Potential Nuclear Localization Signal Research Articles

Multiple types of nuclear localization signals in Entamoeba histolytica

Entamoeba histolytica is a protozoan parasite that belongs to the Amoebozoa supergroup whose study related to the nucleocytoplasmic transport of proteins through the nucleus is poorly studied. In this work, we have performed in silico predictions of the potential nuclear localization signals (NLS) corresponding to the proteome of 8201 proteins from Entamoeba histolytica annotated in the AmoebaDB database. We have found the presence of monopartite nuclear localization signals (MNLSs), bipartite nuclear localization signals (BNLSs), and non-canonical monopartite NLSs with lengths exceeding 20 amino acid residues. Additionally, we detected a new type of NLS consisting of multiple juxtaposed bipartite NLSs (JNLSs) that have not been described in any eukaryotic organism. Also, we have generated consensus sequences for the nuclear import of proteins with the NLSs obtained. Docking experiments between EhImportin α and an MNLS, BNLS, and JNLS outlined the interacting residues between the Importin and cargo proteins, emphasizing their putative roles in nuclear import. By transfecting HA-tagged protein constructs, we assessed the nuclear localization of MNLS (U1A and U2AF1), JMNLS (U2AF2), and non-canonical NLS (N-terminus of Pol ll) in vivo. Our data provide the basis for understanding the nuclear transport process in E. histolytica.

Verticillium dahliae VdPBP1 Transcription Factor Is Required for Hyphal Growth, Virulence, and Microsclerotia Formation.

Verticillium dahliae, a fungal pathogen that affects more than 200 plant species, including tomatoes, requires specific proteins for its early steps in plant infection. One such crucial protein, VdPBP1, exhibits high expression in the presence of tomato roots. Its 313-amino acid C-terminal section restores adhesion in nonadhesive Saccharomyces cerevisiae strains. To uncover its role, we employed a combination of bioinformatics, genetics, and morphological analyses. Our findings underscore the importance of VdPBP1 in fungal growth and pathogenesis. Bioinformatic analysis revealed that the VdPBP1 gene consists of four exons and three introns, encoding a 952-codon reading frame. The protein features a 9aaTAD domain, LsmAD, and PAB1 DNA-binding sites, as well as potential nuclear localization and transmembrane helix signals. Notably, the deletion of a 1.1 kb fragment at the gene's third end impedes microsclerotia formation and reduces pathogenicity. Mutants exhibit reduced growth and slower aerial mycelial development compared to the wild type. The VdPBP1 deletion strain does not induce disease symptoms in tomato plants. Furthermore, VdPBP1 deletion correlates with downregulated microsclerotia formation-related genes, and promoter analysis reveals regulatory elements, including sites for Rfx1, Mig1, and Ste12 proteins. Understanding the regulation and target genes of VdPBP1 holds promise for managing Verticillium wilt disease and related fungal pathogens.

Structural and functional characterization of siadenovirus core protein VII nuclear localization demonstrates the existence of multiple nuclear transport pathways.

Adenovirus protein VII (pVII) plays a crucial role in the nuclear localization of genomic DNA following viral infection and contains nuclear localization signal (NLS) sequences for the importin (IMP)-mediated nuclear import pathway. However, functional analysis of pVII in adenoviruses to date has failed to fully determine the underlying mechanisms responsible for nuclear import of pVII. Therefore, in the present study, we extended our analysis by examining the nuclear trafficking of adenovirus pVII from a non-human species, psittacine siadenovirus F (PsSiAdV). We identified a putative classical (c)NLS at pVII residues 120-128 (120PGGFKRRRL128). Fluorescence polarization and electrophoretic mobility shift assays demonstrated direct, high-affinity interaction with both IMPα2 and IMPα3 but not IMPβ. Structural analysis of the pVII-NLS/IMPα2 complex confirmed a classical interaction, with the major binding site of IMPα occupied by K124 of pVII-NLS. Quantitative confocal laser scanning microscopy showed that PsSiAdV pVII-NLS can confer IMPα/β-dependent nuclear localization to GFP. PsSiAdV pVII also localized in the nucleus when expressed in the absence of other viral proteins. Importantly, in contrast to what has been reported for HAdV pVII, PsSiAdV pVII does not localize to the nucleolus. In addition, our study demonstrated that inhibition of the IMPα/β nuclear import pathway did not prevent PsSiAdV pVII nuclear targeting, indicating the existence of alternative pathways for nuclear localization, similar to what has been previously shown for human adenovirus pVII. Further examination of other potential NLS signals, characterization of alternative nuclear import pathways, and investigation of pVII nuclear targeting across different adenovirus species is recommended to fully elucidate the role of varying nuclear import pathways in the nuclear localization of pVII.

Exploring the surface epitope and nuclear localization analysis of porcine circovirus type 3 capsid protein

Porcine circovirus 3 (PCV3) is a newly emerging virus associated with porcine dermatitis and nephropathy syndrome (PDNS) and reproductive disorders, impacting global pig populations. Porcine circoviruses contain two major open reading frames (ORFs), and the ORF2 encodes the viral capsid protein (Cap). Cap is the most antigenic structural protein and an ideal candidate for the development of vaccines and diagnostic reagents. This study generated a monoclonal antibody (MAb) specific to PCV3 Cap, MAb CCC160, for diagnosis and pathogenesis studies of this novel virus. The MAb specifically recognized PCV3-infected swine lymph node tissue in an immunohistochemical analysis confirming its clinical diagnostic potential. In addition, a novel linear B-cell epitope recognized by MAb CCC160 was identified at the amino acid region 120–134 of Cap. Nuclear localization analysis of PCV3 Cap revealed a potential nuclear localization signal (NLS) in the middle region (aa 131–143) in addition to the dominant N-terminal NLS that is already known. A cell viability assay further demonstrated that the cytotoxicity of PCV3 Cap is correlated with its nuclear localization, indicating a crucial role of Cap in the pathogenic mechanism of PCV3. A full-length construct of PCV3 Cap was successfully expressed using a baculovirus expression system and purified recombinant proteins self-assembled into virus-like particles (VLPs). The protein constitution of the VLPs was confirmed by MAb CCC160 recognition, indicating the correct conformation and specificity of VLP and exhibiting the linear epitope aa 120–134 on the VLP surface. These results provide insights for developing diagnostic tools and potential VLP vaccines for PCV3, revealing its pathogenesis and antigenic properties.

TRIB1 regulates liver regeneration by antagonizing the NRF2-mediated antioxidant response

Robust regenerative response post liver injuries facilitates the architectural and functional recovery of the liver. Intrahepatic redox homeostasis plays a key role in liver regeneration. In the present study, we investigated the contributory role of Tribbles homolog 1 (Trib1), a pseudokinase, in liver regeneration and the underlying mechanism. We report that Trib1 expression was transiently down-regulated in animal and cell models of liver regeneration. Further analysis revealed that hepatocyte growth factor (HGF) repressed Trib1 transcription by evicting liver X receptor (LXRα) from the Trib1 promoter. Knockdown of Trib1 enhanced whereas over-expression of Trib1 suppressed liver regeneration after partial hepatectomy in mice. Of interest, regulation of liver regenerative response by Trib1 coincided with alterations of intracellular ROS levels, GSH levels, and antioxidant genes. Transcriptional assays suggested that Trib1 influenced cellular redox status by attenuating nuclear factor erythroid 2-related factor 2 (Nrf2) activity. Mechanistically, Trib1 interacted with the C-terminus of Nrf2 thus masking a potential nuclear localization signal (NLS) and blocking nuclear accumulation of Nrf2. Finally, correlation between Trib1 expression, Nrf2 nuclear localization, and cell proliferation was identified in liver specimens taken from patients with acute liver failure. In conclusion, our data unveil a novel pathway that depicts Trib1 as a critical link between intracellular redox homeostasis and cell proliferation in liver regeneration.

Identification of type II interferons and receptors in an osteoglossiform fish, the arapaima Arapaima gigas

In mammals, type II interferon (IFN; i.e. IFN-γ) signalling transduces through its specific receptors IFN-γR1 and IFN-γR2. In an osteoglossiform fish, the arapaima Arapaima gigas, three type II IFNs, IFN-γ-like, IFN-γ and IFN-γrel, and their four possible receptor subunits IFN-γR1-1, IFN-γR1-2, IFN-γR2-1 and IFN-γR2-2 were identified in this study. The three type II IFN genes are composed of four exons and three introns, and they all contain IFN-γ signature motif and signal peptide, with the presence of potential nuclear localization signal (NLS) in IFN-γ-like and IFN-γ. The IFN-γR1-1, IFN-γR1-2, IFN-γR2-1 and IFN-γR2-2 are composed of seven exons and six introns, with predicted IFN-γR1-1 and IFN-γR1-2 proteins containing JAK1 and STAT1 binding sites, and IFN-γR2-1 and IFN-γR2-2 containing JAK2 binding sites. Gene synteny analysis showed that the type II IFN and their receptor loci are duplicated in arapaima. All these genes were expressed constitutively in all organs/tissues examined, and responded to the stimulation of polyI:C. The prokaryotic recombinant IFN-γ-like, IFN-γ and IFN-γrel proteins can significantly induce the upregulation of immune-related genes in trunk kidney leucocytes. The ligand-receptor relationship analyses revealed that recombinant IFN-γ-like, IFN-γ, and IFN-γrel transduce downstream signalling through IFN-γR1-1/IFN-γR2-1, IFN-γR1-2/IFN-γR2-2, and IFN-γR1-1, respectively, in xenogeneic cells with the overexpression of original or chimeric receptors. In addition, tyrosine (Y) 366 and Y377 in the intracellular region may be essential for the function of IFN-γR1-2 and IFN-γR1-1, respectively. The finding of type II IFN system in A. gigas thus provides different knowledge in understanding the diversity and evolution of type II IFN ligand-receptor relationships in vertebrates.

A Hydrogen-Sulfide-Repressed Methionine Synthase SlMS1 Acts as a Positive Regulator for Fruit Ripening in Tomato.

Ethylene is a key phytohormone that regulates the ripening of climacteric fruits, and methionine is an indirect precursor of ethylene. However, whether methionine synthase plays a role in fruit ripening in Solanum lycopersicum (tomato) is still unknown. In this study, we find that a tomato methionine synthase (named SlMS1), which could be repressed at the transcriptional level by hydrogen sulfide (H2S), acts as a positive regulator for tomato fruit ripening. By a bioinformatics analysis, it is found that SlMS1 and SlMS2 in tomato are highly homologous to methionine synthases in Arabidopsis thaliana. The expression pattern of SlMS1 and SlMS2 is analyzed in tomato, and SlMS1 expression is up-regulated during fruit ripening, suggesting its potential role in regulating fruit ripening. A potential bipartite nuclear localization signal is found in the amino acid sequence of SlMS1; thus, SlMS1 is tagged with GFP and observed in the leaves of Nicotiana benthamiana. Consistently, SlMS1-GFP shows strong nuclear localization and also cytoplasmic localization. The role of SlMS1 in regulating fruit ripening is investigated in tomato fruit by transient silencing (virus-induced gene silencing, VIGS) and transient overexpression. The results show that SlMS1 silencing causes delayed fruit ripening, evidenced by more chlorophyll and less carotenoid accumulation, while SlMS1 overexpression accelerates fruit ripening significantly compared with control. Further investigation shows that SlMS1 overexpression could up-regulate the expression of carotenoid-synthesis-related genes (PSY1, PDS, ZDS), chlorophyll-degradation-related genes (NYC1, PAO, PPH, SGR1), cell-wall-metabolism-related genes (CEL2, EXP, PG, TBG4, XTH5) and ethylene-synthesis-pathway-related genes (ACO1, ACO3, ACS2), while SlMS1 silencing causes the opposite results. The correlation analysis indicates that SlMS1 expression is negatively correlated with chlorophyll content and positively correlated with carotenoid and ripening-related gene expressions. Taken together, our data suggest that SlMS1 is a positive regulator of tomato fruit ripening and a possible target gene for the ripening-delaying effect of H2S.

BrRH37, a Cabbage (Brassica rapa) DEAD-Box RNA Helicase, Confers Drought Tolerance and ABA Response in Transgenic Arabidopsis Plants

RNA helicases (RHs) are cellular proteins that help in the regulation of post-transcriptional mRNA metabolism, which is vital for the response of crops to abiotic stresses. In this study, we determined the functional role of a cabbage (Brassica rapa) DEAD-box RH, BrRH37, in abiotic stress and abscisic acid (ABA) responses using transgenic Arabidopsis plants. Although BrRH37 contains a putative chloroplast transit peptide and a potential nuclear localization signal, confocal analysis of the expression of the BrRH37-GFP protein in tobacco leaves revealed that BrRH37 is localized in the nucleus. The transcript levels of BrRH37 in cabbage were increased upon drought, cold, or ABA application, whereas its level was decreased by heat or UV stress. The transgenic Arabidopsis plants that constitutively express BrRH37 showed enhanced seedling and root growth or survival rate under drought stress or upon ABA application, whereas their growth and survival were decreased under cold or UV stress. Notably, transcript levels of ABA biosynthesis-related genes, including ABA1 and ABA3, were significantly increased in the transgenic plants compared with that in the wild type. Importantly, BrRH37 was found to recover the growth of the RNA chaperone-deficient Escherichia coli mutant at low temperature and had the ability to break up base pairs in RNA, suggesting that BrRH37 possesses RNA chaperone activity. Altogether, our results indicate that BrRH37 plays diverse functions in plant response to abiotic stress and ABA possibly via its RNA chaperone activity.

Nuclear accumulation of ZFP36L1 is cell cycle-dependent and determined by a C-terminal serine-rich cluster.

ZFP36L1 is an RNA-binding protein responsible for mRNA decay in the cytoplasm. ZFP36L1 has also been suggested as a nuclear-cytoplasmic shuttling protein because it contains a potential nuclear localization signal and a nuclear export signal. However, it remains unclear how the nuclear localization of ZFP36L1 is controlled. In this study, we provide evidence that the nuclear accumulation of ZFP36L1 protein is modulated in a cell cycle-dependent manner. ZFP36L1 protein accumulation in fractionated nuclei was particularly prominent in cellsarrested at G1-/S-phase boundary, while it was downregulated in S-phase cells, and eventually disappeared in G2-phase nuclei. Moreover, forced nuclear targeting of ZFP36L1 revealed marked downregulation of this proteinin S- and G2-phase cells, suggesting that ZFP36L1 can be eliminated in the nucleus. The C-terminal serine-rich cluster of ZFP36L1 is critical for the regulation of its nuclear accumulation because truncation of this probable disordered region enhanced the nuclear localization of ZFP36L1, increased its stability and abolished its cell cycle-dependent fluctuations. These findings provide the first hints to the question of how ZFP36L1 nuclear accumulation is controlled during the course of the cell cycle.

Nuclear MYH9-induced CTNNB1 transcription, targeted by staurosporin, promotes gastric cancer cell anoikis resistance and metastasis.

Rationale: Peritoneal metastasis predicts poor prognosis of gastric cancer (GC) patients, and the underlying mechanisms are poorly understood.Methods: The 2-DIGE, MALDI-TOF/TOF MS and single-cell transcriptome were used to detect differentially expressed proteins among normal gastric mucosa, primary GC and peritoneal metastatic tissues. Lentiviruses carrying shRNA and transcription activator-like effector nuclease technology were used to knock down myosin heavy chain 9 (MYH9) expression in GC cell lines. Immunofluorescence, immune transmission electron microscopy, chromatin fractionation, co-immunoprecipitation, and assays for chromatin immunoprecipitation, dual luciferase reporter, agarose-oligonucleotide pull-down, flow cytometry and cell anoikis were performed to uncover nuclear MYH9-induced β-catenin (CTNNB1) transcription in vitro. Nude mice and conditional transgenic mice were used to investigate the findings in vivo.Results: We observed that MYH9 was upregulated in metastatic GC tissues and was associated with a poor prognosis of GC patients. Mechanistically, we confirmed that MYH9 was mainly localized in the GC cell nuclei by four potential nuclear localization signals. Nuclear MYH9 bound to the CTNNB1 promoter through its DNA-binding domain, and interacted with myosin light chain 9, β-actin and RNA polymerase II to promote CTNNB1 transcription, which conferred resistance to anoikis in GC cells in vitro and in vivo. Staurosporine reduced nuclear MYH9 S1943 phosphorylation to inhibit CTNNB1 transcription, Wnt/β-catenin signaling activation and GC progression in both orthotropic xenograft GC nude mouse and transgenic GC mouse models.Conclusion: This study identified that nuclear MYH9-induced CTNNB1 expression promotes GC metastasis, which could be inhibited by staurosporine, indicating a novel therapy for GC peritoneal metastasis.

Transcriptional and translation shut-off in TBEV infected neural cells and involvement of viral C protein

Tick-borne encephalitis virus (TBEV, Flaviviridae) infection causes severe neurological disease and incapacitates more than 10 000 patients annually in the Eurasian region. Despite extensive studies, some areas of interaction of TBEV with the host cells remain undescribed. Here we investigated the interaction of TBEV and human neural DAOY HTB-186 cells on the transcriptional and translational level. By labelling of nascent RNA and protein molecules in TBEV-infected DAOY cells, we showed that the virus-induced host translational shut-off. Moreover, TBEV interfered also with the expression of host ribosomal RNAs, in particular with the rRNA species transcribed by RNA polymerase I (18S rRNA, 28S rRNA, and their precursor 45-47S pre-rRNA). Synthesis of host rRNAs is an essential host cell process that is localized in the nucleus, namely nucleoli. By searching for virus factor that could be linked with these effects, we described so far unknown nucleolar localization of TBEV capsid protein C. More importantly, preliminary data from transfection of recombinant C protein led to the reduction in nascent protein synthesis indicating the link between TBEV capsid protein and shut-off phenomena which were described. Furthermore, we identified a potential nuclear localization signal, which seems not to be essential for the shut-down effect. Taken together we described a brand new type of interaction between TBEV and host neural cells on the transcriptional and translational level and identified viral factor potentially responsible for the observed phenomena. However, further analyses are needed, and the particular mechanism of action remains still elusive.

Abstract P2037: G Protein-Coupled Receptor 37l1 is Localized on the Nuclear Envelope

G protein-coupled receptors (GPCRs), expressed on the plasma membrane, interact with various types of ligands, which trigger a cascade of signal transduction events leading to different intracellular responses that in turn manifest in physiological changes. Some GPCRs also reside and exert signals from intracellular organelles such as nucleus, endoplasmic reticulum, and Golgi apparatus. Recently, we reported that G protein-coupled receptor 37L1 (GPR37L1) is expressed in the apical membrane of renal proximal tubule cells (RPTCs) and participates in luminal sodium transport and blood pressure regulation by regulating the renal expression of NHE3. However, the mechanism by which GPR37L1 regulates NHE3 expression and function in the RPTC has not been studied. Tandem affinity purification of GPR37L1, combined with mass spectrometry analyses, revealed the association of GPR37L1 with mediators of nuclear importing proteins, such as RAN-GTPase, importin-5, and importin-7. In silico analyses of GPR37L1 amino acid sequence revealed the presence of a potential nuclear localization signal at the N-terminus. Confocal fluorescence imaging of RPTCs expressing GPR37L1 tagged with green fluorescence protein (GPR37L1-GFP) showed distinct nuclear membrane expression of GPR37L1. In addition, fluorescence imaging of nuclei isolated from the RPTCs expressing GPR37L1-GFP showed the expression on the nuclear envelope. Immunoblot analyses confirmed the presence of GPR37L1 in the nuclear protein prepared from the RPTCs expressing GPR37L1-GFP. The purity of the nuclear protein preparations was confirmed by the presence of histone deacetylase 2, a marker for nuclear protein, the absence of Na,K-ATPase, a marker for plasma membrane, and the absence of calnexin, a marker for endoplasmic reticulum. Our results show that GPR37L1 also resides on the nuclear envelope and may play a critical role in the regulation of the expression of genes responsible for maintaining normal electrolyte balance and blood pressure.

Mediated nuclear import and export of TAZ and the underlying molecular requirements

Nucleocytoplasmic distribution of Yap/TAZ is regulated by the Hippo pathway and the cytoskeleton. While interactions with cytosolic and nuclear “retention factors” (14–3–3 and TEAD) are known to control their localization, fundamental aspects of Yap/TAZ shuttling remain undefined. It is unclear if translocation occurs only by passive diffusion or via mediated transport, and neither the potential nuclear localization and efflux signals (NLS, NES) nor their putative regulation have been identified. Here we show that TAZ cycling is a mediated process and identify the underlying NLS and NES. The C-terminal NLS, representing a new class of import motifs, is necessary and sufficient for efficient nuclear uptake via a RAN-independent mechanism. RhoA activity directly stimulates this import. The NES lies within the TEAD-binding domain and can be masked by TEAD, thereby preventing efflux. Thus, we describe a RhoA-regulated NLS, a TEAD-regulated NES and propose an improved model of nucleocytoplasmic TAZ shuttling beyond "retention".

Loss of nucleolar localization of NAT10 promotes cell migration and invasion in hepatocellular carcinoma

NAT10, a nucleolar acetyltransferase, participates in a variety of cellular processes including ribosome biogenesis and DNA damage response. Immunohistochemistry staining showed that cytoplasmic and membranous NAT10 is related to the clinical pathologic characteristics in human cancer tissues. However, the mechanism about how NAT10 translocates from the nucleolus to cytoplasm and membrane is unclear. Here, we obtain a NAT10 deletion mutant localizing in cytoplasm and membrane. Bioinformatics analysis showed that residues 68–75 and 989–1018 are two potential nuclear localization signals (NLS) of NAT10. GFP-NAT10 deletion mutant (Δ989-1018) predominantly translocates into cytoplasm with faint signal retained in the nucleolus, while GFP-NAT10(Δ68-75) still remains in the nucleolus and nucleoplasm, indicating residues 989–1018 is the main nucleolar localization signal (NuLS). GFP-NAT10-D3, with both fragments (residues 68–75 and 989–1018) deleted, completely excludes from the nucleolus and translocates to cytoplasm and membrane. Therefore, complete NuLSs of NAT10 should include residues 68–75 and 989–1018. The cytoplasmic and membranous NAT10 mutant (Flag-NAT10-D3) colocalizes with α-tubulin in cytoplasm and with integrin on cell membrane. Importantly, Flag-NAT10-D3 promotes α-tubulin acetylation and stabilizes microtubules. Consequently, Flag-NAT10-D3 promotes migration and invasion in hepatocellular carcinoma (HCC) cells. Statistical analysis of immunohistochemistry staining of NAT10 in HCC tissues demonstrates that the cytoplasmic NAT10 is correlated with poorer prognosis compared with nuclear NAT10, while the membranous NAT10 predicts the poorest clinical outcome of the patients. We thus provide the evidence for the function of cytoplasmic and membranous NAT10 in the metastasis and prognosis of HCC patients.

Myosin VI in the nucleus of neurosecretory PC12 cells: Stimulation-dependent nuclear translocation and interaction with nuclear proteins

ABSTRACTMyosin VI (MVI) is a unique actin-based motor protein moving towards the minus end of actin filaments, in the opposite direction than other known myosins. Besides well described functions of MVI in endocytosis and maintenance of Golgi apparatus, there are few reports showing its involvement in transcription. We previously demonstrated that in neurosecretory PC12 cells MVI was present in the cytoplasm and nucleus, and its depletion caused substantial inhibition of cell migration and proliferation. Here, we show an increase in nuclear localization of MVI upon cell stimulation, and identification of potential nuclear localization (NLS) and nuclear export (NES) signals within MVI heavy chain. These signals seem to be functional as the MVI nuclear presence was affected by the inhibitors of nuclear import (ivermectin) and export (leptomycin B). In nuclei of stimulated cells, MVI colocalized with active RNA polymerase II, BrUTP-containing transcription sites and transcription factor SP1 as well as SC35 and PML proteins, markers of nuclear speckles and PML bodies, respectively. Mass spectrometry analysis of samples of a GST-pull-down assay with the MVI tail domain as a “bait” identified several new potential MVI binding partners. Among them are proteins involved in transcription and post-transcriptional processes. We confirmed interaction of MVI with heterogeneous nuclear ribonucleoprotein U (hnRNPU) and nucleolin, proteins involved in pre-mRNA binding and transport, and nucleolar function, respectively. Our data provide an insight into mechanisms of involvement of MVI in nuclear processes via interaction with nuclear proteins and support a notion for important role(s) for MVI in gene expression.

Overexpression of a New Zinc Finger Protein Transcription Factor OsCTZFP8 Improves Cold Tolerance in Rice.

Cold stress is one of the most important abiotic stresses in rice. C2H2 zinc finger proteins play important roles in response to abiotic stresses in plants. In the present study, we isolated and functionally characterized a new C2H2 zinc finger protein transcription factor OsCTZFP8 in rice. OsCTZFP8 encodes a C2H2 zinc finger protein, which contains a typical zinc finger motif, as well as a potential nuclear localization signal (NLS) and a leucine-rich region (L-box). Expression of OsCTZFP8 was differentially induced by several abiotic stresses and was strongly induced by cold stress. Subcellular localization assay and yeast one-hybrid analysis revealed that OsCTZFP8 was a nuclear protein and has transactivation activity. To characterize the function of OsCTZFP8 in rice, the full-length cDNA of OsCTZFP8 was isolated and transgenic rice with overexpression of OsCTZFP8 driven by the maize ubiquitin promoter was generated using Agrobacterium-mediated transformation. Among 46 independent transgenic lines, 6 single-copy homozygous overexpressing lines were selected by Southern blot analysis and Basta resistance segregation assay in both T1 and T2 generations. Transgenic rice overexpressing OsCTZFP8 exhibited cold tolerant phenotypes with significantly higher pollen fertilities and seed setting rates than nontransgenic control plants. In addition, yield per plant of OsCTZFP8-expressing lines was significantly (p < 0.01) higher than that of nontransgenic control plants under cold treatments. These results demonstrate that OsCTZFP8 was a C2H2 zinc finger transcription factor that plays an important role in cold tolerance in rice.

Expression and localization of CHI3L1 in monocyte derived dendritic cells

Chitinase-3-like-1 protein (CHI3L1) also called YKL-40, is a 40 kDa mammalian glycoprotein which is a heparin, chitin and collagen binding member of the mammalian chitinase-like proteins. Biological activities of CHI3L1 embrace regulation of cell proliferation, adhesion, angiogenesis, migration and activation. CHI3L1 is produced by variety of cells, including neutrophils, monocytes/macrophages, osteoclasts and Kupffer cells [1]. CHI3L1 secretion is induced by interferon (INF)-g and interleukin (IL)-6 and is an acute phase reactant associated with disease severity and mortality in a variety of infectious [2]. In this study, we have examined the expression and localization of CHI3L1 during the differentiation and maturation of monocyte derived dendritic cells by real time RT-PCR, Western Blot, Confocal Immunofluorescence, and Immunocytochemical assays. Potential nuclear localization signal (NLS) was determinated using the open source software cNLS Mapper and Chimera. Peripheral blood monocytes were differentiated toward immature DCs (iDC) and mature DCs (mDCs) through a combination of factors and cytokines. Our result showed, for the first time, that CHI3L1 is expressed during the process of differentiation and maturation of DCs in time dependent manner. Furthermore, CHI3L1 is evenly distributed in cytoplasm and in the nucleus of both the iDCs and mDCs. In conclusion, the discovery of CHI3L1 expression in DCs has opened new dilemma for designing DC-based cancer immunotherapeutic. In fact, on the light of these results one can’t exclude that as well as activated Tumor-associated macrophages (TAMs) also DCs infiltration could to be a significant unfavorable prognostic factor for cancer patients.

Recombinant Sj16 from Schistosoma japonicum contains a functional N-terminal nuclear localization signal necessary for nuclear translocation in dendritic cells and interleukin-10 production.

Sj16 is a Schistosoma japonicum-derived protein (16kDa in molecular weight) that has been identified as an immune modulation molecule, but the mechanisms of modulation of immune responses are not known. In this report, we aimed to investigate the host immune regulation mechanism by recombinant Sj16 (rSj16) and thus illuminate the molecular mechanism of immune evasion by S. japonicum. The effect of rSj16 and rSj16 mutants on the biology of dendritic cells (DCs) was assessed by examining DC maturation, cytokine production, and expression of surface markers by flow cytometry and enzyme-linked immunosorbent assay. We found that rSj16 significantly stimulated interleukin (IL)-10 production and inhibited LPS-induced bone marrow-derived dendrite cell (BMDC) maturation in a dose-dependent manner. By using antibody neutralization experiments and IL-10-deficient (knockout) mice, we confirmed that the inhibitory effect of rSj16 on LPS-induced BMDCs is due to its induction of IL-10 production. To understand how rSj16 induces the production of IL-10, we analyzed the protein sequence and revealed two potential nuclear localization signals (NLS) in Sj16. The N-terminal NLS (NLS1) is both necessary and sufficient for translocation of rSj16 to the nucleus of BMDCs and is important for subsequent induction of IL-10 production and the inhibition of BMDC maturation by rSj16. The results of our study concluded that the ability of rSj16 to inhibit DC functions is IL-10 dependent which is operated by IL-10R signal pathway. This study also confirmed that NLS is an important domain associated with increased production of IL-10. Our findings will extend the current understanding on host-schistosome relationship and provide insight about bottleneck of parasitic control.

Isolation and Functional Characterization of a Novel Gene Encoding a Dehydration Responsive Element Binding Transcription Factor from Populus euphratica.

Dehydration responsive element binding (DREB) transcription factors (TFs) play a key role in regulating abiotic stress related genes. A new gene (PeDREB2b) encoding an unidentified DRE-binding protein was isolated from 20% PEG6000 treated Populus euphratica Oliv. seedlings by RT-PCR and RACE. Full length of PeDREB2b cDNA was 1110 bp, and an ORF of 870 bp, which encoded 289-amino-acids polypeptide, were included. The deduced amino acid sequence analysis revealed that this protein was a putative AP2/EREBP transcription factor with a conserved AP2/EREBP domain of 64 amino acids and a potential nuclear localization signal (NLS). Based on phylogenetic characterization, PeDREB2b was classified as a member of A-5 group belonged to the DREB family. The PeDREB2b gene is induced by salinization, low temperature, drought and phytohormones GA3, NAA and 6BA, but not by ABA treatment. The fact that the product of PeDREB2b as a DREB transcription factor was verified in our further experiment: the nuclear localization of the gene when it was expressed transiently as a GFP fusion in onion epidermal cells. In addition, PeDREB2b was capable of activating reporter gene expression. To study the salt and drought stress responses for PeDREB2b transgenic Arabidopsis thaliana in detail, integrated physiological, biochemical and genetic approach methods were used. Results indicated that the PeDREB2b gene was over-expressed under stress-inducible rd29A promotor in transgenic plants alleviates the adverse effects of environmental stresses.

Importin α1 Mediates Yorkie Nuclear Import via an N-terminal Non-canonical Nuclear Localization Signal

The Hippo signaling pathway controls organ size by orchestrating cell proliferation and apoptosis. When the Hippo pathway was inactivated, the transcriptional co-activator Yorkie translocates into the nucleus and forms a complex with transcription factor Scalloped to promote the expression of Hippo pathway target genes. Therefore, the nuclear translocation of Yorkie is a critical step in Hippo signaling. Here, we provide evidence that the N-terminal 1-55 amino acids of Yorkie, especially Arg-15, were essential for its nuclear localization. By mass spectrometry and biochemical analyses, we found that Importin α1 can directly interact with the Yorkie N terminus and drive Yorkie into the nucleus. Further experiments show that the upstream component Hippo can inhibit Importin α1-mediated Yorkie nuclear import. Taken together, we identified a potential nuclear localization signal at the N-terminal end of Yorkie as well as a critical role for Importin α1 in Yorkie nuclear import.