Natalia A. Riobo

Nitric oxide inhibits mitochondrial NADH : ubiquinone reductase activity through peroxynitrite formation

This study was aimed at assessing the effects of long-term exposure to NO of respiratory activities in mitochondria from different tissues (with different ubiquinol contents), under conditions that either promote or prevent the formation of peroxynitrite. Mitochondria and submitochondrial particles isolated from rat heart, liver and brain were exposed either to a steady-state concentration or to a bolus addition of NO. NO induced the mitochondrial production of superoxide anions, hydrogen peroxide and peroxynitrite, the latter shown by nitration of mitochondrial proteins. Long-term incubation of mitochondrial membranes with NO resulted in a persistent inhibition of NADH:cytochrome c reductase activity, interpreted as inhibition of NADH:ubiquinone reductase (Complex I) activity, whereas succinate:cytochrome c reductase activity, including Complex II and Complex III electron transfer, remained unaffected. This selective effect of NO and derived species was partially prevented by superoxide dismutase and uric acid. In addition, peroxynitrite mimicked the effect of NO, including tyrosine nitration of some Complex I proteins. These results seem to indicate that the inhibition of NADH:ubiquinone reductase (Complex I) activity depends on the NO-induced generation of superoxide radical and peroxynitrite and that Complex I is selectively sensitive to peroxynitrite. Inhibition of Complex I activity by peroxynitrite may have critical implications for energy supply in tissues such as the brain, whose mitochondrial function depends largely on the channelling of reducing equivalents through Complex I.

The Hedgehog Signal Transduction Network

The Hedgehog network includes canonical and noncanonical signaling modules important for development, homeostasis, and disease. Members of the conserved Hedgehog (Hh) family of secreted proteins play fundamental roles during embryonic development and homeostasis of adult tissues. Hh signal transduction uses an unusual derepression mechanism, in which Hh binding to its receptor component Patched (Ptc) inhibits the capacity of Ptc to repress the heterotrimeric guanine nucleotide–binding protein–coupled receptor Smoothened (Smo). Activation of Smo is accomplished through phosphorylation and subcellular translocation from intracellular vesicles to the plasma membrane and, in vertebrates, also to the primary cilium. This Review focuses primarily on the signaling modules that are set in motion by binding of Hh to Ptc: the “canonical” pathway through which Smo promotes activation of the Gli family of transcription factors, the noncanonical type I pathway in which Ptc modulates cell proliferation and survival independently of Smo, and the noncanonical type II pathway by which Smo regulates the actin cytoskeleton through G inhibitory proteins and small guanosine triphosphatases. Hedgehog (Hh) proteins regulate the development of a wide range of metazoan embryonic and adult structures, and disruption of Hh signaling pathways results in various human diseases. Here, we provide a comprehensive review of the signaling pathways regulated by Hh, consolidating data from a diverse array of organisms in a variety of scientific disciplines. Similar to the elucidation of many other signaling pathways, our knowledge of Hh signaling developed in a sequential manner centered on its earliest discoveries. Thus, our knowledge of Hh signaling has for the most part focused on elucidating the mechanism by which Hh regulates the Gli family of transcription factors, the so-called “canonical” Hh signaling pathway. However, in the past few years, numerous studies have shown that Hh proteins can also signal through Gli-independent mechanisms collectively referred to as “noncanonical” signaling pathways. Noncanonical Hh signaling is itself subdivided into two distinct signaling modules: (i) those not requiring Smoothened (Smo) and (ii) those downstream of Smo that do not require Gli transcription factors. Thus, Hh signaling is now proposed to occur through a variety of distinct context-dependent signaling modules that have the ability to crosstalk with one another to form an interacting, dynamic Hh signaling network.

The Regulation of Mitochondrial Oxygen Uptake by Redox Reactions Involving Nitric Oxide and Ubiquinol

The reversible inhibitory effects of nitric oxide (·NO) on mitochondrial cytochrome oxidase and O2uptake are dependent on intramitochondrial ·NO utilization. This study was aimed at establishing the mitochondrial pathways for ·NO utilization that regulate O⨪2 generation via reductive and oxidative reactions involving ubiquinol oxidation and peroxynitrite (ONOO–) formation. For this purpose, experimental models consisting of intact mitochondria, ubiquinone-depleted/reconstituted submitochondrial particles, and ONOO–-supplemented mitochondrial membranes were used. The results obtained from these experimental approaches strongly suggest the occurrence of independent pathways for ·NO utilization in mitochondria, which effectively compete with the binding of ·NO to cytochrome oxidase, thereby releasing this inhibition and restoring O2 uptake. The pathways for ·NO utilization are discussed in terms of the steady-state levels of ·NO and O⨪2 and estimated as a function of O2 tension. These calculations indicate that mitochondrial ·NO decays primarily by pathways involving ONOO– formation and ubiquinol oxidation and, secondarily, by reversible binding to cytochrome oxidase.

Pathways of signal transduction employed by vertebrate Hedgehogs

Signalling by Hh (Hedgehog) proteins is among the most actively studied receptor-mediated phenomena relevant to development and post-embryonic homoeostatic events. The impact of signalling by the Hh proteins is profound, and work pertaining to the presentation of these proteins and the pathways engaged by them continues to yield unique insights into basic aspects of morphogenic signalling. We review here the mechanisms of signalling relevant to the actions of Hh proteins in vertebrates. We emphasize findings within the past several years on the recognition of, in particular, Sonic hedgehog by target cells, pathways of transduction employed by the seven-pass transmembrane protein Smoothened and end points of action, as manifest in the regulation of the Gli transcription factors. Topics of extended interest are those regarding the employment of heterotrimeric G-proteins and G-protein-coupled receptor kinases by Smoothened. We also address the pathways, insofar as known, linking Smoothened to the expression and stability of Gli1, Gli2 and Gli3. The mechanisms by which Hh proteins signal have few, if any, parallels. It is becoming clear in vertebrates, however, that several facets of signalling are shared in common with other venues of signalling. The challenge in understanding both the actions of Hh proteins and the overlapping forms of regulation will be in understanding, in molecular terms, both common and divergent signalling events.

Activation of heterotrimeric G proteins by Smoothened

The mechanisms by which the activation of Smoothened (Smo), a protein essential to the actions of the Hedgehog family of secreted proteins, is translated into signals that converge on the Gli transcription factors are not fully understood. The seven-transmembrane structure of Smo has long implied the utilization of heterotrimeric GTP-binding regulatory proteins (G proteins); however, evidence in this regard has been indirect and contradictory. In the current study we evaluated the capacity of mammalian Smo to couple to G proteins directly. We found that Smo, by virtue of what appears to be constitutive activity, activates all members of the Gi family but does not activate members of the Gs, Gq, and G12 families. The activation is suppressed by cyclopamine and other inhibitors of Hedgehog signaling and is enhanced by the Smo agonist purmorphamine. Activation of Gi by Smo is essential in the activation of Gli in fibroblasts, because disruption of coupling to Gi with pertussis toxin inhibits the activation of Gli by Sonic hedgehog and a constitutively active form of Smo (SmoM2). However, Gi does not provide a sufficient signal because a truncated form of Smo, although capable of activating Gi, does not effect activation of Gli. Rescue of pertussis toxin-inhibited activation of Gli by Sonic hedgehog can be achieved with a constitutively active Gαi-subunit. The data suggest that Smo is in fact the source of two signals relevant to the activation of Gli: one involving Gi and the other involving events at Smo’s C-tail independent of Gi.

The reaction of nitric oxide with ubiquinol: kinetic properties and biological significance

The reaction of nitric oxide (*NO) with ubiquinol-0 and ubiquinol-2, short-chain analogs of coenzyme Q, was examined in anaerobic and aerobic conditions in terms of formation of intermediates and stable molecular products. The chemical reactivity of ubiquinol-0 and ubiquinol-2 towards *NO differed only quantitatively, the reactions of ubiquinol-2 being slightly faster than those of ubiquinol-0. The ubiquinol/*NO reaction entailed oxidation of ubiquinol to ubiquinone and reduction of *NO to NO-, the latter identified by its reaction with metmyoglobin to form nitroxylmyoglobin and indirectly by measurement of nitrous oxide (N2O) by gas chromatography. Both the rate of ubiquinone accumulation and *NO consumption were linearly dependent on ubiquinol and *NO concentrations. The stoichiometry of *NO consumed per either ubiquinone formed or ubiquinol oxidized was 1.86 A 0.34. The reaction of *NO with ubiquinols proceeded with intermediate formation of ubisemiquinones that were detected by direct EPR. The second order rate constants of the reactions of ubiquinol-0 and ubiquinol-2 with *NO were 0.49 and 1.6 x 10(4) M(-1)s(-1), respectively. Studies in aerobic conditions revealed that the reaction of *NO with ubiquinols was associated with O2 consumption. The formation of oxyradicals - identified by spin trapping EPR- during ubiquinol autoxidation was inhibited by *NO, thus indicating that the O2 consumption triggered by *NO could not be directly accounted for in terms of oxyradical formation or H2O2 accumulation. It is suggested that oxyradical formation is inhibited by the rapid removal of superoxide anion by *NO to yield peroxynitrite, which subsequently may be involved in the propagation of ubiquinol oxidation. The biological significance of the reaction of ubiquinols with *NO is discussed in terms of the cellular O2 gradients, the steady-state levels of ubiquinols and *NO, and the distribution of ubiquinone (largely in its reduced form) in biological membranes with emphasis on the inner mitochondrial membrane.

Hedgehog proteins activate pro-angiogenic responses in endothelial cells through non-canonical signaling pathways.

The Hedgehog (Hh) pathway orchestrates developmental and homeostatic angiogenesis. The three Hh isoforms- Sonic Hedgehog (Shh), Indian Hedgehog (Ihh) and Desert Hedgehog (Dhh)-signal through Patched-1 (PTCH1) and Smoothened (SMO), to activate the Gli transcription factors with a characteristic rank of potency (Shh>>Ihh>Dhh). To dissect the mechanisms through which Hh proteins promote angiogenesis, we analyzed processes inherent to vessel formation in endothelial cells. We found that none of the Hh ligands were able to induce Gli-target genes in human umbilical vein (HUVEC) or human cardiac microvascular endothelial cells (HMVEC), suggesting that endothelial cells do not respond to Hh through the canonical pathway. However, our results show that the three Hh proteins promote endothelial cell tubulogenesis in 3D cultures in a SMO- and Gi protein-dependent manner. Consistent with the required cytoskeletal rearrangement for tubulogenesis, Shh, Ihh, and Dhh all stimulated the small GTPase RhoA and the formation of actin stress fibers. This effect, which was mediated by SMO, Gi proteins, and Rac1, defines a new non-canonical Hh pathway. In addition to regulating the actin cytoskeleton, the Hh ligands promoted survival through inhibition of the pro-apoptotic effect of PTCH1 in a SMO-independent manner. Altogether, our results support the existence of Gli-independent Hh responses in endothelial cells that regulate tubulogenesis and apoptosis. The identification of novel non-canonical responses elicited by Hh proteins in endothelial cells highlights the complexity of the Hh signaling pathway and reveals striking differences in ligand strength for transcriptional and non-transcriptional responses.

Heterotrimeric Gi Proteins Link Hedgehog Signaling to Activation of Rho Small GTPases to Promote Fibroblast Migration

Evidence supporting the functionality of Smoothened (SMO), an essential transducer in most pathways engaged by Hedgehog (Hh), as a Gi-coupled receptor contrasts with the lack of an apparently consistent requirement for Gi in Hh signal transduction. In the present study, we sought to evaluate the role of SMO-Gi coupling in fibroblast migration induced by Sonic Hedgehog (Shh). Our results demonstrate an absolute requirement for Gi in Shh-induced fibroblast migration. We found that Shh acutely stimulates the small Rho GTPases Rac1 and RhoA via SMO through a Gi protein- and PI3K-dependent mechanism, and that these are required for cell migration. These responses were independent of transcription by Gli and of the C-terminal domain of SMO, as we show using a combination of molecular and genetic tools. Our findings provide a mechanistic model for fibroblast migration in response to Shh and underscore the role of SMO-Gi coupling in non-canonical Hh signaling.

Noncanonical Hedgehog signaling.

The notion of noncanonical hedgehog (Hh) signaling in mammals has started to receive support from numerous observations. By noncanonical, we refer to all those cellular and tissue responses to any of the Hh isoforms that are independent of transcriptional changes mediated by the Gli family of transcription factors. In this chapter, we discuss the most recent findings that suggest that Patched1 can regulate cell proliferation and apoptosis independently of Smoothened (Smo) and Gli and the reports that Smo modulates actin cytoskeleton-dependent processes such as fibroblast migration, endothelial cell tubulogenesis, axonal extension, and neurite formation by diverse mechanisms that exclude any involvement of Gli-dependent transcription. We also acknowledge the existence of less stronger evidence of noncanonical signaling in Drosophila.

Oxidation of ubiquinol by peroxynitrite: implications for protection of mitochondria against nitrosative damage

A major pathway of nitric oxide utilization in mitochondria is its conversion to peroxynitrite, a species involved in biomolecule damage via oxidation, hydroxylation and nitration reactions. In the present study the potential role of mitochondrial ubiquinol in protecting against peroxynitrite-mediated damage is examined and the requirements of the mitochondrial redox status that support this function of ubiquinol are established. (1) Absorption and EPR spectroscopy studies revealed that the reactions involved in the ubiquinol/peroxynitrite interaction were first-order in peroxynitrite and zero-order in ubiquinol, in agreement with the rate-limiting formation of a reactive intermediate formed during the isomerization of peroxynitrite to nitrate. Ubiquinol oxidation occurred in one-electron transfer steps as indicated by the formation of ubisemiquinone. (2) Peroxynitrite promoted, in a concentration-dependent manner, the formation of superoxide anion by mitochondrial membranes. (3) Ubiquinol protected against peroxynitrite-mediated nitration of tyrosine residues in albumin and mitochondrial membranes, as suggested by experimental models, entailing either addition of ubiquinol or expansion of the mitochondrial ubiquinol pool caused by selective inhibitors of complexes III and IV. (4) Increase in membrane-bound ubiquinol partially prevented the loss of mitochondrial respiratory function induced by peroxynitrite. These findings are analysed in terms of the redox transitions of ubiquinone linked to both nitrogen-centred radical scavenging and oxygen-centred radical production. It may be concluded that the reaction of mitochondrial ubiquinol with peroxynitrite is part of a complex regulatory mechanism with implications for mitochondrial function and integrity.