Probing the differences between rat liver outer mitochondrial membrane cytochrome b5 and microsomal cytochromes b5

Two distinct forms of cytochrome b5 exist in the rat hepatocyte. One is associated with the membrane of the endoplasmic reticulum (microsomal, or Mc, cyt b5) while the other is associated with the outer membrane of liver mitochondria (OM cyt b5). Rat OM cyt b5, the only OM cyt b5 identified so far, has a significantly more negative reduction potential and is substantially more stable toward chemical and thermal denaturation than Mc cytochromes b5. In addition, hemin is kinetically trapped in rat OM cyt b5 but not in the Mc proteins. As a result, no transfer of hemin from rat OM cyt b5 to apomyoglobin is observed at pH values as low as 5.2, nor can the thermodyamically favored ratio of hemin orientational isomers be achieved under physiologically relevant conditions. These differences are striking given the similarity of the respective protein folds. A combined theoretical and experimental study has been conducted in order to probe the structural basis behind the remarkably different properties of rat OM and Mc cytochromes b5. Molecular dynamics (MD) simulations starting from the crystal structure of bovine Mc cyt b5 revealed a conformational change that exposes several internal residues to the aqueous environment. The new conformation is equivalent to the "cleft-opened" intermediate observed in a previously reported MD simulation of bovine Mc cyt b5 [Storch, E. M., and Daggett, V. (1995) Biochemistry 34, 9682-9693]. The rat OM protein does not adopt a comparable conformation in MD simulations, thus restricting access of water to the protein interior. Subsequent comparisons of the protein sequences and structures suggested that an extended hydrophobic network encompassing the side chains of Ala-18, Ile-32, Leu-36, and Leu-47 might contribute to the inability of rat OM cyt b5 to adopt the cleft-opened conformation and, hence, stabilize its fold relative to the Mc isoforms. A corresponding network is not present in bovine Mc cyt b5 because positions 18, 32, and 47, are occupied by Ser, Leu, and Arg, respectively. To probe the roles played by Ala-18, Ile-32, and Leu-47 in endowing rat OM cyt b5 with its unusual structural properties, we have replaced them with the corresponding residues in bovine Mc cyt b5. Hence, the I32L (single), A18S/L47R (double), and A18S/L47R/I32L (triple) mutants of rat OM cyt b5 were prepared. The stability of these proteins was found to decrease in the following order: WT rat OM > rat OM I32L > rat OM A18S/L47R > rat OM A18S/L47R/I32L > bovine Mc cyt b5. The decrease in stability of the rat OM protein correlates with the extent to which the hydrophobic cluster involving the side chains of residues 18, 32, 36, and 47 has been disrupted. Complete disruption of the hydrophobic network in the triple mutant is confirmed in a 2.0 A resolution crystal structure of the protein. Disruption of the hydrophobic network also facilitates hemin loss at pH 5.2 for the double and triple mutants, with the less stable triple mutant exhibiting the greater rate of hemin transfer to apomyoglobin. Finally, 1H NMR spectroscopy and side-by-side comparisons of the crystal structures of bovine Mc, rat OM, and rat OM A18S/L47R/I32L cyt b5 allowed us to conclude that the nature of residue 32 plays a key role in controlling the relative stability of hemin orientational isomers A and B in rat OM cyt b5. A similar analysis led to the conclusion that Leu-70 and Ser-71 play a pivotal role in stabilizing isomer A relative to isomer B in Mc cytochromes b5.     

IgE receptor type I-dependent regulation of a Rab3D-associated kinase: a possible link in the calcium-dependent assembly of SNARE complexes

Following activation through high affinity IgE receptors (FcepsilonRI), mast cells release, within a few minutes, their granule content of inflammatory and allergic mediators. FcepsilonRI-induced degranulation is a SNARE (soluble N-ethylmaleimide attachment protein receptors)-dependent fusion process. It is regulated by Rab3D, a subfamily member of Rab GTPases. Evidence exists showing that Rab3 action is calcium-regulated although the molecular mechanisms remain unclear. To obtain an understanding of Rab3D function we have searched for Rab3D-associated effectors that respond to allergic triggering through FcepsilonRI. Using the RBL-2H3 mast cell line we detected a Ser/Thr kinase activity, termed here Rak3D (from Rab3D-associated kinase), because it was specifically co-immunoprecipitated with anti-Rab3D antibody. Rak3D activity, as measured by its auto- or transphosphorylation, was maximal in resting cells and decreased upon stimulation. The down-regulation of the observed activity was blocked with EGTA, but not with other degranulation inhibitors, suggesting that its activity functions downstream of calcium influx. We found that Rak3D phosphorylates the NH(2)-terminal regulatory domain of the t-SNARE syntaxin 4, but not syntaxin 2 or 3. The phosphorylation of syntaxin 4 decreased its binding to its partner SNAP23. Thus, we propose a novel phosphorylation-dependent mechanism by which Rab3D controls SNARE assembly in a calcium-dependent manner.     

Amplification and Phylogenetic Relationships of a Subfamily of blood, a Retrotransposable Element of Drosophila

To get a better understanding of the effect of interelement selection on the variation of long terminal repeat retrotransposon families, we have investigated the evolutionary history of blood in the Drosophila melanogaster species complex. We carried out a PCR approach to amplify the 5′ untranslated region from blood in the four species of the complex. This procedure revealed two main classes of size variants. Phylogenetic analyses of nucleotide sequences from these variants and blood elements from the Drosophila Genome Projects database show that elements are grouped according to their size, so that they probably correspond to two subfamilies. These two subfamilies arose prior to the split of the complex, and several facts indicate that the expansion of one of them is leading to the competitive exclusion of the other, at least from the euchromatic regions of the genome. Received: 17 August 2000 / Accepted: 20 November 2000     

Regulation of skeletal muscle tension redevelopment by troponin C constructs with different Ca2+ affinities

In maximally activated skinned fibers, the rate of tension redevelopment (ktr) following a rapid release and restretch is determined by the maximal rate of cross-bridge cycling. During submaximal Ca2+ activations, however, ktr regulation varies with thin filament dynamics. Thus, decreasing the rate of Ca2+ dissociation from TnC produces a higher ktr value at a given tension level (P), especially in the [Ca2+] range that yields less than 50% of maximal tension (Po). In this study, native rabbit TnC was replaced with chicken recombinant TnC, either wild-type (rTnC) or mutant (NHdel), with decreased Ca2+ affinity and an increased Ca2+ dissociation rate (koff). Despite marked differences in Ca2+ sensitivity (>0.5 DeltapCa50), fibers reconstituted with either of the recombinant proteins exhibited similar ktr versus tension profiles, with ktr low (1-2 s-1) and constant up to approximately 50% Po, then rising sharply to a maximum (16 +/- 0.8 s-1) in fully activated fibers. This behavior is predicted by a four-state model based on coupling between cross-bridge cycling and thin filament regulation, where Ca2+ directly affects only individual thin filament regulatory units. These data and model simulations confirm that the range of ktr values obtained with varying Ca2+ can be regulated by a rate-limiting thin filament process.     

Actin filament organization is required for proper cAMP-dependent activation of CFTR

Previous studieshave indicated a role of the actin cytoskeleton in the regulation ofthe cystic fibrosis transmembrane conductance regulator (CFTR) ionchannel. However, the exact molecular nature of this regulation isstill largely unknown. In this report human epithelial CFTR wasexpressed in human melanoma cells genetically devoid of the filaminhomologue actin-cross-linking protein ABP-280 [ABP()]. cAMP stimulation of ABP() cells orcells genetically rescued with ABP-280 cDNA [ABP(+)] waswithout effect on whole cell Cl currents. InABP() cells expressing CFTR, cAMP was also without effect onCl conductance. In contrast, cAMP induced a 10-foldincrease in the diphenylamine-2-carboxylate (DPC)-sensitive whole cellCl currents of ABP(+)/CFTR(+) cells. Further, incells expressing both CFTR and a truncated form of ABP-280 unable tocross-link actin filaments, cAMP was also without effect on CFTRactivation. Dialysis of ABP-280 or filamin through the patch pipette,however, resulted in a DPC-inhibitable increase in the whole cellcurrents of ABP()/CFTR(+) cells. At the single-channel level,protein kinase A plus ATP activated single Clchannels only in excised patches from ABP(+)/CFTR(+) cells.Furthermore, filamin alone also induced Cl channelactivity in excised patches of ABP()/CFTR(+) cells. The presentdata indicate that an organized actin cytoskeleton is required forcAMP-dependent activation of CFTR.

     

The MMP/TIMP system in the nervous system

The matrix metalloproteinases (MMP) belong to a growing family of secreted or membrane-bound (MT-MMP) enzymes that cleave protein components of the extracellular matrix and bioactive factors involved in intercellular signaling. MMP activity is counterbalanced by their four physiological inhibitors, the tissue inhibitors of MMP (TIMPs). Together, MMP and TIMP control cell-cell and cell-matrix interactions associated with physiological processes. However, the breakdown of the protease-inhibitor balance may lead to the loss of tissue homeostasis and the development of degenerative and tumorigenic processes in various tissues. The emerging idea is that the MMP/TIMP system also plays a major role in the pathology and physiology of the nervous system and that mastering MMP activity will set the basis for new and more efficient therapeutic strategies against nervous system disorders.     

Ascorbate distribution during hibernation is independent of ascorbate redox state

Distribution of ascorbate into tissues is an essential process in ascorbate antioxidant defense. Hibernating animals are studied as a model of tolerance to ischemia-reperfusion because of their tolerance to fluctuations in blood flow associated with prolonged torpor and periodic arousal episodes. Throughout hibernation, plasma ascorbate concentration ([Asc](p)) repetitively increases during torpor, then falls during periodic arousal bouts. We previously proposed that high [Asc](p) provides a ready source of antioxidant protection for distribution to the central nervous system and peripheral tissues during arousal. Here we tested whether deliberate oxidation of plasma ascorbate by intravenous administration of ascorbate oxidase (AO), prior to arousal, compromised tissue levels of ascorbate or the other water-soluble antioxidants, glutathione (GSH) and urate. Although AO decreased [Asc](p) to below the level of detection during torpor and after arousal, ascorbate oxidation did not decrease post-arousal tissue levels of reduced ascorbate, glutathione, or urate in any tissue examined, except liver. The data imply that ascorbate is taken up equally well into brain and other tissues as either ascorbate or its oxidized product dehydroascorbate, with subsequent intracellular reduction of dehydroascorbate. Lack of effect of ascorbate oxidation on tissue levels of GSH or urate indicates that dehydroascorbate uptake and reduction do not compromise tissue concentrations of these other water-soluble antioxidants. Thus, we show equal availability of reduced and oxidized plasma ascorbate during metabolically demanding thermogenesis and reperfusion associated with arousal from hibernation.     

Antioxidant effects of water- and lipid-soluble nitroxide radicals in liposomes

Liposomes are today useful tools in different fields of science and technology. A lack of stability due to lipid peroxidation is the main problem in the extension of the use of these formulations. Recent investigative works have reported the protective effects of stable nitroxide radicals against oxidative processes in different media and under different stress conditions. Our group has focused its attention on the natural aging of liposomes and the protection provided by the water- and lipid-soluble nitroxide radicals 2,2,6,6-tetramethylpiperdine-1-oxyl (TEMPO) and doxylstearic acids (5-DSA, 12-DSA, and 16-DSA), respectively. Unilamellar liposomes were incubated under air atmosphere at 37°C, both in the absence and in the presence of these radicals. Conjugated dienes, lipid hydroperoxides, TBARS, membrane fluidity, and nitroxide ESR signal intensity were followed as a function of time. Our results demonstrated that doxylstearic acids were more efficient than TEMPO in retarding lipid peroxidation at all the concentrations tested. The inhibition percentages, depending on the total nitroxide concentration, were not proportional to the lipid–water partition coefficient. Furthermore, time-course ESR signals showed a slower decrease for doxylstearic acids than for TEMPO. No significant differences were found among 5-DSA, 12-DSA, and 16-DSA. We concluded that the nitroxide radical efficiency as antioxidant directly depends on both nitroxide concentration and lipophilicity.     

Peroxynitrite-mediated alpha-tocopherol oxidation in low-density lipoprotein: a mechanistic approach

Previous reports proposed that peroxynitrite (ONOO-) oxidizes alpha-tocopherol (alpha-TOH) through a two-electron concerted mechanism. In contrast, ONOO- oxidizes phenols via free radicals arising from peroxo bond homolysis. To understand the kinetics and mechanism of alpha-TOH and gamma-tocopherol (gamma-TOH) oxidation in low-density lipoprotein (LDL) (direct vs. radical), we exposed LDL to ONOO- added as a bolus or an infusion. Nitric oxide (.NO), ascorbate and CO2 were used as key biologically relevant modulators of ONOO- reactivity. Although approximately 80% alpha-TOH and gamma-TOH depletion occurred within 5 min of incubation of 0.8 microM LDL with a 60 microM bolus of ONOO-, an equimolar infusion of ONOO- over 60 min caused total consumption of both antioxidants. gamma-Tocopherol was preserved relative to alpha-TOH, probably due to gamma-tocopheroxyl radical recycling by alpha-TOH. alpha-TOH oxidation in LDL was first order in ONOO- with approximately 12% of ONOO- maximally available. Physiological concentrations of.NO and ascorbate spared both alpha-TOH and gamma-TOH through independent and additive mechanisms. High concentrations of.NO and ascorbate abolished alpha-TOH and gamma-TOH oxidation. Nitric oxide protection was more efficient for alpha-TOH in LDL than for ascorbate in solution, evidencing the kinetically highly favored reaction of lipid peroxyl radicals with.NO than with alpha-TOH as assessed by computer-assisted simulations. In addition, CO2 (1.2 mM) inhibited both alpha-TOH and lipid oxidation. These results demonstrate that ONOO- induces alpha-TOH oxidation in LDL through a one-electron free radical mechanism; thus the inhibitory actions of.NO and ascorbate may determine low alpha-tocopheryl quinone accumulation in tissues despite increased ONOO- generation.     

Black flower coloration in wild Lisianthius nigrescens: its chemistry and ecological consequences

The major pigments responsible for the flower color within the black flowered Gentianaceae, Lisianthius nigrescens, were characterized by HPLC and chemical analyses HPLC analysis showed one major and one minor anthocyanin and 3 major and 3 minor flavone glycosides. The anthocyanins [delphinidin-3-O-rhamnol(1-6)galactoside and its 5-O-glucoside] comprised an extraordinary 24% of the dry weight of wild collected L. nigrescens corallas, and were accompanied in a 1:1 ratio by a range of apigenin and luteolin 8-C-glucosides and their 7-O-methyl ethers. The high levels of anthocyanins and flavones (and their co-pigmentation) is thought to account for the almost complete absorption of both UV and visible wavebands observed by reflectance photography.