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 中半胱氨酸的功能表征以及生理和病理条件下淋巴细胞氧化新靶点的鉴定。

基本信息

项目摘要

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.
LCK和ZAP-70是协调近端TCR信号的两个关键酪氨酸激酶。最近的数据表明,它们也参与了白血病细胞BCR下游的信号传递。LCK和ZAP-70以及许多其他激酶的激活是通过关键酪氨酸残基的可逆磷酸化来调节的。实验证据表明,除了酪氨酸磷酸化外,半胱氨酸残基的可逆氧化(如亚硫基化)在调节酪氨酸激酶的酶活性中也起着重要作用。然而,LCK和ZAP-70是否以半胱氨酸氧化依赖的方式调节还不完全清楚。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。为此,我们构建了携带C到A取代的载体,并使用Lck或Zap-70缺陷的Jurkat T细胞株进行了功能鉴定。初步数据表明,Lck(C217、C224、C378和C476)和Zap-70(C575)中的半胱氨酸对这些激酶的功能至关重要,因为C到A突变体未能完全重建Jurkat T细胞系中TCR介导的信号转导。该项目的目标是:(I)半胱氨酸突变体的进一步生化和功能表征,(Ii)建立小鼠模型以评估半胱氨酸残基在体内的相关性,以及(Iii)分析Lck和Zap-70半胱氨酸在白血病细胞(如慢性淋巴细胞白血病,CLL)中的功能。除了Lck和ZAP-70,其他信号分子可能以氧化依赖的方式调节。淋巴细胞中磺化的靶点仍然很大程度上是未知的。因此,使用基于二酮的系统,我们探索了来自健康捐赠者以及CLL患者的淋巴细胞中的磺化模式。我们发现,健康的淋巴细胞和白血病细胞都有几种亚硫化的蛋白质。有趣的是,CLL细胞显示出一种特殊的半胱氨酸硫基化模式,这似乎与来自健康捐赠者的细胞不同。该项目的另一个目标是从健康捐赠者和白血病患者的淋巴细胞中鉴定硫基化蛋白(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激活,并治疗自身免疫、免疫缺陷和白血病。

项目成果

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Professor Dr. Luca Simeoni其他文献

Professor Dr. Luca Simeoni的其他文献

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{{ truncateString('Professor Dr. Luca Simeoni', 18)}}的其他基金

Functional characterization of cysteine residues in the regulation of Zap-70 activity in physiology and disease
半胱氨酸残基在生理和疾病中调节 Zap-70 活性的功能表征
  • 批准号:
    416907033
  • 财政年份:
    2019
  • 资助金额:
    --
  • 项目类别:
    Research Grants
Trapping Grb2 within immune cells: The role of transmembrane adaptor proteins
将 Grb2 捕获在免疫细胞内:跨膜接头蛋白的作用
  • 批准号:
    5412690
  • 财政年份:
    2003
  • 资助金额:
    --
  • 项目类别:
    Research Units

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Collaborative Research: TRTech-PGR TRACK: Discovery and characterization of small CRISPR systems for virus-based delivery of heritable editing in plants.
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