Regulation of heme oxygenase-1 by biliverdin reductase

说明(申请人提供)：血红素加氧酶(HO)活性对许多生物学功能至关重要，包括细胞防御机制、神经元活动、血管紧张性和男性生殖。酶系统将血红素分子氧化裂解成胆绿素和一氧化碳，这是一种信号分子。胆绿素被胆绿素还原酶(BVR)还原为抗氧化剂胆红素。我们鉴定了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激酶信号转导通路(如02自由基)以及致癌和病毒感染(如疱疹、艾滋病毒)的环境因素极其敏感。HO-2和HO-3呈结构性表达。我们以前已经证明，HO-1水平在转录本和蛋白质水平上都随着氧化应激的反应而迅速增加。然而，随着刺激措施的取消，这些水平会迅速下降。最近，我们鉴定了HO-1是一种磷酸蛋白。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体内HO活性的抑制剂，也是PKC最有效的抑制剂。综上所述，这些发现使我们推测，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，它激活了PKC/MAP激酶信号通路；作为一种还原酶，它绕过了胆绿素对该信号通路的抑制。我们进一步假设，HO-1的活性和/或稳定性需要磷酸化，因此，控制胆绿素的生产。这些假说将通过以下方面得到验证：1)研究HO-1磷酸化对其活性的意义；以及诱导后HO-1的快速周转是否反映了正常和诱导条件下其磷酸化状态的不同；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激酶途径诱导HO-1基因表达的调节作用；以及4)评估胆绿素对MAP激酶途径激活的影响。H202将被用作02自由基的生成器。以转染腺病毒载体的效应子或其突变的COS细胞为实验模型。

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.