The role of RIPK1 auto-phosphorylation in cell death and inflammation

RIPK1已成为细胞死亡和炎症的重要调节因子。RIPK1通过激酶活性依赖和非依赖功能调控细胞因子和先天免疫受体如TNFR1、TLR3和TLR4下游的信号传导。RIPK1的激酶活性通过激活Caspase-8介导的细胞凋亡和RIPK3-MLKL介导的坏死下垂而诱导细胞死亡，但它也具有依赖于激酶活性的支架功能，有助于激活NF-B和MAPK信号并抑制细胞死亡。在小鼠模型上的研究表明，RIPK1激酶活性介导的细胞死亡是炎症和退行性病变的有力驱动因素，表明RIPK1激酶抑制剂可能有益于人类炎症性疾病的治疗。然而，RIPK1活性诱导细胞死亡和炎症的机制仍然知之甚少。RIPK1的自动磷酸化被认为是诱导细胞死亡的关键，可能是通过调节RIPK1在不同信号复合体中的招募来实现的。然而，目前尚不清楚RIPK1是否有促进细胞死亡的额外底物，或者自动磷酸化是否足以激活细胞死亡信号。在这里，我们旨在利用转基因小鼠模型研究RIPK1自动磷酸化在细胞死亡和炎症中的作用。具体地说，我们将使用表达内源性RIPK1且丝氨酸残基S161和S166上的突变取消或模拟磷酸化的敲入小鼠来评估这些位点的磷酸化如何影响RIPK1信号转导。我们将利用这些小鼠的原代细胞进行体外实验，研究突变对RIPK1依赖的信号的影响，并结合生化分析解决潜在的分子机制。此外，我们将在由RIPK1激酶活性依赖的过程触发的炎症性疾病的小鼠模型中进行研究，以解决RIPK1自磷酸化在体内调节细胞死亡和炎症中的作用。通过揭示RIPK1活性在体外和体内诱导细胞死亡和炎症的机制，这些实验不仅有助于破译先天免疫信号中细胞死亡和炎症途径的复杂调控，而且将有助于更好地理解炎症性疾病的发病机制。

RIPK1 has emerged as an important regulator of cell death and inflammation. RIPK1 controls signalling downstream of cytokine and innate immune receptors, such as TNFR1, TLR3 and TLR4, via kinase activity-dependent and -independent functions. While its kinase activity has been shown to induce cell death by activating caspase-8-mediated apoptosis as well as RIPK3-MLKL-mediated necroptosis, RIPK1 also exhibits kinase activity-independent scaffolding functions that contribute to the activation of NF-B and MAPK signalling and inhibit cell death. Studies in mouse models showed that RIPK1 kinase activity-mediated cell death is a potent driver of inflammatory and degenerative pathologies, suggesting that RIPK1 kinase inhibitors may be beneficial for the treatment of human inflammatory diseases. However, the mechanisms by which RIPK1 kinase activity induces cell death and inflammation remain poorly understood. RIPK1 autophosphorylation is thought to be critical for cell death induction, possibly by regulating the recruitment of RIPK1 in diverse signalling complexes. Yet, it is unclear if RIPK1 has additional substrates that contribute to cell death or if autophosphorylation is sufficient for activation of cell death signalling. Here we aim to study the role of RIPK1 autophosphorylation in cell death and inflammation using genetically modified mouse models. Specifically, we will use knock-in mice expressing endogenous RIPK1 with mutations abrogating or mimicking phosphorylation at serine residues S161 and S166 to assess how phosphorylation of these sites affects RIPK1 signalling. We will employ in vitro experiments in primary cells from these mice to investigate the impact of the mutations on RIPK1-dependent signalling, combined with biochemical assays addressing the underlying molecular mechanisms. In addition, we will perform studies in mouse models of inflammatory diseases that are triggered by RIPK1 kinase activity-dependent processes, in order to address the role of RIPK1 autophosphorylation in the regulation of cell death and inflammation in vivo. By shedding light on the mechanisms by which RIPK1 kinase activity induces cell death and inflammation in vitro and in vivo, these experiments will not only contribute to deciphering the complex regulation of cell death and inflammatory pathways in innate immune signalling but will also lead to the better understanding of the pathogenesis of inflammatory diseases.