Regulation of TNF Receptor-mediated Cell Death

 描述（由申请人提供） TNF与TNFR 1的结合诱导细胞存活或细胞死亡应答。TNF诱导的大多数反应归因于其诱导NF-κB和细胞存活。尽管在40年前就证明了TNF可以杀死细胞（Carswell等，PNAS，1975），但TNF诱导的死亡的体内作用在很大程度上仍然未知。这是由于对TNFR 1如何决定生存与死亡的机械理解不完整，阻碍了我们对死亡占主导地位的途径进行遗传操纵的能力。我们已经确定在TNFR 1通路中存在两个连续的细胞死亡检查点。第一个检查点发生在RIPK 1经历非降解性泛素化时，这是一种转录独立事件。这限制了RIPK 1与死亡信号分子的结合。相反，RIPK 1与生存复合物结合以传播生存信号。第二个检查点发生在促存活基因的NF-κ B依赖性诱导下，这提供了针对死亡的更持久的保护。通过阻断RIPK 1的泛素化来破坏检查点1，释放其诱导凋亡或坏死性凋亡的能力。我们发表的研究表明，检查点1的一个关键要素是通过促生存CASP 8去除CYLD。CYLD是K63连接的泛素特异性的去泛素化酶，其去除导致持续的RIPK 1泛素化和保护免于死亡。然而，当CASP 8介导的CYLD切割被抑制时，CYLD可用于使RIPK 1去泛素化以启动死亡，并且由于CASP 8阻断，唯一可用的选择是坏死性凋亡。这一认识使我们假设CYLD的抑制在检查点1中是关键的，除了蛋白水解外，还存在其他抑制CYLD的机制。在目标1中，我们将测试的假设，线性泛素化是这样的机制之一，它的破坏导致CYLD介导的细胞死亡。在体内，Sharpin（催化线性泛素化的LUBAC E3连接酶的组分）的缺乏导致多器官炎症，并且这被Cyld的化合物缺乏逆转。我们将研究线性泛素化如何抑制CYLD。在目标2中，我们将进行机制研究，以检验CYLD的磷酸化也抑制其活性，当其被破坏时，细胞死亡的假设。在体内，我们将研究是否敲除表皮中的CYLD激酶导致CYLD依赖性坏死性凋亡和随后的皮肤炎症。在目标3中，我们将研究这些抑制CYLD活性的翻译后机制是如何被外部线索控制的。我们将进行机制研究，以测试活化的巨噬细胞中TNFR 2表达的假设，释放这些刹车CYLD，使TNFR 1，然后利用CYLD启动坏死性凋亡。我们将检验TNFR 2诱导的巨噬细胞坏死性凋亡在清除细菌感染中具有有益作用的假设。我们的研究将为LUBAC缺陷患者中描述的病理学提供机制性见解，并可能为TNF介导的疾病如银屑病、IBD、RA和疫苗佐剂的设计提供机制性见解。

 DESCRIPTION (provided by applicant) Binding of TNF to TNFR1 induces either a cell survival or cell death response. Most responses induced by TNF have been attributed to its induction of NF-κB and cell survival. Despite the demonstration that TNF can kill cells 40 years ago (Carswell et al, PNAS, 1975), the in vivo roles of TNF-induced death remain largely unknown. This is due to an incomplete mechanistic understanding of how TNFR1 dictates survival versus death, hampering our ability to genetically manipulate the pathway such that death predominates. We have established that there are two sequential cell death checkpoints in the TNFR1 pathway. The first checkpoint occurs when RIPK1 undergoes non-degradative ubiquitination, a transcription-independent event. This restricts RIPK1 from associating with death-signaling molecules. Instead, RIPK1 associates with a survival complex to propagate the survival signal. The second checkpoint occurs with the NF-κB-dependent induction of pro- survival genes, which provides a longer-lasting protection against death. Disrupting Checkpoint 1 by blocking the ubiquitination of RIPK1 unleashes its ability to induce either apoptosis or necroptosis. Our published studies demonstrated that a critical element of Checkpoint 1 is the removal of CYLD by a pro-survival CASP8. CYLD is a deubiquitinase specific for K63-linked ubiquitin and its removal leads to sustained RIPK1 ubiquitination and protection from death. However when CASP8-mediated cleavage of CYLD is inhibited, CYLD is available to deubiquitinate RIPK1 to initiate death and by virtue of the CASP8 blockade, the only option available is necroptosis. This insight led us to hypothesize that suppression of CYLD is pivotal in Checkpoint 1 and in addition to proteolysis, other mechanisms exist to suppress CYLD. In Aim 1, we will test the hypothesis that linear ubiquitination is one such mechanism and its disruption leads to CYLD-mediated cell death. In vivo, deficiency in Sharpin (a component of the LUBAC E3 ligase that catalyzes linear ubiquitination) leads to multi-organ inflammation, and this is reversed by a compound deficiency in Cyld. We will study how linear ubiquitination suppresses CYLD. In Aim 2, we will conduct mechanistic studies to test the hypothesis that phosphorylation of CYLD also suppresses its activity and when this is disrupted, cells die. In vivo, we will examine whether knocking out the CYLD kinase in the epidermis results in CYLD-dependent necroptosis and subsequent skin inflammation. In Aim 3, we will examine how these post-translational mechanisms that suppress CYLD activity are controlled by external cues. We will conduct mechanistic studies to test the hypothesis that TNFR2 expression in activated macrophages acts to release these brakes on CYLD, enabling TNFR1 then to utilize CYLD to initiate necroptosis. We will test the hypothesis that necroptosis in macrophages enabled by TNFR2 induction has a beneficial role in clearing bacterial infection. Our studies will provide mechanistic insights into the pathologies described in LUBAC-deficient patients and potentially into TNF- mediated disorders such as psoriasis, IBD, RA, and the design of vaccine adjuvants.