Importin Beta Research Articles

Viral Targeting of Importin Alpha-Mediated Nuclear Import to Block Innate Immunity.

Cellular nucleocytoplasmic trafficking is mediated by the importin family of nuclear transport proteins. The well-characterized importin alpha (IMPA) and importin beta (IMPB) nuclear import pathway plays a crucial role in the innate immune response to viral infection by mediating the nuclear import of transcription factors such as IRF3, NFκB, and STAT1. The nuclear transport of these transcription factors ultimately leads to the upregulation of a wide range of antiviral genes, including IFN and IFN-stimulated genes (ISGs). To replicate efficiently in cells, viruses have developed mechanisms to block these signaling pathways. One strategy to evade host innate immune responses involves blocking the nuclear import of host antiviral transcription factors. By binding IMPA proteins, these viral proteins prevent the nuclear transport of key transcription factors and suppress the induction of antiviral gene expression. In this review, we describe examples of proteins encoded by viruses from several different families that utilize such a competitive inhibition strategy to suppress the induction of antiviral gene expression.

Functional analysis of a conserved site mutation in the DNA end processing enzyme PNKP leading to ataxia with oculomotor apraxia type 4 in humans

Polynucleotide kinase 3'-phosphatase (PNKP), an essential DNA end-processing enzyme in mammals with 3'-phosphatase and 5'-kinase activities, plays a pivotal role in multiple DNA repair pathways. Its functional deficiency has been etiologically linked to various neurological disorders. Recent reports have shown that mutation at a conserved glutamine (Gln) in PNKP leads to late-onset ataxia with oculomotor apraxia type 4 (AOA4) in humans and embryonic lethality in pigs. However, the molecular mechanism underlying such phenotypes remains elusive. Here, we report that the enzymatic activities of the mutant versus WT PNKP are comparable; however, cells expressing mutant PNKP and peripheral blood mononuclear cells (PBMCs) of AOA4 patients showed a significant amount of DNA double-strand break accumulation and consequent activation of the DNA damage response. Further investigation revealed that the nuclear localization of mutant PNKP is severely abrogated, and the mutant proteins remain primarily in the cytoplasm. Western blot analysis of AOA4 patient-derived PBMCs also revealed the presence of mutated PNKP predominantly in the cytoplasm. To understand the molecular determinants, we identified that mutation at a conserved Gln residue impedes the interaction of PNKP with importin alpha but not with importin beta, two highly conserved proteins that mediate the import of proteins from the cytoplasm into the nucleus. Collectively, our data suggest that the absence of PNKP in the nucleus leads to constant activation of the DNA damage response due to persistent accumulation of double-strand breaks in the mutant cells, triggering death of vulnerable brain cells-a potential cause of neurodegeneration in AOA4 patients.

Non-transport roles of nuclear import receptors: In need of the right balance.

Nuclear import receptors ensure the recognition and transport of proteins across the nuclear envelope into the nucleus. In addition, as diverse processes as mitosis, post-translational modifications at mitotic exit, ciliogenesis, and phase separation, all share a common need for regulation by nuclear import receptors - particularly importin beta-1 and importin beta-2/transportin - independent on nuclear import. In particular, 1) nuclear import receptors regulate the mitotic spindle after nuclear envelope breakdown, 2) they shield cargoes from unscheduled ubiquitination, regulating their timely proteolysis; 3) they regulate ciliary factors, crucial to cell communications and tissue architecture during development; and 4) they prevent phase separation of toxic proteins aggregates in neurons. The balance of nuclear import receptors to cargoes is critical in all these processes, albeit in opposite directions: overexpression of import receptors, as often found in cancer, inhibits cargoes and impairs downstream processes, motivating the therapeutic design of specific inhibitors. On the contrary, elevated expression is beneficial in neuronal contexts, where nuclear import receptors are regarded as potential therapeutic tools in counteracting the formation of aggregates that may cause neurodegeneration. This paradox demonstrates the amplitude of nuclear import receptors-dependent functions in different contexts and adds complexity in considering their therapeutic implications.

The importin beta superfamily member RanBP17 exhibits a role in cell proliferation and is associated with improved survival of patients with HPV+ HNSCC

BackgroundMore than twenty years after its discovery, the role of the importin beta superfamily member Ran GTP-binding protein (RanBP) 17 is still ill defined. Previously, we observed notable RanBP17 RNA expression levels in head and neck squamous cell carcinoma (HNSCC) cell lines with disruptive TP53 mutations.MethodsWe deployed HNSCC cell lines as well as cell lines from other tumor entities such as HCT116, MDA-MB-231 and H460, which were derived from colon, breast and lung cancers respectively. RNAi was used to evaluate the effect of RanBP17 on cell proliferation. FACS analysis was used for cell sorting according to their respective cell cycle phase and for BrdU assays. Immunocytochemistry was deployed for colocalization studies of RanBP17 with Nucleolin and SC35 (nuclear speckles) domains. TCGA analysis was performed for prognostic assessment and correlation analysis of RanBP17 in HNSCC patients.ResultsRNAi knockdown of RanBP17, significantly reduced cell proliferation in HNSCC cell lines. This effect was also seen in the HNSCC unrelated cell lines HCT116 and MDA-MB-231. Similarly, inhibiting cell proliferation with cisplatin reduced RanBP17 in keratinocytes but lead to induction in tumor cell lines. A similar observation was made in tumor cell lines after treatment with the EGFR kinase inhibitor AG1478. In addition to previous reports, showing colocalization of RanBP17 with SC35 domains, we observed colocalization of RanBP17 to nuclear bodies that are distinct from nucleoli and SC35 domains. Interestingly, for HPV positive but not HPV negative HNSCC, TCGA data base analysis revealed a strong positive correlation of RanBP17 RNA with patient survival and CDKN2A.ConclusionsOur data point to a role of RanBP17 in proliferation of HNSCC and other epithelial cells. Furthermore, RanBP17 could potentially serve as a novel prognostic marker for HNSCC patients. However, we noted a major discrepancy between RanBP17 RNA and protein expression levels with the used antibodies. These observations could be explained by the presence of additional RanBP17 splice isoforms and more so of non-coding circular RanBP17 RNA species. These aspects need to be addressed in more detail by future studies.

LGMD D2 TNPO3-Related: From Clinical Spectrum to Pathogenetic Mechanism.

Limb-girdle muscular dystrophies (LGMDs) are clinically and genetically heterogeneous diseases presenting with a wide clinical spectrum. Autosomal dominant LGMDs represent about 10–15% of LGMDs and include disorders due to defects of DNAJB6, transportin-3 (TNPO3), HNRNPDL, Calpain-3 (CAPN3), and Bethlem myopathy. This review article aims to describe the clinical spectrum of LGMD D2 TNPO3-related, a rare disease due to heterozygous mutation in the TNPO3 gene. TNPO3 encodes for transportin-3, which belongs to the importin beta family and transports into the nucleus serine/arginine-rich (SR) proteins, such as splicing factors, and HIV-1 proteins, thus contributing to viral infection. The purpose of this review is to present and compare the clinical features and the genetic and histopathological findings described in LGMD D2, performing a comparative analytical description of all the families and sporadic cases identified. Even if the causative gene and mutations of this disease have been identified, the pathogenic mechanisms are still an open issue; therefore, we will present an overview of the hypotheses that explain the pathology of LGMD D2 TNPO3-related.

Structural characterization of human importin alpha 7 in its cargo-free form at 2.5\xa0\xc5 resolution

Shuttling of macromolecules between nucleus and cytoplasm is a tightly regulated process mediated through specific interactions between cargo and nuclear transport proteins. In the classical nuclear import pathway, importin alpha recognizes cargo exhibiting a nuclear localization signal, and this complex is transported through the nuclear pore complex by importin beta. Humans possess seven importin alpha isoforms that can be grouped into three subfamilies, with many cargoes displaying specificity towards these importin alpha isoforms. The cargo binding sites within importin alpha isoforms are highly conserved in sequence, suggesting that specificity potentially relies on structural differences. Structures of some importin alpha isoforms, both in cargo-bound and free states, have been previously solved. However, there are currently no known structures of cargo free importin alpha isoforms within subfamily 3 (importin alpha 5, 6, 7). Here, we present the first crystal structure of human importin alpha 7 lacking the IBB domain solved at 2.5 Å resolution. The structure reveals a typical importin alpha architecture comprised of ten armadillo repeats and is most structurally conserved with importin alpha 5. Very little difference in structure was observed between the cargo-bound and free states, implying that importin alpha 7 does not undergo conformational change when binding cargo. These structural insights provide a strong platform for further evaluation of structure–function relationships and understanding how isoform specificity within the importin alpha family plays a role in nuclear transport in health and disease.

Nuclear-localized human respiratory syncytial virus NS1 protein modulates host gene transcription.

SUMMARYHuman respiratory syncytial virus (RSV) is a common cause of lower respiratory tract infections in the pediatric, elderly, and immunocompromised individuals. RSV non-structural protein NS1 is a known cytosolic immune antagonist, but how NS1 modulates host responses remains poorly defined. Here, we observe NS1 partitioning into the nucleus of RSV-infected cells, including the human airway epithelium. Nuclear NS1 coimmunoprecipitates with Mediator complex and is chromatin associated. Chromatin-immunoprecipitation demonstrates enrichment of NS1 that overlaps Mediator and transcription factor binding within the promoters and enhancers of differentially expressed genes during RSV infection. Mutation of the NS1 C-terminal helix reduces NS1 impact on host gene expression. These data suggest that nuclear NS1 alters host responses to RSV infection by binding at regulatory elements of immune response genes and modulating host gene transcription. Our study identifies another layer of regulation by virally encoded proteins that shapes host response and impacts immunity to RSV.

Synthetic hydrogel mimics of the nuclear pore complex for the study of nucleocytoplasmic transport defects in C9orf72 ALS/FTD.

Dipeptide repeats (DPRs) associated with C9orf72 repeat expansions perturb nucleocytoplasmic transport and are implicated in the pathogenesis of amyotrophic lateral sclerosis. We present a synthetic hydrogel platform that can be used to analyze aspects of the molecular interaction of dipeptide repeats and the phenylalanine-glycine (FG) phase of the nuclear pore complex (NPC). Hydrogel scaffolds composed of acrylamide and copolymerized with FG monomers are first formed to recapitulate key FG interactions found in the NPC. With labeled probes, we find evidence that toxic arginine-rich DPRs (poly-GR and poly-PR), but not the non-toxic poly-GP, target NPC hydrogel mimics and block selective entry of a key nuclear transport receptor, importin beta (Impβ). The ease with which these synthetic hydrogel mimics can be adjusted/altered makes them an invaluable tool to dissect complex molecular interactions that underlie cellular transport processes and their perturbation in disease.

P.178A novel mutation in TNPO3 causes congenital limb girdle myopathy with slow progression

We report a second family with autosomal dominant TNPO3/transportinopathy presenting with congenital or early-onset myopathy and slow progression, causing proximal and less pronounced distal muscle weakness. Patients of this Swedish family had clinical examinations, muscle MRI and EMG. Muscle biopsies were used for histopathological and protein expression studies, and TNPO3 constructs were used to study the effect of the mutations in transfected cells. By using the MYOcap targeted exome sequencing gene panel, we identified a novel heterozygous mutation, c.2757delC, in the last part of the Transportin-3/TNPO3 gene in the affected family members. The mutation causes an almost identical frameshift affecting the stop codon and elongating the C-term protein product of the TNPO3 transcript as was previously reported in the first large Spanish-Italian LGMD1F kindred. TNPO3 belongs to the importin beta superfamily, facilitating the nuclear import of Ser/Arg-rich (SR) proteins. TNPO3 plays a role in HIV infection, however, little is known of its normal functions in muscle. We found that the TNPO3 protein was increased in the patient muscle, and accumulated in the subsarcolemmal and perinuclear areas in muscle biopsy. One of the cargo proteins, the splicing factor SRRM2 was normally located in the nucleus, whereas RBM4 showed perinuclear accumulation. In addition, transiently transfected mutant TNPO3 constructs failed to localize to cytoplasmic annulate lamellae pore complexes in cultured cells. We report the clinical, molecular genetic and histopathological features of the second transportinopathy family. The variability of the clinical phenotype together with histopathological findings suggest that several molecular pathways may be involved in the disease pathomechanism, such as nucleo-cytoplasmic shuttling, protein aggregation, and defective protein turnover.

Abstract 5138: Inhibition of kPNB1 and NUPR1 binding increase the anti-cancer drug sensitivity

Abstract Karyopherin beta 1 (KPNB1, known as importin beta 1) is carrier protein, it is known as transporter of protein from cytoplasm to nucleus through nuclear pore complexes (NPCs) using KPNAs as an adapters. The nuclear protein-1 (NUPR1, known as p8/Com-1) is identified as a transcription factor and it is showed multifunctional mechanisms related to cellular stress response such as serum-starvation, oxidative stress and drug stimulation. In this study, we investigated protein-protein interaction between NUPR1 and KPNB1, it might be a new target for cancer therapy.For the evaluation of binding affinity between protein and inhibition effect by compound 1, we processed the single molecule binding assay. Cytotoxicity of combination treatment with doxorubicin and compound 1 were validated several cancer cell lines such as MDA-MB-231, PC-3, SK-OV-3, and SiHa. Gene expression patterns were analyzed RNA-Seq and validated with qRT-PCR and western-blotting. KPNB1 bind to NUPR1 as a high affinity compare to KPNB1 single treatment assay and there was no difference according to concentration of NUPR1. KPNB1 and NUPR1 protein-protein interaction was inhibited by compound 1 according to concentration of compound 1 showed 71%, 44%, and 31% of inhibition rate with 50nM, 10nM, 2.5nM of compound 1, respectively. In doxorubicin single treatment, NUPR1 move to nucleus more than 90% of cells compare to non-treated MDA-MB-231 cells. Otherwise, NUPR1 was exist both nucleus and cytoplasm more than 90% cells, which is indicate that translocation of NUPR1 to the nucleus was blocked by compound 1 treatment. And combination treatment showed synergistic effect of cytotoxicity in various cancer cells such as such as MDA-MB-231, SiHa, PC-3, and SK-OV-3 (1.4 ~ 6 fold). Additionally, NUPR1 related genes were showed change of gene expression in compound 1 combination compared to doxorubicin single treatment. In these results suggest that translocation mechanism of NUPR1 into nucleus bind to KPNB1 and this binding was inhibited by compound 1 which is new candidate drug combination method for cancer therapy. Citation Format: Chan Hee Park, Seung Wook Ham, HyeKyoung Shin, Kyung Soo Oh. Inhibition of kPNB1 and NUPR1 binding increase the anti-cancer drug sensitivity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 5138.

Identification of a nuclear localization signal and importin beta members mediating NUAK1 nuclear import inhibited by oxidative stress.

NUAK1 is a serine/threonine kinase member of the AMPK-α family. NUAK1 regulates several processes in tumorigenesis; however, its regulation and molecular targets are still poorly understood. Bioinformatics analysis predicted that the majority of NUAK1 localizes in the nucleus. However, there are no studies about the regulation of NUAK1 subcellular distribution. Here, we analyzed NUAK1 localization in several human cell lines, mouse embryo fibroblasts, and normal mouse tissues. We found that NUAK1 is located in the nucleus and also in the cytoplasm. Through bioinformatics analysis and studies comparing subcellular localization of wild type and NUAK1 mutants, we identified a conserved bipartite nuclear localization signal at the N-terminal domain of NUAK1. Based on mass spectrometry analysis, we found that NUAK1 interacts with importin-β members including importin-β1 (KPNB1), importin-7 (IPO7), and importin-9 (IPO9). We confirmed that importin-β members are responsible for NUAK1 nuclear import through the inhibition of importin-β by Importazole and the knockdown of either IPO7 or IPO9. In addition, we found that oxidative stress induces NUAK1 cytoplasmic accumulation, indicating that oxidative stress affects NUAK1 nuclear transport. Thus, our study is the first evidence of an active nuclear transport mechanism regulating NUAK1 subcellular localization. These data will lead to investigations of the molecular targets of NUAK1 according to its subcellular distribution, which could be new biomarkers or targets for cancer therapies.

Nuclear import of the DSCAM ‐cytoplasmic domain drives signaling capable of inhibiting synapse formation

DSCAM and DSCAML1 are immunoglobulin and cell adhesion-type receptors serving important neurodevelopmental functions including control of axon growth, branching, neurite self-avoidance, and neuronal cell death. The signal transduction mechanisms or effectors of DSCAM receptors, however, remain poorly characterized. We used a human ORFeome library to perform a high-throughput screen in mammalian cells and identified novel cytoplasmic signaling effector candidates including the Down syndrome kinase Dyrk1a, STAT3, USP21, and SH2D2A. Unexpectedly, we also found that the intracellular domains (ICDs) of DSCAM and DSCAML1 specifically and directly interact with IPO5, a nuclear import protein of the importin beta family, via a conserved nuclear localization signal. The DSCAM ICD is released by γ-secretase-dependent cleavage, and both the DSCAM and DSCAML1 ICDs efficiently translocate to the nucleus. Furthermore, RNA sequencing confirms that expression of the DSCAM as well as the DSCAML1 ICDs alone can profoundly alter the expression of genes associated with neuronal differentiation and apoptosis, as well as synapse formation and function. Gain-of-function experiments using primary cortical neurons show that increasing the levels of either the DSCAM or the DSCAML1 ICD leads to an impairment of neurite growth. Strikingly, increased expression of either full-length DSCAM or the DSCAM ICD, but not the DSCAML1 ICD, significantly decreases synapse numbers in primary hippocampal neurons. Taken together, we identified a novel membrane-to-nucleus signaling mechanism by which DSCAM receptors can alter the expression of regulators of neuronal differentiation and synapse formation and function. Considering that chromosomal duplications lead to increased DSCAM expression in trisomy 21, our findings may help uncover novel mechanisms contributing to intellectual disability in Down syndrome.

Visualization of human karyopherin beta-1/importin beta-1 interactions with protein partners in mitotic cells by co-immunoprecipitation and proximity ligation assays

Karyopherin beta-1/Importin beta-1 is a conserved nuclear transport receptor, acting in protein nuclear import in interphase and as a global regulator of mitosis. These pleiotropic functions reflect its ability to interact with, and regulate, different pathways during the cell cycle, operating as a major effector of the GTPase RAN. Importin beta-1 is overexpressed in cancers characterized by high genetic instability, an observation that highlights the importance of identifying its partners in mitosis. Here we present the first comprehensive profile of importin beta-1 interactors from human mitotic cells. By combining co-immunoprecipitation and proteome-wide mass spectrometry analysis of synchronized cell extracts, we identified expected (e.g., RAN and SUMO pathway factors) and novel mitotic interactors of importin beta-1, many with RNA-binding ability, that had not been previously associated with importin beta-1. These data complement interactomic studies of interphase transport pathways. We further developed automated proximity ligation assay (PLA) protocols to validate selected interactors. We succeeded in obtaining spatial and temporal resolution of genuine importin beta-1 interactions, which were visualized and localized in situ in intact mitotic cells. Further developments of PLA protocols will be helpful to dissect importin beta-1-orchestrated pathways during mitosis.

Survival, bacterial clearance and thrombocytopenia are improved in polymicrobial sepsis by targeting nuclear transport shuttles.

The rising tide of sepsis, a leading cause of death in the US and globally, is not adequately controlled by current antimicrobial therapies and supportive measures, thereby requiring new adjunctive treatments. Severe microvascular injury and multiple organ failure in sepsis are attributed to a “genomic storm” resulting from changes in microbial and host genomes encoding virulence factors and endogenous inflammatory mediators, respectively. This storm is mediated by stress-responsive transcription factors that are ferried to the nucleus by nuclear transport shuttles importins/karyopherins. We studied the impact of simultaneously targeting two of these shuttles, importin alpha 5 (Imp α5) and importin beta 1 (Imp β1), with a cell-penetrating Nuclear Transport Modifier (NTM) in a mouse model of polymicrobial sepsis. NTM reduced nuclear import of stress-responsive transcription factors nuclear factor kappa B, signal transducer and activator of transcription 1 alpha, and activator protein 1 in liver, which was also protected from sepsis-associated metabolic changes. Strikingly, NTM without antimicrobial therapy improved bacterial clearance in blood, spleen, and lungs, wherein a 700-fold reduction in bacterial burden was achieved while production of proinflammatory cytokines and chemokines in blood plasma was suppressed. Furthermore, NTM significantly improved thrombocytopenia, a prominent sign of microvascular injury in sepsis, inhibited neutrophil infiltration in the liver, decreased L-selectin, and normalized plasma levels of E-selectin and P-selectin, indicating reduced microvascular injury. Importantly, NTM combined with antimicrobial therapy extended the median time to death from 42 to 83 hours and increased survival from 30% to 55% (p = 0.022) as compared to antimicrobial therapy alone. This study documents the fundamental role of nuclear signaling mediated by Imp α5 and Imp β1 in the mechanism of polymicrobial sepsis and highlights the potential for targeting nuclear transport as an adjunctive therapy in sepsis management.

Importin-β and CRM1 control a RANBP2 spatiotemporal switch essential for mitotic kinetochore function.

Protein conjugation with small ubiquitin-related modifier (SUMO) is a post-translational modification that modulates protein interactions and localisation. RANBP2 is a large nucleoporin endowed with SUMO E3 ligase and SUMO-stabilising activity, and is implicated in some cancer types. RANBP2 is part of a larger complex, consisting of SUMO-modified RANGAP1, the GTP-hydrolysis activating factor for the GTPase RAN. During mitosis, the RANBP2-SUMO-RANGAP1 complex localises to the mitotic spindle and to kinetochores after microtubule attachment. Here, we address the mechanisms that regulate this localisation and how they affect kinetochore functions. Using proximity ligation assays, we find that nuclear transport receptors importin-β and CRM1 play essential roles in localising the RANBP2-SUMO-RANGAP1 complex away from, or at kinetochores, respectively. Using newly generated inducible cell lines, we show that overexpression of nuclear transport receptors affects the timing of RANBP2 localisation in opposite ways. Concomitantly, kinetochore functions are also affected, including the accumulation of SUMO-conjugated topoisomerase-IIα and stability of kinetochore fibres. These results delineate a novel mechanism through which nuclear transport receptors govern the functional state of kinetochores by regulating the timely deposition of RANBP2.

Control of brown patch (Rhizoctonia solani) in tall fescue (Festuca arundinacea Schreb.) by host induced gene silencing.

Transgenic tall fescue plants expressing RNAi constructs of essential genes of Rhizoctonia solani were resistant to R. solani. Tall fescue (Festuca arundinacea Schreb.) is an important turf and forage grass species widely used for home lawns and on golf courses in North Carolina and other transition zone states in the US. The most serious and frequently occurring disease of tall fescue is brown patch, caused by a basidiomycete fungus, Rhizoctonia solani. This research demonstrates resistance to brown patch disease achieved by the application of host induced gene silencing. We transformed tall fescue with RNAi constructs of four experimentally determined "essential" genes from R. solani (including genes encoding RNA polymerase, importin beta-1 subunit, Cohesin complex subunit Psm1, and a ubiquitin E3 ligase) to suppress expression of those genes inside the fungus and thus inhibit fungal infection. Four gene constructs were tested, and 19 transgenic plants were obtained, among which 12 plants had detectable accumulation of siRNAs of the target genes. In inoculation tests, six plants displayed significantly improved resistance against R. solani. Lesion size was reduced by as much as 90 %. Plants without RNAi accumulation did not show resistance. To our knowledge, this is the first case that RNAi constructs of pathogen genes introduced into a host plant can confer resistance against a necrotrophic fungus.

Antitumor effects of pharmacological EZH2 inhibition on malignant peripheral nerve sheath tumor through the miR-30a and KPNB1 pathway.

BackgroundEnhancer of zeste homolog 2 (EZH2) is a key epigenetic regulator in cancer cell survival, epithelial-mesenchymal transition, and tumorigenesis. Inhibition of EZH2 has become a promising therapeutic option for various human malignancies. Previously, we demonstrated that the EZH2/miR-30d/karyopherin (importin) beta 1 (KPNB1) signaling pathway is critical for malignant peripheral nerve sheath tumor (MPNST) cell survival in vitro and for tumorigenesis in vivo. Here, we sought to determine the antitumor effects of pharmacological inhibition of EZH2 on MPNST in vitro and in vivo.MethodsWe investigated the effects of an EZH2 inhibitor, 3-deazaneplanocin A (DZNep), on MPNST cell cycle, survival and apoptosis in vitro and on MPNST xenograft tumor growth in vivo.ResultsWe found that DZNep treatment impaired MPNST cell viability and proliferation by inducing apoptosis and cell cycle arrest in vitro. Consistently, DZNep treatment also reduced EZH2 and KPNB1 protein levels and upregulated miR-30d expression in MPNST cells. Intraperitoneal administration of DZNep significantly suppressed MPNST tumor initiation and growth rates in a MPNST xenograft mouse model. Immunoblot and immunohistochemical analyses showed that DZNep downregulated EZH2/KPNB1 signaling in vivo, thereby inhibiting MPNST tumor cell proliferation, and induced cell death. We also found that EZH2 inhibited expression of another miR-30 family member, miR-30a, in MPNST cells. Similar to miR-30d, miR-30a inhibited KPNB1 by targeting the KPNB1 3’ untranslated region in MPNST cells. Our data also showed that EZH2 suppressed miR-200b expression and induced epithelial-mesenchymal transition in MPNST cells.ConclusionThese findings demonstrated that DZNep, an inhibitor of S-adenosyl-methionine–dependent methyltransferase, suppressed EZH2/miR-30a,d/KPNB1 signaling and blocked MPNST tumor cell growth and survival in vitro and in vivo. More importantly, our study indicated that pharmacological interference of EZH2 is a potential therapeutic approach for MPNST.Electronic supplementary materialThe online version of this article (doi:10.1186/s12943-015-0325-1) contains supplementary material, which is available to authorized users.

Regulation of Renal Fibrosis by Smad3 Thr388 Phosphorylation

Transforming growth factor-β (TGF-β) promotes tissue fibrosis via receptor-mediated phosphorylation of the receptor-activated Smad2/3, together with Smad4. Of these, Smad3 plays a major profibrotic role in mouse models of tissue fibrosis. Transcriptional activity of the Smad3 protein is regulated by phosphorylation of residues in the C-terminal domain and the linker region. Herein, we examined the role of a novel phosphorylation site within the MH2 domain (T388) in the regulation of Smad3 activity. Confocal microscopy using an Smad3 phosphorylated T388-specific antibody identified phosphorylation of Smad3 T388 in myofibroblasts and tubular epithelial cells in human focal and segmental glomerulosclerosis and mouse models of unilateral ureteric obstruction and diabetic nephropathy, whereas phosphorylated T388 was largely absent in normal kidney. In vitro, TGF-β1 induced phosphorylation of Smad3 T388 in a biphasic pattern. A point mutation of T388/V in an Smad3 construct demonstrated that phosphorylation of T388 promotes Smad3 binding to Smad4 and CDK8, but was not necessary for nuclear translocation. Furthermore, T388 phosphorylation was required for TGF-β-induced collagen I gene promoter activity and extracellular matrix production in cultured fibroblasts. In conclusion, our study identifies phosphorylation of T388 in the Smad3 MH2 domain as an important mechanism that regulates the profibrotic TGF-β/Smad3 signaling pathway, which has direct relevance to human and experimental fibrotic kidney disease.

KPNB1 (karyopherin (importin) beta 1)

Review on KPNB1 (karyopherin (importin) beta 1), with data on DNA, on the protein encoded, and where the gene is implicated.

Calcium mediated regulation of karyopherin nuclear transport receptors

Oxidative stress disrupts the normal function of cells and severe stress situations can result in apoptosis. A specific effect of oxidant stress is an increase in cellular calcium levels, which can be mimicked by treatment with the drug thapsigargin, which inhibits Ca2+ uptake into the endoplasmic reticulum. We sought to determine if changes in cellular calcium levels induced by thapsigargin disrupted nuclear transport by alteration of karyopherin transport receptors and/or the Ran gradient. Altered Ca2+ levels were monitored using a fluorescence based assay. We found that thapsigargin lead to an accumulation of Ran in the cytoplasm, while prolonged exposure led to cell death. However, the disruption of the Ran gradient could be reversed before the induction of apoptosis and rescue was enhanced by the presence of the calcium chelator BAPTA. Thapsigargin treated cells also exhibited altered location of several karyopherin transport receptors including Transportin, Importin alpha, Importin beta, Crm1, and its cofactor RanBP3. These results suggest that alteration of cellular calcium levels causes disruption of the Ran gradient and variation in karyopherin localization, suggesting that there is a role for calcium as a regulator of Ran‐mediated nuclear transport pathways. Therefore, cellular oxidative stress may result in apoptosis due to the calcium induced alteration of nuclear transport pathways.