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Mammalian oocyte maturation is a unique asymmetric division, which is mainly because of actin-based spindle migration to the cortex. In the present study, we report that a kinesin motor KIFC1, which is associated with microtubules for the maintenance of spindle poles in mitosis, is also involved in actin dynamics in murine oocyte meiosis, co-localizing with microtubules during mouse oocyte maturation. Depletion of KIFC1 caused the failure of polar body extrusion, and we found that meiotic spindle formation and chromosome alignment were disrupted. This might be because of the effects of KIFC1 on HDAC6 and NAT10-based tubulin acetylation, which further affected microtubule stability. Mass spectroscopy analysis revealed that KIFC1 also associated with several actin nucleation factors and we found that KIFC1 was essential for the distribution of actin filaments, which further affected spindle migration. Depletion of KIFC1 leaded to aberrant expression of formin 2 and the ARP2/3 complex, and endoplasmic reticulum distribution was also disturbed. Exogenous KIFC1 mRNA supplement could rescue these defects. Taken together, as well as its roles in tubulin acetylation, our study reported a previously undescribed role of kinesin KIFC1 on the regulation of actin dynamics for spindle migration in mouse oocytes.

HIV-1 Rev mediates the nuclear export of intron-containing viral RNA transcripts and is essential for viral replication. Rev is imported into the nucleus by the host protein Importin β (Impβ), but how Rev associates with Impβ is poorly understood. Here we report biochemical, biophysical and structural studies of the Impβ/Rev complex. Gel shift, native mass spectrometry and isothermal titration calorimetry data reveal that Impβ binds two Rev monomers through independent binding sites. Small-angle X-ray scattering (SAXS) data suggest that the HEAT repeats of Impβ retain an extended conformation upon binding Rev, which according to NMR data is primarily recognized through its helical hairpin domain. Peptide scanning data and charge-reversal mutations identify the N-terminal tip of Rev helix α2 within Rev’s Arginine-Rich Motif (ARM) as a primary Impβ binding epitope. Crosslinking mass spectrometry and compensatory mutagenesis data combined with molecular docking simulations suggest a structural model in which one Rev monomer binds to the C-terminal half of Impβ with Rev helix α2 roughly parallel to the HEAT-repeat superhelical axis while the other monomer binds to the N-terminal half. These findings shed light on the molecular basis of Rev recognition by Impβ and highlight an atypical binding behaviour that distinguishes Rev from canonical cellular Impβ cargos.

Harmful effects of high fructose intake on health have been widely reported. Although fructose is known to promote cancer, little is known about the underlying mechanisms. Here, we found that fructose triggers breast cancer metastasis through the ketohexokinase-A signaling pathway. Molecular experiments showed that ketohexokinase-A, rather than ketohexokinase-C, is necessary and sufficient for fructose-induced cell invasion. Ketohexokinase-A-overexpressing breast cancer was found to be highly metastatic in fructose-fed mice. Mechanistically, cytoplasmic ketohexokinase-A enters into the nucleus during fructose stimulation, which is mediated by LRRC59 and KPNB1. In the nucleus, ketohexokinase-A phosphorylates YWHAH at Ser25 and the YWHAH recruits SLUG to the CDH1 promoter, which triggers cell migration. This study provides the effect of nutrition on breast cancer metastasis. High intake of fructose should be restricted in cancer patients to reduce the risk of metastasis. From a therapeutic perspective, the ketohexokinase-A signaling pathway could be a potential target to prevent cancer metastasis., Apart from the enzymatic role of ketohexokinase isoform A (KHK-A) in fructose metabolism the alternative physiological functions of the enzyme are unclear. Here, the authors show that KHK-A mediates fructose-induced metastasis in breast cancer through a nuclear role in repressing the transcriptional activity of the cell-adhesion molecule CDH1.

LGMD D2 is a disease caused by TNPO3 mutation. We describe the expression of TNPO3 and selected proteins, likely modified by TNPO3 mutation, in muscle biopsies of affected patients. We also aim to find other genes involved in pathways correlated to TNPO3. Our morphological study on LGMD D2 muscle described the expression of TNPO3 and SRSF1, a splicing factor transported by TNPO3. Moreover, we investigated some sarcomeric and nuclear proteins, likely altered by TNPO3 mutation. Through an in silico approach we tried to identify genes involved in pathways that include, besides TNPO3 and SRSF1, p62 and Murf-1, altered in LGMD D2. In patients' muscles TNPO3 appeared weaker and randomly organized, with sporadic cytoplasmic aggregates positive for TNPO3; both SRSF1 and sarcomeric alpha actinin showed a different expression, while there were no alterations in the expression of the nuclear proteins. The in silico study lead to identify five genes, all coding for proteins responsible for muscle contraction. Our data suggest a possible interference in the morphology and function of myofibrillar network by mutated TNPO3; these findings are supported by the in silico identification of genes involved in muscle contraction that could help to explain the pathogenic mechanisms of LGMD D2.

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease characterized by the preferential death of motor neurons. Approximately 10% of ALS cases are familial and 90% are sporadic. Fused in sarcoma (FUS) is a ubiquitously expressed RNA-binding protein implicated in familial ALS and frontotemporal dementia (FTD). The physiological function and pathological mechanism of FUS are not well understood, particularly whether post-translational modifications play a role in regulating FUS function. In this study, we discovered that FUS was acetylated at lysine-315/316 (K315/K316) and lysine-510 (K510) residues in two distinct domains. Located in the nuclear localization sequence, K510 acetylation disrupted the interaction between FUS and Transportin-1, resulting in the mislocalization of FUS in the cytoplasm and formation of stress granule-like inclusions. Located in the RNA recognition motif, K315/K316 acetylation reduced RNA binding to FUS and decreased the formation of cytoplasmic inclusions. Treatment with deacetylase inhibitors also significantly reduced the inclusion formation in cells expressing ALS mutation P525L. More interestingly, familial ALS patient fibroblasts showed higher levels of FUS K510 acetylation as compared with healthy controls. Lastly, CREB-binding protein/p300 acetylated FUS, whereas both sirtuins and histone deacetylases families of lysine deacetylases contributed to FUS deacetylation. These findings demonstrate that FUS acetylation regulates the RNA binding, subcellular localization and inclusion formation of FUS, implicating a potential role of acetylation in the pathophysiological process leading to FUS-mediated ALS/FTD.

Several aggregation-prone RNA-binding proteins, including FUS, EWS, TAF15, hnRNP A1, hnRNP A2, and TDP-43, are mutated in neurodegenerative diseases. The nuclear–cytoplasmic distribution of these proteins is controlled by proteins in the karyopherin family of nuclear transport factors (Kaps). Recent studies have shown that Kaps not only transport these proteins but also inhibit their self-association/aggregation, acting as molecular chaperones. This chaperone activity is impaired for disease-causing mutants of the RNA-binding proteins. Here, we review physical data on the mechanisms of self-association of several disease-associated RNA-binding proteins, through liquid–liquid phase separation and amyloid fiber formation. In each case, we relate these data to biophysical, biochemical, and cell biological data on the inhibition of self-association by Kaps. Our analyses suggest that Kaps may be effective chaperones because they contain large surfaces with diverse physical properties that enable them to engage multiple different regions of their cargo proteins, blocking self-association.

TNF-related apoptosis-inducing ligand (TRAIL) and an agonistic antibody against the death-inducing TRAIL receptor 5, DR5, are thought to selectively induce tumor cell death and therefore, have gained attention as potential therapeutics currently under investigation in several clinical trials. However, some tumor cells are resistant to TRAIL/DR5–induced cell death, even though they express DR5. Previously, we reported that DR5 is transported into the nucleus by importin β1, and knockdown of importin β1 upregulates cell surface expression of DR5 resulting in increased TRAIL sensitivity in vitro. Here, we examined the impact of importin β1 knockdown on agonistic anti-human DR5 (hDR5) antibody therapy. Drug-inducible importin β1 knockdown sensitizes HeLa cells to TRAIL-induced cell death in vitro, and exerts an antitumor effect when combined with agonistic anti-hDR5 antibody administration in vivo. Therapeutic importin β1 knockdown, administered via the atelocollagen delivery system, as well as treatment with the importin β inhibitor, importazole, induced regression and/or eradication of two human TRAIL-resistant tumor cells when combined with agonistic anti-hDR5 antibody treatment. Thus, these findings suggest that the inhibition of importin β1 would be useful to improve the therapeutic effects of agonistic anti-hDR5 antibody against TRAIL-resistant cancers.

Renpenning syndrome belongs to a group of X-linked intellectual disability disorders. The Renpenning syndrome–associated protein PQBP1 (polyglutamine-binding protein 1) is intrinsically disordered, associates with several splicing factors, and is involved in pre-mRNA splicing. PQBP1 uses its C-terminal YxxPxxVL motif for binding to the splicing factor TXNL4A (thioredoxin like 4A), but the biological function of this interaction has yet to be elucidated. In this study, using recombinant protein expression, in vitro binding assays, and immunofluorescence microscopy in HeLa cells, we found that a recently reported X-linked intellectual disability–associated missense mutation, resulting in the PQBP1-P244L variant, disrupts the interaction with TXNL4A. We further show that this interaction is critical for the subcellular location of TXNL4A. In combination with other PQBP1 variants lacking a functional nuclear localization signal required for recognition by the nuclear import receptor karyopherin β2, we demonstrate that PQBP1 facilitates the nuclear import of TXNL4A via a piggyback mechanism. These findings expand our understanding of the molecular basis of the PQBP1–TXNL4A interaction and of the etiology and pathogenesis of Renpenning syndrome and related disorders.

Pomalidomide, a derivative of thalidomide, is an effective treatment for multiple myeloma. The drug exerts its effects through CRBN, a component of the E3 ubiquitin ligase complex CRL4CRBN. To search for novel factors involved in the anti-cancer activity of pomalidomide, we performed a genome-wide shRNA library screen and identified 445 genes as those affecting pomalidomide sensitivity. Genes encoding components of the ubiquitin-proteasome pathway, such as subunits of the CRL4CRBN complex, the COP9 signalosome, and the 26S proteasome, were among the pomalidomide-affecting genes. Karyopherin beta 1 (KPNB1) was identified as a novel pomalidomide-affecting gene. KPNB1 was required for the nuclear import of CRBN and for the CRBN-directed, pomalidomide-dependent degradation of a clinically relevant substrate, the transcription factor Aiolos. By contrast, the cytoplasmic translation factor GSPT1 was degraded following treatment with the thalidomide derivative CC-885 only when CRBN was present in the cytoplasm, indicating that subcellular distribution of CRBN is critical for the efficacy of thalidomide-based medications.

KPNA7 is a member of the Importin-α family of nuclear import receptors. KPNA7 forms a complex with Importin-β and facilitates the translocation of signal-containing proteins from the cytoplasm to the nucleus. Exome sequencing of siblings with severe neurodevelopmental defects and clinical features of epilepsy identified two amino acid-altering mutations in KPNA7. Here, we show that the E344Q substitution reduces KPNA7 binding to nuclear localization signals, and that this limits KPNA7 nuclear import activity. The P339A substitution, by contrast, has little effect on KPNA7 binding to nuclear localization signals. Given the neuronal phenotype described in the two patients, we used SILAC labeling, affinity enrichment, and mass spectrometry to identify KPNA7-interacting proteins in human induced pluripotent stem cell-derived neurons. We identified heterogeneous nuclear ribonucleoproteins hnRNP R and hnRNP U as KPNA7-interacting proteins. The E344Q substitution reduced binding and KPNA7-mediated import of these cargoes. The c.1030G > C allele which generates E344Q is within a predicted CTCF binding site, and we found that it reduces CTCF binding by approximately 40-fold. Our data support a role for altered neuronal expression and activity of KPNA7 in a rare type of pediatric epilepsy.

The interferon-inducible myxovirus resistance B (MxB) protein has been reported to inhibit HIV-1 and herpesviruses by blocking the nuclear import of viral DNA. Here, we report a new antiviral mechanism in which MxB restricts the nuclear import of HIV-1 regulatory protein Rev, and as a result, diminishes Rev-dependent expression of HIV-1 Gag protein. Specifically, MxB disrupts the interaction of Rev with the nuclear transport receptor, transportin 1 (TNPO1). Supporting this, the TNPO1-independent Rev variants become less restricted by MxB. In addition, HIV-1 can overcome this inhibition by MxB through increasing the expression of multiply spliced viral RNA and hence Rev protein. Therefore, MxB exerts its anti-HIV-1 function through interfering with the nuclear import of both viral DNA and viral Rev protein.

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Viral structural proteins can have multiple activities. Antivirals that target structural proteins have potential to exhibit multiple antiviral mechanisms. Hepatitis B virus (HBV) core protein (Cp) is involved in most stages of the viral life cycle; it assembles into capsids, packages viral RNA, is a metabolic compartment for reverse transcription, interacts with nuclear trafficking machinery, and disassembles to release the viral genome into the nucleus. During nuclear localization, HBV capsids bind to host importins (e.g., Impβ) via Cp’s C-terminal domain (CTD); the CTD is localized to the interior of the capsid and is transiently exposed on the exterior. We used HAP12 as a representative Cp allosteric modulator (CpAM), a class of antivirals that inappropriately stimulates and misdirects HBV assembly and deforms capsids. CpAM impact on other aspects of the HBV life cycle is poorly understood. We investigate how HAP12 influences the interactions between empty or RNA-filled capsids with Impβ and trypsin in vitro. We show that HAP12 can modulate CTD accessibility and capsid stability, depending on the saturation of HAP12-binding sites. We demonstrate that Impβ synergistically contributes to capsid disruption at high levels of HAP12 saturation, using electron microscopy to visualize the disruption and rearrangement of Cp dimers into aberrant complexes. However, RNA-filled capsids resist the destabilizing effects of HAP12 and Impβ. In summary, we show host protein-induced catalysis of capsid disruption, an unexpected additional mechanism of action for CpAMs. Potentially, untimely capsid disassembly can hamper the HBV life cycle and also cause the virus to become vulnerable to host innate immune responses. IMPORTANCE The HBV core, an icosahedral complex of 120 copies of the homodimeric core (capsid) protein with or without packaged nucleic acid, is transported to the host nucleus by its interaction with host importin proteins. Importin-core interaction requires the core protein C-terminal domain, which is inside the capsid, to “flip” to the capsid exterior. Core protein-directed drugs that affect capsid assembly and stability have been developed recently. We show that these molecules can, synergistically with importins, disrupt capsids. This mechanism of action, synergism with host protein, has the potential to disrupt the virus life cycle and activate the innate immune system.

Efficient and regulated nucleocytoplasmic trafficking of macromolecules to the correct subcellular compartment is critical for proper functions of the eukaryotic cell. The majority of the macromolecular traffic across the nuclear pores is mediated by the Karyopherin-β (or Kap) family of nuclear transport receptors. Work over more than two decades has shed considerable light on how the different Kap family members bring their respective cargoes into the nucleus or the cytoplasm in efficient and highly regulated manners. In this Review, we overview the main features and established functions of Kap family members, describe how Kaps recognize their cargoes and discuss the different ways in which these Kap-cargo interactions can be regulated, highlighting new findings and open questions. We also describe current knowledge of the import and export of the components of three large gene expression machines - the core replisome, RNA polymerase II and the ribosome - pointing out the questions that persist about how such large macromolecular complexes are trafficked to serve their function in a designated subcellular location.

The mating-specific G(alpha) protein of Saccharomyces cerevisiae, Gpa1, stimulates adaptation to pheromone by a mechanism independent of G(beta gamma) sequestration. Genetic evidence suggests that Gpa1 targets the Fus3 mitogen-activated protein kinase, and it has recently been shown that the two proteins interact in cells responding to pheromone. To test the possibility that Gpa1 downregulates the mating signal by affecting the localization of Fus3, we created a Fus3-green fluorescent protein (GFP) fusion protein. In vegetative cells, Fus3-GFP was found in both the cytoplasm and the nucleus. Pheromone stimulated a measurable increase in the ratio of nuclear to cytoplasmic Fus3-GFP. In contrast, the relative level of nuclear Fus3-GFP decreased as cells recovered from pheromone arrest and did not increase when cells adapted to chronic stimulus were challenged again. Accumulation of Fus3-GFP in the nuclei of stimulated cells was also inhibited by overexpression of either wild-type Gpa1, the E364K hyperadaptive mutant form of Gpa1, or the Msg5 dually specific phosphatase. The effects of Gpa1 and Msg5 on Fus3 are partially interdependent. In a genetic screen for adaptive defective mutants, a nonsense allele of the nucleocytoplasmic transport receptor, Kap104, was identified. Truncation of the Kap104 cargo-binding domain blocked the effect of both Gpa1(E364K) and Msg5 on Fus3-GFP localization. Based on these results, we propose that Gpa1 and Msg5 work in concert to downregulate the mating signal and that they do so by inhibiting the pheromone-induced increase of Fus3 in the nucleus. Kap104 is required for the G(alpha)/phosphatase-mediated effect on Fus3 localization.

Summary: Genomic amplification of 3q26.2 locus leads to the increased expression of microRNA 551b-3p (miR551b-3p) in triple-negative breast cancer (TNBC). Our results demonstrate that miR551b-3p translocates to the nucleus with the aid of importin-8 (IPO8) and activates STAT3 transcription. As a consequence, miR551b upregulates the expression of oncostatin M receptor (OSMR) and interleukin-31 receptor-α (IL-31RA) as well as their ligands OSM and IL-31 through STAT3 transcription. We defined this set of genes induced by miR551b-3p as the “oncostatin signaling module,” which provides oncogenic addictions in cancer cells. Notably, OSM is highly expressed in TNBC, and the elevated expression of OSM associates with poor outcome in estrogen-receptor-negative breast cancer patients. Conversely, targeting miR551b with anti-miR551b-3p reduced the expression of the OSM signaling module and reduced tumor growth, as well as migration and invasion of breast cancer cells. : Parashar et al. demonstrate that the mature microRNA-551b-3p translocates to the nucleus for transcriptional activation of STAT3 gene. As a consequence, miR551b activates an autocrine signaling loop through the upregulation of oncostatin family genes, including oncostatin, and its receptors for the growth and metastasis of triple-negative breast cancer. Keywords: miR551b, oncostatin, STAT3, OSMR, miRNA-therapy, IPO8, RNAi

The genotype-first approach has been successfully applied and has elucidated several subtypes of autism spectrum disorder (ASD). However, it requires very large cohorts because of the extensive genetic heterogeneity. We investigate the alternate possibility of whether phenotype-specific genes can be identified from a small group of patients with specific phenotype(s). To identify novel genes associated with ASD and abnormal head circumference using a phenotype-to-genotype approach, we performed whole-exome sequencing on 67 families with ASD and abnormal head circumference. Clinically relevant pathogenic or likely pathogenic variants account for 23.9% of patients with microcephaly or macrocephaly, and 81.25% of those variants or genes are head-size associated. Significantly, recurrent pathogenic mutations were identified in two macrocephaly genes (PTEN, CHD8) in this small cohort. De novo mutations in several candidate genes (UBN2, BIRC6, SYNE1, and KCNMA1) were detected, as well as one new candidate gene (TNPO3,) implicated in ASD and related neurodevelopmental disorders. We identify genotype–phenotype correlations for head-size-associated ASD genes and novel candidate genes for further investigation. Our results also suggest a phenotype-to-genotype strategy would accelerate the elucidation of genotype–phenotype relationships for ASD by using phenotype-restricted cohorts.

Importin α has been described as a nuclear protein transport receptor that enables proteins synthesized in the cytoplasm to translocate into the nucleus. Besides its function in nuclear transport, an increasing number of studies have examined its non-nuclear transport functions. In both nuclear transport and non-nuclear transport, a functional domain called the IBB domain (importin β binding domain) plays a key role in regulating importin α behavior, and is a common interacting domain for multiple binding partners. However, it is not yet fully understood how the IBB domain interacts with multiple binding partners, which leads to the switching of importin α function. In this study, we have distinguished the location and propensities of amino acids important for each function of the importin α IBB domain by mapping the biochemical/physicochemical propensities of evolutionarily conserved amino acids of the IBB domain onto the structure associated with each function. We found important residues that are universally conserved for IBB functions across organisms and families, in addition to those previously known, as well as residues that are presumed to be responsible for the differences in complex-forming ability between families and for functional switching.

Cytoplasmic mislocalization of the TAR-DNA binding protein of 43 kDa (TDP-43) leads to large, insoluble aggregates that are a hallmark of amyotrophic lateral sclerosis and frontotemporal dementia. Here, we study how importin α1/β recognizes TDP-43 bipartite nuclear localization signal (NLS). We find that the NLS makes extensive contacts with importin α1, especially at the minor NLS-binding site. NLS binding results in steric clashes with the C terminus of importin α1 that disrupts the TDP-43 N-terminal domain (NTD) dimerization interface. A putative phosphorylation site in the proximity of TDP-43 R83 at the minor NLS site destabilizes binding to importins by reducing the NLS backbone dynamics. Based on these data, we explain the pathogenic role of several post-translational modifications and mutations in the proximity of TDP-43 minor NLS site that are linked to disease and shed light on the chaperone activity of importin α1/β.

Importin β1 (KPNB1) is a nucleocytoplasmic transport factor with critical roles in both cytoplasmic and nucleocytoplasmic transport, hence there is keen interest in the characterization of its subcellular interactomes. We found limited efficiency of BioID in the detection of importin complex cargos and therefore generated a highly specific and sensitive anti-KPNB1 monoclonal antibody to enable biotinylation by antibody recognition analysis of importin β1 interactomes. The monoclonal antibody recognizes an epitope comprising residues 301-320 of human KPBN1 and strikingly is highly specific for cytoplasmic KPNB1 in diverse applications, with little reaction with KPNB1 in the nucleus. Biotinylation by antibody recognition with this novel antibody revealed numerous new interactors of importin β1, expanding the KPNB1 interactome to cytoplasmic and signaling complexes that highlight potential new functions for the importins complex beyond nucleocytoplasmic transport. Data are available via ProteomeXchange with identifier PXD032728.

Background Hepatocellular carcinoma (HCC) is one of the most intractable tumors in the world due to its high rate of recurrence and heterogeneity. Liver cancer initiating cells also called cancer stem cells (CSCs) play a critical role in resistance against typical therapy and high tumor-initiating potential. However, the role of the novel circular RNA (circRNA) circIPO11 in the maintenance of liver cancer initiating cells remains elusive. Methods CircRNAs highly conserved in humans and mice were identified from 3 primary HCC samples by circRNA array. The expression and function of circIPO11 were further evaluated by Northern blot, limiting dilution xenograft analysis, chromatin isolation by RNA purification-PCR assay (ChIRP) and HCC patient-derived tumor cells (PDC) models. CircIpo11 knockout (KO) mice were generated by a CRISPR/Cas9 technology. Results CircIPO11 is highly expressed in HCC tumor tissues and liver CSCs. CircIPO11 is required for the self-renewal maintenance of liver CSCs to initiate HCC development. Mechanistically, circIPO11 recruits TOP1 to GLI1 promoter to trigger its transcription, leading to the activation of Hedgehog signaling. Moreover, GLI1 is also highly expressed in HCC tumor tissues and liver CSCs, and TOP1 expression levels positively correlate with the metastasis, recurrence and survival of HCC patients. Additionally, circIPO11 knockout in mice suppresses the progression of chemically induced liver cancer development. Conclusion Our findings reveal that circIPO11 drives the self-renewal of liver CSCs and promotes the propagation of HCC via activating Hedgehog signaling pathway. Antisense oligonucleotides (ASOs) against circIPO11 combined with TOP1 inhibitor camptothecin (CPT) exert synergistic antitumor effect. Therefore, circIPO11 and the Hedgehog signaling pathway may provide new potential targets for the treatment of HCC patients.

To infect nondividing cells, HIV-1 needs to cross the nuclear membrane. The importin transportin-SR2 (TRN-SR2 or transportin-3) has been proposed to mediate HIV-1 nuclear import, but the detailed mechanism remains unresolved. The direct interaction of TRN-SR2 with HIV-1 integrase (IN) has been proposed to drive HIV-1 nuclear import. Alternatively, TRN-SR2 may play an indirect role by mediating nuclear import of cleavage and polyadenylation specificity factor 6 (CPSF6). To unravel the role of TRN-SR2, we designed CRISPR/Cas9 guide RNAs targeting different exons of TNPO3. Although this approach failed to generate full knockouts, monoallelic knockout clones were generated with indel mutations. HIV-1 replication was hampered in those clones at the level of HIV-1 nuclear import without an effect on the cellular distribution of the TRN-SR2 cargoes CPSF6 or alternative splicing factor1/pre-mRNA splicing factor SF2 (ASF/SF2). Recombinant ΔV105 TRN-SR2 expressed in clone 15.15 was 2-fold impaired for interaction with HIV-1 IN and classified as an interaction mutant. Our data support a model whereby TRN-SR2 acts as a cofactor of HIV-1 nuclear import without compromising the nuclear import of cellular cargoes. CRISPR/Cas9-induced mutagenesis can be used as a method to generate interface mutants to characterize host factors of human pathogens. IMPORTANCE Combination antiretroviral therapy (cART) effectively controls HIV-1 by reducing viral loads, but it does not cure the infection. Lifelong treatment with cART is a prerequisite for sustained viral suppression. The rapid emergence of drug-resistant viral strains drives the necessity to discover new therapeutic targets. The nuclear import of HIV-1 is crucial in the HIV-1 replication cycle, but the detailed mechanism remains incompletely understood. This study provides evidence that TRN-SR2 directly mediates HIV-1 nuclear import via the interaction with HIV-1 integrase. The interaction between those proteins is therefore a promising target toward a rational drug design which could lead to new therapeutic strategies due to the bottleneck nature of HIV-1 nuclear import. ispartof: MICROBIOLOGY SPECTRUM vol:9 issue:2 ispartof: location:United States status: published

Limb-girdle muscular dystrophy D2 (LGMDD2) is an ultrarare autosomal dominant myopathy caused by mutation of the normal stop codon of the TNPO3 nuclear importin. The mutant protein carries a 15 amino acid C-terminal extension associated with pathogenicity. Here we report the first animal model of the disease by expressing the human mutant TNPO3 gene in Drosophila musculature or motor neurons and concomitantly silencing the endogenous expression of the fly protein ortholog. A similar genotype expressing wildtype TNPO3 served as a control. Phenotypes characterization revealed that mutant TNPO3 expression targeted at muscles or motor neurons caused LGMDD2-like phenotypes such as muscle degeneration and atrophy, and reduced locomotor ability. Notably, LGMDD2 mutation increase TNPO3 at the transcript and protein level in the Drosophila model Upregulated muscle autophagy observed in LGMDD2 patients was also confirmed in the fly model, in which the anti-autophagic drug chloroquine was able to rescue histologic and functional phenotypes. Overall, we provide a proof of concept of autophagy as a target to treat disease phenotypes and propose a neurogenic component to explain mutant TNPO3 pathogenicity in diseased muscles.

Nuclear import receptors (NIRs) not only transport RNA-binding proteins (RBPs) but also modify phase transitions of RBPs by recognizing nuclear localization signals (NLSs). Toxic arginine-rich poly-dipeptides from C9orf72 interact with NIRs and cause nucleocytoplasmic transport deficit. However, the molecular basis for the toxicity of arginine-rich poly-dipeptides toward NIRs function as phase modifiers of RBPs remains unidentified. Here we show that arginine-rich poly-dipeptides impede the ability of NIRs to modify phase transitions of RBPs. Isothermal titration calorimetry and size-exclusion chromatography revealed that proline:arginine (PR) poly-dipeptides tightly bind karyopherin-β2 (Kapβ2) at 1:1 ratio. The nuclear magnetic resonances of Kapβ2 perturbed by PR poly-dipeptides partially overlapped with those perturbed by the designed NLS peptide, suggesting that PR poly-dipeptides target the NLS binding site of Kapβ2. The findings offer mechanistic insights into how phase transitions of RBPs are disabled in C9orf72-related neurodegeneration., Nuclear import receptors (NIRs) regulate self-association of RNA-binding proteins as phase modifiers, while C9orf72-derived arginine-rich polydipeptides lead to aberrant phase transitions. Here the authors show in molecular basis how arginine-rich poly-dipeptides impede the ability of NIRs, particularly Kapβ2.

Kinesin-14 KIFC1 regulates spindle assembly and centrosome clustering in diverse organisms during cell division. KIFC1 proteins are essential for spindle assembly and chromosome alignment in mitosis. However, the roles and mechanisms of KIFC1 proteins in male spermatocytes remain largely unknown. In this study, we reveal that KIFC1 proteins mainly accumulate at the centrosomes and central spindle in mouse spermatocytes both in vitro and in vivo. We utilize two KIFC1 specific inhibitors, AZ82 and CW069, for the inhibition of KIFC1 in mouse spermatogenic cells and cultured GC-2 spd(ts) cells. We find that KIFC1 inhibition results in the increase of spermatocytes with micronuclei, the disorganization of the meiotic spindles, and the formation of multiple centrosomes. Furthermore, we demonstrate that KIFC1 inhibition leads to spindle defects, chromosome misalignment and the formation of aneuploidy in cultured GC-2 spd(ts) cells. In this study, we reveal that KIFC1 proteins are critical for centrosome maintenance and chromosome stability in mouse spermatocytes.

Withaferin A, a natural steroidal lactone found in the extracts of Withania somnifera, is used extensively in traditional medicine and part of an ancient remedy in ayurvedic medicine. Prior investigations into its mode of action have shown withaferin to be a polyfunctional pharmacophore with the covalent engagement of a multitude of therapeutic targets. Herein, we report that withaferin A is also a covalent inhibitor of IPO5, an importin that translocates cargos from the cytosol to the nucleus. We show that withaferin inhibits influenza A replication in epithelial cells (A549). Using a panel of inhibitors that selectively recapitulate part of withaferin A’s pharmacological profile (goyazensolide, withaferin A derivatives, FiVe1, and bardoxolone methyl), we show that IPO5 inhibition contributes to the influenza replication inhibition but is not essential for the observed activity of withaferin A. We show that bardoxolone methyl, a semisynthetic triterpenoid in clinical development to treat chronic kidney disease and that shares some of the pharmacological profile of withaferin, also inhibits influenza A replication effectively. The inhibitory activity against influenza A replication should stimulate further studies to repurpose this therapeutic.

The microtubule-based mitotic spindle is responsible for equally partitioning the genome during each cell division, and its assembly is executed via several microtubule nucleation pathways. Targeting Protein for XKlp2 (TPX2) stimulates the branching microtubule nucleation pathway, where new microtubules are nucleated from preexisting ones within mitotic or meiotic spindles. TPX2, like other spindle assembly factors, is sequestered by binding to nuclear importins-α/β until the onset of mitosis, yet the molecular nature of this regulation remains unclear. Here we demonstrate that TPX2 interacts with importins-α/β with nanomolar affinity in a 1:1:1 monodispersed trimer. We also identify a new nuclear localization sequence in TPX2 that contributes to its high-affinity interaction with importin-α. In addition, we establish that TPX2 interacts with importin-β via dispersed, weak interactions. We show that interactions of both importin-α and -β with TPX2 inhibit its ability to undergo phase separation, which was recently shown to enhance the kinetics of branching microtubule nucleation. In summary, our study informs how importins regulate TPX2 to facilitate spindle assembly, and provides novel insight into the functional regulation of protein phase separation.

RNA abundance is generally sensitive to perturbations in decay and synthesis rates, but crosstalk between RNA polymerase II transcription and cytoplasmic mRNA degradation often leads to compensatory changes in gene expression. Here, we reveal that widespread mRNA decay during early apoptosis represses RNAPII transcription, indicative of positive (rather than compensatory) feedback. This repression requires active cytoplasmic mRNA degradation, which leads to impaired recruitment of components of the transcription preinitiation complex to promoter DNA. Importin α/β- mediated nuclear import is critical for this feedback signaling, suggesting that proteins translocating between the cytoplasm and nucleus connect mRNA decay to transcription. We also show that an analogous pathway activated by viral nucleases similarly depends on nuclear protein import. Collectively, these data demonstrate that accelerated mRNA decay leads to the repression of mRNA transcription, thereby amplifying the shutdown of gene expression. This highlights a conserved gene regulatory mechanism by which cells respond to threats.IMPACT STATEMENTHuman cells respond to cytoplasmic mRNA depletion during early apoptosis by inhibiting RNA polymerase II transcription, thereby magnifying the gene expression shutdown during stress.

International audience; Dysregulated transforming growth factor TGF-beta signaling underlies the pathogenesis of genetic disorders affecting the connective tissue such as Loeys-Dietz syndrome. Here, we report 12 individuals with bi-allelic loss-of-function variants in IPO8 who presented with a syndromic association characterized by cardio-vascular anomalies, joint hyperlaxity, and various degree of dysmorphic features and developmental delay as well as immune dysregulation; the individuals were from nine unrelated families. Importin 8 belongs to the karyopherin family of nuclear transport receptors and was previously shown to mediate TGF-beta-dependent SMADs trafficking to the nucleus in vitro. The important in vivo role of IPO8 in pSMAD nuclear translocation was demonstrated by CRISPR/Cas9-mediated inactivation in zebrafish. Consistent with IPO8's role in BMP/TGF-beta signaling, ipo8(-/-) zebrafish presented mild to severe dorso-ventral patterning defects during early embryonic development. Moreover, ipo8(-/-) zebrafish displayed severe cardiovascular and skeletal defects that mirrored the human phenotype. Our work thus provides evidence that IPO8 plays a critical and non-redundant role in TGF-beta signaling during development and reinforces the existing link between TGF-beta signaling and connective tissue defects.

Nuclear translocation of the p50/p65 heterodimer is essential for NF-κB signaling. In unstimulated cells, p50/p65 is retained by the inhibitor IκBα in the cytoplasm that masks the p65-nuclear localization sequence (NLS). Upon activation, p50/p65 is translocated into the nucleus by the adapter importin α3 and the receptor importin β. Here, we describe a bipartite NLS in p50/p65, analogous to nucleoplasmin NLS but exposed in trans. Importin α3 accommodates the p50- and p65-NLSs at the major and minor NLS-binding pockets, respectively. The p50-NLS is the predominant binding determinant, while the p65-NLS induces a conformational change in the Armadillo 7 of importin α3 that stabilizes a helical conformation of the p65-NLS. Neither conformational change was observed for importin α1, which makes fewer bonds with the p50/p65 NLSs, explaining the preference for α3. We propose that importin α3 discriminates between the transcriptionally active p50/p65 heterodimer and p50/p50 and p65/65 homodimers, ensuring fidelity in NF-κB signaling.

The HIV replication cycle depends on the interaction of viral proteins with proteins of the host. Unraveling host-pathogen interactions during the infection is of great importance for understanding the pathogenesis and the development of antiviral therapies. To date HIV uncoating and nuclear import are the most debated steps of the HIV-1 replication cycle. Despite numerous studies during past decades, there is still much controversy with respect to the identity and the role of viral and host factors involved in these processes. In this review, we provide a comprehensive overview on the role of transportin-SR2 as a host cell factor during active nuclear transport. ispartof: VIRUSES-BASEL vol:13 issue:5 ispartof: location:Switzerland status: published

The nuclear transport receptor importin-beta/karyopherin-beta1 is overexpressed in cancers that display genomic instability. It is regarded as a promising cancer target and inhibitors are being developed. In addition to its role in nucleo-cytoplasmic transport, importin-beta regulates mitosis, but the programmes and pathways in which it operates are defined only in part. To unravel importin-beta's mitotic functions we have developed cell lines expressing either wild-type or a mutant importin-beta form in characterised residues required for nucleoporin binding. Both forms similarly disrupted spindle pole organisation, while only wild-type importin-beta affected microtubule plus-end function and microtubule stability. A proteome-wide search for differential interactors identified a set of spindle regulators sensitive to mutations in the nucleoporin-binding region. Among those, HURP (hepatoma up-regulated protein) is an importin-beta interactor and a microtubule-stabilising factor. We found that induction of wild type, but not mutant importin-beta, under the same conditions that destabilise mitotic microtubules, delocalised HURP, indicating that the spatial distribution of HURP along the spindle requires importin-beta's nucleoporin-binding residues. Concomitantly, importin-beta overexpression sensitises cells to taxanes and synergistically increases mitotic cell death. Thus, the nucleoporin-binding domain is dispensable for importin-beta function in spindle pole organisation, but regulates microtubule stability, at least in part via HURP, and renders cells vulnerable to certain microtubule-targeting drugs.

Endotoxin shock is induced by LPS, one of the most potent virulence factors of the Gram-negative bacteria that cause sepsis. It remains unknown if either proinflammatory stress-responsive transcription factors (SRTFs), ferried to nucleus by importin α5, or lipid-regulating sterol regulatory element binding proteins (SREBPs), transported to the nucleus by importin β1, mediate endotoxin shock. A novel cell-penetrating peptide targeting importin α5 while sparing importin β1 protected 80% of animals from death in response to a high dose of LPS. This peptide suppresses inflammatory mediators, liver glycogen depletion, endothelial injury, neutrophil trafficking, and apoptosis caused by LPS. In d-galactosamine–pretreated mice challenged by 700-times lower dose of LPS, rapid death through massive apoptosis and hemorrhagic necrosis of the liver was also averted by the importin α5–selective peptide. Thus, using a new tool for selective suppression of nuclear transport, we demonstrate that SRTFs, rather than SREBPs, mediate endotoxin shock.

Transfer RNAs (tRNAs) are central players in protein synthesis, which in Eukarya need to be delivered from the nucleus to the cytoplasm by specific transport receptors, most of which belong to the evolutionarily conserved beta-importin family. Based on the available literature, we identified two candidates, Xpo-t and Xpo-5 for tRNA export in Trypanosoma brucei. However, down-regulation of expression of these genes did not disrupt the export of tRNAs to the cytoplasm. In search of alternative pathways, we tested the mRNA export complex Mex67-Mtr2, for a role in tRNA nuclear export, as described previously in yeast. Down-regulation of either exporter affected the subcellular distribution of tRNAs. However, contrary to yeast, TbMex67 and TbMtr2 accumulated different subsets of tRNAs in the nucleus. While TbMtr2 perturbed the export of all the tRNAs tested, silencing of TbMex67, led to the nuclear accumulation of tRNAs that are typically modified with queuosine. In turn, inhibition of tRNA nuclear export also affected the levels of queuosine modification in tRNAs. Taken together, the results presented demonstrate the dynamic nature of tRNA trafficking in T. brucei and its potential impact not only on the availability of tRNAs for protein synthesis but also on their modification status.

Background Karyopherin nuclear transport receptors play important roles in tumour development and drug resistance and have been reported as potential biomarkers and therapeutic targets for tumour treatment. However, IPO5, one of the karyopherin nuclear transport receptor family members, remains largely uncharacterized in tumour progression. Methods The TCGA data, quantitative reverse transcription-PCR (qRT-PCR), western blotting, and IHC analyses were used to detect IPO5 expression in CRC tissues. A series of in vivo and in vitro experiments was utilized to demonstrate the function of IPO5 in CRC tissues. Mass spectrometry (MS), CO-IP technology, subcellular fractionation, and immunofluorescence were utilized to investigate the possible mechanisms of CRC. Results IPO5 was highly expressed and positively correlated with the clinicopathological characteristics of colorectal cancer tissues. Functional experiments indicated that IPO5 could promote the development of CRC. Mechanistically, we screened RASAL2, one cargo of IPO5, and further confirmed that IPO5 bound to the NLS sequence of RASAL2, mediating RASAL2 nuclear translocation and inducing RAS signal activation, thereby promoting the progression of CRC. Conclusions Together, our results indicate that IPO5 is overexpressed in colorectal cancer cells. By transporting RASAL2, IPO5 may play a crucial role in CRC. Electronic supplementary material The online version of this article (10.1186/s13046-019-1290-0) contains supplementary material, which is available to authorized users.

Serine–arginine (SR) proteins are essential splicing factors that promote numerous steps associated with mRNA processing and whose biological function is tightly regulated through multi-site phosphorylation. In the nucleus, the cdc2-like kinases (CLKs) phosphorylate SR proteins on their intrinsically disordered Arg–Ser (RS) domains, mobilizing them from storage speckles to the splicing machinery. The CLKs have disordered N termini that bind tightly to RS domains, enhancing SR protein phosphorylation. The N termini also promote nuclear localization of CLKs, but their transport mechanism is presently unknown. To explore cytoplasmic–nuclear transitions, several classical nuclear localization sequences in the N terminus of the CLK1 isoform were identified, but their mutation had no effect on subcellular localization. Rather, we found that CLK1 amplifies its presence in the nucleus by forming a stable complex with the SR protein substrate and appropriating its NLS for transport. These findings indicate that, along with their well-established roles in mRNA splicing, SR proteins use disordered protein–protein interactions to carry their kinase regulator from the cytoplasm to the nucleus.

The authors describe a system for engineering proteases. They evolved SUMOEu/protease pairs that are compatible with eukaryotic protein expression and used SUMOEu-assisted affinity chromatography to identify Ubc9 as a nuclear import cargo and eIF5A as well as eEF2 as export substrates of Pdr6., Cleavage of affinity tags by specific proteases can be exploited for highly selective affinity chromatography. The SUMO/SENP1 system is the most efficient for such application but fails in eukaryotic expression because it cross-reacts with endogenous proteases. Using a novel selection system, we have evolved the SUMOEu/SENP1Eu pair to orthogonality with the yeast and animal enzymes. SUMOEu fusions therefore remain stable in eukaryotic cells. Likewise, overexpressing a SENP1Eu protease is nontoxic in yeast. We have used the SUMOEu system in an affinity-capture-proteolytic-release approach to identify interactors of the yeast importin Pdr6/Kap122. This revealed not only further nuclear import substrates such as Ubc9, but also Pil1, Lsp1, eIF5A, and eEF2 as RanGTP-dependent binders and thus as export cargoes. We confirmed that Pdr6 functions as an exportin in vivo and depletes eIF5A and eEF2 from cell nuclei. Thus, Pdr6 is a bidirectional nuclear transport receptor (i.e., a biportin) that shuttles distinct sets of cargoes in opposite directions.

Glioblastoma harbors frequent alterations in receptor tyrosine kinases, phosphatidylinositol-3 kinase (PI3K) and phosphatase and tensin homolog (PTEN) that dysregulate phospholipid signaling driven tumor proliferation and therapeutic resistance. Myristoylated alanine-rich C-kinase substrate (MARCKS) is a 32 kDa intrinsically unstructured protein containing a polybasic (+13) effector domain (ED), which regulates its electrostatic sequestration of phospholipid phosphatidylinositol (4,5)-bisphosphate (PIP2), and its binding to phosphatidylserine, calcium/calmodulin, filamentous actin, while also serving as a nuclear localization sequence. MARCKS ED is phosphorylated by protein kinase C (PKC) and Rho-associated protein kinase (ROCK) kinases; however, the impact of MARCKS on glioblastoma growth and radiation sensitivity remains undetermined. In the present study, using a tetracycline-inducible system in PTEN-null U87 cells, we demonstrate that MARCKS overexpression suppresses growth and enhances radiation sensitivity in vivo. A new image cytometer, Xcyto10, was utilized to quantify differences in MARCKS ED phosphorylation on localization and its association with filamentous actin. The overexpression of the non-phosphorylatable ED mutant exerted growth-suppressive and radiation-sensitizing effects, while the pseudo-phosphorylated ED mutant exhibited an enhanced colony formation and clonogenic survival ability. The identification of MARCKS protein-protein interactions using co-immunoprecipitation coupled with tandem mass spectrometry revealed novel MARCKS-associated proteins, including importin-β and ku70. On the whole, the findings of this study suggest that the determination of the MARCKS ED phosphorylation status is essential to understanding the impact of MARCKS on cancer progression.

Accumulating evidences indicates that chronic neuropathic pain is a kind of neuro-immune disorder with enhanced activation of the immune system. Although the prevalence is very high, neuropathic pain remains extremely difficult to cure. miRNAs are a group of short nonprotein coding RNAs, regulating target genes expression via targeting 3'-untranslated region. More and more research indicates that altered miRNAs expression profile relates to the pathogenesis of neuropathic pain. In this study, we firstly detected the expression of six candidate miRNAs in the plasma samples from 23 patients with neuropathic pain and 10 healthy controls. Subsequently, the level of miR-132 and miR-101 was detected in the sural nerve biopsies. We found miR-101 level was significantly repressed in both the plasma samples and sural nerve biopsies from neuropathic pain patients. Predicted by bioinformatics tools and confirmed by dual luciferase assay and immunoblotting, we identified that KPNB1 is a direct target of miR-101. The negative correlation between miR-101 and KPNB1 was also confirmed in the sural nerve biopsies, and miR-101 reduction relates to the activation of NF-κB signaling in vivo and in vitro which contributes to the pathogenesis of neuropathic pain.

The recognition of intrinsically disordered proteins (IDPs) is highly dependent on dynamics owing to the lack of structure. Here we studied the interplay between dynamics and molecular recognition in IDPs with a combination of time‐resolving tools on timescales ranging from femtoseconds to nanoseconds. We interrogated conformational dynamics and surface water dynamics and its attenuation upon partner binding using two IDPs, IBB and Nup153FG, both of central relevance to the nucleocytoplasmic transport machinery. These proteins bind the same nuclear transport receptor (Importinβ) with drastically different binding mechanisms, coupled folding–binding and fuzzy complex formation, respectively. Solvent fluctuations in the dynamic interface of the Nup153FG‐Importinβ fuzzy complex were largely unperturbed and slightly accelerated relative to the unbound state. In the IBB‐Importinβ complex, on the other hand, substantial relative slowdown of water dynamics was seen in a more rigid interface. These results show a correlation between interfacial water dynamics and the plasticity of IDP complexes, implicating functional relevance for such differential modulation in cellular processes, including nuclear transport.

KIFC1 (also called HSET or kinesin-14a) is best known as a multifunctional motor protein essential for mitosis. The present studies are the first to explore KIFC1 in terminally postmitotic neurons. Using RNA interference to partially deplete KIFC1 from rat neurons (from animals of either gender) in culture, pharmacologic agents that inhibit KIFC1, and expression of mutant KIFC1 constructs, we demonstrate critical roles for KIFC1 in regulating axonal growth and retraction as well as growth cone morphology. Experimental manipulations of KIFC1 elicit morphological changes in the axon as well as changes in the organization, distribution, and polarity orientation of its microtubules. Together, the results indicate a mechanism by which KIFC1 binds to microtubules in the axon and slides them into alignment in an ATP-dependent fashion and then cross-links them in an ATP-independent fashion to oppose their subsequent sliding by other motors.SIGNIFICANCE STATEMENTHere, we establish that KIFC1, a molecular motor well characterized in mitosis, is robustly expressed in neurons, where it has profound influence on the organization of microtubules in a number of different functional contexts. KIFC1 may help answer long-standing questions in cellular neuroscience such as, mechanistically, how growth cones stall and how axonal microtubules resist forces that would otherwise cause the axon to retract. Knowledge about KIFC1 may help researchers to devise strategies for treating disorders of the nervous system involving axonal retraction given that KIFC1 is expressed in adult neurons as well as developing neurons.

Prostate cancer (PCa) initiation and progression requires activation of numerous oncogenic signaling pathways. Nuclear-cytoplasmic transport of oncogenic factors is mediated by Karyopherin proteins during cell transformation. However, the role of nuclear transporter proteins in PCa progression has not been well defined. Here, we report that the KPNB1, a key member of Karyopherin beta subunits, is highly expressed in advanced prostate cancers. Further study showed that targeting KPNB1 suppressed the proliferation of prostate cancer cells. The knockdown of KPNB1 reduced nuclear translocation of c-Myc, the expression of downstream cell cycle modulators, and phosphorylation of regulator of chromatin condensation 1 (RCC1), a key protein for spindle assembly during mitosis. Meanwhile, CHIP assay demonstrated the binding of c-Myc to KPNB1 promoter region, which indicated a positive feedback regulation of KPNB1 expression mediated by the c-Myc. In addition, NF-κB subunit p50 translocation to nuclei was blocked by KPNB1 inhibition, which led to an increase in apoptosis and a decrease in tumor sphere formation of PCa cells. Furthermore, subcutaneous xenograft tumor models with a stable knockdown of KPNB1 in C42B PCa cells validated that the inhibition of KPNB1 could suppress the growth of prostate tumor in vivo. Moreover, the intravenously administration of importazole, a specific inhibitor for KPNB1, effectively reduced PCa tumor size and weight in mice inoculated with PC3 PCa cells. In summary, our data established the functional link between KPNB1 and PCa prone c-Myc, NF-kB, and cell cycle modulators. More importantly, inhibition of KPNB1 could be a new therapeutic target for PCa treatment.

CD44 is one of biomarkers of liver cancer stem cells (CSCs). The investigation of mechanism of CD44 translocation helps to uncover new molecular pathways participated in the regulation of various cellular processes in CSCs. In the present study, we observed the translocation of CD44 from cytoplasm to nuclear in the reprogramming process of C3A cells, full-length CD44 presented in the nucleus of liver iCSCs. CD44 was bound with importin β and transportin 1 in liver iCSCs. Inhibition of importin β transport leads to reduction of CD44 in the nucleus. Translocation of CD44 is also influenced by importin α. Besides, overexpression of naïve pluripotent genes, KLF2, KLF5, DNMT3L, GBX2, ZFP42, ESRRB and DPPA4 were found in liver iCSCs. Inhibition of CD44 leads to the reduction of these naïve genes. Luciferase and chromatin immunoprecipitation (ChIP) assays further identified nuclear CD44 bound to the promoter regions of naïve genes, KLF2, KLF5, and ESRRB functioned as transcriptional activators in liver iCSCs. Our present work provides new insight into the dynamic states and functions of CD44 in iCSCs.

The SARS-CoV-2 pandemic has accelerated the study of existing drugs. The mixture of homologs called ivermectin (avermectin-B1a [HB1a] + avermectin-B1b [HB1b]) has shown antiviral activity against SARS-CoV-2 in vitro. However, there are few reports on the behavior of each homolog. We investigated the interaction of each homolog with promising targets of interest associated with SARS-CoV-2 infection from a biophysical and computational-chemistry perspective using docking and molecular dynamics. We observed a differential behavior for each homolog, with an affinity of HB1b for viral structures, and of HB1a for host structures considered. The induced disturbances were differential and influenced by the hydrophobicity of each homolog and of the binding pockets. We present the first comparative analysis of the potential theoretical inhibitory effect of both avermectins on biomolecules associated with COVID-19, and suggest that ivermectin through its homologs, has a multiobjective behavior., Graphical abstract Unlabelled Image

Programmed death-ligand 1 (PD-L1) is an immune checkpoint molecule that negatively regulates anti-tumor immunity. Recent reports indicate that anti-cancer treatments, such as radiation therapy, increase PD-L1 expression on the surface of tumor cells. We previously reported that the nuclear transport receptor karyopherin-β1 (KPNB1) is involved in radiation-increased PD-L1 expression on head-and-neck squamous cell carcinoma cells. However, the mechanisms underlying KPNB1-mediated, radiation-increased PD-L1 expression remain unknown. Thus, the mechanisms of radiation-increased, KPNB1-mediated PD-L1 expression were investigated by focusing on the transcription factor interferon regulatory factor 1 (IRF1), which is reported to regulate PD-L1 expression. Western blot analysis showed that radiation increased IRF1 expression. In addition, flow cytometry showed that IRF1 knockdown decreased cell surface PD-L1 expression of irradiated cells but had a limited effect on non-irradiated cells. These findings suggest that the upregulation of IRF1 after irradiation is required for radiation-increased PD-L1 expression. Notably, immunofluorescence and western blot analyses revealed that KPNB1 inhibitor importazole not only diffused nuclear localization of IRF1 but also decreased IRF1 upregulation by irradiation, which attenuated radiation-increased PD-L1 expression. Taken together, these findings suggest that KPNB1 mediates radiation-increased cell surface PD-L1 expression through both upregulation and nuclear import of IRF1.

Fused in sarcoma (FUS) is a predominantly nuclear RNA-binding protein with key functions in RNA processing and DNA damage repair. Defects in nuclear import of FUS have been linked to severe neurodegenerative diseases; hence, it is of great interest to understand this process and how it is dysregulated in disease. Transportin-1 (TNPO1) and the closely related transportin-2 have been identified as major nuclear import receptors of FUS. They bind to the C-terminal nuclear localization signal of FUS and mediate the protein's nuclear import and at the same time also suppress aberrant phase transitions of FUS in the cytoplasm. Whether FUS can utilize other nuclear transport receptors for the purpose of import and chaperoning has not been examined so far. Here, we show that FUS directly binds to different import receptors in vitro. FUS formed stable complexes not only with TNPO1 but also with transportin-3, importin β, importin 7, or the importin β/7 heterodimer. Binding of these alternative import receptors required arginine residues within FUS-RG/RGG motifs and was weakened by arginine methylation. Interaction with these importins suppressed FUS phase separation and reduced its sequestration into stress granules. In a permeabilized cell system, we further showed that transportin-3 had the capacity to import FUS into the nucleus, albeit with lower efficiency than TNPO1. Our data suggest that aggregation-prone RNA-binding proteins such as FUS may utilize a network of importins for chaperoning and import, similar to histones and ribosomal proteins.

Far upstream element binding protein 1 (FUBP1), a DNA-binding protein, participates in diverse tumor-promoting behaviors by regulating the expression of oncogenes in the nucleus, but the underlying mechanisms remain to be elucidated. In the present study, we found that FUBP1 mRNA and protein expressions were markedly upregulated and closely linked with poor prognosis in cervical cancer. In vitro, functional experiments showed that knockdown of FUBP1 inhibited CC cell proliferation and migration. Therefore, FUBP1 plays a prooncogenic function in CC progression. Further investigations for the first time demonstrated that nuclear localization of FUBP1 regulated the gene expression of immune checkpoint NRP1. Moreover, our work demonstrated that FUBP1 translocated into the nucleus which was mediated by interacting with Transportin-1 (TNPO1). Collectively, this study revealed that FUBP1 might be a potential therapeutic target for the restriction of tumor progression.