Search Results (111)

Novel duck reovirus encodes a new nucleus-localized protein, p18. We aimed to investigate whether the nuclear entry of p18 is dependent on viral replication, identify the cellular proteins that interact with p18, and determine the transporters involved in the nuclear import. The subcellular localization of p18 was observed by confocal microscopy. Cellular proteins interacting with p18 were identified through data-independent acquisition qualitative proteomics. The interaction between p18 and importin α was determined by confocal microscopy, co-immunoprecipitation (Co-IP) and molecular docking. We observed that p18 was localized to the nucleus in transfected cells. Importin α1, α3, α4, α5, and α7 were colocalized and co-immunoprecipitated with p18. The importin α/β1 pathway inhibitor reduced the nuclear distribution of p18. The truncated form of p18, lacking the C-terminal region, was predominantly distributed in the cytoplasm. Collectively, our research suggests that the nuclear entry of p18 is independent of viral infection, importin α is implicated in the nuclear import of p18, and the C-terminal region of p18 is crucial for nuclear localization. Full article

Microtubule-associated proteins (MAPs) are multifunctional tubulin-binding proteins that contribute to essential aspects of mitotic spindle formation. In the present study, loss of the MAP CFDP1 in mice resulted in gastrulation defects and embryonic lethality at e8.5 due to chromosome segregation spindle defects and loss of K-fiber stability. CFDP1 decreased the association of the nuclear transport protein importin α with the essential spindle assembly factor TPX2, thereby promoting Aurora A kinase activation, microtubule nucleation and spindle assembly. Further defining the CFDP1 mode of action, we identified CFDP1 as a bipartite molecule with an acidic N-terminus that harbors a nuclear localization signal essential for importin α dissociation from TPX2 and a basic C-terminus that interacts with tubulin, co-localizes with the mitotic spindle, and promotes microtubule bundling and polymerization. Together, our studies have established CFDP1 as an essential bipartite MAP involved in chromosomal microtubule nucleation in conjunction with TPX2 and Aurora A while also facilitating nuclear TPX2 activation through importin α dissociation. Full article

Importin α (IMPα) proteins are key mediators of nucleocytoplasmic transport and play crucial roles in plant development and stress adaptation. Here, we performed a genome-wide identification of the IMPα gene family in Glycine max, followed by gene structure and conserved motif analyses, chromosomal distribution and duplication inference, synteny and selection (Ka/Ks) analyses, and expression profiling across tissues and stress conditions using public RNA-seq datasets and expression browsers. The GmIMPα genes exhibited diverse gene structures and conserved motifs, suggesting functional diversification within the family. Segmental duplication was identified as the main contributor to family expansion, and most duplicated gene pairs underwent purifying selection. Promoter analysis revealed numerous stress- and hormone-responsive cis-elements, implying complex transcriptional regulation. Expression profiling demonstrated that GmIMPα5 and GmIMPα7 were strongly induced under drought, heat, and salt stresses, indicating potential roles in abiotic stress tolerance. Collectively, our results provide a comprehensive framework for the evolution and functional divergence of the GmIMPα family in soybean and offer candidates for improving stress resilience. Full article

Endothelial cell proliferation, migration, and intercellular interactions for blood vessel formation require coordinated signaling by a myriad of molecules. Following endothelial cell activation by growth factors and cytokines, a variety of signaling molecules are activated on the surface and transported intracellularly by TM4SF1-enriched microdomains (TMEDs), 100–300 nm diameter protein–lipid complexes recruited by the transmembrane protein TM4SF1. TMEDs internalize via microtubules from the cell surface toward the microtubule-organizing center (MTOC) and then enter the nucleus via nuclear pores (see Graphic Illustration). This internalization pathway permits delivery of activated proteins and other signaling molecules from the cell surface to the nucleus, which directly translates extracellular stimuli to modulation of gene expression. Molecules transported by this route include phospholipase C, gamma 1 (PLCγ1), histone deacetylase 6 (HDAC6), and importins. In the absence of TMEDs, endothelial cells lose the ability to divide into cultures in vitro and to support blood vessel formation in mouse embryos in vivo. We liken TMEDs to cable cars, which take in passengers at the cell surface, travel along microtubule cables, and deliver their passengers to various locations, including the “city center”, the nucleus. This commentary aims to elucidate the functions of TMEDs in endothelial cells, to show that cells, like busy cities, need efficient transport systems to deliver molecules to the destinations where they perform their cellular functions. TMEDs offer a novel and curated transport system providing selected molecules with access to the nucleus. Full article

Hand–foot–mouth disease (HFMD), primarily caused by human enteroviruses (EVs), poses a public health challenge, particularly among infants, due to a lack of effective therapies. Elucidating the molecular interplay between EVs and the host immune response is crucial for developing antiviral treatments. Recent studies have highlighted the significance of long non-coding RNAs (lncRNAs) in regulating host–pathogen interactions; however, the mechanisms of lncRNAs in EV infection remain poorly unexplored. Here, we identified a highly inducible nuclear lncRNA, LINC1467, that is upregulated in response to HFMD-related EV infection. Functional analyses revealed that LINC1467 suppresses viral replication. Mechanistically, LINC1467 interacts with nuclear import receptor Importin 8 (IPO8) to form the LINC1467/IPO8/p65 complex, facilitating the phosphorylation and nuclear translocation of p65, thus promoting the expression of pro-inflammatory cytokines and activating the NF-κB pathway. The antiviral function of LINC1467 was further validated in a mouse model of viral infection. These findings uncover a novel lncRNA-mediated regulatory mechanism in the innate immune response and highlight LINC1467 as a promising target for future antiviral strategies against HFMD-related EVs. Full article

Abalone are highly susceptible to haliotid herpesvirus 1 (HaHV1), the causative agent of abalone viral ganglioneuritis (AVG), a re-emerging disease responsible for significant mortality events in both wild and farmed populations. Currently, there are no effective antiviral treatments or preventive measures available against HaHV1, which is partly due to the limited understanding of the immune responses and viral pathogenesis in this non-model marine invertebrate. This highlights the urgent need for novel intervention strategies, including investigations into the molecular mechanisms underlying HaHV1 infection. In other herpesviruses, the large tegument protein UL36 plays a crucial role in transporting the viral capsid to the host cell’s nuclear pore complex (NPC), mediated by N-terminal nuclear localization signals (NLSs). However, the nuclear import mechanism of UL36 homologue (UL36h) in HaHV1 remains largely uncharacterized. In this study, we identified and functionally characterized the NLS motif within HaHV1 UL36h and elucidated its interactions with the importin alpha (IMPα) nuclear import receptor. Through a combination of high-resolution crystallography and quantitative binding assays, we determined the key residues responsible for binding to IMPα and demonstrated isoform-specific variations in binding affinity. Our biochemical and structural analyses confirmed key interactions within the NLS that are essential for IMPα interactions. These findings advance our molecular understanding of HaHV1 host interactions and pave the way for the development of targeted antiviral strategies against abalone herpesvirus infection. Full article

Hypovirus infection is known to reduce the pathogenicity of Cryphonectria parasitica, the causative agent of chestnut blight. Isoforms derived from a viral protein p48 have been discovered in host mitochondria and vesicles, which may contribute to virulence attenuation, as reported in earlier work using two-dimensional electrophoresis (2-DE). In this study, a total of 1739 fungal proteins were identified in fungal vesicles through Tandem Mass Tag (TMT)-based quantitative proteomics. The infection of CHV1-EP713 was associated with 75 up-regulated and 201 down-regulated proteins, predominantly involved in vesicular transport process and related cellular functions, including protein folding, membrane fusion, retrograde transport, autophagy, and ER stress responses. The down-regulation of calnexin, COPI, ArfGAP, importin-β, and Atg8 is consistent with impairments in protein folding, retrograde transport, and autophagy. Meanwhile, the up-regulation of clathrin, dynamin, Vps10p, HSP70, and t-SNAREs indicated enhanced trafficking to vacuoles and increased stress response activity. Overall, our findings indicate that hypoviral infection is associated with extensive alterations in the vesicular transport system of C. parasitica, likely mediated through changes in the abundance of multiple key protein regulators. These alterations may underlie attenuation of virulence by impacting crucial cellular processes. Full article

Several carrier proteins are involved in nuclear translocation from the cytoplasm to the nucleus in eukaryotic cells. We have previously demonstrated the binding of several intact folded and disordered proteins to the human isoform importin α3 (Impα3); furthermore, disordered peptides, corresponding to their nuclear localization signals (NLSs), also interact with Impα3. These proteins and their isolated NLSs also bind to the truncated importin species ∆Impα3, which does not contain the N-terminal disordered importin binding domain (IBB). In this work, we added a further ‘layer’ of conformational disorder to our studies, testing whether the isolated D-enantiomers of NLSs of selected proteins, either folded or unfolded, were capable of binding to both Impα3 and ∆Impα3. The D-enantiomers, like their L-form counterparts, were monomeric and disordered in isolation, as shown by nuclear magnetic resonance (NMR). We measured the ability of such D-enantiomeric NLSs to interact with both importin species by using fluorescence, biolayer interferometry (BLI), isothermal titration calorimetry (ITC), and molecular simulations. In all cases, the binding affinities were within the same range as those measured for their L-isomer counterparts for either Impα3 or ∆Impα3, and the binding locations corresponded to the major NLS binding site of the protein. Thus, the stereoisomeric nature is not important in defining the binding of proteins to the main component of classical cellular translocation machinery, although the primary structure of the hot-spot site for NLS binding of importin is well defined. Full article

Měnglà virus (MLAV) is a member of the genus Dianlovirus in the family Filoviridae, which also includes Ebola virus (EBOV) and Marburg virus (MARV). Whether MLAV poses a threat to human health is uncertain. However, the MLAV VP35 and VP40 proteins can impair IFNα/β gene expression and block IFNα/β-induced Jak-STAT signaling, respectively, suggesting the capacity to counteract human innate immune defenses. In this study, MLAV VP40 is demonstrated to impair the Sendai virus (SeV)-induced activation of the IFNβ promoter. Inhibition is independent of the MLAV VP40 PPPY late-domain motif that interacts with host proteins possessing WW-domains to promote viral budding. Similar IFNβ promoter inhibition was not detected for EBOV or MARV VP40. MLAV VP40 exhibited lesser capacity to inhibit TNFα activation of an NF-κB reporter gene. MLAV VP40 impaired IFNβ promoter activation by an over-expressed, constitutively active form of RIG-I and by the over-expressed IRF3 kinases TBK1 and IKKε. However, MLAV VP40 did not inhibit IFNβ promoter activation by constitutively active IRF3 5D. Consistent with these findings, MLAV VP40 inhibited SeV-induced IRF3 phosphorylation. Although IRF3 phosphorylation occurs in the cytoplasm, MLAV VP40 exhibits substantial nuclear localization, accumulating in foci in HeLa cell nuclei. In contrast, the VP40 of EBOV and MARV exhibited lower degrees of nuclear localization and did not accumulate in foci. MLAV VP40 interacts with importin alpha-1 (IMPα1), suggesting entry via the IMPα/IMPβ nuclear import pathway. Cumulatively, these data identify novel features that distinguish MLAV VP40 from its homologues in EBOV and MARV. Full article

Thyroid carcinogenesis has multiple hallmarks, including evasion of tumor suppressors. Reactivation of wild-type p53 function is the ultimate goal in cancer therapy, which requires an understanding of the p53 suppression mechanism specific to the cancer type. MiR-7-5p and IPO7 are implicated in the pathogenesis of several human diseases. This work aims to investigate the role of miR-7-5p and IPO7 in p53 regulation in papillary thyroid cancer (PTC) cells. Primary cultured thyroid cells and FFPE thyroid tissues from PTC and benign cases were used. Functional experiments were performed by transfection with IPO7 siRNA or miR-7-5p mimic/inhibitor, followed by apoptosis and luciferase reporter assays, immunoblot assays, and RT-PCR. The expression and subcellular localization of IPO7, p53, MDM2, and ribosomal proteins (RPL11 and RPL5) were studied by immunofluorescence staining and confocal microscopy. The results show that IPO7 is overexpressed in PTC and regulated by miR-7-5p. Modulation of IPO7 expression in cultured thyroid cells altered the nucleocytoplasmic shuttling of p53, MDM2, RPL11, and RPL5, in addition to the p53 protein level and activity. The expression pattern of IPO7, p53, and MDM2 in cultured thyroid cells and clinical thyroid tissue specimens confirmed the association between IPO7 overexpression and reduced p53 stability in PTC. In conclusion, the data here show that p53 level and activity are differentially controlled in malignant and benign thyroid cells through miR-7-5P/IPO7-mediated regulation of RP-MDM2-p53 nucleocytoplasmic trafficking. In PTC, downregulation of miR-7-5p with consequent overexpression of IPO7 might be a protective mechanism used by cancer cells to evade p53 growth suppression during carcinogenesis. Full article

Nucleocytoplasmic transport is crucial for neuronal cell physiology and defects are involved in neurodegenerative diseases like amyotrophic lateral sclerosis and Alzheimer’s disease, but also in ageing. Recent studies have suggested, that the classic nuclear import factor adapters KPNA3 (also named importin alpha4) and KPNA4 (also named importin alpha3) could be associated with the development of motor neuron diseases, a condition specifically affecting the neurons projecting from brain to spinal cord or from spinal cord to the muscles. Here we set out to analyze the neuronal function of mice deficient in KPNA3 (Kpna3-KO) or KPNA4 (Kpna4-KO). The motoric abilities and locomotion at different time points in ageing were tested to study the role of these two genes on motor neuron function. While we did not find deficits related to motor neurons in both mouse models, we discovered a hypermotoric phenotype in KPNA4-deficient mice. Attention deficit/hyperactivity disorder (ADHD) is caused by a combination of genetic, environmental and neurobiological factors and a number of genes have been suggested in genome-wide association studies to contribute to ADHD, including KPNA4. Here we provide supportive evidence for KPNA4 as a candidate pathogenic factor in ADHD, by analysing Kpna4-KO mice which show ADHD-like symptoms. Full article

The transcription and replication of the genome of influenza A virus (IAV) take place in the nucleus of infected cells, which is catalyzed by the viral ribonucleoprotein (vRNP) complex. The nuclear import of the vRNP complex and its component proteins is essential for the efficient replication of IAV and is therefore prone to be targeted by host restriction factors. Herein, we found that host cellular protein keratin 6A (KRT6A) is a negative regulator of IAV replication because siRNA-mediated knockdown of KRT6A expression increased the growth titers of IAV, whereas exogenous overexpression of KRT6A reduced viral yields. The nuclear import of incoming vRNP complexes and newly synthesized nucleoprotein (NP) was significantly impaired when KRT6A was overexpressed. Further studies showed that KRT6A interacts with the four vRNP complex proteins—polymerase basic protein 1 (PB1), polymerase basic protein 2 (PB2), polymerase acidic protein (PA), and NP. Notably, the interaction between KRT6A and vRNP complex proteins had no effect on the nuclear import of PB2 or the PB1-PA heterodimer but impaired the interaction between NP and the nuclear import adaptor importin α3, thereby inhibiting the nuclear import of incoming vRNP complexes and newly synthesized NP. Moreover, KRT6A was further shown to suppress the assembly of the vRNP complex and consequently reduce viral polymerase activity. Together, our data uncover a novel role of KRT6A in counteracting the nuclear import and functions of the vRNP complex, thereby restricting the replication of IAV. Full article

Karyopherins, carrier proteins that recognize particular cargo protein patterns known as nuclear localization signals (NLSs), mediate the nuclear translocation of big proteins. In order to better understand the process of nuclear transport of proteins and create the groundwork for the development of innovative treatments that specifically target importins, it is imperative to clarify the intricate interactions between nuclear transporters and their cargo proteins. Until recently, very few NLSs have been documented. In the current work, an in silico method was used to identify NLSs for importin 8. It was determined that the sequence RRKLPVGRS serves as a recognition motif for importin 8 binding a karyopherin that is involved in the nuclear transportation of several important proteins like AGOs, SMADs, RPL23A, and TFE3. The sequence was validated in vitro in the breast cancer cell line T47D. This work subscribes to the effort to clarify the intricate relationships between nuclear transporters and their cargo proteins, in order to better understand the mechanism of nuclear transport of proteins and lay the groundwork for the development of novel therapeutics that target particular importins and have an immediate translational impact. Full article

Background/Objectives: Non-resolving inflammation in macrophage-like cells (MLCs) transdifferentiated from vascular smooth muscle cells and monocyte-derived macrophages aggravates atherosclerosis. We previously showed that polyphenolic protocatechuic acid (PCA) could reduce inflammation burden in monocyte-derived macrophages; however, it remains unknown how this compound affects MLCs inflammation. Methods: MLCs from the transdifferentiation of vascular smooth muscle cells induced by cholesterol and 30-week-old male ApoE^−/− mice fed a semi-purified AIN-93G diet containing either 0.003% (wt:wt) of PCA for a duration of 20 weeks were used to examine the impact of PCA on the inflammatory response of MLCs. Results: Physiologically achievable doses of PCA (0.25–1 μM) dose-dependently inhibited lipopolysaccharide-induced NF-κB activation and simultaneously reduced pro-inflammatory cytokine levels. Mechanistically, this effect was mediated by effecting exportin-1 function, promoting nuclear export of phosphorylated-p65, independent of NF-κB kinase inhibitor α/β/γ, NF-κB inhibitor α, or importin-mediated nuclear import of p-p65. PCA reduced the nucleocytoplasmic ratio of exportin-1 (44%) without altering its abundance. Importantly, dietary supplementation with PCA reduced interleukin-1β content within MLCs in atherosclerotic plaques of ApoE^−/− mice. In addition, dietary PCA reduced MLCs content in atherosclerotic plaques. Conclusions: PCA could attenuate inflammatory response in MLCs by targeting exportin-1 and also could inhibit the transdifferentiation of vascular smooth muscle cells into MLCs within atherosclerotic plaques, which might promote the translation from preclinical studies to clinical trials in patients with atherosclerosis. Full article

Human parvovirus B19 (B19V) is a major human pathogen in which the ssDNA genome is replicated within the nucleus of infected human erythroid progenitor cells (EPCs) through a process involving both cellular and viral proteins, including the non-structural protein (NS)1. We previously characterized the interaction between NS1 classical nuclear localization signal (cNLS: GACHAKKPRIT-182) and host cell importin (IMP)α and proposed it as a potential target for antiviral drug development. Here, we further extend on such findings. First, we demonstrate that NS1 nuclear localization is required for viral production since introducing the K177T substitution in a cloned, infectious viral genome resulted in a non-viable virus. Secondly, we demonstrate that the antiparasitic drug ivermectin (IVM), known to inhibit the IMPα/β dependent nuclear import pathway, could impair the NS1-NLS:IMPα interaction and suppress viral replication in UT7/EpoS1 cells in a dose-dependent manner. We also show that a panel of structurally related avermectins (AVMs) can dissociate the NS1-NLS:IMPα complex with half-maximal inhibitory concentrations in the nanomolar range. Among them, Eprinomectin emerged as the most selective inhibitor of B19V replication, with a selectivity index of c. 5.0. However, when tested in EPCs generated from peripheral blood mononuclear cells, which constitute a cellular population close to the natural target cells in bone marrow, the inhibitory effect of IVM and Eprinomectin was demonstrated to a lesser extent, and both compounds exhibited high toxicity, thus highlighting the need for more specific inhibitors of the NS1-NLS:IMPα interaction. Full article