Regulation of TNF Receptor-mediated Cell Death

TNF与TNFR1结合可诱导细胞存活或细胞死亡反应。TNF诱导的大多数反应归因于其诱导NF-κB和细胞存活。尽管早在40年前就证明TNF可以杀死细胞（Carswell et al， PNAS, 1975），但TNF诱导的死亡在体内的作用仍然很大程度上未知。这是由于对TNFR1如何决定生存与死亡的机制理解不完整，阻碍了我们通过基因操纵途径使死亡占主导地位的能力。我们已经确定在TNFR1通路中有两个连续的细胞死亡检查点。第一个检查点发生在RIPK1经历非降解泛素化时，这是一个转录无关的事件。这限制了RIPK1与死亡信号分子的关联。相反，RIPK1与一个生存复合体结合以传播生存信号。第二个检查点发生在NF-κ b依赖性诱导的促生存基因中，这提供了更持久的抗死亡保护。通过阻断RIPK1的泛素化来破坏检查点1，释放其诱导细胞凋亡或坏死的能力。我们发表的研究表明，检查点1的一个关键因素是促生存CASP8去除CYLD。CYLD是k63连接的泛素特异性的去泛素酶，它的去除导致RIPK1持续泛素化并保护其免于死亡。然而，当CASP8介导的CYLD切割被抑制时，CYLD可用于去泛素化RIPK1以启动死亡，并且由于CASP8阻断，唯一可用的选择是necroptosis。这一发现使我们假设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Aim 1中，我们将检验线性泛素化是这样一种机制的假设，它的破坏导致cyld介导的细胞死亡。在体内，Sharpin （LUBAC E3连接酶的一种成分，催化线性泛素化）的缺乏会导致多器官炎症，而这可以通过Cyld的复合缺乏来逆转。我们将研究线性泛素化如何抑制CYLD。在Aim 2中，我们将进行机制研究来验证CYLD磷酸化也抑制其活性的假设，当这种活性被破坏时，细胞就会死亡。在体内，我们将研究敲除表皮中的CYLD激酶是否会导致CYLD依赖性坏死性下垂和随后的皮肤炎症。在Aim 3中，我们将研究这些抑制CYLD活性的翻译后机制是如何由外部线索控制的。我们将进行机制研究来验证这一假设，即激活的巨噬细胞中TNFR2的表达可以释放这些对CYLD的抑制，使TNFR1能够利用CYLD启动坏死下垂。我们将验证TNFR2诱导的巨噬细胞坏死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缺乏患者的病理描述提供机制见解，并可能为tnf介导的疾病（如牛皮癣、IBD、RA）和疫苗佐剂的设计提供见解。

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.