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FOXO3a is a transcription factor involved in cell growth inhibition and apoptosis. FOXO3a is localized in the cytoplasm in cancer cells, and its nuclear translocation by small molecules is expected to prevent cancer cell growth. In this study, we screened a fungal broth library in HeLa cells using fluorescently labeled FOXO3a and an AI-based imaging system. We identified violaceoid F, which translocates FOXO3a into the nucleus by inhibiting CRM1, which is responsible for nuclear protein export. Violaceoid F was observed to target the reactive cysteine of CRM1 through its α, β-epoxyketone. However, because violaceoid F did not inhibit Crm1 in fission yeast cells, it seems to target cysteine residue(s) other than Cys528 of human CRM1 which are not targeted by other known CRM1 inhibitors, indicating that violaceoid F inhibits CRM1 via a novel mechanism., (© 2024 Federation of European Biochemical Societies.)

Among the known nuclear exportins, CRM1 is the most studied prototype. Dysregulation of CRM1 occurs in many cancers, hence, understanding the role of CRM1 in cancer can help in developing synergistic therapeutics. The study investigates how CRM1 affects prostate cancer growth and survival. It examines the role of CRM1 in regulating androgen receptor (AR) and DNA repair in prostate cancer. Our findings reveal that CRM1 influences AR mRNA and protein stability, leading to a loss of AR protein upon CRM1 inhibition. Furthermore, it highlights the involvement of HSP90 alpha, a known AR chaperone, in the CRM1-dependent regulation of AR protein stability. The combination of CRM1 inhibition with an HSP90 inhibitor demonstrates potent effects on decreasing prostate cancer cell growth and survival. The study further explores the influence of CRM1 on DNA repair proteins and proposes a strategy of combining CRM1 inhibitors with DNA repair pathway inhibitors to decrease prostate cancer growth. Overall, the findings suggest that CRM1 plays a crucial role in prostate cancer growth, and a combination of inhibitors targeting CRM1 and DNA repair pathways could be a promising therapeutic strategy., (© 2025 The Author(s). The FASEB Journal published by Wiley Periodicals LLC on behalf of Federation of American Societies for Experimental Biology.)

CASK, a MAGUK family scaffold protein, regulates gene expression as a transcription co-activator in neurons. However, the mechanism of CASK nucleus translocation and the regulatory function of CASK in myeloid cells remains unclear. Here, we investigated its role in H5N1-infected macrophages. We found that H5N1 triggers CASK nuclear translocation via PKR and SRC signaling. HCK, a SRC family kinase, enhances CASK phosphorylation at S395 via CDK5, facilitating CASK's nuclear entry. Knocking out CASK in myeloid cells specifically reduces interferon-alpha (IFNA) production by hindering the nuclear export of Ifna mRNA, while leaving its mRNA levels unchanged. Myeloid-specific CASK knockout (KO) mice display exacerbated lung inflammation, which correlates with reduced IFNA levels during H5N1 infection. Interactome studies show that H5N1 triggers associations between CASK and CCT4, STIP1, and TNK1. These associations recruit IRF7, POLR2C, TAF15, HNRNPs, and CRM1, enabling the CASK complex to bind to the Ifna promoter, bind co-transcriptionally to Ifna mRNA, and facilitate CRM1-dependent Ifna mRNA export. This underscores CASK's critical role in the antiviral response., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2025 Huang, Sung and Hsieh.)

FOXO3 is a transcription factor that mainly exerts its functions in the cell nucleus. The amino acid sequence of FOXO3 contains a nuclear localization sequence (NLS) and a nuclear export sequence (NES) allowing for nuclear/cytoplasmic shuttling that plays an important role in regulating FOXO3 activity. Nuclear accumulation of FOXO3 proteins can be the result of translocation to the nucleus triggered by upstream regulatory input or trapping of FOXO3 within the nucleus through the inhibition of its nuclear export via the receptor CRM1. In order to distinguish these two modes of FOXO3 activation, we have generated a multiplexed assay. The development of this platform includes a reporter cell line that monitors CRM1 activity by using RFP-labeled HIV-1 Rev. protein with a strong heterologous NES. Simultaneously, the intracellular localization of FOXO3 can be monitored by a second cell line stably expressing GFP-FOXO3. Here we describe a detailed protocol on how to co-culture these reporter cell lines and use them to interrogate compound-induced FOXO3 activation in order to understand the mode of action., (© 2025. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

CRM1 (XPO1) has been well-characterized as a shuttling receptor that mediates the export of protein and RNA cargos to the cytoplasm, and previous analyses have pinpointed several key residues (A541, F572, K568, S1055, and Q742) that modulate CRM1 export activity. CRM1 also has a less studied nuclear function in RNA biogenesis, which is reflected by its localization to the Cajal body and the nucleolus. Here, we have investigated how the mutation of these key residues affects the intranuclear localization of CRM1 and its ability to mediate export of endogenous cargos. We identify A541K as a separation-of-function mutant that reveals the independent nature of the Cajal body and nucleolar localizations of CRM1. We also show that the F572A mutation may have strikingly opposite effects on the export of specific cargos. Importantly, and in contrast to previous claims, our findings indicate that S1055 phosphorylation is not generally required for CRM1 function and that the Q742 is not a function-defining residue in human CRM1. Collectively, our findings provide new insights into an understudied aspect of CRM1 biology and highlight several important issues related to CRM1 function and regulation that need to be re-evaluated and addressed in more detail.

Chromosome Region Maintenance 1 (CRM1), also known as Exportin 1 (XPO1), is a protein that is critical for transport of proteins and RNA to the cytoplasm through the nuclear pore complex. CRM1 inhibition with small molecule inhibitors is currently being studied in many cancers, including leukemias, solid organ malignancies and brain tumors. We review the structure of CRM1, its role in nuclear export, the current availability of CRM1 inhibitors, and the role of CRM1 in a number of distinct cellular processes. A deeper understanding of how CRM1 functions in nuclear export as well as other cellular processes may allow for the development of additional novel CRM1 inhibitors., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Aumann, Kazi, Harrington and Wechsler.)

Plasminogen activator inhibitor-1 (PAI-1/Serpin E1) is classically known for its antifibrinolytic activity via inhibiting uPA and tPA of the fibrinolytic pathway. PAI-1 has a paradoxical role in tumor progression, and its molecular functions are poorly understood. PAI-1 is a widely accepted secretory protease inhibitor, however, a study suggested the localization of PAI-1 in the cytoplasm and the nucleus. Besides the plethora of its biological functions as a secretory protein, intracellular localization, and functions of PAI-1 remain unexplored at the molecular level. In this study, using various in silico approaches, we showed that PAI-1 possesses a nuclear export signal. Using the CRM1-specific inhibitor leptomycin B, we demonstrated that PAI-1 has a functional CRM1-dependent NES, indicating the possibility of its nuclear localization. Further, we confirm that PAI-1 is localized in the nucleus of endothelial cells using fluorescence microscopy and immunoprecipitation. Notably, we identified an unconventional distribution of PAI-1 in the PML bodies of the nucleus of normal endothelial cells, while the protein was restricted in the cytoplasm of slow-growing cells. The data showed that the localization of PAI-1 in PML bodies is highly correlated with the growth potential of endothelial cells. This conditional nucleocytoplasmic shuttling of PAI-1 during the aging of cells could impart a strong link to its age-related functions and tumor progression. Together, this study identifies the novel behavior of PAI-1 that might be linked with cell aging and may be able to unveil the elusive role of PAI-1 in tumor progression., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2024 The Authors.)

Autographa californica multiple nucleopolyhedrovirus (AcMNPV) Ac93 is highly conserved in all sequenced baculovirus genomes, and it plays important roles in both the nuclear egress of nucleocapsids and the formation of intranuclear microvesicles. In this study, we characterized a cellular CRM1-dependent nuclear export signal (NES) of AcMNPV Ac93. Bioinformatic analysis revealed that AcMNPV Ac93 may contain an NES at amino acids 115-125. Green fluorescent protein (GFP) fused to the NES (GFP:NES) of AcMNPV Ac93 is localized to the cytoplasm of transfected cells. Multiple point mutation analysis demonstrated that NES is important for the nuclear export of GFP:NES. Bimolecular fluorescence complementation experiments and co-immunoprecipitation assays confirmed that Ac93 interacts with Spodoptera frugiperda CRM1 (SfCRM1). However, AcMNPV Ac34 inhibits cellular CRM1-dependent nuclear export of GFP:NES. To determine whether the NES in AcMNPV Ac93 is important for the formation of intranuclear microvesicles, an ac93 -null AcMNPV bacmid was constructed; the wild-type and NES-mutated Ac93 were reinserted into the ac93 -null AcMNPV bacmid. Immunofluorescence analysis showed that Ac93 and SfCRM1 were predominantly colocalized at intranuclear microvesicles in infected cells, while the construct containing point mutations at residues 123 and 125 of Ac93 resulted in a defect in budded virus production and the abolishment of intranuclear microvesicles. Together, these data demonstrate that Ac93 contains a functional NES, which is required for the production of progeny viruses and the formation of intranuclear microvesicles.IMPORTANCE Autographa californica multiple nucleopolyhedrovirus (AcMNPV) Ac93 is important for the formation of intranuclear microvesicles. However, how the baculovirus manipulates Ac93 for the formation of intranuclear microvesicles is unclear. In this study, we identified a nuclear export signal (NES) at amino acids 115-125 of AcMNPV Ac93. Our results showed that the NES is required for the interaction between Ac93 and Spodoptera frugiperda CRM1 (SfCRM1). However, AcMNPV Ac34 inhibits the nuclear export of green fluorescent protein fused to the NES. Our analysis revealed that Ac93 and SfCRM1 were predominantly colocalized at intranuclear microvesicles in AcMNPV-infected cells. Together, our results indicate that Ac93 participates in the formation of intranuclear microvesicles via the Ac93 NES-mediated CRM1 pathway., Competing Interests: The authors declare no conflict of interest.

Recent advances in the study of virus-cell interactions have improved our understanding of how viruses that replicate their genomes in the nucleus (e.g., retroviruses, hepadnaviruses, herpesviruses, and a subset of RNA viruses) hijack cellular pathways to export these genomes to the cytoplasm where they access virion egress pathways. These findings shed light on novel aspects of viral life cycles relevant to the development of new antiviral strategies and can yield new tractable, virus-based tools for exposing additional secrets of the cell. The goal of this review is to summarize defined and emerging modes of virus-host interactions that drive the transit of viral genomes out of the nucleus across the nuclear envelope barrier, with an emphasis on retroviruses that are most extensively studied. In this context, we prioritize discussion of recent progress in understanding the trafficking and function of the human immunodeficiency virus type 1 Rev protein, exemplifying a relatively refined example of stepwise, cooperativity-driven viral subversion of multi-subunit host transport receptor complexes., Competing Interests: The authors declare no conflict of interest.

Chlorophyll is one of the key factors for photosynthesis and plays an important role in plant growth and development. We previously isolated an EMS mutagenized rapeseed chlorophyll-reduced mutant ( crm1 ), which had yellow leaf, reduced chlorophyll content and fewer thylakoid stacks. Here, we found that crm1 showed attenuated utilization efficiency of both light energy and CO 2 but enhanced heat dissipation efficiency and greater tolerance to high-light intensity. BSA-Seq analysis identified a single nucleotide change (C to T) and (G to A) in the third exon of the BnaA01G0094500ZS and BnaC01G0116100ZS , respectively. These two genes encode the magnesium chelatase subunit I 1 (CHLI1) that catalyzes the insertion of magnesium into protoporphyrin IX, a pivotal step in chlorophyll synthesis. The mutation sites resulted in an amino acid substitution P144S and G128E within the AAA+ domain of the CHLI1 protein. Two KASP markers were developed and co-segregated with the yellow leaf phenotype in segregating F 2 population. Loss of BnaA01.CHLI1 and BnaC01.CHLI1 by CRISPR/Cas9 gene editing recapitulated the mutant phenotype. BnaA01.CHLI1 and BnaC01.CHLI1 were located in chloroplast and highly expressed in the leaves. Furthermore, RNA-seq analyses revealed the expression of chlorophyll synthesis - related genes were upregulated in the crm1 mutant. These findings provide a new insight into the regulatory mechanism of chlorophyll synthesis in rapeseed and suggest a novel target for improving the photosynthetic efficiency and tolerance to high-light intensity in crops., Supplementary Information: The online version contains supplementary material available at 10.1007/s11032-023-01429-6., Competing Interests: Competing interestsThe authors declare no competing interests., (© The Author(s), under exclusive licence to Springer Nature B.V. 2023. Springer Nature or its licensor (e.g. a society or other partner) holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law.)

Importance: Unlike humans, mice are unable to support HIV-1 infection. This is due, in part, to a constellation of defined minor, species-specific differences in conserved host proteins needed for viral gene expression. Here, we used precision CRISPR/Cas9 gene editing to engineer a "mousified" version of one such host protein, cyclin T1 (CCNT1), in human T cells. CCNT1 is essential for efficient HIV-1 transcription, making it an intriguing target for gene-based inactivation of virus replication. We show that isogenic cell lines engineered to encode CCNT1 bearing a single mouse-informed amino acid change (tyrosine in place of cysteine at position 261) exhibit potent, durable, and broad-spectrum resistance to HIV-1 and other pathogenic lentiviruses, and with no discernible impact on host cell biology. These results provide proof of concept for targeting CCNT1 in the context of one or more functional HIV-1 cure strategies., Competing Interests: The authors declare no conflict of interest.

Importance: Itaconate derivates, as well as the naturally produced metabolite, have been proposed as antivirals against influenza virus. Here, the mechanism behind the antiviral effects of exogenous 4-octyl itaconate (4-OI), a derivative of itaconate, against the influenza A virus replication is demonstrated. The data indicate that 4-OI targets the cysteine at position 528 of the CRM1 protein, resulting in inhibition of the nuclear export of viral ribonucleoprotein complexes in a similar manner as previously described for other selective inhibitors of nuclear export. These results postulate a mechanism not observed before for this immuno-metabolite derivative. This knowledge is helpful for the development of derivatives of 4-OI as potential antiviral and anti-inflammatory therapeutics., Competing Interests: Y.Z. and M.R. are employees and hold stock in AbbVie.

Background: The overactivation of NF-κB signaling is a key hallmark for the pathogenesis of extranodal natural killer/T cell lymphoma (ENKTL), a very aggressive subtype of non-Hodgkin's lymphoma yet with rather limited control strategies. Previously, we found that the dysregulated exportin-1 (also known as CRM1) is mainly responsible for tumor cells to evade apoptosis and promote tumor-associated pathways such as NF-κB signaling., Methods: Herein we reported the discovery and biological evaluation of a potent small molecule CRM1 inhibitor, LFS-1107. We validated that CRM1 is a major cellular target of LFS-1107 by biolayer interferometry assay (BLI) and the knockdown of CRM1 conferred tumor cells with resistance to LFS-1107., Results: We found that LFS-1107 can strongly suppresses the growth of ENKTL cells at low-range nanomolar concentration yet with minimal effects on human platelets and healthy peripheral blood mononuclear cells. Treatment of ENKTL cells with LFS-1107 resulted in the nuclear retention of IkB α and consequent strong suppression of NF-κB transcriptional activities, NF-κB target genes downregulation and attenuated tumor cell growth and proliferation. Furthermore, LFS-1107 exhibited potent activities when administered to immunodeficient mice engrafted with human ENKTL cells., Conclusions: Therefore, LFS-1107 holds great promise for the treatment of ENKTL and may warrant translation for use in clinical trials., Funding: Yang's laboratory was supported by the National Natural Science Foundation of China (Grant: 81874301), the Fundamental Research Funds for Central University (Grant: DUT22YG122) and the Key Research project of 'be Recruited and be in Command' in Liaoning Province (Personal Target Discovery for Metabolic Diseases)., Competing Interests: HL, ML, XT, HW, JG, HL, ZZ, YL, XC, YY No competing interests declared, CL Senior editor, eLife, (© 2023, Liu, Liu, Tian et al.)

NUP98 and NUP214 form chimeric fusion proteins that assemble into phase-separated nuclear bodies containing CRM1, a nuclear export receptor. However, these nuclear bodies' function in controlling gene expression remains elusive. Here, we demonstrate that the nuclear bodies of NUP98::HOXA9 and SET::NUP214 promote the condensation of mixed lineage leukemia 1 (MLL1), a histone methyltransferase essential for the maintenance of HOX gene expression. These nuclear bodies are robustly associated with MLL1/CRM1 and co-localized on chromatin. Furthermore, whole-genome chromatin-conformation capture analysis reveals that NUP98::HOXA9 induces a drastic alteration in high-order genome structure at target regions concomitant with the generation of chromatin loops and/or rearrangement of topologically associating domains in a phase-separation-dependent manner. Collectively, these results show that the phase-separated nuclear bodies of nucleoporin fusion proteins can enhance the activation of target genes by promoting the condensation of MLL1/CRM1 and rearrangement of the 3D genome structure., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Gene expression in eukaryotes begins with transcription in the nucleus, followed by the synthesis of messenger RNA (mRNA), which is then exported to the cytoplasm for its translation into proteins. Along with transcription and translation, mRNA export through the nuclear pore complex (NPC) is an essential regulatory step in eukaryotic gene expression. Multiple factors regulate mRNA export and hence gene expression. Interestingly, proteins from certain types of viruses interact with these factors in infected cells, and such an interaction interferes with the mRNA export of the host cell in favor of viral RNA export. Thus, these viruses hijack the host mRNA nuclear export mechanism, leading to a reduction in host gene expression and the downregulation of immune/antiviral responses. On the other hand, the viral mRNAs successfully evade the host surveillance system and are efficiently exported from the nucleus to the cytoplasm for translation, which enables the continuation of the virus life cycle. Here, we present this review to summarize the mechanisms by which viruses suppress host mRNA nuclear export during infection, as well as the key strategies that viruses use to facilitate their mRNA nuclear export. These studies have revealed new potential antivirals that may be used to inhibit viral mRNA transport and enhance host mRNA nuclear export, thereby promoting host gene expression and immune responses.

O-linked GlcNAc (O-GlcNAc) is an emerging post-translation modification that couples metabolism with cellular signal transduction by crosstalk with phosphorylation and ubiquitination to orchestrate various biological processes. The mechanisms underlying the involvement of O-GlcNAc modifications in N 6 -methyladenosine (m 6 A) regulation are not fully characterized. Herein, we show that O-GlcNAc modifies the m 6 A mRNA reader YTH domain family 1 (YTHDF1) and fine-tunes its nuclear translocation by the exportin protein Crm1. First, we present evidence that YTHDF1 interacts with the sole O-GlcNAc transferase (OGT). Second, we verified Ser196/Ser197/Ser198 as the YTHDF1 O-GlcNAcylation sites, as described in numerous chemoproteomic studies. Then we constructed the O-GlcNAc-deficient YTHDF1-S196A/S197F/S198A (AFA) mutant, which significantly attenuated O-GlcNAc signals. Moreover, we revealed that YTHDF1 is a nucleocytoplasmic protein, whose nuclear export is mediated by Crm1. Furthermore, O-GlcNAcylation increases the cytosolic portion of YTHDF1 by enhancing binding with Crm1, thus upregulating downstream target (e.g. c-Myc) expression. Molecular dynamics simulations suggest that O-GlcNAcylation at S197 promotes the binding between the nuclear export signal motif and Crm1 through increasing hydrogen bonding. Mouse xenograft assays further demonstrate that YTHDF1-AFA mutants decreased the colon cancer mass and size via decreasing c-Myc expression. In sum, we found that YTHDF1 is a nucleocytoplasmic protein, whose cytosolic localization is dependent on O-GlcNAc modification. We propose that the OGT-YTHDF1-c-Myc axis underlies colorectal cancer tumorigenesis., Competing Interests: Conflicts of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Background: During the tumourigenesis and development of colorectal cancer (CRC), the inactivation of tumour suppressor genes is closely involved, although detailed molecular mechanisms remain elusive. Accumulating studies, including ours, have demonstrated that basic leucine zipper transcription factor ATF (activating transcription factor)-like 2 (BATF2) is a capable tumour suppressor that localises in the nucleus. However, its different subcellular localisation, potential functions and underlying mechanisms are unclear., Methods: The translocation of BATF2 and its clinical relevance were detected using CRC samples, cell lines and xenograft nude mice. Candidate BATF2-binding proteins were screened using co-immunoprecipitation, quantitative label-free liquid chromatography-tandem mass spectrometry proteomic analysis, Western blotting and immunofluorescence. Recombinant plasmids, point mutations and siRNAs were applied to clarify the binding sites between BATF2 and chromosome region maintenance 1 (CRM1)., Results: The present study found that BATF2 was mainly localised in the cytoplasm, rather than nucleus, of CRC cells in vitro and in vivo, while cytoplasmic BATF2 expression was inversely correlated with the prognosis of CRC patients. Furthermore, we identified the nuclear export and subsequent ubiquitin-mediated degradation of BATF2 in CRC cells. Mechanistically, a functional nuclear export sequence (any amino acid) was characterised in BATF2 protein, through which BATF2 bound to CRM1 and translocated out of nucleus, ultimately enhancing CRC growth via inducing activator protein 1 (AP-1)/cyclin D1/phosphorylated retinoblastoma protein (pRb) signalling pathway. Additionally, nuclear export of BATF2 can be retarded by the mutation of NES in BATF2 or the knockdown of CRM1, whereas CRM1 expression was negatively associated with nuclear BATF2 expression and the prognosis of CRC patients., Conclusion: These findings revealed the biological effects and underlying mechanisms of cytoplasmic localisation of BATF2. Furthermore, suppressing nuclear export of BATF2 via mutating its NES region or inhibiting CRM1 expression may serve as a promising therapeutic strategy against CRC., (© 2023 The Authors. Clinical and Translational Medicine published by John Wiley & Sons Australia, Ltd on behalf of Shanghai Institute of Clinical Bioinformatics.)

Nuclear epidermal growth factor receptor (EGFR) has been shown to be correlated with drug resistance and a poor prognosis in patients with cancer. Previously, we have identified a tripartite nuclear localization signal (NLS) within EGFR. To comprehensively determine the functions and underlying mechanism of nuclear EGFR and its clinical implications, we aimed to explore the nuclear export signal (NES) sequence of EGFR that is responsible for interacting with the exportins. We combined in silico prediction with site-directed mutagenesis approaches and identified a putative NES motif of EGFR, which is located in amino acid residues 736-749. Mutation at leucine 747 (L747) in the EGFR NES led to increased nuclear accumulation of the protein via a less efficient release of the exportin CRM1. Interestingly, L747 with serine (L747S) and with proline (L747P) mutations were found in both tyrosine kinase inhibitor (TKI)-treated and -naïve patients with lung cancer who had acquired or de novo TKI resistance and a poor outcome. Reconstituted expression of the single NES mutant EGFR L747P or EGFR L747S , but not the dual mutant along with the internalization-defective or NLS mutation, in lung cancer cells promoted malignant phenotypes, including cell migration, invasiveness, TKI resistance, and tumor initiation, supporting an oncogenic role of nuclear EGFR. Intriguingly, cells with germline expression of the NES L747 mutant developed into B cell lymphoma. Mechanistically, nuclear EGFR signaling is required for sustaining nuclear activated STAT3, but not for Erk. These findings suggest that EGFR functions are compartmentalized and that nuclear EGFR signaling plays a crucial role in tumor malignant phenotypes, leading to tumorigenesis in human cancer., Competing Interests: None., (AJCR Copyright © 2023.)

Antiretroviral therapy (ART) reduces human immunodeficiency virus type 1 (HIV-1) infection, but selection of treatment-refractory variants remains a major challenge. HIV-1 encodes 16 canonical proteins, a small number of which are the singular targets of nearly all antiretrovirals developed to date. Cellular factors are increasingly being explored, which may present more therapeutic targets, more effectively target certain aspects of the viral replication cycle, and/or limit viral escape. Unlike most other positive-sense RNA viruses that encode at least one helicase, retroviruses are limited to the host repertoire. Accordingly, HIV-1 subverts DEAD-box helicase 3X (DDX3X) and numerous other cellular helicases of the Asp-Glu-x-Asp/His (DExD/H)-box family to service multiple aspects of its replication cycle. Here we review DDX3X and other DExD/H-box helicases in HIV-1 replication and their inhibition., Competing Interests: Declaration of interests The authors declare no conflicts of interest., (Copyright © 2022 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Sentrin/small ubiquitin-like modifier (SUMO) has emerged as a powerful mediator regulating biological processes and participating in pathophysiological processes that cause human diseases, such as cancer, myocardial fibrosis and neurological disorders. Sumoylation has been shown to play a positive regulatory role in keloids. However, the sumoylation mechanism in keloids remains understudied. We proposed that sumoylation regulates keloids via a complex. RanGAP1 acted as a synergistic, functional partner of SUMOs in keloids. Nuclear accumulation of Smad4, a TGF-β/Smad pathway member, was associated with RanGAP1 after SUMO1 inhibition. RanGAP1*SUMO1 mediated the nuclear accumulation of Smad4 due to its impact on nuclear export and reduction in the dissociation of Smad4 and CRM1. We clarified a novel mechanism of positive regulation of sumoylation in keloids and demonstrated the function of sumoylation in Smad4 nuclear export. The NPC-associated RanGAP1*SUMO1 complex functions as a disassembly machine for the export receptor CRM1 and Smad4. Our research provides new perspectives for the mechanisms of keloids and nucleocytoplasmic transport., (© 2022 The Authors. Journal of Cellular and Molecular Medicine published by Foundation for Cellular and Molecular Medicine and John Wiley & Sons Ltd.)

Circadian clocks are self-sustained molecular oscillators controlling daily changes of behavioral activity and physiology. For functional reliability and precision, the frequency of these molecular oscillations must be stable at different environmental temperatures, known as "temperature compensation." Despite being an intrinsic property of all circadian clocks, this phenomenon is not well understood at the molecular level. Here, we use behavioral and molecular approaches to characterize a novel mutation in the period (per) clock gene of Drosophila melanogaster, which alters a predicted nuclear export signal (NES) of the PER protein and affects temperature compensation. We show that this new per I530A allele leads to progressively longer behavioral periods and clock oscillations with increasing temperature in both clock neurons and peripheral clock cells. While the mutant PER I530A protein shows normal circadian fluctuations and post-translational modifications at cool temperatures, increasing temperatures lead to both severe amplitude dampening and hypophosphorylation of PER I530A . We further show that PER I530A displays reduced repressor activity at warmer temperatures, presumably because it cannot inactivate the transcription factor CLOCK (CLK), indicated by temperature-dependent altered CLK post-translational modification in per I530A flies. With increasing temperatures, nuclear accumulation of PER I530A within clock neurons is increased, suggesting that wild-type PER is exported out of the nucleus at warm temperatures. Downregulating the nuclear export factor CRM1 also leads to temperature-dependent changes of behavioral rhythms, suggesting that the PER NES and the nuclear export of clock proteins play an important role in temperature compensation of the Drosophila circadian clock., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Hutchinson-Gilford progeria syndrome (HGPS) is a rare premature aging disorder caused by the expression of progerin, a mutant variant of Lamin A. Recently, HGPS studies have gained relevance because unraveling its underlying mechanism would help to understand physiological aging. We previously reported that the CRM1-mediated nuclear protein export pathway is exacerbated in HGPS cells, provoking the mislocalization of numerous protein targets of CRM1. We showed that normalization of this mechanism by pharmacologically inhibiting CRM1 with LMB (specific CRM1 inhibitor), mitigates the senescent phenotype of HGPS cells. Since mitochondrial dysfunction is a hallmark of HGPS, in this study we analyze the effect of LMB on mitochondrial function. Remarkably, LMB treatment induced the recovery of mitochondrial function in HGPS cells, as shown by the improvement in mitochondrial morphology, mitochondrial membrane potential, and ATP levels, which consequently impeded the accumulation of ROS but not mitochondrial superoxide. We provide evidence that the beneficial effect of LMB is mechanistically based on a combinatory effect on mitochondrial biogenesis via upregulation of PGC-1α expression (master transcription cofactor of mitochondrial genes), and mitophagy through the recovery of lysosomal content. The use of exportin CRM1 inhibitors constitutes a promising strategy to treat HGPS and other diseases characterized by mitochondrial impairment.

Breast cancer stem-like cells (BCSCs) have been suggested as the underlying cause of tumor recurrence, metastasis and drug resistance in triple-negative breast cancer (TNBC). Here, we report the discovery and biological evaluation of a highly potent small-molecule antagonist of exportin-1, LFS-1107. We ascertained that exportin-1 (also named as CRM1) is a main cellular target of LFS-1107 by nuclear export functional assay, bio-layer interferometry binding assay and C528S mutant cell line. We found that LFS-1107 significantly inhibited TNBC tumor cells at low-range nanomolar concentration and LFS-1107 can selectively eliminate CD44 + CD24 - enriched BCSCs. We demonstrated that LFS-1107 can induce the nuclear retention of Survivin and consequent strong suppression of STAT3 transactivation abilities and the expression of downstream stemness regulators. Administration of LFS-1107 can strongly inhibit tumor growth in mouse xenograft model and eradicate BCSCs in residual tumor tissues. Moreover, LFS-1107 can significantly ablate the patient-derived tumor organoids (PDTOs) of TNBC as compared to a few approved cancer drugs. Lastly, we revealed that LFS-1107 can enhance the killing effects of chemotherapy drugs and downregulate multidrug resistance related protein targets. These new findings provide preclinical evidence of defining LFS-1107 as a promising therapeutic agent to deplete BCSCs for the treatment of TNBC., (Copyright © 2022 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Tilapia Lake Virus (TiLV) is an emerging virus lethal to tilapia, which threatens the global tilapia aquaculture with severe implications for food security. TiLV possesses similar features to orthomyxoviruses but is classified in the sole and the monotypic genus Tilapinevirus of the family Amnoonviridae. TiLV enveloped virions encapsidate a genome comprising ten segments of single-stranded, negative RNA. Remarkably, nine of TiLV's ten major proteins lack sequence homology to any known viral or cellular proteins. The mode of TiLV entry into tilapia cells is not known. Following the measurement of the entry window of TiLV (∼3 h), we applied a panel of inhibitors of known regulators of endocytic functions to map the molecular requirements for TiLV entry. We identified productive entry by quantification of TiLV nucleoprotein expression and the generation of infectious particles. Inhibition of dynamin activity with dynasore or dynole, or depletion of cholesterol with methyl-β-cyclodextrin, strongly inhibited TiLV protein synthesis and infectious virion production. Moreover, inhibition of actin cytoskeleton polymerization with latrunculin A or microtubule polymerization with nocodazole within the entry window resulted in partial inhibition of TiLV infection. In contrast, inhibitors of endosomal acidification (NH 4 Cl, bafilomycin A1, or chloroquine), an inhibitor of clathrin-coated pit assembly (pitstop 2), and erlotinib-an inhibitor of the endocytic Cyclin G-associated kinase (GAK), did not affect TiLV entry. Altogether, these results suggest that TiLV enters via dynamin-mediated endocytosis in a cholesterol-, cytoskeleton-dependent manner, and clathrin-, pH-independent manner. Thus, despite being an orthomyxo-like virus, when compared to the prototypical orthomyxovirus (influenza A virus), TiLV shows a distinct set of requirements for entry into cells., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Abu Rass, Kembou-Ringert, Zamostiano, Eldar, Ehrlich and Bacharach.)

The nuclear export factor CRM1-mediated pathway is known to be important for the nuclear egress of progeny parvovirus capsids in the host cells with virus-mediated cell cycle arrest at G2/M. However, it is still unclear whether this is the only pathway by which capsids exit the nucleus. Our studies show that the nuclear egress of DNA-containing full canine parvovirus. capsids was reduced but not fully inhibited when CRM1-mediated nuclear export was prevented by leptomycin B. This suggests that canine parvovirus capsids might use additional routes for nuclear escape. This hypothesis was further supported by our findings that nuclear envelope (NE) permeability was increased at the late stages of infection. Inhibitors of cell cycle regulatory protein cyclin-dependent kinase 1 (Cdk1) and pro-apoptotic caspase 3 prevented the NE leakage. The change in NE permeability could be explained by the regulation of the G2/M checkpoint which is accompanied by early mitotic and apoptotic events. The model of G2/M checkpoint activation was supported by infection-induced nuclear accumulation of cyclin B1 and Cdk1. Both NE permeability and nuclear egress of capsids were reduced by the inhibition of Cdk1. Additional proof of checkpoint function regulation and promotion of apoptotic events was the nucleocytoplasmic redistribution of nuclear transport factors, importins, and Ran, in late infection. Consistent with our findings, post-translational histone acetylation that promotes the regulation of several genes related to cell cycle transition and arrest was detected. In conclusion, the model we propose implies that parvoviral capsid egress partially depends on infection-induced G2/M checkpoint regulation involving early mitotic and apoptotic events., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Mattola, Mäntylä, Aho, Salminen, Leclerc, Oittinen, Salokas, Järvensivu, Hakanen, Ihalainen, Viiri and Vihinen-Ranta.)

Chromosomal region maintenance 1 (CRM1 also known as Xpo1 and exportin-1) is the receptor for the nuclear export controlling the intracellular localization and function of many cellular and viral proteins that play a crucial role in viral infections and cancer. The inhibition of CRM1 has emerged as a promising therapeutic approach to interfere with the lifecycle of many viruses, for the treatment of cancer, and to overcome therapy resistance. Recently, selinexor has been approved as the first CRM1 inhibitor for the treatment of multiple myeloma, providing proof of concept for this therapeutic option with a new mode of action. However, selinexor is associated with dose-limiting toxicity and hence, the discovery of alternative small molecule leads that could be developed as less toxic anticancer and antiviral therapeutics will have a significant impact in the clinic. Here, we report a CRM1 inhibitor discovery platform. The development of this platform includes reporter cell lines that monitor CRM1 activity by using red fluorescent protein or green fluorescent protein-labeled HIV-1 Rev protein with a strong heterologous nuclear export signal. Simultaneously, the intracellular localization of other proteins, to be interrogated for their capacity to undergo CRM1-mediated export, can be followed by co-culturing stable cell lines expressing fluorescent fusion proteins. We used this platform to interrogate the mode of nuclear export of several proteins, including PDK1, p110α, STAT5A, FOXO1, 3, 4 and TRIB2, and to screen a compound collection. We show that while p110α partially relies on CRM1-dependent nuclear export, TRIB2 is exported from the nucleus in a CRM1-independent manner. Compound screening revealed the striking activity of an organoselenium compound on the CRM1 nuclear export receptor., (© 2022 The Authors. Traffic published by John Wiley & Sons Ltd.)

An appropriate balance between testicular testosterone and estradiol is required for spermatogenesis. Excess estradiol is often identified in the semen and serum of infertile men; however, the mechanisms behind this observation remain unclear. This study indicates the relationship between heat stress and aromatase synthesis in Leydig cells. We used R2C rat Leydig tumor cells, which can synthesize both testosterone and estradiol. Aromatase transcription was regulated by the PⅡ promoter with or without heat stress. Heat stress at 40 °C increased aromatase expression and decreased testosterone to estradiol ratio and nuclear DAX-1 (dosage-sensitive sex reversal, adrenal hypoplasia critical region, on chromosome X, gene 1), which is a suppressor of steroidogenic factor 1 (SF-1). Leptomycin B and KPT-185, a nuclear export inhibitor, prevented nuclear DAX-1 deficiency induced by heat stress and inhibited aromatase transcription. These results indicate that heat stress interferes with DAX-1-SF-1 interaction and induces SF-1-dependent aromatase transcription., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Ataxia telangiectasia mutated (ATM) is a serine-threonine protein kinase and important regulator of the DNA damage response (DDR). One critical ATM target is the structural subunit A (PR65-S401) of protein phosphatase 2A (PP2A), known to regulate diverse cellular processes such as mitosis and cell growth as well as dephosphorylating many proteins during the recovery from the DDR. We generated mouse embryonic fibroblasts expressing PR65-WT, -S401A (cannot be phosphorylated), and -S401D (phospho-mimetic) transgenes. Significantly, S401 mutants exhibited extensive chromosomal aberrations, impaired DNA double-strand break (DSB) repair and underwent increased mitotic catastrophe after radiation. Both S401A and the S401D cells showed impaired DSB repair (nonhomologous end joining and homologous recombination repair) and exhibited delayed DNA damage recovery, which was reflected in reduced radiation survival. Furthermore, S401D cells displayed increased ERK and AKT signaling resulting in enhanced growth rate further underscoring the multiple roles ATM-PP2A signaling plays in regulating prosurvival responses. Time-lapse video and cellular localization experiments showed that PR65 was exported to the cytoplasm after radiation by CRM1, a nuclear export protein, in line with the very rapid pleiotropic effects observed. A putative nuclear export sequence (NES) close to S401 was identified and when mutated resulted in aberrant PR65 shuttling. Our study demonstrates that the phosphorylation of a single, critical PR65 amino acid (S401) by ATM fundamentally controls the DDR, and balances DSB repair quality, cell survival and growth by spatiotemporal PR65 nuclear-cytoplasmic shuttling mediated by the nuclear export receptor CRM1., (© 2022. The Author(s).)

Single-cell imaging has emerged as a powerful means to study viral replication dynamics and identify sites of virus−host interactions. Multivariate aspects of viral replication cycles yield challenges inherent to handling large, complex imaging datasets. Herein, we describe the design and implementation of an automated, imaging-based strategy, “Human Immunodeficiency Virus Red-Green-Blue” (HIV RGB), for deriving comprehensive single-cell measurements of HIV-1 unspliced (US) RNA nuclear export, translation, and bulk changes to viral RNA and protein (HIV-1 Rev and Gag) subcellular distribution over time. Differentially tagged fluorescent viral RNA and protein species are recorded using multicolor long-term (>24 h) time-lapse video microscopy, followed by image processing using a new open-source computational imaging workflow dubbed “Nuclear Ring Segmentation Analysis and Tracking” (NR-SAT) based on ImageJ plugins that have been integrated into the Konstanz Information Miner (KNIME) analytics platform. We describe a typical HIV RGB experimental setup, detail the image acquisition and NR-SAT workflow accompanied by a step-by-step tutorial, and demonstrate a use case wherein we test the effects of perturbing subcellular localization of the Rev protein, which is essential for viral US RNA nuclear export, on the kinetics of HIV-1 late-stage gene regulation. Collectively, HIV RGB represents a powerful platform for single-cell studies of HIV-1 post-transcriptional RNA regulation. Moreover, we discuss how similar NR-SAT-based design principles and open-source tools might be readily adapted to study a broad range of dynamic viral or cellular processes.

Hepatitis B virus (HBV) is a global pathogen. We report here that the cellular CRM1 machinery can mediate nuclear export of entire HBV core (HBc) particles containing encapsidated viral RNAs. Two CRM1-mediated nuclear export signals (NES CRM1 ) cluster at the conformationally flexible spike tips of HBc particles. Mutant NES CRM1 capsids exhibit strongly reduced associations with CRM1 and nucleoporin358 in vivo. CRM1 and NXF1 machineries mediate nuclear export of HBc particles independently. Inhibition of nuclear export has pleiotropic consequences, including nuclear accumulation of HBc particles, a significant reduction of encapsidated viral RNAs in the cytoplasm but not in the nucleus, and barely detectable viral DNA. We hypothesize an HBV life cycle where encapsidation of the RNA pregenome can initiate early in the nucleus, whereas DNA genome maturation occurs mainly in the cytoplasm. We identified a druggable target for HBV by blocking its intracellular trafficking., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2022 The Author(s). Published by Elsevier Inc. All rights reserved.)

After receptor-mediated endocytosis and endosomal escape, adenoviral capsids can travel via microtubule organizing centers to the nuclear envelope. Upon capsid disassembly, viral genome import into nuclei of interphase cells then occurs through nuclear pore complexes, involving the nucleoporins Nup214 and Nup358. Import also requires the activity of the classic nuclear export receptor CRM1, as it is blocked by the selective inhibitor leptomycin B. We have now used artificially enucleated as well as mitotic cells to analyze the role of an intact nucleus in different steps of the viral life cycle. In enucleated U2OS cells, viral capsids traveled to the microtubule organizing center, whereas their removal from this complex was blocked, suggesting that this step required nuclear factors. In mitotic cells, on the other hand, CRM1 promoted capsid disassembly and genome release, suggesting a role of this protein that does not require intact nuclear envelopes or nuclear pore complexes and is distinct from its function as a nuclear export receptor. Similar to enucleation, inhibition of CRM1 by leptomycin B also leads to an arrest of adenoviral capsids at the microtubule organizing center. In a small-scale screen using leptomycin B-resistant versions of CRM1, we identified a mutant, CRM1 W142A P143A, that is compromised with respect to adenoviral capsid disassembly in both interphase and mitotic cells. Strikingly, this mutant is capable of exporting cargo proteins out of the nucleus of living cells or digitonin-permeabilized cells, pointing to a role of the mutated region that is not directly linked to nuclear export. IMPORTANCE A role of nucleoporins and of soluble transport factors in adenoviral genome import into the nucleus of infected cells in interphase has previously been established. The nuclear export receptor CRM1 promotes genome import, but its precise function is not known. Using enucleated and mitotic cells, we showed that CRM1 does not simply function by exporting a crucial factor out of the nucleus that would then trigger capsid disassembly and genome import. Instead, CRM1 has an export-independent role, a notion that is also supported by a mutant, CRM1 W142A P143A, which is export competent but deficient in viral capsid disassembly, in both interphase and mitotic cells.

Ovarian cancer (OC) is the main type of cancer that affects the female reproductive system and has a high morbidity and mortality rate. This study aimed to explore the regulatory effect of the chromosomal region maintenance 1 (CRM1)-survivin axis on the progression of OC. Ovarian cancer cells were transfected with pcDNA3.1-survivin and short hairpin RNA (sh)-CRM1. Cell proliferation was analyzed by cell counting kit-8 (CCK8), 5-ethynyl-2´-deoxyuridine (EdU) staining, and colony formation assays. Apoptosis was detected using flow cytometry. Quantitative real-time polymerase chain reaction (qRT-PCR) and Western blotting were performed to analyze the expression of RNA and protein, respectively. qRT-PCR and prognostic correlation analyses revealed that CRM1 is highly expressed in OC cells and related to survival. The results of qRT-PCR, CCK8, colony formation test, EdU staining, flow cytometry, and Western blotting showed that CRM1 silencing inhibited the proliferation and colony formation of OVCAR 3 and SKOV3 cells and promoted cell apoptosis by promoting Caspase-3 activation. Survivin was positively regulated by CRM1 and promoted the development of OC. The results of the rescue experiment showed that overexpression of survivin reversed the inhibitory effect of CRM1 knockdown on the proliferation of ovarian cancer cells and its inhibitory effect on apoptosis. Our findings confirm the role of the CRM1-survivin signal transduction axis in OC by regulating the proliferation and apoptosis of OC cells, and may thus serve as a potential therapeutic target for OC.

The Karyopherin protein CRM1 or XPO1 is the major nuclear export receptor that regulates nuclear exit of thousands of macromolecules in the cell. CRM1 recognizes protein cargoes by binding to their 8-15 residue-long nuclear export signals (NESs). A ternary CRM1-Ran-RanBP1 complex engineered to be suitable for crystallization has enabled structure determination by X-ray crystallography of CRM1 bound to many NES peptides and small-molecule inhibitors. Here, we present a protocol for the purification of the individual proteins, formation of the ternary CRM1-Ran-RanBP1 complex and crystallization of this complex for X-ray crystallography., (© 2022. Springer Science+Business Media, LLC, part of Springer Nature.)

CRM1 recognizes hundreds to thousands of protein cargoes by binding to the eight to fifteen residue-long nuclear export signals (NESs) within their polypeptide chains. Various assays to measure the binding affinity of NESs for CRM1 have been developed. CRM1 binds to NESs with a wide range of binding affinities, with dissociation constants that span from low nanomolar to tens of micromolar. An optimized binding affinity assay with improved throughput was recently developed to measure binding affinities of NES peptides for CRM1 in the presence of excess RanGTP. The assay can measure affinities, with multiple replicates, for up to seven different NES peptides per screening plate. Here, we present a protocol for the purification of the necessary proteins and for measuring CRM1-NES binding affinities., (© 2022. Springer Science+Business Media, LLC, part of Springer Nature.)

As the central node between nutrition signaling input and the metabolic pathway, AMP-activated protein kinase (AMPK) is tightly regulated to maintain energy homeostasis. Subcellular compartmentalization of AMPK is one of the critical regulations that enables AMPK to access proper targets and generate appropriate responses to specific perturbations and different levels of stress. One of the characterized localization mechanisms is RanGTPase-driven CRM1 that recognizes the nuclear export sequence (NES) on the α subunit to translocate AMPK into the cytoplasm. Nuclear localization putatively employs RanGTPase-driven importin that might recognize the nuclear localization signal (NLS) present on the AMPKα2 kinase domain. Nucleo-cytoplasmic shuttling of AMPK is influenced by multiple factors, such as starvation, exercise, heat shock, oxidant, cell density, and circadian rhythm. Tissue-specific localization, which distributes AMPK trimers with different combinations, has also been shown to be vital in maintaining tissue-specific metabolism. Tissue-specific and subcellular distribution of AMPK might be attributed to differences in the expression of the subunit, the stabilization by protein regulators, tissue activity, and the localization of AMPK activators. Considering the importance of AMPK localization in coordinating signaling and metabolism, further research is due to fully elucidate the largely unknown complex mechanism underlying this regulation.

Autographa californica multiple nucleopolyhedrovirus (AcMNPV) assembles its nucleocapsids and occlusion-derived virions (ODVs) in the nucleus, which requires AcMNPV regulation for viral structural proteins to accumulate in the nucleus during its replication in cells. It is generally accepted that the nuclear import receptor plays a predominant role in this process. CRM1 is a nuclear export receptor that forms an export complex with its cargo protein to exit the nucleus. We previously discovered that AcMNPV inhibited CRM1-dependent nuclear export by the viral protein Ac34. This finding suggested that Ac34 could sequester CRM1-dependent proteins in the nucleus and play a novel role in the nuclear accumulation of viral structural proteins. Using the CRM1 inhibitor leptomycin B (LMB), we demonstrated that CRM1 inhibition promoted AcMNPV replication, as LMB treatment readily increased the virus titer, and even functionally surrogate Ac34 to rescue the infectivity of an ac34-knockout virus. To elucidate whether CRM1 inhibition contributes to the nuclear accumulation of viral structural proteins, we systematically analyzed the impact of CRM1 inhibition on viral protein spatial distribution patterns. We found that the nucleocapsid protein Ac102 and ODV envelope protein E26 were retained in the nucleus in response to CRM1 inhibition by Ac34. This finding indicates that AcMNPV is evolving to simultaneously exploit bidirectional nucleocytoplasmic trafficking to assist in viral replication., (Copyright © 2021. Published by Elsevier B.V.)

The novel coronavirus disease 2019 (COVID-19) caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is responsible for the recent global pandemic. The nuclear export protein (XPO1) has a direct role in the export of SARS-CoV proteins including ORF3b, ORF9b, and nucleocapsid. Inhibition of XPO1 induces anti-inflammatory, anti-viral, and antioxidant pathways. Selinexor is an FDA-approved XPO1 inhibitor. Through bioinformatics analysis, we predicted nuclear export sequences in the ACE-2 protein and confirmed by in vitro testing that inhibition of XPO1 with selinexor induces nuclear localization of ACE-2. Administration of selinexor inhibited viral infection prophylactically as well as therapeutically in vitro. In a ferret model of COVID-19, selinexor treatment reduced viral load in the lungs and protected against tissue damage in the nasal turbinates and lungs in vivo. Our studies demonstrated that selinexor downregulated the pro-inflammatory cytokines IL-1β, IL-6, IL-10, IFN-γ, TNF-α, and GMCSF, commonly associated with the cytokine storm observed in COVID-19 patients. Our findings indicate that nuclear export is critical for SARS-CoV-2 infection and for COVID-19 pathology and suggest that inhibition of XPO1 by selinexor could be a viable anti-viral treatment option., (Copyright © 2021 The Authors. Published by Elsevier B.V. All rights reserved.)

Epstein-Barr virus (EBV), the pathogen of several human malignancies, encodes many proteins required to be transported into the nucleus for viral DNA reproduction and nucleocapsids assembly in the lytic replication cycle. Here, fluorescence microscope, mutation analysis, interspecies heterokaryon assays, co-immunoprecipitation assay, RNA interference, and Western blot were performed to explore the nuclear import mechanism of EBV encoded BLLF2 protein. BLLF2 was shown to be a nucleocytoplasmic shuttling protein neither by a chromosomal region maintenance 1 (CRM1)- nor by a transporter associated with antigen processing (TAP)-dependent pathway. Yet, BLLF2's two functional nuclear localization signals (NLSs), NLS1 ( 16 KRQALETVPHPQNRGR 31 ) and NLS2 ( 44 RRPRPPVAKRRRFPR 58 ), were identified, whereas the predicted NES was nonfunctional. Finally, BLLF2 was proven to transport into the nucleus via a Ran-dependent and importin β1-dependent pathway. This mechanism may contribute to a more extensive insight into the assembly and synthesis of EBV virions in the nucleus, thus affording a new direction for the treatment of viruses., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Li, Guo, Deng, Hu, Li, Deng, Zhong, Xie, Shi, Hong, Zheng, Cai and Li.)

Dysregulation of the nuclear export machinery mediated by chromosomal maintenance 1 (CRM1, also known as exportin-1), is closely associated with various human disorders, such as breast cancer. Previously, we identified sulforaphene and its synthetic analogues as covalent inhibitors of CRM1. Herein, we describe the discovery and biological evaluation of another sulforaphene synthetic analogue, LFS-31, as a potential CRM1 inhibitor. In addition, we investigated the reversible binding mechanism of LFS-31 with CRM1 through molecular simulations coupled with bio-layer interferometry (BLI) and found relatively high binding affinity (KD = 43.1 ± 35.3 nM) between the LFS-31 and CRM1 groups. We found that LFS-31 exhibited a stronger growth suppression of triple-negative breast cancer (TNBC) cells than non-TNBC cells, and had minimal effect on normal breast cells. Pharmacological treatment of TNBC cells with LFS-31 at nanomolar concentrations led to the nuclear retention of IkBα resulting in strong suppression of NF-κB transcriptional activity and attenuated cell growth and proliferation, which collectively contributed to the antitumor responses. To the best of our knowledge, this is the first study to demonstrate the use of a sulforaphene analogue as a potent CRM1 inhibitor that targets the NF-κB signaling pathway for the targeted therapy of TNBC., (© 2021 John Wiley & Sons A/S . Published by John Wiley & Sons Ltd.)

Background: The nuclear pore complexes (NPCs) are built of about 30 different nucleoporins and act as key regulators of molecular traffic between the cytoplasm and the nucleus for sizeable proteins (> 40 kDa) which must enter the nucleus. Various nuclear transport receptors are involved in import and export processes of proteins through the nuclear pores. The most prominent nuclear export receptor is chromosome region maintenance 1 (CRM1), also known as exportin 1 (XPO1). One of its cargo proteins is the prolyl hydroxylase 2 (PHD2) which is involved in the initiation of the degradation of hypoxia-inducible factors (HIFs) under normoxia. HIFs are proteins that regulate the cellular adaptation under hypoxic conditions. They are involved in many aspects of cell viability and play an important role in the hypoxic microenvironment of cancer. In cancer, CRM1 is often overexpressed thus being a putative target for the development of new cancer therapies. The newly FDA-approved pharmaceutical Selinexor (KPT-330) selectively inhibits nuclear export via CRM1 and is currently tested in additional Phase-III clinical trials. In this study, we investigated the effect of CRM1 inhibition on the subcellular localization of HIF-1α and radiosensitivity., Methods: Human hepatoma cells Hep3B and human osteosarcoma cells U2OS were treated with Selinexor. Intranuclear concentration of HIF-1α protein was measured using immunoblot analysis. Furthermore, cells were irradiated with 2-8 Gy after treatment with Selinexor compared to untreated controls., Results: Selinexor significantly reduced the intranuclear level of HIF-1α protein in human hepatoma cells Hep3B and human osteosarcoma cells U2OS. Moreover, we demonstrated by clonogenic survival assays that Selinexor leads to dose-dependent radiosensitization in Hep3B-hepatoma and U2OS-osteosarcoma cells., Conclusion: Targeting the HIF pathway by Selinexor might be an attractive tool to overcome hypoxia-induced radioresistance.

Introduction: Peptides can be rationally designed as non-covalent inhibitors for molecularly targeted therapy. However, it remains challenging to efficiently deliver the peptides into the targeted cells, which often severely affects their therapeutic efficiency., Methods: Herein, we created a novel non-covalent peptide inhibitor against nuclear export factor CRM1 by a structure-guided drug design method and targetedly delivered the peptide into cancer cells by a nanoparticle-mediated gene expression system for use as a cancer therapy., Results: The nuclear export signal (NES)-optimized CRM1 peptide inhibitor colocalized with CRM1 to the nuclear envelope and inhibited nuclear export in cancer cell lines in vitro. The crystal structures of the inhibitors complexed with CRM1 were solved. In contrast to the covalent inhibitors, the peptides were similarly effective against cells harboring the CRM1 C528S mutation. Moreover, a plasmid encoding the peptides was delivered by a iRGD-modified nanoparticle to efficiently target and transfect the cancer cells in vivo after intravenous administration. The peptides could be selectively expressed in the tumor, resulting in the efficient inhibition of subcutaneous melanoma xenografts without obvious systemic toxicity., Discussion: This work provides an effective strategy to design peptide-based molecularly targeted therapeutics, which could lead to the development of future targeted therapy., Competing Interests: QS, YL, MS, MG, and MX have applied patents on the designed peptides in this study. The authors report no other conflicts of interest in this work., (© 2021 Sui et al.)

Nup214 is a major nucleoporin on the cytoplasmic side of the nuclear pore complex with roles in late steps of nuclear protein and mRNA export. It interacts with the nuclear export receptor CRM1 (also known as XPO1) via characteristic phenylalanine-glycine (FG) repeats in its C-terminal region. Here, we identify a classic nuclear export sequence (NES) in Nup214 that mediates Ran-dependent binding to CRM1. Nup214 versions with mutations in the NES, as well as wild-type Nup214 in the presence of the selective CRM1 inhibitor leptomycin B, accumulate in the nucleus of Nup214-overexpressing cells. Furthermore, physiological binding partners of Nup214, such as Nup62 and Nup88, are recruited to the nucleus together with Nup214. Nuclear export of mutant Nup214 can be rescued by artificial nuclear export sequences at the C-terminal end of Nup214, leading also to a correct localization of Nup88. Our results suggest a function of the Nup214 NES in the biogenesis of the nuclear pore complex and/or in terminal steps of CRM1-dependent protein export., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2021. Published by The Company of Biologists Ltd.)

Background: Nrf2-Bach1 antioxidant signaling pathway is considered as one of the most important mechanisms of cellular resistance to oxidative injury. The effect of hyperoside (Hyp) on the expression and distribution of Bach1, the relationship of Hyp's antioxidative effect and the influence of Bach1 remains unclear., Purpose: The aim of this study was to investigate the role and mechanisms of Bach1 in the protective effect of Hyp on oxidative liver injury., Methods: The protective effect of Hyp on oxidative stress injury was observed in vivo and in vitro. Next, the influence of Hyp on Bach1 expression and distribution, and competitive combination of Nrf2-Bach1 with ARE in H 2 O 2 -induced L02 cell was studied by Western blot, RT-PCR, immunofluorescence and CHIP assay. Finally, the expressions of Crm1, ERK and p38 and their roles on Hyp mediated nuclear export of Bach1 were investigated by Western blot., Results: Hyp ameliorated the pathological damage, reduced the liver index, AST, ALT and MDA activities, and increased SOD and GSH levels in the CCl 4 -induced acute liver injury mouse model. Hyp attenuated H 2 O 2 -induced oxidative stress injury in L02 cells. Hyp promoted the early rapid redistribution of Bach1 from nucleus to cytoplasm. CHIP analyses demonstrated that Hyp enhanced the levels of Nrf2-ARE complex, and weakened the levels of Bach1-ARE complex within three hours. In addition, Hyp enhanced transport protein Crm1 expression and ERK1/2 activity. And LMB, a Crm1 inhibitor, attenuated the effect of Hyp on Bach1 nuclear export and anti-oxidation. U0126, an ERK1/2 inhibitor, reduced the effect of Hyp on Crm1 expression and the Bach1 redistribution., Conclusion: The hepatoprotective mechanism of Hyp was related to improve Bach1 nuclear export depending on ERK1/2-Crm1 to upregulate the level of Nrf2 binding to ARE., Competing Interests: The authors report no conflicts of interest in this work., (© 2021 Cai et al.)

Hepatitis B virus (HBV) core protein (Cp) can be found in the nucleus and cytoplasm of infected hepatocytes; however, it preferentially segregates to a specific compartment correlating with disease status. Regulation of this intracellular partitioning of Cp remains obscure. In this paper, we report that cellular compartments are filled and vacated by Cp in a time- and concentration-dependent manner in both transfections and infections. At early times after transfection, Cp, in a dimeric state, preferentially localizes to the nucleolus. Later, the nucleolar compartment is emptied and Cp progresses to being predominantly nuclear, with a large fraction of the protein in an assembled state. Nuclear localization is followed by cell-wide distribution, and then Cp becomes exclusively cytoplasmic. The same trend in Cp movement is seen during an infection. Putative nucleolar retention signals have been identified and appear to be structure dependent. Export of Cp from the nucleus involves the CRM1 exportin. Time-dependent flux can be recapitulated by modifying Cp concentration, suggesting transitions are regulated by reaching a threshold concentration. IMPORTANCE HBV is an endemic virus. More than 250 million people suffer from chronic HBV infection and about 800,000 die from HBV-associated disease each year. HBV is a pararetrovirus; in an infected cell, viral DNA in the nucleus is the template for viral RNA that is packaged in nascent viral capsids in the cytoplasm. Inside those capsids, while resident in cytoplasm, the linear viral RNA is reverse transcribed to form the circular double-stranded DNA (dsDNA) of the mature virus. The HBV core (or capsid) protein plays a role in almost every step of the viral life cycle. Here, we show the core protein appears to follow a programmed, sequential localization from cytoplasmic translation then into the nucleolus, to the nucleus, and back to the cytoplasm. Localization is primarily a function of time, core protein concentration, and assembly. This has important implications for our understanding of the mechanisms of antivirals that target HBV capsid assembly., (Copyright © 2021 Nair and Zlotnick.)

Jacob is a synapto-nuclear messenger protein that couples NMDAR activity to CREB-dependent gene expression. In this study, we investigated the nuclear distribution of Jacob and report a prominent targeting to the nuclear envelope that requires NMDAR activity and nuclear import. Immunogold electron microscopy and proximity ligation assay combined with STED imaging revealed preferential association of Jacob with the inner nuclear membrane where it directly binds to LaminB1, an intermediate filament and core component of the inner nuclear membrane (INM). The association with the INM is transient; it involves a functional nuclear export signal in Jacob and a canonical CRM1-RanGTP-dependent export mechanism that defines the residing time of the protein at the INM. Taken together, the data suggest a stepwise redistribution of Jacob within the nucleus following nuclear import and prior to nuclear export.

Spastin, a microtubule-severing AAA ATPase, regulates microtubule dynamics and plays important roles in cell division and neurogenesis. Mutations in the spastin-coding gene SPAST lead to neurodegenerative disorders and cause spastic paraplegia type 4. Spastin has two main isoforms, M1 and M87, that differ only in the presence or absence of 86 N-terminal amino acids and have alternative splicing variants that lack exon4. The N-terminal region of M1 contains a hydrophobic domain, nuclear localization signal (NLS), and nuclear export signal (NES), which partly explains the differences in the two isoforms' localization. However, the mechanisms involved in regulating isoform localization, and the effects of localization on spastin functions are not fully understood. We found endogenous M1 and M87 shuttled between the nucleus and cytoplasm during the cell cycle. We identified a NES (amino acids 195-204) that spans the microtubule-interacting and endosomal-trafficking domain and exon4 region. Furthermore, the NES sequence contains both the coiled-coil and exon4 region of spastin isoforms. Highly conserved leucine 195 in exon3 and the two residues in exon4 are crucial for predicted coiled-coil formation. Mutations in NES or leptomycin B treatment reduced cytoplasmic localization and microtubule fragmentation in M87 rather than in M1. Phosphomimetic mutation of threonine 306 adjacent to the NLS (amino acids 309-312) inhibited nuclear transport of M87. Our results indicate that the newly identified NES in the spastin isoforms containing exon4 regulates the subcellular localization of spastin in coordination with NLS controlled by the phosphorylation state of spastin, and is involved in microtubule severing., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Export of mRNAs from the nucleus to the cytoplasm is a key regulatory step in the expression of proteins. mRNAs are transported through the nuclear pore complex (NPC). Export of mRNAs responds to a variety of cellular stimuli and stresses. Revelations of the specific effects elicited by NPC components and associated co-factors provides a molecular basis for the export of selected RNAs, independent of bulk mRNA export. Aberrant RNA export has been observed in primary human cancer specimens. These cargo RNAs encode factors involved in nearly all facets of malignancy. Indeed, the NPC components involved in RNA export as well as the RNA export machinery can be found to be dysregulated, mutated, or impacted by chromosomal translocations in cancer. The basic mechanisms associated with RNA export with relation to export machinery and relevant NPC components are described. Therapeutic strategies targeting this machinery in clinical trials is also discussed. These findings firmly position RNA export as a targetable feature of cancer along with transcription and translation.

Xenopus: egg extracts are a powerful in vitro tool for studying complex biological processes, including nuclear reconstitution, nuclear membrane and pore assembly, and spindle assembly. Extracts have been further used to demonstrate a moonlighting regulatory role for nuclear import receptors or importins on these cell cycle assembly events. Here we show that exportins can also play a role in these events. Addition of Crm1, Exportin-t, or Exportin-5 decreased nuclear pore assembly in vitro. RanQ69L-GTP, a constitutively active form of RanGTP, ameliorated inhibition. Both Crm1 and Exportin-t inhibited fusion of nuclear membranes, again counteracted by RanQ69L-GTP. In mitotic extracts, Crm1 and Exportin-t negatively impacted spindle assembly. Pulldowns from the extracts using Crm1- or Exportin-t-beads revealed nucleoporins known to be essential for both nuclear pore and spindle assembly, with RanQ69L-GTP decreasing a subset of these target interactions. This study suggests a model where exportins, like importins, can regulate major mitotic assembly events.

Background: Osteosarcoma is a rare but aggressive bone cancer that occurs primarily in children. Like other rare cancers, treatment advances for osteosarcoma have stagnated, with little improvement in survival for the past several decades. Developing new treatments has been hampered by extensive genomic heterogeneity and limited access to patient samples to study the biology of this complex disease., Methods: To overcome these barriers, we combined the power of comparative oncology with patient-derived models of cancer and high-throughput chemical screens in a cross-species drug discovery pipeline., Results: Coupling in vitro high-throughput drug screens on low-passage and established cell lines with in vivo validation in patient-derived xenografts we identify the proteasome and CRM1 nuclear export pathways as therapeutic sensitivities in osteosarcoma, with dual inhibition of these pathways inducing synergistic cytotoxicity., Conclusions: These collective efforts provide an experimental framework and set of new tools for osteosarcoma and other rare cancers to identify and study new therapeutic vulnerabilities.

The estrogen receptor alpha (ERα) is a ligand-activated transcription factor whose activity is modulated by its interaction with multiple protein complexes. In this work, we have identified the protein interferon alpha inducible protein 27 (IFI27/ISG12) as a novel ERα-associated protein. IFI27/ISG12 transcription is regulated by interferon and estradiol and its overexpression is associated to reduced overall survival in ER+ breast cancer patients but its function in mammary gland tissue remains elusive. In this study we showed that overexpression of IFI27/ISG12 in breast cancer cells attenuates ERα transactivation activity and the expression of ERα-dependent genes. Our results demonstrated that IFI27/ISG12 overexpression in MCF-7 cells reduced their proliferation rate in 2-D and 3-D cell culture assays and impaired their ability to migrate in a wound-healing assay. We show that IFI27/ISG12 downregulation of ERα transactivation activity is mediated by its ability to facilitate the interaction between ERα and CRM1/XPO1 that mediates the nuclear export of large macromolecules to the cytoplasm. IFI27/ISG12 overexpression was shown to impair the estradiol-dependent proliferation and tamoxifen-induced apoptosis in breast cancer cells. Our results suggest that IFI27/ISG12 may be an important factor in regulating ERα activity in breast cancer cells by modifying its nuclear versus cytoplasmic protein levels. We propose that IFI27/ISG12 may be a potential target of future strategies to control the growth and proliferation of ERα-positive breast cancer tumors., (Copyright © 2020 Cervantes-Badillo, Paredes-Villa, Gómez-Romero, Cervantes-Roldán, Arias-Romero, Villamar-Cruz, González-Montiel, Barrios-García, Cabrera-Quintero, Rodríguez-Gómez, Cancino-Villeda, Zentella-Dehesa and León-Del-Río.)