Nuclear transport proteins: structure, function, and disease relevance.

Proper subcellular localization is crucial for the functioning of biomacromolecules, including proteins and RNAs. Nuclear transport is a fundamental cellular process that regulates the localization of many macromolecules within the nuclear or cytoplasmic compartments. In humans, approximately 60 proteins are involved in nuclear transport, including nucleoporins that form membrane-embedded nuclear pore complexes, karyopherins that transport cargoes through these complexes, and Ran system proteins that ensure directed and rapid transport. Many of these nuclear transport proteins play additional and essential roles in mitosis, biomolecular condensation, and gene transcription. Dysregulation of nuclear transport is linked to major human diseases such as cancer, neurodegenerative diseases, and viral infections. Selinexor (KPT-330), an inhibitor targeting the nuclear export factor XPO1 (also known as CRM1), was approved in 2019 to treat two types of blood cancers, and dozens of clinical trials of are ongoing. This review summarizes approximately three decades of research data in this field but focuses on the structure and function of individual nuclear transport proteins from recent studies, providing a cutting-edge and holistic view on the role of nuclear transport proteins in health and disease. In-depth knowledge of this rapidly evolving field has the potential to bring new insights into fundamental biology, pathogenic mechanisms, and therapeutic approaches.

Drosophila screen connects nuclear transport genes to DPR pathology in c9ALS/FTD.

Hexanucleotide repeat expansions in C9orf72 are the most common cause of amyotrophic lateral sclerosis (ALS) and frontotemporal degeneration (FTD) (c9ALS/FTD). Unconventional translation of these repeats produces dipeptide repeat proteins (DPRs) that may cause neurodegeneration. We performed a modifier screen in Drosophila and discovered a critical role for importins and exportins, Ran-GTP cycle regulators, nuclear pore components, and arginine methylases in mediating DPR toxicity. These findings provide evidence for an important role for nucleocytoplasmic transport in the pathogenic mechanism of c9ALS/FTD.

The transcriptional repression activity of STAF65γ is facilitated by promoter tethering and nuclear import of class IIa histone deacetylases.

Aberrant expression levels of transcriptional regulators result in alterations in transcriptional control. STAF65γ is a structural subunit of the GCN5 transcriptional co-activator complex. Reports showed that STAF65γ is highly expressed in several human cancer cells, but the consequences of this aberrant expression pattern remain elusive. Here, we show that the STAF65γ protein is highly expressed in lung adenocarcinoma patients and high levels of STAF65γ correlate with poor prognosis. High levels of STAF65γ cause repression of the c-Myc oncogene through physical association with transcription factor YY1 and co-repressors HDACs. Physical interactions between STAF65γ and class IIa HDACs facilitate nuclear enrichment and regulate the assembly of HDAC complexes. Moreover, SUMOylation of STAF65γ is necessary for maintaining the co-repressor complex containing YY1 and class IIa HDACs at the promoter. Our findings reveal a distinct role of STAF65γ in nuclear import, transcriptional repression, and cell cycle regulation at high levels of expression, which is associated with poor clinical outcomes of lung adenocarcinoma.

Wogonoside induces cell cycle arrest and differentiation by affecting expression and subcellular localization of PLSCR1 in AML cells.

Wogonoside is the main flavonoid component derived from the root of Scutellaria baicalensis Georgi. It is a popular Chinese herbal medicine with the potential to treat hematologic malignancies. In this study, we investigated the anticancer effects of wogonoside in acute myeloid leukemia (AML) cell lines and primary patient-derived AML cells. Wogonoside exerted antiproliferative properties both in vitro and in vivo. Furthermore, it efficiently inhibited the proliferation of U937 and HL-60 cells through the induction of G1 phase arrest and the promotion of differentiation. We also demonstrated that wogonoside significantly increased the transcription of phospholipid scramblase 1 (PLSCR1) due to its influence on the expression of cell cycle- and differentiation-related genes, including the upregulation of p21waf1/cip1 and downregulation of the oncogenic protein c-Myc. Wogonoside also promoted PLSCR1 trafficking into the nucleus and facilitated its binding to the inositol 1,4,5-trisphosphate receptor 1 (IP3R1) promoter, thus increasing the expression of IP3R1. Finally, inhibition of PLSCR1 expression with small interfering RNA partially blocked wogonoside-induced cell cycle arrest and differentiation and disturbed the wogonoside-associated molecular events. The results of this study therefore suggest that wogonoside may represent a therapeutic candidate for the treatment of AML.

Nuclear factor κB subunits RelB and cRel negatively regulate Toll-like receptor 3-mediated ß-interferon production via induction of transcriptional repressor protein YY1.

The induction of ß-interferon (IFN-ß) is a key anti-viral response to infection by RNA viruses. Virus-induced expression of IFN-ß requires the co-operative action of the transcription factors IRF-3/7, NF-κB, and ATF-2/c-Jun on the IFN-ß promoter leading to the orderly recruitment of chromatin remodeling complexes. Although viruses strongly activate NF-κB and promote its binding to the IFN-ß promoter, recent studies have indicated that NF-κB is not essential for virus-induced expression of IFN-ß. Herein, we examined the role of NF-κB in regulating IFN-ß expression in response to the viral-sensing Toll-like receptor 3 (TLR3). Intriguingly pharmacological inhibition of the NF-κB pathway augments late phase expression of IFN-ß expression in response to TLR3 stimulation. We show that the negative effect of NF-κB on IFN-ß expression is dependent on the induction of the transcriptional repressor protein YinYang1. We demonstrate that the TLR3 ligand polyriboinosinic:polyribocytidylic acid (poly(I:C)) induces expression and nuclear translocation of YinYang1 where it interacts with the IFN-ß promoter and inhibits the binding of IRF7 to the latter. Evidence is also presented showing that the NF-κB subunits c-Rel and RelB are the likely key drivers of these negative effects on IFN-ß expression. These findings thus highlight for the first time a novel self-regulatory mechanism that is employed by TLR3 to limit the level and duration of IFN-ß expression.

Identification of NR4A2 as a transcriptional activator of IL-8 expression in human inflammatory arthritis.

Expression of the orphan nuclear receptor NR4A2 is controlled by pro-inflammatory mediators, suggesting that NR4A2 may contribute to pathological processes in the inflammatory lesion. This study identifies the chemoattractant protein, interleukin 8 (IL-8/CXCL8), as a molecular target of NR4A2 in human inflammatory arthritis and examines the mechanism through which NR4A2 modulates IL-8 expression. In TNF-alpha-activated human synoviocyte cells, enhanced expression of IL-8 mRNA and protein correspond to temporal changes in NR4A2 transcription and nuclear distribution. Ectopic expression of NR4A2 leads to robust changes in endogenous IL-8 mRNA levels and co-treatment with TNF-alpha results in significant (p<0.001) secretion of IL-8 protein. Transcriptional effects of NR4A2 on the human IL-8 promoter are enhanced in the presence of TNF-alpha, suggesting molecular crosstalk between TNF-alpha signalling and NR4A2. A dominant negative IkappaB kinase antagonizes the combined effects of NR4A2 and TNF-alpha on IL-8 promoter activity. Co-expression of NR4A2 and the p65 subunit of NF-kappaB enhances IL-8 transcription and functional studies indicate that transactivation occurs independently of NR4A2 binding to DNA or heterodimerization with additional nuclear receptors. The IL-8 minimal promoter region is sufficient to support NR4A2 and NF-kappaB/p65 co-operative activity and NR4A2 can interact with NF-kappaB/p65 on a 39bp sequence within this region. In patients treated with methotrexate for active inflammatory arthritis, a reduction in NR4A2 synovial tissue levels correlate significantly (n=10, r=0.73, p=0.002) with changes in IL-8 expression. Collectively, these data delineate an important role for NR4A2 in modulating IL-8 expression and reveal novel transcriptional responses to TNF-alpha in human inflammatory joint disease.

Fusion of OTT to BSAC results in aberrant up-regulation of transcriptional activity.

OTT/RBM15-BSAC/MAL/MKL1/MRTF-A was identified as a fusion transcript generated by t(1;22)(p13;q13) in acute megakaryoblastic leukemia. Previous studies have shown that BSAC (basic, SAP, and coiled-coil domain) activates the promoters containing CArG boxes via interaction with serum response factor, and OTT (one twenty-two) negatively regulates the development of megakaryocytes and myeloid cells. However, the mechanism by which OTT-BSAC promotes leukemia is largely unknown. Here we show that OTT-BSAC, but not BSAC or OTT strongly activates several promoters containing a transcription factor Yin Yang 1-binding sequence. In addition, although BSAC predominantly localizes in the cytoplasm and its nuclear translocation is considered to be regulated by the Rho-actin signaling pathway, OTT-BSAC exclusively localizes in the nucleus. Moreover, OTT interacts with histone deacetylase 3, but this interaction is abolished in OTT-BSAC. Collectively, these functional and spatial changes of OTT and BSAC caused by the fusion might perturb their functions, culminating in the development of acute megakaryoblastic leukemia.

A high soy diet reduces programmed cell death and enhances bcl-xL expression in experimental stroke.

Soy phytoestrogens have been proposed as an alternative to estrogen replacement therapy and have demonstrated potential neuroprotective effects in the brain. We have shown that a high soy diet significantly reduces infarct size following permanent middle cerebral artery occlusion (MCAO). Here, we tested the hypothesis that a high soy diet would attenuate programmed cell death after stroke. Adult female Sprague-Dawley rats were ovariectomized and fed either an isoflavone-reduced diet (IFP) or a high soy diet (SP) for 2 weeks before undergoing 90 min of transient middle cerebral artery occlusion (tMCAO) followed by 22.5 h reperfusion. Infarct size, as assessed by triphenyltetrazolium chloride staining, was significantly reduced by a high soy diet (P<0.05). Apoptosis in the ischemic cortex, measured by TUNEL staining, was significantly reduced by the high soy diet. The number of active caspase-3 positive cells and caspase-mediated alpha-spectrin cleavage were also significantly decreased in the ischemic cortex of SP rats. Furthermore, nuclear translocation of apoptosis-inducing factor (AIF) was significantly reduced in the ischemic cortex of SP rats. Soy significantly increased bcl-x(L) mRNA and protein expression in the ischemic cortex compared with IFP rats. Immunohistochemistry revealed increased neuronal expression of bcl-2 and bcl-x(L) in the ischemic cortex of both IFP and SP rats following tMCAO. These results suggest that a high soy diet decreases both caspase-dependent and caspase-independent programmed cell death following tMCAO. Further, a high soy diet enhances expression of the cell survival factor bcl-x(L) following tMCAO, contributing to the neuroprotective effects of soy in the ischemic cortex.

The CREB coactivator TORC2 functions as a calcium- and cAMP-sensitive coincidence detector.

Elevations in circulating glucose and gut hormones during feeding promote pancreatic islet cell viability in part via the calcium- and cAMP-dependent activation of the transcription factor CREB. Here, we describe a signaling module that mediates the synergistic effects of these pathways on cellular gene expression by stimulating the dephosphorylation and nuclear entry of TORC2, a CREB coactivator. This module consists of the calcium-regulated phosphatase calcineurin and the Ser/Thr kinase SIK2, both of which associate with TORC2. Under resting conditions, TORC2 is sequestered in the cytoplasm via a phosphorylation-dependent interaction with 14-3-3 proteins. Triggering of the calcium and cAMP second messenger pathways by glucose and gut hormones disrupts TORC2:14-3-3 complexes via complementary effects on TORC2 dephosphorylation; calcium influx increases calcineurin activity, whereas cAMP inhibits SIK2 kinase activity. Our results illustrate how a phosphatase/kinase module connects two signaling pathways in response to nutrient and hormonal cues.

A novel interaction between calreticulin and ubiquitin-like nuclear protein in rice.

Calreticulin (CRT), a major Ca2+ -sequestering protein, has been implicated in a variety of cellular functions such as Ca2+ storage, signaling and chaperone activity within the cytoplasm and endoplasmic reticulum. To investigate the biological role of CRT in rice, 21 partial cDNAs, encoding proteins that interacted with rice CRT in a yeast two-hybrid interaction-cloning system, were characterized and the nucleotide sequences were found to be identical to each other. A full-length cDNA of 3.5 kb, obtained from rice genomic sequence data and 5' RACE, codes for a novel protein of 966 amino acid residues and was designated as CRTintP (CRT interacting protein). Primary sequence analysis of CRTintP showed no sequence homology with the known functional proteins; however, a potential ubiquitin-like domain at the N-terminal together with a putative leucine zipper, a nuclear localization signal and several sites for serine/threonine kinases were evident. Cellular localization of CRTintP demonstrated its role in directing green fluorescent protein to the nucleus in onion epidermal cells. Northern and immunoblot analysis showed increased expression of CRT and CRTintP in response to cold stress. Co-immunoprecipitation using anti-CRT antibodies confirmed the existence of the CRT-CRTintP complex in vivo in the stressed leaf tissue, suggesting their potential role in regulating stress response.

Cyclin B1 and CDK1: nuclear localization and upstream regulators.

Formation of an active nuclear cyclin B1-CDK1 complex is a highly intricate procedure requiring many different levels of regulation. Each of these regulatory steps represents a potential target for controlling cell proliferation. Accumulation of threshold levels of cyclin B1 protein at the G2/M transition requires the cooperation of various promoter elements, possibly the activation of several transcription factors, enhanced cyclin B1 mRNA stability and, in some cases, translational activation of dormant mRNA. Binding of cyclin B1 to its inactive partner, CDK1, initiates conformational changes allowing CDK1 to alter its phosphorylation status and to become an active kinase. Lastly, the active cyclin B1-CDK1 complex must translocate to the nucleus to begin phosphorylating nuclear substrates. These phosphorylation events are necessary for mitotic onset. While cyclin B1 is capable of shuttling from the nucleus to the cytoplasm throughout interphase, mitotic onset requires phosphorylation of cyclin B1 within the CRS region, thereby enhancing import and inhibiting export of the cyclin B1-CDK1 complex. Elucidating the role of mediators controlling cyclin B1-CDK1 translocation at the onset of mitosis is essential in developing drug targets for cell cycle control.

T cell hyporesponsiveness induced by oral administration of ovalbumin is associated with impaired NFAT nuclear translocation and p27kip1 degradation.

Oral tolerance is an important physiological component of the immune system whereby the organism avoids dangerous reactions such as hypersensitivity to ingested food proteins and other luminal Ags which may cause tissue damage and inflammation. In addition, it has been shown in animal models and in humans that oral tolerance can be applied to controlling undesired immune responses, including autoimmune diseases, allergies, and organ transplant rejections. However, the molecular mechanisms of oral tolerance have been poorly defined. In this study, we investigated the molecular basis underlying the hyporesponsiveness of orally tolerant CD4 T cells using a TCR transgenic mouse system in which oral tolerance was induced by long-term feeding with high dose Ag. We demonstrate that the hyporesponsive state of the CD4 T cells was maintained by a selective impairment in the TCR-induced calcium/NFAT signaling pathway and in the IL-2R-induced degradation of p27(kip1) and cell cycle progression. Thus, physiological mucosal tolerance is revealed to be associated with a unique type of T cell hyporesponsiveness which differs from previously described anergic T cells.