Intracellular delivery of peptides via association with ubiquitin or SUMO-1 coupled to protein transduction domains
© Vitte and Jalinot; licensee BioMed Central Ltd. 2008
Received: 01 August 2007
Accepted: 29 February 2008
Published: 29 February 2008
We previously developed small hybrid proteins consisting of SUMO-1 linked to an heptapeptide fused to the Tat protein transduction domain (PTD). The heptapeptide motif was selected from a library of random sequences to specifically bind HIV-1 regulatory proteins Tat or Rev. These constructs, named SHP, are able to enter primary lymphocytes and some of them inhibit HIV-1 replication. Considering these positive results and other data from the literature, we further tested the ability of ubiquitin or SUMO-1 linked to various PTD at their N-terminus to deliver within cells proteins or peptides fused downstream of their diglycine motif. In this system it is expected that the intracellular ubiquitin or SUMO-1 hydrolases cleave the PTD-Ub or PTD-SUMO-1 modules from the cargo polypeptide, thereby allowing its delivery under an unmodified form.
Several bacterial expression vectors have been constructed to produce modular proteins containing from the N- to the C-terminus: the FLAG epitope, a cleavage site for a protease, a PTD, human ubiquitin or SUMO-1, and either GFP or the HA epitope. Nine different PTDs were tested, including the Tat basic domain, wild type or with various mutations, and stretches of arginine or lysine. It was observed that some of these PTDs, mainly the Tat PTD and seven or nine residues long polyarginine motifs, caused association of the hybrid proteins with cells, but none of these constructs were delivered to the cytosol. This conclusion was derived from biochemical and immunofluorescence studies, and also from the fact that free cargo protein resulting from cleavage by proteases after ubiquitin or SUMO-1 was never observed. However, in agreement with our previous observations, mutation of the diglycine motif into alanine-arginine, as in the SHP constructs, allows cytosol entry demonstrated by immunofluorescence observations on living cells and by cell fractionation analyses. This process results from a non-endocytic pathway.
Our observations indicate that fusion of SUMO-1 to a peptide-PTD module allows generation of a stable hybrid protein that is easily produced in bacteria and which efficiently enters into cells but this property necessitates mutation of the diglycine motif at the end of SUMO-1, thereby impairing delivery of the peptide alone.
The rapid progress in the understanding of protein networks underlying biological functions, as well as of the specific roles played by particular polypeptides in human pathologies such as cancer, has fuelled the search for means to deliver peptides or proteins into cells within a therapeutic perspective. Exciting developments originated from previous studies on the viral transactivator Tat, as well as the antennapedia transcription factor [1–3]. Characterization of the capacity of these proteins to enter cells led to the mapping of peptidic domains of limited size responsible for this property which turned out to be transferable by linkage to various peptides or proteins [4–6]; for a recent review see ). These so-called protein transduction domains (PTD) or cell-penetrating peptides raised the possibility of delivering an exogenous protein component into cells. This has been established for many different proteins or peptides ex vivo and has also been shown to work in the whole animal . However, several recent studies have raised doubts concerning the veritable capacity of such hybrid proteins to enter cells [7, 9–12]. For immunofluorescence studies in particular, the fixation step has been shown to cause possible artefacts. In some cases reported cellular entry is therefore questionable, but in others the observed biological effects are difficult to explain without authentic cellular delivery [13–15]. The exact molecular mechanism that allows penetration within cells is also confusing. This property has been shown in some instances to be independent of energy consumption but in others to involve various forms of endocytosis [7, 12, 16–19]. A detailed study with a hybrid TAT-CRE construct has shown that cellular entry was achieved through macropinocytosis . From the published data it appears that the exact mechanism involved depends on the precise nature of the protein and of the PTD. The cell type is also probably important.
A potential problem with peptides or proteins to be delivered into cells is their instability. Association with a folded stable domain can increase this stability. Ubiquitin or members of this protein family can be interesting in this perspective. Several expression systems in bacteria or eukaryotes have benefited from this property favouring the production of otherwise poorly-expressed proteins [20–24]. Indeed, fusion of ubiquitin to their N-terminus can allow the production of such proteins. This is also the case with SUMO-1. In addition, association with ubiquitin or SUMO-1 allows easy cleavage after the diglycine motif which terminates the protein. This possibility has been used in particular for the experimental system which allowed characterization of the N-end rule which states that the stability of a protein depends on the nature of its amino-terminal residue .
Considering these notions, we designed a system to deliver proteins into cells without any addition by creating fusions with PTD-ubiquitin or PTD-SUMO-1 hybrids. Although the system allows efficient expression in bacteria and easy purification, it appeared that these hybrid proteins do not allow efficient delivery into cells. By contrast, mutation of the diglycine motif and association with a peptide motif linked to the Tat or poly arginine PTD permits efficient entry and the mechanism sustaining this entry is energy-independent.
Results and discussion
Design of fusion proteins with the capability of delivering a given protein
We next tested the efficiency of these PTDs in primary lymphocytes. This was done with the ubiquitin and SUMO-1 GFP fusion proteins bearing the Tat and polyR PTDs. These proteins were detected in lymphocytes lysed in SDS and this association was clearly reinforced when lymphocytes were activated (Figure 2c, compare upper and lower panels). The efficiency of the various PTDs was also tested without the presence of ubiquitin or SUMO-1. In the FPG constructs the PTD is directly upstream of the GFP protein. Similarly to what was observed with the ubiquitin fusion proteins, PTDs 1 and 5 mediated association of GFP with cells (Figure 2d, lanes 1 and 2). PTD 6 was weakly active compared to the Tat and polyR motifs (Figure 2d, lane 3). Interestingly, PTD 7 which corresponds to a stretch of seven arginines was as active as PTD 5 which has nine arginines (Figure 2d, compare lanes 4 and 2), but by contrast PTD 8 with eleven arginines showed no activity (Figure 2e, lane 3). A weak cell association was also detected with PTD 9 which corresponds to nine lysines (Figure 2e, lane 4).
Taken together these results show that the Tat and polyR7 or polyR9 were the most efficient motifs under these conditions. In agreement with previous observations, the polyR motif efficiency requires an optimum size of seven to nine residues and increasing this length leads to loss of the effect .
Ubiquitin and SUMO-1 fusion proteins do not enter cells
Veritable cell penetration by the SHPR proteins
Taken together these observations firmly establish that SHPR proteins are able to penetrate within cells via a mechanism that is energy-independent. Hence this is likely to occur by direct passage through the cellular membrane. The intracellular SUMO-1-peptide fusion is mostly nuclear, probably as consequence of the nucleic acid binding properties of the Tat PTD but also possibly due to the SUMO-1 domain. Indeed, it has been reported for several proteins that SUMO-1 addition triggers nuclear entry. This indicates that our system is probably appropriate for targeting nuclear proteins but not cytoplasmic factors. In the nucleus the SHPs show a diffuse localization. These observations are in agreement with the biological effect of SHPR142 and SHPR190 which are able to block replication of HIV-1 in primary lymphocytes or macrophages when added to the culture medium . The results of the fractionation experiments also support the previous conclusion that the ubiquitin GFP proteins are unable to penetrate within the intracellular milieu. This is in agreement with a previously published report that similar ubiquitin-peptide and ubiquitin-protein constructs are not able to enter the cytosol . However, these authors interestingly showed that dendritic cells were able to uptake and process such hybrid proteins, at least to some extent. Hence, in future studies it will be interesting to test if our ubiquitin or SUMO-1 hybrids can be processed in such cells. An intriguing aspect of our observations is the role of the diglycine motif. Indeed, its presence seems to have a strong negative effect on detection of the SUMO-1-peptide hybrid in cells. As the presence of ubiquitin proteases in the cellular membrane has been reported, it is possible that cleavage after the C-terminal diglycine motif occur simultaneously to crossing of the membrane. However, we did not observe cleavage products of our hybrid proteins, even under conditions of proteasome activity blockage [30, 31]. It remains possible that after cleavage both parts are routed towards endosomes and further degraded in lysosomes. Structural studies have established the importance of the diglycine motif in the interaction of SUMO-1 or ubiquitin with C-terminal hydrolases or isopeptidase . Hence, an expected effect of the mutation of this sequence to alanine-arginine is loss of interaction with these enzymes. It is possible that this event explains the efficient capacity of the SHP proteins to enter cells.
The results presented in this report clarify the mechanism of the cellular entry of SUMO-1-peptide-PTD constructs and confirm that these constructs can efficiently deliver a peptide into cells. Unexpectedly, they show that it is important to block the cleavage that normally occurs at the junction of SUMO-1 and the peptide by mutating the diglycine motif. These constructs which can be easily produced in bacteria potentially offer an interesting means of delivering a peptide able to act as an agonist or antagonist with respect to a pivotal cellular protein.
As a first step both 5'-CATGGGCGATTATAAAGATGACGATAAAGGCGGTCA-3' and 5'-TATGACCGCCTTTATCGTCATCTTTATAATCGCC-3' oligonucleotides were annealed and inserted between the Nco I and Nde I restriction sites of vector pET15b giving vector pET-FLAG. The ubiquitin coding sequence was amplified using the following sense and antisense primers: 5'-GAAGATCTCATATGGGCGGTACCCAAATCTTCGTGAAAACCC-3', 5'-GAAGATCTGCGGCCGCCCGGGATCCATACCACCTCTCAGACGC-3'. The amplified DNA fragment was digested by the Nde I and BamH I restriction enzymes and inserted between Nde I and BamH I restriction sites of pET-FLAG, generating vector pET-FU. The GFP coding sequence was obtained from vector pEGFP-1 (Clontech) by Not I – BamH I digestion and was inserted between the Not I and BamH I restriction sites of vector pET-FUG. The various PTDs were included in pET-FUG by annealing appropriate oligonucleotides and inserting them between the NdeI and KpnI restriction sites of pET-FUG, giving vectors pET-FPUG. In some of these latter vectors the sequence encoding ubiquitin was replaced by that of SUMO-1 by digesting pET-FPUG by Kpn I and BamH I. The SUMO-I sequence was obtained by PCR amplification and inserted between these two restriction sites, giving vectors pET-FPSG. Replacement of the GFP coding sequence by that of the HA epitope was made by digesting vectors pET-FPUG and pET-FPSG by BamH I and Not I and inserting annealed sense and antisense following oligonucleotides: 5'-GATCCTGTCTACCCATACGACGTCCCAGACTACGCTGGTAAGTAAGC-3'; 5'-GGCCGCTTACTTACCAGCGTAGTCTGGGACGTCGTATGGGTAGACAG-3'.
Protein production in bacteria
BL21(DE3)codon+ E. coli were transformed with plasmids encoding the different fusion proteins. Transformants were selected on LB plates containing ampicillin and colonies were then grown overnight at 37°C in LB broth supplemented with 100 mg/ml ampicillin with shaking at 250 rpm. The overnight culture was diluted 25-fold with fresh LB medium complemented with ampicillin and cultured at 37°C until an OD at 600 nm of 0.9 was reached. Protein expression was then induced by addition of 1 mM IPTG (isopropyl-1-thio-β-D-galactopyranoside, Uptima) and incubation at 24°C for 3 h. Bacteria were collected by centrifugation and lysed by sonication in a buffer containing 20 mM NaCl, 0.1 M Tris-HCl pH 7.4, 10 mM MgCl2, lysozyme, endonuclease and antiprotease agents (Complete, Roche). After removal of cell debris by centrifugation, the extract was dialysed in TBS (20 mM Tris pH 7.6, 137 mM NaCl,) and loaded onto a M2 Anti-FLAG column (anti-FLAG M2 Affinity Gel, Sigma). The column was washed first with TBS supplemented with 200 mM NaCl and then with TBS. The fusion proteins were eluted by competition with 100 μg/ml of FLAG peptide diluted in TBS, followed by washing with TBS. The fusion protein fractions were then combined and dialysed in PBS (Phosphate-Buffered-Saline) supplemented with 0.5 mM β-mercaptoethanol. Following dialysis, proteins were sterilized by filtration through a 0.025 μm membrane. All purified fusion proteins were dissolved in PBS containing 0.5 mM β-mercaptoethanol then aliquoted and stored at -80°C. Fusion protein purity was checked by 12% SDS-PAGE followed by staining with Coomassie Brilliant Blue. Protein concentrations were determined by densitometry analysis using bovine serum albumin (BSA) as the standard ("Bradford test", Biorad).
Cells were incubated at 37°C in a 5% CO2-humidified atmosphere. HeLa cells were cultured in Dulbecco's modified Eagle's medium and Jurkat cells in RPMI 1640 medium supplemented with 5 and 10% foetal calf serum, respectively. Human peripheral blood mononuclear cells (PBMC) were isolated from the blood of healthy donors using Ficoll density gradients. Activation of the lymphocytes was performed by incubating the cells with 1 mg/ml phytohemagglutinin (PHA) and 20 IU/ml IL-2 for 48 h.
Immunoblot and immunofluorescence
For immunoblot analysis, the samples were loaded onto a 12% SDS-PAGE and proteins were electrotransferred on a PVDF membrane (Amersham), which was then blocked with PBS Tween 0.1% supplemented with 5% dry milk. Membranes were probed with either mouse monoclonal anti-FLAG M2 antibody (Sigma, dilution 1:1000 in PBS Tween 0.1%), a mouse monoclonal anti-GFP antibody (dilution 1:1000 in PBS Tween 0.1%) or a mouse monoclonal anti-SUMO-1 antibody (Zymed, dilution 1:2000 in PBS Tween 0.1%). Mouse anti-β actin (Sigma, dilution 1:5000) and rabbit polyclonal anti-RRM2 antibody were also used to verify the extracts homogeneity or fractionation purity, respectively. Sheep anti-mouse or anti-rabbit immunoglobulins coupled to peroxidase (Amersham, dilution 1:6000) were used as secondary antibodies. Revelation was performed by chemiluminescence using the ECL or ECL plus reagent (Amersham Biosciences). Alternatively (Figure 2d and 2e), the membrane was incubated with anti-mouse immunoglobulins coupled to cyanin 5 (dilution 1:500), and the signal visualized using a STORM 860 apparatus.
For immunofluorescence, cells treated with the different fusion proteins were washed 3 times with PBS and then placed in HBS culture medium (10 mM Hepes, pH 7.3; 10 mM D-glucose; 135 mM NaCl; 5 mM KCl; 2 mM MgCl2; 2 mM CaCl2). The cells were then directly loaded onto glass slides coated with 1 mg/ml polylysin by incubation during 5 min followed by a wash with H2O. When GFP fusion proteins were used in the experiment, cells were directly observed for GFP fluorescence by confocal microscopy. Alternatively, SHP proteins, which do not include GFP, were coupled to the Alexa Fluor 488 dye using the Molecular Probes protein labelling kit (A-10235) according to the manufacturer's instructions.
green fluorescent protein
hepes buffer saline
human immunodeficiency virus type 1
protein transduction domain
small ubiquitin-related modifier
SUMO-1 heptapeptide PTD
SUMO-1 heptapeptide PTD Rev
SUMO-1 heptapeptide PTD Tat
sodium dodecyl sulfate
We are grateful to Armelle Roisin and Jean-Philippe Robin for help with cell culture and protein purification. We also wish to thank Robin Buckland for critical reading of the manuscript. We also thank the Agence Nationale de Recherche contre le SIDA (ANRS), for a grant and an A-L V fellowship, and Sidaction, for an A-L V fellowship.
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