Tubulin polyglutamylation stimulates spastin-mediated microtubule severing.

Posttranslational glutamylation of tubulin is present on selected subsets of microtubules in cells. Although the modification is expected to contribute to the spatial and temporal organization of the cytoskeleton, hardly anything is known about its functional relevance. Here we demonstrate that glutamylation, and in particular the generation of long glutamate side chains, promotes the severing of microtubules. In human cells, the generation of long side chains induces spastin-dependent microtubule disassembly and, consistently, only microtubules modified by long glutamate side chains are efficiently severed by spastin in vitro. Our study reveals a novel control mechanism for microtubule mass and stability, which is of fundamental importance to cellular physiology and might have implications for diseases related to microtubule severing.

Targeting cyclin B1 through peptide-based delivery of siRNA prevents tumour growth.

The development of short interfering RNA (siRNA), has provided great hope for therapeutic targeting of specific genes responsible for pathological disorders. However, the poor cellular uptake and bioavailability of siRNA remain a major obstacle to their clinical development and most strategies that propose to improve siRNA delivery remain limited for in vivo applications. In this study, we report a novel peptide-based approach, MPG-8 an improved variant of the amphipathic peptide carrier MPG, that forms nanoparticles with siRNA and promotes their efficient delivery into primary cell lines and in vivo upon intra-tumoral injection. Moreover, we show that functionalization of this carrier with cholesterol significantly improves tissue distribution and stability of siRNA in vivo, thereby enhancing the efficiency of this technology for systemic administration following intravenous injection without triggering any non-specific inflammatory response. We have validated the therapeutic potential of this strategy for cancer treatment by targeting cyclin B1 in mouse tumour models, and demonstrate that tumour growth is compromised. The robustness of the biological response achieved through this approach, infers that MPG 8-based technology holds a strong promise for therapeutic administration of siRNA.

A new potent secondary amphipathic cell-penetrating peptide for siRNA delivery into mammalian cells.

RNA interference constitutes a powerful tool for biological studies, but has also become one of the most challenging therapeutic strategies. However, small interfering RNA (siRNA)-based strategies suffer from their poor delivery and biodistribution. Cell-penetrating peptides (CPPs) have been shown to improve the intracellular delivery of various biologically active molecules into living cells and have more recently been applied to siRNA delivery. To improve cellular uptake of siRNA into challenging cell lines, we have designed a secondary amphipathic peptide (CADY) of 20 residues combining aromatic tryptophan and cationic arginine residues. CADY adopts a helical conformation within cell membranes, thereby exposing charged residues on one side, and Trp groups that favor cellular uptake on the other. We show that CADY forms stable complexes with siRNA, thereby increasing their stability and improving their delivery into a wide variety of cell lines, including suspension and primary cell lines. CADY-mediated delivery of subnanomolar concentrations of siRNA leads to significant knockdown of the target gene at both the mRNA and protein levels. Moreover, we demonstrate that CADY is not toxic and enters cells through a mechanism which is independent of the major endosomal pathway. Given its biological properties, we propose that CADY-based technology will have a significant effect on the development of fundamental and therapeutic siRNA-based applications.

A new generation of peptide-based inhibitors targeting HIV-1 reverse transcriptase conformational flexibility.

The biologically active form of human immunodeficiency virus (HIV) type 1 reverse transcriptase (RT) is a heterodimer. The formation of RT is a two-step mechanism, including a rapid protein-protein interaction "the dimerization step," followed by conformational changes "the maturation step," yielding the biologically active form of the enzyme. We have previously proposed that the heterodimeric organization of RT constitutes an interesting target for the design of new inhibitors. Here, we propose a new class of RT inhibitors that targets protein-protein interactions and conformational changes involved in the maturation of heterodimeric reverse transcriptase. Based on a screen of peptides derived from the thumb domain of this enzyme, we have identified a short peptide P(AW) that inhibits the maturation step and blocks viral replication at subnanomolar concentrations. P(AW) only binds dimeric RT and stabilizes it in an inactive/non-processive conformation. From a mechanistic point of view, P(AW) prevents proper binding of primer/template by affecting the structural dynamics of the thumb/fingers of p66 subunit. Taken together, these results demonstrate that HIV-1 RT maturation constitutes an attractive target for AIDS chemotherapeutics.

A non-covalent peptide-based carrier for in vivo delivery of DNA mimics.

The dramatic acceleration in identification of new nucleic-acid-based therapeutic molecules has provided new perspectives in pharmaceutical research. However, their development is limited by their poor cellular uptake and inefficient trafficking. Here we describe a short amphipathic peptide, Pep-3, that combines a tryptophan/phenylalanine domain with a lysine/arginine-rich hydrophilic motif. Pep-3 forms stable nano-size complexes with peptide-nucleic acid analogues and promotes their efficient delivery into a wide variety of cell lines, including primary and suspension lines, without any associated cytotoxicity. We demonstrate that Pep-3-mediated delivery of antisense-cyclin B1-charged-PNA blocks tumour growth in vivo upon intratumoral and intravenous injection. Moreover, we show that PEGylation of Pep-3 significantly improves complex stability in vivo and consequently the efficiency of antisense cyclin B1 administered intravenously. Given the biological characteristics of these vectors, we believe that peptide-based delivery technologies hold a true promise for therapeutic applications of DNA mimics.

First step of the cell-penetrating peptide mechanism involves Rac1 GTPase-dependent actin-network remodelling.

BACKGROUND INFORMATION: Application of CPPs (cell-penetrating peptides) constitutes a promising strategy for the intracellular delivery of therapeutic molecules. The non-covalent approach based on the amphipathic peptide MPG has been successfully used to improve the delivery of biologically active macromolecules, both in cellulo and in vivo, through a mechanism independent of the endosomal pathway and mediated by the membrane potential. RESULTS: In the present study, we have investigated the first step of the cellular uptake mechanism of MPG and shown that both MPG and MPG-cargo complexes interact with the extracellular matrix through the negatively charged heparan sulfate proteoglycans. We demonstrated that initiation of cellular uptake constitutes a highly dynamic mechanism where the binding of MPG or the MPG-cargo to the extracellular matrix is rapidly followed by a remodelling of the actin network associated with the activation of the GTPase Rac1. We suggest that MPG-induced clustering of the glycosaminoglycan platform constitutes the 'onset' of the cellular uptake mechanism, thereby increasing membrane dynamics and membrane fusion processes. This process favours cell entry of MPG or MPG-DNA complexes, which is further controlled by the ability of MPG to induce a local membrane destabilization. CONCLUSIONS: Although CPPs are taken up through different pathways and mechanisms, the initial step involves electrostatic interactions with the glycosaminoglycan platform, and the dynamics of associated membrane microdomains can be generalized to most non-viral delivery systems.

A non-covalent peptide-based strategy for protein and peptide nucleic acid transduction.

The development of therapeutic peptides and proteins is limited by the poor permeability and the selectivity of the cell membrane. The discovery of protein transduction domains has given a new hope for administration of large proteins and peptides in vivo. We have developed a non-covalent strategy for protein transduction based on an amphipathic peptide, Pep-1, that consists of a hydrophobic domain and a hydrophilic lysine-rich domain. Pep-1 efficiently delivers a variety of fully biologically active peptides and proteins into cells, without the need for prior chemical cross-linking or chemical modifications. The mechanism through which Pep-1 delivers active macromolecules does not involve the endosomal pathway and the dissociation of the Pep-1/macromolecule particle occurs immediately after it crosses the cell membrane. Pep-1 has been successfully applied to the screening of therapeutic peptides in vivo and presents several advantages: stability in physiological buffer, lack of toxicity and of sensitivity to serum. In conclusion, Pep-1 technology could contribute significantly to the development of fundamental and therapeutic applications and be an alternative to covalent protein transduction domain-based technologies.

Insight into the mechanism of a peptide inhibitor of HIV reverse transcriptase dimerization.

The biologically active forms of human immunodeficiency viruses type 1 and 2 reverse transcriptase (RT) found in infectious virions are heterodimers. We have previously shown that the dimeric nature of reverse transcriptase represents an important target for the design of a new class of antiviral agents and have designed a short peptide (Pep-7) derived from the tryptophan-rich motif of the connection subdomain that blocks dimerization of reverse transcriptase in vitro and abolishes viral infection. In the present work, we have investigated the mechanism through which this peptide inhibits RT dimerization and consequently viral propagation. We demonstrate that Pep-7 interacts preferentially with the p51 subunit within the heterodimeric reverse transcriptase, which destabilizes reverse transcriptase dimer conformation, thereby triggering dissociation. We have identified two residues Trp(24) and Phe(61), located on the fingers subdomain of p51, required for Pep-7 binding. Selective mutation of these residues on p51 to a glycine dramatically alters the stability of the RT-heterodimer suggesting that the fingers subdomain of p51 is also involved in stabilization of reverse transcriptase. We propose that the binding site of Pep-7 is located in a cleft between the fingers and the connection subdomains of p51 that contains the two highly conserved residues Phe(61) and Trp(24).

Design of a novel class of peptide inhibitors of cyclin-dependent kinase/cyclin activation.

Cyclin dependent kinases (CDKs) are key regulators of the cell cycle progression and therefore constitute excellent targets for the design of anticancer agents. Most of the inhibitors identified to date inhibit kinase activity by interfering with the ATP-binding site of CDKs. We recently proposed that the protein/protein interface and conformational changes required in the molecular mechanism of CDK2-cyclin A activation were potential targets for the design of specific inhibitors of cell cycle progression. To this aim, we have designed and characterized a small peptide, termed C4, derived from amino acids 285-306 in the alpha5 helix of cyclin A. We demonstrate that this peptide does not interfere with complex formation but forms stable complexes with CDK2-cyclin A. The C4 peptide significantly inhibits kinase activity of complexes harboring CDK2 in a competitive fashion with respect to substrates but does not behave as an ATP antagonist. Moreover, when coupled with the protein transduction domain of Tat, the C4 peptide blocks the proliferation of tumor cell lines, thereby constituting a potent lead for the development of specific CDK-cyclin inhibitors.

On the mechanism of non-endosomial peptide-mediated cellular delivery of nucleic acids.

Recently, we described a new strategy for the delivery of nucleic acids into mammalian cells, based on an amphipathic peptide of 27 residues called MPG, which was designed on the basis of a hydrophobic domain derived from a fusion sequence associated with a nuclear localization sequence and separated by a linker. This peptide carrier constitutes a powerful tool for the delivery of nucleic acids in cultured cells, without requiring any covalent coupling. We have examined the conformational states of MPG in its free form and complexed with a cargo, as well as its ability to interact with phospholipids, and have investigated the structural consequences of these interactions. In spite of its similarity to the similarly designed cell-penetrating peptide Pep-1, MPG behaves significantly differently from the conformational point of view. Circular dichroism (CD) analysis reveals a transition from a nonstructured to a beta-sheet conformation upon interaction with phospholipids. We propose that the membrane crossing process involves formation of a transient transmembrane pore-like structure. Partial conformational change of MPG is associated with formation of a complex with its cargo, and an increase in sheet content occurs upon association with the cell membrane.

Regulation of human SRY subcellular distribution by its acetylation/deacetylation.

SRY, a Y chromosome-encoded DNA-binding protein, is required for testis organogenesis in mammals. Expression of the SRY gene in the genital ridge is followed by diverse early cell events leading to Sertoli cell determination/differentiation and subsequent sex cord formation. Little is known about SRY regulation and its mode of action during testis development, and direct gene targets for SRY are still lacking. In this study, we demonstrate that interaction of the human SRY with histone acetyltransferase p300 induces the acetylation of SRY both in vitro and in vivo at a single conserved lysine residue. We show that acetylation participates in the nuclear localisation of SRY by increasing SRY interaction with importin beta, while specific deacetylation by HDAC3 induces a cytoplasmic delocalisation of SRY. Finally, by analysing p300 and HDAC3 expression profiles during both human or mouse gonadal development, we suggest that acetylation and deacetylation of SRY may be important mechanisms for regulating SRY activity during mammalian sex determination.

Primary amphipathic cell-penetrating peptides: structural requirements and interactions with model membranes.

To identify rules for the design of efficient cell-penetrating peptides that deliver therapeutic agents into subcellular compartments, we compared the properties of two closely related primary amphipathic peptides that mainly differ by their conformational state. On the basis of a peptide Pbeta that is nonstructured in water and that promotes efficient cellular uptake of nucleic acids through noncovalent association, we have designed a peptide [Palpha] that is predicted to adopt a helical conformation. We show that [Pbeta] undergoes a lipid-induced conformational transition into a sheet structure, while [Palpha] remains helical. Penetration experiments show that both peptides can spontaneously insert into phospholipid membranes. Analysis of compression isotherms indicates that both peptides interact with phospholipids in the liquid expanded and liquid condensed states. AFM observations reveal that the peptides strongly disrupt the lipid organization of the monolayers and that the conformational state can influence the uptake by model membranes.