Residues within the Foot-and-Mouth Disease Virus 3Dpol Nuclear Localization Signal Affect Polymerase Fidelity.

Many RNA viruses encode a proof-reading deficient, low-fidelity RNA-dependent polymerase (RdRp), which generates genetically diverse populations that can adapt to changing environments and thwart antiviral therapies. 3Dpol, the RdRp of the foot-and-mouth disease virus (FMDV), is responsible for replication of viral genomes. The 3Dpol N terminus encodes a nuclear localization signal (NLS) sequence,MRKTKLAPT, important for import of the protein to host nucleus. Previous studies showed that substitutions at residues 18 and 20 of the NLS are defective in proper incorporation of nucleotides and RNA binding. Here, we use a systematic alanine scanning mutagenesis approach to understand the role of individual residues of the NLS in nuclear localization and nucleotide incorporation activities of 3Dpol We identify two residues of 3Dpol NLS, T19 and L21, that are important for the maintenance of enzyme fidelity. The 3Dpol NLS alanine substitutions of T19 and L21 results in aberrant incorporation of nucleoside analogs, conferring a low fidelity phenotype of the enzyme. A molecular dynamics simulation of RNA- and mutagen (RTP)-bound 3Dpol revealed that the T19 residue participates in a hydrogen bond network, including D165 in motif F and R416 at the C terminus of the FMDV 3Dpol and RNA template-primer. Based on these findings and previous studies, we conclude that at least the first six residues of theMRKTKLAPT sequence motif play a vital role in the maintenance of faithful RNA synthesis activity (fidelity) of FMDV 3Dpol, suggesting that the role of the NLS motif in similar viral polymerases needs to be revisited.IMPORTANCE In this study, we employed genetic and molecular dynamics approaches to analyze the role of individual amino acids of the FMDV 3Dpol nuclear localization signal (NLS). The NLS residues were mutated to alanine using a type A full-genome cDNA clone, and the virus progeny was analyzed for defects in growth and in competition with the parental virus. We identified two mutants in 3Dpol, T19A and L21A, that exhibited high rate of mutation, were sensitive to nucleotide analogs, and displayed reduced replicative fitness compared to the parental virus. Using molecular dynamics simulation, we demonstrated that residues T19 and L21 played a role in the structural configuration of the interaction network at the 3Dpol palm subdomain. Cumulatively, our data suggest that the T19 and L21 3Dpol amino acids are important for maintaining the fidelity of the FMDV polymerase and ensuring faithful replication of the FMDV genome.

Nuclear pore targeting of the yeast Pom33 nucleoporin depends on karyopherin and lipid binding.

Pom33 is an integral membrane protein of the yeast nuclear pore complex (NPC), and it is required for proper NPC distribution and assembly. To characterize the Pom33 NPC-targeting determinants, we performed immunoprecipitation experiments followed by mass spectrometry analyses. This identified a new Pom33 partner, the nuclear import factor Kap123. In vitro experiments revealed a direct interaction between the Pom33 C-terminal domain (CTD) and Kap123. In silico analysis predicted the presence of two amphipathic α-helices within Pom33-CTD. Circular dichroism and liposome co-flotation assays showed that this domain is able to fold into α-helices in the presence of liposomes and preferentially binds to highly curved lipid membranes. When expressed in yeast, under conditions abolishing Pom33-CTD membrane association, this domain behaves as a Kap123-dependent nuclear localization signal (NLS). Although deletion of Pom33 C-terminal domain (Pom33(ΔCTD)-GFP) impaired Pom33 stability and NPC targeting, mutants affecting either Kap123 binding or the amphipathic properties of the α-helices did not display any detectable defect. However, combined impairment of lipid and Kap123 binding affects targeting of Pom33 to NPCs. These data highlight the requirement of multiple determinants and mechanisms for proper NPC localization of Pom33.

Low-Molecular Weight Polyethylenimine Modified with Pluronic 123 and RGD- or Chimeric RGD-NLS Peptide: Characteristics and Transfection Efficacy of Their Complexes with Plasmid DNA.

To solve the problem of transfection efficiency vs. cytotoxicity and tumor-targeting ability when polyethylenimine (PEI) was used as a nonviral gene delivery vector, new degradable PEI polymers were synthesized via cross-linking low-molecular-weight PEI with Pluronic P123 and then further coupled with a targeting peptide R4 (RGD) and a bifunctional R11 (RGD-NLS), which were termed as P123-PEI-R4 and P123-PEI-R11, respectively. Agarose gel electrophoresis showed that both P123-PEI-R4 and P123-PEI-R11 efficaciously condense plasmid DNA at a polymer-to-pDNA w/w ratio of 3.0 and 0.4, respectively. The polyplexes were stable in the presence of serum and could protect plasmid DNA against DNaseI. They had uniform spherical nanoparticles with appropriate sizes around 100-280 nm and zeta-potentials about +40 mV. Furthermore, in vitro experiments showed that these polyplexes had lower cytotoxicity at any concentration compared with PEI 25 kDa, thus giving promise to high transfection efficiency as compared with another P123-PEI derivate conjugated with trifunctional peptide RGD-TAT-NLS (P123-PEI-R18). More importantly, compared with the other polymers, P123-PEI-R11 showed the highest transfection efficiency with relatively lower cytotoxicity at any concentration, indicating that the new synthetic polymer P123-PEI-R11 could be used as a safe and efficient gene deliver vector.

Polymer-peptide delivery platforms: effect of oligopeptide orientation on polymer-based DNA delivery.

The success of nonviral transfection using polymers hinges on efficient nuclear uptake of nucleic acid cargo and overcoming intra- and extracellular barriers. By incorporating PKKKRKV heptapeptide pendent groups as nuclear localization signals (NLS) on a polymer backbone, we demonstrate protein expression levels higher than those obtained from JetPEI and Lipofectamine 2000, the latter being notorious for coupling high transfection efficiency with cytotoxicity. The orientation of the NLS peptide grafts markedly affected transfection performance. Polymers with the sequence attached to the backbone from the valine residue achieved a level of nuclear translocation higher than the levels of those having the NLS groups attached in the opposite orientation. The differences in nuclear localization and DNA complexation strength between the two orientations correlated with a striking difference in protein expression, both in cell culture and in vivo. Polyplexes formed from these comb polymer structures exhibited transfection efficiencies superior to those of Lipofectamine 2000 but with greatly reduced toxicity. Moreover, these novel polymers, when administered by intramuscular ultrasound-mediated delivery, allowed a high level of reporter gene expression in mice, demonstrating their therapeutic promise in vivo.

Proximity labelling of pro-interleukin-1α reveals evolutionary conserved nuclear interactions.

Interleukin-1α is a suggested dual-function cytokine that diverged from interleukin-1ß in mammals potentially by acquiring additional biological roles that relate to highly conserved regions in the pro-domain of interleukin-1α, including a nuclear localisation sequence and histone acetyltransferase-binding domains. Why evolution modified pro-interleukin-1α's subcellular location and protein interactome, and how this shaped interleukin-1α's intracellular role, is unknown. Here we show that TurboID proximity labelling with pro-interleukin-1α suggests a nuclear role for pro-interleukin-1α that involves interaction with histone acetyltransferases, including EP300. We also identify and validate inactivating mutations in the pro-interleukin-1α nuclear localisation sequence of multiple mammalian species, including toothed whales, castorimorpha and marsupials. However, histone acetyltransferase-binding domains are conserved in those species that have lost pro-interleukin-1α nuclear localisation. Together, these data suggest that histone acetyltransferase binding and nuclear localisation occurred together, and that while some species lost the nuclear localisation sequence in their pro-interleukin-1α, histone acetyltransferase binding ability was maintained. The nuclear localisation sequence was lost from several distinct species at different evolutionary times, suggesting convergent evolution, and that the loss of the nuclear localisation sequence confers some important biological outcome.

Facial asymmetry and clinical manifestations in patients with novel insertion of the TCOF1 gene.

This study explored the role of TCOF1 insertion mutations in Taiwanese patients with craniofacial anomalies. Twelve patients with single or multiple, asymmetrical congenital craniofacial anomalies were enrolled. Genomic DNA was prepared from leukocytes; the coding regions of TCOF1 were analyzed by polymerase chain reaction and direct sequencing. Clinical manifestations were correlated to the TCOF1 mutation. Six of 12 patients diagnosed with hemifacial microsomia exhibited a novel insertion mutation 4127 ins G (frameshift) in exon 24 in the TCOF1 gene. All six patients were diagnosed with anomalies on the left side. In addition, four of these six patients had hearing impairment; three had other major anomalies; and two had developmental delay. The insertion caused a frameshift, an early truncation, the loss of two putative nuclear localization signals (residues 1404-1420 and 1424-1440), and the loss of coiled coil domain (1406-1426) in treacle protein. These findings support the existence of two regulators of growth of the mandibular condyles.

Multifunctional REDV-G-TAT-G-NLS-Cys peptide sequence conjugated gene carriers to enhance gene transfection efficiency in endothelial cells.

Rapid endothelialization on small diameter artificial blood vessels is an effective strategy to facilitate long-term patency and inhibit thrombosis. The gene delivery can enhance the proliferation and migration of endothelial cells (ECs), which is beneficial for rapid endothelialization. REDV-G-TAT-G-NLS-Cys (abbreviated as TP-G) peptide could weakly condense pEGFP-ZNF580 (pZNF580) and transfect ECs, but its transfection efficiency was still very low because of its low positive charge, low stability and weak endosome escape ability. In order to develop more stable and efficient gene carriers with low cytotoxicity, in the present study, we conjugated different amounts of TP-G peptide onto poly(lactide-co-glycolide)-g-polyethylenimine (PLGA-g-PEI) amphiphilic copolymers via a hetero-poly(ethylene glycol) spacer (OPSS-PEG-NHS). The TP-G peptide and PEI could cooperatively and strongly condense pZNF580. The carrier's cytotoxicity was reduced by the introduction of poly(ethylene glycol) spacer. They condensed pZNF580 to form gene complexes (PPP-TP-G/pZNF580) with suitable size and positive zeta potential for gene delivery. The transfected ECs promoted their migration ability as demonstrated by cell migration assay. The results of cellular uptake and confocal laser scanning microscopy showed significantly high internalization efficiency, endosomal/lysosomal escape and nucleus location of pZNF580 by this multifunctional TP-G peptide sequence conjugated gene delivery system. Furthermore, several inhibitors were used to study the cellular uptake pathways of PPP-TP-G/pZNF580 complexes. The results showed that PPP-TP-G2/Cy5-oligonucleotide complexes exhibited the optimized endocytosis pathways which facilitated for cellular uptake. In conclusion, the multifunctional TP-G peptide conjugated gene carriers could promote the transfection efficiency due to the multifunction of REDV, cell-penetrating peptide and nuclear localization signal in the peptide sequence, which could be a suitable gene carrier for endothelialization.

Nuclear localization of the parafibromin tumor suppressor protein implicated in the hyperparathyroidism-jaw tumor syndrome enhances its proapoptotic function.

Parafibromin is a tumor suppressor protein encoded by HRPT2, a gene recently implicated in the hereditary hyperparathyroidism-jaw tumor syndrome, parathyroid cancer, and a subset of kindreds with familial isolated hyperparathyroidism. Human parafibromin binds to RNA polymerase II as part of a PAF1 transcriptional regulatory complex. The mechanism by which loss of parafibromin function can lead to neoplastic transformation is poorly understood. Because the subcellular localization of parafibromin is likely to be critical for its function with the nuclear PAF1 complex, we sought to experimentally define the nuclear localization signal (NLS) of parafibromin and examine its potential role in parafibromin function. Using site-directed mutagenesis, we define a dominant bipartite NLS and a secondary NLS, both in the NH(2)-terminal region of parafibromin whose combined mutation nearly abolishes nuclear targeting. The NLS-mutant parafibromin is significantly impaired in its association with endogenous Paf1 and Leo1. We further report that overexpression of wild-type but not NLS-mutant parafibromin induces apoptosis in transfected cells. Inhibition of endogenous parafibromin expression by RNA interference inhibits the basal rate of apoptosis and apoptosis resulting from DNA damage induced by camptothecin, a topoisomerase I inhibitor. These experiments identify for the first time a proapoptotic activity of endogenous parafibromin likely to be important in its role as a tumor suppressor and show a functional role for the NLS of parafibromin in this activity.

Multimerization via its myosin domain facilitates nuclear localization and inhibition of core binding factor (CBF) activities by the CBFbeta-smooth muscle myosin heavy chain myeloid leukemia oncoprotein.

In CBFbeta-SMMHC, core binding factor beta (CBFbeta) is fused to the alpha-helical rod domain of smooth muscle myosin heavy chain (SMMHC). We generated Ba/F3 hematopoietic cells expressing a CBFbeta-SMMHC variant lacking 28 amino acids homologous to the assembly competence domain (ACD) required for multimerization of skeletal muscle myosin. CBFbeta-SMMHC(DeltaACD) multimerized less effectively than either wild-type protein or a variant lacking a different 28-residue segment. In contrast to the control proteins, the DeltaACD mutant did not inhibit CBF DNA binding, AML1-mediated reporter activation, or G(1) to S cell cycle progression, the last being dependent upon activation of CBF-regulated genes. We also linked the CBFbeta domain to 149 or 83 C-terminal CBFbeta-SMMHC residues, retaining 86 or 20 amino acids N-terminal to the ACD. CBFbeta-SMMHC(149C) multimerized and slowed Ba/F3 proliferation, whereas CBFbeta-SMMHC(83C) did not. The majority of CBFbeta-SMMHC and CBFbeta-SMMHC(149C) was detected in the nucleus, whereas the DeltaACD and 83C variants were predominantly cytoplasmic, indicating that multimerization facilitates nuclear retention of CBFbeta-SMMHC. When linked to the simian virus 40 nuclear localization signal (NLS), a significant fraction of CBFbeta-SMMHC(DeltaACD) entered the nucleus but only mildly inhibited CBF activities. As NLS-CBFbeta-SMMHC(83C) remained cytoplasmic, we directed the ACD to CBF target genes by linking it to the AML1 DNA binding domain or to full-length AML1. These AML1-ACD fusion proteins did not affect Ba/F3 proliferation, in contrast to AML1-ETO, which markedly slowed G(1) to S progression dependent upon the integrity of its DNA-binding domain. Thus, the ACD facilitates inhibition of CBF by mediating multimerization of CBFbeta-SMMHC in the nucleus. Therapeutics targeting the ACD may be effective in acute myeloid leukemia cases associated with CBFbeta-SMMHC expression.

Nuclear targeting of non-viral gene carriers using psoralen-nuclear localization signal (NLS) conjugates.

A nuclear localization signal was non-covalently attached to DNA for the purpose of enhancing transfection efficiencies of non-viral gene carriers. Psoralen, a nucleic acid-intercalating agent, was chemically attached to a signal peptide. The conjugate spontaneously intercalated into DNA and then poly(ethyleneimine) [PEI] was added to prepare a DNA/PEI complex containing the signal peptide moieties. The existence of the conjugate did not alter the complexation process between DNA and PEI, which was confirmed by dynamic light scattering. The conjugate was slowly released from the DNA/PEI complex for 24h, while a burst release was examined when the conjugated was added to DNA without PEI. The complex containing a signal peptide moiety increased transfection efficiencies on COS-1 cells, compared to a mutant signal peptide or a control. Cytotoxicity of the conjugate slowly increased as the amount of the conjugate increased, however, the cytotoxic effect of the conjugate was not significant at the effective concentration of the conjugate for transfections. Therefore, the psoralen-nuclear localization signal is expected to be a potent transfection enhancing agent without a covalent modification of transgenes.

Development and in vitro validation of a targeted delivery vehicle for DNA vaccines.

Usage of DNA vaccination has been limited by inefficient cellular expression of plasmid constructs used in DNA vaccines. We describe a novel system for enhancing delivery of DNA vaccine plasmids into cells and their nuclei. This delivery system uses recombinant reovirus type 3 sigma1 attachment protein genetically modified with a nuclear localization sequence (sigma1-NLS) as a targeting ligand. Purified sigma1-NLS was covalently conjugated to the polycation polyethyleneimine (PEI) using a carboxyl-reactive cross-linking agent and complexed with plasmid DNA. The benefit of the NLS in enhancement of protein delivery into the nucleus was demonstrated by liposome-mediated loading of cells with sigma1 or sigma1-NLS. In L929 fibroblasts loaded with sigma1-NLS, 69% of the internalized protein was recovered in the nuclear fraction after 6 h compared to just 10% when using unmodified sigma1. Transfection of L929 cells with sigma1-NLS-conjugated PEI complexed with a luciferase expression plasmid resulted in a mean 16-fold increase in luciferase activity over complexes made with unmodified PEI, compared to a mean 3-fold boost obtained using sigma1-conjugated PEI. These results suggest that sigma1-NLS is a useful bifunctional targeting ligand suitable for enhancing DNA delivery and subsequent gene expression for both DNA vaccine applications and nonviral gene therapy.

Nuclear Localization and Gene Expression Modulation by a Fluorescent Sequence-Selective p-Anisyl-benzimidazolecarboxamido Imidazole-Pyrrole Polyamide.

Synthetic pyrrole (P)-imidazole (I) containing polyamides can target predetermined DNA sequences and modulate gene expression by interfering with transcription factor binding. We have previously shown that rationally designed polyamides targeting the inverted CCAAT box 2 (ICB2) of the topoisomerase IIα (topo IIα) promoter can inhibit binding of transcription factor NF-Y, re-inducing expression of the enzyme in confluent cells. Here, the A/T recognizing fluorophore, p-anisylbenzimidazolecarboxamido (Hx) was incorporated into the hybrid polyamide HxIP, which fluoresces upon binding to DNA, providing an intrinsic probe to monitor cellular uptake. HxIP targets the 5'-TACGAT-3' sequence of the 5' flank of ICB2 with high affinity and sequence specificity, eliciting an ICB2-selective inhibition/displacement of NF-Y. HxIP is readily taken up by NIH3T3 and A549 cells, and detected in the nucleus within minutes. Exposure to the polyamide at confluence resulted in a dose-dependent upregulation of topo IIα expression and enhanced formation of etoposide-induced DNA strand breaks.

Modular design of non-viral vectors with bioactive components.

Inefficient gene delivery continues to limit gene therapy applications to both basic and applied sciences. Approaches for engineering vectors increasingly include bioactive components that bind cellular receptors, disrupt membranes, or enhance nuclear transport. Recently, a novel cationic lipid was developed by modifying the glucocorticoid dexamethasone. This cationic corticosteroid condenses plasmids for gene delivery, while also modulating inflammation. Modular vectors containing bioactive components that target various cellular processes can overcome the barriers limiting gene transfer.

Coupling of importin beta binding peptide on plasmid DNA: transfection efficiency is increased by modification of lipoplex's physico-chemical properties.

BACKGROUND: Non-viral vectors for gene transfer are less immunogenic than viral vectors but also less efficient. Significant effort has focused on enhancing non-viral gene transfer efficiency by increasing nuclear import of plasmid DNA, particularly by coupling nuclear localization peptidic sequences to plasmid DNA. RESULTS: We have coupled a 62-aminoacid peptide derived from hSRP1alpha importin beta binding domain, called the IBB peptide to plasmid DNA by using the heterobifunctional linker N-(4-azido-2,3,5,6 tetrafluorobenzyl)-6-maleimidyl hexanamide (TFPAM-6). When covalently coupled to plasmid DNA, IBB peptide did not increase the efficiency of cationic lipid mediated transfection. The IBB peptide was still able to interact with its nuclear import receptor, importin beta, but non-specifically. However, we observed a 20-fold increase in reporter gene expression with plasmid DNA / IBB peptide complexes under conditions of inefficient transfection. In which case, IBB was associated with plasmid DNA through self assembling ionic interaction. CONCLUSIONS: The improvement of transfection activity was not due to an improved nuclear import of DNA, but rather by the modification of physicochemical properties of IBB peptide / plasmid complexes. IBB peptide increased lipoplex size and these larger complexes were more efficient for gene transfer.

NLS copy-number variation governs efficiency of nuclear import--case study on dUTPases.

Nucleocytoplasmic trafficking of large macromolecules requires an active transport machinery. In many cases, this is initiated by binding of the nuclear localization signal (NLS) peptide of cargo proteins to importin-α molecules. Fine orchestration of nucleocytoplasmic trafficking is of particularly high importance for proteins involved in maintenance of genome integrity, such as dUTPases, which are responsible for prevention of uracil incorporation into the genome. In most eukaryotes, dUTPases have two homotrimeric isoforms: one of these contains three NLSs and is present in the cell nucleus, while the other is located in the cytoplasm or the mitochondria. Here we focus on the unusual occurrence of a pseudo-heterotrimeric dUTPase in Drosophila virilis that contains one NLS, and investigate its localization pattern compared to the homotrimeric dUTPase isoforms of Drosophila melanogaster. Although the interaction of individual NLSs with importin-α has been well characterized, the question of how multiple NLSs of oligomeric cargo proteins affect their trafficking has been less frequently addressed in adequate detail. Using the D. virilis dUTPase as a fully relevant physiologically occurring model protein, we show that NLS copy number influences the efficiency of nuclear import in both insect and mammalian cell lines, as well as in D. melanogaster and D. virilis tissues. Biophysical data indicate that NLS copy number determines the stoichiometry of complexation between importin-α and dUTPases. The main conclusion of our study is that, in D. virilis, a single dUTPase isoform efficiently reproduces the cellular dUTPase distribution pattern that requires two isoforms in D. melanogaster.

Molecular weight-dependent genetic information transfer with disulfide-linked polyethylenimine-based nonviral vectors.

One strategy for improving gene vector properties of polyethylenimine is to facilitate individual transfection mechanism steps. This study investigates (i) improving transfection efficiency by attaching peptide nuclear localization signals (nuclear localization signals: SV40 large T antigen nuclear localization signal or C-terminus of histone H1) to polyethylenimine (10 kDa) and (ii) using disulfide linkages, which are expected to be stable during polyplex formation, but cleaved inside cells giving improved gene release. Nuclear localization signal-containing polyplexes exhibited low cytotoxicity, whereas transfection efficiency with high molecular weight plasmid DNA increased up to 3.6 times that of underivatized polyethylenimine in Neuro2A cells at higher molar ratio of polyethylenimine-nitrogen to DNA-phosphate (N/P) ratios. However, with luciferase-specific low molecular weight small interfering RNA in Neuro2A/EGFPLuc cells, nuclear localization signal-containing polyplexes with disulfide linkages caused substantial cytotoxicity at N/P ratios >15 and no consistent significant reduction in luciferase expression. Possible explanations for molecular weight-dependent differences in genetic information transfer by polyplexes containing disulfide-linked nuclear localization signals are discussed.

Identification and functional characterization of three NoLS (nucleolar localisation signals) mutations of the CDC73 gene.

Hyperparathyroidism Jaw-Tumour Syndrome (HPT-JT) is characterized by primary hyperparathyroidism (PHPT), maxillary/mandible ossifying fibromas and by parathyroid carcinoma in 15% of cases. Inactivating mutations of the tumour suppressor CDC73/HRPT2 gene have been found in HPT-JT patients and also as genetic determinants of sporadic parathyroid carcinoma/atypical adenomas and, rarely, typical adenomas, in familial PHPT. Here we report the genetic and molecular analysis of the CDC73/HRPT2 gene in three patients affected by PHPT due to atypical and typical parathyroid adenomas, in one case belonging to familial PHPT. Flag-tagged WT and mutant CDC73/HRPT2 proteins were transiently transfected in HEK293 cells and functional assays were performed in order to investigate the effect of the variants on the whole protein expression, nuclear localization and cell overgrowth induction. We identified four CDC73/HRPT2 gene mutations, three germline (c.679_680delAG, p.Val85_Val86del and p.Glu81_Pro84del), one somatic (p.Arg77Pro). In three cases the mutation was located within the Nucleolar Localisation Signals (NoLS). The three NoLS variants led to instability either of the corresponding mutated protein or mRNA or both. When transfected in HEK293 cells, NoLS mutated proteins mislocalized with a predeliction for cytoplasmic or nucleo-cytoplasmic localization and, finally, they resulted in overgrowth, consistent with a dominant negative interfering effect in the presence of the endogenous protein.

New insights on the barrel medic MtOGG1 and MtFPG functions in relation to oxidative stress response in planta and during seed imbibition.

In plants, 8-oxoguanine DNA glycosylase/lyase (OGG1) and formamidopyrimidine-DNA glycosylase (FPG) play similar roles within the base excision repair (BER) pathway involved in the removal of oxidized bases, e.g. 7,8-dihydro-8-oxoguanine (8-oxo-dG) and formamidopyrimidine (FAPy) lesions. To date, it is not clear why plants have retained both the OGG1 and FPG functions. In the present work, we have investigated the possible roles played in planta by MtOGG1 and MtFPG genes from Medicago truncatula Gaertn. (barrel medic). Bioinformatic investigation revealed the presence of putative mitochondrial and nuclear localization signals in the MtOGG1 and MtFPG amino acid sequences, respectively, thus suggesting for different subcellular fates. The expression profiles of both genes were evaluated by Quantitative Real-Time PCR (QRT-PCR) in barrel medic plantlets grown in vitro under oxidative stress conditions induced by copper (CuCl(2), 0.05, 0.1 and 0.2 mM) and polyethylene glycol (PEG6000, 50, 100 and 150 g L(-1)). The MtOGG1 and MtFPG genes were up-regulated in response to stress agents, at different levels, depending on treatment and tissue. As for copper, MtOGG1 showed significant up-regulation (up to 1.2- and 1.7-fold) only in roots while the MtFPG mRNA significantly increased (up to 1.3- and 2.8-fold, respectively) in roots and aerial parts. In response to PEG, the MtOGG1 expression was significantly enhanced in aerial parts (up to 1.3-fold) while the MtFPG showed significant (1.2-fold) up-regulation in roots. The expression profiles of MtOGG1 and MtFPG genes were also evaluated during seed imbibition, a physiological process which is characterized by Reactive Oxygen Species (ROS) accumulation and requires active DNA repair.

A phosphatidic acid binding/nuclear localization motif determines lipin1 function in lipid metabolism and adipogenesis.

Lipins are phosphatidic acid phosphatases with a pivotal role in regulation of triglyceride and glycerophospholipid metabolism. Lipin1 is also an amplifier of PGC-1α, a nuclear coactivator of PPAR-α responsive gene transcription. Lipins do not contain recognized membrane-association domains, but interaction of these enzymes with cellular membranes is necessary for access to their phospholipid substrate. We identified a role for a conserved polybasic amino acid motif in an N-terminal domain previously implicated as a determinant of nuclear localization in selective binding of lipin1ß to phosphatidic acid, using blot overlay assays and model bilayer membranes. Studies using lipin1ß polybasic motif variants establish that this region is also critical for nuclear import and raise the possibility that nuclear/cytoplasmic shuttling of lipin1ß is regulated by PA. We used pharmacological agents and lipin1ß polybasic motif mutants to explore the role of PA-mediated membrane association and nuclear localization on lipin1ß function in phospholipid metabolism and adipogenic differentiation. We identify a role for the lipin1 polybasic motif as both a lipid binding motif and a primary nuclear localization sequence. These two functions are necessary for full expression of the biological activity of the protein in intracellular lipid metabolism and transcriptional control of adipogenesis.

Effect of polyethyleneglycol spacer on the binding properties of nuclear localization signal-modified liposomes to isolated nucleus.

The nuclear delivery process is a crucial barrier to successful gene delivery, especially in non-dividing cells. We previously proposed a novel strategy for the nuclear delivery of plasmid DNA (pDNA), in which the pDNA is encapsulated in lipid bilayers that had been modified with nucleus-targeting signals, including nuclear localizing signals derived from SV40 (NLS) or sugar units. In the present study, we report on an investigation of the effect of the topology of the liposome-modified NLS on its ability to bind to the isolated nucleus. NLS was directly attached to a liposome (NLS-Lip) by incorporating stearylated NLS (STR-NSL), or by modification with a polyethyleneglycol (PEG) spacer (NLS-PEG-Lip). NLS-unmodified liposomes (PEG-Lip) were used as a control. The liposomes, after labeling with 7-nitrobenz-2-oxa-1,3-diazole (NBD), were incubated with a cell homogenate derived from JAWS II cells, followed by isolation of the nuclear fraction by centrifugation. The PEG-Lip preparation showed negligible binding to the nucleus. In contrast, the binding of NLS-Lips to the nucleus gradually increased in a STR-NLS density-dependent manner. Interestingly, the binding of NLS-PEG-Lips to the nucleus is highly effective even at low density, suggesting that the presence of the PEG spacer is an important factor in improving the binding activity of NLS-modified liposomes to the nucleus. This information will be useful for the design of nucleus-targeting carriers.