Nuclear Localization Research Articles

AYAP1-2 contributes to bFGF-induced proliferation In gastric cancer.

Gastric cancer (GC) is one of the leading causes of cancer-related deaths in humans worldwide. Fibroblast growth factor family (FGFs) and the Hippo signaling pathway play an important role in the epithelial-mesenchymal transition (EMT) process of GC. YAP1, a key mediator of the Hippo pathway, plays an important role in tumor genesis. Alternative splicing of human YAP1 mRNA results in two major isoforms: YAP1-1, which contains a single WW domain, and YAP1-2, which contains two WW domains, respectively. There are significant differences in post-transcriptional regulation and function. Basic FGF (bFGF) treatment promoted the EMT process of most GC cell lines, and the proliferation ability was enhanced. This process may be related to the upregulation of YAP1, the proliferation ability of GC was significantly alleviated upon YAP1 knockdown. bFGF treatment can induce EMT of GC through YAP1-2 and enhance their proliferative ability. In this process, bFGF may enhance the nuclear localization of YAP1-2.In the mouse model of intraperitoneal implantation tumorigenesis, it was shown that under the action of bFGF, the expressing YAP1-2 cell lines could form larger tumors than the expressing YAP1-1, but both of them were larger than the YAP1 knockdown. Our results show that YAP1-2 is the main subtype of bFGF-induced EMT and proliferation of GC cells.

TPR is required for cytoplasmic chromatin fragment formation during senescence.

During oncogene-induced senescence there are striking changes in the organisation of heterochromatin in the nucleus. This is accompanied by activation of a pro-inflammatory gene expression programme - the senescence associated secretory phenotype (SASP) - driven by transcription factors such as NF-κB. The relationship between heterochromatin re-organisation and the SASP has been unclear. Here we show that TPR, a protein of the nuclear pore complex basket required for heterochromatin re-organisation during senescence, is also required for the very early activation of NF-κB signalling during the stress-response phase of oncogene-induced senescence. This is prior to activation of the SASP and occurs without affecting NF-κB nuclear import. We show that TPR is required for the activation of innate immune signalling at these early stages of senescence and we link this to the formation of heterochromatin-enriched cytoplasmic chromatin fragments thought to bleb off from the nuclear periphery. We show that HMGA1 is also required for cytoplasmic chromatin fragment formation. Together these data suggest that re-organisation of heterochromatin is involved in altered structural integrity of the nuclear periphery during senescence, and that this can lead to activation of cytoplasmic nucleic acid sensing, NF-κB signalling, and activation of the SASP.

KPNA3 regulates histone locus body formation by modulating condensation and nuclear import of NPAT.

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.

Ubiquitin Ligase U-Box51 Positively Regulates Drought Stress in Potato (Solanum tuberosum L.)

The ubiquitin-proteasome system (UPS) is a key protein degradation pathway in eukaryotes, in which E3 ubiquitin ligases mediate protein ubiquitination, directly or indirectly targeting substrate proteins to regulate various biological processes, including plant growth, hormone signaling, immune responses, and adaptation to abiotic stress. In this study, we identified plant U-box protein 51 in Solanum tuberosum (StPUB51) as an E3 ubiquitin ligase through transcriptomic analysis, and used it as a candidate gene for gene-function analysis. Quantitative real-time PCR (qRT-PCR) was used to examine StPUB51 expression across different tissues, and its expression patterns under simulated drought stress induced by polyethylene glycol (PEG 6000) were assessed. Transgenic plants overexpressing StPUB51 and plants with down-regulated StPUB51 expression were generated to evaluate drought tolerance. The activities of key antioxidant enzymes-superoxide dismutase (SOD), catalase (CAT), and peroxidase (POD) as well as malondialdehyde (MDA) content in transgenic plants’ leaves were measured under drought conditions. Protein–protein interactions involving StPUB51 were explored via yeast two-hybrid (Y2H) screening, with interaction verification by bimolecular fluorescence complementation (BiFC). StPUB51 was predominantly expressed in stems, with lower expression observed in tubers, and its expression was significantly upregulated in response to 20% PEG-6000 simulated drought. Subcellular localization assays revealed nuclear localization of the StPUB51 protein. Under drought stress, StPUB51-overexpressing plants exhibited enhanced SOD, POD, and CAT activities and reduced MDA levels, in contrast to plants with suppressed StPUB51 expression. Y2H and BiFC analyses identified two interacting proteins, StSKP2A and StGATA1, which may be functionally linked to StPUB51. Collectively, these findings suggest that StPUB51 plays a positive regulatory role in drought tolerance, enhancing resilience in potato growth and stress adaptation.

Feedback control of the heat shock response by spatiotemporal regulation of Hsp70.

Cells maintain homeostasis via dynamic regulation of stress response pathways. Stress pathways transiently induce response regulons via negative feedback loops, but the extent to which individual genes provide feedback has not been comprehensively measured for any pathway. Here, we disrupted the induction of each gene in the Saccharomyces cerevisiae heat shock response (HSR) and quantified cell growth and HSR dynamics following heat shock. The screen revealed a core feedback loop governing the expression of the chaperone Hsp70 reinforced by an auxiliary feedback loop controlling Hsp70 subcellular localization. Mathematical modeling and live imaging demonstrated that multiple HSR targets converge to promote Hsp70 nuclear localization via its release from cytosolic condensates. Following ethanol stress, a distinct set of factors similarly converged on Hsp70, suggesting that nonredundant subsets of the HSR regulon confer feedback under different conditions. Flexible spatiotemporal feedback loops may broadly organize stress response regulons and expand their adaptive capacity.

The RNA-dependent association of phosphatidylinositol 4,5-bisphosphate with intrinsically disordered proteins contribute to nuclear compartmentalization.

The RNA content is crucial for the formation of nuclear compartments, such as nuclear speckles and nucleoli. Phosphatidylinositol 4,5-bisphosphate (PIP2) is found in nuclear speckles, nucleoli, and nuclear lipid islets and is involved in RNA polymerase I/II transcription. Intriguingly, the nuclear localization of PIP2 was also shown to be RNA-dependent. We therefore investigated whether PIP2 and RNA cooperate in the establishment of nuclear architecture. In this study, we unveiled the RNA-dependent PIP2-associated (RDPA) nuclear proteome in human cells by mass spectrometry. We found that intrinsically disordered regions (IDRs) with polybasic PIP2-binding K/R motifs are prevalent features of RDPA proteins. Moreover, these IDRs of RDPA proteins exhibit enrichment for phosphorylation, acetylation, and ubiquitination sites. Our results show for the first time that the RDPA protein Bromodomain-containing protein 4 (BRD4) associates with PIP2 in the RNA-dependent manner via electrostatic interactions, and that altered PIP2 levels affect the number of nuclear foci of BRD4 protein. Thus, we propose that PIP2 spatiotemporally orchestrates nuclear processes through association with RNA and RDPA proteins and affects their ability to form foci presumably via phase separation. This suggests the pivotal role of PIP2 in the establishment of a functional nuclear architecture competent for gene expression.

Clinical spectrum and molecular basis in 19 Chinese patients with 46, XY disorder of sexual development caused by NR5A1 mutations

BackgroundNuclear receptor subfamily 5 group A member 1 (NR5A1) plays pivotal roles in steroidogenesis and gonadal development. 46, XY disorder of sexual development (DSD) caused by NR5A1 mutations is a rare genetic condition. This study aimed to provide a comprehensive analysis of the clinical characteristics and molecular defects observed in 19 Chinese patients with NR5A1 variants, including assessing the deleterious effects of novel variants in vitro and evaluating their functional impact on the gonad and adrenal glands in vivo.Materials and methodsSubjects with NR5A1 variants were identified from 223 Chinese 46, XY DSD patients via next-generation sequencing. In-silico analysis and functional assays were performed to evaluate the transcriptional activity, expression levels and nuclear localization of novel NR5A1 variants. The histological structure of the gonads was evaluated via immunohistochemistry (IHC).ResultsPatients with NR5A1 gene variants presented with serious conditions, including micropenis, cryptorchidism, azoospermia, and radiological abnormalities of the spleen. Five novel NR5A1 variants were identified, including heterozygous p.Y5*, p.Q42E and p.L359_L363del, as well as copy number variation (CNV) of chr9:127213317–127570245_del and an exon 6 duplication. A total of 63.2% (12/19) of patients harbored additional variants other than NR5A1. Defective transcriptional regulatory activities and abnormal protein expression levels were observed in NR5A1 variants. The reduced levels of DHEA-S and 11-oxygenated steroids indicate a mild impairment in adrenal function among certain patients. The IHC analysis of the testis revealed intact expression levels of SOX9 in Sertoli cells, while significant differences were observed in the expression pattern of CYP17A1 in Leydig cells among patients. The preserved maturation of adult Leydig cells in the patients may trigger spontaneous puberty.ConclusionsPatients with NR5A1 mutations exhibit complex phenotypes. The observed clinical heterogeneity may be attributed to oligogenic mutations, dysregulated Leydig cell function, as well as the impaired ability to modulate the transcription of target genes.

VOZ‐dependent priming of salicylic acid‐dependent defense against Rhizopus stolonifer by β‐aminobutyric acid requires the TCP protein TCP2 in peach fruit

SUMMARYVascular plant one‐zinc finger (VOZ) transcription factors (TFs) play crucial roles in plant immunity. Nevertheless, how VOZs modulate defense signaling in response to elicitor‐induced resistance is not fully understood. Here, the defense elicitor β‐aminobutyric acid (BABA) resulted in the visible suppression of Rhizopus rot disease of peach fruit caused by Rhizopus stolonifer. Defense priming by BABA was notably associated with increased levels of salicylic acid (SA) and SA‐dependent gene expression. Data‐independent acquisition proteomic analysis revealed that two VOZ proteins (PpVOZ1 and PpVOZ2) were substantially upregulated in BABA‐induced resistance (BABA‐IR). Furthermore, the interaction of PpVOZ1 and PpVOZ2 and their potential target of the TEOSINTE‐BRANCHED1/CYCLOIDEA/PCF (TCP)‐family protein PpTCP2 screened from protein–protein interaction networks was confirmed by yeast two‐hybrid (Y2H), luciferase complementation imaging and glutathione S‐transferase pull‐down assays. Furthermore, subcellular localization, yeast one‐hybrid, electrophoretic mobility shift assay and dual‐luciferase reporter assays demonstrated that nuclear localization of both PpVOZ1 and PpVOZ2 was critical for their contribution to BABA‐IR, as these proteins potentiated the PpTCP2‐mediated transcriptional activation of isochorismate synthase genes (ICS1/2). The overexpression of both PpVOZ1 and PpVOZ2 could activate the transcription of SA‐dependent genes and provide disease resistance in transgenic Arabidopsis. In contrast, the ppvoz1cas9 and ppvoz2cas9 loss‐of‐function mutations and the voz1cas9 voz2cas9 double mutation attenuated BABA‐IR against R. stolonifer. Therefore, the three identified positive TFs, PpVOZ1, PpVOZ2, and PpTCP2, synergistically contribute to the BABA‐activated priming of systemic acquired resistance in postharvest peach fruit by a VOZ‐TCP‐ICS regulatory module.

SIRT7 stabilizes β-catenin and promotes canonical Wnt activation via upregulating FZD7

AimsThe dysregulated Wnt/β-Catenin signaling pathway leads to occurrence of various diseases, and abnormal activation of β-Catenin is a major characteristic of human HCC. FZD7 is a positive regulator of the Wnt/β-catenin signaling pathway, and its upregulation is related to increase of β-catenin expression and carcinogenesis in human HCC. However, mechanisms underlying FZD7 upregulation in HCC remain elusive. Main methodsNuclear cytosol fractionation, immunofluorescence and Top-Flash were used to detect the activation of β-Catenin. Protein half-life and ubiquitination assays were applied to evaluate protein stability. RNA-seq combined with qRT-PCR was used to evaluate differential gene expressions after SIRT7 knockdown. Wound healing and transwell assays were used to measure cancer cell migration. Key findingsSIRT7-mediated FZD7 expression is essential for stability and activation of β-catenin. Knockdown SIRT7 in HCC cells resulted in enhanced binding of β-catenin to the DC, decreased its stability, nuclear localization and activation. Knockdown FZD7 reversed SIRT7 overexpression mediated β-catenin stabilization and impairment of binding of β-catenin to the DC. At molecular level, SIRT7 promotes FZD7 expression via upregulating transcription factor PU.1, knockdown PU.1 abolished SIRT7-mediated upregulation of FZD7. Finally, we confirmed that FZD7 was responsible for SIRT7-mediated β-catenin stabilization and HCC cells migration. By using clinical samples, we observed strong positive correlations between SIRT7 and PU.1, FZD7, p-GSK3β and β-Catenin in human HCC. SignificanceOur results thus revealed a previously undisclosed role of SIRT7 in regulating the canonical Wnt/β-catenin signaling pathway, thereby offering additional evidence that SIRT7 holds promise as a novel therapeutic target for human HCC.

Maternal exposure to 4-tert-octylphenol causes alterations in the morphology and function of microglia in the offspring mouse brain

4-tert-Octylphenol (OP), an endocrine disrupting chemical is widely used in the production of industrial products. Prenatal exposure to endocrine-disrupting chemicals negatively affects the brain. However, the influence of OP exposure during neurodevelopment in adult offspring remains unclear. Thus, in the present study, we investigated the effects of maternal OP exposure on brain development in adult offspring by analyzing primary glial cell cultures and mice. Our findings revealed that OP exposure led to a specific increase in the mRNA expression of the ionized calcium-binding adapter molecule 1 (Iba-1) and the proportion of amoeboid microglia in the primary glial cell culture and adult offspring mice. Exposure to OP increased the transcriptional activation of Iba-1 and estrogen response element, which were counteracted by estrogen receptor antagonists ICI 182,780. Moreover, OP exposure increased the nuclear localization of the estrogen receptor. Remarkably, OP exposure decreased the mRNA expression levels of proinflammatory cytokines and genes associated with immune response in the brains of the offspring. OP exposure upregulated actin filament-related genes and altered cytoskeletal gene expression, as demonstrated by microarray analysis. The morphological changes in microglia did not result in an inflammatory response following lipopolysaccharide treatment. Taken together, the effects of OP exposure during neurodevelopment persist into adulthood, resulting in microglial dysfunction mediated by estrogen receptor signaling pathways in the brains of adult offspring mice.

Detection of Hepatitis C Virus Infection from Patient Sera in Cell Culture Using Semi-Automated Image Analysis

The study of hepatitis C virus (HCV) replication in cell culture is mainly based on cloned viral isolates requiring adaptation for efficient replication in Huh7 hepatoma cells. The analysis of wild-type (WT) isolates was enabled by the expression of SEC14L2 and by inhibitors targeting deleterious host factors. Here, we aimed to optimize cell culture models to allow infection with HCV from patient sera. We used Huh7-Lunet cells ectopically expressing SEC14L2, CD81, and a GFP reporter with nuclear translocation upon cleavage by the HCV protease to study HCV replication, combined with a drug-based regimen for stimulation of non-modified wild-type isolates. RT-qPCR-based quantification of HCV infections using patient sera suffered from a high background in the daclatasvir-treated controls. We therefore established an automated image analysis pipeline based on imaging of whole wells and iterative training of a machine learning tool, using nuclear GFP localization as a readout for HCV infection. Upon visual validation of hits assigned by the automated image analysis, the method revealed no background in daclatasvir-treated samples. Thereby, infection events were found for 15 of 34 high titer HCV genotype (gt) 1b sera, revealing a significant correlation between serum titer and successful infection. We further show that transfection of viral RNA extracted from sera can be used in this model as well, albeit with so far limited efficiency. Overall, we generated a robust serum infection assay for gt1b isolates using semi-automated image analysis, which was superior to conventional RT-qPCR-based quantification of viral genomes.

Andrographolide attenuates SARS-CoV-2 infection via an up-regulation of glutamate-cysteine ligase catalytic subunit (GCLC)

BackgroundAndrographolide is a medicinal compound which possesses anti-SARS-CoV-2 activity. A number of cellular targets of andrographolide have been identified by target predictions and computational studies. PurposeHowever, a potential cellular target of andrographolide has never been explored in SARS-CoV-2 infected lung epithelial cells. We aimed to identify cellular pathways involved in andrographolide-mediated anti-SARS-CoV-2 activity. MethodsThe viral infection was determined by immunofluorescence staining, enzyme-linked immunosorbent assay and focus-forming assay. Proteomic analysis was employed to identify cellular pathways and key proteins controlled by andrographolide in the human lung epithelial cells Calu-3 infected by SARS-CoV-2. Immunofluorescence staining was used to test protein expression and localization. Western blot and realtime PCR were utilized to elucidate gene expression. Cellular glutathione level was examined by a reduced/oxidized glutathione assay. An ectopic gene expression was delivered by plasmid transfection. ResultsGene ontology analysis indicates that proteins involved in nuclear factor erythroid 2-related factor 2 (NRF2)-regulated pathways were differentially expressed by andrographolide. Notably, andrographolide increased expression and nuclear localization of the transcription factor NRF2. In addition, transcriptional expression of GCLC and glutamate-cysteine ligase modifier subunit (GCLM), which are NRF2 target genes, were induced by andrographolide. We further find that infection of SARS-CoV-2 resulted in a reduction of glutathione level in Calu-3; the effect that was rescued by andrographolide. Moreover, andrographolide also induced expression of the glutathione producing enzyme GCLC in SARS-CoV-2 infected lung epithelial cells. Importantly, an ectopic over-expression of GCLC or treatment of N-acetyl-L-cysteine in Calu-3 cells led to a decrease in SARS-CoV-2 infection. ConclusionCollectively, our findings suggest the interplay between GCLC-mediated glutathione biogenesis induced by andrographolide and the anti-SARS-CoV-2 activity. The glutathione biogenesis and recycling pathways should be further exploited as a targeted therapy against SARS-CoV-2 infection.

Elucidating loss-of-function mechanisms of monoallelic EPAS1 mutations underlying congenital hypoplastic anaemia in a paediatric anaemia cohort.

HIF-2α, encoded by EPAS1, plays a dominant role in regulating erythropoietin (EPO) production, maintaining the dynamic balance of erythropoiesis. Gain-of-function mutations in EPAS1 cause erythrocytosis. However, anaemia caused by EPAS1 loss-of-function mutations has been confined to only one case report, and the underlying mechanism remains unclear. Herein, the reanalysis of high-throughput sequencing data from 311 patients with anaemia identified three monoallelic EPAS1 variants from three unrelated families in a paediatric anaemia cohort. The probands showed highly consistent clinical phenotypes with normocytic and normochromic anaemia, reticulocytopenia and relative deficiency of serum EPO, characterised as congenital hypoplastic anaemia. Invitro studies suggested that defects in steady-state protein abundance, nuclear localisation and binding with co-activator in EPAS1 variants lead to impaired EPO transcriptional activation. Therefore, loss-of-function mutations in EPAS1 can cause erythroid hypoplasia in an EPO-dependent manner. This study identified a new causative gene for congenital hypoplastic anaemia and clarified the molecular aetiology of loss-of-function EPAS1 mutations.

Golodirsen restores DMD transcript imbalance in Duchenne Muscular Dystrophy patient muscle cells

BackgroundAntisense oligonucleotides (AON) represent a promising treatment for Duchenne muscular dystrophy (DMD) carrying out-of-frame deletions, but also show limitations. In a completed clinical trial golodirsen, approved by FDA to induce skipping of DMD gene exon 53 in eligible patients, we demonstrated increase in DMD expression and protein production, albeit with inter-patient variability.MethodsHere, we investigate further the golodirsen mechanism of action using myotubes derived from MyoD transfected fibroblasts isolated from DMD patients at the baseline of the clinical trial SRP-4053.ResultsWe confirm golodirsen’s selectivity and efficiency in removing only exon 53. For the first time in human cells, we revealed a significant reduction in the so called DMD “transcript imbalance”, in golodirsen-treated DMD muscle cultures. The transcript imbalance is a unique DMD phenomenon characterized by non-homogeneous transcript expression along its entire length and responsible for the reduced stability of the transcript. Our in-vivo study also showed that the efficiency of exon skipping did not always correspond to a proportional restoration of the dystrophin protein. Predominant nuclear localization of the DMD transcript, observed in patients and animal models, persists even after exon skipping.ConclusionAll these findings suggest challenges other than AON delivery for high level of protein restoration in DMD, highlighting the importance of investigating the biological mechanisms upstream of protein production to further enhance the efficiency of any AON treatment in this condition.

A rare genetic variant in APEX1 is associated with familial amyotrophic lateral sclerosis with slow progression.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder characterized by loss of motor neurons and progressive muscle weakness. We aimed to identify the pathogenic genetic variants in familial ALS (fALS) pedigrees and to elucidate their impact on the disease phenotype. Through the analysis of whole-genome sequencing data of 34 fALS probands that screened negative for mutations in the most common ALS-causing genes, we identified a rare missense variant in APEX1 (NM_001641.4: c.22G > A, p.Gly8Arg) associated with ALS in one pedigree. Fluorescence microscopy images using green fluorescent protein (GFP)-fusion proteins suggested that this amino acid substitution could cause an impairment in nuclear localization of the protein. We described the clinical characteristics of this cohort analyzed and found that patients carrying this variant exhibit lower motor neuron onset and prolonged survival. The relation between APEX1 and ALS occurrence has been elusive despite evidence of a neuroprotective role for the gene. This study provides evidence linking an APEX1 variant with fALS and information on the distinct clinical manifestation. This study contributes to the understanding of the genetic basis of ALS, as well as a potential mechanism leading to loss of neurons, highlighting possible opportunities of targeted treatment harnessing the DNA repair process or ameliorating the oxidative stress.

The Shh-p38-NFATc1 signaling pathway is essential for osteoclastogenesis during tooth eruption

Tooth eruption, a critical stage in tooth development, is related to osteoclastogenesis. Intraperitoneal injection of Shh agonists into neonatal mice promoted tooth eruption at postnatal day (PN) 15, whereas treatment with the Shh inhibitor (LDE225) suppressed this process. When RAW264.7 osteoclast precursor cells were treated with RANKL, NFATc1 translocated from the cytoplasm to the nucleus and induced cell differentiation into TRAP+ osteoclasts; this process was activated by Shh but inhibited by LDE225. Treating RAW264.7 cells with the p38 inhibitor, BIRB796, also inhibited NFATc1 nuclear localization. p-p38 expression in the alveolar bone of PN3 and PN5 mice was decreased by treatment with LDE225, and RAW264.7 cell differentiation was reduced by BIRB796, regardless of treatment with Shh. Furthermore, Shh activated p38 signaling pathway in RAW264.7 cells, while p38 phosphorylation was reduced by LDE225, which ultimately inhibited osteoclast precursor differentiation. Therefore, we concluded that Shh promotes osteoclast precursor differentiation via the p38-NFATc1 signaling pathway.

Mapping of Nuclear Localization Signal in Secreted Liver-Specific Protein 2 of Plasmodium falciparum.

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.

KPNB1-ATF4 induces BNIP3-dependent mitophagy to drive odontoblastic differentiation in dental pulp stem cells

BackgroundDifferentiating dental pulp stem cells (DPSCs) into odontoblasts is a critical process for tooth self-repair and dentine‒pulp engineering strategies in the clinic. However, the mechanism underlying the regulation of DPSC odontoblastic differentiation remains largely unknown. Here, we demonstrated that BCL-2 interacting protein 3 (BNIP3)-dependent mitophagy is associated with importin subunit beta-1 (KPNB1)-activating transcription factor 4 (ATF4), which promotes DPSC odontoblastic differentiation.MethodsThe key genes involved in DPSC odontogenic differentiation were identified via bioinformatics. Stable silencing or overexpression of BNIP3 was performed to investigate its impact on DPSC differentiation in vitro (n ≥ 3). To explore the role of BNIP3 in vivo, tooth root fragments loaded with the hydrogel-transfected DPSC complex were implanted into nude mice (n ≥ 6). Dual-luciferase reporter assays and chromatin immunoprecipitation (ChIP) polymerase chain reaction (PCR) were conducted to explore the binding site of ATF4 to the BNIP3 promoter (n ≥ 3). Mitochondrial function experiments were performed to investigate the impact of ATF4-BNIP3 on mitochondria (n ≥ 3). Immunoprecipitation (IP) mass spectrometry (MS) was used to investigate the interaction between ATF4 and its binding protein, KPNB1. Plasmids containing wild-type (WT)/mutant (MUT)-nuclear localization signal (NLS) forms of ATF4 were constructed to determine the specific amino acid residues recognized by KPNB1 and their effects on DPSC odontoblastic differentiation (n ≥ 3).ResultsCompared with those in the control group, the levels of autophagy and mitophagy, especially BNIP3-dependent mitophagy, were greater in the DPSC odontoblastic differentiation group (P < 0.05). Genetic silencing or overexpression of BNIP3 demonstrated that BNIP3 expression was positively correlated with the transition of DPSCs into odontoblasts both in vitro and in vivo (P < 0.05). ATF4 regulates the expression of BNIP3 by directly binding to approximately −1292 to −1279 bp and approximately −1185 to −1172 bp within the BNIP3 promoter region, which is associated with mitophagy and mitochondrial reactive oxygen species (mtROS) levels (P < 0.05). Moreover, ATF4 increased mitophagy, mitochondrial function, and cell differentiation potential via BNIP3 (P < 0.05). Mechanistically, KPNB1 is a novel interacting protein of ATF4 that specifically recognizes amino acids (aa) 280–299 within ATF4 to control its translocation into the nucleus and subsequent transcription and differentiation processes (P < 0.05).ConclusionsWe reported that the critical role of KPNB1/ATF4/BNIP3 axis-dependent mitophagy could provide new cues for the regeneration of the dental pulp‒dentin complex in DPSCs.Graphical

Identification of SLC35A1 as an essential host factor for the transduction of multi-serotype recombinant adeno-associated virus (AAV) vectors.

We conducted a genome-wide CRISPR/Cas9 screen in suspension 293 F cells transduced with rAAV5. The highly selected genes revealed after two rounds of screening included the previously reported KIAA0319L, TM9SF2, and RNF121, along with a cluster of genes involved in glycan biogenesis, Golgi apparatus localization, and endoplasmic reticulum penetration. In this report, we focused on solute carrier family 35 member A1 (SLC35A1), a Golgi apparatus-localized cytidine 5'-monophosphate-sialic acid (CMP-SIA) transporter. We confirmed that SLC35A1 knockout (KO) significantly decreased rAAV5 transduction to a level lower than that observed in KIAA0319L or TM9SF2 KO cells. Although SLC35A1 KO drastically reduced the expression of α2,6-linked SIA on the cell surface, the expression of α2,3-linked SIA, as well as the cell binding and internalization of rAAV5, was only moderately affected. Moreover, SLC35A1 KO significantly diminished the transduction of AAV multi-serotypes, including rAAV2 and rAAV3, which do not utilize SIAs for primary attachment. Notably, the SLC35A1 KO markedly increased transduction of rAAV9 and rAAV11, which primarily attach to cells via binding to galactose. Further analyses revealed that SLC35A1 KO significantly decreased vector nuclear import. More importantly, although the C-terminal cytoplasmic tail deletion (∆C Tail) mutant of SLC35A1 did not drastically decrease SIA expression, it significantly decreased rAAV transduction, as well as vector nuclear import, suggesting that the C-tail is critical in these processes. Furthermore, the T128A mutant significantly decreased SIA expression but still supported rAAV transduction and nuclear import. These findings highlight the involvement of the CMP-SIA transporter in the intracellular trafficking of rAAV vectors post-internalization.IMPORTANCErAAV is an essential tool for gene delivery in the treatment of genetic disorders; however, the mechanisms of rAAV transduction remain partially understood. GPR108 is vital for the transduction of most rAAV vectors, but not for rAAV5. We aimed to identify host factors that impact AAV5 transduction akin to GPR108. Using a genome-wide CRISPR/Cas9 screen in 293 F cells, we identified SLC35A1, a Golgi apparatus-localized CMP-sialic acid transporter that transports CMP-sialic acid from the cytoplasm into the Golgi apparatus for sialylation, is essential to rAAV transduction. Further studies across various AAV serotypes showed SLC35A1 significantly affects vector nuclear import post-internalization. These results underscore the crucial role of SLC35A1 in intracellular trafficking beyond the initial cell attachment of rAAV.

Structurally Distinct Nurr1 Ligands Exhibit Different Pharmacological Characteristics in Regulating Inflammatory Responses of Microglial BV-2 Cells.

Nurr1 (NR4A2) is a member of nuclear receptor superfamily that regulates gene transcription in midbrain dopaminergic neurons and also inhibits nuclear factor-κB-mediated inflammatory responses in brain microglial cells. To date, various compounds have been reported to stimulate transcriptional activity of Nurr1 on neuronal genes, but their anti-inflammatory actions are poorly characterized. The present study examined the effects of three kinds of Nurr1 ligands, amodiaquine, 1,1-bis(3'-indolyl)-1-(p-chlorophenyl)-methane (C-DIM12) and 5-chloronaphthalen-1-amine (5-CNA), on inflammatory responses of microglial BV-2 cells. Lipopolysaccharide (LPS)-induced upregulation of interleukin-1β mRNA and tumor necrosis factor α mRNA was inhibited by all three compounds, whereas upregulation of interleukin-6 mRNA and inducible nitric oxide synthase (iNOS) mRNA was significantly inhibited only by 5-CNA. On the other hand, LPS-induced nuclear translocation of p65 subunit of nuclear factor-κB was prevented only by amodiaquine. C-DIM12 increased nuclear localization of Nurr1 and transiently upregulated Nurr1 protein expression, whereas amodiaquine and 5-CNA had no effect on these parameters. Notably, inhibitory effect of 5-CNA on iNOS mRNA upregulation was reversed by co-application of amodiaquine. Conversely, inhibitory effect of amodiaquine on p65 nuclear translocation was cancelled by 5-CNA. These results reveal distinct characteristics of anti-inflammatory actions of Nurr1 ligands.