研究泛素黏合酶Cullin3的受質接受子BtbVII對Hedgehog訊息傳遞之調控

Abstract

泛素化和其後的蛋白質降解，是由泛素黏合酶的受質接受子來調控此步驟的專一性。在果蠅中，已知Cullin3 (Cul3)為基底的泛素黏合酶複合物透過Roadkill (Rdx)受質接受子，辨識Hedgehog (Hh)訊息傳遞的作用分子Cubitus interruptus (Ci)，並對其進行泛素化。然而，rdx的突變細胞所展現出Ci累積的表現型，其強度和範圍並不如Cul3突變時對Ci所造成的效果，因此我們認為除了Rdx外，應該還有其他的受質接受子存在。此外，在本實驗室先前的研究中發現，Cul3的突變也會對Ci進入細胞核的機制發生影響，所以我們認為Cul3在Hh訊息傳遞中具有多重的調控功能，而不單是調節Ci的蛋白質量。在本研究中，我們發現一個含有BTB區塊的蛋白BtbVII，它在in vivo和in vitro實驗中都能和Cul3接合，並且有遺傳交互作用，因此我們認為BtbVII有可能作為Cul3泛素黏合酶複合物中的受質接受子。此外，我們發現Costal2 (Cos2)和Ci可能會是BtbVII所辨識的受質分子，其線索來自於一個酵母菌雙雜交系統資料庫PIMRider，其中報導了BtbVII和Cos2、Ci之間的交互作用，我們更進一步以GST pull-down實驗證實了BtbVII和Cos2之間的接合能力。除此之外，在大量表現BtbVII的細胞中，Cos2和Ci的量都有明顯的下降。因此，我們推論BtbVII可能會在Cul3為基底的泛素黏合酶中扮演辨識受質的角色，而且可能會透過辨識Cos2和Ci並促進其泛素化，來調控Hh訊息傳遞。

The specificity of ubiquitination as well as the subsequent protein degradation is controlled by the substrate-specific adaptors that recruit substrates to the core ubiquitin ligase (E3) complexes. In Drosophila, it is known that the Hedgehog (Hh) pathway effector, Cubitus interruptus (Ci), is targeted by the adaptor protein Roadkill (Rdx) to Cullin3 (Cul3)-based E3 for ubiquitination. However, the existence of additional adaptor proteins is suggested by the less extent of Ci accumulation in the rdx mutant cells compared to that of Cul3 mutants. Besides, the Ci subcellular localization change in the Cul3 mutant cells implies that Cul3 regulates Hh pathway in multiple aspects. Here, I identified a potential adaptor protein, BtbVII, which binds Cul3 in vitro and in vivo. In a yeast two-hybrid database PIMRider, BtbVII has been shown to interact with two Hh pathway components Ci and Costal2 (Cos2), which liberates Ci to the nucleus in response to high Hh signals. We have confirmed the binding of BtbVII to Cos2 by means of GST pull-down. Moreover, Cos2 and Ci protein levels are reduced in the BtbVII overexpressing cells. Hence BtbVII is likely the linker that recruits Cos2 to Cul3 for ubiquitination; it may explain the Cul3-mediated control of Ci nuclear translocation. Taken together, I demonstrate that BtbVII is a potential substrate-specific adaptor for Cul3-based ubiquitin ligases, and it may regulate Hh signaling pathway by targeting Cos2 and Ci for ubiquitination.