Crystal structure of human CRM1, covalently modified by 2-mercaptoethanol on Cys528, in complex with RanGTP.

CRM1 is a nuclear export receptor that has been intensively targeted over the last decade for the development of antitumor and antiviral drugs. Structural analysis of several inhibitor compounds bound to CRM1 revealed that their mechanism of action relies on the covalent modification of a critical cysteine residue (Cys528 in the human receptor) located in the nuclear export signal-binding cleft. This study presents the crystal structure of human CRM1, covalently modified by 2-mercaptoethanol on Cys528, in complex with RanGTP at 2.58 Šresolution. The results demonstrate that buffer components can interfere with the characterization of cysteine-dependent inhibitor compounds.

The export receptor Crm1 forms a dimer to promote nuclear export of HIV RNA.

The HIV Rev protein routes viral RNAs containing the Rev Response Element (RRE) through the Crm1 nuclear export pathway to the cytoplasm where viral proteins are expressed and genomic RNA is delivered to assembling virions. The RRE assembles a Rev oligomer that displays nuclear export sequences (NESs) for recognition by the Crm1-Ran(GTP) nuclear receptor complex. Here we provide the first view of an assembled HIV-host nuclear export complex using single-particle electron microscopy. Unexpectedly, Crm1 forms a dimer with an extensive interface that enhances association with Rev-RRE and poises NES binding sites to interact with a Rev oligomer. The interface between Crm1 monomers explains differences between Crm1 orthologs that alter nuclear export and determine cellular tropism for viral replication. The arrangement of the export complex identifies a novel binding surface to possibly target an HIV inhibitor and may point to a broader role for Crm1 dimerization in regulating host gene expression.

The interaction of CRM1 and the nuclear pore protein Tpr.

While much has been devoted to the study of transport mechanisms through the nuclear pore complex (NPC), the specifics of interactions and binding between export transport receptors and the NPC periphery have remained elusive. Recent work has demonstrated a binding interaction between the exportin CRM1 and the unstructured carboxylic tail of Tpr, on the nuclear basket. Strong evidence suggests that this interaction is vital to the functions of CRM1. Using molecular dynamics simulations and a newly refined method for determining binding regions, we have identified nine candidate binding sites on CRM1 for C-Tpr. These include two adjacent to RanGTP--from which one is blocked in the absence of RanGTP--and three next to the binding region of the cargo Snurportin. We report two additional interaction sites between C-Tpr and Snurportin, suggesting a possible role for Tpr import into the nucleus. Using bioinformatics tools we have conducted conservation analysis and functional residue prediction investigations to identify which parts of the obtained binding sites are inherently more important and should be highlighted. Also, a novel measure based on the ratio of available solvent accessible surface (RASAS) is proposed for monitoring the ligand/receptor binding process.

Ran GTPase in nuclear envelope formation and cancer metastasis.

Ran is a small ras-related GTPase that controls the nucleocytoplasmic exchange of macromolecules across the nuclear envelope. It binds to chromatin early during nuclear formation and has important roles during the eukaryotic cell cycle, where it regulates mitotic spindle assembly, nuclear envelope formation and cell cycle checkpoint control. Like other GTPases, Ran relies on the cycling between GTP-bound and GDP-bound conformations to interact with effector proteins and regulate these processes. In nucleocytoplasmic transport, Ran shuttles across the nuclear envelope through nuclear pores. It is concentrated in the nucleus by an active import mechanism where it generates a high concentration of RanGTP by nucleotide exchange. It controls the assembly and disassembly of a range of complexes that are formed between Ran-binding proteins and cellular cargo to maintain rapid nuclear transport. Ran also has been identified as an essential protein in nuclear envelope formation in eukaryotes. This mechanism is dependent on importin-ß, which regulates the assembly of further complexes important in this process, such as Nup107-Nup160. A strong body of evidence is emerging implicating Ran as a key protein in the metastatic progression of cancer. Ran is overexpressed in a range of tumors, such as breast and renal, and these perturbed levels are associated with local invasion, metastasis and reduced patient survival. Furthermore, tumors with oncogenic KRAS or PIK3CA mutations are addicted to Ran expression, which yields exciting future therapeutic opportunities.

Recognition of mono-ADP-ribosylated ARTD10 substrates by ARTD8 macrodomains.

ADP-ribosyltransferases (ARTs) catalyze the transfer of ADP-ribose from NAD(+) onto substrates. Some ARTs generate in an iterative process ADP-ribose polymers that serve as adaptors for distinct protein domains. Other ARTs, exemplified by ARTD10, function as mono-ADP-ribosyltransferases, but it has been unclear whether this modification occurs in cells and how it is read. We observed that ARTD10 colocalized with ARTD8 and defined its macrodomains 2 and 3 as readers of mono-ADP-ribosylation both in vitro and in cells. The crystal structures of these two ARTD8 macrodomains and isothermal titration calorimetry confirmed their interaction with ADP-ribose. These macrodomains recognized mono-ADP-ribosylated ARTD10, but not poly-ADP-ribosylated ARTD1. This distinguished them from the macrodomain of macroH2A1.1, which interacted with poly- but not mono-ADP-ribosylated substrates. Moreover, Ran, an ARTD10 substrate, was also read by ARTD8 macrodomains. This identifies readers of mono-ADP-ribosylated proteins, defines their structures, and demonstrates the presence of this modification in cells.

Antileukemic activity of nuclear export inhibitors that spare normal hematopoietic cells.

Drugs that target the chief mediator of nuclear export, chromosome region maintenance 1 protein (CRM1) have potential as therapeutics for leukemia, but existing CRM1 inhibitors show variable potencies and a broad range of cytotoxic effects. Here, we report the structural analysis and antileukemic activity of a new generation of small-molecule inhibitors of CRM1. Designated selective inhibitors of nuclear export (SINE), these compounds were developed using molecular modeling to screen a small virtual library of compounds against the nuclear export signal (NES) groove of CRM1. The 2.2-Å crystal structure of the CRM1-Ran-RanBP1 complex bound to KPT-251, a representative molecule of this class of inhibitors, shows that the drug occupies part of the groove in CRM1 that is usually occupied by the NES, but penetrates much deeper into the groove and blocks CRM1-directed protein export. SINE inhibitors exhibit potent antileukemic activity, inducing apoptosis at nanomolar concentrations in a panel of 14 human acute myeloid leukemia (AML) cell lines representing different molecular subtypes of the disease. When administered orally to immunodeficient mice engrafted with human AML cells, KPT-251 had potent antileukemic activity with negligible toxicity to normal hematopoietic cells. Thus, KPT-SINE CRM1 antagonists represent a novel class of drugs that warrant further testing in AML patients.

Impairment of glioma stem cell survival and growth by a novel inhibitor for Survivin-Ran protein complex.

PURPOSE: Glioblastoma multiforme (GBM) is a devastating disease. Recent studies suggest that the stem cell properties of GBM contribute to the development of therapy resistance. EXPERIMENTAL DESIGN: The expression of Survivin and Ran was evaluated by immunohistochemistry with GBM tissues, and quantitative reverse transcriptase (qRT)-PCR and immunocytochemistry with patient-derived GBM sphere cultures. With a computational structure-based drug design, 11 small-molecule compounds were designed, synthesized, and evaluated as inhibitor candidates for the molecular interaction of Survivin protein. The molecular mechanism of the lead compound, LLP-3, was determined by Western blot, ELISA, in situ proximity ligation assay, and immunocytochemistry. The effects of LLP-3 treatment on GSCs were evaluated both in vitro and in vivo. Quantitative immunohistochemistry was carried out to compare Survivin expression in tissues from 44 newly diagnosed and 31 recurrent post-chemoradiation GBM patients. Lastly, the sensitivities of temozolomide-resistant GBM spheres to LLP-3 were evaluated in vitro. RESULTS: Survivin and Ran were strongly expressed in GBM tissues, particularly in the perivasculature, and also in patient-derived GSC cultures. LLP-3 treatment disrupted the Survivin-Ran protein complex in cancer cells and abolished the growth of patient-derived GBM spheres in vitro and in vivo. This inhibition was dependent on caspase activity and associated with p53 status of cells. Immunohistochemistry showed that Survivin expression is significantly increased in recurrent GBM compared with newly diagnosed tumors, and temozolomide-resistant GBM spheres exhibited high sensitivities to LLP-3 treatment. CONCLUSIONS: Disruption of the Survivin-Ran complex by LLP-3 abolishes survival and growth of GSCs both in vitro and in vivo, indicating an attractive novel therapeutic approach for GBM.

The interaction of Epac1 and Ran promotes Rap1 activation at the nuclear envelope.

Epac1 (exchange protein directly activated by cyclic AMP [cAMP]) couples intracellular cAMP to the activation of Rap1, a Ras family GTPase that regulates cell adhesion, proliferation, and differentiation. Using mass spectrometry, we identified the small G protein Ran and Ran binding protein 2 (RanBP2) as potential binding partners of Epac1. Ran is a small G protein best known for its role in nuclear transport and can be found at the nuclear pore through its interaction with RanBP2. Here we demonstrate that Ran-GTP and Epac1 interact with each other in vivo and in vitro. This binding requires a previously uncharacterized Ras association (RA) domain in Epac1. Surprisingly, the interaction of Epac1 with Ran is necessary for the efficient activation of Rap1 by Epac1. We propose that Ran and RanBP2 anchor Epac1 to the nuclear pore, permitting cAMP signals to activate Rap1 at the nuclear envelope.

The mitotic arrest deficient protein MAD2B interacts with the small GTPase RAN throughout the cell cycle.

BACKGROUND: Previously, we identified the mitotic arrest deficient protein MAD2B (MAD2L2) as a bona fide interactor of the renal cell carcinoma (RCC)-associated protein PRCC. In addition, we found that fusion of PRCC with the transcription factor TFE3 in t(X;1)(p11;q21)-positive RCCs results in an impairment of this interaction and, concomitantly, an abrogation of cell cycle progression. Although MAD2B is thought to inhibit the anaphase promoting complex (APC) by binding to CDC20 and/or CDH1(FZR1), its exact role in cell cycle control still remains to be established. METHODOLOGY/PRINCIPAL FINDINGS: Using a yeast two-hybrid interaction trap we identified the small GTPase RAN, a well-known cell cycle regulator, as a novel MAD2B binding protein. Endogenous interaction was established in mammalian cells via co-localization and co-immunoprecipitation of the respective proteins. The interaction domain of RAN could be assigned to a C-terminal moiety of 60 amino acids, whereas MAD2B had to be present in its full-length conformation. The MAD2B-RAN interaction was found to persist throughout the cell cycle. During mitosis, co-localization at the spindle was observed. CONCLUSIONS/SIGNIFICANCE: The small GTPase RAN is a novel MAD2B binding protein. This novel protein-protein interaction may play a role in (i) the control over the spindle checkpoint during mitosis and (ii) the regulation of nucleocytoplasmic trafficking during interphase.

Redox regulation of Ran GTPase.

Ran, a small Ras-like GTP-binding nuclear protein, plays a key role in modulation of various cellular signaling events including the cell cycle. This study shows that a cellular redox agent (nitrogen dioxide) facilitates Ran guanine nucleotide dissociation, and identifies a unique Ran redox architecture involved in that process. Sequence analysis suggests that Dexras1 and Rhes GTPases also possess the Ran redox architecture. As Ran releases an intact nucleotide, the redox regulation mechanism of Ran is likely to differ from the radical-based guanine nucleotide modification mechanism suggested for Ras and Rho GTPases. These results provide a mechanistic reason for the previously observed oxidative stress-induced perturbation of the Ran-mediated nuclear import, and suggest that oxidative stress could be a factor in the regulation of cell signal transduction pathways associated with Ran.

New peptide vaccine candidates for epithelial cancer patients with HLA-A3 supertype alleles.

We previously identified 2 cancer-associated antigens, immediate early response gene X-1 (IEX) and small GTPase (Ran), and their 5 epitopes using human leukocyte antigen (HLA)-A33-restricted and tumor-infiltrating T cells from a colon cancer patient. In this study, we examined whether or not these peptides can induce cytotoxic T lymphocytes (CTLs) in HLA-A11+ or HLA-A31+ epithelial cancer patients because the HLA-A11, HLA-A31, and HLA-A33 alleles share binding motifs as an HLA-A3 supertype family, which is widely distributed in many ethnic populations. Among them, the 2 peptides, IEX 47-56 and IEX 61-69, induced peptide-specific CTLs from peripheral blood mononuclear cells of cancer patients with the HLA-A11 and HLA-A31 alleles more efficiently than the other 3 peptides. Antibody blocking and cold inhibition experiments revealed that the cytotoxicity of peptide-induced CTLs against cancer cells was attributable to peptide-specific and CD8+ T cells. Together with our previous findings, these results indicate that the 2 IEX peptides could be appropriate vaccine candidates for HLA-A11, HLA-A31, and HLA-A33 positive epithelial cancer patients. This information could expand the chance of a peptide-based cancer vaccine for epithelial cancer patients of many ethnic populations.

Significant proportions of nuclear transport proteins with reduced intracellular mobilities resolved by fluorescence correlation spectroscopy.

Nuclear transport requires freely diffusing nuclear transport proteins to facilitate movement of cargo molecules through the nuclear pore. We analyzed dynamic properties of importin alpha, importin beta, Ran and NTF2 in nucleus, cytoplasm and at the nuclear pore of neuroblastoma cells using fluorescence correlation spectroscopy. Mobile components were quantified by global fitting of autocorrelation data from multiple cells. Immobile components were quantified by analysis of photobleaching kinetics. Wild-type Ran was compared to various mutant Ran proteins to identify components representing GTP or GDP forms of Ran. Untreated cells were compared to cells treated with nocodazole or latrunculin to identify components associated with cytoskeletal elements. The results indicate that freely diffusing importin alpha, importin beta, Ran and NTF2 are in dynamic equilibrium with larger pools associated with immobile binding partners such as microtubules in the cytoplasm. These findings suggest that formation of freely diffusing nuclear transport intermediates is in competition with binding to immobile partners. Variation in concentrations of freely diffusing nuclear transport intermediates among cells indicates that the nuclear transport system is sufficiently robust to function over a wide range of conditions.

Polo-like kinase 1-mediated phosphorylation of the GTP-binding protein Ran is important for bipolar spindle formation.

Polo-like kinase functions are essential for the establishment of a normal bipolar mitotic spindle, although precisely how Plk1 regulates the spindle is uncertain. In this study, we report that the small GTP/GDP-binding protein Ran is associated with Plk1. Plk1 is capable of phosphorylating co-immunoprecipitated Ran in vitro on serine-135 and Ran is phosphorylated in vivo at the same site during mitosis when Plk1 is normally activated. Cell cultures over-expressing a Ran S135D mutant have significantly higher numbers of abnormal mitotic cells than those over-expressing either wild-type or S135A Ran. The abnormalities in S135D mutant cells are similar to cells over-expressing Plk1. Our data suggests that Ran is a physiological substrate of Plk1 and that Plk1 regulates the spindle organization partially through its phosphorylation on Ran.

Abnormal centrosome amplification in cells through the targeting of Ran-binding protein-1 by the human T cell leukemia virus type-1 Tax oncoprotein.

Human T cell leukemia virus type-1 (HTLV-1) is an oncogenic retrovirus etiologically causal of adult T cell leukemia. The virus encodes a Tax oncoprotein that functions in transcriptional regulation, cell cycle control, and transformation. Because adult T cell leukemia like many other human cancers is a disease of genomic instability with frequent gains and losses of chromosomes, to understand this disease it is important to comprehend how HTLV-1 engenders aneuploidy in host cells. In this regard, loss of cell cycle checkpoints permits tolerance of aneuploidy but does not explain how aneuploidy is created. We show here that HTLV-1 Tax causes abnormal centrosome fragmentation in the mitotic phase of the cell cycle. We report that Tax directly binds Ran and Ran-binding protein-1, locates to centrosomes/spindle poles, and causes supernumerary centrosomes.

Loading and unloading: orchestrating centrosome duplication and spindle assembly by Ran/Crm1.

The cell cycle is an intricate process of DNA replication and cell division that concludes with the formation of two genetically equivalent daughter cells. In this progression, the centrosome is duplicated once and only once during the G1/S transition to produce the bipolar spindle and ensure proper chromosome segregation. The presence of multiple centrosomes in cancer cells suggests that this process is mis-regulated during carcinogenesis. This suggests that certain factors exist that license the progression of centrosome duplication and serve to inhibit further duplications during a single cell cycle. Recent studies suggest that the Ran/Crm1 complex not only regulates nucleocytoplasmic transport but is also independently involved in mitotic spindle assembly. Factors that are capable of interacting with Ran/Crm1 through their nuclear export sequences, such as cyclins/cdks, p53 and Brca1/2, may potentially function as centrosome checkpoints akin to the G1/S and G2/M checkpoints of the cell cycle. Our recent findings indicate that nucleophosmin, a protein whose trafficking is mediated by the Ran/Crm1 network, is one of such checkpoint factors for maintaining proper centrosome duplication. We propose that Ran/Crm1 may act as a 'loading dock' to coordinate various checkpoint factors in regulating the fidelity of centrosome duplication during cell cycle progression, and the disruption of these processes may lead to genomic instability and an acceleration of oncogenesis.

Human Ran cysteine 112 oxidation by pervanadate regulates its binding to keratins.

We used a proteomic approach to identify proteins that associate with keratins 8 or 18 (K8/K18) in a pervanadate-dependent manner. Pervanadate triggers Ran-K8/K18 binding and a gel-migration-shift of Ran from 25 to 27 kDa, which does not occur upon exposure to H2O2 or vanadate or if pervanadate is excluded during cell solubilization. Generation of 27-kDa Ran is not related to hyperphosphorylation, is heat-insensitive, but occurs upon conversion of Ran cysteines to cysteic acid. The pervanadate-mediated Ran cysteine --> cysteic acid oxidation and its related gel migration shift affects other proteins including actin. Mutation of the three Ran cysteines (Cys-85, -112, and -120) showed that Ran Cys-112 oxidation generates 27-kDa Ran and accounts for its keratin binding. Proteasome inhibition accentuates Ran-keratin binding after cell exposure to pervanadate. Therefore, cell-free exposure to pervanadate causes cysteine to cysteic acid oxidation of Ran and several other proteins and Ran-K8/K18 association. In cells, stabilization of oxidized Ran by proteasome inhibition promotes Ran-keratin interaction. Keratin sequestration of oxidized Ran may provide a back-up protective mechanism in some cases of oxidative injury.

Carrier-independent nuclear import of the transcription factor PU.1 via RanGTP-stimulated binding to Nup153.

PU.1 is a transcription factor of the Ets family with important functions in hematopoietic cell differentiation. Using green fluorescent protein-PU.1 fusions, we show that the Ets DNA binding domain of PU.1 is necessary and sufficient for its nuclear localization. Fluorescence and ultrastructural nuclear import assays showed that PU.1 nuclear import requires energy but not soluble carriers. PU.1 interacted directly with two nucleoporins, Nup62 and Nup153. The binding of PU.1 to Nup153, but not to Nup62, increased dramatically in the presence of RanGMPPNP, indicating the formation of a PU.1.RanGTP.Nup153 complex. The Ets domain accounted for the bulk of the interaction of PU.1 with Nup153 and RanGMPPNP. Because Nup62 is located close to the midplane of the nuclear pore complex whereas Nup153 is at its nuclear side, these findings suggest a model whereby RanGTP propels PU.1 toward the nuclear side of the nuclear pore complex by increasing its affinity for Nup153. This notion was confirmed by ultrastructural studies using gold-labeled PU.1 in permeabilized cells.

Aurora kinase inhibitor ZM447439 blocks chromosome-induced spindle assembly, the completion of chromosome condensation, and the establishment of the spindle integrity checkpoint in Xenopus egg extracts.

The Aurora family kinases contribute to accurate progression through several mitotic events. ZM447439 ("ZM"), the first Aurora family kinase inhibitor to be developed and characterized, was previously found to interfere with the mitotic spindle integrity checkpoint and chromosome segregation. Here, we have used extracts of Xenopus eggs, which normally proceed through the early embryonic cell cycles in the absence of functional checkpoints, to distinguish between ZM's effects on the basic cell cycle machinery and its effects on checkpoints. ZM clearly had no effect on either the kinetics or amplitude in the oscillations of activity of several key cell cycle regulators. It did, however, have striking effects on chromosome morphology. In the presence of ZM, chromosome condensation began on schedule but then failed to progress properly; instead, the chromosomes underwent premature decondensation during mid-mitosis. ZM strongly interfered with mitotic spindle assembly by inhibiting the formation of microtubules that are nucleated/stabilized by chromatin. By contrast, ZM had little effect on the assembly of microtubules by centrosomes at the spindle poles. Finally, under conditions where the spindle integrity checkpoint was experimentally induced, ZM blocked the establishment, but not the maintenance, of the checkpoint, at a point upstream of the checkpoint protein Mad2. These results show that Aurora kinase activity is required to ensure the maintenance of condensed chromosomes, the generation of chromosome-induced spindle microtubules, and activation of the spindle integrity checkpoint.

An ATP-dependent activity that releases RanGDP from NTF2.

The small GTPase Ran functions in several critical processes in eukaryotic cells including nuclear transport, nuclear envelope formation, and spindle formation. A RanGDP-binding protein, NTF2, facilitates translocation of RanGDP through the nuclear pore complex and also acts to stabilize RanGDP against nucleotide exchange. Here, we identify a novel activity that stimulates release of GDP from Ran in the presence of NTF2. Hydrolyzable ATP enhances the GDP dissociation activity, and this enhancement is inhibited by nonhydrolyzable ATP analogues. In contrast, neither hydrolyzable ATP nor nonhydrolyzable ATP analogues affect GDP dissociation from Ran catalyzed by recombinant RCC1 or inhibition of GDP dissociation from Ran by recombinant NTF2. The ATP-dependent RanGDP dissociation activity therefore has the properties of a RanGDP dissociation inhibitor (GDI) displacement factor (RanGDF) where the GDI is NTF2. A protein phosphatase inhibitor mixture stimulates the RanGDF activity, suggesting the activity is regulated by phosphorylation. We propose that the ATP-dependent NTF2 releasing factor may have a role in the RanGDP/GTP cycle.

Crystal structure of the PsbP protein of photosystem II from Nicotiana tabacum.

PsbP is a membrane-extrinsic subunit of the water-oxidizing complex photosystem II (PS II). The evolutionary origin of PsbP has long been a mystery because it specifically exists in higher plants and green algae but not in cyanobacteria. We report here the crystal structure of PsbP from Nicotiana tabacum at a resolution of 1.6 A. Its structure is mainly composed of beta-sheet, and is not similar to any structures in cyanobacterial PS II. However, the electrostatic surface potential of PsbP is similar to that of cyanobacterial PsbV (cyt c(550)), which has a function similar to PsbP. A structural homology search with the DALI algorithm indicated that the folding of PsbP is very similar to that of Mog1p, a regulatory protein for the nuclear transport of Ran GTPase. The structure of PsbP provides insight into its novel function in GTP-regulated metabolism in PS II.