Study on molecules that negatively regulate ROS production in human granulocyes

我们研究了Btk和Btk相关分子在调控活性氧（ROS）产生中的作用。导致呼吸爆发的酶是NADPH氧化酶。在缺乏Btk的情况下，接头Mal与质膜上的PI(3)K和PTKs相关，而在对照静息中性粒细胞中，Btk与细胞质中的Mal相互作用并将其限制在细胞质中。这些激酶被过度磷酸化并被激活。我们通过观察PTK的负调节因子（如Csk/Cbp和磷酸酶），深入探讨了Btk缺失时PTK激活的机制。我们还生成了野生型Mal和突变型Mal，以检查Mal的膜易位是否对中性粒细胞的启动至关重要。Btk还参与了嗜酸性细胞ROS过度产生的调控。我们现在正在研究来自Btk缺陷患者的肥大细胞，以检查Btk在乳糜酶和胰蛋白酶产生中的作用。Itk，主要表达于T细胞的Tec家族激酶，在T细胞中调控ROS产生的作用目前正在研究中。我们的数据证明了Btk（和Mal）在负调控粒细胞ROS过度产生中的重要性。到目前为止，我们的数据表明，ph结构域缺陷Mal可能抑制中性粒细胞的启动，因此该蛋白可能用于预防中性粒细胞在脓毒症等疾病中的过度激活。

We studied the roles of Btk and Btk-associated molecules in regulation of reactive oxygen species (ROS) production. The enzyme responsible for the respiratory burst is NADPH oxidase. In the absence of Btk, the adaptor Mal was associated with PI(3)K and PTKs at the plasma membrane, whereas in control resting neutrophils, Btk interacted with and confined Mal in the cytoplasm. These kinases were hyper-phosphorylated and were activated. We delved into the mechanism of PTK activation in the absence of Btk by looking into the negative regulator of PTKs (e.g. Csk/Cbp and phosphatases). We also generated wild type Mal and mutant Mal to check whether membrane translocation of Mal is critical for priming of neutrophils.Btk was also involved in regulation of over-production of ROS in eosinophils. We are now working on mast cells derived from Btk deficient patients to examine the role of Btk in production of chymase and tryptase. The role of Itk, Tec family kinase expressed mainly in T cells, in the regulation of ROS production in T cells is currently under investigation.Our data demonstrated the importance of Btk (and Mal) in negative regulation of ROS hyperproduction in granulocytes. Our data so far indicate that PH-domain defective Mal may inhibit priming of neutrophils, and thus the protein may be used for the prevention of hyper-activation of neutrophils in conditions such as sepsis.