DEGRADATION OF IMMUNOMODULATORY PROTEINS BY KSHV K5

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 泛素化的靶向细胞表面受体的机制是复杂的。K5是一种March家族的E3泛素连接酶，可通过促进细胞表面蛋白的内吞和降解来下调其表达。在这篇报道中，我们提出了这种E3连接酶介导ICAM-1、B7.2和MHC I调节的多种分子机制。内化分析表明，每个靶点的下调可以通过增加细胞表面的内吞作用来发生。然而，K5中一个保守的基于酪氨酸的内吞基序的突变导致了一个蛋白质缺乏指导MHC I或ICAM-1内吞的能力，但仍然能够以一种普遍存在的独立于内吞的方式下调B7.2。此外，两个酸性簇的突变消除了K5介导的MHC I降解，而仅略微减少了ICAM-1或B7.2蛋白的破坏。这个突变体取消了所有靶标的可检测泛素化。最后，除了它在引导靶蛋白泛素化方面的关键作用外，对含有环-CH突变体的K5的共聚焦显微镜表明，该结构域在细胞内定位中起着关键作用。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. The mechanisms involved in targeting cell surface receptors for ubiquitylation are complex. K5 is a MARCH family E3 ubiquitin ligase that can down regulate a variety of cell surface proteins through enhancement of their endocytosis and degradation. In this report, we present data that there are multiple molecular mechanisms of ICAM-1, B7.2 and MHC I modulation mediated by this E3 ligase. Internalization assays demonstrate that down regulation of each target can occur through increased endocytosis from the cell surface. However, mutation of a conserved tyrosine-based endocytosis motif in K5 resulted in a protein lacking the ability to direct MHC I or ICAM-1 endocytosis, but still able to down regulate B7.2 in a ubiquitindependant, but endocytosis-independent manner. Further, mutation of two acidic clusters abolished K5-mediated MHC I degradation while only slightly decreasing ICAM-1 or B7.2 protein destruction. This same mutant abolished detectable ubiquitylation of all targets. Finally, in addition to its critical role in directing target protein ubiquitylation, confocal microscopy of K5 containing a RING-CH mutant demonstrated that this domain plays a key role in intracellular localization.