanomolar affinity, intriguingly being able to compete with Impß1 for binding to Gli1, through the fact that the sites for SuFu and Impß1 binding overlap at the Gli1 N-terminus. The results indicate for the first time that the relative intracellular concentrations of SuFu and Impß1 are likely to determine the localization of Gli1, with implications for its action in cancer, as well as in developmental systems.

Binding of p110 retinoblastoma protein inhibits nuclear import of simian virus SV40 large tumor antigen.

Nuclear import of the simian virus 40 large tumor antigen (T-ag) is dependent on its nuclear localization signal (NLS) within amino acids 126-132 that is recognized by the importin alpha/beta1 heterodimer, as well as a protein kinase CK2 site at serine 112 upstream of the NLS, which enhances the interaction approximately 50-fold. Here we show for the first time that T-ag nuclear import is negatively regulated by N-terminal sequences (amino acids 102-110), which represent the binding site (BS) for the retinoblastoma (Rb) tumor suppressor protein (p110(Rb)). Quantitative confocal laser scanning microscopic analysis of the transport properties of T-ag constructs with or without Rb binding site mutations in living transfected cells or in a reconstituted nuclear transport system indicates that the presence of the RbBS significantly reduces nuclear accumulation of T-ag. A number of approaches, including the analysis of T-ag nuclear import in an isogenic cell pair with and without functional p110(Rb) implicate p110(Rb) binding as being responsible for the reduced nuclear accumulation, with the Ser(106) phosphorylation site within the RbBS appearing to enhance the inhibitory effect. Immunoprecipitation experiments confirmed association of T-ag and p110(Rb) and dependence thereof on negative charge at Ser(106). The involvement of p110(Rb) in modulating T-ag nuclear transport has implications for the regulation of nuclear import of other proteins from viruses of medical significance that interact with p110(Rb), and how this may relate to transformation.

Crystal structure of a Legionella pneumophila ecto -triphosphate diphosphohydrolase, a structural and functional homolog of the eukaryotic NTPDases.

Many pathogenic bacteria have sophisticated mechanisms to interfere with the mammalian immune response. These include the disruption of host extracellular ATP levels that, in humans, is tightly regulated by the nucleoside triphosphate diphosphohydrolase family (NTPDases). NTPDases are found almost exclusively in eukaryotes, the notable exception being their presence in some pathogenic prokaryotes. To address the function of bacterial NTPDases, we describe the structures of an NTPDase from the pathogen Legionella pneumophila (Lpg1905/Lp1NTPDase) in its apo state and in complex with the ATP analog AMPPNP and the subtype-specific NTPDase inhibitor ARL 67156. Lp1NTPDase is structurally and catalytically related to eukaryotic NTPDases and the structure provides a basis for NTPDase-specific inhibition. Furthermore, we demonstrate that the activity of Lp1NTPDase correlates directly with intracellular replication of Legionella within macrophages. Collectively, these findings provide insight into the mechanism of this enzyme and highlight its role in host-pathogen interactions.

The respiratory syncytial virus matrix protein possesses a Crm1-mediated nuclear export mechanism.

The respiratory syncytial virus (RSV) matrix (M) protein is localized in the nucleus of infected cells early in infection but is mostly cytoplasmic late in infection. We have previously shown that M localizes in the nucleus through the action of the importin beta1 nuclear import receptor. Here, we establish for the first time that M's ability to shuttle to the cytoplasm is due to the action of the nuclear export receptor Crm1, as shown in infected cells, and in cells transfected to express green fluorescent protein (GFP)-M fusion proteins. Specific inhibition of Crm1-mediated nuclear export by leptomycin B increased M nuclear accumulation. Analysis of truncated and point-mutated M derivatives indicated that Crm1-dependent nuclear export of M is attributable to a nuclear export signal (NES) within residues 194 to 206. Importantly, inhibition of M nuclear export resulted in reduced virus production, and a recombinant RSV carrying a mutated NES could not be rescued by reverse genetics. That this is likely to be due to the inability of a nuclear export deficient M to localize to regions of virus assembly is indicated by the fact that a nuclear-export-deficient GFP-M fails to localize to regions of virus assembly when expressed in cells infected with wild-type RSV. Together, our data suggest that Crm1-dependent nuclear export of M is central to RSV infection, representing the first report of such a mechanism for a paramyxovirus M protein and with important implications for related paramyxoviruses.

Human cytomegalovirus DNA polymerase catalytic subunit pUL54 possesses independently acting nuclear localization and ppUL44 binding motifs.

The catalytic subunit of human cytomegalovirus (HCMV) DNA polymerase pUL54 is a 1242-amino-acid protein, whose function, stimulated by the processivity factor, phosphoprotein UL44 (ppUL44), is essential for viral replication. The C-terminal residues (amino acids 1220-1242) of pUL54 have been reported to be sufficient for ppUL44 binding in vitro. Although believed to be important for functioning in the nuclei of infected cells, no data are available on either the interaction of pUL54 with ppUL44 in living mammalian cells or the mechanism of pUL54 nuclear transport and its relationship with that of ppUL44. The present study examines for the first time the nuclear import pathway of pUL54 and its interaction with ppUL44 using dual color, quantitative confocal laser scanning microscopy on live transfected cells and quantitative gel mobility shift assays. We showed that of two nuclear localization signals (NLSs) located at amino acids 1153-1159 (NLSA) and 1222-1227 (NLSB), NLSA is sufficient to confer nuclear localization on green fluorescent protein (GFP) by mediating interaction with importin alpha/beta. We also showed that pUL54 residues 1213-1242 are sufficient to confer ppUL44 binding abilities on GFP and that pUL54 and ppUL44 can be transported to the nucleus as a complex. Our work thus identified distinct sites within the HCMV DNA polymerase, which represent potential therapeutic targets and establishes the molecular basis of UL54 nuclear import.

Nuclear import of the respiratory syncytial virus matrix protein is mediated by importin beta1 independent of importin alpha.

The matrix (M) protein of respiratory syncytial virus (RSV) plays an important role in virus assembly through specific interactions with RSV nucleocapsids and envelope glycoproteins in the cytoplasm as well as with the host cell membrane. We have previously shown that M localizes to the nucleus of infected cells at an early stage in the RSV infection cycle, where it may be instrumental in inhibiting host cell processes. The present study uses transient expression of M as well as a truncated green fluorescent protein (GFP) fusion derivative to show for the first time that M is able to localize in the nucleus in the absence of other RSV gene products, through the action of amino acids 110-183, encompassing the nucleic acid binding regions of the protein, that are sufficient to target GFP to the nucleus. Using native PAGE, ELISA-based binding assays, a novel Alphascreen assay, and an in vitro nuclear transport assay, we show that M is recognized directly by the importin beta1 nuclear import receptor, which mediates its nuclear import in concert with the guanine nucleotide-binding protein Ran. Retention of M in the nucleus through binding to nuclear components, probably mediated by the putative zinc finger domain of M, also contributes to M nuclear accumulation. This is the first report of the importin binding and nuclear import properties of a gene product from a negative sense RNA virus, with implications for the function of RSV M and possibly other viral M proteins in the nucleus of infected cells.

Quantitative analysis of protein-protein interactions by native page/fluorimaging.

We have developed a new quantitative native PAGE mobility shift assay, which allows for the measurement of binding affinities for interacting protein pairs, one of which is fluorescently labelled. We have used it to examine recognition of the Simian virus 40 (SV40) large tumour T-antigen (T-ag) nuclear localisation sequence (NLS) by members of the importin (Imp) superfamily of nuclear transport proteins. We demonstrate that the T-ag NLS binds to the Imp alpha/beta heterodimer in NLS-dependent manner, determining that it binds with eight-fold higher affinity (340 nM), when compared to Imp alpha alone, consistent with autoinhibition of Imp alpha when not complexed with Imp beta. The mobility shift assay is able to detect nM binding affinities, making it a sensitive and useful tool to analyse protein-protein interactions in solution.

Apoptin nuclear accumulation is modulated by a CRM1-recognized nuclear export signal that is active in normal but not in tumor cells.

Tumor cell-specific activity of chicken anemia virus viral protein 3 (VP3 or apoptin) is believed to be dependent on its ability to localize in the nucleus of transformed but not of primary or nontransformed cells. The present study characterizes the signals responsible for the novel nucleocytoplasmic trafficking properties of VP3 using two isogenic tumor/nontumor cell pairs. In addition to the tumor cell-specific nuclear targeting signal, comprising two stretches of basic amino acids in the VP3 COOH terminus which are highly efficient in tumor but not in normal cells, we define the CRM1-recognized nuclear export sequence (NES) within the VP3 tumor cell-specific nuclear targeting signal for the first time. Intriguingly, the NES (amino acids 97-105) is functional in normal but not in tumor cells through the action of the threonine 108 phosphorylation site adjacent to the NES which inhibits its action. In addition, we characterize a leucine-rich sequence (amino acids 33-46) that assists VP3 nuclear accumulation by functioning as a nuclear retention sequence, conferring association with promyelocytic leukemia nuclear bodies. This unique combination of signals is the basis of the tumor cell-specific nuclear targeting abilities of VP3.

Role of prodomain in importin-mediated nuclear localization and activation of caspase-2.

Caspase-2 is unique among mammalian caspases because it localizes to the nucleus in a prodomain-dependent manner. The caspase-2 prodomain also regulates caspase-2 activity via a caspase recruitment domain that mediates oligomerization of procaspase-2 molecules and their subsequent autoactivation. In this study we sought to map specific functional regions in the caspase-2 prodomain that regulate its nuclear transport and also its activation. Our data indicate that caspase-2 contains a classical nuclear localization signal (NLS) at the C terminus of the prodomain which is recognized by the importin alpha/beta heterodimer. The mutation of a conserved Lys residue in the NLS abolishes nuclear localization of caspase-2 and binding to the importin alpha/beta heterodimer. Although caspase-2 is imported into the nucleus, mutants lacking the NLS were still capable of inducing apoptosis upon overexpression in transfected cells. We define a region in the prodomain that regulates the ability of caspase-2 to form dot- and filament-like structures when ectopically expressed, which in turn promotes cell killing. Our data provides a mechanism for caspase-2 nuclear import and demonstrate that association of procaspase-2 into higher order structures, rather than its nuclear localization, is required for caspase-2 activation and its ability to induce apoptosis.