Engineering chromosome region maintenance 1 fragments that bind to nuclear export signals.
Ontology highlight
ABSTRACT:
SUBMITTER: Lei Y
PROVIDER: S-EPMC7255508 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Leucine-rich nuclear export signals (NESs) mediate rapid nuclear export of proteins via interaction with CRM1. This interaction is stimulated by RanGTP but remains of a relatively low affinity. In order to identify strong signals, we screened a 15-mer random peptide library for CRM1 binding, both in the presence and absence of RanGTP. Under each condition, strikingly similar signals were enriched, conforming to the NES consensus sequence. A derivative of an NES selected in the absence of RanGTP exhibits very high affinity for CRM1 in vitro and stably binds without the requirement of RanGTP. Localisation studies and RNA interference demonstrate inefficient CRM1-mediated export and accumulation of CRM1 complexed with the high-affinity NES at nucleoporin Nup358. These results provide in vivo evidence for a nuclear export reaction intermediate. They suggest that NESs have evolved to maintain low affinity for CRM1 to allow efficient export complex disassembly and release from Nup358.
| S-EPMC517610 | biostudies-other
Project description: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.
| S-EPMC5358978 | biostudies-literature
Project description:Nucleocytoplasmic trafficking of proteins/RNAs is essential to normal cellular function. Indeed, accumulating evidence suggests that cancer cells escape anti-neoplastic mechanisms and benefit from pro-survival signals via the dysregulation of this system. The nuclear exporter chromosome region maintenance 1 (CRM1) protein is the only protein in the karyopherin-β protein family that contributes to the trafficking of numerous proteins and RNAs from the nucleus. It is considered to be an oncogenic, anti-apoptotic protein in transformed cells, since it reportedly functions as a gatekeeper for cell survival, including affecting p53 function, and ribosomal biogenesis. Furthermore, abnormally high expression of CRM1 is correlated with poor patient prognosis in various malignancies. Therapeutic targeting of CRM1 has emerged as a novel cancer treatment strategy, starting with a clinical trial with leptomycin B, the original specific inhibitor of CRM1, followed by development of several next-generation small molecules. KPT-330, a novel member of the CRM1-selective inhibitors of nuclear export (SINE) class of compounds, is currently undergoing clinical evaluation for the therapy of various malignancies. Results from these trials suggest that SINE compounds may be particularly useful against hematological malignancies, which often become refractory to standard chemotherapeutic agents.
| S-EPMC4526315 | biostudies-literature
Project description:Neutrophils are deprived of proliferative capacity and have a tightly controlled lifespan to avoid their persistence at the site of injury. We have recently described that the proliferating cell nuclear antigen (PCNA), a nuclear factor involved in DNA replication and repair of proliferating cells, is a key regulator of neutrophil survival. In neutrophils, PCNA was localized exclusively in the cytoplasm due to its nuclear-to-cytoplasmic relocalization during granulocytic differentiation. We showed here that leptomycin B, an inhibitor of the chromosome region maintenance 1 (CRM1) exportin, inhibited PCNA relocalization during granulocytic differentiation of HL-60 and NB4 promyelocytic cell lines and of human CD34(+) primary cells. Using enhanced green fluorescent protein fusion constructs, we have demonstrated that PCNA relocalization involved a nuclear export signal (NES) located from Ile-11 to Ile-23 in the PCNA sequence. However, this NES, located at the inner face of the PCNA trimer, was not functional in wild-type PCNA, but instead, was fully active and leptomycin B-sensitive in the monomeric PCNAY114A mutant. To test whether a defect in PCNA cytoplasmic relocalization would affect its antiapoptotic activity in mature neutrophils, a chimeric PCNA fused with the SV40 nuclear localization sequence (NLS) was generated to preclude its cytoplasmic localization. As expected, neutrophil-differentiated PLB985 cells expressing ectopic SV40NLS-PCNA had an increased nuclear PCNA as compared with cells expressing wild-type PCNA. Accordingly, the nuclear PCNA mutant did not show any antiapoptotic activity as compared with wild-type PCNA. Nuclear-to-cytoplasmic relocalization that occurred during myeloid differentiation is essential for PCNA antiapoptotic activity in mature neutrophils and is dependent on the newly identified monomerization-dependent PCNA NES.
| S-EPMC3460476 | biostudies-literature
Project description:BACKGROUND: Chikungunya virus (CHIKV) is an alphavirus of the Togaviridae family. After autoproteolytic cleavage, the CHIKV capsid protein (CP) is involved in RNA binding and assembly of the viral particle. The monomeric CP is approximately 30 kDa in size and is small enough for passive transport through nuclear pores. Some alphaviruses are found to harbor nuclear localization signals (NLS) and transport of these proteins between cellular compartments was shown to be energy dependent. The active nuclear import of cytoplasmic proteins is mediated by karyopherins and their export by exportins. As nuclear and cytoplasmic trafficking may play a role in the life cycle of CHIKV, we have sought to identify nuclear localization and nuclear export signals in CHIKV CP in a virus-free system. METHODS: EGFP-fusion proteins of CHIKV CP and mutants thereof were created and used to monitor their intracellular localization. Binding of cellular proteins was confirmed in pull-down assays with purified CP using co-immuoprecipitation. Nuclear localization was demonstrated in a virus-free system using fluorescence microscopy. RESULTS: Here we show that CHIKV CP is a nuclear-cytoplasmic shuttling protein with an active NLS that binds to karyopherin ? (Kar?) for its nuclear translocation. We also found that the Kar?4 C-terminal NLS binding site is sufficient for this interaction. We further demonstrate that CHIKV CP interacts directly with the export receptor CRM1 to transport this viral protein out of the nucleus via a nuclear export signal (NES). The CHIKV CP NES was mapped between amino acids 143 and 155 of CP. Deduced from in silico analyses we found that the NES has a mode of binding similar to the snurportin-1 CRM1 complex. CONCLUSIONS: We were able to show that in a virus-free system that the CHIKV capsid protein contains both, a NLS and a NES, and that it is actively transported between the cytoplasma and the nucleus. We conclude that CHIKV CP has the ability to shuttle via interaction with karyopherins for its nuclear import and, vice versa, by CRM1-dependent nuclear export.
| S-EPMC3765696 | biostudies-literature
Project description:The Rsp5 ubiquitin ligase regulates numerous cellular processes. Rsp5 is mainly localized to the cytoplasm but nuclear localization was also reported. A potential nuclear export signal was tested for activity by using a GFP(2) reporter. The 687-LIGGIAEIDI-696 sequence located in the Hect domain was identified as a nuclear export signal active in a Crm1-dependent manner, and its importance for the localization of Rsp5 was documented by using fluorescence microscopy and a lacZ-based reporter system. Analysis of the cellular location of other Rsp5 fragments fused with GFP(2) indicated two independent potential nuclear localization signals, both located in the Hect domain. We also uncovered Rsp5 fragments that are important to targeting/tethering Rsp5 to various regions in the cytoplasm. The presented data indicate that Rsp5 ligase is a shuttling protein whose distribution within the cytoplasm and partitioning between cytoplasmic and nuclear locations is determined by a balance between the actions of several targeting sequences and domains.
| S-EPMC3167943 | biostudies-literature
Project description:Thyroid hormone receptor (TR) is a member of the nuclear receptor superfamily that shuttles between the cytosol and nucleus. The fine balance between nuclear import and export of TR has emerged as a critical control point for modulating thyroid hormone-responsive gene expression; however, sequence motifs of TR that mediate shuttling are not fully defined. Here, we characterized multiple signals that direct TR shuttling. Along with the known nuclear localization signal in the hinge domain, we identified a novel nuclear localization signal in the A/B domain of thyroid hormone receptor α1 that is absent in thyroid hormone receptor β1 and inactive in the oncoprotein v-ErbA. Our prior studies showed that thyroid hormone receptor α1 exits the nucleus through two pathways, one dependent on the export factor CRM1 and the other CRM1-independent. Here, we identified three novel CRM1-independent nuclear export signal (NES) motifs in the ligand-binding domain as follows: a highly conserved NES in helix 12 (NES-H12) and two additional NES sequences spanning helix 3 and helix 6, respectively. Mutations predicted to disrupt the α-helical structure resulted in a significant decrease in NES-H12 activity. The high degree of conservation of helix 12 suggests that this region may function as a key NES in other nuclear receptors. Furthermore, our mutagenesis studies on NES-H12 suggest that altered shuttling of thyroid hormone receptor β1 may be a contributing factor in resistance to thyroid hormone syndrome. Taken together, our findings provide a detailed mechanistic understanding of the multiple signals that work together to regulate TR shuttling and transcriptional activity, and they provide important insights into nuclear receptor function in general.
| S-EPMC3438959 | biostudies-literature
Project description:LIM-kinase 1 (LIMK1) is a serine/threonine kinase that phosphorylates cofilin and regulates actin-filament dynamics. LIMK1, which contains two LIM domains and a single PDZ domain, localizes predominantly in the cytoplasm, but its mutant, deleted with the PDZ domain, localizes mainly in the nucleus, thereby indicating that the PDZ domain plays a role in the cytoplasmic localization of LIMK1. Here we provide evidence that the PDZ domain of LIMK1 contains two functional leucine-rich nuclear-export signals (NESs). The PDZ domain of LIMK1 fused with glutathione S-transferase (GST-PDZ), when injected into the nucleus, was rapidly excluded from the nucleus, but its mutant with replacements of conserved hydrophobic residues in two putative NESs by alanines remained in the nucleus. The nuclear export of GST-PDZ was sensitive to leptomycin B (LMB), a specific inhibitor of nuclear export mediated by leucine-rich NESs. Malfunctional mutation of two NESs or LMB treatment prevented the nuclear export of full-length LIMK1 and induced its nuclear accumulation. These results suggest that the predominant localization of LIMK1 in the cytoplasm is supported by two NESs within the PDZ domain and that LIMK1 normally shuttles between the cytoplasm and the nucleus. We also provide evidence that a short basic cluster sequence within the protein-kinase domain is involved in the nuclear import of LIMK1.
| S-EPMC1220118 | biostudies-other
Project description:In eukaryotic cells, most mRNAs are exported from the nucleus by the transcription export (TREX) complex, which is loaded onto mRNAs after their splicing and capping. We have studied in mammalian cells the nuclear export of mRNAs that code for secretory proteins, which are targeted to the endoplasmic reticulum membrane by hydrophobic signal sequences. The mRNAs were injected into the nucleus or synthesized from injected or transfected DNA, and their export was followed by fluorescent in situ hybridization. We made the surprising observation that the signal sequence coding region (SSCR) can serve as a nuclear export signal of an mRNA that lacks an intron or functional cap. Even the export of an intron-containing natural mRNA was enhanced by its SSCR. Like conventional export, the SSCR-dependent pathway required the factor TAP, but depletion of the TREX components had only moderate effects. The SSCR export signal appears to be characterized in vertebrates by a low content of adenines, as demonstrated by genome-wide sequence analysis and by the inhibitory effect of silent adenine mutations in SSCRs. The discovery of an SSCR-mediated pathway explains the previously noted amino acid bias in signal sequences and suggests a link between nuclear export and membrane targeting of mRNAs.
| S-EPMC2100149 | biostudies-literature
Project description:Germline missense mutations affecting a single BRCA2 allele predispose humans to cancer. Here we identify a protein-targeting mechanism that is disrupted by the cancer-associated mutation, BRCA2(D2723H), and that controls the nuclear localization of BRCA2 and its cargo, the recombination enzyme RAD51. A nuclear export signal (NES) in BRCA2 is masked by its interaction with a partner protein, DSS1, such that point mutations impairing BRCA2-DSS1 binding render BRCA2 cytoplasmic. In turn, cytoplasmic mislocalization of mutant BRCA2 inhibits the nuclear retention of RAD51 by exposing a similar NES in RAD51 that is usually obscured by the BRCA2-RAD51 interaction. Thus, a series of NES-masking interactions localizes BRCA2 and RAD51 in the nucleus. Notably, BRCA2(D2723H) decreases RAD51 nuclear retention even when wild-type BRCA2 is also present. Our findings suggest a mechanism for the regulation of the nucleocytoplasmic distribution of BRCA2 and RAD51 and its impairment by a heterozygous disease-associated mutation.
| S-EPMC3796201 | biostudies-literature