scholarly journals Nuclear localization signals in phage terminal proteins provide a novel gene delivery tool in mammalian cells

2013 ◽  
Vol 6 (2) ◽  
pp. e22829 ◽  
Author(s):  
Modesto Redrejo-Rodríguez ◽  
Daniel Muñoz-Espín ◽  
Isabel Holguera ◽  
Mario Mencía ◽  
Margarita Salas
Keyword(s):  
Gene Delivery ◽  
Nuclear Localization ◽  
Mammalian Cells ◽  
Nuclear Localization Signals ◽  
Novel Gene ◽  
Terminal Proteins

scholarly journals The Fanconi Anemia Proteins FAA and FAC Function in Different Cellular Compartments to Protect Against Cross-Linking Agent Cytotoxicity

Blood ◽  
1998 ◽  
Vol 92 (7) ◽  
pp. 2229-2236 ◽  
Author(s):  
Frank A.E. Kruyt ◽  
Hagop Youssoufian
Keyword(s):  
Nuclear Localization ◽  
Fanconi Anemia ◽  
Nuclear Export ◽  
Mammalian Cells ◽  
Bone Marrow Failure ◽  
Cross Linking ◽  
Disease Genes ◽  
Macromolecular Complex ◽  
Subcellular Compartment ◽  
Nuclear Localization Signals

Abstract Fanconi anemia (FA) is an autosomal recessive disease characterized by chromosomal instability, bone marrow failure, and a high risk of developing malignancies. Although the disorder is genetically heterogeneous, all FA cells are defined by their sensitivity to the apoptosis-inducing effect of cross-linking agents, such as mitomycin C (MMC). The cloned FA disease genes, FAC and FAA, encode proteins with no homology to each other or to any known protein. We generated a highly specific antibody against FAA and found the protein in both the cytoplasm and nucleus of mammalian cells. By subcellular fractionation, FAA is also associated with intracellular membranes. To identify the subcellular compartment that is relevant for FAA activity, we appended nuclear export and nuclear localization signals to the carboxy terminus of FAA and enriched its localization in either the cytoplasm or the nucleus. Nuclear localization of FAA was both necessary and sufficient to correct MMC sensitivity in FA-A cells. In addition, we found no evidence for an interaction between FAA and FAC either in vivo or in vitro. Together with a previous finding that FAC is active in the cytoplasm but not in the nucleus, our results indicate that FAA and FAC function in separate subcellular compartments. Thus, FAA and FAC, if functionally linked, are more likely to be in a linear pathway rather than form a macromolecular complex to protect against cross-linker cytotoxicity.

scholarly journals The Fanconi Anemia Proteins FAA and FAC Function in Different Cellular Compartments to Protect Against Cross-Linking Agent Cytotoxicity

Blood ◽  
1998 ◽  
Vol 92 (7) ◽  
pp. 2229-2236 ◽  
Author(s):  
Frank A.E. Kruyt ◽  
Hagop Youssoufian
Keyword(s):  
Nuclear Localization ◽  
Fanconi Anemia ◽  
Nuclear Export ◽  
Mammalian Cells ◽  
Bone Marrow Failure ◽  
Cross Linking ◽  
Disease Genes ◽  
Macromolecular Complex ◽  
Subcellular Compartment ◽  
Nuclear Localization Signals

Fanconi anemia (FA) is an autosomal recessive disease characterized by chromosomal instability, bone marrow failure, and a high risk of developing malignancies. Although the disorder is genetically heterogeneous, all FA cells are defined by their sensitivity to the apoptosis-inducing effect of cross-linking agents, such as mitomycin C (MMC). The cloned FA disease genes, FAC and FAA, encode proteins with no homology to each other or to any known protein. We generated a highly specific antibody against FAA and found the protein in both the cytoplasm and nucleus of mammalian cells. By subcellular fractionation, FAA is also associated with intracellular membranes. To identify the subcellular compartment that is relevant for FAA activity, we appended nuclear export and nuclear localization signals to the carboxy terminus of FAA and enriched its localization in either the cytoplasm or the nucleus. Nuclear localization of FAA was both necessary and sufficient to correct MMC sensitivity in FA-A cells. In addition, we found no evidence for an interaction between FAA and FAC either in vivo or in vitro. Together with a previous finding that FAC is active in the cytoplasm but not in the nucleus, our results indicate that FAA and FAC function in separate subcellular compartments. Thus, FAA and FAC, if functionally linked, are more likely to be in a linear pathway rather than form a macromolecular complex to protect against cross-linker cytotoxicity.

scholarly journals Application of a Saccharomyces cerevisiae Model To Study Requirements for Trafficking of Yersinia pestis YopM in Eucaryotic Cells

2003 ◽  
Vol 71 (2) ◽  
pp. 937-947 ◽  
Author(s):  
Elżbieta Skrzypek ◽  
Tanya Myers-Morales ◽  
Sidney W. Whiteheart ◽  
Susan C. Straley
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Yersinia Pestis ◽  
Nuclear Localization ◽  
Mammalian Cells ◽  
Fluorescent Protein ◽  
Vesicular Trafficking ◽  
Leader Sequence ◽  
Host Cells ◽  
Permissive Temperature ◽  
Nuclear Localization Signals

ABSTRACT YopM is a leucine-rich repeat (LRR) virulence protein that is delivered into host cells when any of the three human-pathogenic species of Yersinia binds to mammalian cells. It exhibits heterogeneity of size and sequence among the yersiniae, but the functional consequences of this variability are not yet known. Yersinia pestis YopM was previously shown to accumulate in the nuclei of infected HeLa cells by a mechanism that requires vesicular trafficking. In this study, we characterized the trafficking of Y. pestis YopM in a Saccharomyces cerevisiae model previously found to support nuclear localization of YopM from an enteropathogenic Yersinia strain (C. F. Lesser and S. I. Miller, EMBO J. 20:1840-1849, 2001). Y. pestis YopM was N-terminally fused to the yeast enhanced green fluorescent protein (yEGFP) and inducibly expressed in the cytoplasm. yEGFP-YopM localized to the yeast nucleus, showing that this property is conserved for YopMs so far tested and that infection and the presence of other Yops are not required for its trafficking. When expressed in S. cerevisiae that is temperature sensitive for vesicular transport, YopM failed to accumulate in the nucleus at the nonpermissive temperature but did accumulate when the permissive temperature was restored. This shows that vesicular trafficking also is required in yeast for normal localization of YopM. YopM consists of a 71-residue leader sequence, 15 LRRs, and a 32-residue tail. Deletion analysis revealed that the leader sequence or tail is alone insufficient to direct YopM to the nucleus, showing that the LRR structure is required. Both the N-terminal and C-terminal halves of YopM localized to the nucleus, indicating the possible presence of two nuclear localization signals (NLSs) in YopM or domains in YopM where an NLS-containing protein might bind; this fits with the presence of two highly conserved regions among Yersinia YopMs. yEGFP-YopM lacking LRRs 4 to 7 or 7 to 10 accumulated in the nucleus in yeast, and YopM lacking these LRRs concentrated normally in the HeLa cell nucleus after delivery by Yersinia infection, showing that these LRRs are not essential for YopM trafficking in eucaryotic cells. However, because Y. pestis carrying either of these YopMs is strongly compromised in virulence in mice, these findings revealed that LRRs 4 to 10 map a region of YopM or support a conformation of YopM that is necessary for a pathogenic effect.

scholarly journals Permeabilized mammalian cells as an experimental system for nuclear import of geminiviral karyophilic proteins and of synthetic peptides derived from their nuclear localization signal regions

2006 ◽  
Vol 87 (9) ◽  
pp. 2709-2720 ◽  
Author(s):  
Gideon Kass ◽  
Gabriel Arad ◽  
Joseph Rosenbluh ◽  
Yedidya Gafni ◽  
Adolf Graessmann ◽  
...  
Keyword(s):  
Nuclear Localization ◽  
Nuclear Import ◽  
Synthetic Peptides ◽  
Mammalian Cells ◽  
Specific Binding ◽  
Nuclear Localization Signals ◽  
Direct Demonstration ◽  
Bipartite Genome ◽  
Monopartite Genome

The plant-infecting geminiviruses deliver their genome and viral proteins into the host cell nucleus. Members of the family Geminiviridae possess either a bipartite genome composed of two ∼2.6 kb DNAs or a monopartite genome of ∼3.0 kb DNA. The bipartite genome of Bean dwarf mosaic virus (BDMV) encodes several karyophilic proteins, among them the capsid protein (CP) and BV1 (nuclear shuttle protein). A CP is also encoded by the monopartite genome of Tomato yellow leaf curl virus (TYLCV). Here, an in vitro assay system was used for direct demonstration of nuclear import of BDMV BV1 and TYLCV CP, as well as synthetic peptides containing their putative nuclear localization signals (NLSs). Full-length recombinant BDMV BV1 and TYLCV CP mediated import of conjugated fluorescently labelled BSA molecules into nuclei of permeabilized mammalian cells. Fluorescently labelled and biotinylated BSA conjugates bearing the synthetic peptides containing aa 3–20 of TYLCV CP (CP-NLS) or aa 84–106 of BDMV BV1 (BV1-NLS) were also imported into the nuclei of permeabilized cells. This import was blocked by the addition of unlabelled BSA–NLS peptide conjugates or excess unlabelled free NLS peptides. The CP- and BV1-NLS peptides also mediated nuclear import of fluorescently labelled BSA molecules into the nuclei of microinjected mesophyll cells of Nicotiana benthamiana leaves, demonstrating their biological function in intact plant tissue. BV1-NLS and CP-NLS were shown to mediate specific binding to importin α, both in vitro and in vivo. These results are consistent with a common nuclear-import pathway for CP and BV1, probably via importin α.

Nuclear localization of Ku antigen is promoted independently by basic motifs in the Ku70 and Ku80 subunits

2001 ◽  
Vol 114 (1) ◽  
pp. 89-99
Author(s):  
J. Bertinato ◽  
C. Schild-Poulter ◽  
R.J. Hache
Keyword(s):  
Amino Acids ◽  
Nuclear Localization ◽  
Nuclear Import ◽  
Nuclear Localization Signal ◽  
Mammalian Cells ◽  
Full Length ◽  
Telomere Maintenance ◽  
Nuclear Localization Signals ◽  
Localization Signal ◽  
Ku Antigen

The Ku antigen is a heteromeric (Ku70/Ku80), mostly nuclear protein. Ku participates in multiple nuclear processes from DNA repair to V(D)J recombination to telomere maintenance to transcriptional regulation and serves as a DNA binding subunit and allosteric regulator of DNA-dependent protein kinase. While some evidence suggests that subcellular localization of Ku may be subject to regulation, how Ku gains access to the nucleus is poorly understood. In this work, using a combination of indirect immunofluorescence and direct fluorescence, we have demonstrated that transfer of the Ku heterodimer to the nucleus is determined by basic nuclear localization signals in each of the Ku subunits that function independently. A bipartite basic nuclear localization signal between amino acids 539–556 of Ku70 was observed to be required for nuclear import of full-length Ku70 monomer, while a short Ku80 motif of four amino acids from 565–568 containing three lysines was required for the nuclear import of full-length Ku80. Ku heterodimers containing only one nuclear localization signal accumulated in the nucleus as efficiently as wild-type Ku, while site directed mutagenesis inactivating the basic motifs in each subunit, resulted in a Ku heterodimer that was completely localized to the cytoplasm. Lastly, our results indicate that mutations in Ku previously proposed to abrogate Ku70/Ku80 heterodimerization, markedly reduced the accumulation of Ku70 without affecting heterodimer formation in mammalian cells.

scholarly journals An Effective Cationic Human Serum Albumin-based Gene-Delivery Carrier Containing the Nuclear Localization Signal

Pharmaceutics ◽  
2019 ◽  
Vol 11 (11) ◽  
pp. 608 ◽  
Author(s):  
Guan ◽  
Song ◽  
Zhang ◽  
Chen ◽  
Hu ◽  
...  
Keyword(s):  
Human Serum Albumin ◽  
Gene Delivery ◽  
Serum Albumin ◽  
Human Serum ◽  
Delivery System ◽  
Nuclear Localization ◽  
Transfection Efficiency ◽  
P53 Protein ◽  
Nuclear Localization Signals ◽  
Nutritional Effect

Considerable effort has been devoted to the development of gene carriers over the years. However, toxicity, immunogenicity, and low transfection efficiency are still major barriers. How to overcome these obstacles has become a burning question in gene delivery. In the present study, a simple cationic human serum albumin (CHSA)-based gene-delivery system containing nuclear localization signals (NLSs) was constructed to conquer the limitations. CHSA/NLS/plasmid DNA (pDNA) complexes were prepared and characterized by Hoechst 33258 intercalation, gel retardation assay, morphological analysis, circular dichroism (CD) spectroscopy, particle size, and zeta potential measurements. Results showed that CHSA/NLS/pDNA complexes were able to condense and protect pDNA with high encapsulation efficiency. The complexes displayed a nutritional effect on cells at a low concentration and there was no significant cytotoxicity or immunogenicity. In addition, CHSA/NLS/pDNA complexes exhibited excellent cellular uptake rates and the mechanism was mainly the clathrin or macropinocytosis-dependent endocytosis pathway. Furthermore, CHSA/NLS/pDNA significantly enhanced gene expression efficiency in vitro. More importantly, CHSA/NLS/pDNA complexes showed a desired antitumor effect in vivo, exhibiting the highest inhibition rate (57.3%) and significant upregulation in p53 protein. All these results confirm that CHSA/NLS/pDNA complexes have a bright future as a safe and effective delivery system for gene therapy.

scholarly journals Functional eukaryotic nuclear localization signals are widespread in terminal proteins of bacteriophages

2012 ◽  
Vol 109 (45) ◽  
pp. 18482-18487 ◽  
Author(s):  
M. Redrejo-Rodriguez ◽  
D. Munoz-Espin ◽  
I. Holguera ◽  
M. Mencia ◽  
M. Salas
Keyword(s):  
Nuclear Localization ◽  
Nuclear Localization Signals ◽  
Terminal Proteins

Effect of peptides bearing nuclear localization signals on therapeutic ultrasound mediated gene delivery

2008 ◽  
Vol 10 (10) ◽  
pp. 1150-1159 ◽  
Author(s):  
Maayan Duvshani-Eshet ◽  
Hadas Keren ◽  
Shira Oz ◽  
Inna S. Radzishevsky ◽  
Amram Mor ◽  
...  
Keyword(s):  
Gene Delivery ◽  
Nuclear Localization ◽  
Therapeutic Ultrasound ◽  
Nuclear Localization Signals
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