Functional characterization of cysteines in Lck and Zap-70 and identification of new targets of oxidation in lymphocytes under physiological and pathological conditions.

Lck和Zap-70是协调近端TCR信号传导的两种关键酪氨酸激酶。最近的数据表明，它们也参与白血病细胞中BCR下游的信号传导。Lck和Zap-70以及许多其他激酶的激活通过关键酪氨酸残基的可逆磷酸化来调节。实验证据表明，除了酪氨酸磷酸化之外，半胱氨酸残基的可逆氧化（例如亚磺酰化）也在酪氨酸激酶的酶活性的调节中起重要作用。然而，Lck和Zap-70是否以半胱氨酸氧化依赖性方式调节尚未完全了解。本项目的目的是研究Lck和Zap-70中半胱氨酸残基在生理和病理条件下的功能作用。为了这个目的，我们产生了携带C到A取代的构建体，并使用Lck-或Zap-70-缺陷型Jurkat T细胞系进行功能表征。初步数据显示，Lck（C217、C224、C378和C476）和Zap-70（C575）内的半胱氨酸对于这些激酶的功能至关重要，因为C至A突变体未能在Jurkat T细胞系中完全重建TCR介导的信号传导。该项目的目标是：（i）半胱氨酸突变体的进一步生物化学和功能表征，（ii）生成小鼠模型以评估体内半胱氨酸残基的相关性，以及（iii）分析白血病细胞（例如慢性淋巴细胞白血病，CLL）中Lck和Zap-70半胱氨酸的功能。除了Lck和Zap-70之外，其他信号分子可以以氧化依赖性方式调节。淋巴细胞中亚磺酰化的靶点在很大程度上仍是未知的。因此，使用基于双甲酮的系统，我们已经探索了来自健康供体以及来自CLL患者的淋巴细胞中的亚磺酰化模式。我们发现健康淋巴细胞和白血病细胞都显示出几种亚磺酰化蛋白。有趣的是，CLL细胞显示出蛋白质半胱氨酸亚磺酰化的特定模式，这似乎与来自健康供体的细胞不同。该项目的另一个目的是鉴定来自健康供体和白血病患者的淋巴细胞中的亚磺酰化蛋白（氧化还原体）。我们希望我们的研究将有助于开发新的分子和药理学工具来调节淋巴细胞活化和治疗自身免疫，免疫缺陷和白血病。

Lck and Zap-70 are two key tyrosine kinases that orchestrate proximal TCR signaling. Recent data have shown that they are also involved in signaling downstream of the BCR in leukemic cells. Activation of Lck and Zap-70, as well as of many other kinases, is regulated via reversible phosphorylation of crucial tyrosine residues. The experimental evidence suggests that, in addition to tyrosine phosphorylation, also reversible oxidation (e.g. sulfenylation) of cysteine residues, plays an important role in the regulation of the enzymatic activity of tyrosine kinases. However, whether Lck and Zap-70 are regulated in a cysteine oxidation-dependent manner is not yet completely understood. The aim of this project is to investigate the functional role of cysteine residues within Lck and Zap-70 under both physiological and pathological conditions. To this aim we generated constructs carrying C to A substitutions and performed functional characterization using Lck- or Zap-70-deficient Jurkat T-cell lines. Preliminary data show that cysteines within Lck (C217, C224, C378, and C476) and Zap-70 (C575) are crucial for the function of these kinases, as C to A mutants failed to fully reconstitute TCR-mediated signaling in the Jurkat T-cell lines. The goals of this project are: (i) further biochemical and functional characterization of the cysteine mutants, (ii) generation of mouse models to assess the relevance of the cysteine residues in vivo, and (iii) analysis of the function of Lck and Zap-70 cysteines in leukemic cells (e.g. chronic lymphocytic leukemia, CLL). In addition to Lck and Zap-70, other signaling molecules may be regulated in an oxidation-dependent fashion. Targets of sulfenylation in lymphocytes are still largely unknown. Therefore, using dimedone-based systems, we have explored the sulfenylation pattern in lymphocytes from healthy donors as well as from CLL patients. We have found that both healthy lymphocytes and leukemic cells show several sulfenylated proteins. Interestingly, CLL cells display a specific pattern of protein cysteine sulfenylation, which appears to be different from that of the cells from healthy donors. One additional aim of this project is to identify sulfenylated proteins (redoxome) in lymphocytes from both healthy donors and leukemia patients. We hope that our studies will contribute to the development of new molecular and pharmacological tools to modulate lymphocyte activation and to treat autoimmunity, immunodeficiency, and leukemia.