Gene Regulation In The Immune System

免疫系统中的基因调控

• 批准号: 8149271
• 负责人: Keiko Ozato
• 金额: $ 80.02万
• 依托单位: EUNICE KENNEDY SHRIVER NATIONAL INSTITUTE OF CHILD HEALTH & HUMAN DEVELOPMENT
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/8149271
• 关键词: Gene Expression Regulation; Immune system

Sequestosome 1/p62 (p62) is a scaffold/adaptor protein with multiple functions implicated for neuronal and bone diseases. It carries a ubiquitin binding domain through which it mediates proteasome-dependent proteolysis. In addition, p62 is reported to regulate NF- B activity in some cells. To date, however, the role of p62 in innate immunity has not been fully elucidated. In this study, we found that IFN- plus TLR signaling stimulates late expression of p62 in murine M. Overexpression of p62 inhibited expression of multiple cytokines, IL-12p40, TNF- , IL-1β, IL-6, and IFN-β, whereas p62 underexpression by small hairpin RNA markedly elevated their expression, indicating that p62 is a broad negative regulator of cytokine expression in stimulated M. We have shown that p62 interacts with IRF8 and Ro52, the transcription factor and ubiquitin E3 ligase that are important for IL-12p40 expression. This interaction, detectable at a late stage in stimulated M, led to increased polyubiquitination and destabilization of IRF8. Upon M stimulation, p62 bound to TNFR-associated factor 6, another E3 ligase important for NF- B activation, but later this interaction was replaced by the recruitment of the deubiquitinating enzyme, cylindromatosis, an inhibitor of NF- B activity. Recruitment of cylindromatosis coincided with reduced TNFR-associated factor 6 autoubiquitination and lower NF- B activation. Our results indicate that p62 orchestrates orderly regulation of ubiquitin modification processes in M to ensure attenuation of cytokine transcription postactivation. Together, p62 may provide a mechanism by which to control excessive inflammatory responses after M activation. Previously we have shown that IRF3 and IRF7, as well as IRF8 are conjugated to a small ubiquitin like peptides SUMO in response to toll like receptor (TLR) and retinoic acid like receptor (RLR) signaling. This is catalyzed by However, a SUMO E3 ligase that catalyzes covalent SUNO conjugation to the IRF proteins has not been identified. Toward this end we have begun analyzing the role of Piasy in ype I IFN transcription. PIASy is a member of the PIAS family that includes four SUMO ligases. We found that PIASy among other family members is a likely candidate involved in regulating type I IFN transcription, because when ectopically expressed Piasy repressed IRF7 mediated IFN promoter activity most strongly relative to other PIAS members. In line with this result, Piasy-/- embryonic fibloblasts (MEFs) induced type I IFNs at much higher levels than wild type cells, when stimulated by viruses, TLR and RLR signals. Furthermore IRF3 and IRF7 were SUMOylated by both PIAS1 and PIASy. PIASy and other PIAS members have several domains including a protein-protein interaction domain, the catalytic domain containing a zinc finger motif and SUMO interaction motif. Our results show that while the N-terminal domain is not required, the central SUMO catalytic domain and the C-terminal SUMO binding motif play important role in regulating IFN transcription.

隔离1/p62（p62）是一种支架/适配器蛋白，具有多种功能，涉及神经元和骨骼疾病。它带有泛素结合结构域，通过该结构域介导蛋白酶体依赖性蛋白水解。此外，据报道P62调节某些细胞中的NF-B活性。但是，迄今为止，尚未完全阐明p62在先天免疫中的作用。在这项研究中，我们发现IFN-Plus TLR信号传导刺激了鼠M中p62的晚期表达。 p62的过表达抑制了多种细胞因子IL-12P40，TNF-，IL-1β，IL-6和IFN-β的表达，而小型发夹RNA的p62 p62不足以显着升高它们的表达，表明p62是刺激M的细胞动力学表现的宽片负调节剂。我们已经表明，p62与IRF8和RO52相互作用，IRF8和RO52是对于IL-12P40表达很重要的转录因子和泛素E3连接酶。这种相互作用可在刺激的m中后期发现，导致IRF8的多泛素化和不稳定。 M刺激后，p62与TNFR相关因子6结合，这是另一个对NF-B激活重要的E3连接酶，但后来通过募集去泛素化酶的募集来代替这种相互作用。与TNFR相关的因子6自荧光和降低的NF-B激活相一致的囊肿性肿瘤病的募集。我们的结果表明，p62在M中策划了有序调节泛素修饰过程，以确保激活后细胞因子转录的衰减。 p62一起可以提供一种机制，可以通过该机制来控制M激活后过度的炎症反应。 以前，我们已经表明，IRF3和IRF7以及IRF8被偶然地与小泛素（如肽Sumo）相结合，以响应Toll（例如受体（TLR））和视黄酸（例如受体（RLR）信号）。然而，这是由Sumo E3连接酶催化的，即尚未鉴定出与IRF蛋白的共价共轭。 为此，我们已经开始分析piasy在YPE I IFN转录中的作用。 Piasy是PIAS家族的成员，其中包括四个相扑连接酶。我们发现，在其他家庭成员中，Piasy可能是参与调节I型IFN转录的候选人，因为当异位表达的PIASY抑制IRF7介导的IFN启动子活性相对于其他PIAS成员最强烈。与此结果一致，当病毒，TLR和RLR信号刺激时，piasy - / - 胚胎fiblosts（MEFS）诱导的I型IFN的水平比野生型细胞高得多。此外，IRF3和IRF7被PIAS1和PIASY均汇总。 Piasy和其他PIAS成员具有多个域，包括蛋白质 - 蛋白质相互作用结构域，含有锌指基序和SUMO相互作用基序的催化结构域。 我们的结果表明，尽管不需要N末端结构域，但中央SUMO催化结构域和C-末端SUMO结合基序在调节IFN转录中起重要作用。