Distinct modulation of the endothelin-1 pathway in iNOS-/- and eNOS-/- mice

We hypothesized that constitutive endothelial NO synthase (eNOS) and inducible NO synthase (iNOS) have opposite effects on the regulation of endothelin and its receptors. We therefore sought to determine whether deletions of iNOS or eNOS genes in mice modulate pressor responses to endothelin and the expression of ETA and ETB receptors in a similar fashion. Despite unchanged baseline hemodynamic parameters, anesthetized iNOS-/- mice displayed reduced pressor responses to endothelin-1, but not to that of IRL-1620, a selective ETB agonist. Protein content of cardiac ETA receptors was reduced in iNOS-/- mice compared with wild-type mice, but that of ETB receptors was unchanged. Anesthetized eNOS-/- mice presented a hypertensive state, accompanied by an enhanced pressor response to intravenous endothelin-1, whereas the pressor response to IRL-1620 was reduced. Protein levels were also found to be increased for ETA receptors, but reduced for ETB receptors, in cardiac tissues of eNOS-/- mice. In conscious animals, both strains responded equally to the hypotensive effect of an ETA antagonist, ABT-627, whereas orally administered A-192621, an ETB antagonist, increased MAP to a greater extent in eNOS-/- than in wild-type mice. Furthermore, significant levels of immunoreactive endothelin were found in mesenteric arteries in eNOS-/- but not in iNOS-/- or wild-type congeners. Our study shows that repression of iNOS or eNOS has differential effects on endothelin-1 and its receptors. We have also shown that the heart is the main organ in which iNOS or eNOS repression induces important alterations in protein content of endothelin receptors in adult mice.     

Mitochondrial adaptations to steatohepatitis induced by a methionine- and choline-deficient diet

Nonalcoholic fatty liver disease (NAFLD) has become common liver disease in Western countries. There is accumulating evidence that mitochondria play a key role in NAFLD. Nevertheless, the mitochondrial consequences of steatohepatitis are still unknown. The bioenergetic changes induced in a methionine- and choline-deficient diet (MCDD) model of steatohepatitis were studied in rats. Liver mitochondria from MCDD rats exhibited a higher rate of oxidative phosphorylation with various substrates, a rise in cytochrome oxidase (COX) activity, and an increased content in cytochrome aa3. This higher oxidative activity was associated with a low efficiency of the oxidative phosphorylation (ATP/O, i.e., number of ATP synthesized/natom O consumed). Addition of a low concentration of cyanide, a specific COX inhibitor, restored the efficiency of mitochondria from MCDD rats back to the control level. Furthermore, the relation between respiratory rate and protonmotive force (in the nonphosphorylating state) was shifted to the left in mitochondria from MCDD rats, with or without cyanide. These results indicated that, in MCDD rats, mitochondrial ATP synthesis efficiency was decreased in relation to both proton pump slipping at the COX level and increased proton leak although the relative contribution of each phenomenon could not be discriminated. MCDD mitochondria also showed a low reactive oxygen species production and a high lipid oxidation potential. We conclude that, in MCDD-fed rats, liver mitochondria exhibit an energy wastage that may contribute to limit steatosis and oxidative stress in this model of steatohepatitis.     

Overexpression of the atypical protein kinase C zeta reduces topoisomerase II catalytic activity,cleavable complexes formation,and drug-induced cytotoxicity in monocytic U937 leukemia cells

In this study, we evaluated the influence of protein kinase C zeta (PKC zeta) on topoisomerase II inhibitor-induced cytotoxicity in monocytic U937 cells. In U937-zeta J and U937-zeta B cells, enforced PKC zeta expression, conferred by stable transfection of PKC zeta cDNA, resulted in total inhibition of VP-16- and mitoxantrone-induced apoptosis and decreased drug-induced cytotoxicity, compared with U937-neo control cells. In PKC zeta-overexpressing cells, drug resistance correlated with decreased VP-16-induced DNA strand breaks and DNA protein cross-links measured by alkaline elution. Kinetoplast decatenation assay revealed that PKC zeta overexpression resulted in reduced global topoisomerase II activity. Moreover, in PKC zeta-overexpressing cells, we found that PKC zeta interacted with both alpha and beta isoforms of topoisomerase II, and these two enzymes were constitutively phosphorylated. However, when human recombinant PKC zeta (rH-PKC zeta) was incubated with purified topoisomerase II isoforms, rH-PKC zeta interacted with topoisomerase II beta but not with topoisomerase II alpha. PKC zeta/topoisomerase II beta interaction resulted in phosphorylation of this enzyme and in decrease of its catalytic activity. Finally, this report shows for the first time that topoisomerase II beta is a substrate for PKC zeta, and that PKC zeta may significantly influence topoisomerase II inhibitor-induced cytotoxicity by altering topoisomerase II beta activity through its kinase function.     

Role of two dileucine-like motifs in insulin receptor anchoring to microvilli

In the absence of ligand, the insulin receptor is maintained on microvilli on the cell surface. A dileucine motif (LL(986-987)) is necessary but not sufficient for this anchoring, which also required the presence of additional sequence(s) downstream of position 1000. The aim of the present study was to identify this (these) additional sequence(s). First, exons 16 or 17 were fused to the extracellular and transmembrane domains of complement receptor 1 and stably expressed in Chinese hamster ovary cells. Results obtained indicate that exon 17 is sufficient for anchoring to microvilli. Second, analysis of insulin receptor mutants truncated within exon 17 demonstrated that whereas receptors truncated at position 1000 showed no preferential association with microvilli, receptors truncated at position 1012 displayed a level of association identical to that of the full-length insulin receptor. Third, mutation of a diisoleucine motif (II(1006-1007)) present within this 12-amino acid stretch abrogated the preferential association of the receptor with microvilli. These results indicate that the domain required for association of insulin receptor with microvilli is contained within the region encoded by exon 17 and that, within this sequence, two dileucine-like motifs (LL(986-987) and II(1006-1007)) play a crucial role.     

Filamins but Not Janus Kinases Are Substrates of the ASB2α Cullin-Ring E3 Ubiquitin Ligase in Hematopoietic Cells

The ASB2α protein is the specificity subunit of an E3 ubiquitin ligase complex involved in hematopoietic differentiation and is proposed to exert its effects by regulating the turnover of specific proteins. Three ASB2α substrates have been described so far: the actin-binding protein filamins, the Mixed Lineage Leukemia protein, and the Janus kinases 2 and 3. To determine the degradation of which substrate drives ASB2α biological effects is crucial for the understanding of ASB2α functions in hematopoiesis. Here, we show that neither endogenous nor exogenously expressed ASB2α induces degradation of JAK proteins in hematopoietic cells. Furthermore, we performed molecular modeling to generate the first structural model of an E3 ubiquitin ligase complex of an ASB protein bound to one of its substrates.     

The CXCL12gamma chemokine displays unprecedented structural and functional properties that make it a paradigm of chemoattractant proteins

The CXCL12gamma chemokine arises by alternative splicing from Cxcl12, an essential gene during development. This protein binds CXCR4 and displays an exceptional degree of conservation (99%) in mammals. CXCL12gamma is formed by a protein core shared by all CXCL12 isoforms, extended by a highly cationic carboxy-terminal (C-ter) domain that encompass four overlapped BBXB heparan sulfate (HS)-binding motifs. We hypothesize that this unusual domain could critically determine the biological properties of CXCL12gamma through its interaction to, and regulation by extracellular glycosaminoglycans (GAG) and HS in particular. By both RT-PCR and immunohistochemistry, we mapped the localization of CXCL12gamma both in mouse and human tissues, where it showed discrete differential expression. As an unprecedented feature among chemokines, the secreted CXCL12gamma strongly interacted with cell membrane GAG, thus remaining mostly adsorbed on the plasmatic membrane upon secretion. Affinity chromatography and surface plasmon resonance allowed us to determine for CXCL12gamma one of the higher affinity for HS (K(d) = 0.9 nM) ever reported for a protein. This property relies in the presence of four canonical HS-binding sites located at the C-ter domain but requires the collaboration of a HS-binding site located in the core of the protein. Interestingly, and despite reduced agonist potency on CXCR4, the sustained binding of CXCL12gamma to HS enabled it to promote in vivo intraperitoneal leukocyte accumulation and angiogenesis in matrigel plugs with much higher efficiency than CXCL12alpha. In good agreement, mutant CXCL12gamma chemokines selectively devoid of HS-binding capacity failed to promote in vivo significant cell recruitment. We conclude that CXCL12gamma features unique structural and functional properties among chemokines which rely on the presence of a distinctive C-ter domain. The unsurpassed capacity to bind to HS on the extracellular matrix would make CXCL12gamma the paradigm of haptotactic proteins, which regulate essential homeostatic functions by promoting directional migration and selective tissue homing of cells.     

HIV-1 entry into T-cells is not dependent on CD4 and CCR5 localization to sphingolipid-enriched,detergent-resistant,raft membrane domains

The contribution of raft domains to human immunodeficiency virus (HIV) 1 entry was assessed. In particular, we asked whether the CD4 and CCR5 HIV-1 receptors need to associate with sphingolipid-enriched, detergent-resistant membrane domains (rafts) to allow viral entry into primary and T-cell lines. Based on Triton X-100 solubilization and confocal microscopy, CD4 was shown to distribute partially to rafts. In contrast, CCR5 did not associate with rafts and localized in nonraft plasma membrane domains. HIV-1-receptor partitioning remained unchanged upon viral adsorption, suggesting that viral entry probably takes place outside rafts. To directly investigate this possibility, we targeted CD4 to nonraft domains of the membrane by preventing CD4 palmitoylation and interaction with p56(lck). Directed mutagenesis of both targeting signals significantly prevented association of CD4 with rafts, but did not suppress the HIV-1 receptor function of CD4. Collectively, these results strongly suggest that the presence of HIV-1 receptors in rafts is not required for viral infection. We show, however, that depleting plasma membrane cholesterol inhibits HIV-1 entry. We therefore propose that cholesterol modulates the HIV-1 entry process independently of its ability to promote raft formation.     

C-terminal half of human centrin 2 behaves like a regulatory EF-hand domain

Human centrin 2 (HsCen2) is an EF-hand protein that plays a critical role in the centrosome duplication and separation during cell division. We studied the structural and Ca(2+)-binding properties of two C-terminal fragments of this protein: SC-HsCen2 (T94-Y172), covering two EF-hands, and LC-HsCen2 (M84-Y172), having 10 additional residues. Both fragments are highly disordered in the apo state but become better structured (although not conformationally homogeneous) in the presence of Ca(2+) and depending on the nature of the cations (K(+) or Na(+)) in the buffer. Only the longer C-terminal domain, in the Ca(2+)-saturated state and in the presence of Na(+) ions, was amenable to structure determination by nuclear magnetic resonance. The solution structure of LC-HsCen2 reveals an open two EF-hand structure, similar to the conformation of related Ca(2+)-saturated regulatory domains. Unexpectedly, the N-terminal helix segment (F86-T94) lies over the exposed hydrophobic cavity. This unusual intramolecular interaction increases considerably the Ca(2+) affinity and constitutes a useful model for the target binding.