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Gilles Divita

Publications and source records attributed to Gilles Divita.

At least 19 recordsLinked to original sources
PubMed2007

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.

Actins↗
PubMed2006

Ubiquitin-associated domain of Mex67 synchronizes recruitment of the mRNA export machinery with transcription.

The mRNA nuclear export receptor Mex67/Mtr2 is recruited to mRNAs through RNA-binding adaptors, including components of the THO/TREX complex that couple transcription to mRNA export. Here we show that the ubiquitin-associated (UBA) domain of Mex67 is not only required for proper nuclear export of mRNA but also contributes to recruitment of Mex67 to transcribing genes. Our results reveal that the UBA domain of Mex67 directly interacts with polyubiquitin chains and with Hpr1, a component of the THO/TREX complex, which is regulated by ubiquitylation in a transcription-dependent manner. This interaction transiently protects Hpr1 from ubiquitin/proteasome-mediated degradation and thereby coordinates recruitment of the mRNA export machinery with transcription and early messenger ribonucleoproteins assembly.

Biological Transport↗
PubMed2006

Formation of transmembrane ionic channels of primary amphipathic cell-penetrating peptides. Consequences on the mechanism of cell penetration.

The ability of three primary amphipathic Cell-Penetrating Peptides (CPPs) CH3-CO-GALFLGFLGAAGSTMGAWSQPKKKRKV-NH-CH2-CH2-SH, CH3-CO-GALFLAFLAAALS LMGLWSQPKKKRKV-NH-CH2-CH2-SH, and CH3-CO-KETWWETWWTEWSQPKKKRKV-NH-CH2-CH2-SH called Pbeta, Palpha and Pep-1, respectively, to promote pore formation is examined both in Xenopus oocytes and artificial planar lipid bilayers. A good correlation between pore formation and their structural properties, especially their conformational versatility, was established. This work shows that the cell-penetrating peptides Pbeta and Pep-1 are able to induce formation of transmembrane pores in artificial bilayers and that these pores are most likely at the basis of their ability to facilitate intracellular delivery of therapeutics. In addition, their behaviour provides some information concerning the positioning of the peptides with respect to the membrane and confirms the role of the membrane potential in the translocation process.

Animals↗
PubMed2006

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.

Animals↗
PubMed2006

Nucleotide excision repair and template-independent addition by HIV-1 reverse transcriptase in the presence of nucleocapsid protein.

During HIV replication, reverse transcriptase (RT), assisted by the nucleocapsid protein (NC), converts the genomic RNA into proviral DNA. This process appears to be the major source of genetic variability, as RT can misincorporate nucleotides during minus and plus strand DNA synthesis. To investigate nucleotide addition or substitution by RT, we set up in vitro models containing HIV-1 RNA, cDNA, NC, and various RTs. We used the wild type RT and azidothymidine- and didanosine-resistant RTs, because they represent the major forms of resistant RTs selected in patients undergoing therapies. Results show that all RTs can add nucleotides in a non-template fashion at the cDNA 3'-end, a reaction stimulated by NC. Nucleotide substitutions were examined using in vitro systems where 3'-mutated cDNAs were extended by RT on an HIV-1 RNA template. With NC, RT extension of the mutated cDNAs was efficient, and surprisingly, mutations were frequently corrected. These results suggest for the first time that RT has excision-repair activity that is triggered by NC. Chaperoning of RT by NC might be explained by the fact that NC stabilizes an RT-DNA binary complex. In conclusion, RT-NC interactions appear to play critical roles in HIV-1 variability.

DNA Primers↗
PubMed2006

Light controllable siRNAs regulate gene suppression and phenotypes in cells.

Small interfering RNA (siRNA) is widely recognized as a powerful tool for targeted gene silencing. However, siRNA gene silencing occurs during transfection, limiting its use is in kinetic studies, deciphering toxic and off-target effects and phenotypic assays requiring temporal, and/or spatial regulation. We developed a novel controllable siRNA (csiRNA) that is activated by light. A single photo removable group is coupled during oligonucleotide synthesis to the 5' end of the antisense strand of the siRNA, which blocks the siRNA's activity. A low dose of light activates the siRNA, independent of transfection resulting in knock down of specific target mRNAs and proteins (GAPDH, p53, survivin, hNuf2) without stimulating non-specific effects such as regulated protein kinase PKR and induction of the interferon response. We demonstrate survivin and hNuf2 csiRNAs temporally knockdown their mRNAs causing multinucleation and cell death by mitotic arrest, respectively. Furthermore, we demonstrate a dose-dependent light regulation of hNuf2 csiRNA activity and resulting phenotype. The light controllable siRNAs are introduced into cells using commercially available reagents including the MPG peptide based delivery system. The csiRNAs are comparable to standard siRNAs in their transfection efficiency and potency of gene silencing. This technology should be of interest for phenotypic assays such as cell survival, cell cycle regulation, and cell development.

Biological Assay↗
PubMed2006

Interactions of amphipathic carrier peptides with membrane components in relation with their ability to deliver therapeutics.

To identify rules for the design of efficient CPPs that can deliver therapeutic agents such as nucleic acids (DNAs, siRNAs) or proteins and PNAs into subcellular compartments, we compared the properties of several primary and secondary amphipathic CPPs. Studies performed with lipid monolayers at the air-water interface have enabled identification of the nature of the lipid-peptide interactions and characterization of the influence of phospholipids on the ability of these peptides to penetrate into lipidic media. Penetration and compression experiments reveal that both peptides interact strongly with phospholipids, and observations on Langmuir-Blodgett transfers indicate that they can modify the lipid organization. Conformational investigations indicate that the lipid-peptide interactions govern the conformational state(s) of the peptides. On the basis of the ability of both peptides to promote ion permeation through both natural and artificial membranes, models illustrating the translocation processes have been proposed. One is based on the formation of a beta-barrel pore-like structure while another is based on the association of helices.

Amino Acid Sequence↗
PubMed2005

Interactions of amphipathic CPPs with model membranes.

We have investigated the interactions between two carrier peptides and model membrane systems as well as the conformational consequences of these interactions. Studies performed with lipid monolayers at the air-water interface have enabled identification of the nature of the lipid-peptide interactions and characterization of the influence of phospholipids on the ability of these peptides to penetrate into lipidic media. Penetration experiments reveal that both peptides interact strongly with phospholipids. Conformational investigations indicate that the lipid-peptide interaction govern the conformational state of the peptides. Based on the ability of both peptides to promote ion permeabilization of both natural and artificial membranes, we propose a model illustrating the translocation process. For MPG, it is based on the formation of a beta-barrel pore-like structure, while for Pep-1, it is based on association of helices.

Amino Acid Sequence↗
PubMed2005

Biochemical characterization and crystal structure of a Dim1 family associated protein: Dim2.

The U4/U6*U5 tri-snRNP complex is the catalytic core of the pre-mRNA splicing machinery. The thioredoxin-like protein hDim1 (U5-15 kDa) constitutes an essential component of the U5 particle, and its functions have been reported to be highly conserved throughout evolution. Recently, the Dim1-like protein (DLP) family has been extended to other proteins harboring similar sequence motifs. Here we report the biochemical characterization and crystallographic structure of a 149 amino acid protein, hDim2, which shares 38% sequence identity with hDim1. The crystallographic structure of hDim2 solved at 2.5 A reveals a classical thioredoxin-fold structure. However, despite the similarity in the thioredoxin fold, hDim2 differs from hDim1 in many significant features. The structure of hDim2 contains an extra alpha helix (alpha3) and a beta strand (beta5), which stabilize the protein, suggesting that they may be involved in interactions with hDim2-specific partners. The stability and thermodynamic parameters of hDim2 were evaluated by combining circular dichroism and fluorescence spectroscopy together with chromatographic and cross-linking approaches. We have demonstrated that, in contrast to hDim1, hDim2 forms stable homodimers. The dimer interface is essentially stabilized by electrostatic interactions and involves tyrosine residues located in the alpha3 helix. Structural analysis reveals that hDim2 lacks some of the essential structural motifs and residues that are required for the biological activity and interactive properties of hDim1. Therefore, on the basis of structural investigations we suggest that, in higher eukaryotes, although both hDim1 and hDim2 are involved in pre-mRNA splicing, the two proteins are likely to participate in different multisubunit complexes and biological processes.

Amino Acid Sequence↗
PubMed2005

Selective modification of alternative splicing by indole derivatives that target serine-arginine-rich protein splicing factors.

The prevalence of alternative splicing as a target for alterations leading to human genetic disorders makes it highly relevant for therapy. Here we have used in vitro splicing reactions with different splicing reporter constructs to screen 4,000 chemical compounds for their ability to selectively inhibit spliceosome assembly and splicing. We discovered indole derivatives as potent inhibitors of the splicing reaction. Importantly, compounds of this family specifically inhibit exonic splicing enhancer (ESE)-dependent splicing, because they interact directly and selectively with members of the serine-arginine-rich protein family. Treatment of cells expressing reporter constructs with ESE sequences demonstrated that selected indole derivatives mediate inhibition of ESE usage in vivo and prevent early splicing events required for HIV replication. This discovery opens the exciting possibility of a causal pharmacological treatment of aberrant splicing in human genetic disorders and development of new antiviral therapeutic approaches.

Alternative Splicing↗
PubMed2005

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).

Amino Acid Motifs↗
PubMed2005

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.

Amino Acid Sequence↗
PubMed2005

Interactions of primary amphipathic cell penetrating peptides with model membranes: consequences on the mechanisms of intracellular delivery of therapeutics.

This review focuses on two peptides, MPG and Pep-1, which facilitate the transfer of nucleic acids and proteins, respectively, into subcellular compartments. We have investigated the interactions between these two carrier peptides and model membrane systems as well as the conformational consequences of these interactions. Studies performed with lipid monolayers at the air-water interface have enabled identification of the nature of the lipid-peptide interactions and characterization of the influence of phospholipids on the ability of these peptides to penetrate into lipidic media. Penetration and compression experiments reveal that both peptides interact strongly with phospholipids, and observations on Langmuir-Blodgett transfers indicate that they can modify the lipid organization. Conformational investigations indicate that the lipid-peptide interaction govern the conformational state of the peptides. Based on the ability of both peptides to promote ion permeabilization of both natural and artificial membranes, we propose a model illustrating the translocation process. For MPG it is based on the formation of a beta-barrel pore-like structure while for Pep-1 it is based on association of helices.

Amino Acid Sequence↗
PubMed2004

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.

Amino Acid Sequence↗
PubMed2004

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.

Adsorption↗
PubMed2004

Insight into the mechanism of internalization of the cell-penetrating carrier peptide Pep-1 through conformational analysis.

Recently, we described a new strategy for the delivery of proteins and peptides into mammalian cells, based on an amphipathic peptide of 21 residues, Pep-1, which was designed on the basis of a protein-interacting domain associated with a nuclear localization sequence and separated by a linker. This peptide carrier constitutes a powerful tool for the delivery of active proteins or peptides both in cultured cells and in vivo, without requiring any covalent coupling. We have examined the conformational states of Pep-1 in its free form and complexed with a cargo peptide and have investigated their ability to interact with phospholipids and the structural consequences of these interactions. From the conformational point of view, Pep-1 behaves significantly differently from other similarly designed cell-penetrating peptides. CD analysis revealed a transition from a nonstructured to a helical conformation upon increase of the concentration. Determination of the structure by NMR showed that in water, its alpha-helical domain extends from residues 4-13. CD and FTIR indicate that Pep-1 adopts a helical conformation in the presence of phospholipids. Adsorption measurements performed at the air-water interface are consistent with the helical form. Pep-1 does not undergo conformational changes upon formation of a particle with a cargo peptide. In contrast, we observe a partial conformational transition when the complex encounters phospholipids. We propose that the membrane crossing process involves formation of a transient transmembrane pore-like structure. Conformational change of Pep-1 is not associated with complexation with its cargo but is induced upon association with the cell membrane.

Air↗
PubMed2003

Insight into the mechanism of the peptide-based gene delivery system MPG: implications for delivery of siRNA into mammalian cells.

The improvement of non-viral-based gene delivery systems is of prime importance for the future of gene and antisense therapies. We have previously described a peptide-based gene delivery system, MPG, derived from the fusion peptide domain of HIV-1 gp41 protein and the nuclear localisation sequence (NLS) of SV40 large T antigen. MPG forms stable non-covalent complexes with nucleic acids and improves their delivery. In the present work, we have investigated the mechanism through which MPG promotes gene delivery. We demonstrate that cell entry is independent of the endosomal pathway and that the NLS of MPG is involved in both electrostatic interactions with DNA and nuclear targeting. MPG/DNA particles interact with the nuclear import machinery, however, a mutation which affects the NLS of MPG disrupts these interactions and prevents nuclear delivery of DNA. Nevertheless, we show that this mutation yields a variant of MPG which is a powerful tool for delivery of siRNA into mammalian cells, enabling rapid release of the siRNA into the cytoplasm and promoting robust down-regulation of target mRNA. Taken together, these results support the potential of MPG-like peptides for therapeutic applications and suggest that specific variations in the sequence may yield carriers with distinct targeting features.

Active Transport, Cell Nucleus↗