In murine embryonic fibroblasts, N-acetyl-L-cysteine (NAC), a GSH generating agent, enhances hypoxic apoptosis by blocking the NFB survival pathway (Qanungo, S., Wang, M., and Nieminen, A. L. (2004) J. Biol. Chem. 279, 50455-50464). Here, we examined sulfhydryl modifications of the p65 subunit of NFB that are responsible for NFB inactivation. In MIA PaCa-2 pancreatic cancer cells, hypoxia increased p65-NFB DNA binding and NFB transactivation by 2.6-and 2.8-fold, respectively. NAC blocked these events without having an effect on p65-NFB protein levels and p65-NFB nuclear translocation during hypoxia. Pharmacological inhibition of the NFB pathway also induced hypoxic apoptosis, indicating that the NFB signaling pathway is a major protective mechanism against hypoxic apoptosis. In cell lysates after hypoxia and treatment with N-ethylmaleimide (thiol alkylating agent), dithiothreitol (disulfide reducing agent) was not able to increase binding of p65-NFB to DNA, suggesting that most sulfhydryls in p65-NFB protein were in reduced and activated forms after hypoxia, thereby being blocked by N-ethylmaleimide. In contrast, with hypoxic cells that were also treated with NAC, dithiothreitol increased p65-NFB DNA binding. Glutaredoxin (GRx), which specifically catalyzes reduction of protein-SSG mixed disulfides, reversed inhibition of p65-NFB DNA binding in extracts from cells treated with hypoxia plus NAC and restored NFB activity. This finding indicated that p65-NFB-SSG was formed in situ under hypoxia plus NAC conditions. In cells, knock-down of endogenous GRx1, which also promotes protein glutathionylation under hypoxic radical generating conditions, prevented NAC-induced NFB inactivation and hypoxic apoptosis. The results indicate that GRx-dependent S-glutathionylation of p65-NFB is most likely responsible for NAC-mediated NFB inactivation and enhanced hypoxic apoptosis.Tumor hypoxia is strongly associated with tumor propagation, malignant progression, and resistance to chemo-and radiation therapy (1). NFB 2 is a redox-regulated transcription factor that is activated during hypoxia (2, 3). NFB belongs to the Rel family, which includes five mammalian Rel/NFB proteins: RelA (p65), c-Rel, RelB, NFB1 (p50/p105), and NFB2 (p52/ p100) (4). The inactive form of NFB is localized in the cytoplasm as p65:p50 (the most abundant form) or p50:cRel heterodimers through interaction with IB repressor proteins (IB␣, IB␤, IB␥, and IB⑀) (5). Once activated, NFB translocates to the nucleus, where it binds to DNA and activates various target genes including Bcl-xL, Bcl-2, a hematopoieticspecific Bcl-2 homologue A1, caspase-8-FADD-like interleukin-1␤-converting enzyme inhibitory protein, tumor necrosis factor receptor-associated factors 1 and 2, cellular inhibitors of apoptosis, and X chromosome-linked inhibitor of apoptosis (XIAP/hILP) (6, 7).NFB family proteins have a conserved domain of ϳ300 amino acids in the amino-terminal region known as the Rel homology region. The Rel homology region consists of a DNA binding domain, a dimerization d...

Glutaredoxins (GRx) catalyze reversible protein glutathionylation. They are implicated in sulfhydryl homeostasis and regulation of redox signal transduction, controlling various cellular processes like DNA synthesis, defense against oxidative stress, apoptosis signaling, and DNA-binding of transcription factors. Two isoforms of GRx are well characterized in mammals: GRx1, the "cytosolic" form, and GRx2, the "mitochondrial" form. Here we report documentation of GRx1 in mitochondria, localized exclusively in the intermembrane space and segregated from GRx2, localized exclusively in the mitochondrial matrix. We hypothesize that GRx1 and GRx2 in their unique locations regulate different functions of the mitochondria via reversible S-glutathionylation.

To understand the physiological function of glutaredoxin, a thiotransferase catalyzing the reduction of mixed disulfides of protein and glutathione (protein-SSG), we generated a line of knockout mice deficient in the cytosolic glutaredoxin 1 (Grx1). To our surprise, mice deficient in Grx1 were not more susceptible to acute oxidative insults in models of heart and lung injury induced by ischemia/ reperfusion and hyperoxia, respectively; suggesting that changes in S-glutathionylation status of cytosolic proteins are not the major cause of such tissue injury. On the other hand, mouse embryonic fibroblasts (MEFs) isolated from Grx1-deficient mice displayed an increased vulnerability to diquat and paraquat, but they were not more susceptible to cell death induced by hydrogen peroxide (H 2 O 2 ) and diamide. A deficiency in Grx1 also sensitized MEFs to protein S-glutathionylation in response to H 2 O 2 treatment and retarded deglatuthionylation of the S-glutathionylated proteins, especially evident for an unspecified protein of approximately 44 kDa. Additional experiments showed that MEFs lacking Grx1 were more tolerant to apoptosis induced by tumor necrosis factor α plus actinomycin D. These findings suggest that different oxidants may damage the cells via distinct mechanisms in which Grx1-dependent de-glutathionylation may or may not be protective, and Grx1 may exert its function on specific target proteins.

A frameshift mutation 138delT generates an open reading frame in the pseudogene, cytochrome P4502D7 (CYP2D7), and an alternate spliced functional transcript of CYP2D7 containing partial inclusion of intron 6 was identified in human brain but not in liver or kidney from the same individual. mRNA and protein of the brain variant CYP2D7 were detected in 6 of 12 human autopsy brains. Genotyping revealed the presence of the frameshift mutation 138delT only in those human subjects who expressed the brain variant CYP2D7. Genomic DNA analysis in normal volunteers revealed the presence of functional CYP2D7 in 4 of 8 individuals. In liver, the major organ involved in drug metabolism, a minor metabolic pathway mediated by CYP2D6 metabolizes codeine (pro-drug) to morphine (active drug), whereas norcodeine is the major metabolite. In contrast, when expressed in Neuro2a cells, brain variant CYP2D7 metabolized codeine to morphine with greater efficiency compared with the corresponding activity in cells expressing CYP2D6. Morphine binds to -opioid receptors in certain regions of the central nervous system, such as periaqueductal gray, and produces pain relief. The brain variant CYP2D7 and -opioid receptor colocalize in neurons of the periaqueductal gray area in human brain, indicating that metabolism of codeine to morphine could occur at the site of opioid action. Histiospecific isoforms of P450 generated by alternate splicing, which mediate selective metabolism of pro-drugs within tissues, particularly the brain, to generate active drugs may play an important role in drug action and provide newer insights into the genetics of metabolism.Cytochrome P450 (EC 1.14.14.1; P450) 1 and associated monooxygenases, a family of heme proteins, are the principal class of drug-metabolizing enzymes. A supergene family encodes them, and the member proteins exist as multiple forms having distinct yet overlapping substrate specificities. Multiple forms of P450, which are selectively induced or inhibited by a variety of drugs, are known to exist in liver, the major organ involved in P450-mediated metabolism (1). However, the potential to generate active metabolite(s) at the site of action has generated interest in extrahepatic P450. This has prompted extensive investigations into the xenobiotic metabolizing capability of extrahepatic organs, such as lung, kidney, skin, and nasal epithelium, and the far reaching consequences of such metabolism, in situ, within specific cells in target organs have been recognized in laboratory animals (2) and humans (3). The preferential localization of drug-metabolizing enzymes within specific cell types in these organs renders such cells significantly capable of metabolizing drugs (4). Thus, even minor metabolic pathways of xenobiotic metabolism can produce major effects if they take place at the site of action. These observations have prompted investigation into P450-associated monooxygenases in brain with an effort to determine the capability of the brain to metabolize psychoactive drugs (5, 6). P450-mediated ...

CD6 is associated with T-cell modulation and is implicated in several autoimmune diseases. We previously demonstrated that Itolizumab, a CD6 domain 1 (CD6D1) specific humanized monoclonal antibody, inhibited the proliferation and cytokine production by T lymphocytes stimulated with anti-CD3 antibody or when co-stimulated with ALCAM. Aberrant IL-17 producing CD4+ helper T-cells (Th17) have been identified as pivotal for the pathogenesis of certain inflammatory autoimmune disorders, including psoriasis. Itolizumab has demonstrated efficacy in human diseases known to have an IL-17 driven pathogenesis. Here, in in vitro experiments we show that by day 3 of human PBMC activation using anti-CD3 and anti-CD28 co-stimulation in a Th17 polarizing milieu, 15–35% of CD4+ T-cells overexpress CD6 and there is an establishment of differentiated Th17 cells. Addition of Itolizumab reduces the activation and differentiation of T cells to Th17 cells and decreases production of IL-17. These effects are associated with the reduction of key transcription factors pSTAT3 and RORγT. Further, transcription analysis studies in these conditions indicate that Itolizumab suppressed T cell activation by primarily reducing cell cycle, DNA transcription and translation associated genes. To understand the mechanism of this inhibition, we evaluated the effect of this anti-human CD6D1 mAb on ALCAM-CD6 as well as TCR-mediated T cell activation. We show that Itolizumab but not its F(ab’)2 fragment directly inhibits CD6 receptor hyper-phosphorylation and leads to subsequent decrease in associated ZAP70 kinase and docking protein SLP76. Since Itolizumab binds to CD6 expressed only on human and chimpanzee, we developed an antibody binding specifically to mouse CD6D1. This antibody successfully ameliorated the incidence of experimental autoimmune encephalitis in the mice model. These results position CD6 as a key molecule in sustaining the activation and differentiation of T cells and an important target for modulating autoimmune diseases.