We have examined the role of NAD(P)H:quinone oxidoreductase 1 (NQO1) in the bioreductive metabolism of 17-allylamino-demethoxygeldanamycin (17-AAG). High-performance liquid chromatography (HPLC) analysis of the metabolism of 17-AAG by recombinant human NQO1 revealed the formation of a more polar metabolite 17-AAGH 2 . The formation of 17-AAGH 2 was NQO1 dependent, and its formation could be inhibited by the addition of 5-methoxy-1,2-dimethyl-3-[(4-nitrophenoxy)methyl]indole-4,7-dione (ES936), a mechanismbased (suicide) inhibitor of NQO1. The reduction of 17-AAG to the corresponding hydroquinone 17-AAGH 2 was confirmed by tandem liquid chromatography-mass spectrometry. 17-AAGH 2 was relatively stable and only slowly underwent autooxidation back to 17-AAG over a period of hours. To examine the role of NQO1 in 17-AAG metabolism in cells, we used an isogenic pair of human breast cancer cell lines differing only in NQO1 levels. MDA468 cells lack NQO1 due to a genetic polymorphism, and MDA468/NQ16 cells are a stably transfected clone that express high levels of NQO1 protein. HPLC analysis of 17-AAG metabolism using cell sonicates and intact cells showed that 17-AAGH 2 was formed by MDA468/NQ16 cells, and formation of 17-AAGH 2 could be inhibited by ES936. No 17-AAGH 2 was detected in sonicates or intact MDA468 cells. Following a 4-hour treatment with 17-AAG, the MDA468/NQ16 cells were 12-fold more sensitive to growth inhibition compared with MDA468 cells. More importantly, the increased sensitivity of MDA468/NQ16 cells to 17-AAG could be abolished if the cells were pretreated with ES936. Cellular markers of heat shock protein (Hsp) 90 inhibition, Hsp70 induction, and Raf-1 degradation were measured by immunoblot analysis. Marked Hsp70 induction and Raf-1 degradation was observed in MDA468/NQ16 cells but not in MDA468 cells. Similarly, downstream Raf-1 signaling molecules mitogen-activated protein kinase/extracellular signal-regulated kinase (ERK) kinase and ERK also showed decreased levels of phosphorylation in MDA468/NQ16 cells but not in MDA468 cells. The ability of 17-AAG and 17-AAGH 2 to inhibit purified yeast and human Hsp90 ATPase activity was examined. Maximal 17-AAG-induced ATPase inhibition was observed in the presence of NQO1 and could be abrogated by ES936, showing that 17-AAGH 2 was a more potent Hsp90 inhibitor compared with 17-AAG. Molecular modeling studies also showed that due to increased hydrogen bonding between the hydroquinone and the Hsp90 protein, 17-AAGH 2 was bound more tightly to the ATP-binding site in both yeast and human Hsp90 models. In conclusion, these studies have shown that reduction of 17-AAG by NQO1 generates 17-AAGH 2 , a relatively stable hydroquinone that exhibits superior Hsp90 inhibition. (Cancer Res 2005; 65(21): 10006-15)

4-Hydroxynonenal (4-HNE) is an endogenous product of lipid peroxidation known to play a role in cellular signaling through protein modification and is a major precursor for protein carbonyl adducts found in alcoholic liver disease (ALD). In the present study, a greater than 2-fold increase in protein carbonylation of sirtuin 3 (SIRT3), a mitochondrial class III histone deacetylase, is reported in liver mitochondrial extracts of ethanol consuming mice. The consequence of this in vivo carbonylation on SIRT3 deacetylase activity is unknown. Interestingly, mitochondrial protein hyperacetylation was observed in a time-dependent increase in a model of chronic ethanol consumption; however, the underlying mechanisms for this remain unknown. Tandem mass spectrometry was used to identify and characterize the in vitro covalent modification of rSIRT3 by 4-HNE at Cys280, a critical zinc-binding residue, and the resulting inhibition of rSIRT3 activity via pathophysiologically relevant concentrations of 4-HNE. Computational-based molecular modeling simulations indicate that 4-HNE modification alters the conformation of the zinc-binding domain inducing minor changes within the active site, resulting in the allosteric inhibition of SIRT3 activity. These conformational data are supported by the calculated binding energies derived from molecular docking studies suggesting the substrate peptide of acetyl-CoA synthetase 2 (AceCS2-Kac), displays a greater affinity for native SIRT3 compared with the 4-HNE adducted protein. The results of this study characterize altered mitochondrial protein acetylation in a mouse model of chronic ethanol ingestion and thiol-specific allosteric inhibition of rSIRT3 resulting from 4-HNE adduction.

No abstract

Inhibition of Wee1 is emerging as a novel therapeutic strategy for cancer, and some data suggest that cells with dysfunctional p53 are more sensitive to Wee1 inhibition combined with conventional chemotherapy than those with functional p53. We and others found that Wee1 inhibition sensitizes leukemia cells to cytarabine. Thus, we sought to determine whether chemosensitization by Wee1 inhibition is dependent on p53 dysfunction and whether combining Wee1 inhibition is tolerable and effective in vivo. Synergistic inhibition of proliferation with a Wee1 inhibitor in clinical development, MK1775, and cytarabine was observed in all acute myeloid leukemia (AML) cell lines tested, regardless of p53 functionality. Mechanistic studies indicate that inhibition of Wee1 abrogates the S-phase checkpoint and augments apoptosis induced by cytarabine. In AML and lung cancer cell lines, genetic disruption of p53 did not alter the cells' enhanced sensitivity to antimetabolites with Wee1 inhibition. Lastly, mice with AML were treated with cytarabine and/or MK1775. The combination of MK1775 and cytarabine was well-tolerated in mice and enhanced the anti-leukemia effects of cytarabine, including survival. Thus, inhibition of Wee1 sensitizes hematologic and solid tumor cell lines to antimetabolite chemotherapeutics, whether p53 is functional or not, suggesting that the use of p53 mutation as a predictive biomarker for response to Wee1 inhibition may be restricted to certain cancers and/or chemotherapeutics. These data provide preclinical justification for testing MK1775 and cytarabine in patients with leukemia.

Cigarette smoke is a potent inhibitor of pulmonary T cell responses, resulting in decreased immune surveillance and an increased incidence of respiratory tract infections. The ␣,␤-unsaturated aldehydes in cigarette smoke (acrolein and crotonaldehyde) inhibited production of interleukin-2 (IL-2), IL-10, granulocyte-macrophage colony-stimulating factor, interferon-␥, and tumor necrosis factor-␣ by human T cells but did not inhibit production of IL-8. The saturated aldehydes (acetaldehyde, propionaldehyde, and butyraldehyde) in cigarette smoke were inactive. Acrolein inhibited induction of NF-B DNA binding activity after mitogenic stimulation of T cells but had no effect on induction of NFAT or AP-1. Acrolein inhibited NF-B1 (p50) binding to the IL-2 promoter in a chromatin immunoprecipitation assay by >99%. Using purified recombinant p50 in an electrophoretic mobility shift assay, we demonstrated that acrolein was 2000-fold more potent than crotonaldehyde in blocking DNA binding to an NF-B consensus sequence. Matrix-assisted laser desorption/ionization time-offlight and tandem mass spectrometry demonstrated that acrolein alkylated two amino acids (Cys-61 and Arg-307) in the DNA binding domain. Crotonaldehyde reacted with Cys-61, but not Arg-307, whereas the saturated aldehydes in cigarette smoke did not react with p50. These experiments demonstrate that aldehydes in cigarette smoke can regulate gene expression by direct modification of a transcription factor.Cigarette smoke produces profound suppression of pulmonary immunity, resulting in an increased incidence and severity of respiratory tract infections. A recent Institute of Medicine study concluded that smoking increased the incidence of influenza and bacterial pneumonia and accounted for 19,000 smoking-related deaths per year (1). Children infected with Mycobacterium tuberculosis are five times more likely to develop pulmonary tuberculosis if exposed to cigarette smoke (2), and smoking doubles the risk of developing Pneumocystis carinii pneumonia in human immunodeficiency virus-infected individuals (3). Several studies have demonstrated that smoking suppresses T and B cell responses in the lungs without affecting cells in the peripheral blood (4 -7), but little research has been done to elucidate the underlying mechanism behind this phenomenon.We have recently identified two classes of immunosuppressive compounds in cigarette smoke. The dihydroxyphenols (hydroquinone and catechol) in the particulate phase inhibit T cell proliferation by blocking cell cycle progression in late G 1 and S phase (8 -12). In addition, the ␣,␤-unsaturated aldehydes, acrolein (CH 2 ACHCHO) and crotonaldehyde (CH 3 CHACHCHO) in the gas phase of cigarette smoke inhibit the production of several proinflammatory cytokines including IL-2, 4 tumor necrosis factor-␣, and granulocyte-macrophage colony-stimulating factor, with an IC 50 of 3 and 6 M, respectively (13). The saturated aldehydes (acetaldehyde, propionaldehyde, and butyraldehyde) have IC 50 values of Ͼ1500 M. The typical cigarett...

Proliferating cells consume more glucose to cope with the bioenergetics and biosynthetic demands of rapidly dividing cells as well as to counter a shift in cellular redox environment. This study investigates the hypothesis that manganese superoxide dismutase (MnSOD) regulates cellular redox flux and glucose consumption during the cell cycle. A direct correlation was observed between glucose consumption and percentage of S-phase cells in MnSOD wild type fibroblasts, which was absent in MnSOD homozygous knockout fibroblasts. Results from electron paramagnetic resonance spectroscopy and flow cytometry assays showed a significant increase in cellular superoxide levels in S-phase cells, which was associated with an increase in glucose and oxygen consumption, and a decrease in MnSOD activity. Mass spectrometry results showed a complex pattern of MnSOD-methylation at both lysine (68, 89, 122, and 202) and arginine (197 and 216) residues. MnSOD protein carrying a K89A mutation had significantly lower activity compared to wild type MnSOD. Computational-based simulations indicate that lysine and arginine methylation of MnSOD during quiescence would allow greater accessibility to the enzyme active site as well as increase the positive electrostatic potential around and within the active site. Methylation-dependent changes in the MnSOD conformation and subsequent changes in the electrostatic potential around the active site during quiescence vs. proliferation could increase the accessibility of superoxide, a negatively charged substrate. These results support the hypothesis that MnSOD regulates a “metabolic switch” during progression from quiescent through the proliferative cycle. We propose MnSOD as a new molecular player contributing to the “Warburg effect.”

Peroxiredoxin 6 (PRX6) belongs to the 1-Cys class of peroxiredoxins and is recognized as an important antioxidant protein in tissues such as cardiac muscle, skin, and lung. Preliminary in vivo proteomic data have revealed that PRX6 is adducted by 4-hydroxynonenal (4HNE) in the livers of rats chronically fed an ethanol-containing diet. The goals of this study were to evaluate the in vitro effect of aldehyde adduction on PRX6 peroxidase activity, identify specific sites of aldehyde modification using mass spectrometry, and predict conformational changes due to adduction using molecular modeling. PRX6 was found to be resistant to inactivation via aldehyde modification; however, Western blots of adducted protein revealed that both 4HNE and 4-oxononenal (4ONE) caused extensive cross-linking, resulting in high molecular mass species. Tandem mass spectrometry (ESI-LC-MS/MS) analysis demonstrated multiple sites of modification, but adduction of the active site Cys47 was not observed. Molecular modeling simulations indicated that adduction at Cys91 results in a change in protein active site conformation, which potentially restricts access of 4-HNE to Cys47. The Cys91-Lys209 cross-linked adducts could provide the conformational changes required to inactivate the protein by either restricting access to electrophiles or preventing important amino acid interactions within the catalytic triad.

b S Supporting Information O xidative modification of proteins by reactive aldehydes in the liver has been implicated in an increasing number of disease states, including primary biliary cirrhosis, hepatitis C, and chronic alcoholic liver disease (ALD). 1À4 A primary marker for measuring increased oxidative stress in cells is the presence of 4-hydroxy-2-nonenal (4-HNE), the level of which is increased in chronic metabolic liver diseases such as non-alcoholic steatohepatitis (NASH) and ALD. 5,6 Reactive aldehydes such as 4-HNE arise from peroxidation of lipids, including linoleic acid within membranes. 7 Although overall concentrations of 4-HNE are probably in the micromolar or submicromolar range, it has been calculated that concentrations within lipid bilayers could approach 3.8À100 mM. 8,9 4-HNE is an electrophile that will react with protein nucleophiles, including Cys, Lys, and His residues of proteins such as phosphatase and tensin homologue from which chromosome 10 has been deleted (PTEN), tubulin, and peroxiredoxin 6. 10À12 The Akt kinase pathway plays a critical role in the regulation of cell growth, survival, and proliferation. There are three different mammalian isoforms of Akt [Akt1, Akt2, and Akt3 (PKBR, respectively)]. Although these isoforms are encoded on different genes, they are more than 80% identical in amino acid