Regulation of heme oxygenase-1 by biliverdin reductase

描述（由申请人提供）：血红素加氧酶（HO）活性对许多生物功能至关重要，包括细胞防御机制、神经元活性、血管张力和男性生殖。酶系统将血红素分子氧化分解为胆绿素和一氧化碳（一种信号分子）。胆绿素被胆绿素还原酶（BVR）还原为胆红素，一种抗氧化剂。我们已经鉴定出3种HO同工酶：HO-1 (HSP-32)， HO-2和HO-3。HO-1对引起氧化应激和激活MAP激酶信号转导途径（如02自由基）以及致癌和病毒感染（如疱疹、HIV）的环境因子非常敏感。HO-2和HO-3本构表达。我们之前已经表明，在转录物和蛋白质水平上，HO-1水平在氧化应激反应中迅速增加。然而，随着刺激措施的取消，这一水平迅速下降。最近我们发现HO-1是一种磷酸化蛋白。这种类型的蛋白质修饰对蛋白质的稳定性和活性具有重要意义。此外，我们还发现BVR是一种丝氨酸/苏氨酸激酶和蛋白激酶C （PKC）的激活剂。PKC位于MAP激酶信号通路的上游。另一方面，我们发现血红素降解产物胆绿素是HO活性的体内抑制剂，是PKC最有效的抑制剂。综上所述，我们推测环境氧化应激调节HO-1表达的机制涉及BVR的激酶和还原酶活性：作为一种激酶，它激活PKC/MAP激酶信号通路；并且，作为一种还原酶，它绕过胆绿素对信号通路的抑制。我们进一步假设磷酸化是HO-1活性和/或稳定性所必需的；因此，控制胆绿素的产生。这些假设将通过以下方式得到验证：1)研究HO-1磷酸化对其活性的意义；检查诱导HO-1的快速周转是否反映了正常和诱导条件下HO-1磷酸化状态的差异；2)确定HO-1是否是BVR和/或PKC的磷酸化底物；3)分析BVR通过激活MAP激酶通路诱导HO-1基因表达的调控作用；和;4)评估胆绿素对MAP激酶通路激活的影响。H202将被用作02自由基的产生源。将效应物腺病毒载体或其突变对应物转染COS细胞作为实验模型。

DESCRIPTION (provided by applicant): Heme oxygenase (HO) activity is vital to a host of biological functions including cellular defense mechanisms, neuronal activity, vascular tone and male reproduction. The enzyme system oxidatively cleaves the heme molecule to biliverdin and carbon monoxide, a signal molecule. Biliverdin is reduced to bilirubin, an antioxidant, by biliverdin reductase (BVR). We have identified 3 HO isozymes: HO-1 (HSP-32), HO-2 and HO-3. HO-1 is exquisitely sensitive to environmental agents that cause oxidative stress and activate MAP kinase signal transduction pathways (e.g. 02 free radicals) as well as carcinogenesis and viral infections (e.g. Herpes, HIV). HO-2 and HO-3 are constitutively expressed. We have previously shown that HO-1 levels rapidly increase in response to oxidative stress both at the transcript and protein levels. The levels, however, rapidly decline with removal of the stimulus. Recently we identified HO-1 as a phosphoprotein. This type of protein modification can be of significance to protein stability and activity. Also, we discovered BVR to be a serine/threonine kinase and an activator of protein kinase C (PKC). PKC is upstream of the MAP kinase signaling pathway. On the other hand, we found that heme degradation product, biliverdin, is an in vivo inhibitor of HO activity and, a most effective inhibitor of PKC. Collectively, these findings lead us to hypothesize that a mechanism for the regulation of HO-1 expression by environmental oxidative stress involves both the kinase and the reductase activities of BVR: As a kinase, it activates the PKC/MAP kinase signaling pathway; and, as a reductase, it circumvents inhibition of the signaling pathway by biliverdin. We further hypothesize that phosphorylation is required for HO-1 activity and/or stability; hence, control of biliverdin production. These hypotheses will be tested by: 1) investigating the significance of HO-1 phosphorylation to its activity; and, examining whether rapid turnover of the induced HO-1 reflects a difference in its phosphorylation state under normal and induced conditions; 2) determining whether HO-1 is a phosphorylation substrate for BVR and/or PKC; 3) analyzing the regulatory role of BVR on induction of HO-1 gene expression through activation of the MAP kinase pathway; and; 4) assessing the effect of biliverdin on MAP kinase pathway activation. H202 will be utilized as the generator of 02 radicals. COS cells transfected with adenoviral vectors of effectors or their mutated counterparts will be used as the experimental model.