Tài liệu Báo cáo khoa học: Different mechanisms for cellular internalization of the HIV-1

Different mechanisms for cellular internalization of the HIV-1

Tat-derived cell penetrating peptide and recombinant proteins

fused to Tat

Michelle Silhol1

, Mudit Tyagi2

, Mauro Giacca2

, Bernard Lebleu1 and Eric ViveÁ s

1

1

Institut de GeÂneÂtique MoleÂculaire de Montpellier, CNRS UMR 5124, BP5051, Montpellier, France; 2

Molecular Medicine Laboratory, International Centre for Genetic Engineering and Biotechnology, Trieste, Italy

Translocation through the plasma membrane is a major

limiting step for the cellular delivery of macromolecules.

A promising strategy to overcome this problem consists in

the chemical conjugation (or fusion) to cell penetrating

peptides (CPP) derived from proteins able to cross the

plasma membrane. A large number of di€erent cargo mol￾ecules such as oligonucleotides, peptides, peptide nucleic

acids, proteins or even nanoparticles have been internalized

in cells by this strategy. One of these translocating peptides

was derived from the HIV-1 Tat protein. The mechanisms by

which CPP enter cells remain unknown. Recently, convinc￾ing biochemical and genetic ®ndings has established that the

full-length Tat protein was internalized in cells via the

ubiquitous heparan sulfate (HS) proteoglycans. We dem￾onstrate here that the short Tat CPP is taken up by a route

that does not involve the HS proteoglycans.

Keywords: Tat; cell penetrating peptide (CPP); cellular

uptake; heparan sulfate.

Several cell-penetrating peptides (CPP) allowing the ef®cient

internalization of various nonpermeant drugs in different

cell lines have been recently described. A covalent link had

to be created between the CPP and the cargo molecule to

promote ef®cient membrane translocation of the chimera

[1±7]. A 16-mer peptide derived from the Antennapedia

protein homeodomain [8] and a 13-mer peptide derived

from the HIV-1 Tat protein [9] have been extensively

studied. In our initial experiments using the short Tat basic

domain, we demonstrated the uptake of chemically conju￾gated nonpermeant peptides [10]. Then, several peptides

showing a cellular activity were successfully vectorized either

with the Antennapedia peptide [11] or the Tat peptide [12±

14]. Along the same lines, antisense oligonucleotides (ON)

were coupled chemically to the Antennapedia peptide [1], or

to the short Tat peptide [2,15]. Ef®cient internalization and

biological activity of the ONs were observed. Peptide nucleic

acids (PNAs) were also taken up by cells after coupling to

Transportan or to the Antennapedia peptide [3], or to the

Tat peptide (E. ViveÁs & B. Lebleu, unpublished observa￾tions). Regulation of the galanin receptor expression by a

sequence speci®c antisense activity was observed after

incubation of cells with the chimera [3]. The cellular

internalization of proteins such as b-galactosidase, horse￾radish peroxidase or Fab antibody fragment was also

reported. In these cases, the carrier Tat peptide and the

transported protein were associated either by chemical

coupling [4,5,16] or by genetic construction leading to a

fusion protein expressing the 13-amino-acid CPP moiety at

its N-terminus [6,7].

We have focused on the short HIV-1 Tat derived

peptide. Indeed it was initially shown that the maximum

rate of internalization was reached when three to four

molecules of a 35-amino-acid Tat peptide were chemically

coupled to the transported protein [4]. In this case, the use

of shorter peptides appeared to reduce the uptake process.

A structure±function relationship study of the peptide

encompassing this 35-amino-acid region then allowed

delineation of the translocating activity domain to a

13-mer amino-acid sequence [9]. This sequence contains

six arginine residues and two lysine residues within a linear

sequence of 13 amino acids, conferring a highly cationic

character on this peptide . It was later shown that arginine

residues were essential for translocation as deletion (or

replacement by alanine) of a single arginine severely

reduced internalization [10,17].

The mechanism by which these cell penetrating peptides

(and their conjugates) enter cells is not yet determined,

although endocytosis does not seem to be required [9,18].

First, it was shown for the Antennapedia peptide that

structural requirements were not involved in the uptake

process as the inverso D-isomer form of the peptide [19] or

insertion of proline residues within the primary sequence

[18] did not impair cell uptake. Tat behaviour is very similar

to Antennapedia as the Tat peptide with all D-amino acids

(48GRKKRRQRRRPPQ60C) still enters cells [20] and the

retro-inverso form of the Tat peptide (57RRRQRRKKR49

with all D-amino acids) is even more ef®ciently translocated

than the corresponding native peptide [17]. Second, both

peptides are internalized at 4 °C [9,18], a temperature which

abolishes active transport mechanisms involving endocyto￾sis. Third, both peptides were found to be taken up in

Correspondence to E. Vives, Institut de GeÂneÂtique MoleÂculaire de

Montpellier, CNRS UMR 5124, BP5051, 1919 route de Mende, 34033

Montpellier cedex 1, France. Fax: + 33 467 040231,

Tel.: + 33 467 613661, E-mail: [email protected]

Abbreviations: CPP, cell penetrating peptides; HS, heparan sulfate;

PNA, peptide nucleic acid; GST, gluthathione S-transferase; GFP,

green ¯uorescent protein; FHV, ¯ock house virus.

(Received 7 September 2001, accepted 14 November 2001)

Eur. J. Biochem. 269, 494±501 (2002) Ó FEBS 2002