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Yuh Min Chook - One of the best experts on this subject based on the ideXlab platform.
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Correlation of CRM1-NES affinity with Nuclear Export activity.
Molecular biology of the cell, 2018Co-Authors: Ho Yee Joyce Fung, Tolga Cagatay, Jordan M. Baumhardt, Yuh Min ChookAbstract:CRM1 (Exportin1/XPO1) Exports hundreds of broadly functioning protein cargoes out of the cell nucleus by binding to their classical Nuclear Export signals (NESs). The 8- to 15-amino-acid-long NESs contain four to five hydrophobic residues and are highly diverse in both sequence and CRM1-bound structure. Here we examine the relationship between Nuclear Export activities of 24 different NES peptides in cells and their CRM1-NES affinities. We found that binding affinity and Nuclear Export activity are linearly correlated for NESs with dissociation constants ( Kds) between tens of nanomolar to tens of micromolar. NESs with Kds outside this range have significantly reduced Nuclear Export activities. These include two unusually tight-binding peptides, one from the nonstructural protein 2 of murine minute virus (MVM NS2) and the other a mutant of the protein kinase A inhibitor (PKI) NES. The crystal structure of CRM1-bound MVM NS2NES suggests that extraordinarily tight CRM1 binding arises from intramolecular contacts within the NES that likely stabilizes the CRM1-bound conformation in free peptides. This mechanistic understanding led to the design of two novel peptide inhibitors that bind CRM1 with picomolar affinity.
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Nuclear Export receptor CRM1 recognizes diverse conformations in Nuclear Export signals.
eLife, 2017Co-Authors: Ho Yee Joyce Fung, Yuh Min ChookAbstract:Nuclear Export receptor CRM1 binds highly variable Nuclear Export signals (NESs) in hundreds of different cargoes. Previously we have shown that CRM1 binds NESs in both polypeptide orientations (Fung et al., 2015). Here, we show crystal structures of CRM1 bound to eight additional NESs which reveal diverse conformations that range from loop-like to all-helix, which occupy different extents of the invariant NES-binding groove. Analysis of all NES structures show 5-6 distinct backbone conformations where the only conserved secondary structural element is one turn of helix that binds the central portion of the CRM1 groove. All NESs also participate in main chain hydrogen bonding with human CRM1 Lys568 side chain, which acts as a specificity filter that prevents binding of non-NES peptides. The large conformational range of NES backbones explains the lack of a fixed pattern for its 3-5 hydrophobic anchor residues, which in turn explains the large array of peptide sequences that can function as NESs.
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Structural determinants of Nuclear Export signal orientation in binding to Exportin CRM1
eLife, 2015Co-Authors: Ho Yee Joyce Fung, Chad A. Brautigam, Yuh Min ChookAbstract:Many organisms keep their DNA within a structure inside their cells called the nucleus. Two layers of membrane surround the nucleus and keep the DNA separate from the rest of the cell's contents. Yet, proteins and other molecules can move in and out of the nucleus by passing through small pores in this Nuclear membrane. To travel through these pores, larger molecules such as proteins rely on the assistance of transport receptors, including one called CRM1. This transport receptor helps to Export hundreds of different proteins from the nucleus by recognizing a part of their structure called the ‘Nuclear Export signal’. Earlier work has shown that three different Nuclear Export signals interact with CRM1 in a similar ways by binding to a groove on its outer surface. But, there are several different types of Nuclear Export signal, and many are predicted to have three-dimensional structures that would seem to prevent them from binding to CRM1 in this way. As yet, it remains unknown how these diverse signals interact with this important transporter receptor. Protein crystallization is a technique that is used to visualize a protein's three-dimensional structure. Fung et al. have now used this approach to investigate how a particular class of Nuclear Export signals (called ‘class 3’) bind to CRM1. First, a modified form of CRM1 was crystallized once it had bound to a small fragment of protein that contains a class 3 Nuclear Export signal. The protein's molecular structure was then revealed by performing X-ray diffraction on the crystals. The results show, unexpectedly, that two different Nuclear Export signals in class 3 bind to the groove of CRM1 in the opposite direction to that reported previously. Additional biochemical and structural experiments then identified a particular feature or motif in the Nuclear Export signals that determines which way round they bind to CRM1. This discovery advances our understanding of how these signals work, which will allow us to more accurately identify new Nuclear Export signals from genome sequences. As more CRM1-binding Nuclear Export signals are discovered in the future, the experimental data sets used to train the computational programs that are currently used to locate these signals in genomic sequences will be diversified and improved.
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structural basis for leucine rich Nuclear Export signal recognition by crm1
Nature, 2009Co-Authors: Xiuhua Dong, Anindita Biswas, Katherine E Suel, Laurie K Jackson, Rita Martinez, Hongmei Gu, Yuh Min ChookAbstract:CRM1 (also known as XPO1 and Exportin 1) mediates Nuclear Export of hundreds of proteins through the recognition of the leucine-rich Nuclear Export signal (LR-NES). Here we present the 2.9 A structure of CRM1 bound to snurportin 1 (SNUPN). Snurportin 1 binds CRM1 in a bipartite manner by means of an amino-terminal LR-NES and its nucleotide-binding domain. The LR-NES is a combined α-helical-extended structure that occupies a hydrophobic groove between two CRM1 outer helices. The LR-NES interface explains the consensus hydrophobic pattern, preference for intervening electronegative residues and inhibition by leptomycin B. The second Nuclear Export signal epitope is a basic surface on the snurportin 1 nucleotide-binding domain, which binds an acidic patch on CRM1 adjacent to the LR-NES site. Multipartite recognition of individually weak Nuclear Export signal epitopes may be common to CRM1 substrates, enhancing CRM1 binding beyond the generally low affinity LR-NES. Similar energetic construction is also used in multipartite Nuclear localization signals to provide broad substrate specificity and rapid evolution in Nuclear transport. CRM1 is a Nuclear transport receptor that mediates Export of a large number of proteins out of the nucleus through recognition of leucine-rich Nuclear Export signals (LR-NES). In this study, Chook and colleagues present the crystal structure of CRM1 in complex with a substrate called snurportin1. Snurportin1 binds CRM1 in a bipartite manner through an N-terminal LR-NES and its nucleotide-binding domain. Further analysis reveals a second NES epitope in the nucleotide-binding domain of snurportin1. The authors propose that multipartite recognition of individually weak NES epitopes may be a common feature of CRM1 binding. The crystal structure of CRM1 in complex with a substrate called snurportin 1 is presented. Snurportin 1 binds CRM1 in a bipartite manner by means of an amino-terminal leucine-rich Nuclear Export signal (LR-NES) and its nucleotide-binding domain. Further analysis reveals a second NES epitope in the nucleotide-binding domain of snurportin 1, and multipartite recognition of individually weak NES epitopes may be a common feature of CRM1 binding.
Carl G. Maki - One of the best experts on this subject based on the ideXlab platform.
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Regulation of p53 Nuclear Export through sequential changes in conformation and ubiquitination.
The Journal of biological chemistry, 2007Co-Authors: Linghu Nie, Mark M. Sasaki, Carl G. MakiAbstract:Wild-type p53 is a conformationally labile protein that undergoes Nuclear-cytoplasmic shuttling. MDM2-mediated ubiquitination promotes p53 Nuclear Export by exposing or activating a Nuclear Export signal (NES) in the C terminus of p53. We observed that cancer-derived p53s with a mutant (primary antibody 1620-/pAb240+) conformation localized in the cytoplasm to a greater extent and displayed increased susceptibility to ubiquitination than p53s with a more wild-type (primary antibody 1620+/pAb240-) conformation. The cytoplasmic localization of mutant p53s required the C-terminal NES and an intact ubiquitination pathway. Mutant p53 ubiquitination occurred at lysines in both the DNA-binding domain (DBD) and C terminus. Interestingly, Lys to Arg mutations that inhibited ubiquitination restored Nuclear localization to mutant p53 but had no apparent effect on p53 conformation. Further studies revealed that wild-type p53, like mutant p53, is ubiquitinated by MDM2 in both the DBD and C terminus and that ubiquitination in both regions contributes to its Nuclear Export. MDM2 binding can induce a conformational change in wild-type p53, but this conformational change is insufficient to promote p53 Nuclear Export in the absence of MDM2 ubiquitination activity. Taken together, these results support a stepwise model for mutant and wild-type p53 Nuclear Export. In this model, the conformational change induced by either the cancer-derived mutation or MDM2 binding precedes p53 ubiquitination. The addition of ubiquitin to DBD and C-terminal lysines then promotes Nuclear Export via the C-terminal NES.
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The MDM2 RING-finger domain is required to promote p53 Nuclear Export
Nature cell biology, 2000Co-Authors: Rory K. Geyer, Carl G. MakiAbstract:MDM2 can bind to p53 and promote its ubiquitination and subsequent degradation by the proteasome. Current models propose that Nuclear Export of p53 is required for MDM2-mediated degradation, although the function of MDM2 in p53 Nuclear Export has not been clarified. Here we show that MDM2 can promote the Nuclear Export of p53 in transiently transfected cells. This activity requires the Nuclear-Export signal (NES) of p53, but not the NES of MDM2. A mutation within the MDM2 RING-finger domain that inhibits p53 ubiquitination also inhibits the ability of MDM2 to promote p53 Nuclear Export. Finally, inhibition of Nuclear Export stabilizes wild-type p53 and leads to accumulation of ubiquitinated p53 in the nucleus. Our results indicate that MDM2-mediated ubiquitination, or other activities associated with the RING-finger domain, can stimulate the Export of p53 to the cytoplasm through the activity of the p53 NES.
Ed Hurt - One of the best experts on this subject based on the ideXlab platform.
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Nuclear Export of tRNA.
Results and problems in cell differentiation, 2002Co-Authors: George Simos, Helge Grosshans, Ed HurtAbstract:Exit of tRNA from the nucleus was shown, long time ago, to be a saturable and carrier-mediated process. Nevertheless, only recently, progress in the field of nucleocytoplasmic transport gave first insight into the mechanism of tRNA Nuclear Export. A Nuclear Export receptor for tRNA (Los1p/Xpo-t), belonging to the importin β (karyopherin) family, has been characterized in yeast and mammalian cells. Mature tRNA molecules can associate with Los1p/ Xpo-t and the GTP-bound form of the small GTPase Ran to form an Export complex in the nucleus. This complex translocates through the Nuclear-pore complexes and dissociates upon GTP hydrolysis in the cytoplasm. Genetic studies in yeast have, however, shown thatLOS1 is not essential, unless additional steps in the tRNA biogenesis pathway are impaired, suggesting the existence of additional tRNA Nuclear Export routes. Furthermore, modification and aminoacylation of tRNA may also be important for efficient transport of tRNA into the cytoplasm.
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Nuclear Export of 60s ribosomal subunits depends on xpo1p and requires a Nuclear Export sequence containing factor nmd3p that associates with the large subunit protein rpl10p
Molecular and Cellular Biology, 2001Co-Authors: Olivier Gadal, Daniela Straus, Jacques J Kessl, Bernard L Trumpower, David Tollervey, Ed HurtAbstract:Nuclear Export of ribosomes requires a subset of nucleoporins and the Ran system, but specific transport factors have not been identified. Using a large subunit reporter (Rpl25p-eGFP), we have isolated several temperature-sensitive ribosomal Export (rix) mutants. One of these corresponds to the ribosomal protein Rpl10p, which interacts directly with Nmd3p, a conserved and essential protein associated with 60S subunits. We find that thermosensitive nmd3 mutants are impaired in large subunit Export. Strikingly, Nmd3p shuttles between the nucleus and cytoplasm and is Exported by the Nuclear Export receptor Xpo1p. Moreover, we show that Export of 60S subunits is Xpo1p dependent. We conclude that Nuclear Export of 60S subunits requires the Nuclear Export sequence-containing nonribosomal protein Nmd3p, which directly binds to the large subunit protein Rpl10p.
Joyce M. Slingerland - One of the best experts on this subject based on the ideXlab platform.
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CRM1/Ran-mediated Nuclear Export of p27(Kip1) involves a Nuclear Export signal and links p27 Export and proteolysis.
Molecular biology of the cell, 2003Co-Authors: Michael K. Connor, Rouslan Kotchetkov, Sandrine Cariou, Ansgar Resch, Rafaella Lupetti, Richard G. Beniston, Frauke Melchior, Ludger Hengst, Joyce M. SlingerlandAbstract:We show that p27 localization is cell cycle regulated and we suggest that active CRM1/RanGTP-mediated Nuclear Export of p27 may be linked to cytoplasmic p27 proteolysis in early G1. p27 is Nuclear in G0 and early G1 and appears transiently in the cytoplasm at the G1/S transition. Association of p27 with the Exportin CRM1 was minimal in G0 and increased markedly during G1-to-S phase progression. Proteasome inhibition in mid-G1 did not impair Nuclear import of p27, but led to accumulation of p27 in the cytoplasm, suggesting that Export precedes degradation for at least part of the cellular p27 pool. p27-CRM1 binding and Nuclear Export were inhibited by S10A mutation but not by T187A mutation. A putative Nuclear Export sequence in p27 is identified whose mutation reduced p27-CRM1 interaction, Nuclear Export, and p27 degradation. Leptomycin B (LMB) did not inhibit p27-CRM1 binding, nor did it prevent p27 Export in vitro or in heterokaryon assays. Prebinding of CRM1 to the HIV-1 Rev Nuclear Export sequence did not inhibit p27-CRM1 interaction, suggesting that p27 binds CRM1 at a non-LMB-sensitive motif. LMB increased total cellular p27 and may do so indirectly, through effects on other p27 regulatory proteins. These data suggest a model in which p27 undergoes active, CRM1-dependent Nuclear Export and cytoplasmic degradation in early G1. This would permit the incremental activation of cyclin E-Cdk2 leading to cyclin E-Cdk2-mediated T187 phosphorylation and p27 proteolysis in late G1 and S phase.
Pablo Wappner - One of the best experts on this subject based on the ideXlab platform.
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Central Role of the Oxygen-dependent Degradation Domain of Drosophila HIFα/Sima in Oxygen-dependent Nuclear Export
Molecular biology of the cell, 2009Co-Authors: Maximiliano Irisarri, Sofía Lavista-llanos, Nuria M. Romero, Lazaro Centanin, Andrés Dekanty, Pablo WappnerAbstract:The Drosophila HIFα homologue, Sima, is localized mainly in the cytoplasm in normoxia and accumulates in the nucleus upon hypoxic exposure. We have characterized the mechanism governing Sima oxygen-dependent subcellular localization and found that Sima shuttles continuously between the nucleus and the cytoplasm. We have previously shown that Nuclear import depends on an atypical bipartite Nuclear localization signal mapping next to the C-terminus of the protein. We show here that Nuclear Export is mediated in part by a CRM1-dependent Nuclear Export signal localized in the oxygen-dependent degradation domain (ODDD). CRM1-dependent Nuclear Export requires both oxygen-dependent hydroxylation of a specific prolyl residue (Pro850) in the ODDD, and the activity of the von Hippel Lindau tumor suppressor factor. At high oxygen tension rapid Nuclear Export of Sima occurs, whereas in hypoxia, Sima Nuclear Export is largely inhibited. HIFα/Sima nucleo-cytoplasmic localization is the result of a dynamic equilibrium between Nuclear import and Nuclear Export, and Nuclear Export is modulated by oxygen tension.
