Nitric oxide suppresses the expression of Bcl-2 binding protein BNIP3 in hepatocytes

Nitric oxide (NO) is not only an important signaling molecule, but it also regulates the expression of a number of genes in the liver. We have previously shown that apoptosis in hepatocytes exposed to tumor necrosis factor-alpha and actinomycin D is prevented by NO derived from the inducible nitric-oxide synthase (iNOS), by mechanisms that are both dependent on and independent of modulation of cyclic guanosine monophosphate (cGMP) subsequent to activation of soluble guanylyl cyclase (sGC). We hypothesize that one mechanism by which NO exerts these effects is by regulating the expression of genes involved in apoptosis. We used differential display-polymerase chain reaction to isolate NO-regulated genes in hepatocytes from iNOS knockout mice (to eliminate endogenous inducible NO production). Using this analysis, we identified a NO-suppressed gene fragment homologous with the pro-apoptotic Bcl-2 binding protein BNIP3. Northern analysis confirmed the NO-dependent suppression of BNIP3 in cultured cells. Similarly, the NO donor S-nitroso-N-acetyl-dl-penicillamine (1-1000 microm) down-regulated the expression of BNIP3 in both iNOS knockout and wild-type hepatocytes. This effect of NO was reversed by the sGC inhibitor 1H-(1,2,4)-oxadiazole[4,3-a]quinoxalon-1-one (ODQ),suggesting the involvement of the sGC/cGMP pathway in the modulation of BNIP3 by NO. We propose that suppression of BNIP3 expression is one sGC/cGMP-dependent mechanism by which NO might affect the process of hepatocyte apoptosis.     

Vibrio vulnificus cytolysin induces superoxide anion-initiated apoptotic signaling pathway in human ECV304 cells

Previous studies showed that exposure to Vibrio vulnificus cytolysin (VVC) caused characteristic morphologic changes and dysfunction of vascular structures in lung. VVC showed cytotoxicity for mammalian cells in culture and acted as a vascular permeability factor. In this study, the underlying mechanisms of VVC-induced cytotoxicity was investigated on ECV304 cell, a human vascular endothelial cell line. When cells were exposed to 0.4 hemolytic units (HU) of VVC, consecutive apoptotic events were observed; the elevation of superoxide anion (O (-.)(2)), the release of cytochrome c, the activation of caspase-3, the cleavage of poly(ADP-ribose) polymerase, and the DNA fragmentation. The pretreatment with 4-hydroxy-2,2,6,6-tetramethylpiperidine-N-oxyl (TEMPO), O(-.) 2) scavenger, completely abolished O(-.)(2) levels and downstream apoptotic events. Moreover, pretreatment with cyclosporin A (CsA), a mitochondrial permeability transition inhibitor, was capable of attenuating O(-.)(2)-mediated cytochrome c release and caspase-3 activation, and consequent apoptosis. Apoptosis, as demonstrated by oligonucleosomal DNA fragmentation and fluorescence microscopy, was induced 24 h after VVC treatment, which was also prevented by caspase-3 inhibitor, Ac-DEVD-CHO. Caspase-1 inhibitor, Ac-YVAD-CHO, did not protect ECV 304 cells from apoptosis. These results suggest a scenario where VVC-induced apoptosis is triggered by the generation of O(-.)(2), release of cytochrome c from mitochondria, activation of caspase-3, degradation of poly(ADP-ribose) polymerase, and DNA fragmentation. The induction of apoptosis in endothelial cells by VVC may provide a pivotal mechanism for understanding the pathophysiology of septicemia.     

J-domain protein, Jac1p, of yeast mitochondria required for iron homeostasis and activity of Fe-S cluster proteins

J-proteins are molecular chaperones with a characteristic domain predicted to mediate interaction with Hsp70 proteins. We have previously isolated yeast mutants of the mitochondrial Hsp70, Ssq1p, in a genetic screen for mutants with altered iron homeostasis. Here we describe the isolation of mutants of the J-domain protein, Jac1p, using the same screen. Mutant jac1 alleles predicted to encode severely truncated proteins (lacking 70 or 152 amino acids) were associated with phenotypes strikingly similar to the phenotypes of ssq1 mutants. These phenotypes include activation of the high affinity cellular iron uptake system and iron accumulation in mitochondria. In contrast to iron accumulation, Fe-S proteins of mitochondria were specifically deficient. In jac1 mutants, like in ssq1 mutants, processing of the Yfh1p precursor protein from intermediate to mature forms was delayed. In the genetic backgrounds used in this study, jac1 null mutants were found to be viable, permitting analysis of genetic interactions. The Deltajac1 Deltassq1 double mutant was more severely compromised for growth than either single mutant, suggesting a synthetic or additive effect of these mutations. Overexpression of Jac1p partially suppressed ssq1 slow growth and vice versa. Similar mitochondrial localization and similar mutant phenotypes suggest that Ssq1p and Jac1p are functional partners in iron homeostasis.     

NADPH-cytochrome P450 oxidoreductase. Structural basis for hydride and electron transfer

NADPH-cytochrome P450 oxidoreductase catalyzes transfer of electrons from NADPH, via two flavin cofactors, to various cytochrome P450s. The crystal structure of the rat reductase complexed with NADP(+) has revealed that nicotinamide access to FAD is blocked by an aromatic residue (Trp-677), which stacks against the re-face of the isoalloxazine ring of the flavin. To investigate the nature of interactions between the nicotinamide, FAD, and Trp-677 during the catalytic cycle, three mutant proteins were studied by crystallography. The first mutant, W677X, has the last two C-terminal residues, Trp-677 and Ser-678, removed; the second mutant, W677G, retains the C-terminal serine residue. The third mutant has the following three catalytic residues substituted: S457A, C630A, and D675N. In the W677X and W677G structures, the nicotinamide moiety of NADP(+) lies against the FAD isoalloxazine ring with a tilt of approximately 30 degrees between the planes of the two rings. These results, together with the S457A/C630A/D675N structure, allow us to propose a mechanism for hydride transfer regulated by changes in hydrogen bonding and pi-pi interactions between the isoalloxazine ring and either the nicotinamide ring or Trp-677 indole ring. Superimposition of the mutant and wild-type structures shows significant mobility between the two flavin domains of the enzyme. This, together with the high degree of disorder observed in the FMN domain of all three mutant structures, suggests that conformational changes occur during catalysis.     

Lipoprotein secretion and triglyceride stores in the heart

The genes for apolipoprotein B and microsomal triglyceride transfer protein are expressed in mouse and human heart tissue. Why the heart would express these "lipoprotein assembly" genes has been unclear. Here we demonstrate that the beating mouse heart actually secretes spherical lipoproteins. Moreover, increased cardiac production of lipoproteins (e.g., in mice that express a human apolipoprotein B transgene) was associated with increased triglyceride secretion from the heart and decreased stores of triglycerides within the heart. Increased cardiac production of lipoproteins also reduced the pathological accumulation of triglycerides that occurs in the hearts of mice lacking long-chain acyl coenzyme A dehydrogenase. In contrast, blocking heart lipoprotein secretion (e.g., in heart-specific microsomal triglyceride transfer protein knockout mice) increased cardiac triglyceride stores. Thus, heart lipoprotein secretion helps regulate cardiac triglyceride stores and may protect the heart from the detrimental effects of surplus lipids.     

In vitro transactivation of Bacillus subtilis RNase P RNA

We have partially characterised an alpha4-fucosyltransferase (alpha4-FucT) from Vaccinium myrtillus, which catalysed the biosynthesis of the Lewis(a) adhesion determinant. The enzyme was stable up to 50 degrees C. The optimum pH was 7.0, both in the presence and in the absence of Mn(2+). The enzyme was inhibited by Mn(2+) and Co(2+), and showed resistance towards inhibition with N-ethylmaleimide. It transferred fucose to N-acetylglucosamine in the type I Galbeta3GlcNAc motif from oligosaccharides linked to a hydrophobic tail and glycoproteins (containing the type I motif). Sialylated oligosaccharides containing the type II Galbeta4GlcNAc motif were not acceptors. The catalytic mechanism of the plant alpha4-FucT possibly involves a His residue, and it must have arisen by convergent evolution relative to its mammalian counterparts.     

Interlineage distribution and characteristics of the structure of two subfamilies of Drosophila melanogaster MDG4 (gypsy) retrotransposon

The distribution of two variants of MDG4 (gypsy) was analyzed in several Drosophila melanogaster strains. Southern blot hybridization revealed the inactive variant of MDG4 in all strains examined and active MDG4 only in some of them. Most of the strains harboring the active MDG4 variant were recently isolated from natural populations. It is of interest that the active MDG4 prevailed over the inactive one only in strains carrying the mutant flamenco gene. Several lines were analyzed in more detail. The number of MDG4 sites on salivary-gland polytene chromosomes was established via in situ hybridization, and MDG4 was tested for transposition using the ovoD test.     

Signaling system in Porphyromonas gingivalis based on a LuxS protein

The luxS gene of quorum-sensing Vibrio harveyi is required for type 2 autoinducer production. We identified a Porphyromonas gingivalis open reading frame encoding a predicted peptide of 161 aa that shares 29% identity with the amino acid sequence of the LuxS protein of V. harveyi. Conditioned medium from a late-log-phase P. gingivalis culture induced the luciferase operon of V. harveyi, but that from a luxS insertional mutant did not. In P. gingivalis, the expression of luxS mRNA was environmentally controlled and varied according to the cell density and the osmolarity of the culture medium. In addition, differential display PCR showed that the inactivation of P. gingivalis luxS resulted in up-regulation of a hemin acquisition protein and an arginine-specific protease and reduced expression of a hemin-regulated protein, a TonB homologue, and an excinuclease. The data suggest that the luxS gene in P. gingivalis may function to control the expression of genes involved in the acquisition of hemin.     

Lipase and its modulator from Pseudomonas sp. strain KFCC 10818: proline-to-glutamine substitution at position 112 induces formation of enzymatically active lipase in the absence of the modulator

A lipase gene, lipK, and a lipase modulator gene, limK, of Pseudomonas sp. strain KFCC 10818 have been cloned, sequenced, and expressed in Escherichia coli. The limK gene is located immediately downstream of the lipK gene. Enzymatically active lipase was produced only in the presence of the limK gene. The effect of the lipase modulator LimK on the expression of active lipase was similar to those of the Pseudomonas subfamily I.1 and I.2 lipase-specific foldases (Lifs). The deduced amino acid sequence of LimK shares low homology (17 to 19%) with the known Pseudomonas Lifs, suggesting that Pseudomonas sp. strain KFCC 10818 is only distantly related to the subfamily I.1 and I.2 Pseudomonas species. Surprisingly, a lipase variant that does not require LimK for its correct folding was isolated in the study to investigate the functional interaction between LipK and LimK. When expressed in the absence of LimK, the P112Q variant of LipK formed an active enzyme and displayed 63% of the activity of wild-type LipK expressed in the presence of LimK. These results suggest that the Pro(112) residue of LipK is involved in a key step of lipase folding. We expect that the novel finding of this study may contribute to future research on efficient expression or refolding of industrially important lipases and on the mechanism of lipase folding.     

Genetic and structural organization of the aminophenol catabolic operon and its implication for evolutionary process

The aminophenol (AP) catabolic operon in Pseudomonas putida HS12 mineralizing nitrobenzene was found to contain all the enzymes responsible for the conversion of AP to pyruvate and acetyl coenzyme A via extradiol meta cleavage of 2-aminophenol. The sequence and functional analyses of the corresponding genes of the operon revealed that the AP catabolic operon consists of one regulatory gene, nbzR, and the following nine structural genes, nbzJCaCbDGFEIH, which encode catabolic enzymes. The NbzR protein, which is divergently transcribed with respect to the structural genes, possesses a leucine zipper motif and a MarR homologous domain. It was also found that NbzR functions as a repressor for the AP catabolic operon through binding to the promoter region of the gene cluster in its dimeric form. A comparative study of the AP catabolic operon with other meta cleavage operons led us to suggest that the regulatory unit (nbzR) was derived from the MarR family and that the structural unit (nbzJCaCbDGFEIH) has evolved from the ancestral meta cleavage gene cluster. It is also proposed that these two functional units assembled through a modular type gene transfer and then have evolved divergently to acquire specialized substrate specificities (NbzCaCb and NbzD) and catalytic function (NbzE), resulting in the creation of the AP catabolic operon. The evolutionary process of the AP operon suggests how bacteria have efficiently acquired genetic diversity and expanded their metabolic capabilities by modular type gene transfer.