Bipartite Nuclear Localization Signal Research Articles

Structures of the Karyopherins Kap121p and Kap60p Bound to the Nuclear Pore-Targeting Domain of the SUMO Protease Ulp1p

The budding yeast small ubiquitin-like modifier (SUMO) protease Ulp1p catalyzes both the processing of newly synthesized SUMO to its mature form and the deconjugation of SUMO from target proteins, thereby regulating a wide range of cellular processes including cell division, DNA repair, DNA replication, transcription, and mRNA quality control. Ulp1p is localized primarily at the nuclear pore complex (NPC) through interactions involving the karyopherins Kap121p and Kap95p–Kap60p heterodimer and a subset of nuclear pore-associated proteins. The sequestration of Ulp1p at the nuclear periphery is crucial for the proper control of protein desumoylation. To gain insights into the role of the karyopherins in regulating the localization of Ulp1p, we have determined the crystal structures of Kap121p and Kap60p bound to the N-terminal non-catalytic domain of Ulp1p that is necessary and sufficient for NPC targeting. Contrary to a previous proposal that Ulp1p is tethered to the transport channel of the NPC through unconventional interactions with the karyopherins, our structures reveal that Ulp1p has canonical nuclear localization signals (NLSs): (1) an isoleucine-lysine-NLS (residues 51–55) that binds to the NLS-binding site of Kap121p, and (2) a classical bipartite NLS (residues 154–172) that binds to the major and minor NLS-binding sites of Kap60p. Ulp1p also binds Kap95p directly, and the Ulp1p–Kap95p binding is enhanced by the importin-β-binding domain of Kap60p. GTP-bound Gsp1p (the yeast Ran ortholog) and the exportin Cse1p cooperate to release Ulp1p from the karyopherins, indicating that the stable sequestration of Ulp1p to the NPC would require a karyopherin-independent mechanism to anchor Ulp1p at the NPC.

Nuclear Import of Janus Kinase 1 Is Mediated By Multiple Alpha Importins and Is Required for the Survival of Diffuse Large B-Cell Lymphoma

Janus kinases (JAKs) are non-receptor tyrosine kinases that are generally found bound to cytokine receptors. JAKs have well established roles in activating signal transducers and activators of transcription (STATs) in response to cytokine stimulation in the cytoplasm. However, the epigenetic regulation of gene transcription by nuclear JAK2 through phosphorylation of tyrosine 41 on the histone protein H3 has recently been discovered. Our very recent work demonstrated that this histone targeting mechanism applies to another member of the JAK family, JAK1, which is activated by the autocrine cytokines IL6 and IL10 in activated B-cell-like diffuse large B cell lymphoma (ABC DLBCL). Genome-wide mapping of the H3Y41p histone mark revealed 2956 potential JAK1 target genes, 91% of which do not have a STAT motif in their promoter region. Here we investigated the JAK1 nuclear transfer mechanism in ABC DLBCL. Using cell fractionation and immunofluorescence assays, we detected a nuclear pool of JAK1 in both HEK 293T cells and ABC DLBCL cell lines. In searching for a putative nuclear localization signal (NLS) of JAK1, we identified a basic amino acid enriched sequence (KRKK…KHKK, starting from amino acid 342) that was reminiscent of a classical bipartite NLS. Immunofluorescence with GFP-tagged JAK1 truncations and full length JAK1 with mutations in putative NLS revealed that the first group of amino acids (KRKK) was the NLS that was responsible for the nuclear transport of JAK1. Our results, however, indicate that the active transport of JAK1 into the nucleus is independent of its activation status, since nucleocytoplasmic distribution did not change either in HEK293T cells when constitutively activated JAK1 was expressed or in ABC DLBCL cells with IL-6 stimulation or in GCB DLBCL cells that lack cytokine signaling. To identify potential importins that recognize the NLS of JAK1 and subsequently transport JAK1 into the nucleus, we used immunoprecipitation and GST pull down assays. We found that importin a4, a5 and a7 (encoded by KPNA3, KPNA1, and KPNA6 respectively) physically interacted with the NLS of JAK1 and these interactions were abolished when the 4 amino acids in the NLS were mutated to alanine, suggesting these alpha importins recognize the NLS of JAK1 (KRKK) and mediate the nuclear transport of JAK1. To test whether the NLS-mediated nuclear transfer that allows JAK1 to function in the nucleus is important for the fitness of ABC DLBCL cells, we performed JAK1 knockdown and rescue experiments in TMD8 and OCI-Ly10 cells, in which endogenous JAK1 was knocked down by an shRNA and an shRNA-resistant cDNA of either wild type or NLS-defective JAK1 was constantly expressed. The results revealed that knockdown of JAK1 led to reduced viable cells in the culture, suggesting that JAK1 expression is required for cancer cell survival. As expected, overexpression of wild type JAK1 completely reversed the toxicity due to knockdown of endogenous JAK1. However, overexpression of the NLS-defective JAK1 did not rescue the toxic effects of JAK1 shRNA. Thus, the findings suggest that nuclear transport of JAK1 is an essential process to maintain the survival of ABC DLBCL, and targeting the JAK1 NLS might be a potential targeted therapeutic strategy. Disclosures No relevant conflicts of interest to declare.

Designed inhibitor for nuclear localization signal of polo-like kinase 1 induces mitotic arrest.

Polo-like kinase 1 (Plk1), a member of polo-like kinase family, regulates multiple essential steps of the cell cycle progression. Plk1 is overexpressed in multiple cancer cell lines and considered to be a prime anticancer target. Plk1 accumulates in the nucleus during S and G2 phases by its bipartite nuclear localization signal (NLS) sequence, which is crucial for Plk1 regulation during normal cell cycle progression. Here, through combined computational and experimental studies, we identified compound D110, which inhibits Plk1 kinase activity with an IC50 of 85nm and blocks the nuclear localization of Plk1 during S and G2 phases. D110-treated cancer cells were arrested at mitosis with monopolar spindle, indicating the inhibition of the Plk1 kinase activity in cell. As D110 interacts with both the ATP site and the NLS in Plk1, it demonstrates good selectivity toward Plk2 and Plk3. The strategy of simultaneously inhibiting kinase activity and its subcellular translocations offers a novel approach for selective kinase inhibitor design.

Testis-Specific GTPase (TSG): An oligomeric protein.

BackgroundRas-related proteins in brain (Rab)-family proteins are key members of the membrane trafficking pathway in cells. In addition, these proteins have been identified to have diverse functions such as cross-talking with different kinases and playing a role in cellular signaling. However, only a few Rab proteins have been found to have a role in male germ cell development. The most notable functions of this process are performed by numerous testis-specific and/or germ cell-specific genes. Here, we describe a new Rab protein that is specifically expressed in male germ cells, having GTPase activity.ResultsTestis-specific GTPase (TSG) is a male-specific protein that is highly expressed in the testis. It has an ORF of 1593 base pairs encoding a protein of 530 amino acids. This protein appears in testicular cells approximately 24 days postpartum and is maintained thereafter. Immunohistochemistry of testicular sections indicates localized expression in germ cells, particularly elongating spermatids. TSG has a bipartite nuclear localization signal that targets the protein to the nucleus. The C-terminal region of TSG contains the characteristic domain of small Rab GTPases, which imparts GTPase activity. At the N-terminal region, it has a coiled-coil motif that confers self-interaction properties to the protein and allows it to appear as an oligomer in the testis.ConclusionTSG, being expressed in the male gonad in a developmental stage-specific manner, may have a role in male germ cell development. Further investigation of TSG function in vivo may provide new clues for uncovering the secrets of spermatogenesis.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-016-3145-9) contains supplementary material, which is available to authorized users.

The Matrix Protein of Nipah Virus Targets the E3-Ubiquitin Ligase TRIM6 to Inhibit the IKKε Kinase-Mediated Type-I IFN Antiviral Response.

For efficient replication, viruses have developed mechanisms to evade innate immune responses, including the antiviral type-I interferon (IFN-I) system. Nipah virus (NiV), a highly pathogenic member of the Paramyxoviridae family (genus Henipavirus), is known to encode for four P gene-derived viral proteins (P/C/W/V) with IFN-I antagonist functions. Here we report that NiV matrix protein (NiV-M), which is important for virus assembly and budding, can also inhibit IFN-I responses. IFN-I production requires activation of multiple signaling components including the IκB kinase epsilon (IKKε). We previously showed that the E3-ubiquitin ligase TRIM6 catalyzes the synthesis of unanchored K48-linked polyubiquitin chains, which are not covalently attached to any protein, and activate IKKε for induction of IFN-I mediated antiviral responses. Using co-immunoprecipitation assays and confocal microscopy we show here that the NiV-M protein interacts with TRIM6 and promotes TRIM6 degradation. Consequently, NiV-M expression results in reduced levels of unanchored K48-linked polyubiquitin chains associated with IKKε leading to impaired IKKε oligomerization, IKKε autophosphorylation and reduced IFN-mediated responses. This IFN antagonist function of NiV-M requires a conserved lysine residue (K258) in the bipartite nuclear localization signal that is found in divergent henipaviruses. Consistent with this, the matrix proteins of Ghana, Hendra and Cedar viruses were also able to inhibit IFNβ induction. Live NiV infection, but not a recombinant NiV lacking the M protein, reduced the levels of endogenous TRIM6 protein expression. To our knowledge, matrix proteins of paramyxoviruses have never been reported to be involved in innate immune antagonism. We report here a novel mechanism of viral innate immune evasion by targeting TRIM6, IKKε and unanchored polyubiquitin chains. These findings expand the universe of viral IFN antagonism strategies and provide a new potential target for development of therapeutic interventions against NiV infections.

Characterization of the nuclear import signal of herpes simplex virus 1 UL31.

The herpes simplex virus 1 (HSV-1) UL31 protein is a multifunctional nucleoprotein that is important for viral infection; however, little is known concerning its subcellular localization signal. Here, by transfection with a series of HSV-1 UL31 deletion mutants fused to enhanced yellow fluorescent protein (EYFP), a bipartite nuclear localization signal (NLS) was identified and mapped to amino acids (aa) 1 to 27 (MYDTDPHRRGSRPGPYHGKERRRSRSS). Additionally, fluorescence results showed that the predicted nuclear export signal (NES) might be nonfunctional, and the functional NES of UL31 might require a specific conformation. Taken together, these results would provide significant information for the study of the biological function of UL31 during HSV-1 infection.

The Pseudorabies Virus DNA Polymerase Accessory Subunit UL42 Directs Nuclear Transport of the Holoenzyme.

Pseudorabies virus (PRV) DNA replication occurs in the nuclei of infected cells and requires the viral DNA polymerase. The PRV DNA polymerase comprises a catalytic subunit, UL30, and an accessory subunit, UL42, that confers processivity to the enzyme. Its nuclear localization is a prerequisite for its enzymatic function in the initiation of viral DNA replication. However, the mechanisms by which the PRV DNA polymerase holoenzyme enters the nucleus have not been determined. In this study, we characterized the nuclear import pathways of the PRV DNA polymerase catalytic and accessory subunits. Immunofluorescence analysis showed that UL42 localizes independently in the nucleus, whereas UL30 alone predominantly localizes in the cytoplasm. Intriguingly, the localization of UL30 was completely shifted to the nucleus when it was coexpressed with UL42, demonstrating that nuclear transport of UL30 occurs in an UL42-dependent manner. Deletion analysis and site-directed mutagenesis of the two proteins showed that UL42 contains a functional and transferable bipartite nuclear localization signal (NLS) at amino acids 354–370 and that K354, R355, and K367 are important for the NLS function, whereas UL30 has no NLS. Coimmunoprecipitation assays verified that UL42 interacts with importins α3 and α4 through its NLS. In vitro nuclear import assays demonstrated that nuclear accumulation of UL42 is a temperature- and energy-dependent process and requires both importins α and β, confirming that UL42 utilizes the importin α/β-mediated pathway for nuclear entry. In an UL42 NLS-null mutant, the UL42/UL30 heterodimer was completely confined to the cytoplasm when UL42 was coexpressed with UL30, indicating that UL30 utilizes the NLS function of UL42 for its translocation into the nucleus. Collectively, these findings suggest that UL42 contains an importin α/β-mediated bipartite NLS that transports the viral DNA polymerase holoenzyme into the nucleus in an in vitro expression system.

Structural and Calorimetric Studies Demonstrate that Xeroderma Pigmentosum Type G (XPG) Can Be Imported to the Nucleus by a Classical Nuclear Import Pathway via a Monopartite NLS Sequence

Xeroderma pigmentosum type G (XPG) proteins are involved in DNA lesion recognition and promotion of nucleotide excision repair. Specific mutations in these proteins may lead to Cockayne syndrome, in which the patients may display severe developmental retardation and neurological abnormalities. No structural information is available for their spacer region or the C-terminal domain, which are important, respectively, for specific nucleotide excision repair activity and substrate specificity, as well as nuclear translocation. Immunofluorescence studies suggested two specific regions of the XPG C-terminus as potential bipartite nuclear localization sequences, which would be responsible for its translocation to the nucleus by the classical nuclear import pathway mediated by the importin‐α (Impα). Thus, in order to test these hypotheses and gain insight into the structural basis for the nuclear import process for the XPG protein, we solved the crystal structures of complexes formed by the Impα and peptides corresponding to both putative nuclear localization signal (NLS) sequences (XPG1 and XPG2) and performed isothermal titration calorimetry assays to determine their binding affinities. Structural experiments confirm the binding of both NLS peptides to Impα but, unexpectedly, they bind to the receptor as monopartite NLSs. The isothermal titration calorimetry assays demonstrated that XPG1 and XPG2 peptides bind to two separate binding sites, but with high affinity to the major NLS-binding site of the Impα, resembling classical monopartite SV40 TAg NLS. The results lead to insights about what distinguishes monopartite and bipartite NLSs, as well as the differential roles of XPG1 and XPG2 NLSs in the nuclear localization of XPG.

RepA Protein Encoded by Oat dwarf virus Elicits a Temperature-Sensitive Hypersensitive Response-Type Cell Death That Involves Jasmonic Acid-Dependent Signaling.

The hypersensitive response (HR) is a component of disease resistance that is often induced by pathogen infection, but essentially no information is available for members of the destructive mastreviruses. We have investigated an HR-type response elicited in Nicotiana species by Oat dwarf virus (ODV) and have found that expression of the ODV RepA protein but not other ODV-encoded proteins elicits the HR-type cell death associated with a burst of H2O2. Deletion mutagenesis indicates that the first nine amino acids (aa) at the N terminus of RepA and the two regions located between aa residues 173 and 195 and between aa residues 241 and 260 near the C terminus are essential for HR-type cell-death elicitation. Confocal and electron microscopy showed that the RepA protein is localized in the nuclei of plant cells and might contain bipartite nuclear localization signals. The HR-like lesions mediated by RepA were inhibited by temperatures above 30°C and involvement of jasmonic acid (JA) in HR was identified by gain- and loss-of-function experiments. To our knowledge, this is the first report of an elicitor of HR-type cell death from mastreviruses.

Down-regulation of the zinc-finger homeobox protein TSHZ2 releases GLI1 from the nuclear repressor complex to restore its transcriptional activity during mammary tumorigenesis

Although breast cancer is one of the most common malignancies, the molecular mechanisms underlying its development and progression are not fully understood. To identify key molecules involved, we screened publicly available microarray datasets for genes differentially expressed between breast cancers and normal mammary glands. We found that three of the genes predicted in this analysis were differentially expressed among human mammary tissues and cell lines. Of these genes, we focused on the role of the zinc-finger homeobox protein TSHZ2, which is down-regulated in breast cancer cells. We found that TSHZ2 is a nuclear protein harboring a bipartite nuclear localization signal, and we confirmed its function as a C-terminal binding protein (CtBP)-dependent transcriptional repressor. Through comprehensive screening, we identified TSHZ2-suppressing genes such as AEBP1 and CXCR4, which are conversely up-regulated by GLI1, the downstream transcription factor of Hedgehog signaling. We found that GLI1 forms a ternary complex with CtBP2 in the presence of TSHZ2 and that the transcriptional activity of GLI1 is suppressed by TSHZ2 in a CtBP-dependent manner. Indeed, knockdown of TSHZ2 increases the expression of AEBP1 and CXCR4 in TSHZ2-expressing immortalized mammary duct epithelium. Concordantly, immunohistochemical staining of mammary glands revealed that normal duct cells expresses GLI1 in the nucleus along with TSHZ2 and CtBP2, whereas invasive ductal carcinoma cells, which does not express TSHZ2, show the increase in the expression of AEBP1 and CXCR4 and in the cytoplasmic localization of GLI1. Thus, we propose that down-regulation of TSHZ2 is crucial for mammary tumorigenesis via the activation of GLI1.

Human RNA lariat Debranching Enzyme Protein 1 –A surveillant for branch RNAs for degradation

Splicing is a processs to remove introns from precursor of mRNAs (pre-mRNAs). Introns are excised as a lariat form, and they should be debranched before degradation. This reaction is conferred by a RNA lariat debranching enzyme 1 (Dbr1) protein. The Dbr1 protein is evolutionarily conserved among many species and shares GNHE motif for debranching activity that is identical to protein phosphatase activity center. The human Dbr1 protein has a bipartite type nuclear localization signal, and it shuttles between the nucleus and the cytoplasm, suggesting novel function(s) in the cytoplasm. The human Dbr1 protein interacts with two proteins, Xab2 and hDrn1. Since Xab2 is involved in not only splicing but also transcription-coupled DNA repair (TCR), hDbr1 may also have a role in TCR. Although the function of hDrn1 is not known yet, this protein specifically interact with carboxy terminal of hDbr1 and it is also a nucleo-cytoplasmic shuttling protein. A heterodimer of hDbr1-hDrn1 may have role(s) in both in the nucleus and the cytoplasm of human cells.

Tip-to-nucleus migration dynamics of the asexual development regulator FlbB in vegetative cells.

In Aspergillus nidulans, asexual differentiation requires the presence of the transcription factor FlbB at the cell tip and apical nuclei. Understanding the relationship between these two pools is crucial for elucidating the biochemical processes mediating conidia production. Tip-to-nucleus communication was demonstrated by photo-convertible FlbB::Dendra2 visualization. Tip localization of FlbB depends on Cys382 in the C-terminus and the bZIP DNA-binding domain in the N-terminus. FlbE, a critical FlbB interactor, binds the bZIP domain. Furthermore, the absence of FlbE results in loss of tip localization but not nuclear accumulation. flbE deletion also abrogates transcriptional activity indicating that FlbB gains transcriptional competence from interactions with FlbE at the tip. Finally, a bipartite nuclear localization signal is required for nuclear localization of FlbB. Those motifs of FlbB may play various roles in the sequence of events necessary for the distribution and activation of this transcriptionally active developmental factor. The tip accumulation, FlbE-dependent activation, transport and nuclear import sketch out a process of relaying an environmentally triggered signal from the tip to the nuclei. As the first known instance of transcription factor-mediated tip-to-nucleus communication in filamentous fungi, this provides a general framework for analyses focused on elucidating the set of molecular mechanisms coupling apical signals to transcriptional events.

Nuclear Localization of the DNA Repair Scaffold XRCC1: Uncovering the Functional Role of a Bipartite NLS.

We have characterized the nuclear localization signal (NLS) of XRCC1 structurally using X-ray crystallography and functionally using fluorescence imaging. Crystallography and binding studies confirm the bipartite nature of the XRCC1 NLS interaction with Importin α (Impα) in which the major and minor binding motifs are separated by >20 residues, and resolve previous inconsistent determinations. Binding studies of peptides corresponding to the bipartite NLS, as well as its major and minor binding motifs, to both wild-type and mutated forms of Impα reveal pronounced cooperative binding behavior that is generated by the proximity effect of the tethered major and minor motifs of the NLS. The cooperativity stems from the increased local concentration of the second motif near its cognate binding site that is a consequence of the stepwise binding behavior of the bipartite NLS. We predict that the stepwise dissociation of the NLS from Impα facilitates unloading by providing a partially complexed intermediate that is available for competitive binding by Nup50 or the Importin β binding domain. This behavior provides a basis for meeting the intrinsically conflicting high affinity and high flux requirements of an efficient nuclear transport system.

Abstract 2036: Keratin 17 mediates p27KIP1-nuclear export, proliferative signaling and tumor growth

Abstract One of the most fundamental traits of cancer cells is the ability to sustain proliferation by circumventing cell cycle regulatory programs normally controlled by tumor suppressors. Keratin 17 (K17) is not expressed in most somatic tissues but is overexpressed and is a negative prognostic marker in cervical, breast, ovarian, and gastric carcinomas. The mechanisms by which K17 contributes to cancer-related signaling, however, remain unknown. Here, we report that nuclear-localized K17 interacts with tumor suppressor p27KIP1 and mediates its nuclear export, maintaining proliferation. After validating K17 is a prognostic marker in cervical cancer, independent of tumor stage, we performed in vitro and in vivo experiments to investigate the role of K17 in proliferative signaling and tumor growth using loss- and gain- of function approaches in cervical cancer and other cancer-derived cell lines. We found that K17 expression maintains proliferation, while silencing K17 induces G1 arrest by nuclear accumulation and stabilization of tumor suppressors p27KIP1 and pRb. During G1, K17 localizes in the nucleus, mediated by a classical bipartite nuclear localization signal (NLS) (AA385-400) that was identified by in silico analysis. This NLS is specific among keratins and present only in primates but not in other species. To our knowledge, this is the first report that a keratin has a NLS and is capable of undergoing nuclear translocation. During G1, p27KIP1is exported from the nucleus in a CRM1-dependent manner and is subsequently degraded, triggering G1/S transition. p27KIP1 lacks the classical leucine-rich nuclear export signal (NES) and requires an adaptor for CRM1-exportin binding. After confirming the binding of K17 and p27KIP1 within the nucleus, we performed in silico analyses and identified a leucine-rich NES required for CRM1-binding in K17 (AA194-199), which was further validated by a 3-fold nuclear retention of K17 and p27KIP1 after leptomycin B treatment. We introduced point mutations within the K17-NES (mNES) and the K17-NLS (mNLS). Nuclear p27KIP1 was lost in cells expressing wild-type K17. In contrast, nuclear p27KIP1 levels were 3-fold higher in cells expressing either mNLS or mNES. Furthermore, nuclear localization of K17 was abolished in mNLS cells. Xenografts of cervical cancer cells showed that tumors derived from K17 expressing cells were 3-times larger than those derived from K17 knockdown cells, the latter showing increased nuclear p27KIP1 and decreased PCNA and Ki67 expression. Finally, we identified an inverse correlation between K17 and p27KIP1 expression in human cervical cancer specimens, intertumorally and intratumorally. These data suggest a model in which nuclear-localized K17 acts as an oncoprotein promoting G1/S transition in cancer cells, by mediating exportin binding and nuclear translocation of tumor suppressor p27KIP1, contributing to sustained proliferation, tumor aggressiveness and poor-patient outcome. Citation Format: Luisa F. Escobar-Hoyos, Ruchi Shah, Lucia Roa-Peña, Nilofar Najafian, Elizabeth Vanner, Anna Banach, Erik Nielsen, Ramsey Al-Khalil, Ali Akalin, David Talmage, Kenneth Shroyer. Keratin 17 mediates p27KIP1-nuclear export, proliferative signaling and tumor growth. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2036. doi:10.1158/1538-7445.AM2015-2036

Characterization of the nuclear import and export signals of pseudorabies virus UL31.

The pseudorabies virus (PRV) UL31 protein (pUL31) is a homologue of the herpes simplex virus 1 pUL31, which is a multifunctional protein that is important for HSV-1 infection. However, little is known concerning the subcellular localization signal of PRV UL31. Here, by transfection with a series of PRV UL31 deletion mutants fused to an enhanced yellow fluorescent protein (EYFP) gene, a bipartite nuclear localization signal (NLS) and a PY motif NLS of UL31 were identified and mapped to amino acids (aa) 4 to 20 (RRRLLRRKSSAARRKTL) and aa 21 to 34 (TRAARDRYAPYFAY), respectively. Additionally, the predicted nuclear export signal (NES) was shown to be nonfunctional. Taken together, this information opens up new avenues for investigating the biological functions of UL31 during PRV infection.

Introgression of the SbASR-1 gene cloned from a halophyte Salicornia brachiate enhances salinity and drought endurance in transgenic groundnut (arachis hypogaea)and acts as a transcription factor [corrected

The SbASR-1 gene, cloned from a halophyte Salicornia brachiata, encodes a plant-specific hydrophilic and stress responsive protein. The genome of S. brachiata has two paralogs of the SbASR-1 gene (2549 bp), which is comprised of a single intron of 1611 bp, the largest intron of the abscisic acid stress ripening [ASR] gene family yet reported. In silico analysis of the 843-bp putative promoter revealed the presence of ABA, biotic stress, dehydration, phytohormone, salinity, and sugar responsive cis-regulatory motifs. The SbASR-1 protein belongs to Group 7 LEA protein family with different amino acid composition compared to their glycophytic homologs. Bipartite Nuclear Localization Signal (NLS) was found on the C-terminal end of protein and localization study confirmed that SbASR-1 is a nuclear protein. Furthermore, transgenic groundnut (Arachis hypogaea) plants over-expressing the SbASR-1 gene constitutively showed enhanced salinity and drought stress tolerance in the T1 generation. Leaves of transgenic lines exhibited higher chlorophyll and relative water contents and lower electrolyte leakage, malondialdehyde content, proline, sugars, and starch accumulation under stress treatments than wild-type (Wt) plants. Also, lower accumulation of H2O2 and O2 .- radicals was detected in transgenic lines compared to Wt plants under stress conditions. Transcript expression of APX (ascorbate peroxidase) and CAT (catalase) genes were higher in Wt plants, whereas the SOD (superoxide dismutase) transcripts were higher in transgenic lines under stress. Electrophoretic mobility shift assay (EMSA) confirmed that the SbASR-1 protein binds at the consensus sequence (C/G/A)(G/T)CC(C/G)(C/G/A)(A/T). Based on results of the present study, it may be concluded that SbASR-1 enhances the salinity and drought stress tolerance in transgenic groundnut by functioning as a LEA (late embryogenesis abundant) protein and a transcription factor.

Bipartite Nuclear Localization Signal Controls Nuclear Import and DNA-Binding Activity of IFN Regulatory Factor 3.

Accurate cellular localization plays a crucial role in the effective function of most signaling proteins, and nuclear trafficking is central to the function of transcription factors. IFN regulatory factor (IRF)3 is a master transcription factor responsible for the induction of type I IFN, which plays a crucial role in host antiviral innate immune responses. However, the mechanisms for control and regulation of IRF3 nuclear import largely remain to be elucidated. In our study, we identified a bipartite nuclear localization signal (NLS) in IRF3, with two interdependent basic clusters separated by a 7-aa linker. Our study further demonstrated that the bipartite NLS of IRF3 is also critical for IRF3 DNA-binding activity, indicating that the two functions of this region are integrated, which is in contrast to other IRFs. Furthermore, the IFN bioassay and infection studies suggest that IRF3 NLS is essential to the IRF3-mediated IFN responses and antiviral immunity. Overall, our results reveal a previously unrecognized bipartite NLS for IRF3 that contains both DNA-binding activity and nuclear import function, and they shed light on the regulatory mechanisms of IRF3 activation and IRF3-mediated antiviral responses.

The Herpes Simplex Virus Protein pUL31 Escorts Nucleocapsids to Sites of Nuclear Egress, a Process Coordinated by Its N-Terminal Domain.

Progeny capsids of herpesviruses leave the nucleus by budding through the nuclear envelope. Two viral proteins, the membrane protein pUL34 and the nucleo-phosphoprotein pUL31 form the nuclear egress complex that is required for capsid egress out of the nucleus. All pUL31 orthologs are composed of a diverse N-terminal domain with 1 to 3 basic patches and a conserved C-terminal domain. To decipher the functions of the N-terminal domain, we have generated several Herpes simplex virus mutants and show here that the N-terminal domain of pUL31 is essential with basic patches being critical for viral propagation. pUL31 and pUL34 entered the nucleus independently of each other via separate routes and the N-terminal domain of pUL31 was required to prevent their premature interaction in the cytoplasm. Unexpectedly, a classical bipartite nuclear localization signal embedded in this domain was not required for nuclear import of pUL31. In the nucleus, pUL31 associated with the nuclear envelope and newly formed capsids. Viral mutants lacking the N-terminal domain or with its basic patches neutralized still associated with nucleocapsids but were unable to translocate them to the nuclear envelope. Replacing the authentic basic patches with a novel artificial one resulted in HSV1(17+)Lox-UL31-hbpmp1mp2, that was viable but delayed in nuclear egress and compromised in viral production. Thus, while the C-terminal domain of pUL31 is sufficient for the interaction with nucleocapsids, the N-terminal domain was essential for capsid translocation to sites of nuclear egress and a coordinated interaction with pUL34. Our data indicate an orchestrated sequence of events with pUL31 binding to nucleocapsids and escorting them to the inner nuclear envelope. We propose a common mechanism for herpesviral nuclear egress: pUL31 is required for intranuclear translocation of nucleocapsids and subsequent interaction with pUL34 thereby coupling capsid maturation with primary envelopment.

Cleavage of bovine adenovirus type 3 non-structural 100K protein by protease is required for nuclear localization in infected cells but is not essential for virus replication

The L6 region of bovine adenovirus type 3 (BAdV-3) encodes a non-structural protein named 100K. Rabbit antiserum raised against BAdV-3 100K recognized a protein of 130 kDa at 12-24 h and proteins of 130, 100, 95 and 15 kDa at 36-48 h after BAdV-3 infection. The 100K species localized to the nucleus and the cytoplasm of BAdV-3-infected cells. In contrast, 100K localized predominantly to the cytoplasm of the transfected cells. However, BAdV-3 infection of cells transfected with 100K-enhanced yellow fluorescent protein-expressing plasmid detected fluorescent protein in the nucleus of the cells, suggesting that other viral proteins may be required for the nuclear localization of 100K. Interaction of BAdV-3 100K with BAdV-3 33K protein did not alter the cytoplasmic localization of 100K. However, co-expression of BAdV-3 100K and BAdV-3 protease localized 100K to the nucleolus of the transfected cells. Subsequent analysis suggested that BAdV-3 protease cleaves 100K at two identified potential protease cleavage sites (aa 740-745 and 781-786) in transfected or BAdV-3-infected cells. The cleaved C terminus (107 aa) was localized to the nucleolus of the transfected cells. Further analysis suggested that the cleaved C terminus contains a bipartite nuclear localization signal and utilizes import receptor importin-α3 of the classical importin-α/β transport pathway for nuclear transport. Successful isolation of recombinant BAdV-3 expressing mutant 100K (substitution of alanine for glycine in the potential protease cleavage site) suggested that cytoplasmic cleavage of BAdV-3 100K by adenoviral protease is not essential for virus replication.

Cloning and expression analysis of HSP70 gene from mangrove plant Kandelia obovata under cold stress.

Heat shock protein 70 (HSP70), the primary member of the HSPs that play various stress-protective roles in plants. In this study, a hsp70 gene of Kandelia obovata (KoHsp70) was cloned by rapid amplification of cDNA ends (RACE). The full-length of KoHsp70 was 2255 bp, consisting of a 5'-terminal untranslated region (UTR) of 118 bp, a 3'-terminal UTR of 178 bp, and an open reading frame (ORF) of 1959 bp. The ORF (KoHSP70) was predicted to encode a polypeptide of 652 amino acids with a theoretical molecular weight (MW) of 71.40 kDa and a pI of 5.16. The amino acid sequence analysis revealed that the KoHSP70 contained three conserved regions of HSP70 family, a bipartite nuclear localization signal sequences (NLS), an ATP/GTP-binding site motif and a cytoplasmic characteristic motif (EEVD). Homology analysis indicated that KoHSP70 shared 96.0 % identity with the known HSP70 (Gossypium hirsutum). Bioinformatics analysis indicated that the KoHSP70 was hydrophilic and had no signal peptide or transmembrane region. The mRNA expression of KoHsp70 kept relatively stable at first and then increased significantly after 48 h cold stress, and reached the highest level at 168 h after cold treatment. The results indicated that the KoHsp70 was a stress-inducible gene that might play a role in cold stress-protective response and in coping with environmental and biological stresses for K. obovata. This study provided a basis to further study the mechanism of anti-adverseness and expression characteristics under stress conditions of K. obovata.