Induction of nuclear transport with a synthetic peptide homologous to the SV40 T antigen transport signal

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 13-mer synthetic peptide containing seven amino 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 (Mr 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.     

Comparison of diverse transport signals in synthetic peptide-induced nuclear transport

Several investigations have demonstrated the ability of synthetic peptides homologous to the nuclear transport signal of simian virus 40 large T antigen to induce the nuclear transport of nonnuclear carrier proteins. To determine the generality of peptide-induced transport, six peptides with sequences derived from four previously identified nuclear transport signals were synthesized and examined for their ability to induce the transport of mouse immunoglobulin G following microinjection into the cytoplasm of mammalian cells. Peptides containing transport signals from simian virus 40 T antigen, Xenopus nucleoplasmin, and adenovirus E1A proteins were highly efficient at peptide-induced transport, while a peptide homologous to yeast MAT alpha 2 protein was incapable of inducing transport. A short nucleoplasmin peptide that contained only the basic amino acid domain was capable of inducing transport but yielded a much slower rate of transport than a long nucleoplasmin peptide encompassing the previously identified minimal transport signal. The short nucleoplasmin signal exhibited a greater capacity for transport than a peptide homologous to the cytoplasmic mutant T antigen signal when conjugates with a low number of signals coupled per carrier protein were examined. However, the short nucleoplasmin peptide was only marginally more effective than the T antigen mutant peptide when conjugates with a high number of signals coupled per carrier protein were examined.     

Identification of the human c-myc protein nuclear translocation signal.

We identified and characterized two regions of the human c-myc protein that target proteins into the nucleus. Using mutant c-myc proteins and proteins that fuse portions of c-myc to chicken muscle pyruvate kinase, we found that residues 320 to 328 (PAAKRVKLD; peptide M1) induced complete nuclear localization, and their removal from c-myc resulted in mutant proteins that distributed in both the nucleus and cytoplasm but retained rat embryo cell cotransforming activity. Residues 364 to 374 (RQRRNELKRSP; peptide M2) induced only partial nuclear targeting, and their removal from c-myc resulted in mutant proteins that remained nuclear but were cotransformationally inactive. We conjugated synthetic peptides containing M1 or M2 to human serum albumin and microinjected the conjugate into the cytoplasm of Vero cells. The peptide containing M1 caused rapid and complete nuclear accumulation, whereas that containing M2 caused slower and only partial nuclear localization. Thus, M1 functions as the nuclear localization signal of c-myc, and M2 serves some other and essential function.     

A long synthetic peptide containing a nuclear localization signal and its flanking sequences of SV40 T-antigen directs the transport of IgM into the nucleus efficiently

Synthetic short peptides containing only the nuclear localization signal (NLS) direct the transport of nonnuclear proteins into the nucleus. As a conjugate of the synthetic peptide with immunoglobulin M (IgM) did not enter the nucleus, there was believed to be a size limit for nuclear transport of NLS-conjugated proteins. However, we found that IgM conjugated with purified nucleoplasmin, a nuclear protein of Xenopus oocytes, rapidly accumulated in the nucleus. For direct comparison with the short peptide, we prepared a long peptide containing the NLS and its flanking sequences of SV40 large T-antigen and its mutated long peptide, in which possible phosphorylation sites located at the amino terminal of the NLS were changed to alanine. Kinetic experiments showed that wild-type long peptide-IgM conjugates were almost entirely taken up into the nucleus within 30 min after their injection, whereas almost 60 min was required for the mutated long peptide-IgM conjugates to enter the nucleus of all the cells examined, and there was no apparent accumulation of short peptide-IgM conjugates in the nucleus within 60 min. These results indicate that even when the kinetics of transport are affected by amino acid substitutions, the long peptide directs the transport of large molecules such as IgM into the nucleus.     

Effect of basic and nonbasic amino acid substitutions on transport induced by simian virus 40 T-antigen synthetic peptide nuclear transport signals.

A previous study demonstrated the ability of a synthetic peptide homologous to the simian virus 40 T-antigen nuclear transport signal to induce the nuclear transport of carrier proteins and the dependence of peptide-induced transport on a positive charge at the lysine corresponding to amino acid 128 of T antigen. In this investigation synthetic peptides were utilized to examine the effect on transport of amino acid substitutions within the T-antigen nuclear transport signal. Nuclear transport was evaluated by immunofluorescence after microinjection of protein-peptide conjugates into the cytoplasm of mammalian cells. Substitution of other basic amino acids at position 128 revealed a hierarchy for nuclear transport. The rate of nuclear transport was most rapid when a lysine was at position 128 followed in descending order by arginine, D-lysine, ornithine, and p-aminophenylalanine. Peptide-induced nuclear transport was dependent upon a positively charged amino acid at positions 128 and 129, since substitutions of neutral asparagines at these positions abolished transport. However, partial transport was observed with the peptide having an asparagine at position 128 when a high number of peptides were conjugated to the carrier protein.     

The trimethylguanosine cap structure of U1 snRNA is a component of a bipartite nuclear targeting signal

The ability of series of U1 snRNAs and U6 snRNAs to migrate into the nucleus of Xenopus oocytes after injection into the cytoplasm was analyzed. The U snRNAs were made either by injecting U snRNA genes into the nucleus of oocytes or, synthetically, by T7 RNA polymerase, incorporating a variety of cap structures. The results indicate that nuclear targeting of U1 snRNA requires both a trimethylguanosine cap structure and binding of at least one common U snRNP protein. Using synthetic U6 snRNAs, it is further demonstrated that the trimethylguanosine cap structure can act in nuclear targeting in the absence of the common U snRNP proteins. These results imply that U snRNP nuclear targeting signals are of a modular nature.     

Identification of a short basic peptide motif able to drive copy-number dependent nuclear accumulation of a linked protein

The repetitive [RTRG](6) peptide was fortuitously identified as a potent nuclear localization signal when linked to the green fluorescent reporter protein. Replacing the arginines by lysines, or the threonines by glycines, both resulted in a decreased nuclear targeting ability of the peptide within this context. By contrast, the sequence [RT](12) proved able to drive nuclear accumulation of the linked protein as efficiently as the starting peptide. Remarkably, [RTRG](n) peptides where n=2 to 6 showed a gradual, copy-number dependent, increase in their ability to target the green fluorescent protein to the cell nucleus. As a consequence, the nuclear to cytoplasmic concentration ratio of the linked protein within the cell could be adjusted to different values depending on the number of repeats used in the fusion. Our observation may open the way to the use of [RTRG](n) repeats of given lengths (n=2 to 6) for fixing the nuclear-cytoplasmic partition of shuttling protein domains in the course of their functional study.     

Synthetic peptides containing a region of SV40 large T-antigen involved in nuclear localization direct the transport of proteins into the nucleus

We studied the mechanism of transport of proteins into the nucleus using synthetic peptides containing the nuclear location signal sequence of Simian virus 40 (SV 40) large T-antigen. When chick erythrocytes containing a synthetic large T-antigen nuclear translocation signal were fused with SV 40-transformed human fibroblasts, the migration of native large T-antigen into the chick nuclei was suppressed. Migration of proteins detected by human specific antinuclear autoimmune antibody was not blocked. An analog of the nuclear location signal peptide did not inhibit entry of large T-antigen into the chick nuclei. This result suggests that the peptide blocked the migration of only native large T-antigen into the nucleus, and that the signal of the majority of nuclear proteins for nuclear transport is not the same as that of the large T-antigen. The synthetic peptide was conjugated chemically with bovine serum albumin (BSA) and introduced into the cytoplasm of cultured human cells by red blood cell ghost-mediated microinjection. The BSA-synthetic peptide conjugate was found predominantly in the nucleus within 2 h after its introduction into the cells. BSA conjugated with the cross-linking reagent alone was not transported into the nucleus. Acetylated synthetic peptide was not effective in promoting nuclear localization of BSA. Mild trypsin treatment of the BSA-synthetic peptide conjugate suppressed nuclear localization. Conjugates of the synthetic peptide with phycoerythrin (Mr about 150 kD) and with secretory IgA (Mr about 380 kD) were both found in the nucleus very shortly after their introduction into the cytoplasm. These results suggest that the synthetic peptide containing the nuclear location signal sequence provides exogenous proteins with the ability to migrate into the nucleus. But, since a conjugate of the synthetic peptide with IgM (Mr about 940 kD) did not migrate into the nucleus after its microinjection, there may be a size limit in nuclear transport of conjugated proteins.     

The effects of variations in the number and sequence of targeting signals on nuclear uptake

To determine if the number of targeting signals affects the transport of proteins into the nucleus, Xenopus oocytes were injected with colloidal gold particles, ranging in diameter from 20 to 280 A, that were coated with BSA cross-linked with synthetic peptides containing the SV-40 large T-antigen nuclear transport signal. Three BSA conjugate preparations were used; they had an average of 5, 8, and 11 signals per molecule of carrier protein. In addition, large T-antigen, which contains one signal per monomer, was used as a coating agent. The cells were fixed at various times after injection and subsequently analyzed by electron microscopy. Gold particles coated with proteins containing the SV-40 signal entered the nucleus through central channels located within the nuclear pores. Analysis of the intracellular distribution and size of the tracers that entered the nucleus indicated that the number of signals per molecule affect both the relative uptake of particles and the functional size of the channels available for translocation. In control experiments, gold particles coated with BSA or BSA conjugated with inactive peptides similar to the SV-40 transport signal were virtually excluded from the nucleus. Gold particles coated with nucleoplasmin, an endogenous karyophilic protein that contains five targeting signals per molecule, was transported through the nuclear pores more effectively than any of the BSA-peptide conjugates. Based on a correlation between the peri-envelope density of gold particles and their relative uptake, it is suggested that the differences in the activity of the two targeting signals is related to their binding affinity for envelope receptors. It was also determined, by performing coinjection experiments, that individual pores are capable of recognizing and transporting proteins that contain different nuclear targeting signals.     

The intracellular and nuclear-targeted delivery of an antiandrogen drug by carrier peptides

Cell permeable carrier peptides are currently of interest for their potential to improve the delivery of bioactive molecules into cells and to specific cellular compartments. We have investigated the activity of a derivative of the antiandrogen drug, bicalutamide, attached to the cell-permeable carrier peptide penetratin(R). We have used both disulfide (labile) and thioether (nonlabile) linkages to attach the bicalutamide derivative to the peptide in order to assess whether one type of chemistry has advantages over the other. In addition we have added a nuclear localization sequence (NLS) to the carrier peptide to investigate whether localization of the drug to the nucleus of the cell affects the activity of the drug. Biotin-labeled peptides were used to demonstrate that the carrier peptide is rapidly accumulated inside cultured cells, and that the incorporation of an NLS in the sequence results in its nuclear targeting. The bicalutamide derivative linked to carrier peptides via a disulfide-linkage exerted no greater antiproliferative effect in LNCaP cells, than the bicalutamide derivative alone. The bicalutamide derivative linked to the carrier peptide by a non-labile thioether linkage showed a similar activity profile. When the construct includes a nuclear targeting sequence, however, a markedly increased antiproliferative effect was observed. This study has thus shown that the activity of bicalutamide may be enhanced by the nonlabile attachment of a cell-permeable and nuclear-targeted peptide, which has implications for the development of novel antiandrogens for the treatment of prostate cancer.     

Peptide vaccine against canine parvovirus: identification of two neutralization subsites in the N terminus of VP2 and optimization of the amino acid sequence.

The N-terminal domain of the major capsid protein VP2 of canine parvovirus was shown to be an excellent target for development of a synthetic peptide vaccine, but detailed information about number of epitopes, optimal length, sequence choice, and site of coupling to the carrier protein was lacking. Therefore, several overlapping peptides based on this N terminus were synthesized to establish conditions for optimal and reproducible induction of neutralizing antibodies in rabbits. The specificity and neutralizing ability of the antibody response for these peptides were determined. Within the N-terminal 23 residues of VP2, two subsites able to induce neutralizing antibodies and which overlapped by only two glycine residues at positions 10 and 11 could be discriminated. The shortest sequence sufficient for neutralization induction was nine residues. Peptides longer than 13 residues consistently induced neutralization, provided that their N termini were located between positions 1 and 11 of VP2. The orientation of the peptides at the carrier protein was also of importance, being more effective when coupled through the N terminus than through the C terminus to keyhole limpet hemocyanin. The results suggest that the presence of amino acid residues 2 to 21 (and probably 3 to 17) of VP2 in a single peptide is preferable for a synthetic peptide vaccine.     

Localization and topogenesis studies of cytoplasmic and vacuolar homologs of the Galanthus nivalis agglutinin

The Galanthus nivalis agglutinin (GNA) is synthesized as a preproprotein. To corroborate the role of the different targeting peptides in the topogenesis of GNA and related proteins, different constructs were made whereby both the complete original GNA gene and different truncated sequences were coupled to the enhanced green fluorescent protein (EGFP). In addition, a GNA ortholog from rice that lacks the signal peptide and C-terminal propeptide sequence was fused to EGFP. These fusion constructs were expressed in tobacco BY-2 cells and their localization analyzed by confocal fluorescence microscopy. We observed that the processed preproprotein of GNA was directed towards the vacuolar compartment, whereas both the truncated forms of GNA corresponding to the mature lectin polypeptide and the rice ortholog of GNA were located in the nucleus and the cytoplasm. It can be concluded, therefore, that removal of the C-terminal propeptide and the signal peptide is sufficient to change the subcellular targeting of a normally vacuolar protein to the nuclear/cytoplasmic compartment of the BY-2 cells. These findings support the proposed hypothesis that cytoplasmic/nuclear GNA-like proteins and their vacuolar homologs are evolutionarily related and that the classical GNA-related lectins might have evolved from cytoplasmic orthologs through an evolutionary event involving the insertion of a signal peptide and a C-terminal propeptide.     

Peptide-based targeting of fluorophores to organelles in living cells

Peptides carrying organelle-specific import or retention sequences can target the fluorophore BODIPY(581/591) to the nucleus, peroxisomes, endoplasmic reticulum (ER), or the trans-Golgi network (TGN). The peroxisomal peptide contains the PTS1 sequence AKL. For targeting to the ER or TGN, the peptides carry the retention sequences KDEL and SDYQRL, respectively. A peptide carrying the nuclear leader sequence of the simian virus SV40 large tumor antigen, KKKRK, was used to direct the fluorophore to the nucleus. The fluorescent peptides for peroxisomes, ER, and the TGN spontaneously incorporate into living fibroblasts at 37 degrees C and accumulate in their target organelles within minutes. The uptake is still significant at 4 degrees C, indicating that endocytosis is not required for internalization. The highly charged nuclear peptide (net charge +4) does not spontaneously internalize. However, by transient permeabilization of the plasma membrane, this fluorescent peptide was found to rapidly accumulate in the nucleus. These fluorescent peptides open new opportunities to follow various aspects of specific organelles such as their morphology, biogenesis, dynamics, degradation, and their internal parameters (pH, redox).     

Factors determining the efficacy of nuclear delivery of antisense oligonucleotides by gold nanoparticles

The present study investigates the applicability of nanoparticle delivery vectors for two-stage targeting that involves both cell entry by endocytosis and nuclear targeting using viral peptide signals. A nanoparticle vector consists of four components: a carrier nanoparticle, a stabilizer, targeting peptides, and a therapeutic cargo. Extensive study of bovine serum albumin (BSA)-peptide stabilized nanoparticle conjugates demonstrated limitations of these systems due to colloidal instability when oligonucleotides and multiple peptides were attached to the BSA protein. We found that the widely used protein streptavidin (SA) was an appropriate alternative to BSA for cell-targeting experiments. Targeting peptides and gene splicing oligonucleotides were attached to SA-nanoparticles using biotin labels. The present study uses a gene-splicing assay as a test for oligonucleotide delivery to the cell nucleus. Successful modification of gene splicing by an antisense oligonucleotide indicates that the latter must have crossed the plasma membrane, entered the nucleus, found the target sequence in the newly transcribed pre-mRNA, and hybridized to it in the spliceosome strongly enough to displace the splicing factors designed to interact with the target sequence. Targeting nanoparticles that carry gene-splicing oligonucleotides were compared with a control experiment that used lipofectamine (LF). While enhanced activity was observed in the control experiment, in the presence of LF, no gene splicing was observed for the nanoparticle targeting vectors without LF. We conclude that sequestration of cargo from the harsh conditions of the endosome is a desirable strategy for cell-targeting nanoparticles.     

A method for the large-scale cloning of nuclear proteins and nuclear targeting sequences on a functional basis

We describe here a selection strategy allowing the cloning of sequences that contain a functional nuclear targeting signal. Our method relies on the use of green fluorescent protein fusion proteins to identify nuclear targeting sequences. Transfected cells expressing nuclear protein fusions were isolated on the basis of their nuclear fluorescence using flow cytometry and the transfected DNAs were recovered after bacterial transformation with total DNA from pools of sorted cells. Starting from a cDNA expression library, in which only 1% of the expressed proteins were nuclear, we obtained a 70-fold enrichment in nuclear protein-encoding clones after a single round of selection. Among the 63 clones that have been partially sequenced to date, 25 (40%) corresponded to known nuclear proteins and 13 (20%) to previously uncharacterized sequences. Despite their ability to target the green fluorescent protein marker to the cell nucleus, about half of the cloned sequences did not encode canonical basic or bipartite nuclear localization signals. The method can thus be applied to the large-scale cloning of functional nuclear targeting sequences, which opens the way to a wide investigation of nuclear import mechanisms and to the identification of previously unknown nuclear proteins.     

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SV40 Large T-antigen nuclear signal analogues: Successful nuclear targeting with bovine serum albumin but not low molecular weight fluorescent conjugates

The signal sequence of a nuclear-directed protein encodes the necessary information for targeting the attached proteins to the cell nucleus. The sequence/structural requirements for a functional transport signal were explored with a series of peptides derived from the simian virus 40 large T-antigen nuclear signal 126–134 (CPKKKRKVED-NH2, wild type) conjugated to bovine serum albumin (BSA) through an N-terminal Cys (1) with m-maleimidobenzoyl-N-hydroxysuccinimide ester. Nuclear accumulation was virtually complete 15 min after microinjection into green monkey kidney cells (TC-7). Peptides with Asn, Orn, and Gln substituted for Lys¹²⁸, the reverse wild-type peptide (DEVKRKKKPC-NH2) and the long 34-residue wild-type analogue (CYDDEATADSQHSTPPKKKRKVEDPKDFESELLS-NH2), were synthesized and conjugated similarly to BSA. The Orn peptide and the 34-residue wild-type analogue conjugated to BSA also transported to the nucleus but at a slower rate than 1. The reverse wild-type, Asn- and Gln-BSA conjugates of these signal analogues did not show transport to the nucleus after 6 h of incubation. In an effort to learn if such signal sequences would also target a small molecule such as a fluorescent tag to the nucleus, 1 fluorescently tagged with monobromobimane was prepared and microinjected into TC-7 cells. The peptide was distributed throughout the cell. These results support the notion that a positively charged residue at position 128 is needed for rapid nuclear transport and that the intracellular transport machinery has spatial recognition. The results with fluorophore-peptide conjugates suggest nuclear localization of these low molecular weight peptides will be difficult to attain even if attached to a functional nuclear localization sequence.     

A nuclear localization signal binding protein in the nucleolus

We used functional wild-type and mutant synthetic nuclear localization signal peptides of SV-40 T antigen cross-linked to human serum albumin (peptide conjugates) to assay their binding to proteins of rat liver nuclei on Western blots. Proteins of 140 and 55 kD (p140 and p55) were exclusively recognized by wild-type peptide conjugates. Free wild-type peptides competed for the wild-type peptide conjugate binding to p140 and p55 whereas free mutant peptides, which differed by a single amino acid from the wild type, competed less efficiently. The two proteins were extractable from nuclei by either low or high ionic strength buffers. We purified p140 and raised polyclonal antibodies in chicken against the protein excised from polyacrylamide gels. The anti-p140 antibodies were monospecific as judged by their reactivity with a single nuclear protein band of 140 kD on Western blots of subcellular fractions of whole cells. Indirect immunofluorescence microscopy on fixed and permeabilized Buffalo rat liver (BRL) cells with anti-p140 antibodies exhibited a distinct punctate nucleolar staining. Rhodamine- labeled wild-type peptide conjugates also bound to nucleoli in a similar pattern on fixed and permeabilized BRL cells. Based on biochemical characterization, p140 is a novel nucleolar protein. It is possible that p140 shuttles between the nucleolus and the cytoplasm and functions as a nuclear import carrier.     

Organelle-targeted delivery of biological macromolecules using the protein transduction domain: potential applications for Peptide aptamer delivery into the nucleus

Extensive effort is currently being expended on the innovative design and engineering of new molecular carrier systems for the organelle-targeted delivery of biological cargoes (e.g., peptide aptamers or biological proteins) as tools in cell biology and for developing novel therapeutic approaches. Although cell-permeable Tat peptides are useful carriers for delivering biological molecules into the cell, much internalized Tat-fused cargo is trapped within macropinosomes and thus not delivered into organelles. Here, we devised a novel intracellular targeting technique to deliver Tat-fused cargo into the nucleus using an endosome-disruptive peptide (hemagglutinin-2 subunit) and a nuclear localization signal peptide. We show for the first time that Tat-conjugated peptide aptamers can be selectively delivered to the nucleus by using combined hemagglutinin-2 subunit and nuclear localization signal peptides. This nuclear targeting technique resulted in marked enhancement of the cytostatic activity of a Tat-fused p53-derived peptide aptamer against human MDM2 (mouse double minute 2) that inhibits p53-MDM2 binding. Thus, our technique provides a unique methodology for the development of novel therapeutic approaches based on intracellular targeting.     

Electroporation-Mediated Delivery of Nucleolar Targeting Sequences from Semliki Forest Virus Nucleocapsid Protein

Abstract

Electroporation was used as a powerful and simple method to probe to the intracellular distribution and trafficking of signal sequences. By coupling synthetic peptides to carrier reporter groups, specific amino acid sequences responsible for nucleolar targeting of Semliki Forest virus (SFV) Core (C) protein were found out. In the N-terminal part of the C protein the sequences ⁶⁶KPKKKKTTKPKPKTQPKK^{83 92}KKKDKQADKKKKP¹⁰⁵ are able to situate BSA or KLH as reporter proteins in the nucleolus, suggesting that SFV C protein contains at least two independent nucleolar targeting sequences.