Solution structure of NOD1 CARD and mutational analysis of its interaction with the CARD of downstream kinase RICK

NOD1 is a cytosolic signalling host pattern-recognition receptor composed of a caspase-activating and recruitment domain (CARD), a nucleotide-binding and oligomerization domain (NOD) and leucine-rich repeats. It plays a crucial role in innate immunity by activating the NF-kappaB pathway via its downstream effector the kinase RICK (RIP2) following the recognition of a specific bacterial ligand. RICK is recruited by NOD1 through interaction of their respective CARDs. Here we present the high resolution NMR structure of the NOD1 CARD. It is generally similar to other CARDs of known structure, consisting of six tightly packed helices, although the length and orientation of the last helix is unusual. Mutations in both the NOD1 and RICK CARD domains were assayed by immuno-precipitation of cell lysates and in vivo NF-kappaB activation in order to define residues important for CARD-CARD interaction and downstream signalling. The results show that the interaction is critically dependent on three acidic residues on NOD1 CARD and three basic residues on RICK CARD and thus is likely to have a strong electrostatic component, similar to other characterised CARD-CARD interactions.     

Interactions between substrates and the haem-bound nitric oxide of ferric and ferrous bacterial nitric oxide synthases

We report here the resonance Raman spectra of the FeIII-NO and FeII-NO complexes of the bacterial NOSs (nitric oxide synthases) from Staphylococcus aureus and Bacillus subtilis. The haem-NO complexes of these bacterial NOSs displayed Fe-N-O frequencies similar to those of the mammalian NOSs, in presence and absence of L-arginine, indicating that haem-bound NO and L-arginine had similar haem environments in bacterial and mammalian NOSs. The only notable difference between the two types of NOS was the lack of change in Fe-N-O frequencies of the FeIII-NO complexes upon (6R) 5,6,7,8-tetrahydro-L-biopterin binding to bacterial NOSs. We report, for the first time, the characterization of NO complexes with NOHA (N(omega)-hydroxy-L-arginine), the substrate used in the second half of the catalytic cycle of NOSs. In the FeIII-NO complexes, both L-arginine and NOHA induced the Fe-N-O bending mode at nearly the same frequency as a result of a steric interaction between the substrates and the haem-bound NO. However, in the FeII-NO complexes, the Fe-N-O bending mode was not observed and the nu(Fe-NO) mode displayed a 5 cm(-1) higher frequency in the complex with NOHA than in the complex with L-arginine as a result of direct interactions that probably involve hydrogen bonds. The different behaviour of the substrates in the FeII-NO complexes thus reveal that the interactions between haem-bound NO and the substrates are finely tuned by the geometry of the Fe-ligand structure and are relevant to the use of the FeII-NO complex as a model of the oxygenated complex of NOSs.     

New 1,2,3,4-tetrahydropyrrolo[3,4-b]indole derivatives as selective CB2 receptor agonists

The preparation and evaluation of a novel class of CB2 agonists based on a 1,2,3,4-tetrahydropyrrolo[3,4-b]indole moiety are reported. They showed binding affinities up to 4.2 nM toward CB2 with sub-nanomolar EC(50) values. They also showed moderate to good (>350-fold) selectivity over the CB1 receptor.     

Bax activation and mitochondrial insertion during apoptosis

The mitochondrial apoptotic pathway is a highly regulated biological mechanism which determines cell fate. It is defined as a cascade of events, going from an apoptotic stimulus to the MOM permeabilization, resulting in the activation of the so-called executive phase. This pathway is very often altered in cancer cells.The mitochondrial permeabilization is under the control of the Bcl-2 family of proteins (pBcls). These proteins share one to four homology domains (designed BH1-4) with Bcl-2, and are susceptible of homo- and/or hetero-dimerization. In spite of a poor amino-acid sequence homology, these proteins exhibit very similar tertiary structures. Strikingly, while some of these proteins are anti-apoptotic, the others are pro-apoptotic. Pro-apoptotic proteins are further divided in two sub-classes: multi-domains proteins, among which Bax and Bak, which exhibit BH1-3 domains, and BH3-only proteins (or BOPs). Schematically, BOPs and anti-apoptotic proteins antagonistically regulate the activation of the multi-domain proteins Bax and Bak and their oligomerization in the MOM, the latter process being responsible for the apoptotic mitochondrial permeabilization.Considering the critical role of Bax in cancer cells apoptosis, we focus in this review on the molecular events of Bax activation through its interaction with the other proteins from the Bcl-2 family. The mechanism by which Bax triggers the MOM permeabilization once activated will be discussed in some other reviews in this special issue.     

Role of cardiolipin on tBid and tBid/Bax synergistic effects on yeast mitochondria

The apoptotic effector Bid regulates cell death at the level of mitochondria. Under its native state, Bid is a soluble cytosolic protein that undergoes proteolysis and yields a 15 kDa-activated form tBid (truncated Bid). tBid translocates to mitochondria and participates in cytochrome c efflux by a still unclear mechanism, some of them at least mediated by Bax. Using mitochondria isolated from wild-type and cardiolipin (CL)-synthase-less yeast strains, we observed that tBid perturbs mitochondrial bioenergetics by inhibiting state-3 respiration and ATP synthesis and that this effect was strictly dependent on the presence of CL. In a second set of experiments, heterologous coexpression of tBid and Bax in wild-type and CL-less yeast strains showed that (i) tBid binding and the subsequent alteration of mitochondrial bioenergetics increased Bax-induced cytochrome c release and (ii) the absence of CL favors Bax effects independently of the presence of t-Bid. These data support recent views suggesting a dual function of CL in mitochondria-dependent apoptosis.     

tBid interaction with cardiolipin primarily orchestrates mitochondrial dysfunctions and subsequently activates Bax and Bak

TNFR1/Fas engagement results in the cleavage of cytosolic Bid to truncated Bid (tBid), which translocates to mitochondria. We demonstrate that recombinant tBid induces in vitro immediate destabilization of the mitochondrial bioenergetic homeostasis. These alterations result in mild uncoupling of mitochondrial state-4 respiration, associated with an inhibition the adenosine diphosphate (ADP)-stimulated respiration and phosphorylation rate. tBid disruption of mitochondrial homeostasis was inhibited in mitochondria overexpressing Bcl-2 and Bcl-XL. The inhibition of state-3 respiration is mediated by the reorganization of cardiolipin within the mitochondrial membranes, which indirectly affects the activity of the ADP/ATP translocator. Cardiolipin-deficient yeast mitochondria did not exhibit any respiratory inhibition by tBid, proving the absolute requirement for cardiolipin for tBid binding and activity. In contrast, the wild-type yeast mitochondria underwent a similar inhibition of ADP-stimulated respiration associated with reduced ATP synthesis. These events suggest that mitochondrial lipids rather than proteins are the key determinants of tBid-induced destabilization of mitochondrial bioenergetics.     

Plant sterols and stanols: effects on mixed micellar composition and LXR (target gene) activation

Plant stanols and sterols of the 4-desmethyl family (e.g., sitostanol and sitosterol) effectively decrease LDL cholesterol concentrations, whereas 4,4-dimethylsterols (alpha-amyrin and lupeol) do not. Serum carotenoid concentrations, however, are decreased by both plant sterol families. The exact mechanisms underlying these effects are not known, although effects on micellar composition have been suggested. With a liver X receptor (LXR) coactivator peptide recruitment assay, we showed that plant sterols and stanols from the 4-desmethylsterol family activated both LXRalpha and LXRbeta, whereas 4,4-dimethyl plant sterols did not. In fully differentiated Caco-2 cells, the functionality of this effect was shown by the increased expression of ABCA1, one of the known LXR target genes expressed by Caco-2 cells in measurable amounts. The LXR-activating potential of the various plant sterols/stanols correlated positively with ABCA1 mRNA expression. Reductions in serum hydrocarbon carotenoids could be explained by the effects of the 4-desmethyl family and 4,4-dimethylsterols on micellar carotenoid incorporation. Our findings indicate that the decreased intestinal absorption of cholesterol and carotenoids by plant sterols and stanols is caused by two distinct mechanisms.     

Lipid oxidation and autophagy in yeast

Autophagy, a process involved in the degradation and the recycling of long-lived proteins and organelles to survive nitrogen starvation, is generally non-selective. However, recent data suggest that selective forms of autophagy exist, that are able to specifically target several organelles, including mitochondria. Conversely, mitochondrial alterations could trigger autophagy. Such a selective form of autophagy might be involved in the elimination of damaged mitochondria. We reported previously that, mitochondria were early targets of rapamycin-induced autophagy. Here we report that rapamycin-induced autophagy is accompanied by the early production of reactive oxygen species and by the early oxidation of mitochondrial lipid. Inhibition of these oxidative effects by resveratrol largely impaired autophagy of both cytosolic proteins and mitochondria, and delayed subsequent cell death. These results support a role of mitochondrial oxidation events in the activation of autophagy.