Nuclear Localization Signal Motif Research Articles

HNF1A-MODY Mutations in Nuclear Localization Signal Impair HNF1A-Import Receptor KPNA6 Interactions.

Mutations in hepatocyte nuclear factor (HNF)1A gene cause the most common form of Maturity-onset diabetes of the young (MODY), a monogenic subtype of diabetes mellitus. Functional characterization of mutant proteins reveals that mutations may disrupt DNA binding capacity, transactivation ability and nuclear localization of HNF1A depending on the position of the mutation. Previously identified Arg271Trp and Ser345Tyr mutations in HNF1A were found to be defective in nuclear localization. Arg271 residue resides in a region similar to classical nuclear localization signal (NLS) motif, while Ser345 does not. Importin α family members recognize NLS motifs on cargo proteins and subsequently translocate them into nucleus. Here, we first investigated the nuclear localization mechanism of wild type HNF1A protein. For this purpose, we analyzed the interaction of HNF1A with three mouse homolog importin α proteins (KPNA2, KPNA4 and KPNA6) by co-immunoprecipitation assay and molecular docking simulation. Hereby, KPNA6 was identified as the main import receptor, which is responsible for the transport of HNF1A into the nucleus. Immunolocalization studies in mouse pancreatic cells (Min6) also confirmed the co-localization of HNF1A and KPNA6 in the cytoplasm. Secondly, the interaction between KPNA6 and mutant HNF1A proteins (Arg271Trp and Ser345Tyr) was assessed. Co-immunoprecipitation studies revealed a reduced interaction compared to wild type HNF1A. Our study demonstrated for the first time that HNF1A transcription factor is recognized and transported by importin/karyopherin import family, and mutations in NLS motifs may disrupt the interaction leading to nuclear localization abnormalities and MODY phenotype.

Trypanosoma cruzi Importin α: ability to bind to a functional classical nuclear localization signal of the bipartite type.

Importin α, a transport factor in the classical pathway of nuclear transport of proteins in eukaryotes, has not been experimentally studied in trypanosomatids. A chimeric fluorescent version of this protein (TcImportin α-EGFP) expressed in transfected epimastigotes of Trypanosoma cruzi is characterized here. Initially, the cellular localization of the tagged protein was analysed in exponentially growing and non-growing quiescent cells in a stationary phase. In growing epimastigotes, the fluorescence signal appeared to be mostly localized in the nucleolus, with additional minor fluorescent dots observed close to the nuclear periphery. In the stationary phase, both aged epimastigotes and metacyclic trypomastigotes presented with dispersed fluorescence of a granular form within the nucleoplasm of the cells that predominantly localized in poorly DAPI-stained regions. On the other hand, the ability of a tagged (6×His) version of TcImportin α to bind the nuclear protein cargo TcRPA31 (TcRPA31-EGFP) was determined by pull-down assays of co-transfected cultures. In addition, the results from the in vitro analyses with these tagged recombinant proteins showed that the functional nuclear localization signal (NLS) previously mapped to TcRPA31 was sufficient to sustain binding to TcImportin α. Moreover, the second cluster of basic amino acids within this bipartite NLS (formerly termed element B) was found to be essential for complex formation, as previously described for the nuclear translocation of these fluorescent chimeras. To our knowledge, this approach is the first in which Importin α was experimentally researched in kinetoplastids. The ability of TcImportin α to bind the NLS motif analysed here, is an essential feature expected for its potential functional role as a soluble transport factor.

Resolving the Interactome of the Human Macrophage Immunometabolism Regulator (MACIR) with Enhanced Membrane Protein Preparation and Affinity Proteomics.

Expression of the macrophage immunometabolism regulator gene (MACIR) is associated with severity of autoimmune disease pathology and with the regulation of macrophage biology through unknown mechanisms. The encoded 206 amino acid protein lacks homology to any characterized protein sequence and is a disordered protein according to structure prediction algorithms. To identify interactions of MACIR with proteins from all subcellular compartments, a membrane solubilization buffer is employed, that together with a high affinity EF hand based pull down method, increases the resolution of quantitative mass spectrometry analysis with significant enrichment of interactions from membrane bound nuclear and mitochondrial compartments compared to samples prepared with radioimmunoprecipitation assay buffer. A total of 63 significant interacting proteins are identified and interaction with the nuclear transport receptor TNPO1 and the trafficking proteins UNC119 homolog A and B are validated by immunoprecipitation. Mutational analysis in two candidate nuclear localization signal motifs in the MACIR amino acid sequence shows the interaction with TNPO1 is likely via a non-classical proline/tyrosine-nuclear localization signal motif (aa98-117). It is shown that employing a highly specific and high affinity pull down method that performs efficiently in this glycerol and detergent rich buffer is a powerful approach for the analysis of uncharacterized protein interactomes.

BRCA1-associated protein induced proliferation and migration of gastric cancer cells through MAPK pathway

BRCA1-associated protein (BRAP) was first found to bind to the nuclear localization signal motifs of BRCA1. In this study, we investigated the role of BRAP in gastric cancer. The cancer genome atlas(TCGA) data were obtained from UALCAN. We downregulated and upregulated the level of BRAP in gastric cancer cells by transfection with shRNAs and plasmids. Then, we evaluated the expression of BRAP by qRT-PCR and investigated the expression of important proteins by Western blot analysis. We conducted a microarray analysis to identify the function of BRAP in gastric cancer cells. Then, we investigated the effect of BRAP on proliferation and migration by CCK-8 assays, colony formation assays, wound healing assays and an extreme limiting dilution analysis. The analysis of TCGA data showed that BRAP was significantly overexpressed in gastric cancer tissues compared to that in normal gastric mucosal tissues (P < 0.001). A hybridization-based microarray assay was used to analyze MGC-803 cells and BRAP-downregulated MGC-803 cells. We found 22,199 protein-coding RNAs that were differentially expressed. The genes in the two groups were analyzed with the Kyoto Encyclopedia of Genes and Genomes (KEGG) database, and both the focal adhesion and MAPK pathways were significantly enriched. The results of Cell Counting Kit-8(CCK-8) assays, colony formation assays, wound healing assays and the extreme limiting dilution analysis showed that the knockdown of BRAP reduced gastric cancer cell proliferation and migration and inhibited the process of epithelial-mesenehymal transition (EMT). The overexpression of BRAP induced gastric cancer cell proliferation, migration and the process of EMT. To verify the function of the mitogen-activated protein kinase (MAPK) signaling pathway, we performed a Western blot analysis. The results showed that the downregulation of BRAP decreased the levels of p-ERK and p-Raf1, thereby decreasing the activity of the MAPK signaling pathway. The use of Honokiol increased the levels of p-ERK and p-Raf1, rescuing the function of BRAP downregulation in the MAPK pathway. Xenograft tumor transplantation experiments in nude mice further confirmed the role of BRAP in gastric cancer progression and metastasis.

Discovering nuclear targeting signal sequence through protein language learning and multivariate analysis

Nuclear localization signals (NLSs) are peptides that target proteins to the nucleus by binding to carrier proteins in the cytoplasm that transport their cargo across the nuclear membrane. Accurate identification of NLSs can help elucidate the functions of nuclear protein complexes. The currently known NLS predictors are usually specific to certain species or largely dependent on prior knowledge of NLS basic residues. Thus, a more general predictor is highly desired to reduce the potentially high false positives or false negatives in discovering new NLSs. Here, we report a new method, INSP (Identification Nucleus Signal Peptide), to effectively identify NLS mainly based on statistical knowledge and machine learning algorithms. In our NLS machine learning model, we considered the query protein sequence as text and extracted the sequence context features using a natural language model. These word-vector features encode discriminative knowledge of NLS motif frequency and are thus useful for model recognition. The output of the machine learning model will be fused with statistical knowledge of the query sequence to build a final multivariate regression model for NLS peptide identification. The experimental results demonstrate a promising performance of the new INSP approach. INSP is freely available at: www.csbio.sjtu.edu.cn/bioinf/INSP/for academic use.

Retinoic Acid Binding Leads to CRABP2 Rigidification and Dimerization.

Cellular retinoic acid-binding protein 2 (CRABP2) delivers all-trans retinoic acid (atRA) to retinoic acid receptors (RARs), allowing for the activation of specific gene transcription. The structural similarities between free and atRA-bound CRABP2 raise the questions of how atRA binding occurs and how the atRA:CRABP2 complex is recognized by downstream binding partners. Thus, to gain insights into these questions, we conducted a detailed atRA-CRABP2 interaction study using nuclear magnetic resonance spectroscopy. The data showed that free CRABP2 displays widespread intermediate-time scale dynamics that is effectively suppressed upon atRA binding. This effect is mirrored by the fast-time scale dynamics of CRABP2. Unexpectedly, CRABP2 rigidification in response to atRA binding leads to the stabilization of a homodimerization interface, which encompasses residues located on helix α2 and the βC-βD loop as well as residues on strands βI-βA and the βH-βI loop. Critically, this rigidification also affects CRABP2's nuclear localization signal and RAR-binding motif, suggesting that the loss of conformational entropy upon atRA binding may be the key for the diverse cellular functions of CRABP2.

Functional characterization of IRF8 regulation of type II IFN in golden pompano (Trachinotus ovatus)

Interferon regulatory factor 8 (IRF8) increases type I IFN transcription levels by binding to IFN promoters, thereby playing a role in innate immunity. Nevertheless, the detailed mechanism through which IRF8 regulates type II IFN in fish remains ambiguous. In the present study, two genes from the golden pompano (Trachinotus ovatus), IRF8 (ToIRF8) and IFN gamma (ToIFNγ), were identified in the IFN/IRF-based signalling pathway. The full-length ToIRF8 cDNA was composed of 2,141 bp and encoded a 421 amino acid polypeptide; the genomic DNA was 2,917 bp in length and consisted of 8 exons and 7 introns. The putative protein showed the highest sequence identity (90–92%) with fish IRF8 and possessed a DNA-binding domain (DBD), an IRF-association domain (IAD) and a nuclear localization signal (NLS) motif consistent with those of IRF8 in other vertebrates. Furthermore, the ToIRF8 transcripts were expressed in all examined tissues of healthy fish, with higher levels observed in the central nervous and immune relevant tissues. They were upregulated by polyinosinic acid: polycytidylic acid [poly (I: C)], lipopolysaccharide (LPS) and flagellin treatments in the blood, liver, intestine and kidney. The results from assays of subcellular localization showed that ToIRF8 was localized to the cytoplasm. Moreover, to investigate whether ToIRF8 was a regulator of ToIFNγ, a promoter analysis was performed using progressive deletion mutations of ToIFNγ. The results indicated that the region from −601 bp to −468 bp includes the core promoter. Mutation analyses indicated that the activity of the ToIFNγ promoter significantly decreased after the targeted mutation of the M1-M3 binding sites. Additionally, overexpressed ToIRF8 in vitro notably increased the expression of several IFN/IRF-based signalling pathway genes. These results suggest that IRF8 is vital in the defence of T. ovatus against bacterial infection and contributes to a better understanding of the transcriptional mechanisms of ToIRF8 on type II IFN in fish.

LlDREB1G, a novel DREB subfamily gene from Lilium longiflorum, can enhance transgenic Arabidopsis tolerance to multiple abiotic stresses

In plants, most DREB1s/CBFs proteins with a single AP2/ERF domain participate in responses to abiotic stresses. Here, we cloned a DREB subfamily gene from Lilium longiflorum (designated as LlDREB1G). Sequence analyses revealed that it is composed of 219 amino acids, and contains an AP2/ERF domain, a putative nuclear localization signal (NLS), a DSAW/R and LWSY motif. LlDREB1G, which was localized to the nucleus, showed strongly induced expression under low temperature, and was also stimulated by mechanical agitation, heat shock, ABA and H2O2 stresses. Moreover, the LlDREB1G gene had an obvious circadian rhythm and organ specificity in expression. The LlDREB1G transcription factor was functional in the heterologous system (Arabidopsis). Transgenic Arabidopsis plants with higher transcription of LlDREB1G gene exhibited increased acquired thermotolerance and resistance to freezing and dehydration stresses. Besides, the transgenic seeds were insensitive to exogenous ABA treatment than those of the wild type (WT) in germination experiments. Compared with the WT seedlings, the seedlings of transgenic Arabidopsis overexpressing LlDREB1G showed strongly upregulated expression of stress-related genes after cold, drought or acclimation to heat shock treatments. Furthermore, overexpression of LlDREB1G increased the proline levels and survival rates, but decreased the electrolyte leakage and H2O2 accumulation in the transgenic plants after freezing, dehydration and high temperature treatments. These results imply that LlDREB1G gene may be involved in the responses to multiple abiotic stresses in an ABA-dependent and -independent manner. LlDREB1G gene, a DREB subfamily gene from Lilium longiflorum, may be involved in the responses to multiple abiotic stresses in an ABA-dependent and -independent manner.

Identification of nuclear localization signal and nuclear export signal of VP1 from the chicken anemia virus and effects on VP2 shuttling in cells

BackgroundVP1 of the chicken anemia virus (CAV) is a structural protein that is required for virus encapsulation. VP1 proteins are present both in the nucleus and cytoplasm; however, the functional nuclear localization signal (NLS) and nuclear export signal (NES) of VP1 are still unknown. This study aimed to characterize the NLS and NES motifs of VP1 using bioinformatics methods and multiple-site fragment deletions, and investigate shuttling of VP2 from nucleus to cytoplasm by co-transfection with VP1.MethodsTwo putative NLS motifs were predicted by the WoLF PSORT and NLStradamus programs from the amino acid sequence of VP1. Three NES motifs of VP1 were predicted by the NetNES 1.1 Server and ELM server programs. All mutants were created by multiple-site fragment deletion mutagenesis. VP1 and VP2 were co-expressed in cells using plasmid transfection.ResultsA functional NLS motif was identified at amino acid residues 3 to 10 (RRARRPRG) of VP1. Critical amino acids 3 to 10 were significantly involved in nuclear import in cells and were evaluated using systematic deletion mutagenesis. Three NES motifs of VP1 were predicted by the NetNES 1.1 Server and ELM server programs. A functional NES was identified at amino acid residues 375 to 388 (ELDTNFFTLYVAQ). Leptomycin B (LMB) treatment demonstrated that VP1 export from nucleus to cytoplasm occurred through a chromosome region maintenance 1 (CRM1)-dependent pathway. With co-expression of VP1 and VP2 in cells, we observed that VP1 may transport VP2 from nucleus to cytoplasm.ConclusionOur data showed that VP1 of CAV contained functional NLS and NES motifs that modulated nuclear import and export through a CRM1-dependent pathway. Further, VP1 may play a role in the transport of VP2 from nucleus to cytoplasm.

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.

Characterization and in silico proteomic analysis of C2 and C3 proteins of squash leaf curl China virus associated with pumpkin leaf curl disease in Assam, India.

The pumpkin leaf curl disease is an emerging disease of pumpkin in Assam, India. Symptomatic pumpkin leaf samples from different locations were immunologically tested using Begomovirus specific antibody. PCR with the ELISA-positive samples, using Geminivirus universal primers amplified 1.4 kb virus-specific fragments. Sequence of these amplicons showed around 95% identity with squash leaf curl China virus-[Pumpkin: Varanasi] (SLCCV-Pumpkin: Varanasi EU573715). To investigate the possible functions of the viral proteins present in the fragment, the full-length C2 and C3 genes were conceptually translated and were subjected to in silico proteomic analyses. The phylogenetic analysis of both the proteins divulged the relationship of our isolate with related viruses and isolates. Multiple sequence alignment (MSA) of the proteins revealed the presence of the known viral conserved motifs, viz., zinc-finger (ZNF) motif [36CXCX(7)CX(6)H53], the arginine-rich nuclear localization signal (NLS) motif (28RRRR31) as well as the minimal activation domain in C2 protein. In the C3 protein, the 91LKYLD95 and the replication enhancer motif (30YFK32) were found to be conserved. Finally, 3-D models of the two proteins were predicted via ab initio approach and subsequently, the models were validated. To our knowledge, this study is a pioneering attempt to construct the ab initio 3-D models of two begomoviral proteins taking a SLCCV isolate as a model. Keywords: begomovirus; ELISA; ZNF motif; NLS motif; ab initio modelling.

Evidence for Stabilizing Selection Driving Mutational Turnover of Short Motifs in the Eukaryotic Complementary Sex Determiner (Csd) Protein.

Short linear motifs (SLiMs) can play pivotal functional roles in proteins, such as targeting proteins to specific subcellular localizations, modulating the efficiency of translation and tagging proteins for degradation. Until recently we had little knowledge about SLiM evolution. Only a few amino acids in these motifs are functionally important, making them likely to evolve ex nihilo and suggesting that they can play key roles in protein evolution. Several reports now suggest that these motifs can appear and disappear while their function in the protein is preserved, a process sometimes referred to as “turnover”. However, there has been a lack of specific experiments to determine whether independently evolved motifs do indeed have the same function, which would conclusively determine whether the process of turnover actually occurs. In this study, we experimentally detected evidence for such a mutational turnover process for nuclear localization signals (NLS) during the post-duplication divergence of the Complementary sex determiner (Csd) and Feminizer (Fem) proteins in the honeybee (Apis mellifera) lineage. Experiments on the nuclear transport activity of protein segments and those of the most recent common ancestor (MRCA) sequences revealed that three new NLS motifs evolved in the Csd protein during the post-duplication divergence while other NLS motifs were lost that existed before duplication. A screen for essential and newly evolved amino acids revealed that new motifs in the Csd protein evolved by one or two missense mutations coding for lysine. Amino acids that were predating the duplication were also essential in the acquisition of the C1 motif suggesting that the ex nihilo origin was constrained by preexisting amino acids in the physical proximity. Our data support a model in which stabilizing selection maintains the constancy of nuclear transport function but allowed mutational turnover of the encoding NLS motifs.

Nuclear localization of Hif‐3α requires two redundant NLS motifs in its unique C‐terminal region

Hif-3α, a member of the hypoxia-inducible factor (HIF) family, enters the nucleus and regulates gene expression in response to hypoxia. The molecular basis of its nuclear localization is not clear. HIF-1α and HIF-2α use a bipartite nuclear localization signal (NLS) to enter the nucleus. This motif is not conserved in Hif-3α. Although there is a conserved Arg/Lys rich motif in the Hif-3α N-terminal region, deletion of this region has minimal effect on Hif-3α nuclear localization. Here, we mapped the functional NLS to the unique C-terminal region of Hif-3α and identified two clusters of basic residues critical for its nuclear localization. The two NLS motifs are functionally redundant. Our results, thus, suggest that Hif-3α nuclear localization is mediated through two redundant NLS motifs located in its unique C-terminal region.

Intracellular Localization of Blattella germanica Densovirus (BgDV1) Capsid Proteins.

Densovirus genome replication and capsid assembly take place in the nucleus of the infected cells. However, the mechanisms underlying such processes as the delivery of virus proteins to the nucleus and the export of progeny virus from the nucleus remain elusive. It is evident that nuclear transport signals should be involved in these processes. We performed an in silico search for the putative nuclear localization signal (NLS) and nuclear export signal (NES) motifs in the capsid proteins of the Blattella germanica Densovirus 1 (BgDV1) densovirus. A high probability NLS motif was found in the common C-terminal of capsid proteins together with a NES motif in the unique N-terminal of VP2. We also performed a global search for the nuclear traffic signals in the densoviruses belonging to five Densovirinae genera, which revealed high diversity in the patterns of NLSs and NESs. Using a heterologous system, the HeLa mammalian cell line expressing GFP-fused BgDV1 capsid proteins, we demonstrated that both signals are functionally active. We suggest that the NLS shared by all three BgDV1 capsid proteins drives the trafficking of the newly-synthesized proteins into the nucleus, while the NES may play a role in the export of the newly-assembled BgDV1 particles into the cytoplasm through nuclear pore complexes.

Identification of DEAD-Box RNA Helicase DDX41 as a Trafficking Protein That Involves in Multiple Innate Immune Signaling Pathways in a Zebrafish Model.

DDX41 is an important sensor for host recognition of DNA viruses and initiation of nuclear factor-κB (NF-κB) and IFN signaling pathways in mammals. However, its occurrence and functions in other vertebrates remain poorly defined. Here, a DDX41 ortholog [Danio rerio DDX41 (DrDDX41)] with various conserved structural features to its mammalian counterparts was identified from a zebrafish model. This DrDDX41 was found to be a trafficking protein distributed in the nucleus of resting cells but transported into the cytoplasm under DNA stimulation. Two nuclear localization signal motifs were localized beside the coiled-coil domain, whereas one nuclear export signal motif existed in the DEADc domain. DrDDX41 acts as an initiator for the activation of NF-κB and IFN signaling pathways in a Danio rerio STING (DrSTING)-dependent manner through its DEADc domain, which is a typical performance of mammalian DDX41. These observations suggested the conservation of DDX41 proteins throughout the vertebrate evolution, making zebrafish an alternative model in understanding DDX41-mediated immunology. With this model system, we found that DrDDX41 contributes to DrSTING–Danio rerio STAT6 (DrSTAT6)-mediated chemokine (Danio rerio CCL20) production through its DEADc domain. To the best of our knowledge, this work is the first report showing that DDX41 is an upstream initiator in this newly identified signaling pathway. The DrDDX41-mediated signaling pathways play important roles in innate antibacterial immunity because knockdown of either DrDDX41 or DrSTING/DrSTAT6 significantly reduced the survival of zebrafish under Aeromonas hydrophilia or Edwardsiella tarda infection. Our findings would enrich the current knowledge of DDX41-mediated immunology and the evolutionary history of the DDX41 family.

The 164 K, 165 K and 167 K residues in 160YPVVKKPKLTEE171 are required for the nuclear import of goose parvovirus VP1

goose parvovirus (GPV) belongs to the Dependoparvovirus genus in Parvovirinae subfamily within Parvoviridae family, is the etiological agent of Derzsy's disease. Nuclear localization signal (NLS) is important for parvovirus lifecycle in the delivery of genomes and the structural protein of progeny virus into the nucleus. Here, NLS was first identified in GPV. By using the PSORT II program, a basic region (BR, 160YPVVKKPKLTEE171) in the N-terminus of VP1 was found, which predicted as putative NLS motif of goose parvovirus capsid. The GPV BR could transfer both small reporter proteins (EGFP) and large reporter protein (β-galactosidase) into the nucleus by Immunofluorescence assay. Furthermore, the K164A, or K165A, or K167A substitutions mutation of GPV VP1 did abolish its nuclear localization, suggesting that the 164 K, 165 K and 167 K residues in the 160YPVVKKPKLTEE171 are required for its for nuclear import. Our finding may help us to gain a better understand of GPV lifecycle.

A novel nuclear localization signal spans the linker of the two DNA-binding subdomains in the conserved paired domain of Pax6.

Paired box (Pax) 6, a member of the Pax family of transcription factors, contains two DNA-binding domains, called the paired domain (PD) and the homeodomain (HD), and plays pivotal roles in development of structures such as the eye, central nervous system and pancreas. Pax6 is a major developmental switching molecule because, for example, ectopic expression of the Pax6 gene can induce ectopic whole eye development. Intensive research has been devoted to elucidating the molecular mechanism(s) involved in the function(s) of Pax6, but many issues remain unexplained. One of the important issues is to identify the nuclear localization signal (NLS) in the PD of Pax6, which is predicted to have a stronger NLS activity than that in the HD. We produced expression plasmid constructs that encode the chick Pax6 protein modified to delete the entire PD except for fragments containing putative NLS sequences, and electroporated them in ovo into the developing chick midbrain to define the NLS of the PD. The results show that the NLS in the PD of chick Pax6 consists of an unusually long sequence of 36 amino acid residues. Within this long NLS motif, the central 18 amino acids comprising two consecutive nine-residue segments showed highest NLS activity; this central area corresponds to the C-terminal half of the third α-helix of the PAI subdomain and the subsequent 11 amino acids of a 16-residue linker between PAI and the adjacent RED subdomain. This information helps to elucidate the molecular mechanism by which Pax6 plays a pivotal role during ontogeny.

The Novel Secreted Meloidogyne incognita Effector MiISE6 Targets the Host Nucleus and Facilitates Parasitism in Arabidopsis.

Meloidogyne incognita is highly specialized parasite that interacts with host plants using a range of strategies. The effectors are synthesized in the esophageal glands and secreted into plant cells through a needle-like stylet during parasitism. In this study, based on RNA-seq and bioinformatics analysis, we predicted 110 putative Meloidogyne incognita effectors that contain nuclear localization signals (NLSs). Combining the Burkholderia glumae–pEDV based screening system with subcellular localization, from 20 randomly selected NLS effector candidates, we identified an effector MiISE6 that can effectively suppress B. glumae-induced cell death in Nicotiana benthamiana, targets to the nuclei of plant cells, and is highly expressed in early parasitic J2 stage. Sequence analysis showed that MiISE6 is a 157-amino acid peptide, with an OGFr_N domain and two NLS motifs. Hybridization in situ verified that MiISE6 is expressed in the subventral esophageal glands. Yeast invertase secretion assay validated the function of the signal peptide harbored in MiISE6. Transgenic Arabidopsis thaliana plants expressing MiISE6 become more susceptible to M. incognita. Inversely, the host-derived RNAi of MiISE6 of the nematode can decrease its parasitism on host. Based on transcriptome analysis of the MiISE6 transgenic Arabidopsis samples and the wild-type samples, we obtained 852 differentially expressed genes (DEGs). Integrating Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses, we found that expression of MiISE6 in Arabidopsis can suppress jasmonate signaling pathway. In addition, the expression of genes related to cell wall modification and the ubiquitination proteasome pathway also have detectable changes in the transgenic plants. Results from the present study suggest that MiISE6 is involved in interaction between nematode-plant, and plays an important role during the early stages of parasitism by interfering multiple signaling pathways of plant. Moreover, we found homologs of MiISE6 in other sedentary nematodes, Meloidogyne hapla and Globodera pallida. Our experimental results provide evidence to decipher the molecular mechanisms underlying the manipulation of host immune defense responses by plant parasitic nematodes, and transcriptome data also provide useful information for further study nematode–plant interactions.

Plasmodium circumsporozoite protein suppresses the growth of A549 cells via inhibiting nuclear transcription factor κB

Blocking the activation of nuclear factor κB (NF-κB) is a promising strategy for the treatment of non-small cell lung cancer. The circumsporozoite protein (CSP), a key component of the sporozoite stage of the malaria parasite, was previously reported to block NF-κB activation in hepatocytes. Therefore, in the present study, the effect of CSP on the growth of the human lung cancer cell line, A549, was investigated. It was demonstrated that transfection with a recombinant plasmid expressing CSP was able to inhibit the proliferation of A549 cells in a dose-dependent manner and induce the apoptosis of A549 cells. A NF-κB gene reporter assay indicated that CSP and its nuclear localization signal (NLS) motif were able to equally suppress the activation of NF-κB following stimulation with human recombinant tumor necrosis factor (TNF)-α in A549 cells. Furthermore, western blot analysis indicated that NLS did not affect the phosphorylation and degradation of IκB, but was able to markedly inhibit the nuclear translocation of NF-κB in TNF-α stimulated A549 cells. Therefore, the data suggest that CSP may be investigated as a potential novel NF-κB inhibitor for the treatment of lung cancer.

Non-equivalence of nuclear import among nuclei in multinucleated skeletal muscle cells.

Skeletal muscle is primarily composed of large myofibers containing thousands of post-mitotic nuclei distributed throughout a common cytoplasm. Protein production and localization in specialized myofiber regions is crucial for muscle function. Myonuclei differ in transcriptional activity and protein accumulation, but how these differences among nuclei sharing a cytoplasm are achieved is unknown. Regulated nuclear import of proteins is one potential mechanism for regulating transcription spatially and temporally in individual myonuclei. The best-characterized nuclear localization signal (NLS) in proteins is the classical NLS (cNLS), but many other NLS motifs exist. We examined cNLS and non-cNLS reporter protein import using multinucleated muscle cells generated in vitro, revealing that cNLS and non-cNLS nuclear import differs among nuclei in the same cell. Investigation of cNLS nuclear import rates in isolated myofibers ex vivo confirmed differences in nuclear import rates among myonuclei. Analyzing nuclear import throughout myogenesis revealed that cNLS and non-cNLS import varies during differentiation. Taken together, our results suggest that both spatial and temporal regulation of nuclear import pathways are important in muscle cell differentiation and protein regionalization in myofibers.