Véronique Blais

Inducible Dimerization and Inducible Cleavage Reveal a Requirement for Both Processes in Caspase-8 Activation

Caspase-8 is a cysteine protease activated by membrane-bound receptors at the cytosolic face of the cell membrane, initiating the extrinsic pathway of apoptosis. Caspase-8 activation relies on recruitment of inactive monomeric zymogens to activated receptor complexes, where they produce a fully active enzyme composed of two catalytic domains. Although in vitro studies using drug-mediated affinity systems or kosmotropic salts to drive dimerization have indicated that uncleaved caspase-8 can be readily activated by dimerization alone, in vivo results using mouse models have reached the opposite conclusion. Furthermore, in addition to interdomain autoprocessing, caspase-8 can be cleaved by activated executioner caspases, and reports of whether this cleavage event can lead to activation of caspase-8 have been conflicting. Here, we address these questions by carrying out studies of the activation characteristics of caspase-8 mutants bearing prohibitive mutations at the interdomain cleavage sites both in vitro and in cell lines lacking endogenous caspase-8, and we find that elimination of these cleavage sites precludes caspase-8 activation by prodomain-driven dimerization. We then further explore the consequences of interdomain cleavage of caspase-8 by adapting the tobacco etch virus protease to create a system in which both the cleavage and the dimerization of caspase-8 can be independently controlled in living cells. We find that unlike the executioner caspases, which are readily activated by interdomain cleavage alone, neither dimerization nor cleavage of caspase-8 alone is sufficient to activate caspase-8 or induce apoptosis and that only the coordinated dimerization and cleavage of the zymogen produce efficient activation in vitro and apoptosis in cellular systems.

Caspase-7 uses an exosite to promote poly(ADP ribose) polymerase 1 proteolysis

During apoptosis, hundreds of proteins are cleaved by caspases, most of them by the executioner caspase-3. However, caspase-7, which shares the same substrate primary sequence preference as caspase-3, is better at cleaving poly(ADP ribose) polymerase 1 (PARP) and Hsp90 cochaperone p23, despite a lower intrinsic activity. Here, we identified key lysine residues (K38KKK) within the N-terminal domain of caspase-7 as critical elements for the efficient proteolysis of these two substrates. Caspase-7s N-terminal domain binds PARP and improves its cleavage by a chimeric caspase-3 by ∼30-fold. Cellular expression of caspase-7 lacking the critical lysine residues resulted in less-efficient PARP and p23 cleavage compared with cells expressing the wild-type peptidase. We further showed, using a series of caspase chimeras, the positioning of p23 on the enzyme providing us with a mechanistic insight into the binding of the exosite. In summary, we have uncovered a role for the N-terminal domain (NTD) and the N-terminal peptide of caspase-7 in promoting key substrate proteolysis.

Processing of proendothelin‐1 by members of the subtilisin‐like pro‐protein convertase family

Endothelial cells (ECs) secrete numerous bioactive peptides that are initially synthesized as inactive precursor proteins. One of these, proendothelin‐1 (proET‐1), undergoes proteolysis at specific pairs of basic amino acids. Here, we wished to examine the role of mammalian convertases in this event. Northern blot analysis shows that only furin and PC7 are expressed in ECs. In vitro cleavage of proET‐1 by furin or PC7 demonstrated that both enzymes efficiently and specifically process proET‐1. These data reveal that furin and PC7 have similar specificities towards proET‐1 and suggest that both enzymes may participate in the maturation of proET‐1 in ECs.

Type 1 inositol-1,4,5-trisphosphate receptor is a late substrate of caspases during apoptosis.

Apoptosis is characterized by the proteolytic cleavage of hundreds of proteins. One of them, the type 1 inositol‐1,4,5‐trisphosphate receptor (IP3R‐1), a multimeric receptor located on the endoplasmic reticulum (ER) membrane that is critical to calcium homeostasis, was reported to be cleaved during staurosporine (STS) induced‐apoptosis in Jurkat cells. Because the reported cleavage site separates the IP3 binding site from the channel moiety, its cleavage would shut down a critical signaling pathway that is common to several cellular processes. Here we show that IP3R‐1 is not cleaved in 293 cells treated with STS, TNFα, Trail, or ultra‐violet (UV) irradiation. Further, it is not cleaved in Hela or Jurkat cells induced to undergo apoptosis with Trail, TNFα, or UV. In accordance with previous reports, we demonstrate that it is cleaved in a Jurkat cell line treated with STS. However its cleavage occurs only after poly(ADP‐ribose) polymerase (PARP), which cleavage is a hallmark of apoptosis, and p23, a poor caspase‐7 substrate, are completely cleaved, suggesting that IP3R‐1 is a relatively late substrate of caspases. Nevertheless, the receptor is fully accessible to proteolysis in cellulo by ectopically overexpressed caspase‐7 or by the tobacco etch virus (TEV) protease. Finally, using recombinant caspase‐3 and microsomal fractions enriched in IP3R‐1, we show that the receptor is a poor caspase‐3 substrate. Consequently, we conclude that IP3R‐1 is not a key death substrate. J. Cell. Biochem. 113: 2775–2784, 2012.

Molecular determinants involved in activation of caspase 7

During apoptosis, initiator caspases (8, 9 and 10) activate downstream executioner caspases (3, 6 and 7) by cleaving the IDC (interdomain connector) at two sites. Here, we demonstrate that both activation sites, site 1 and site 2, of caspase 7 are suboptimal for activation by initiator caspases 8 and 9 in cellulo, and in vitro using recombinant proteins and activation kinetics. Indeed, when both sites are replaced with the preferred motifs recognized by either caspase 8 or 9, we found an up to 36-fold improvement in activation. Moreover, cleavage at site 1 is preferred to site 2 because of its location within the IDC, since swapping sites does not lead to a more efficient activation. We also demonstrate the important role of Ile195 of site 1 involved in maintaining a network of contacts that preserves the proper conformation of the active enzyme. Finally, we show that the length of the IDC plays a crucial role in maintaining the necessity of proteolysis for activation. In fact, although we were unable to generate a caspase 7 that does not require proteolysis for activity, shortening the IDC of the initiator caspase 8 by four residues was sufficient to confer a requirement for proteolysis, a key feature of executioner caspases. Altogether, the results demonstrate the critical role of the primary structure of caspase 7s IDC for its activation and proteolytic activity.

Synthesis of Gly-ψ[(Z)CF═CH]-Phe, a Fluoroalkene Dipeptide Isostere, and Its Incorporation into a Leu-enkephalin Peptidomimetic.

A new Leu-enkephalin peptidomimetic designed to explore the hydrogen bond acceptor ability of the third peptide bond has been prepared and studied. This new analog is produced by replacing the third amide of Leu-enkephalin with a fluoroalkene. An efficient and innovative synthesis of the corresponding dipeptide surrogate Fmoc-Gly-ψ[(Z)CF═CH]-Phe-OH is described. The key step involves the alkylation of a tin dienolate from the less hindered face of its chiral sulfonamide auxiliary derived from camphor. Once its synthesis was complete, its incorporation into the peptidomimetic sequence was achieved on a solid support with chlorotrityl resin following the Fmoc strategy. The peptidomimetic was characterized using competition binding with [125I]-deltorphin I on membrane extracts of HEK293 cells expressing the mouse delta opioid receptor (DOPr) and based on its abilities to inhibit the electrically induced contractions of the mouse vas deferens and to activate the ERK1/2 signaling pathway in DRGF11/DOPr-GFP cells. Together with our previous observations, our findings strongly suggest that the third amide bond of Leu-enkephalin primarily acts as a hydrogen bond acceptor in DOPr. Consequently, this amide bond can be successfully replaced by an ester, a thioamide, or a fluoroalkene without greatly impacting the binding or biological activity of the corresponding analogs. The lipophilicity (LogD7.4) of the active analog was also measured. It appears that fluoroalkenes are almost as efficient at increasing the lipophilicity as normal alkenes.

Involvement of the coatomer protein complex I in the intracellular traffic of the delta opioid receptor

&NA; The delta opioid receptor (DOPr) is known to be mainly expressed in intracellular compartments. It remains unknown why DOPr is barely exported to the cell surface, but it seems that a substantial proportion of the immature receptor is trapped within the endoplasmic reticulum (ER) and the Golgi network. In the present study, we performed LC‐MS/MS analysis to identify putative protein partners involved in the retention of DOPr. Analysis of the proteins co‐immunoprecipitating with Flag‐DOPr in transfected HEK293 cells revealed the presence of numerous subunits of the coatomer protein complex I (COPI), a vesicle‐coating complex involved in recycling resident proteins from the Golgi back to the ER. Further analysis of the amino acid sequence of DOPr identified multiple consensus di‐lysine and di‐arginine motifs within the intracellular segments of DOPr. Using cell‐surface ELISA and GST pulldown assays, we showed that DOPr interacts with COPI through its intracellular loops 2 and 3 (ICL2 and ICL3, respectively) and that the mutation of the K164AK166 (ICL2) or K250EK252 (ICL3) putative COPI binding sites increased the cell‐surface expression of DOPr in transfected cells. Altogether, our results indicate that COPI is a binding partner of DOPr and provide a putative mechanism to explain why DOPr is highly retained inside the cells. HighlightsDOPr colocalizes with &bgr;‐COP in the Golgi.COPI interacts with DOPr.Retention of DOPr in the Golgi and the ER is regulated by COPI.Di‐lysine binding motifs within the second and third intracellular loops of DOPr are involved in the interaction with COPI.

Adeno-associated virus 2/9 delivery of Cre recombinase in mouse primary afferents

Genetically-modified animal models have significantly increased our understanding of the complex central nervous system circuits. Among these models, inducible transgenic mice whose specific gene expression can be modulated through a Cre recombinase/LoxP system are useful to study the role of specific peptides and proteins in a given population of cells. In the present study, we describe an efficient approach to selectively deliver a Cre-GFP to dorsal root ganglia (DRG) neurons. First, mice of different ages were injected in both hindpaws with a recombinant adeno-associated virus (rAAV2/9-CBA-Cre-GFP). Using this route of injection in mice at 5 days of age, we report that approximately 20% of all DRG neurons express GFP, 6 to 8 weeks after the infection. The level of infection was reduced by 50% when the virus was administered at 2 weeks of age. Additionally, the virus-mediated delivery of the Cre-GFP was also investigated via the intrathecal route. When injected intrathecally, the rAAV2/9-CBA-Cre-GFP virus infected a much higher proportion of DRG neurons than the intraplantar injection, with up to 51.6% of infected lumbar DRG neurons. Noteworthy, both routes of injection predominantly transduced DRG neurons over spinal and brain neurons.

Exploration of the fifth position of leu-enkephalin and its role in binding and activating delta (DOP) and mu (MOP) opioid receptors

Enkephalins are pentapeptidic endogenous ligands that regulate nociception by binding to mu (MOP) and delta (DOP) opioid receptors. To further explore the role of the leucine residue of Leu‐enkephalin, 12 peptidomimetic analogs were synthesized by systematically replacing this residue with non‐natural amino acids. The analogs were tested for their ability to bind DOP and MOP. We also investigated the potency of these analogs to inhibit cAMP production and to recruit β‐arrestin 2 via both receptors. We found that replacement of the leucine residue by substituted non‐natural amino acid derivatives of alanine, cycloleucine, or isoleucine was generally well tolerated. By contrast, substituting leucine with homoproline greatly reduced the affinity for DOP and, to a lesser extent, for MOP. Interestingly, when compared to Leu‐enkephalin, analogs containing either aza‐β‐homoleucine or cycloleucine showed a bias toward inhibition of cAMP production through the activation of DOP but not MOP. By contrast, derivatives containing 4,5‐dehydroleucine or d‐allo‐isoleucine conferred a bias toward β‐arrestin 2 at MOP, but not DOP. Our results suggest that position 5 in Leu‐enkephalin analogs can be further exploited to develop compounds with the potential to produce bias toward G protein or β‐arrestin 2.

Synthesis and Evaluation of a 64Cu-Conjugate, a Selective δ-Opioid Receptor Positron Emission Tomography Imaging Agent

Given the putative selectivity of the antagonist TIPP (Tyr-Tic-Phe-Phe) for δ-opioid receptors (DOP), this compound was selected for the design of a novel 64Cu-radiolabeled potent and selective DOP positron emission tomography (PET) imaging agent. Ex vivo autoradiography of TIPPD-PEG-K(NOTA/64Cu)-NH2 on rat brain sections produced a distribution pattern consistent with the known expression of DOP. Taken together, the in vitro and ex vivo data indicate that this 64Cu-tracer holds promise for studying the DOP by means of PET.