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Results for “Nuclear Localization Signals”

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PubMed2006

Characterization of the nuclear localization signal of high risk HPV16 E2 protein.

The E2 protein of high risk human papillomavirus type 16 (HPV16) contains an amino-terminal (N) domain, a hinge (H) region and a carboxyl-terminal (C) DNA-binding domain. Using enhanced green fluorescent protein (EGFP) fusions with full length E2 and E2 domains in transfection assays in HeLa cells, we found that the C domain is responsible for the nuclear localization of E2 in vivo, whereas the N and H domains do not contain additional nuclear localization signals (NLSs). Deletion analysis of EGFP-E2 and EGFP-cE2 determined that the C domain contains an alpha helix cNLS that overlaps with the DNA-binding region. Mutational analysis revealed that the arginine and lysine residues in this cNLS are essential for nuclear localization of HPV16 E2. Interestingly, these basic amino acid residues are well conserved among the E2 proteins of BPV-1 and some high risk HPV types but not in the low risk HPV types, suggesting that there are differences between the NLSs and corresponding nuclear import pathways between these E2 proteins.

Animals↗

PubMed2002

Targeting of nonkaryophilic cell-permeable peptides into the nuclei of intact cells by covalently attached nuclear localization signals.

Dermaseptins are a family of antimicrobial peptides that lyse target bacterial cells by destabilization of their membranes. Here we present a novel application of a peptide derived from the dermaseptin S4, S4(13). At nontoxic concentrations, fluorescently labeled S4(13) was able to penetrate intact cultured HeLa cells but essentially failed to enter their nuclei despite its low molecular weight. Covalent attachment of nuclear localization signal (NLS) motifs of the SV40-T-antigen and of the HIV-1 Rev protein (ARM) conferred karyophilic properties upon the S4(13). The resulting peptides, which were designated as PV-S4(13) and RR-S4(13) penetrated into intact HeLa cells and were able to accumulate within the cells' nuclei. In studies with digitonin-permeabilized cells, nuclear uptake of the PV-S4(13) and the RR-S4(13) peptides showed the same features that characterize active nuclear import. Nuclear import was observed at 37 degrees C, was ATP-dependent, and was inhibited by the free peptides bearing the SV40 NLS and the Rev and Tat ARMs. Microinjected S4(13) remained in the cytoplasm while microinjected RR-S4(13) was translocated into the cells' nuclei. The new type of cell-permeable "karyophilic" peptides described here may be of potential application as a lead compound for therapeutic purposes, as a tool to study nucleocytoplasmic shuttling in intact cells, and for the delivery of peptides to the nucleus.

Amino Acid Sequence↗

PubMed2006

N-terminal acylation of the SV40 nuclear localization signal peptide enhances its oligonucleotide binding and membrane translocation efficiencies.

Octanoyl and palmitoyl groups were coupled to the N-terminus of an analog of the SV40 nuclear localization signal peptide, SV126-133(Ser128), to study the effect of the fatty acid chain length on the complex formation with a single-stranded antisense oligodeoxynucleotide (ODN) and on the cellular uptake of the complex. The strongest binding affinity was observed for the palmitoylated peptide, indicating the better accessibility of the positively charged lysyl and arginyl side-chains to the phosphate groups due to the turn structures stabilized by the palmitoyl group. On increase of the peptide to ODN molar ratio (rM), gradual unstacking of the bases was observed, the maximal rate being reached at rM=10. At rM>10 restacking of the nucleotide bases was detected and the ODN was completely encapsulated in a liposome-like structure made up of palmitoylated peptides. Cell translocation experiments revealed a highly efficient cell transport of the ODN by palmitoylated SV40 peptide at rM>10.

Acylation↗

PubMed1996

The nuclear localization signal of lymphoid enhancer factor-1 is recognized by two differentially expressed Srp1-nuclear localization sequence receptor proteins.

Proteins are directed to the nucleus by their nuclear localization sequences (NLSs) in a multistep process. The first step, which is to dock the NLS-containing protein to the nuclear pore, is carried out in part by a recently identified NLS receptor named Srp1/importin-alpha. Using the high mobility group (HMG) DNA binding domain of human lymphoid enhancer factor-1 (hLEF-1) as bait in a yeast two-hybrid screen, we have identified two different mouse Srp1 proteins (pendulin/importin-alpha and mSrp1) that each bind to a 9-amino acid sequence in hLEF-1 called the B box. We show that the B box of hLEF-1, a region essential for high affinity DNA binding, is also necessary and sufficient for nuclear localization, lending support to the model that NLSs can function both in nuclear transport and DNA binding. Pendulin and mSrp1 are the mouse homologues of hRch1/hSrp1alpha/importin-alpha and hSrp1/karyopherin alpha/NPI-1, respectively, and show considerable sequence divergence from each other. We find a surprising and significant difference in the expression pattern of pendulin and mSrp1 mRNA, suggesting that these two Srp1 proteins are distinguishable in function as well as sequence.

Amino Acid Sequence↗

PubMed1998

The N-terminal domain of IkappaB alpha masks the nuclear localization signal(s) of p50 and c-Rel homodimers.

Members of the Rel/NF-kappaB family of transcription factors are related to each other over a region of about 300 amino acids called the Rel Homology Domain (RHD), which governs DNA binding, dimerization, and binding to inhibitor. At the C-terminal end of the RHD, each protein has a nuclear localization signal (NLS). The crystal structures of the p50 and RelA family members show that the RHD consists of two regions: an N-terminal section which contains some of the DNA contacts and a C-terminal section which contains the remaining DNA contacts and controls dimerization. In unstimulated cells, the homo- or heterodimeric Rel/NF-kappaB proteins are cytoplasmic by virtue of binding to an inhibitor protein (IkappaB) which somehow masks the NLS of each member of the dimer. The IkappaB proteins consist of an ankyrin-repeat-containing domain that is required for binding to dimers and N- and C-terminal domains that are dispensable for binding to most dimers. In this study, we examined the interaction between IkappaB alpha and Rel family homodimers by mutational analysis. We show that (i) the dimerization regions of p50, RelA, and c-Rel are sufficient for binding to IkappaB alpha, (ii) the NLSs of RelA and c-Rel are not required for binding to IkappaB alpha but do stabilize the interaction, (iii) the NLS of p50 is required for binding to IkappaB alpha, (iv) only certain residues within the p50 NLS are required for binding, and (v) in a p50-IkappaB alpha complex or a c-Rel-IkappaB alpha complex, the N terminus of IkappaB alpha either directly or indirectly masks one or both of the dimer NLSs.

Binding Sites↗

PubMed2005

Specific 3'-terminal modification of DNA with a novel nucleoside analogue that allows a covalent linkage of a nuclear localization signal and enhancement of DNA stability.

We report a straightforward method for the site-specific modification of long double-stranded DNA by using a maleimide adduct of deoxycytidine. This novel nucleoside analogue was efficiently incorporated at the 3'-termini of DNA by terminal deoxynucleotidyl transferase (TdT). Thiol-containing compounds can be covalently linked to the maleimide moieties. We added a nuclear localization signal peptide to the 3'-terminal of a 350 bp-long DNA that encoded short-hairpin RNA, and these modifications resulted in the enhancement of silencing activity by RNA interference. This enhancement is mainly attributed to increased stability of the template DNA.

Amino Acid Sequence↗

PubMed2002

A nuclear localization signal and a membrane association domain contribute to the cellular localization of the Tobacco mosaic virus 126-kDa replicase protein.

A transient expression system using onion epidermal cells was used to investigate domains of the Tobacco mosaic virus (TMV) 126-kDa replicase protein involved in cellular localization. Initially, a nuclear localization signal (NLS), identified within the amino-terminus of the 126-kDa protein, was investigated for its functionality using fusion constructs containing the green fluorescent protein (GFP). Fusion of the amino-terminal 70 amino acids of the 126-kDa protein, containing the NLS, to a beta-glucuronidase-GFP open reading frame (ORF), directed the accumulation of fluorescence to the nucleus. In contrast, similar constructs lacking the NLS or containing a mutated NLS sequence failed to accumulate within the nucleus. Additional investigations using GFP fusion constructs containing the first 178 or 388 amino acids of the 126-kDa protein also displayed nuclear localization. However, fusion constructs encoding the first 781 amino acids or the entire 126-kDa ORF did not accumulate within the nucleus but instead associated with the endoplasmic reticulum (ER), forming spot-like inclusions. Thus, a dominant ER association domain exists between amino acids 388 and 781 of the 126-kDa protein. Interestingly, a full-length 126-kDa GFP fusion construct encoding a nonfunctional NLS mutation also localized to the ER but did not form inclusions. Furthermore, a TMV mutant containing the same nonfunctional NLS mutation failed to replicate in protoplasts. Together these findings suggest that both the NLS and the ER retention domain contribute to the functional localization of the 126-kDa protein.

Amino Acid Sequence↗

PubMed1992

Comparison between synthetic nuclear localization signal peptides from the steroid/thyroid hormone receptors superfamily.

The main objective of the study is to demonstrate that short basic peptides from the steroid/thyroid hormone receptors superfamily act as Nuclear Localization Signals out of receptors context. Such synthesized peptides, chemically coupled to Bovine Serum Albumin, were shown to enable the corresponding BSA-conjugate to be transported to the nucleus. A second objective is to demonstrate the utility of viral cointernalization as a good method for rapid quantitation, comparison and competition in nuclear entry.

Adenoviridae↗

PubMed2001

A novel isoform of human lymphoid enhancer-binding factor-1 (LEF-1) gene transcript encodes a protein devoid of HMG domain and nuclear localization signal.

Lymphoid enhancer-binding factor-1 (LEF-1), a member of the high mobility group (HMG) family of proteins, regulates expression of T-cell receptor-alpha gene and is one of the key regulatory molecules in the epithelial-mesenchymal interactions during embryonic development. Among others, LEF-1 regulates expression of cytokeratin genes involved in formation of hair follicles and the gene encoding the cell-adhesion molecule E-cadherin. Transcription factor LEF-1, which acts as a dimer, binds beta-catenin and is involved in signal transduction by the wnt pathway. We have cloned and sequenced a novel isoform of human LEF-1 gene transcript. This isoform encodes a truncated protein devoid of HMG domain and nuclear localization signal but retaining beta-catenin binding domain. This isoform might either act in a dominant-negative manner by interfering with native LEF-1, or might bind beta-catenin in the cytosol, which would result in attenuation of the signals transmitted by the LEF-beta-catenin pathway.

Amino Acid Sequence↗

PubMed2002

Utilization of synthetic peptides containing nuclear localization signals for nonviral gene transfer systems.

The ability of nonviral gene delivery systems to overcome extracellular and intracellular barriers is a critical issue for future clinical applications. In recent years, several efforts were focused on the elucidation of the gene transfer mechanisms and on the development of multicomponent systems in order to improve both targeted gene delivery and transfection efficiency. The transport of the therapeutic DNA from the cytoplasm into the nucleus is an inefficient process and is considered as the major limiting step in nondividing cells. One of the strategies to improve nuclear uptake of DNA is taking advantage of the cellular nuclear import machinery. Synthetic peptides containing a nuclear localization signal (NLS) are bound to the DNA so that the resulting DNA-NLS complex can be recognized as a nuclear import substrate by specific intracellular receptor proteins. In this review, we critically summarize recent studies applying this approach with a particular focus on NLS-sequence specificity. Implications of the observed results are also discussed in regards to future developments of this technology.

Active Transport, Cell Nucleus↗

PubMed2005

Oxadiazols: a new class of rationally designed anti-human immunodeficiency virus compounds targeting the nuclear localization signal of the viral matrix protein.

Despite recent progress in anti-human immunodeficiency virus (HIV) therapy, drug toxicity and emergence of drug-resistant isolates during long-term treatment of HIV-infected patients necessitate the search for new targets that can be used to develop novel antiviral agents. One such target is the process of nuclear translocation of the HIV preintegration complex. Previously we described a class of arylene bis(methylketone) compounds that inhibit HIV-1 nuclear import by targeting the nuclear localization signal (NLS) in the matrix protein (MA). Here we report a different class of MA NLS-targeting compounds that was selected using computer-assisted drug design. The leading compound from this group, ITI-367, showed potent anti-HIV activity in cultures of T lymphocytes and macrophages and also inhibited HIV-1 replication in ex vivo cultured lymphoid tissue. The virus carrying inactivating mutations in MA NLS was resistant to ITI-367. Analysis by real-time PCR demonstrated that the compound specifically inhibited nuclear import of viral DNA, measured by two-long terminal repeat circle formation. Evidence of the existence of this mechanism was provided by immunofluorescent microscopy, using fluorescently labeled HIV-1, which demonstrated retention of the viral DNA in the cytoplasm of drug-treated macrophages. Compounds inhibiting HIV-1 nuclear import may be attractive candidates for further development.

Anti-HIV Agents↗

PubMed1996

An epitope on Ki antigen recognized by autoantibodies from lupus patients shows homology with the SV40 large T antigen nuclear localization signal.

OBJECTIVE: Epitopes on Ki antigen were analyzed using synthetic peptides, including KILT, a 16-mer peptide with an amino acid sequence homologous to the SV40 large T antigen nuclear localization signal (SV40 T NLS). METHODS: In addition to KILT, 4 synthetic peptides, all potential epitopes on Ki antigen according to computer analysis, were prepared and tested for reactivity with 49 anti-Ki-positive lupus sera by enzyme-linked immunosorbent assay. RESULTS: Eighteen sera reacted with KILT, but not with other peptides. The reaction of anit-Ki sera with KILT was specifically inhibited by recombinant Ki antigen. Eight of 49 anti-Ki sera reacted with a 7-mer synthetic peptide of SV40 T NLS, and the reaction was specifically inhibited by KILT. CONCLUSION: The 16-mer Ki peptide containing the sequence homologous to the SV40 T NLS is one of the antigenic epitopes recognized by anti-Ki antibodies in lupus sera.

Amino Acid Sequence↗

PubMed2005

Nuclear localization signal of ING4 plays a key role in its binding to p53.

ING4, a novel member of ING family, is recently reported to interact with tumor suppressor p53 and negatively regulate the cell growth with significant G2/M arrest of cell cycle in HepG2 cells through upregulation of p53-inducible gene p21. However, which region of ING4 could have contributed to the binding to p53 remains largely unclear. Herein, the GST-pulldown experiments revealed that the middle region of ING4, a potential bipartite nuclear localization signal (NLS), could be involved in the binding to p53. Furthermore, the interaction of ING4 to p53 was abrogated in vitro and in vivo when certain mutations or the entire deletion of the NLS domain occurred. More interestingly, the mutations of the NLS domain could alter the ING4 nuclear localization, disrupt the interaction of ING4 with p53, and even, deregulate the p53-inducible gene p21 in MCF-7 cells. All data indicated that the NLS domain of ING4 is essential for the binding of ING4 to p53 and the function of ING4 associated with p53.

Amino Acid Sequence↗

PubMed2000

Characterization of human herpesvirus 7 U27 gene product and identification of its nuclear localization signal.

A monoclonal antibody, 5H4, that recognizes human herpesvirus 7 (HHV-7) was used in Western analysis to probe HHV-7-infected SupT1 cells. This antibody recognizes a 40-kDa virus-specific polypeptide that is expressed in the absence of viral DNA synthesis. By screening a lambdagt11 HHV-7 cDNA library, the gene encoding the protein was identified as the U27 open reading frame previously reported [J. Virol. (1996) 70, 5975-5989]. Immunofluorescent studies showed a punctate nuclear localization of the protein in both HHV-7-infected cells and transfected cells. A computer program predicted two classic nuclear localization signals (NLSs) in the middle and C-terminal regions of the protein. A C-terminal deletion mutant of the protein could not enter the nucleus, whereas green fluorescent protein or maltose binding protein fused to the C-terminal region of the protein was transported into the nucleus. These findings demonstrate that the predicted C-terminal, but not middle, NLS of the protein actually function as NLS. In addition, nuclear transport of a maltose binding protein-fusion protein containing the C-terminal NLS of the U27 protein was inhibited by both wheat germ agglutinin and a Q69L Ran-GTP mutant, indicating that the U27 protein is transported into the nucleus from the cytoplasm by means of classic nuclear transport machinery. Interestingly, this NLS motif is highly conserved at the C-termini of all herpesvirus DNA polymerase processivity factors that have been examined.

Amino Acid Sequence↗

PubMed2002

Cellular import mediated by nuclear localization signal Peptide sequences.

The cellular delivery of therapeutic agents and their localization within cells is currently a great challenge in medicinal chemistry. A few cationic peptides have shown a strong propensity to cross the cytoplasmic membrane and enter cells. Nuclear localization signal (NLS) sequences are a class of highly cationic peptides that may be exploited for cellular import of linked cargo. A series of NLS sequence peptides were investigated for entry into different cancer cell lines by flow cytometry and confocal microscopy. All NLS peptides demonstrated rapid accumulation within cells when added to the cellular media. Covalent adducts of proteins and oligonucleotides with NLS peptides were also effectively imported within cells. An understanding of the structural and mechanistic properties of these sequences will provide great potential for the rational design of efficient and selective peptidic delivery systems.

Cell Compartmentation↗

PubMed2005

EGF-stimulation activates the nuclear localization signal of SHP-1.

Protein tyrosine phosphatase SHP-1 plays a critical role in the regulation of a variety of intracellular signaling pathways. SHP-1 is predominantly expressed in the cells of hematopoietic origin, and is recognized as a negative regulator of lymphocyte development and activation. SHP-1 consists of two Src homology 2 (SH2) domains and one protein tyrosine phosphatase (PTP) domain followed by a highly basic C-terminal tail containing tyrosyl phosphorylation sites. It is unclear how the C-terminal tail regulates SHP-1 function. We report the examination of the subcellular localization of a variety of truncated or mutated SHP-1 proteins fused with enhanced green fluorescent protein (EGFP) protein at either the N-terminal or the C-terminal end in different cell lines. Our data demonstrate that a nuclear localization signal (NLS) is located in the C-terminal tail of SHP-1 and the signal is primarily defined by three amino-acid residues (KRK) at the C-terminus. This signal is generally blocked in the native protein and can be exposed by fusing EGFP at the appropriate position or by domain truncation. We have also revealed that this NLS of SHP-1 is triggered by epidermal growth factor (EGF) stimulation and mediates translocation of SHP-1 from the cytosol to the nucleus in COS7 cell lines. These results not only demonstrate the importance of the C-terminal tail of SHP-1 in the regulation of nuclear localization, but also provide insights into its role in SHP-1-involved signal transduction pathways.

Amino Acid Sequence↗

PubMed2002

Sequence-specific gene cleavage in intact mammalian cells by 125I-labeled triplex-forming oligonucleotides conjugated with nuclear localization signal peptide.

Triplex-forming oligonucleotides (TFO) are designed to bind sequence specifically to their DNA targets without a significant disturbance of the double helix. They have been proposed to deliver DNA-reactive agents to specific DNA sequences for gene targeting applications. We suggested the use of 125I-labeled TFO for delivery of the energy of radioiodine decay to specific genes. This approach is called antigene radiotherapy. Here we demonstrate the ability of 125I-labeled TFO to produce sequence-specific breaks within a target in the human mdrl gene in cultured cells. TFO and TFO conjugated with a nuclear localization signal peptide (NLS) were delivered into cells using cationic liposomes. This was done either alone or in the presence of an excess of a "ballast" oligonucleotide with an unrelated sequence. In all cases, nuclear localization of TFO and survival of the cells after treatment has been confirmed. Breaks in the gene target were analyzed by restriction enzyme digestion of the DNA recovered from the TFO-treated cells followed by Southern hybridization with DNA probes flanking the target sequence. We have found that TFO/NLS conjugates cleave the target in a concentration-dependent manner regardless of the presence of the "ballast" oligonucleotide. In contrast, TFO without NLS cleaved the target only in the presence of an excess of the "ballast." We hypothesize that TFO and TFO/NLS are delivered into the nucleus by different pathways. These results provide a new insight into the mechanism of intracellular transport of oligonucleotides and open new avenues for improvement of the efficacy of antigene therapies.

Amino Acid Sequence↗

PubMed1991

Identification of the nuclear localization signal of human papillomavirus type 16 L1 protein.

Human papillomavirus type 16(HPV16) L1 and L2 capsid proteins can be detected only in the nucleus of infected cells. For other nuclear proteins, specific sequences of basic amino acids(aa) termed nuclear localization signals (NLS) direct the protein from the cytoplasm to the nucleus. We used a series of deletion and substitution mutations of the HPV16 L1 protein, produced by recombinant vaccinia virus (rVV), to identify NLS within HPV16 L1 and showed that HPV16 L1 contains two NLS sequences, each containing basic aa clusters. One NLS consisted of 6 basic amino acids (KRKKRK from aa 525 to 530) at the carboxy terminal end of L1. The other NLS contained 2 basic aa clusters(KRK from aa 510 to 512 and KR at aa 525, 526) separated by 12 amino acids. Mutations in either NLS did not alter nuclear localization of L1 when the other remained intact, but mutations to both prevented nuclear localization of L1. The L1 NLS could be overridden by introduction of a membrane binding sequence at the amino terminal end of the protein. A databases search showed that all sequenced papillomaviruses are predicted to have L1 and L2 capsid proteins with sequences of basic amino acids homologous with one or both NLS of HPV16 L1.

Amino Acid Sequence↗