探討IVNS1ABP對Cullin3-KLHL20泛素接合酶之調控機轉

Abstract

目前已知在真核細胞中，泛素系統參與並調控著許多重要的細胞功能與訊號傳遞，在此系統中，Cullin-RING 接合酶酵素(CRLs)是最大群的泛素接合酶酵素，CRLs透過各自不同的受質辨識器來決定受質專一性，然而，對於Cullin-RING接合酶酵素的調控機制尚須進一步的研究。在此篇研究中，我們找到一蛋白質IVNS1ABP能與Cul3-KLHL20此接合酶酵素之受質辨識器KLHL20結合，IVNS1ABP藉由自身的Kelch repeats直接與KLHL20的Kelch repeats結合，但是IVNS1ABP不是此Cul3-KLHL20的受質，有趣的是，雖然IVNS1ABP和KLHL20一樣同是BTB-Kelch family的一員，但因缺少幾個重要的氨基酸導致IVNS1ABP無法與Cul3結合，相反地，透過進一步的研究，我們發現IVNS1ABP會干擾KLHL20和受質如PML與DAPK之間的結合，此外，IVNS1BP也會透過抑制KLHL20與Cul3的結合進而影響了Cul3-KLHL20接合酶酵素的形成，透過這兩個機制，IVNS1ABP可以減少KLHL20對於受質如PML與DAPK的泛素化以及降解，接著，我們也發現當細胞缺少IVNS1ABP的時候，PML和DAPK的蛋白質含量降低，同時也伴隨著PML和DAPK的泛素化程度之提升，顯示IVNS1ABP對於Cul3-KLHL20接合酶酵素之調控是具有生理上的意義，此外實驗也證實IVNS1ABP可以透過和KLHL20的結合來抑制KLHL20功能，進而恢復PML-IV引起的細胞生長遲緩與DAPK引發的細胞凋亡。總而言之，我們的研究指出IVNS1ABP藉由與KLHL20的結合來負向調控Cul3-KLHL20接合酶酵素。

Ubiquitin system serves as a dominant regulator of biological process and propagation of information in the eukaryotic cell. In this system, the Cullin-RING E3 ligases (CRLs), the largest class of E3 ligase family, function with their hundreds of different substrate receptors, which are responsible for substrate recognition and specificity. However, the regulation of this family of E3 ligases is largely unknown. In this thesis, we identified IVNS1ABP as a novel interacting protein of KLHL20, a substrate adaptor of Cul3 based E3 ligase. IVNS1ABP directly binds to KLHL20 through kelch-kelch interaction. However, IVNS1ABP is not a substrate of Cul3-KLHL20 complex. Interestingly, IVNS1ABP is itself a BTB-kelch protein but cannot interact with Cul3 due to the lack of several conserved residues important for Cul3 binding. In contrast, IVNS1ABP competes with substrates, such as PML and DAPK, for binding to KLHL20 and also inhibits the recruitment of KLHL20 to Cul3. Through these two mechanisms, IVNS1ABP attenuates the KLHL20-mediated ubiquitination and degradation of the substrates including PML and DAPK. Furthermore, loss of IVNS1ABP increases substrates ubiquitination and degradation, supporting its physiological function in KLHL20 mediated regulation. Consistent with our finding, IVNS1ABP is able to rescue the inhibitory effects of KLHL20 on the antiproliferative function of PML and apoptotic function of DAPK. Together, our study identifies IVNS1ABP as a negative regulator of the Cul3-KLHL20 E3 ligase via its interaction with KLHL20.