Synthetic peptides as nuclear localization signals

Goldfarb, David S.; Gariépy, Jean; Schoolnik, Gary K.; Kornberg, Roger D.

doi:10.1038/322641a0

Cited by 458 publications

(274 citation statements)

References 22 publications

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“…For an import substrate, we used the well-characterized conjugate of the SV40 large T-antigen NLS coupled to HSA ( Goldfarb et al, 1986;Silver et al, 1989;Newmeyer and Wilson, 1991). In addition, the substrate was labeled with the fluorochrome lissamine rhodamine.…”

Section: Semi-intact Yeast Cells Import Proteins Into the Nucleusmentioning

confidence: 99%

“…The process of nuclear protein uptake occurs in at least two steps; NLS-dependent binding at the nuclear envelope followed by ATP-dependent translocation through the nuclear pore complex (Newmeyer and Forbes, 1988;Richardson et al, 1988;Akey and Goldfarb, 1989). Because of the apparent saturability (Goldfarb et al, 1986) and specificity of NLS function (e.g., Kalderon et al, 1984), receptors that recognize NLS-bearing proteins were postulated to exist. Several candidate proteins have been found in both the cytosol and associated with the nucleus (Adam et al, 1989;Lee and Melese, 1989;Li and Thomas, 1989;Silver et al, 1989;Meier and Blobel, 1990; Please address all correspondence to Dr. Pamela Silver.…”

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confidence: 99%

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Reconstitution of nuclear protein transport with semi-intact yeast cells.

Schlenstedt

Hurt

Doye

et al. 1993

The Journal of Cell Biology

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Abstract.We have developed an in vitro nuclear protein import reaction from semi-intact yeast cells. The reaction uses cells that have been permeabilized by freeze-thaw after spheroplast formation. Electron microscopic analysis and antibody-binding experiments show that the nuclear envelope remains intact but the plasma membrane is perforated. In the presence of ATP and cytosol derived from yeast or mammalian cells, a protein containing the nuclear localization sequence (NLS) of SV40 large T-antigen is transported into the nucleus. Proteins with mutant NLSs are not imported. In the absence of cytosol, binding of NLScontaining proteins occurs at the nuclear envelope. N-ethylmaleimide treatment of the cytosol as well as antibodies to the nuclear pore protein Nspl inhibit import but not binding to the nuclear envelope. Yeast mutants defective in nuclear protein transport were tested in the in vitro import reaction. Semi-intact cells from temperature-sensitive nspl mutants failed to import but some binding to the nuclear envelope was observed. On the other hand, no binding and thus no import into nuclei was observed in semi-intact nsp49 cells which are mutated in another nuclear pore protein. Np13 mutants, which are defective for nuclear protein import in vivo, were also deficient in the binding step under the in vitro conditions. Thus, the transport defect in these mutants is at the level of the nucleus and the point at which nuclear transport is blocked can be defined.M ANY nuclear-destined proteins contain short stretches of amino acids (termed NLS ~ for nuclear localization sequences) that target the protein to the nucleus (for review see Silver, 1991;Goldfarb and Michaud, 1991;Nigg et al., 1991;Garcia-Bustos et al., 1991a). The process of nuclear protein uptake occurs in at least two steps; NLS-dependent binding at the nuclear envelope followed by ATP-dependent translocation through the nuclear pore complex (Newmeyer and Forbes, 1988;Richardson et al., 1988;Akey and Goldfarb, 1989). Because of the apparent saturability (Goldfarb et al., 1986) and specificity of NLS function (e.g., Kalderon et al., 1984), receptors that recognize NLS-bearing proteins were postulated to exist. Several candidate proteins have been found in both the cytosol and associated with the nucleus (Adam et al

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Section: Semi-intact Yeast Cells Import Proteins Into the Nucleusmentioning

confidence: 99%

mentioning

confidence: 99%

Reconstitution of nuclear protein transport with semi-intact yeast cells.

Schlenstedt

Hurt

Doye

et al. 1993

The Journal of Cell Biology

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“…A signal sequence domain contained within the cargo proteins allows for the recognition of the protein by the transport receptor (8). The classical and by far most well characterized nuclear localization sequence (NLS), which consists of multiple basic amino acids including lysine and arginine residues, was originally identified in the SV40 large T antigen (9). This NLS, which utilizes an importin ␣/␤ heterodimer as the import receptor, is both necessary and sufficient for protein transport across the NPC.…”

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confidence: 99%

Arginine Methylation of RNA Helicase A Determines Its Subcellular Localization

Smith¹,

Schurter²,

Wong‐Staal³

et al. 2004

Journal of Biological Chemistry

111

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RNA helicase A (RHA) undergoes nuclear translocation via a classical import mechanism utilizing karyopherin beta. The nuclear transport domain (NTD) of RHA is known to be necessary and sufficient for its bi-directional nuclear trafficking. We report here that arginine methylation is a novel requirement for NTDmediated nuclear import. Nuclear translocation of glutathione S-transferase (GST)-NTD fusion proteins is abrogated by arginine-methylation inhibitors. However, in vitro arginine-methylation of GST-NTD prior to injection allows the fusion protein to localize to the nucleus in the presence of methylation inhibitors. Removal of the arginine-rich C-terminal region negates the effects of the methylation inhibitors on NTD import, suggesting that methylation of the NTD C terminus the relieves the cytoplasmic retention of RHA. The NTD physically interacts with PRMT1, the major protein arginine methyltransferase. These findings provide evidence for a novel arginine methylation-dependent regulatory pathway controlling the nuclear import of RHA.Arginine di-methylation, which is restricted to eukaryotic cells, occurs frequently in the context of RGG tripeptides (1, 2). Two classes of protein arginine methyltransferases (PRMT) 1 have been characterized and classified based on the symmetry of their reaction products. Type I PRMTs account for the formation of asymmetric N G ,N G -dimethylarginine, whereas type II enzymes catalyze the formation of symmetric N G ,N G Ј-dimethylarginine. Five related enzymes that catalyze asymmetric arginine methylation have thus far been identified. The majority of Type I PRMT activity in eukaryotic cells appears to be accounted for by PRMT1 and its functional yeast homologue Hmt1/Rmt1 (3). Several recent studies have illustrated an important function of arginine methylation in the regulation of protein function. Methylation of Sam68, a proline-rich srckinase substrate known to interact with WW or Src homology 3 domain containing signaling proteins, decreases its affinity for Src homology 3 domains but does not alter binding to WW motif-containing proteins (4). Similarly, arginine methylation of the STAT1 (signal transducer and activator of transcription) transcription factor decreases its affinity for its inhibitor PIAS1 (protein inhibitor of activated STAT 1), thereby modulating interferon-induced gene transcription (5). A positive modulatory effect of arginine methylation on protein-protein interaction is observed with the Type II PRMT substrates SmD1 and SmD3, which require methylation for efficient binding to the spinal muscular atrophy gene product, SMN (6,7).Proteins within eukaryotic cells that shuttle across the nuclear envelope to facilitate proper cell function undergo nucleocytoplasmic transport by utilizing specific nuclear transport receptors, which ferry the cargo proteins through the nuclear pore complex (NPC). A signal sequence domain contained within the cargo proteins allows for the recognition of the protein by the transport receptor (8). The classical and by far most we...

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“…Nuclear localization sequences (NLS), which consist of roughly 10 amino acids and possess a high cationic net charge as well as inherent cell-permeation properties [62], belong to the above-mentioned class of signal peptides. Nuclear localization and transfection efficiency, important properties for gene therapy, have been repeatedly demonstrated for NLS-mediated [59,[63][64][65] delivery of not only polymer or phage particle encapsulated DNA [66,67], but also of gold nanoparticles [37] and proteins [68]. Signal peptides also enabled efficient targeting of mitochondria, which are of great interest for drug delivery, as mitochondria house the protein complexes of the electron transport chain [69], generate reactive oxygen species and are therefore essential for cell proliferation [70] and intracellular signaling [71,72].…”

Section: Cell-compartment Targeting With Cppsmentioning

confidence: 99%

Cell-penetrating peptides for nanomedicine – how to choose the right peptide

2015

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More than two decades ago, a group of peptides, now known as cell-penetrating peptides, sparked the hope that the ultimate carrier molecules have been found. The high expectations for these peptides, which are reflected in their bold name, led to many disappointments due to the controversial results their utilization entailed and nowadays even their effectiveness has been called into question. In this review, we discuss the uptake mechanism and application of cell penetrating peptides as mediators for organelle specific delivery of nanocarriers, pointing out the possibilities as well as strategies of their successful utilization. Additionally, we provide an overview of the conjugation techniques usually employed for the attachment of cell penetrating peptides to quantum dots, as well as silver and gold nanoparticles, and we address the various aspects that need to be considered for the successful implementation of cell penetrating peptides for organelle-specific delivery of nanoparticles into cells.Keywords: cell penetrating peptide; drug delivery; gold nanoparticle; nanomedicine; polymeric nanoparticle; quantum dot. Highlights-We discuss the mechanisms of cell penetrating peptide's uptake and briefly introduce the different types of peptides in order to present a more complete picture of the intracellular fate of the peptide-cargo conjugates. -We highlight the recent applications of cell penetrating peptides in the targeted delivery of quantum dots as well as gold, silver and polymeric nanoparticles into mammalian cells in the context of organelle specificity. -We give a short overview of the conjugation techniques usually utilized for the attachment of cell penetrating peptides to quantum dots as well as silver and gold nanoparticles.

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Synthetic peptides as nuclear localization signals

Cited by 458 publications

References 22 publications

Reconstitution of nuclear protein transport with semi-intact yeast cells.

Reconstitution of nuclear protein transport with semi-intact yeast cells.

Arginine Methylation of RNA Helicase A Determines Its Subcellular Localization

Cell-penetrating peptides for nanomedicine – how to choose the right peptide

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