Specific targeting of caspase-9/PP2A interaction as potential new anti-cancer therapy.

PURPOSE: PP2A is a serine/threonine phosphatase critical to physiological processes, including apoptosis. Cell penetrating peptides are molecules that can translocate into cells without causing membrane damage. Our goal was to develop cell-penetrating fusion peptides specifically designed to disrupt the caspase-9/PP2A interaction and evaluate their therapeutic potential in vitro and in vivo. EXPERIMENTAL DESIGN: We generated a peptide containing a penetrating sequence associated to the interaction motif between human caspase-9 and PP2A (DPT-C9h), in order to target their association. Using tumour cell lines, primary human cells and primary human breast cancer (BC) xenografts, we investigated the capacity of DPT-C9h to provoke apoptosis in vitro and inhibition of tumour growth (TGI) in vivo. DPT-C9h was intraperitoneally administered at doses from 1 to 25 mg/kg/day for 5 weeks. Relative Tumour Volume (RTV) was calculated. RESULTS: We demonstrated that DPT-C9h specifically target caspase-9/PP2A interaction in vitro and in vivo and induced caspase-9-dependent apoptosis in cancer cell lines. DPT-C9h also induced significant TGI in BC xenografts models. The mouse-specific peptide DPT-C9 also induced TGI in lung (K-Ras model) and breast cancer (PyMT) models. DPT-C9h has a specific effect on transformed B cells isolated from chronic lymphocytic leukemia patients without any effect on primary healthy cells. Finally, neither toxicity nor immunogenic responses were observed. CONCLUSION: Using the cell-penetrating peptides blocking caspase-9/PP2A interactions, we have demonstrated that DPT-C9h had a strong therapeutic effect in vitro and in vivo in mouse models of tumour progression.

HIV rebound after discontinuation of antiretroviral therapy increases and expands HIV-specific CD8+ responses but has no impact on its functionality.

A higher functionality of CD8(+) T cells might contribute to low-level HIV replication in long-term nonprogressors (LTNPs). However, the contrary could also be true, being the function of CD8(+) T cells modulated by HIV replication. We tested whether enhanced HIV replication following antiretroviral therapy interruption could modify the functional profile of HIV-specific CD8(+) responses. Production of MIP-1beta, IL-2, TNF-alpha, and CD107 expression by CD8(+) T cells in response to Gag and Nef optimal peptide pools was analyzed using polychromatic flow cytometry in nine HIV-infected individuals followed for 12 months after discontinuation of antiretroviral therapy. At baseline, CD8(+) T cell subsets with the greatest contribution to response were MIP-beta(+)TNF-alpha(-)IL-2(-)CD107(+) and MIP-beta(+)TNF-alpha(-)IL-2(-)CD107. Most responses were mediated by subsets expressing only one or two molecules. After 12 months of discontinuing antiretroviral therapy, no significant differences were observed in the functional profile of Gag- and Nef-specific CD8(+) responses. However, viral rebound induced a significant increase in the heterogeneity of Gag-specific CD8(+) responses. In summary, viral replication following discontinuation of antiretroviral therapy has no significant impact on qualitative aspects of HIV-specific CD8(+) responses. Thus, a higher functionality of CD8(+) responses does not seem to be the consequence of low-level virus replication.

Membrane-perturbing properties of three peptides corresponding to the ectodomain of hepatitis C virus E2 envelope protein.

Based on the predicted capacity to interact with membranes at the interface, we have found three regions in the ectodomain of the hepatitis C virus envelope glycoprotein E2 (430-449, 543-560 and 603-624) with the ability to destabilize membranes. Three peptides corresponding to the sequence of these regions have been synthesized and their interaction with liposomes have been characterized. The three peptides were able to insert deeply into the hydrophobic core of negatively charged phospholipids as stated by fluorescence depolarization of the probe 1,6-diphenyl-1,3,5-hexatriene. Peptides E2(430-449) and E2(603-624) were able to induce aggregation of phosphatidylglycerol vesicles in a concentration-dependent manner both at neutral and acidic pH while peptide E2(543-560) did not induce any increase of optical density at 360 nm in the concentration range studied. The three peptides induced lipid mixing and the release of the internal contents in a dose-dependent manner when acidic phospholipids were used. Fourier transformed infrared spectroscopy indicated that the peptides adopted mainly a beta-sheet conformation which is not modified by the presence of acidic phospholipids. Taken together, our results point out to the involvement of these three regions in the fusion mechanism of HCV at the plasma membrane level.

Influence of acylation of a Peptide corresponding to the amino-terminal region of endothelial nitric oxide synthase on the interaction with model membranes.

Covalent attachment of fatty acids to proteins is a common form of protein modification which has been shown to influence both structure and interaction with membranes. Endothelial nitric oxide synthase (eNOS) is dually acylated by the fatty acids myristate and palmitate. We have synthesized four peptides corresponding to the first 28 amino acids of the N-terminal region of eNOS. Besides the nonacylated eNOS sequence, three additional peptides with different degrees of acylation have been obtained: myristoylated, doubly palmitoylated, and dually myristoylated and doubly palmitoylated. Acylation itself, myristic and/or palmitic, confers the peptide the ability to adopt extended conformations, indicated by the fact that the CD spectrum of all acylated peptides has a minimum at approximately 215 nm characteristic of beta-sheet structure. The nonacylated sequence interacts with model membranes composed of acidic phospholipids probably through ionic interactions with the polar headgroup of the phospholipids. However, the acylated peptides are able to insert deeply into the hydrophobic core of both neutral and acidic phospholipids, maintaining the spectral features of extended conformations. When DMPC vesicles containing cholesterol and sphingomyelin at 10% were used, the insertion of the triacylated peptide almost completely canceled the thermal transition, although the interaction of the other acylated peptides also reduced the transition amplitude but to a much lower extent and affected only the acyl chains in the fluid state.

New insights in protein phosphorylation: a signature for protein phosphatase 1 interacting proteins.

Protein phosphatase 1 is regulated by the interaction between a catalytic subunit (PP1c) and multiple interacting proteins that allow the specific dephosphorylation of diverse cellular targets. This communication proposes to use the simultaneous presence of distinct consensus PP1c docking motifs R/K-x(0,1)-V-x-F and F-x-x-R/K-x-R/K as a signature to identify proteins putatively interacting with the PP1c. To develop this concept, we propose a new website, http://pp1 signature.pasteur.fr, which allows the identification of putative PP1-interacting proteins containing the two distinct PP1c docking consensus motifs represented in the Swissprot library. To validate the new concept of signature, we were able to characterise, by co-immunoprecipitation, four new PP1c interacting proteins randomly selected from the database in our website.

Proteomic identification of brain proteins that interact with dynein light chain LC8.

Cytoplasmic dynein is a large minus end-directed microtubule motor that translocates cargos towards the minus end of microtubules. Light chain 8 of the dynein machinery (LC8) has been reported to interact with a large variety of proteins that possess K/RSTQT or GIQVD motifs in their sequence, hence permitting their transport in a retrograde manner. Yeast two-hybrid analysis has revealed that in brain, LC8 associates directly with several proteins such as neuronal nitric oxide synthase, guanylate kinase domain-associated protein and gephyrin. In this work, we report the identification of over 40 polypeptides, by means of a proteomic approach, that interact with LC8 either directly or indirectly. Many of the neuronal proteins that we identified cluster at the post-synaptic terminal, and some of them such as phosphofructokinase, lactate dehydrogenase or aldolase are directly involved in glutamate metabolism. Other pool of proteins identified displayed the LC8 consensus binding motif. Finally, recombinant LC8 was produced and a library of overlapping dodecapeptides (pepscan) was employed to map the LC8 binding site of some of the proteins that were previously identified using the proteomic approach, hence confirming binding to the consensus binding sites.

Rafts: a simple way to control apoptosis by subcellular redistribution.

Apoptosis is an essential feature of development and homeostasis in higher organisms. Lipid rafts are subdomains of the plasma membrane that contain high concentrations of cholesterol and sphingolipids. In response to intra or extracellular stimuli, lipid rafts can include or exclude proteins to variable extents. This favors specific protein-protein interactions and modulates the activity of signaling cascades. Recently, a number of proteins involved in apoptotic signals have been located in lipid rafts. Among these proteins is included Bad, a pro-apoptotic molecule belonging to the Bcl-2 family. Bad is attached to lipid rafts in proliferating cells while associated to mitochondria in apoptotic cells, suggesting that the interaction of Bad with rafts is a dynamic process involved in the control of apoptosis. In this review, we briefly summarize the structure of rafts and illustrate their contribution to the control of apoptosis.

Recognition of novel viral sequences that associate with the dynein light chain LC8 identified through a pepscan technique.

Recent data from multiple laboratories indicate that upon infection, many different families of viruses hijack the dynein motor machinery and become transported in a retrograde manner towards the cell nucleus. In certain cases, one of the dynein light chains, LC8, is involved in this interaction. Using a library of overlapping dodecapeptides synthesized on a cellulose membrane (pepscan technique) we have analyzed the interaction of the dynein light chain LC8 with 17 polypeptides of viral origin. We demonstrate the strong binding of two herpesvirus polypeptides, the human adenovirus protease, vaccinia virus polymerase, human papillomavirus E4 protein, yam mosaic virus polyprotein, human respiratory syncytial virus attachment glycoprotein, human coxsackievirus capsid protein and the product of the AMV179 gene of an insect poxvirus to LC8. Our data corroborate the manipulation of the dynein macromolecular complex of the cell during viral infection and point towards the light chain LC8 as one of the most frequently used targets of virus manipulation.