Faculty Bibliography

Vitamin K1, K2, and K3 are essential nutrients associated with blood clotting and bone metabolism. Quinone oxidoreductases [NAD(P)H:quinone oxidoreductase 1 (NQO1) and NRH:quinone oxidoreductase 2 (NQO2)] are among the selected enzymes that catalyze reduction of vitamin K to vitamin K hydroquinone. NQO1 catalyzes high affinity reduction of vitamin K3 but has only weak affinity for reduction of vitamin K1 and K2. Vitamin K hydroquinone serves as a cofactor for vitamin K gamma-carboxylase that catalyzes gamma-carboxylation of specific glutamic acid residues in Gla-factors/proteins leading to their activation and participation in blood clotting and bone metabolism. Concomitant with Gla modification, a reduced vitamin K molecule is converted to vitamin K epoxide, which is converted back to vitamin K by the enzyme vitamin K epoxide reductase to complete vitamin K cycle. Vitamin K is also redox cycled. One-electron reduction of vitamin K3 leads to the formation of semiquinone that in the presence of oxygen is oxidized back to vitamin K3. Oxygen is reduced to generate reactive oxygen species (ROS) that causes oxidative stress and cytotoxicity. Vitamin K is used as radiation sensitizer or in mixtures with other chemotherapeutic drugs to treat several types of cancer. ROS generated in redox cycling contributes to anticancer activity of vitamin K. NQO1 competes with enzymes that redox cycle vitamin K and catalyzes two-electron reduction of vitamin K3 to hydroquinone. This skips formation of semiquinone and ROS. Therefore, NQO1 metabolically detoxifies vitamin K3 and protects cells against oxidative stress and other adverse effects. On the contrary, NQO2 catalyzes metabolic activation of vitamin K3 leading to cytotoxicity. The role of NQO1 and NQO2 in metabolic detoxification and/or activation of vitamin K1 and K2 remains to be determined. Future studies are also required to identify the enzymes that catalyze high affinity reduction of vitamin K1 and K2 to hydroquinone for use in gamma-carboxylation reactions.

Successful pregnancy depends on well coordinated developmental events involving both maternal and embryonic components. Although a host of signaling pathways participate in implantation, decidualization, and placentation, whether there is a common molecular link that coordinates these processes remains unknown. By exploiting genetic, molecular, pharmacological, and physiological approaches, we show here that the nuclear transcription factor peroxisome proliferator-activated receptor (PPAR) delta plays a central role at various stages of pregnancy, whereas maternal PPARdelta is critical to implantation and decidualization, and embryonic PPARdelta is vital for placentation. Using trophoblast stem cells, we further elucidate that a reciprocal relationship between PPARdelta-AKT and leukemia inhibitory factor-STAT3 signaling pathways serves as a cell lineage sensor to direct trophoblast cell fates during placentation. This novel finding of stage-specific integration of maternal and embryonic PPARdelta signaling provides evidence that PPARdelta is a molecular link that coordinates implantation, decidualization, and placentation crucial to pregnancy success. This study is clinically relevant because deferral of on time implantation leads to spontaneous pregnancy loss, and defective trophoblast invasion is one cause of preeclampsia in humans.

The conjunctive presence of mechanical stress and active transforming growth factor beta1 (TGF-beta1) is essential to convert fibroblasts into contractile myofibroblasts, which cause tissue contractures in fibrotic diseases. Using cultured myofibroblasts and conditions that permit tension modulation on the extracellular matrix (ECM), we establish that myofibroblast contraction functions as a mechanism to directly activate TGF-beta1 from self-generated stores in the ECM. Contraction of myofibroblasts and myofibroblast cytoskeletons prepared with Triton X-100 releases active TGF-beta1 from the ECM. This process is inhibited either by antagonizing integrins or reducing ECM compliance and is independent from protease activity. Stretching myofibroblast-derived ECM in the presence of mechanically apposing stress fibers immediately activates latent TGF-beta1. In myofibroblast-populated wounds, activation of the downstream targets of TGF-beta1 signaling Smad2/3 is higher in stressed compared to relaxed tissues despite similar levels of total TGF-beta1 and its receptor. We propose activation of TGF-beta1 via integrin-mediated myofibroblast contraction as a potential checkpoint in the progression of fibrosis, restricting autocrine generation of myofibroblasts to a stiffened ECM.

BACKGROUND: The aim of this study was to determine whether neocortical long-term potentiation (LTP) is deficient in patients with Alzheimer's disease (AD) and in amyloid precursor protein (APP)/presenilin-1 (PS1) mice, an AD animal model. We then ascertained whether this deficit might be paralleled by functional abnormalities of N-methyl-D-aspartate (NMDAR) glutamate receptors. METHODS: We studied neocortical LTP-like plasticity in 10 patients with mild-to-moderate AD and 10 age-matched normal controls using paired associative stimulation (PAS). We assessed neocortical (medial prefrontal cortex and primary motor cortex) and hippocampal LTP in brain slices of symptomatic APP/PS1 mice. NMDAR composition and signaling as well as synaptic calcium influx were determined in motor, prefrontal and hippocampal cortices of APP/PS1 mice. RESULTS: Both AD patients and transgenic animals showed a deficit in NMDAR-dependent forms of neocortical plasticity. Biochemical analysis showed impaired NMDAR function in symptomatic APP/PS1 mice. CONCLUSIONS: Neocortical plasticity is impaired in both patients with AD and APP/PS1 mice. The results of our biochemical studies point to impaired NMDAR function as the most likely cause for the neocortical plasticity deficit in AD

INrf2:Nrf2 are sensors of chemical/radiation stress. Nrf2 dissociates from INrf2 in response to a stress and translocates in the nucleus. This leads to induction of a battery of antioxidant genes that protect cells. Nrf2 is then exported out and degraded. INrf2 functions as an adaptor of ubiquitin ligase for ubiquitination and degradation of Nrf2. Here we demonstrate the presence of a novel feedback autoregulatory loop between INrf2 and Nrf2 that controls cellular abundance of INrf2 and Nrf2. Nrf2 controls its own degradation by regulating expression and induction of the INrf2 gene. The antioxidant treatment of cells led to nuclear localization and stabilization of Nrf2 and induction of INrf2 gene expression. Mutagenesis, transfection, and chromatin immunoprecipitation assays identified an antioxidant-response element in the reverse strand of the proximal INrf2 promoter that binds to Nrf2 and regulates expression and antioxidant induction of the INrf2 gene. In addition, short interfering RNA inhibition or overexpression of Nrf2 led to a respective decrease and increase in INrf2 gene expression. These results implicated Nrf2 in the regulation of expression and induction of INrf2. The induction of INrf2 followed ubiquitination and degradation of Nrf2 and suppression of INrf2 gene expression. In conclusion, Nrf2 regulates INrf2 by controlling its transcription, and INrf2 controls Nrf2 by degrading it.

Helicases are enzymes that couple ATP hydrolysis to the unwinding of double-stranded (ds) nucleic acids. The bacteriophage T4 helicase (gp41) is a hexameric helicase that promotes DNA replication within a highly coordinated protein complex termed the replisome. Despite recent progress, the gp41 unwinding mechanism and regulatory interactions within the replisome remain unclear. Here we use a single tethered DNA hairpin as a real-time reporter of gp41-mediated dsDNA unwinding and single-stranded (ss) DNA translocation with 3-base pair (bp) resolution. Although gp41 translocates on ssDNA as fast as the in vivo replication fork ( approximately 400 bp/s), its unwinding rate extrapolated to zero force is much slower ( approximately 30 bp/s). Together, our results have two implications: first, gp41 unwinds DNA through a passive mechanism; second, this weak helicase cannot efficiently unwind the T4 genome alone. Our results suggest that important regulations occur within the replisome to achieve rapid and processive replication.