Jean Mery

A peptide carrier for the delivery of biologically active proteins into mammalian cells

The development of peptide drugs and therapeutic proteins is limited by the poor permeability and the selectivity of the cell membrane. There is a growing effort to circumvent these problems by designing strategies to deliver full-length proteins into a large number of cells. A series of small protein domains, termed protein transduction domains (PTDs), have been shown to cross biological membranes efficiently and independently of transporters or specific receptors, and to promote the delivery of peptides and proteins into cells. TAT protein from human immunodeficiency virus (HIV-1) is able to deliver biologically active proteins in vivo and has been shown to be of considerable interest for protein therapeutics. Similarly, the third α-helix of Antennapedia homeodomain, and VP22 protein from herpes simplex virus promote the delivery of covalently linked peptides or proteins into cells. However, these PTD vectors display a certain number of limitations in that they all require crosslinking to the target peptide or protein. Moreover, protein transduction using PTD–TAT fusion protein systems may require denaturation of the protein before delivery to increase the accessibility of the TAT–PTD domain. This requirement introduces an additional delay between the time of delivery and intracellular activation of the protein. In this report, we propose a new strategy for protein delivery based on a short amphipathic peptide carrier, Pep-1. This peptide carrier is able to efficiently deliver a variety of peptides and proteins into several cell lines in a fully biologically active form, without the need for prior chemical covalent coupling or denaturation steps. In addition, this peptide carrier presents several advantages for protein therapy, including stability in physiological buffer, lack of toxicity, and lack of sensitivity to serum. Pep-1 technology should be extremely useful for targeting specific protein–protein interactions in living cells and for screening novel therapeutic proteins.

A New Potent HIV-1 Reverse Transcriptase Inhibitor A SYNTHETIC PEPTIDE DERIVED FROM THE INTERFACE SUBUNIT DOMAINS

The biologically relevant and active forms of human immunodeficiency viruses type 1 and 2 reverse transcriptase found in infectious virions are heterodimers produced in a two-step dimerization process. Dimerization involves first the rapid association of the two subunits, followed by a slow conformational change yielding a fully active form. We have shown that the dimeric nature of reverse transcriptase represents a important target for the design of a new class of antiviral agents. In this work, we propose a new strategy for its inhibition by targeting protein/protein interactions during viral formation in infected cells. From the screening of peptides derived from the tryptophan cluster at the interface of the connection subdomain, we have designed a short peptide (10 residues) corresponding to residues 395–404, which can block dimerization of reverse transcriptase in vitro and in infected cells. This peptide is highly efficient in abolishing the production of viral particles, without any adverse toxic side effects, when transduced into human immunodeficiency virus type 1-infected cells together with a new peptide carrier.

Identification of the first Rho–GEF inhibitor, TRIPα, which targets the RhoA-specific GEF domain of Trio

The Rho–guanine nucleotide exchange factors (Rho–GEFs) remodel the actin cytoskeleton via their Rho–GTPase targets and affect numerous physiological processes such as transformation and cell motility. They are therefore attractive targets to design specific inhibitors that may have therapeutic applications. Trio contains two Rho–GEF domains, GEFD1 and GEFD2, which activate the Rac and RhoA pathways, respectively. Here we have used a genetic screen in yeast to select in vivo peptides coupled to thioredoxin, called aptamers, that could inhibit GEFD2 activity. One aptamer, TRIAPα (TRio Inhibitory APtamer), specifically blocks GEFD2‐exchange activity on RhoA in vitro. The corresponding peptide sequence, TRIPα, inhibits TrioGEFD2‐mediated activation of RhoA in intact cells and specifically reverts the neurite retraction phenotype induced by TrioGEFD2 in PC12 cells. Thus TRIPα is the first Rho–GEF inhibitor isolated so far, and represents an important step in the design of inhibitors for the expanding family of Rho–GEFs.

Interactions of Primary Amphipathic Vector Peptides with Membranes. Conformational Consequences and Influence on Cellular Localization

Abstract. The conformations of two peptides produced by the combinations of a nuclear localization sequence and a sequence issued from the fusion protein gp41 of HIV 1 have been analyzed both in solution and in membranes or in membrane mimicking environments. Both are shown to be nonordered in water, α-helical when incorporated into SDS micelles where the helical domain concerns the hydrophobic part of the peptides. Interactions with lipids induce the formation of β-sheet and the lipid-peptide interactions are governed by the nature of the lipid polar headgroups. A monolayer study shows that replacement of the sequence separating the two sequences with an arginine favors the lipid-peptide interactions which may contribute to the understanding of the different, nuclear and membrane associated, cellular localizations of the peptides.

Solid-phase synthesis using a new polyacrylic resin : Synthesis of the fragment 14–21 of the amino acid sequence of histone H4

Abstract The solid-phase synthesis of the octapeptide 1 AcGly-Ala-Lys-Arg-His-Arg-Lys-ValOMe, which represents the fragment 14-21 of the amino acid sequence of the chromosomal histone H4, as well as of the structurally related nonapeptide 2 AcGly-Ala-Lys-Leu-Arg-His-Arg-Lys-ValOMe, is described using a new polyacrylic resin containing a glycolamide ester linkage(resin-NHCO-CH 2 -OCO-peptide) acting as a labile anchoring moiety between the resin and the peptide. After elongation of the polypeptide chain using classical protecting groups, i.e. t-butyloxycarbonyl for the α-NH 2 function, benzyloxycarbonyl, nitro and 2,4-dinitrophenyl groups for the side-chains of Lys, Arg and His respectively, both peptides 1 and 2 were obtained in good yields and with a high purity as shown by high-pressure liquid chromatography, by amino-acid analysis and by high-field proton NMR spectroscopy. This work demonstrates the ability of the newly introduced polyacrylic resin to act as a convenient support for solid-phase peptide synthesis.

Model studies in relation to the molecular structure of chromatin

Summary The paper reports a thermal denaturation analysis of calf thymus DNA associated with synthetic basic peptides (di-, tri-, and tetra-) including the X-X structure (basic doublet, X = Arg of Lys) which appears to be characteristic of the primary structure of all the known histones. Analogies in thermal behaviour between peptides with a basic doublet structure and aliphatic diamines of the series H 2 N (CH 2 ) n NH 2 , suggest that a positive doubly charged structure of defined length is crucial for the interaction with double stranded DNA. A model is proposed for the interaction between histones and DNA on the basis of the above-mentioned intercharge distance requirement (diamine model), which is detailed in the case of the arginine-rich histone F2a1. The role of the enzymatic acetylation and deacetylation of specific lysine residues in the case of arginine-rich histones is discussed in terms of the described diamine model. A possible role of the residue His 18 of histone F2a1 is considered in connection with this model. Proton magnetic resonance studies of DNA associated with two model compounds, malouetine (a steroidal bis quaternary ammonium salt) and histidinamide, demonstrate that accurate description (magnetic, thermodynamic and kinetic) of the complexes can be obtained. Preliminary results, using the analytical ultracentrifugation technique, in the case of some diamine complexes with circulary closed PM2 DNA are also reported.

Enzymatic deacetylation of a synthetic peptide fragment of histone H4

Acetylation and deacetylation of the ~-amino groups of specific histone lysyl residues occurs in the course of the cell cycle. The overall level of acetylation at any given time involves an equilibrium between acetate uptake catalysed by nuclear acetyltransferases [ 1,2] and acetate removal catalysed by nuclear deacetylases [3-51. All the histones that make up the chromatin core particle, i.e., histones H2A, H2B, H3 and H4 are subject, in vivo, to acetylation and deacetylation but no acetylation sites have been observed on the very lysine-rich histone Hl [2]. On the basis of sequence analogies, a classification of the acetylation sites into two types (A,B) was proposed [2]. In type A there is a single lysyl residue surrounded by two non-basic residues and in type B a basic doublet such as Lys-Lys, Lys-Arg and ArgLys. This classification suggests that only a small fragment of the histone molecule is necessary for enzymatic recognition. However, chemical studies on the activity of a calf thymus deacetylase using histone H4 fragments, including the monoacetylated peptide (15-2 1) H-Ala-Lys”j (Ac)-Arg-His-Arg-LysVal-OH, led to the conclusion that a rather long sequence of the histone molecule is required for enzyme recognition [6]. Here we report the enzymatic deacetylation of the diacetylated histone H4 fragment (14-21) Ac-GlyAla-Lys-(AC)-Arg-His-Arg-Lys(Ac)-Val-NH?, with the Nand C-terminal groups protected by an acetyl and an amide group, respectively, in conditions similar to those used for the deacetylation of the whole histone H4 molecule. 2. Materials and methods

Design and synthesis of a peptide derived from positions 195-244 of human cdc25C phosphatase.

We have designed, synthesized and purified a 51 amino acid peptide derived from an essential domain of human cdc25C phosphatase. In vivo, differential phosphorylation of this domain regulates either the induction of mitotic processes, or the checkpoint arrest of eukaryotic cells in response to DNA damage. Peptide synthesis was achieved using the stepwise Fmoc strategy and resulted in an important yield of highly pure peptide. The final peptide was identified by amino acid analysis, electrospray mass spectrometry and nuclear magnetic resonance, which revealed that one of the two methionines within the peptide was oxidized into its sulphoxide derivative We investigated whether this 51 amino acid peptide folded into secondary structures in solution by circular dichroism and observed the formation of alpha helices in TFE. Finally, we verified that this peptide could bind to its biologically relevant 14‐3‐3 partner in vitro by fluorescence spectroscopy. Copyright

Chemical studies on histone acetylation using a synthetic peptide fragment of histone H4

The enzymatic acetylation of e-NH2 groups of lysyl residues of histones may be important in the control of the structure and the function of chromatin [ 11. All the histones that make up the chromatin core particle, i.e., histones H2A, H2B, H3 and H4, are subject to enzymatic acetylation [2]. Primary structure studies of histones show that only a few specific lysyl residues are involved and that the extent of acetylation depends on cellular activity [2-41. The chemical modification of a specific lysyl residue by acetylation has been described as a molecular process which is able to regulate the structure of DNA in chromatin [5]. On the basis of sequence analogies, a classification into two types has been proposed [2] for the acetylation sites of histones: (i) Type A with a single lysyl residue surrounded by two non-basic residues; (ii) Type B comprising a basic doublet such as LysLys,Lys-Arg and Arg-Lys. This classification suggests that only a small fragment of the histone molecule is necessary for enzymatic recognition. This work shows that an acetyltransferase acting on histone H4 is also able to acetylate the synthetic octapeptide AcCly-Ala-Lys-Arg-His-Arg-LysValNH? which corresponds to the sequence fragment of histone H4 between residues 14 and 2 1 and includes two lysyl residues, one of which Lys16 is the major site of acetylation in calf thymus histone H4. Peptide [ 14-2 l] contains two basic doublets, i.e., Lys16Arg and Arg-Lys”, which correspond to acetylation sites of type B. The ability of the acetyltransferase to recognize either both sites or one of them selectively has been investigated.

Capping and dynamic relation between domains 1 and 2 of gelsolin.

Gelsolin is a protein that severs and caps actin filaments. The two activities are located in the N‐terminal half of the gelsolin molecules. Severing and subsequent capping requires the binding of domains 2 and 3 (S2–3) to the side of the filaments to position the N‐terminal domain 1 (S1) at the barbed end of actin (actin subdomains 1 and 3). The results provide a structural basis for the gelsolin capping mechanism. The effects of a synthetic peptide derived from the sequence of a binding site located in gelsolin S2 on actin properties have been studied. CD and IR spectra indicate that this peptide presented a secondary structure in solution which would be similar to that expected for the native full length gelsolin molecule. The binding of the synthetic peptide induces conformational changes in actin subdomain 1 and actin oligomerization. An increase in the polymerization rate was observed, which could be attributed to a nucleation kinetics effect. The combined effects of two gelsolin fragments, the synthetic peptide derived from an S2 sequence and the purified segment 1 (S1), were also investigated as a molecule model. The two fragments induced nucleation enhancement and inhibited actin depolymerization, two characteristic properties of capping. In conclusion, for the first time it is reported that the binding of a small synthetic fragment is sufficient to promote efficient capping by S1 at the barbed end of actin filaments. ©1998 European Peptide Society and John Wiley & Sons, Ltd.