Regulation of TNF Receptor-mediated Cell Death

肿瘤坏死因子与肿瘤坏死因子受体结合可诱导细胞存活或细胞死亡反应。肿瘤坏死因子诱导的大多数反应被归因于其诱导的NF-κB和细胞存活。尽管40年前已经证明了肿瘤坏死因子可以杀死细胞(Carswell等人，PNAS，1975)，但肿瘤坏死因子诱导的死亡在体内的作用仍然很大程度上是未知的。这是由于对TNFR1如何决定生存和死亡的机械理解不完整，阻碍了我们从基因上操纵这一途径的能力，从而使死亡占主导地位。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经历非降解性泛素化时，这是一个转录无关的事件。这限制了RIPK1与死亡信号分子的联系。相反，RIPK1与生存复合体结合来传播生存信号。第二个检查点发生在依赖于NF-κB的促生存基因的诱导下，这提供了对死亡的更持久的保护。通过阻断RIPK1的泛素化来扰乱检查点1，可以释放其诱导细胞凋亡或坏死性下垂的能力。我们发表的研究表明，检查点1的一个关键要素是通过支持生存的CASP8去除CyLD。CyLD是一种脱泛素素酶，专用于K63连接的泛素，它的去除会导致RIPK1持续的泛素化和对死亡的保护。然而，当CASP8介导的CyLD的切割被抑制时，CyLD可以去泛素化RIPK1以启动死亡，并且由于CASP8的阻断，唯一可用的选择是坏死性下垂。这一认识使我们假设，抑制CyLD在检查点1中是关键的，除了蛋白分解之外，还有其他机制可以抑制CyLD。在目标1中，我们将检验这样的假设，即线性泛素化是一种这样的机制，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连接酶的一种成分)的缺乏会导致多器官炎症，而CyLD的复合缺陷可以逆转这种情况。我们将研究线性泛素化是如何抑制CyLD的。在目标2中，我们将进行机制研究，以检验CyLD的磷酸化也会抑制其活性的假设，当这种作用被破坏时，细胞死亡。在体内，我们将检查敲除表皮中的CyLD激酶是否会导致依赖于CyLD的坏死性下垂和随后的皮肤炎症。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来验证这一假说，即激活的巨噬细胞中TNFR2的表达作用于释放CyLD上的这些刹车，从而使TNFR1能够利用CyLD启动坏死性下垂。我们将测试这一假设，即由TNFR2诱导的巨噬细胞坏死性下垂在清除细菌感染方面具有有益的作用。我们的研究将提供对LUBAC缺乏患者所描述的病理的机械性见解，以及潜在的肿瘤坏死因子介导的疾病，如牛皮癣、IBD、RA，以及疫苗佐剂的设计。

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.