Effect of basic and nonbasic amino acid substitutions on transport induced by simian virus 40 T-antigen synthetic peptide nuclear transport signals (original) (raw)
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Cell, 1986
A system was developed for the analysis of protein transport to the nucleus. Carrier proteins cross-linked to synthetic peptides were microinjected into the cytoplasm of mammalian cells, and protein transport was evaluated by immunofluorescence staining of fixed cells. A Wmer synthetic peptide containing seven ammo acids homologous to SV40 T antigen was capable of inducing nuclear transport, but no transport was observed when proteins were coupled with a synthetic peptide homologous to a nuclear-transport-defective T antigen. The largest protein-peptide conjugate efficiently transported was ferritin (M, 465,000). The rate of transport was influenced by the number of peptides per molecule of carrier protein and, to some degree, by the size of the carrier protein. Transport of some conjugates was almost complete in 15 min at room temperature.
Journal of Cell Science, 1989
Summary In this paper, progress towards the goal of understanding communication between the nucleus and cytoplasm using an in vitro system is reviewed. To probe the mechanism of nuclear targeting, we developed an in vitro transport system and have begun to dissect the highly selective process of nuclear transport. The basic parameters of transport were defined using an easily isolated nuclear protein, nucleoplasmin. To study the interaction of nuclear targeting signals with the pore, an artificial nuclear transport substrate was constructed, which consists of human serum albumin coupled to the signal sequence of the SV40 T-antigen. A similar peptide–protein conjugate was made using a mutant signal sequence. These conjugates were fluorescently labeled and/or tagged with gold and tested for transport in the in vitro system. High levels of nuclear transport of the wild-type signal sequence-containing protein were observed, while no transport of the mutant signal sequence-containing pro...
Bioconjugate Chemistry, 2004
Nonviral gene delivery is limited by inefficient transfer of DNA from the cytoplasm to the nucleus. Nuclear localization sequence (NLS) peptides have been widely used to exploit intracellular transport mechanisms and promote nuclear uptake of DNA. However, the exact conditions to successfully utilize the properties of NLS peptides are still unclear. In the present study a panel of NLS peptides that bind different transport receptors were compared for their ability to enhance nonviral gene transfer. Several factors such as method of incorporating the NLS peptide, type of NLS peptide, DNA morphology, and proper characterization of NLS peptide/DNA conjugates were identified as important considerations in utilizing NLS peptides to enhance gene transfer. In particular, it was shown that a peptide derived from human T cell leukaemia virus type 1 (HTLV) was able to effectively condense DNA into discrete particles and mediate levels of transgene expression up to 32-fold greater than polylysine-based polyplexes. This is the first study to demonstrate efficient transfection mediated by an importin -binding peptide based on the HTLV sequence. Promising results were also achieved with a 7-fold increase in gene expression using a NLS peptide/DNA conjugate formed by site-specific linkage of an extended SV40 peptide via a peptide nucleic acid (PNA) clamp. Altogether, the results from this study should help to define the requirements for successful NLS-enhanced transfection.
Molecular and cellular biology, 1989
The transport of proteins into the nucleus requires not only the presence of a nuclear transport signal on the targeted protein but also the signal recognition proteins and the nuclear pore translocation apparatus. Complicating the search for the signal recognition proteins is the fact that the nuclear transport signals identified share little obvious homology. In this study, synthetic peptides homologous to the nuclear transport signals from the simian virus 40 large T antigen, Xenopus oocyte nucleoplasmin, adenovirus E1A, and Saccharomyces cerevisiae MAT alpha 2 proteins were coupled to a UV-photoactivable cross-linker and iodinated for use in an in vitro cross-linking reaction with cellular lysates. Four proteins, p140, p100, p70, and p55, which specifically interacted with the nuclear transport signal peptides were identified. Unique patterns of reactivity were observed with closely related pairs of nuclear transport signal peptides. Competition experiments with labeled and unla...
Sequence requirements for synthetic peptide-mediated translocation to the nucleus
Molecular and cellular biology, 1989
The abilities of 18 synthetic peptides to target a carrier protein to the nucleus following microinjection into the cytoplasm of HeLa cells were determined. Eight of the sequences chosen for synthesis were based on published nuclear targeting regions as determined by gene fusion and deletion experiments. Six of these sequences were found to be effective when mimicked by a synthetic peptide and conjugated to a carrier protein. One additional peptide was based on a region of lamin L1, a nuclear protein from Xenopus laevis, in which the nuclear targeting region had not been previously investigated. This peptide was also able to target a carrier protein to the nucleus. Eight other peptides which resemble the known targeting signals had little or no nuclear targeting ability. Peptides which were able to target a carrier protein to the nucleus did so within 45 min of injection into the cytoplasm. Two peptides with little or no apparent nuclear targeting ability after 45 min were examined ...
The nucleocytoplasmic transport of viral proteins
Virologica Sinica, 2010
Molecules can enter the nucleus by passive diffusion or active transport mechanisms, depending on their size. Small molecules up to size of 50-60 kDa or less than 10 nm in diameter can diffuse passively through the nuclear pore complex (NPC), while most proteins are transported by energy driven transport mechanisms. Active transport of viral proteins is mediated by nuclear localization signals (NLS), which were first identified in Simian Virus 40 large T antigen and had subsequently been identified in a large number of viral proteins. Usually they contain short stretches of lysine or arginine residues. These signals are recognized by the importin super-family (importin α and β) proteins that mediate the transport across the nuclear envelope through Ran-GTP. In contrast, only one class of the leucine-rich nuclear export signal (NES) on viral proteins is known at present. Chromosome region maintenance 1 (CRM1) protein mediates nuclear export of hundreds of viral proteins through the r...
Bioconjugate Chemistry, 2006
Cell-penetrating peptides (CPPs) are characterized by their ability to be internalized in mammalian cells. To investigate the relative potency of CPPs as carriers of medicinally relevant cargo, a positive read-out assay based on the ability of a peptide nucleic acid (PNA) oligomer to promote correct expression of a recombinant luciferase gene was employed. Seven different CPPs were included in the study: Transportan, oligo-arginine (R 7-9 ), pTat, Penetratin, KFF, SynB3, and NLS. The CPP-PNA conjugates were synthesized by different conjugation chemistries: continuous synthesis, maleimide coupling, and ester or disulfide linkage. Under serum-free conditions PNA-SS-Transportan-amide (ortho)-PNA was found to be the most potent conjugate, resulting in maximum luciferase signal at a concentration of 1-2 µM. (D-Arg) 9 -PNA showed optimal efficacy at 5 µM but gave rise to only one-third of the luciferase signal obtained with the Transportan conjugate. The pTat-and KFF-PNA conjugates showed significantly lower efficacy. The penetratin-, SynB3-. and NLS-PNA conjugates showed only minimal or no activity. Serum was found to have a drastic negative impact on CPP-driven cellular uptake. PNA-SS-Transportan-acid (ortho) and (D-Arg) 9 -PNA were least sensitive to the presence of serum. Both the chemical nature and, in the case of Transportan, the position of the peptide PNA coupling were found to have a major impact on the transport capacity of the peptides. However, no simple relationship between linker type and antisense activity of the conjugates could be deduced from the data.
Journal of Molecular Biology, 1999
The HIV-1 auxiliary protein Vif contains a basic domain within its sequence. This basic region, 90 RKKR 93 , is similar to the prototypic nuclear localization signal (NLS). However, Vif is not a nuclear protein and does not function in the nucleus. Here we have studied the karyophilic properties of this basic region. We have synthesized peptides corresponding to this positively charged NLS-like region and observed that these peptides inhibited nuclear transport via the importin pathway in vitro with IC 50 values in the micromolar range. Inhibition was observed only with peptides derived from the positively charged region, but not from other regions of the Vif protein, showing sequence speci®city. On the other hand, the Vif inhibitory peptide Vif88-98 did not confer karyophilic properties when conjugated to BSA. The inactive Vif conjugate and the active SV40-NLS-BSA conjugate both contained a similar number of peptides conjugated to each BSA molecule, as was determined by amino acid analysis of the peptide-BSA conjugates. Thus, the lack of nuclear import of the Vif peptide-BSA conjugate cannot be attributed to insuf®cient number of conjugated peptide molecules per BSA molecule. Our results suggest that the HIV-1 Vif protein carries an NLS-like sequence that inhibits, but does not mediate, nuclear import via the importin pathway. We have termed such signals as nuclear transport inhibitory signals (NTIS). The possible role of NTIS in controlling nuclear uptake, and speci®cally during virus infection, is discussed herein. Our results raise the possibility that NLS-like sequences of certain low molecular weight viral proteins may serve as regulators of nucleocytoplasmic traf®cking and not neccessarily as mediators of nuclear import.
Biochemical and Biophysical Research Communications, 1998
eral attempts have been reported and beside the lipo-Peptides containing a hydrophobic motif associated some fusion process they are based on two different with a nuclear localization signal separated by various approaches. The first one involves the use of peptides linkers were synthesized in solid phase. The hydrophocorresponding (5) or related (6) to the homeodomain of bic sequence corresponds either to a signal peptide antennapedia. Indeed, it was shown that these peptides sequence or to a fragment of the fusion peptide of GP41 are able to reach the nucleus of neuronal cells. The while the hydrophilic sequence is that of a nuclear second approach is based on the fact that peptides conlocalization signal. The C-termini of these peptides taining an oligolysine sequence (7,8) can be easily interbear a cysteamide group that was linked to a fluoresnalized by cells and thus this type of peptide can act cent probe. This allowed the cellular localization of as a drug carrier. These approaches, however, suffer the probe to be determined as a function of the peptide from some weaknesses as they deal with particular cell sequences. The labeled peptides were then incubated types and/or lead to slow migration processes. In order with fibroblasts. Using N-biotinylated derivatives we to improve the efficiency of such carriers and enlarge confirmed by indirect immunofluorescence that the the scope of target cell types as well as to reduce the observed localizations corresponds to those of the pepdelay in the delivery of the transported materials into tides. The presence of a linker appears to play a role in the cellular localization. One of these peptides was the nucleus we tentatively designed new types of pepsuccessfully used to target fluorescent oligodeoxy-tidic carriers which contain two distinct peptide senucleotides into living cells demonstrating improved quences. The role of each sequence within the peptide cell delivery of peptide-oligodeoxynucleotide conjuis the following : one is involved in the facilitation of gates. ᭧ 1998 Academic Press lipid anchoring and associated translocation through the membrane while the other is implicated in nuclear addressing. The biological interest of these new families of peptides would be to facilitate cellular internal-The efficiency of drug translocation into cell nuclei ization of therapeutic agents. In addition, conjugation is an important aspect in drug design and is still probof these peptides to oligonucleotides (9,10) might be lematic mainly due to possible degradation of both the applicable and usefull for antisense strategies. Here carrier and the drug. Up to now the most powerful we report the design of these peptides, their synthesis methods that facilitate nuclear access include microinand their linking to fluorescent probes. Furthermore, jection, liposome fusion or electroporation of conjugates we describe the cellular localization of these probes containing a nuclear localization signal (NLS) and a after incubation of the conjugates with living cells, as drug (1-4). However, such procedures do not appear well as the potential applications of these peptides in appropriate when a large number of cells are directed antisense ODN (oligodeoxynucleotide) delivery techat nor for non-accessible cells. It is therefore important nology. to design new types of conjugates which are able to reach the intracellular compartments. In this field sev-MATERIALS AND METHODS Peptide synthesis. The synthesis of all peptides was performed by solid phase peptide synthesis according to the procedure reported