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Shuttling from the cytoplasm to the nucleus is a regulated cellular process which involves the recognition of nuclear localization signal-containing proteins by importins. Nuclear-cytoplasmic protein transport is found aberrant in cancer, which impacts subcellular localization of proteins that modulate drug responses and cell growth. We have previously demonstrated that the classically cytoplasmic antiapoptotic XIAP protein is associated with breast cancer chemoresistance and poorer clinical outcomes, when mis localized in the nucleus. Nevertheless, little is known about the mechanisms of XIAP nuclear translocation. In this study, we compared importin expression and response to importin inhibitors in cancer cellular models with distinct drug sensitivity phenotypes and subcellular localization of XIAP. Remarkably, importins α1, α5 and β1 were found differentially expressed among drug sensitive and resistant cell lines, as well as primary breast tumors compared to normal tissues. Interestingly, nuclear XIAP-expressing cancer cells exhibiting resistance to both docetaxel and doxorubicin have shown pronounced sensitivity to importin inhibition. Pharmacological intervention of nuclear transport revealed that XIAP can shuttle from the cytoplasm to the nucleus dependently on the importins α/β1 classical pathway. Last, we have shown that INI-43-mediated inhibition of importins α/β1 potentiates the cytotoxic effects of chemotherapy in drug refractory cells. These findings indicate that targeting protein nuclear import via importins α and β1 might be of potential clinical benefit for drug resistance tumors, particularly when combined with conventional chemotherapy., Competing Interests: Declaration of competing interest 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., (Copyright © 2025 Elsevier Inc. All rights reserved.)

Osteoporosis is mainly caused by an imbalance in osteoclast and osteoblast regulation, resulting in an imbalance in bone homeostasis. Ginsenoside Rg3 (Rg3) has been reported to have a therapeutic effect on alleviating osteoporosis. Nonetheless, the underlying mechanisms have not been completely elucidated. Herein, the molecular mechanism of Rg3 alleviation in osteoporosis was further explored. An in vitro model was established utilising the receptor activator of nuclear factor-kappaB ligand (RANKL) to induce osteoclast differentiation of RAW264.7 cells. RNA-sequencing results showed that karyopherin subunit alpha 2 (KPNA2) is one of the significantly differentially expressed genes regulated by Rg3 in RANKL-induced RAW264.7 cells. Basic experiments further suggested that KPNA2 is up-regulated in a time-dependent manner in the RANKL-induced RAW264.7 cells, while Rg3 treatment reduced its expression in a dose- and time-dependent manner. Knockdown of KPNA2 inhibited osteoclast formation and the expression of related molecules, including those in the nuclear factor kappa-B (NF-κB) pathway. The NF-κB inhibitor, JSH-23, partially abolished the impact of KPNA2 overexpression on osteoclast formation, indicating KPNA2 activates NF-κB. Furthermore, KPNA2 overexpression partially abolished the inhibitory impact of Rg3 on osteoclast formation, indicating that KPNA2 is a target of Rg3. These results suggest that KPNA2 plays a role in how Rg3 influences on osteoclast differentiation and osteoporosis through the NF-κB pathway., (© 2025 John Wiley & Sons Australia, Ltd.)

Background: Recent studies have illuminated the complexities of treating advanced bladder cancer (BCa), underscoring the importance of comprehending its molecular mechanisms for creating novel therapies. While the role of Karyopherin a2 (KPNA2) in promoting BCa growth is established, the precise mechanism remains elusive., Methods: To investigate the regulatory role of KPNA2 in BCa, we employed a comprehensive approach integrating clinical case data and bioinformatics analysis to evaluate the expression of KPNA2 in BCa tissues. Mechanisms promoting cancer by KPNA2 were examined using both in vivo and in vitro models., Results: Our research reveals that miR-26b-5p acts as an anticancer factor by targeting and inhibiting KPNA2 expression. Furthermore, we have observed that the interaction between KPNA2 and Kinesin Family Member C1 (KIFC1) facilitates the transition of BCa cells into the G2/M phase, thereby promoting tumor advancement via activation of the Phosphoinositide 3-kinase (PI3K)- Protein Kinase B (AKT) pathway. Importantly, this investigation is the first to identify KPNA2 expression in exosomes originating from BCa tissues. Plasma exosomes from patients with BCa exhibited notably increased levels of KPNA2 compared with healthy controls, suggesting KPNA2 as a potential new tumor indicator. Additionally, KPNA2 from BCa cells triggered the conversion of fibroblasts into cancer-associated fibroblasts (CAFs), which secreted elevated levels of interleukin-6 (IL-6), contributing to a tumor-supporting environment., Conclusions: These findings suggest that KPNA2 is a key gene that promotes BCa progression, can potentially be a novel tumor marker, and may serve as a new therapeutic target for BCa., Competing Interests: Declarations. Ethics approval and consent to participate: Ethical approval for this study was obtained from the Human and Animal Research Ethics Committee of the First Affiliated Hospital of Shenzhen University (Shenzhen, China) (No. 2024SYY-010, Date of approval: 18 April 2024). Animal experiments were approved by the Animal Ethics Committee of the First Affiliated Hospital of Shenzhen University (Shenzhen, China) (no. 20240060, Date of approval: 15 April 2024). The ethics committee of the First Affiliated Hospital of Shenzhen University follows the provisions of the Declaration of Helsinki and the Basel Declaration. Consent for publication: All authors have read the manuscript and provided their consent for the submission. Competing interests: The authors declare no competing interests., (© 2025. The Author(s).)

Importin α is a crucial player in the nucleocytoplasmic transport of nuclear localization signal (NLS)-containing cargo proteins and is suggested to bind to DNA directly. We hypothesized that importin α, after binding to DNA, may move along DNA via sliding or hopping. We investigated the movement dynamics of importin αs fused to AcGFP along DNA using single-molecule fluorescence microscopy and single-tethered DNA arrays. Single-molecule data demonstrated importin α diffuses along DNA in fast and slow mobility modes. The diffusion by importin α in the fast mobility mode did not depend on salt concentration, suggesting sliding motion with continuous contact with DNA. The sliding was supported by restricted diffusion of importin α in Cas9 obstacles bound to DNA. Next, we tested whether importin α can transport a cargo molecule along DNA. Two-color imaging data established that importin α co-slides along DNA with SV40 TAg-NLS as a model cargo. We found that importin β1 together with RanGTP significantly enhanced the DNA binding of importin α and the recruitment of a model cargo TRIM28 to DNA, suggesting that importin β1/RanGTP are involved in the switching of importin α/cargo from the nuclear transport pathway to DNA sliding. Single-molecule and in vivo immunofluorescence assay demonstrates importin α assists in accumulating TRIM28 within nuclear chromatin regions. Thus, we present novel findings on the sliding dynamics and the cargo transport of importin α along DNA. The relatively faster sliding by importin α allows efficient delivery of cargo proteins to their target sites, even on long genomic DNA., Competing Interests: Declaration of competing interest The authors declare no competing financial interests., (Copyright © 2025 Elsevier Inc. All rights reserved.)

Background: Nuclear import, dependent on the transporter importin α (IMPα), is a drug target for apicomplexan parasites Plasmodium falciparum and Toxoplasma gondii. Indeed, a panel of small molecule inhibit interactions between IMPα and nuclear localization signals (NLSs) in vitro and the growth of rapidly dividing stages (P. falciparum blood stages and T. gondii tachyzoites) in culture., Objectives: As new drugs targeting multiple life cycle stages of both parasites are required, the panel of IMPα inhibitors was tested for their ability to inhibit nuclear transport in the rapidly dividing stages and the maturation of differentiated stages (P. falciparum gametocytes and T. gondii bradyzoites)., Methods: Using biophysical assays, Bay 11-7082, a Bay 11-7085 structural analogue, was tested for inhibition of IMPα:NLS interactions. The effect of the panel of inhibitors on the nuclear localization of reporter proteins was analysed in both parasites using transfections and microscopy. Also, using microscopy, the effect of inhibitors on differentiated stages of both parasites was tested., Results: Bay 11-7085 can inhibit nuclear transport in tachyzoites, while GW5074 and Caffeic Acid Phenethyl Ester (CAPE) can inhibit nuclear transport in the blood stages. Interestingly, CAPE can strongly inhibit gametocyte maturation, and Bay 11-7082 and Bay 11-7085 weakly inhibit bradyzoite differentiation., Conclusions: As differentiation of gametocytes and bradyzoites is dependent on the activation of gene expression triggered by the nuclear translocation of transcription factors, our work provides a 'proof of concept' that targeting nuclear import is a viable strategy for the development of therapeutics against multiple stages of apicomplexan parasites, some of which are recalcitrant to existing drugs., (© The Author(s) 2024. Published by Oxford University Press on behalf of British Society for Antimicrobial Chemotherapy. All rights reserved. For commercial re-use, please contact reprints@oup.com for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site—for further information please contact journals.permissions@oup.com.)

Hexanucleotide repeat expansions in C9orf72 are the most common genetic mutation associated with amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) (C9-ALS/FTD). Dipeptide repeat (DPR) proteins, such as poly(proline-arginine) (polyPR) generated from G4C2 repeat expansions, have been shown to be highly toxic. In this study, PR20 was labeled with fluorescein isothiocyanate (FITC) to track its cellular localization. Several cell lines demonstrated survival under PR20 treatment by sequestering PR20 in the cytoplasm. Treatment with JQ-1 or Ivermectin (Iver) translocated PR20 into the nucleus, leading to cell death. Mechanistically, KPNA2/KPNB1 interacted with PR20 in the cytoplasm and hindered PR20 from entering the cell nucleus. Genetic silencing of KPNA2/KPNB1 converted PR20-resistant cells into PR20-sensitive cells. Treatment with JQ1 significantly reduced the protein levels of KPNA2/KPNB1, allowing PR20 to enter the nucleus. Overexpression of KPNA2 or KPNB1 effectively blocked cell death induced by co-treatment with JQ-1 and PR20. Our results indicate that super-enhancers shield cells from PR20 toxicity by upregulating the expression of KPNA2/KPNB1., (© 2025 John Wiley & Sons Ltd.)

Background: Breast cancer is the most frequently diagnosed cancer among women worldwide with high morbidity and mortality. Previous studies have indicated that RNA-binding motif protein-15 (RBM15), an N6-methyladenosine (m6A) writer, is implicated in the growth of breast cancer cells. Herein, we aimed to explore the function and detailed mechanism of RBM15 in breast cancer., Methods: In this research, UALCAN databases were applied to analyze the expression of RBM15 or Karyopherin-2 alpha (KPNA2) in BRCA. RBM15 and KPNA2 mRNA levels were determined using real-time quantitative polymerase chain reaction (RT-qPCR) assay. RBM15, KPNA2, and Programmed cell death ligand 1 (PD-L1) protein levels were measured using western blot. Cell proliferation, migration, and invasion were assessed using 5-ethynyl-2'-deoxyuridine (EdU) and Transwell assays. The biological role of RBM15 on breast cancer tumor growth was verified using the xenograft tumor model in vivo. Effects of breast cancer cells on the proliferation and apoptosis of CD8 + T cells were analyzed using flow cytometry. Interaction between RBM15 and KPNA2 was validated using methylated RNA immunoprecipitation (MeRIP) and dual-luciferase reporter assays., Results: RBM15 and KPNA2 were highly expressed in breast cancer tissues and cell lines. Furthermore, RBM15 silencing might suppress breast cancer cell proliferation, migration, invasion, and lymphocyte immunity in vitro, as well as block tumor growth in vivo. At the molecular level, RBM15 might improve the stability and expression of KPNA2 mRNA via m6A methylation., Conclusion: RBM15 might contribute to the malignant progression and immune escape of breast cancer cells partly by modulating the stability of KPNA2 mRNA, providing a promising therapeutic target for breast cancer., Competing Interests: Disclosure The authors have stated that they have no conflicts of interest., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Adeno-associated viruses (AAVs) are the most extensively researched viral vectors for gene therapy globally. The AAV viral protein 1 (VP1) N-terminus controls the capsid's ability to translocate into the cell nucleus; however, the exact mechanism of this process is largely unknown. In this study, we sought to elucidate the precise interactions between AAV serotype 6 (AAV6), a promising vector for immune disorders, and host transport receptors responsible for vector nuclear localization. Focusing on the positively charged basic areas within the N-terminus of AAV6 VP1, we identified a 53-amino acid region that interacts with nuclear import receptors. We measured the binding affinities between this region and various nuclear import receptors, discovering a notably strong interaction with IMPα5 and IMPα7 in the low nanomolar range. We also elucidated the X-ray crystal structure of this region in complex with an importin alpha (IMPα) isoform, uncovering its binding as a bipartite nuclear localization signal (NLS). Furthermore, we show that using this bipartite NLS, AAV6 VP1 capsid protein can localize to the nucleus of mammalian cells in a manner dependent on the IMPα/IMPβ nuclear import pathway. This study provides detailed insights into the interaction between the AAV6 VP1 capsid protein and nuclear import receptors, deepening our knowledge of AAV nuclear import mechanisms and establishing a basis for the improvement of AAV6-based gene therapy vectors.IMPORTANCEAAVs, recognized as the most extensively researched viral vectors for gene therapy globally, offer significant advantages over alternatives due to their small size, non-pathogenic nature, and innate ability for tissue-specific targeting. AAVs are required to localize to the nucleus to perform their role as a gene therapy vector; however, the precise mechanisms that facilitate this process remain unknown. Despite sharing overt genomic similarities with AAV1 and AAV2, AAV6 is a unique serotype. It is currently recognized for its ability to effectively transduce hematopoietic cell lineages and, consequently, is considered promising for the treatment of immune disorders. Identifying the exact mechanisms that permit AAV6 to access the nucleus can open up new avenues for gene therapy vector engineering, which can ultimately lead to increased therapeutic benefits., Competing Interests: The authors declare no conflict of interest.

The glucose metabolism homeostasis in the follicular fluid microenvironment plays an important role in follicular maturation and ovulation, and excessively high or low glucose concentrations have adverse effects on the differentiation of follicular granulosa cells (GCs). However, a limited number of microRNAs (miRNA) have been reported to be involved in glucose-stimulated GCs differentiation. In this study, we characterized the miRNA expression profiles of sheep ovarian GCs cultured in high-glucose and optimal glucose concentrations and focused on a differentially expressed miRNA: miR-17-5p , which may be involved in regulating high-glucose-induced GC apoptosis by targeting KPNA2 . We found that overexpression of miR-17-5p significantly promoted GCs proliferation and inhibited cell apoptosis, while downregulated the mRNA and protein expression of apoptosis-related makers (Bax, Caspase-3, Caspase-9, and Bcl-2). In contrast to the classical mechanism of miRNA silencing target gene expression, miR-17-5p overexpression significantly upregulated the expression of target gene KPNA2 . A dual luciferase reporter gene assay verified the targeted binding relationship between miR-17-5p and KPNA2 promoter. Meanwhile, overexpression of KPNA2 further promoted the downregulation of apoptosis-related genes driven by miR-17-5p mimics. Knockdown of KPNA2 blocked the inhibitory effect of miR-17-5p mimics on the expression of apoptosis-related genes. Our results demonstrated that miR-17-5p activated the KPNA2 promoter region and upregulated KPNA2 expression, thereby inhibiting GCs apoptosis under high glucose.

Background: Prostate cancer (PCa) is commonly occurred among males worldwide and its prognosis could be influenced by biochemical recurrence (BCR). MicroRNAs (miRNAs) are functional regulators in carcinogenesis, and miR-221-3p was reported as one of the significant candidates deregulated in PCa. However, its regulatory pattern in PCa BCR across literature reports was not consistent, and the targets and mechanisms in PCa malignant transition and BCR are less explored., Methods: In this study, an evidence-based and knowledge-guided approach was proposed to decipher the role and mechanism of miR-221-3p in PCa development. First, the literature-reported inconsistency between miR-221-3p and PCa BCR was quantitatively measured by meta-analysis. Then a knowledge-guided network strategy was applied to prioritize key targets of miR-221-3p in PCa progression based both on topological and functional characterization of genes in multi-omics miRNA-mRNA and protein-protein interaction networks. Finally, a key gene was computationally identified and experimentally validated using cell line and clinical samples through EdU assay, scratch assay, transwell assay, dual-luciferase reporter assay and the epithelial-to-mesenchymal transition (EMT)-related analysis., Results: Down-regulation of miR-221-3p was correlated with a lower biochemical recurrence-free survival (BRFS) in PCa (HR: 0.72, 95%, CI: 0.64-0.81, P < 0.00001). A significant down-regulation of miR-221-3p was observed in most of the PCa cells compared with the normal control. KPNA2 was identified as a key target of miR-221-3p and it was over-expressed in all the PCa cells and human PCa tissues. Moreover, elevated miR-221-3p inhibited the proliferation, migration, invasion, and EMT of PCa cells in vitro via directly and negatively mediating KPNA2 expression., Conclusions: miR-221-3p down-regulation was a risk factor for PCa BRFS, and its over-expression could inhibit the malignant phenotype and EMT of PCa cells by directly targeting KPNA2. Translational and personalized applications of the findings will be conducted in the future., Competing Interests: Declaration. Ethics approval and consent to participate: The study was conducted in accordance with the Declaration of Helsinki (revised in 2013). This study was approved by the Ethics Committee of The First Affiliated Hospital of Soochow University with the number of 2014 − 312, and all patients had signed informed consent before surgery. Consent for publication: Not applicable. Competing interests: The authors declare no competing interests., (© 2025. The Author(s).)

The histone locus body (HLB) is a membraneless organelle that determines the transcription of replication-dependent histones. However, the mechanisms underlying the appropriate formation of the HLB in the nucleus but not in the cytoplasm remain unknown. HLB formation is dependent on the scaffold protein NPAT. We identify KPNA3 as a specific importin that drives the nuclear import of NPAT by binding to the nuclear localization signal (NLS) sequence. NPAT undergoes phase separation, which is inhibited by KPNA3-mediated impairment of self-association. In this, a C-terminal self-interaction facilitator (C-SIF) motif, proximal to the NLS, binds the middle 431-1,030 sequence to mediate the self-association of NPAT. Mechanistically, the anchoring of KPNA3 to the NPAT-NLS sterically blocks C-SIF motif-dependent NPAT self-association. This leads to the suppression of aberrant NPAT condensation in the cytoplasm. Collectively, our study reveals a previously unappreciated role of KPNA3 in modulating HLB formation and delineates a steric hindrance mechanism that prevents inappropriate cytoplasmic NPAT condensation., (© 2024 Xu et al.)

Controlling biomolecular condensate formation within the nucleus is critical for genome function. In this issue, Xu et al. (https://doi.org/10.1083/jcb.202401036) report that KPNA3 promotes histone locus body formation and expression of replication-dependent histone genes by both importing NPAT into the nucleus and preventing NPAT condensation from improperly occurring in the cytoplasm., (© 2024 Geisler et al.)

Proteins in the importin α (IMPA) family play pivotal roles in intracellular nucleocytoplasmic transport. Arabidopsis thaliana possesses nine IMPA members, with diverse tissue-specific expression patterns. Among these nine IMPAs, IMPA1, IMPA2, and IMPA4 cluster together phylogenetically, suggesting potential functional redundancy. To explore this redundancy, we analyzed single and multiple T-DNA mutants for these genes and discovered severe growth defects in the impa1 impa2 impa4 triple knockout mutant but not in the single or double mutants. Complementation with IMPA1, IMPA2, or IMPA4 fused to green fluorescent protein (GFP) rescued the growth defects observed in the impa1 impa2 impa4 mutant, indicating the functional redundancy of these three IMPAs. The IMPA-GFP fusion proteins were localized in the nucleus and nuclear envelope, suggesting their involvement in nucleocytoplasmic transport processes. Comparative transcriptomics revealed that salicylic acid (SA)-responsive genes were significantly upregulated in the impa1 impa2 impa4 triple mutant. Consistent with this observation, impa1 impa2 impa4 mutant plants accumulated SA and reactive oxygen species to high levels compared with wild-type plants. We also found enhanced resistance to the anthracnose pathogen Colletotrichum higginsianum in the impa1 impa2 impa4 mutants, suggesting that defense responses were constitutively activated in the impa1 impa2 impa4 mutant. Our findings shed light on the redundant roles of IMPA1, IMPA2, and IMPA4 in suppressing the autoimmune responses and suggest avenues of research to clarify their potentially unique roles., (© 2024 Society for Experimental Biology and John Wiley & Sons Ltd.)

Histone lysine lactylation (Kla) has recently been reported to participate in various biological processes, regulating transcription, inflammation, and immune-related diseases. However, the mechanism of histone Kla in innate immunity and viral infection remains largely unknown. Here, we observed fluorescent Kla signals in all four histones (H2A, H2B, H3, and H4) in PK-15 cells. Immunoprecipitation analysis showed prominent histone Kla protein bands, with H2B being the most abundant. We generated the H2B K16R mutant plasmid and identified K16 as one of the Kla modification sites in H2B. Further exploration revealed increased global H2B Kla and H2BK16la levels upon classical swine fever virus (CSFV) infection. By employing the Kla agonist (L-lactate), inhibitor (oxamate), or siLDHA, we demonstrated that H2BK16la and pan Kla in PK-15 cells rely on the LDHA-lactate axis, which is also crucial for CSFV-induced H2BK16la and pan Kla levels. Moreover, our data proved the interaction between H2B and CSFV NS4A protein. Notably, H2B Kla can modulate CSFV proliferation. Mechanistically, H2BK16la and pan Kla activate the nuclear factor kappa B (NF-κB) pathway by mediating p65 nuclear translocation via karyopherin α2 (KPNA2), thereby inducing type III interferon (IFN-λ) expression and inhibiting CSFV replication. In conclusion, our study unveils the role of H2B Kla in regulating the NF-κB pathway during viral infection, presenting a novel mechanism. These findings significantly contribute to understanding the pathogenic mechanisms during viral infection and hold promise for the development of viral therapeutic strategies., Competing Interests: Declaration of competing interest The authors declare no conflict of interest., (Copyright © 2025 Elsevier B.V. All rights reserved.)

The secretory proteome of Plasmodium exhibits differential spatial and functional activity within host cells. Plasmodium secretes proteins that translocate into the human host cell nucleus. Liver-specific protein 2 of Plasmodium falciparum ( Pf- LISP2) shows nuclear accumulation in human hepatocytes during the late liver stage of malaria parasite development. However, the nuclear translocation mechanism for Pf- LISP2 remains largely uncharacterized. Here, we identified a classical bipartite nuclear localization signal (NLS) located in the C-terminal region of Pf- LISP2. Phylogenetic analysis revealed that this NLS is unique to Plasmodium falciparum and its close relative Plasmodium reichenowi , suggesting an evolutionary adaptation linked to their shared primate hosts. Functional assays confirmed the NLS's nuclear import activity, as fusion constructs of the Pf- LISP2 NLS with Pf- aldolase ( Pf- aldolase-NLS-EGFP) localized exclusively to the nucleus of HepG2 cells. Mutation analysis of key lysine and arginine residues in the bipartite NLS demonstrated that the basic amino acid clusters are essential for nuclear localization. Importin-α/β interaction was found to be crucial for Pf- LISP2 nuclear transport, as coexpression of the NLS constructs with the importin-α/β inhibitor mCherry-Bimax2 significantly blocked nuclear translocation. Specific interactions between the lysine and arginine residues of Pf- LISP2's NLS and the conserved tryptophan and asparagine residues of human importin-α1 facilitate the cytosol-to-nuclear translocation of Pf- LISP2. Additionally, LISP2 lacks any nuclear export signal. These results provide new insights into the mechanisms of nuclear transport in Plasmodium falciparum , potentially contributing to the understanding of its pathogenicity and host-cell interactions during liver-stage infection.

We aim to clarify the specific role of Karyopherin α2 (KPNA2) in the progression of laryngeal cancer, a kind of malignant tumor with a poor curative effect. We performed the bioinformatic analysis to obtain the ferroptosis-related differentially expressed genes. KPNA2 was screened out. Then the CCK-8 assay, wound healing assay, and transwell assay were used to clarify the changes in the proliferation, migration, and invasion abilities of laryngeal cancer cells after silencing KPNA2. The concentrations of iron ions, glutathione, superoxide dismutase, and malondialdehyde were evaluated by the corresponding detection kits. The expression levels of cyclooxygenase 2, Acyl-CoA synthetase long-chain family member 4, glutathione peroxidase 4, forkhead box O (FoxO)1a and FoxO3a were determined by Western Blot. A total of 45 ferroptosis-related differentially expressed genes in laryngeal cancer were obtained, and KPNA2 was selected after bioinformatic analysis. In ferroptosis-induced laryngeal cancer cells, the cell viability, migration rate, invasion ability, and the expression of glutathione peroxidase 4, glutathione, and superoxide dismutase were further decreased and the expression of cyclooxygenase 2, Acyl-CoA synthetase long-chain family member 4, iron ions, and malondialdehyde were further increased after silencing KPNA2. The expression levels of FoxO1a and FoxO3a in laryngeal cancer cells were increased by silencing KPNA2. KPNA2 may be a promising therapeutic target for laryngeal cancer. Down-regulation of KPNA2 can promote ferroptosis in laryngeal cancer by stimulating the FoxO signaling pathway., Competing Interests: Declarations. Competing Interests: The authors declare no competing interests., (© 2024. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

The Orthopoxvirus (OPXV) genus of the Poxviridae includes human pathogens variola virus (VARV), monkeypox virus (MPXV), vaccinia virus (VACV), and a number of zoonotic viruses. A number of Bcl-2-like proteins of VACV are involved in escaping the host innate immunity. However, little work has been devoted to the evolution and function of their orthologues in other OPXVs. Here, we found that MPXV protein P2, encoded by the P2L gene, and P2 orthologues from other OPXVs, such as VACV protein N2, localize to the nucleus and antagonize interferon (IFN) production. Exceptions to this were the truncated P2 orthologues in camelpox virus (CMLV) and taterapox virus (TATV) that lacked the nuclear localization signal (NLS). Mechanistically, the NLS of MPXV P2 interacted with karyopherin α-2 (KPNA2) to facilitate P2 nuclear translocation, and competitively inhibited KPNA2-mediated IRF3 nuclear translocation and downstream IFN production. Deletion of the NLS in P2 or orthologues significantly enhanced IRF3 nuclear translocation and innate immune responses, thereby reducing viral replication. Moreover, deletion of NLS from N2 in VACV attenuated viral replication and virulence in mice. These data demonstrate that the NLS-mediated translocation of P2 is critical for P2-induced inhibition of innate immunity. Our findings contribute to an in-depth understanding of the mechanisms of OPXV P2 orthologue in innate immune evasion.

Purpose: Adrenocortical carcinoma (ACC) afflicts both pediatric and adult populations and is characterized by dismal prognosis and elevated mortality. Given the inconsistent therapeutic benefits and significant side effects associated with the conventional chemotherapy agent, mitotane, and the nascent stage of immunotherapy and targeted treatments, there is an urgent need to identify novel prognostic biomarkers and therapeutic targets in ACC., Methods: Utilizing multi-omic datasets from The Cancer Genome Atlas (TCGA) and the Gene Expression Omnibus (GEO), we employed Weighted Gene Co-expression Network Analysis (WGCNA), Cox regression, Receiver Operating Characteristic (ROC) curves, and survival analyses to sift for potential prognostic biomarkers. We subsequently validated these findings through immunohistochemistry and cell assays, and delved into the biological role of KPNA2 in ACC through functional enrichment analysis, mutational landscape, and immune cell infiltration., Results: A total of 77 progression-associated genes with aberrant chromosomal accessibility were discerned within the TCGA-ACC dataset. By integrating ROC and Cox regression from GEO datasets, KPNA2 emerged as an independent risk factor portending poor outcomes in ACC. ATAC-seq analysis revealed attenuated chromatin accessibility of KPNA2 in cases with unfavorable prognosis. Immunohistochemistry corroborated elevated KPNA2 expression, which was linked to enhanced proliferation and invasion. Elevated KPNA2 levels were found to activate oncogenic pathways while simultaneously suppressing immunological responses. Immune infiltration analysis further revealed a decrement in CD8+ T-cell infiltration in KPNA2-high cohorts., Conclusion: This study demonstrates the clinical and biological significance of KPNA2 in ACC and suggests that KPNA2 could serve as a promising biomarker for predicting prognosis and immunotherapeutic responses in pediatric and adult ACC patients.

Ethnopharmacological Relevance: Weifuchun (WFC) is a Traditional Chinese Medicine commonly used for treating atrophic gastritis and intestinal metaplasia. Till date, its antitumor effect on gastric cancer (GC) and the underlying mechanisms of the effect remains unelucidated., Aim of the Study: We aim to investigate if WFC can suppress the malignancy of stomach cancer cells and dissect the molecular basis and the associated molecular and cellular features., Materials and Methods: Stomach cancer cell lines and normal gastric epithelial cells were treated with WFC. CCK8 assay, caspase-3 activity assay, adhesion assay, microRNA database analysis, transfection, RT-PCR, Western Blotting, signaling pathway analysis, and in vivo GC model were employed to examine the changes in the features of the gastric cancer cells and the molecular mechanisms of the effect of WFC., Results: Here we present data demonstrating that WFC suppresses the malignant cellular phenotypes of GC and this inhibitory effect is mediated by downregulating the expression of oncogenic KPNA2. Furthermore, WFC downregulates KPNA2 through miR-26a-5p-mediated gene silencing and the deactivated phosphorylation dynamics of mitogen-activated protein kinase (MAPK). The suppressive effect of WFC on stomach cancer cell behavior was further confirmed in animal model., Conclusion: Therefore, WFC can exert inhibitory effect on the malignancy of GC cells by reducing the levels of KPNA2. Moreover, the miR-26a-5p rescue and the deactivation MAPK pathway induced by WFC result in the downregulation of KPNA2 expression. Thus, our findings suggest WFC as a potential treatment option against GC., Competing Interests: Declaration of competing interest Lvli Ma, Wei Zhang, Daqing Qi, and Linhui Chen were employed by Hangzhou Huqingyu Hall Pharmaceutical Co., Ltd, Hangzhou, China. All authors declare no other competing interests., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Plant pathogens secrete nuclear effectors into the host nuclei to modulate the host immune system. Although several nuclear effectors of fungal pathogens have been recently reported, the molecular mechanism of NLS-associated transport vehicles of nuclear effectors and the roles of NLS in transcriptional reprogramming of host immunity genes remain enigmatic. We previously reported the MoHTR1, a nuclear effector of the rice blast fungus, Magnaporthe oryzae. MoHTR1 is translocated to rice nuclei but not in fungal nuclei. Here, we identify the core NLS (RxKK) responsible for MoHTR1's nuclear localization. MoHTR1 is translocated in the host nucleus through interaction with rice importin α. MoHTR1 NLS empowers it to translocate the cytoplasmic effectors of M. oryzae into rice nuclei. Furthermore, other nuclear effector candidates of the blast pathogen and rice proteins which have RxKK also exhibit nuclear localization, highlighting the crucial role of RxKK in this process. We also unveil the importance of SUMOylation in the stability of MoHTR1 and translocation of MoHTR1 to host nuclei. Moreover, MoHTR1 NLS is essential for the pathogenicity of M. oryzae by reprogramming immunity-associated genes in the host. Our findings provide insights into the significance of plant-specific NLS on fungal nuclear effectors and its role in plant-pathogen interactions., Competing Interests: Competing interests The authors declare no competing interests., (© 2024. The Author(s).)

GLABRA2 (GL2), a class IV homeodomain leucine-zipper (HD-Zip IV) transcription factor from Arabidopsis, is a developmental regulator of specialized cell types in the epidermis. GL2 contains a monopartite nuclear localization sequence (NLS) that is conserved in most HD-Zip IV members across the plants. We demonstrate that NLS mutations affect nuclear transport and result in a loss-of-function phenotypes. NLS fusions to enhanced yellow fluorescent protein (EYFP) show that it is sufficient for nuclear localization in roots and trichomes. Despite partial overlap of the NLS with the homeodomain, genetic dissection indicates that nuclear localization and DNA binding are separable functions. Affinity purification of GL2 from plants followed by MS-based proteomics identified importin α (IMPα) isoforms as potential GL2 interactors. NLS structural prediction and molecular docking studies with IMPα-3 revealed major interacting residues. Cytosolic yeast two-hybrid assays and co-immunoprecipitation experiments with recombinant proteins verified NLS-dependent interactions between GL2 and several IMPα isoforms. IMPα triple mutants (impα-1,2,3) exhibit abnormal trichome formation and defects in GL2 nuclear localization in trichomes, consistent with tissue-specific and redundant functions of IMPα isoforms. Taken together, our findings provide mechanistic evidence for IMPα-dependent nuclear localization of GL2 in Arabidopsis, a process that is critical for cell type differentiation of the epidermis., (© The Author(s) 2024. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved. For commercial re-use, please contact reprints@oup.com for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site—for further information please contact journals.permissions@oup.com.)

The nuclear transport of proteins is mediated by karyopherins and has been implicated to be crucial for germ cell and embryonic development. Deletion of distinct members of the karyopherin alpha family has been shown to cause male and female infertility in mice. Using a genetrap approach, we established mice deficient for KPNA2 (KPNA2 KO) and investigated the role of this protein in male germ cell development and fertility. Breeding of male KPNA2 KO mice leads to healthy offsprings in all cases albeit the absence of KPNA2 resulted in a reduction in sperm number by 60%. Analyses of the KPNA2 expression in wild-type mice revealed a strong KPNA2 presence in meiotic germ cells of all stages while a rapid decline is found in round spermatids. The high KPNA2 expression throughout all meiotic stages of sperm development suggests a possible function of KPNA2 during this phase, hence in its absence the spermatogenesis is not completely blocked. In KPNA2 KO mice, a higher portion of sperms presented with morphological abnormalities in the head and neck region, but a severe spermiogenesis defect was not found. Thus, we conclude that the function of KPNA2 in round spermatids is dispensable, as our mice do not show any signs of infertility. Our data provide evidence that KPNA2 is not crucial for male germ cell development and fertility., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Rother et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Cytoplasmic aggregation of the TAR-DNA binding protein of 43 kDa (TDP-43) is the hallmark of sporadic amyotrophic lateral sclerosis (ALS). Most ALS patients with TDP-43 aggregates in neurons and glia do not have mutations in the TDP-43 gene but contain aberrantly post-translationally modified TDP-43. Here, we found that a single acetylation-mimetic mutation (K82Q) near the TDP-43 minor Nuclear Localization Signal (NLS) box, which mimics a post-translational modification identified in an ALS patient, can lead to TDP-43 mislocalization to the cytoplasm and irreversible aggregation. We demonstrate that the acetylation mimetic disrupts binding to importins, halting nuclear import and preventing importin α1/β anti-aggregation activity. We propose that perturbations near the NLS are an additional mechanism by which a cellular insult other than a genetically inherited mutation leads to TDP-43 aggregation and loss of function. Our findings are relevant to deciphering the molecular etiology of sporadic ALS., Competing Interests: Declaration of competing interest 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., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Middle East Respiratory Syndrome Coronavirus (MERS-CoV) is an enveloped, positive-sense RNA virus that emerged in 2012, causing sporadic cases and localized outbreaks of severe respiratory illness with high fatality rates. A characteristic feature of the immune response to MERS-CoV infection is low type I IFN induction, despite its importance in viral clearance. The non-structural proteins (nsps) of other coronaviruses have been shown to block IFN production. However, the role of nsp5 from MERS-CoV in IFN induction of human respiratory cells is unclear. In this study, we elucidated the role of MERS-CoV-nsp5, the viral main protease, in modulating the host's antiviral responses in human bronchial epithelial BEAS 2b cells. We found that overexpression of MERS-CoV-nsp5 had a dose-dependent inhibitory effect on IFN-β promoter activation and cytokine production induced by HMW-poly(I:C). It also suppressed IFN-β promoter activation triggered by overexpression of key components in the RIG-I-like receptor (RLR) pathway, including RIG-I, MAVS, IKK-ε and IRF3. Moreover, the overexpression of MERS-CoV-nsp5 did not impair expression or phosphorylation of IRF3, but suppressed the nuclear translocation of IRF3. Further investigation revealed that MERS-CoV-nsp5 specifically interacted with IRF3. Using docking and molecular dynamic (MD) simulations, we also found that amino acids on MERS-CoV-nsp5, IRF3, and KPNA4 may participate in protein-protein interactions. Additionally, we uncovered protein conformations that mask the nuclear localization signal (NLS) regions of IRF3 and KPNA4 when interacting with MERS-CoV-nsp5, suggesting a mechanism by which this viral protein blocks IRF3 nuclear translocation. Of note, the IFN-β expression was restored after administration of protease inhibitors targeting nsp5, indicating this suppression of IFN-β production was dependent on the enzyme activity of nsp5. Collectively, our findings elucidate a mechanism by which MERS-CoV-nsp5 disrupts the host's innate antiviral immunity and thus provides insights into viral pathogenesis., (© 2024. The Author(s).)

Importin α4, which is encoded by the Kpna4 gene, is a well-characterized nuclear-cytoplasmic transport factor known to mediate transport of transcription factors including NF-κB. Here, we report that Kpna4 knock-out (KO) mice exhibit psychiatric disorder-related behavioral abnormalities such as anxiety-related behaviors, decreased social interaction, and sensorimotor gating deficits. Contrary to a previous study predicting attenuated NF-κB activity as a result of Kpna4 deficiency, we observed a significant increase in expression levels of NF-κB genes and proinflammatory cytokines such as TNFα, Il-1β or Il-6 in the prefrontal cortex or basolateral amygdala of the KO mice. Moreover, examination of inflammatory responses in primary cells revealed that Kpna4 deficient cells have an increased inflammatory response, which was rescued by addition of not only full length, but also a nuclear transport-deficient truncation mutant of importin α4, suggesting contribution of its non-transport functions. Furthermore, RNAseq of sorted adult microglia and astrocytes and subsequent transcription factor analysis suggested increases in polycomb repressor complex 2 (PRC2) activity in Kpna4 KO cells. Taken together, importin α4 deficiency induces psychiatric disorder-related behavioral deficits in mice, along with an increased inflammatory response and possible alteration of PRC2 activity in glial cells., (© 2024. The Author(s).)

Adenoviral pVII proteins are multifunctional, highly basic, histone-like proteins that can bind to and transport the viral genome into the host cell nucleus. Despite the identification of several nuclear localization signals (NLSs) in the pVII protein of human adenovirus (HAdV)2, the mechanistic details of nuclear transport are largely unknown. Here we provide a full characterization of the nuclear import of precursor (Pre-) pVII protein from an ancient siadenovirus, frog siadenovirus 1 (FrAdV1), using a combination of structural, functional, and biochemical approaches. Two strong NLSs (termed NLSa and NLSd) interact with importin (IMP)β1 and IMPα, respectively, and are the main drivers of nuclear import. A weaker NLS (termed NLSb) also contributes, together with an additional signal (NLSc) which we found to be important for nucleolar targeting and intranuclear binding. Expression of wild-type and NLS defective derivatives Pre-pVII in the presence of selective inhibitors of different nuclear import pathways revealed that, unlike its human counterpart, FrAdV1 Pre-pVII nuclear import is dependent on IMPα/β1 and IMPβ1, but not on transportin-1 (IMPβ2). Clearly, AdVs evolved to maximize the nuclear import pathways for the pVII proteins, whose subcellular localization is the result of a complex process. Therefore, our results pave the way for an evolutionary comparison of the interaction of different AdVs with the host cell nuclear transport machinery., (© 2024 The Author(s). Traffic published by John Wiley & Sons Ltd.)

Evidence shows that tropomodulin 1 (TMOD1) is a powerful diagnostic marker in the progression of several cancer types. However, the regulatory mechanism of TMOD1 in tumor progression is still unclear. Here, we showed that TMOD1 was highly expressed in acute myeloid leukemia (AML) specimens, and TMOD1-silencing inhibited cell proliferation by inducing autophagy in AML THP-1 and MOLM-13 cells. Mechanistically, the C-terminal region of TMOD1 directly bound to KPNA2, and TMOD1-overexpression promoted KPNA2 ubiquitylation and reduced KPNA2 levels. In contrast, TMOD1-silencing increased KPNA2 levels and facilitated the nuclear transfer of KPNA2, then subsequently induced autophagy and inhibited cell proliferation by increasing the nucleocytoplasmic transport of p53 and AMPK activation. KPNA2/p53 inhibitors attenuated autophagy induced by silencing TMOD1 in AML cells. Silencing TMOD1 also inhibited tumor growth by elevating KPNA2-mediated autophagy in nude mice bearing MOLM-13 xenografts. Collectively, our data demonstrated that TMOD1 could be a novel therapeutic target for AML treatment., Competing Interests: Declaration of Competing Interest The authors declare that they have no conflicts of interest to report regarding the present study., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

RNA-binding proteins (RBPs) play critical roles in genome regulation. In this study, we explored the latent function of KPNA2, which is an essential member of the RBP family, in the regulation of alternative splicing (AS) in gastric cancer (GC). We analyzed the role of KPNA2 in regulating differential expression and AS via RNA sequencing (RNA-seq) and improved RNA immunoprecipitation sequencing (iRIP-seq). Clinical specimens were used to analyze the associations between KPNA2 expression and clinicopathological characteristics. CCK8 assays, transwell assays and wound healing assays were performed to explore the effect of KPNA2/WDR62 on GC cell progression. KPNA2 was shown to be highly expressed in GC cells and tissues and associated with lymph node metastases. KPNA2 promoted the proliferation, migration and invasion of GC cells and primarily regulated exon skipping, alternative 3's splice sites (A3SSs), alternative 5' splice sites (A5SSs), and cassette exons. We further revealed that KPNA2 participated in biological processes related to cell proliferation, and the immune response in GC via the regulation of transcription. In addition, KPNA2 preferentially bound to intron regions. Notably, KPNA2 regulated the A3SS AS mode of WDR62, and upregulation of WDR62 reversed the KPNA2 downregulation-induced inhibition of GC cell proliferation, migration and invasion. Finally, we discovered that the AS of immune-related molecules could be regulated by KPNA2. Overall, our results demonstrated for the first time that KPNA2 functions as an oncogenic splicing factor in GC that regulated the AS and differential expression of GC-related genes, and KPNA2 may be a potential target for GC treatment., (© 2024. The Author(s).)

In this issue, the discovery by Yang et al. (https://doi.org/10.1083/jcb.202308013) that folded WW domains of YAP1 and other proteins bind to Impα introduces a new class of globular NLS, contrasting with the extensively studied linear NLS motifs. This finding underscores the versatility of importins in recognizing their cargo proteins., (© 2024 Bernardes and Chook.)

The nuclear transport of proteins plays an important role in mediating the transition from egg to embryo and distinct karyopherins have been implicated in this process. Here, we studied the impact of KPNA2 deficiency on preimplantation embryo development in mice. Loss of KPNA2 results in complete arrest at the 2cell stage and embryos exhibit the inability to activate their embryonic genome as well as a severely disturbed nuclear translocation of Nucleoplasmin 2. Our findings define KPNA2 as a new maternal effect gene., (© 2024 The Authors. The FASEB Journal published by Wiley Periodicals LLC on behalf of Federation of American Societies for Experimental Biology.)

Adeno-associated viruses (AAV) are one of the world's most promising gene therapy vectors and as a result, are one of the most intensively studied viral vectors. Despite a wealth of research into these vectors, the precise characterisation of AAVs to translocate into the host cell nucleus remains unclear. Recently we identified the nuclear localization signals of an AAV porcine strain and determined its mechanism of binding to host importin proteins. To expand our understanding of diverse AAV import mechanisms we sought to determine the mechanism in which the Cap protein from a bat-infecting AAV can interact with transport receptor importins for translocation into the nucleus. Using a high-resolution crystal structure and quantitative assays, we were able to not only determine the exact region and residues of the N-terminal domain of the Cap protein which constitute the functional NLS for binding with the importin alpha two protein, but also reveal the differences in binding affinity across the importin-alpha isoforms. Collectively our results allow for a detailed molecular view of the way AAV Cap proteins interact with host proteins for localization into the cell nucleus.

Barley yellow dwarf viruses (BYDVs) cause widespread damage to global cereal crops. Here we report a novel strategy for elevating resistance to BYDV infection. The 17K protein, a potent virulence factor conserved in BYDVs, interacted with barley IMP-α1 and -α2 proteins that are nuclear transport receptors. Consistently, a nuclear localization signal was predicted in 17K, which was found essential for 17K to be transported into the nucleus and to interact with IMP-α1 and -α2. Reducing HvIMP-α1 and -α2 expression by gene silencing attenuated BYDV-elicited dwarfism, accompanied by a lowered nuclear accumulation of 17K. Among the eight common wheat CRISPR mutants with two to four TaIMP-α1 and -α2 genes mutated, the triple mutant α1 aaBBDD /α2 AAbbdd and the tetra-mutant α1 aabbdd /α2 AAbbDD displayed strong BYDV resistance without negative effects on plant growth under field conditions. The BYDV resistance exhibited by α1 aaBBDD /α2 AAbbdd and α1 aabbdd /α2 AAbbDD was correlated with decreased nuclear accumulation of 17K and lowered viral proliferation in infected plants. Our work uncovers the function of host IMP-α proteins in BYDV pathogenesis and generates the germplasm valuable for breeding BYDV-resistant wheat. Appropriate reduction of IMP-α gene expression may be broadly useful for enhancing antiviral resistance in agricultural crops and other economically important organisms., (© 2023 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd.)

KPNA1 is a mediator of nucleocytoplasmic transport that is abundantly expressed in the mammalian brain and regulates neuronal differentiation and synaptic function. De novo mutations in Kpna1 have been identified using genome-wide association studies in humans with schizophrenia; however, it remains unclear how KPNA1 contributes to schizophrenia pathogenesis. Recent studies have suggested a complex combination of genetic and environmental factors that are closely related to psychiatric disorders. Here, we found that subchronic administration of phencyclidine, a psychotropic drug, induced vulnerability and behavioral abnormalities consistent with the symptoms of schizophrenia in Kpna1-deficient mice. Microarray assessment revealed that the expression levels of dopamine d1/d2 receptors, an RNA editing enzyme, and a cytoplasmic dynein component were significantly altered in the nucleus accumbens brain region in a gene-environment (G × E) interaction-dependent manner. Our findings demonstrate that Kpna1-deficient mice may be useful as a G × E interaction mouse model for psychiatric disorders and for further investigation into the pathogenesis of such diseases and disorders., (© 2024. The Author(s).)

NEIL glycosylases, including NEIL1, NEIL2, and NEIL3, play a crucial role in the base excision DNA repair pathway (BER). The classical importin pathway mediated by importin α/β and cargo proteins containing nuclear localization sequences (NLS) is the most common transport mechanism of DNA repair proteins to the nucleus. Previous studies have identified putative NLSs located at the C-terminus of NEIL3 and NEIL1. Crystallographic, bioinformatics, calorimetric (ITC), and fluorescence assays were used to investigate the interaction between NEIL1 and NEIL3 putative NLSs and importin-α (Impα). Our findings showed that NEIL3 contains a typical cNLS, with medium affinity for the major binding site of Impα. In contrast, crystallographic analysis of NEIL1 NLS revealed its binding to Impα, but with high B-factors and a lack of electron density at the linker region. ITC and fluorescence assays indicated no detectable affinity between NEIL1 NLS and Impα. These data suggest that NEIL1 NLS is a non-classical NLS with low affinity to Impα. Additionally, we compared the binding mode of NEIL3 and NEIL1 with Mus musculus Impα to human isoforms HsImpα1 and HsImpα3, which revealed interesting binding differences for HsImpα3 variant. NEIL3 is a classical medium affinity monopartite NLS, while NEIL1 is likely to be an unclassical low-affinity bipartite NLS. The base excision repair pathway is one of the primary systems involved in repairing DNA. Thus, understanding the mechanisms of nuclear transport of NEIL proteins is crucial for comprehending the role of these proteins in DNA repair and disease development., Competing Interests: Declaration of Competing Interest 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., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Nucleocytoplasmic transport regulates the passage of proteins between the nucleus and cytoplasm. In the best characterized pathway, importin (IMP) α bridges cargoes bearing basic, classical nuclear localization signals (cNLSs) to IMPβ1, which mediates transport through the nuclear pore complex. IMPα recognizes three types of cNLSs via two binding sites: the major binding site accommodates monopartite cNLSs, the minor binding site recognizes atypical cNLSs, while bipartite cNLSs simultaneously interact with both major and minor sites. Despite the growing knowledge regarding IMPα-cNLS interactions, our understanding of the evolution of cNLSs is limited. We combined bioinformatic, biochemical, functional, and structural approaches to study this phenomenon, using polyomaviruses (PyVs) large tumor antigens (LTAs) as a model. We characterized functional cNLSs from all human (H)PyV LTAs, located between the LXCXE motif and origin binding domain. Surprisingly, the prototypical SV40 monopartite NLS is not well conserved; HPyV LTA NLSs are extremely heterogenous in terms of structural organization, IMPα isoform binding, and nuclear targeting abilities, thus influencing the nuclear accumulation properties of full-length proteins. While several LTAs possess bipartite cNLSs, merkel cell PyV contains a hybrid bipartite cNLS whose upstream stretch of basic amino acids can function as an atypical cNLS, specifically binding to the IMPα minor site upon deletion of the downstream amino acids after viral integration in the host genome. Therefore, duplication of a monopartite cNLS and subsequent accumulation of point mutations, optimizing interaction with distinct IMPα binding sites, led to the evolution of bipartite and atypical NLSs binding at the minor site., (© 2023 The Authors. Protein Science published by Wiley Periodicals LLC on behalf of The Protein Society.)

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) surfaced on 31 December, 2019, and was identified as the causative agent of the global COVID-19 pandemic, leading to a pneumonia-like disease. One of its accessory proteins, ORF6, has been found to play a critical role in immune evasion by interacting with KPNA2 to antagonize IFN signaling and production pathways, resulting in the inhibition of IRF3 and STAT1 nuclear translocation. Since various mutations have been observed in ORF6, therefore, a comparative binding, biophysical, and structural analysis was used to reveal how these mutations affect the virus's ability to evade the human immune system. Among the identified mutations, the V9F, V24A, W27L, and I33T, were found to have a highly destabilizing effect on the protein structure of ORF6. Additionally, the molecular docking analysis of wildtype and mutant ORF6 and KPNA2 revealed the docking score of - 53.72 kcal/mol for wildtype while, -267.90 kcal/mol, -258.41kcal/mol, -254.51 kcal/mol and -268.79 kcal/mol for V9F, V24A, W27L, and I33T respectively. As compared to the wildtype the V9F showed a stronger binding affinity with KPNA2 which is further verified by the binding free energy (-42.28 kcal/mol) calculation. Furthermore, to halt the binding interface of the ORF6-KPNA2 complex, we used a computational molecular search of potential natural products. A multi-step virtual screening of the African natural database identified the top 5 compounds with best docking scores of -6.40 kcal/mol, -6.10 kcal/mol, -6.09 kcal/mol, -6.06 kcal/mol, and -6.03 kcal/mol for tophit1-5 respectively. Subsequent all-atoms simulations of these top hits revealed consistent dynamics, indicating their stability and their potential to interact effectively with the interface residues. In conclusion, our study represents the first attempt to establish a foundation for understanding the heightened infectivity of new SARS-CoV-2 variants and provides a strong impetus for the development of novel drugs against them., 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 Suleman, Said, Khan, Rehman, Alshammari, Crovella and Yassine.)

Background: Circular RNAs (circRNAs) have a vital role in the occurrence of numerous cancers. However, its function and pattern of expression in cervical cancer (CC) remain unclear. This research aims to investigate the hsa&#95;circ&#95;000002's regulatory mechanism in CC., Methods: Hsa&#95;circ&#95;0000021, miR-3940-3p, and KPNA2 expression levels were estimated through qRT-PCR. Nuclear/cytoplasmic separation was conducted to find the subcellular location of hsa&#95;circ&#95;0000021. Western blot was done to estimate the levels of KPNA2 protein. CCK-8, BrdU, wound healing, transwell, and tumor xenograft assays were performed to study how hsa&#95;circ&#95;0000021/miR-3940-3P/KPNA2 function affect CC. Hsa&#95;circ&#95;0000021's targeting relationships with miR-3940-3p and KPNA2 were ascertained through RIP and luciferase experiments., Results: Hsa&#95;circ&#95;0000021 and KPNA2 were overexpressed and inversely associated with the levels of miR-3940-3p in CC. Knocking down either hsa&#95;circ&#95;0000021 or KPNA2 repressed the growth of CC tumors as well as the proliferation, invasion, and migration of CC cells. Silencing miR-3940-3p promoted the malignant proliferation of CC cells. Regarding its mechanism, hsa&#95;circ&#95;0000021 affected the malignant CC cell proliferation via the sponging of miR-3940-3p, which targeted KPNA2., Conclusion: Hsa&#95;circ&#95;0000021 regulates the miR-3940-3p/KPNA2 axis to promote CC occurrence. This potentially is a novel target for CC treatment., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

Nuclear transport is the basis for the biological reaction of eukaryotic cells, as it is essential to coordinate nuclear and cytoplasmic events separated by nuclear envelope. Although we currently understand the basic molecular mechanisms of nuclear transport in detail, many unexplored areas remain. For example, it is believed that the regulations and biological functions of the nuclear transport receptors (NTRs) highlights the significance of the transport pathways in physiological contexts. However, physiological significance of multiple parallel transport pathways consisting of more than 20 NTRs is still poorly understood, because our knowledge of each pathway, regarding their substrate information or how they are differently regulated, is still limited. In this report, we describe studies showing how nuclear transport systems in general are affected by temperature rises, namely, thermal stress or heat stress. We will then focus on Importin α family members and unique transport factor Hikeshi, because these two NTRs are affected in heat stress. Our present review will provide an additional view to point out the importance of diversity of the nuclear transport pathways in eukaryotic cells., (© 2024. The Author(s), under exclusive license to Springer Nature Singapore Pte Ltd.)

Karyopherins, the nucleocytoplasmic transporters, participate in multiple RNA silencing stages by transporting associated proteins into the nucleus. Importin α is a member of karyopherins and has been reported to facilitate virus infection via nuclear import of viral proteins. Unlike other RNA viruses, silencing of importin α2 (α2i) by virus-induced gene silencing (VIGS) boosted the titre of bamboo mosaic virus (BaMV) in protoplasts, and inoculated and systemic leaves of Nicotiana benthamiana. The enhanced BaMV accumulation in importin α2i plants was linked to reduced levels of RDR6-dependent secondary virus-derived small-interfering RNAs (vsiRNAs). Small RNA-seq revealed importin α2 silencing did not affect the abundance of siRNAs derived from host mRNAs but significantly reduced the 21 and 22 nucleotide vsiRNAs in BaMV-infected plants. Deletion of BaMV TGBp1, an RNA silencing suppressor, compromised importin α2i-mediated BaMV enhancement. Moreover, silencing of importin α2 upregulated NbAGO10a, a proviral protein recruited by TGBp1 for BaMV vsiRNAs clearance, but hindered the nuclear import of NbAGO10a. Taken together, these results indicate that importin α2 acts as a negative regulator of BaMV invasion by controlling the expression and nucleocytoplasmic shuttling of NbAGO10a, which removes vsiRNAs via the TGBp1-NbAGO10a-SDN1 pathway. Our findings reveal the hidden antiviral mechanism of importin α2 in countering BaMV infection in N. benthamiana., (© 2024 The Authors. Molecular Plant Pathology published by British Society for Plant Pathology and John Wiley & Sons Ltd.)

Introduction: Sirtuin 1 (SIRT1) is a key modulator in several types of cancer, including colorectal cancer (CRC). Here, we probed into the molecular mechanism of SIRT1 regulating the development and chemoresistance of CRC., Methods: Differentially expressed genes related to the growth, metastasis and chemoresistance of CRC were identified by bioinformatics analysis. The expression of SIRT1 in clinical tissues from CRC patients and CRC cell lines was detected by RT-qPCR. Interactions among SIRT1, p53, miR-101 and KPNA3 were analyzed. The effect of SIRT1 on the cell viability, migration, invasion, epithelial-mesenchymal transformation and chemoresistance to 5-FU was evaluated using loss-function investigations in CRC cells. Finally, a xenograft model of CRC and a metastasis model were constructed for further exploration of the roles of SIRT1 in vivo., Results: SIRT1 was elevated in CRC tissues and cell lines. SIRT1 decreased p53 via deacetylation, and consequently downregulated the expression of miR-101 while increasing that of the miR-101 target gene KPNA3. By this mechanism, SIRT1 enhanced the proliferation, migration, invasion, epithelial-mesenchymal transformation, and resistance to 5-FU of CRC cells. In addition, in vivo data also showed that SIRT1 promoted the growth, metastasis and chemoresistance to 5-FU of CRC cells via regulation of the p53/miR-101/KPNA3 axis., Conclusions: In conclusion, SIRT1 can function as an oncogene in CRC by accelerating the growth, metastasis and chemoresistance to 5-FU of CRC cells through the p53/miR-101/KPNA3 axis.

While importin-α is well studied in mammals, the knowledge in avian species is still limited. In this study, we compared the mRNA expression patterns of five importin-α isoforms in the respiratory tract, liver, and spleen of chickens, turkeys, and pekin ducks in two different age-groups. In addition, we determined the distribution of importin-α in selected tissue of conchae, trachea, and lung of post-hatch chickens at all cellular levels by immunohistochemical staining. Our results indicate that importin-α3 is the most abundant isoform in the respiratory tract of chickens, turkeys, and pekin ducks. Moreover, importin-α is expressed as a gradient with lowest mRNA levels in the conchae and highest levels in the lung. The mRNA expression levels of most isoforms were higher in tissues from post-hatch chickens and turkeys in comparison to the corresponding embryos. In contrast to that, duck embryos mostly show higher mRNA expression levels of importin-α than post-hatch ducks., Competing Interests: Declaration of Competing Interest The authors declare no conflict of interest., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Microtubule transport and nuclear import are functionally connected, and the nuclear pore complex (NPC) can interact with microtubule motors. For several alphaherpesvirus proteins, nuclear localization signals (NLSs) and their interactions with specific importin-α proteins have been characterized. Here, we review recent insights on the roles of microtubule motors, capsid-associated NLSs, and importin-α proteins for capsid transport, capsid docking to NPCs, and genome release into the nucleoplasm, as well as the role of importins for nuclear viral transcription, replication, capsid assembly, genome packaging, and nuclear capsid egress. Moreover, importin-α proteins exert antiviral effects by promoting the nuclear import of transcription factors inducing the expression of interferons (IFN), cytokines, and IFN-stimulated genes, and the IFN-inducible MxB restricts capsid docking to NPCs., Competing Interests: Declaration of Competing Interest Nothing to declare., (Copyright © 2023 Elsevier B.V. All rights reserved.)

We herein discuss the impacts of miR-101-3p on the tumorigenesis-related cell behaviors in lung squamous cell carcinoma (LUSC) by repressing KPNA2. TCGA database was utilized to measure miR-101-3p and KPNA2 levels in LUSC tissues and cells. The interaction of miR-101-3p and KPNA2-3'UTR was determined by dual luciferase assay. Western blot evaluated the protein level of KPNA2. MiR-101-3p was under-expressed in LUSC cells while KPNA2 was overexpressed. Western blot confirmed the impact of KPNA2 expression on cancer cell progression. The negative regulatory impact of miR-101-3p on KPNA2 was also verified. In vitro cell function assays revealed the suppressing effect of high miR-101-3p expression on cell invasion, migration and viability, as well as its promoting effect on apoptosis. Up-regulated miR-101-3p weakened the promoting effect of overexpressed KPNA2 on LUSC malignant progression. To conclude, miR-101-3p repressed viability, invasion, and migration, and facilitated cell apoptosis in LUSC by suppressing KPNA2., (©The Author(s) 2023. Open Access. This article is licensed under a Creative Commons CC-BY International License.)

We now recognize that with aging, hematopoietic stem and progenitor cells (HSPCs) acquire mutations that confer a fitness advantage and clonally expand in a process now termed clonal hematopoiesis (CH). Because CH predisposes to a variety of health problems, including cancers, cardiovascular diseases, and inflammatory conditions, there is intense interest in the inherited alleles associated with the development of CH. DNA variants near TERT, SMC4, KPNA4, IL12A, CD164, and ATM confer the strongest associations. In this review, we discuss our current state of knowledge regarding germline predisposition to CH., Competing Interests: Declaration of Competing Interest The authors have no conflict of interest regarding this review., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Background: Circ&#95;0000376 could promote non-small cell lung cancer (NSCLC) progression; however, the role of circ&#95;0000376 in paclitaxel (PTX) resistance of NSCLC is still unknown., Methods: Quantitative real-time polymerase chain reaction (qRT-PCR) and western blot were applied for measuring circ&#95;0000376, microRNA-1298-5p (miR-1298-5p), and karyopherin subunit alpha 4 (KPNA4) expression. The half inhibitory concentration (IC50) of PTX was assessed by cell counting kit-8 assay. 5-ethynyl-2'-deoxyuridine assay, wound healing assay, transwell assay, and flow cytometry were performed to measure the proliferation, migration, invasion, and apoptosis of cells. The regulatory mechanisms of circ&#95;0000376, miR-1298-5p, and KPNA4 were validated by dual-luciferase reporter assay, RNA immunoprecipitation assay, and rescue experiments. Xenograft assay was used for the functional analysis of circ&#95;0000376 in vivo., Results: Circ&#95;0000376 and KPNA4 expressions were upregulated, while miR-1298-5p expression was downregulated in PTX-resistant tissues and cells. Circ&#95;0000376 depletion promoted PTX sensitivity and apoptosis, while suppressing the proliferation, migration, and invasion of PTX-resistant NSCLC cells. Furthermore, circ&#95;0000376 could modulate KPNA4 expression by sponging miR-1298-5p. Rescue experiments showed that miR-1298-5p inhibition reversed the circ&#95;0000376 depletion-mediated anticancer effects and PTX sensitivity. Moreover, miR-1298-5p inhibited PTX resistance and tumorigenesis of PTX-resistant cells, which were abolished by KPNA4 overexpression. In addition, circ&#95;0000376 knockdown suppressed tumor growth and enhanced PTX sensitivity in vivo., Conclusion: Circ&#95;0000376 facilitated PTX resistance and tumorigenesis of NSCLC by miR-1298-5p/KPNA4 axis, suggesting an underlying therapeutic strategy for NSCLC., (© 2023 The Authors. Thoracic Cancer published by China Lung Oncology Group and John Wiley & Sons Australia, Ltd.)

Importin α is a nuclear transporter that binds to nuclear localization signals (NLSs), consisting of 7-20 positively charged amino acids found within cargo proteins. In addition to cargo binding, intramolecular interactions also occur within the importin α protein due to binding between the importin β-binding (IBB) domain and the NLS-binding sites, a phenomenon called auto-inhibition. The interactions causing auto-inhibition are driven by a stretch of basic residues, similar to an NLS, in the IBB domain. Consistent with this, importin α proteins that do not have some of these basic residues lack auto-inhibition; a naturally occurring example of such a protein is found in the apicomplexan parasite Plasmodium falciparum. In this report, we show that importin α from another apicomplexan parasite, Toxoplasma gondii, harbors basic residues (KKR) in the IBB domain and exhibits auto-inhibition. This protein has a long, unstructured hinge motif (between the IBB domain and the NLS-binding sites) that does not contribute to auto-inhibition. However, the IBB domain may have a higher propensity to form an α-helical structure, positioning the wild-type KKR motif in an orientation that results in weaker interactions with the NLS-binding site than a KRR mutant. We conclude that the importin α protein from T. gondii shows auto-inhibition, exhibiting a different phenotype from that of P. falciparum importin α. However, our data indicate that T. gondii importin α may have a low strength of auto-inhibition. We hypothesize that low levels of auto-inhibition may confer an advantage to these important human pathogens., (© 2023. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

The disordered expression of ZNF143 is closely related to the malignant progression of tumours. However, the basic control mechanism of ZNF143 in glioma has not yet been clarified. Therefore, we tried to find a new pathway to illustrate the function of ZNF143 in glioma. To explore the function of KPNA2 in the development of glioma, we used survival analysis by the Kaplan‒Meier method to assess the overall survival (OS) of patients with low and high KPNA2 expression in the TCGA and CGGA cohorts. Western blotting assays and RT‒PCR assays were utilized to determine the expression level of KPNA2 in glioma cells. The interaction between ZNF143 and KPNA2 was confirmed by ChIP assays. Proliferation was assessed by CCK-8 assays, and migration was evaluated by wound healing and Transwell assays. Apoptosis was determined by flow cytometry, and the expression level of YAP/TAZ was visualized using an immunofluorescence assay. The expression levels of LATS1, LATS2, YAP1, and p-YAP1 were determined. Patients with low KPNA2 expression showed a better prognosis than those with high KPNA2 expression. KPNA2 was found to be upregulated in human glioma cells. ZNF143 can bind to the promoter region of KPNA2. Downregulation of ZNF143 and KPNA2 can activate the Hippo signalling pathway and reduce YAP/TAZ expression in human glioma cells, thus inducing apoptosis of human glioma cells and weakening their proliferation, migration and invasion. In conclusion, ZNF143 mediates the Hippo/YAP signalling pathway and inhibits the growth and migration of glioma cells by regulating KPNA2., (© 2023. The Author(s).)

Hepatocellular carcinoma is the most common type of liver cancer and associated with a high fatality rate. This disease poses a major threat to human health worldwide. A considerable number of genetic and epigenetic factors are involved in the development of hepatocellular carcinoma. However, the molecular mechanism underlying the progression of hepatocellular carcinoma remains unclear. Karyopherin subunit alpha 2 (KPNA2), also termed importin α1, is a member of the nuclear transporter family. In recent years, KPNA2 has been gradually linked to the nuclear transport pathway for a variety of tumor-associated proteins. Furthermore, it promotes tumor development by participating in various pathophysiological processes such as cell proliferation, apoptosis, immune response, and viral infection. In hepatocellular carcinoma, it has been found that KPNA2 expression is significantly higher in liver cancer tissues versus paracancerous tissues. Moreover, it has been identified as a marker of poor prognosis and early recurrence in patients with hepatocellular carcinoma. Nevertheless, the role of KPNA2 in the development of hepatocellular carcinoma remains to be determined. This review summarizes the current knowledge on the pathogenesis and role of KPNA2 in hepatocellular carcinoma, and provides new directions and strategies for the diagnosis, treatment, and prediction of prognosis of this disease., Competing Interests: Declaration of Competing Interest The authors declare that they have no competing interests., (Copyright © 2023. Published by Elsevier Masson SAS.)

Karyopherin subunit alpha 2 (KPNA2, importin α1) is a nucleoplasmic protein responsible for the nuclear import of proteins with classical nuclear localization signals. Aberrant nuclear accumulation of KPNA2 has been observed in numerous cancer tissues. AMP-activated protein kinase (AMPK) is involved in the phosphorylation and acetylation of KPNA2 in enterocytes. However, the impact of these post-translational modifications on modulating the nucleocytoplasmic distribution of KPNA2 and its oncogenic role remain unclear. Unlike nuclear accumulation of wild-type KPNA2, which promoted lung cancer cell migration, KPNA2 Lys22 acetylation-mimicking mutations (K22Q and K22Q/S105A) prevented nuclear localization of KPNA2 and reduced the cell migration ability. Cytosolic KPNA2 K22Q interacted with and restricted the nuclear entry of E2F transcription factor 1 (E2F1), an oncogenic cargo protein of KPNA2, in lung cancer cells. Intriguingly, the AMPK activator EX229 promoted the nuclear export of KPNA2 S105A. However, the CBP/p300 inhibitor CCS-1477 abolished this phenomenon, suggesting that CBP/p300-mediated acetylation of KPNA2 promoted KPNA2 nuclear export in lung cancer cells. Collectively, our findings suggest that the CBP/p300 positively regulates KPNA2 acetylation, which enhances its cytosolic localization and suppresses its oncogenic activity in lung cancer., Competing Interests: Declaration of competing interest 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., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Human parvovirus B19 (B19V) is a major human pathogen causing a variety of diseases, characterized by a selective tropism to human progenitor cells in bone marrow. In similar fashion to all Parvoviridae members, the B19V ssDNA genome is replicated within the nucleus of infected cells through a process which involves both cellular and viral proteins. Among the latter, a crucial role is played by non-structural protein (NS)1, a multifunctional protein involved in genome replication and transcription, as well as modulation of host gene expression and function. Despite the localization of NS1 within the host cell nucleus during infection, little is known regarding the mechanism of its nuclear transport pathway. In this study we undertake structural, biophysical, and cellular approaches to characterize this process. Quantitative confocal laser scanning microscopy (CLSM), gel mobility shift, fluorescence polarization and crystallographic analysis identified a short sequence of amino acids (GACHAKKPRIT-182) as the classical nuclear localization signal (cNLS) responsible for nuclear import, mediated in an energy and importin (IMP) α/β-dependent fashion. Structure-guided mutagenesis of key residue K177 strongly impaired IMPα binding, nuclear import, and viral gene expression in a minigenome system. Further, treatment with ivermectin, an antiparasitic drug interfering with the IMPα/β dependent nuclear import pathway, inhibited NS1 nuclear accumulation and viral replication in infected UT7/Epo-S1 cells. Thus, NS1 nuclear transport is a potential target of therapeutic intervention against B19V induced disease., Competing Interests: Declaration of competing interest 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., (Copyright © 2023 Elsevier B.V. All rights reserved.)