A novel gamma-N-methylaminobutyrate demethylating oxidase involved in catabolism of the tobacco alkaloid nicotine by Arthrobacter nicotinovorans pAO1

Nicotine catabolism, linked in Arthrobacter nicotinovorans to the presence of the megaplasmid pAO1, leads to the formation of gamma-N-methylaminobutyrate from the pyrrolidine ring of the alkaloid. Until now the metabolic fate of gamma-N-methylaminobutyrate has been unknown. pAO1 carries a cluster of ORFs with similarity to sarcosine and dimethylglycine dehydrogenases and oxidases, to the bifunctional enzyme methylenetetrahydrofolate dehydrogenase/cyclohydrolase and to formyltetrahydrofolate deformylase. We cloned and expressed the gene carrying the sarcosine dehydrogenase-like ORF and showed, by enzyme activity, spectrophotometric methods and identification of the reaction product as gamma-aminobutyrate, that the predicted 89 395 Da flavoprotein is a demethylating gamma-N-methylaminobutyrate oxidase. Site-directed mutagenesis identified His67 as the site of covalent attachment of FAD and confirmed Trp66 as essential for FAD binding, for enzyme activity and for the spectral properties of the wild-type enzyme. A K-m of 140 mum and a k(cat) of 800 s(-1) was determined when gamma-N-methylaminobutyrate was used as the substrate. Sarcosine was also turned over by the enzyme, but at a rate 200-fold slower than gamma-N-methylaminobutyrate. This novel enzyme activity revealed that the first step in channelling the gamma-N-methylaminobutyrate generated from nicotine into the cell metabolism proceeds by its oxidative demethylation

ERK1/2 signalling protects against apoptosis following endoplasmic reticulum stress but cannot provide long-term protection against BAX/BAK-independent cell death

Disruption of protein folding in the endoplasmic reticulum (ER) causes ER stress. Activation of the unfolded protein response (UPR) acts to restore protein homeostasis or, if ER stress is severe or persistent, drive apoptosis, which is thought to proceed through the cell intrinsic, mitochondrial pathway. Indeed, cells that lack the key executioner proteins BAX and BAK are protected from ER stress-induced apoptosis. Here we show that chronic ER stress causes the progressive inhibition of the extracellular signal-regulated kinase (ERK1/2) signalling pathway. This is causally related to ER stress since reactivation of ERK1/2 can protect cells from ER stress-induced apoptosis whilst ERK1/2 pathway inhibition sensitises cells to ER stress. Furthermore, cancer cell lines harbouring constitutively active BRAFV600E are addicted to ERK1/2 signalling for protection against ER stress-induced cell death. ERK1/2 signalling normally represses the pro-death proteins BIM, BMF and PUMA and it has been proposed that ER stress induces BIM-dependent cell death. We found no evidence that ER stress increased the expression of these proteins; furthermore, BIM was not required for ER stress-induced death. Rather, ER stress caused the PERK-dependent inhibition of cap-dependent mRNA translation and the progressive loss of pro-survival proteins including BCL2, BCLXL and MCL1. Despite these observations, neither ERK1/2 activation nor loss of BAX/BAK could confer long-term clonogenic survival to cells exposed to ER stress. Thus, ER stress induces cell death by at least two biochemically and genetically distinct pathways: a classical BAX/BAK-dependent apoptotic response that can be inhibited by ERK1/2 signalling and an alternative ERK1/2- and BAX/BAK-independent cell death pathway

Free Cysteine Modulates the Conformation of Human C/EBP Homologous Protein

The C/EBP Homologous Protein (CHOP) is a nuclear protein that is integral to the unfolded protein response culminating from endoplasmic reticulum stress. Previously, CHOP was shown to comprise extensive disordered regions and to self-associate in solution. In the current study, the intrinsically disordered nature of this protein was characterized further by comprehensive in silico analyses. Using circular dichroism, differential scanning calorimetry and nuclear magnetic resonance, we investigated the global conformation and secondary structure of CHOP and demonstrated, for the first time, that conformational changes in this protein can be induced by the free amino acid l-cysteine. Addition of l-cysteine caused a significant dose-dependent decrease in the protein helicity – dropping from 69.1% to 23.8% in the presence of 1 mM of l-cysteine – and a sequential transition to a more disordered state, unlike that caused by thermal denaturation. Furthermore, the presence of small amounts of free amino acid (80 µM, an 8∶1 cysteine∶CHOP ratio) during CHOP thermal denaturation altered the molecular mechanism of its melting process, leading to a complex, multi-step transition. On the other hand, high levels (4 mM) of free l-cysteine seemed to cause a complete loss of rigid cooperatively melting structure. These results suggested a potential regulatory function of l-cysteine which may lead to changes in global conformation of CHOP in response to the cellular redox state and/or endoplasmic reticulum stress

KRIT1 Regulates the Homeostasis of Intracellular Reactive Oxygen Species

KRIT1 is a gene responsible for Cerebral Cavernous Malformations (CCM), a major cerebrovascular disease characterized by abnormally enlarged and leaky capillaries that predispose to seizures, focal neurological deficits, and fatal intracerebral hemorrhage. Comprehensive analysis of the KRIT1 gene in CCM patients has suggested that KRIT1 functions need to be severely impaired for pathogenesis. However, the molecular and cellular functions of KRIT1 as well as CCM pathogenesis mechanisms are still research challenges. We found that KRIT1 plays an important role in molecular mechanisms involved in the maintenance of the intracellular Reactive Oxygen Species (ROS) homeostasis to prevent oxidative cellular damage. In particular, we demonstrate that KRIT1 loss/down-regulation is associated with a significant increase in intracellular ROS levels. Conversely, ROS levels in KRIT1−/− cells are significantly and dose-dependently reduced after restoration of KRIT1 expression. Moreover, we show that the modulation of intracellular ROS levels by KRIT1 loss/restoration is strictly correlated with the modulation of the expression of the antioxidant protein SOD2 as well as of the transcriptional factor FoxO1, a master regulator of cell responses to oxidative stress and a modulator of SOD2 levels. Furthermore, we show that the KRIT1-dependent maintenance of low ROS levels facilitates the downregulation of cyclin D1 expression required for cell transition from proliferative growth to quiescence. Finally, we demonstrate that the enhanced ROS levels in KRIT1−/− cells are associated with an increased cell susceptibility to oxidative DNA damage and a marked induction of the DNA damage sensor and repair gene Gadd45α, as well as with a decline of mitochondrial energy metabolism. Taken together, our results point to a new model where KRIT1 limits the accumulation of intracellular oxidants and prevents oxidative stress-mediated cellular dysfunction and DNA damage by enhancing the cell capacity to scavenge intracellular ROS through an antioxidant pathway involving FoxO1 and SOD2, thus providing novel and useful insights into the understanding of KRIT1 molecular and cellular functions

FOXO3A Regulates Peroxiredoxin III Expression in Human Cardiac Fibroblasts*

Human cardiac fibroblasts are protected from oxidative stress triggered by inflammation after myocardial injury (Li, P. F., Dietz, R., and von Harsdorf, R. (1999) FEBS Lett. 448, 206–210) by expressing potent antioxidant defenses such as superoxide dismutases, catalases, glutathione-peroxidases, and peroxiredoxins. Recently the transcription factor FOXO3A has been shown to increase resistance to oxidative stress by up-regulation of mitochondrial superoxide dismutase and peroxisomal catalase (Kops, G. J., Dansen, T. B., Polderman, P. E., Saarloos, I., Wirtz, K. W., Coffer, P. J., Huang, T. T., Bos, J. L., Medema, R. H., and Burgering, B. M. (2002) Nature 419, 316–321; Nemoto, S., and Finkel, T. (2002) Science 295, 2450–2452). We hypothesized that FOXO3A also regulates the expression of Prx III, the mitochondrial peroxiredoxin, in human cardiac fibroblasts. We found that depletion of FOXO3A leads to a dramatic reduction of Prx III mRNA and protein in serum-deprived human cardiac fibroblasts. These data suggest that endogenous FOXO3A is necessary for base-line expression of Prx III. Next, we identified two putative FOXO3A DNA binding sites in Prx III promoter at –267 and –244 nucleotides relative to the start codon. We demonstrated that both sequences are required for binding of endogenous FOXO3A to the Prx III promoter by performing electromobility shift assays and chromatin immunoprecipitation assays. Inhibition of endogenous FOXO3A by insulin growth factor 1 prevented binding of FOXO3A to Prx III promoter. In contrast, overexpression of FOXO3A increased Prx III promoter activity. Furthermore, depletion of Prx III was associated with enhanced apoptosis and oxidative stress after serum deprivation. We conclude that FOXO3A mediates Prx III expression, and this may play a critical role in the resistance to oxidative stress in cardiac fibroblasts