利用鄰近標記探討 PTPN9 的生理地位

Abstract

PTPN9是一種非受體型蛋白酪氨酸磷酸酶。PTPN9和其他蛋白酪氨酸磷酸酶最大的不同在於PTPN9擁有一個CRAL-TRIO脂質結合結構域。之前的研究指出，PTPN9能夠調控生物體對於胰島素的反應性，或者是在免疫系統中扮演一個重要的角色。儘管如此，PTPN9的生理地位仍未被定義清楚，因為PTPN9的受質並沒有被好好地探索。鄰近標記的發明是為了提供一個更好的，來取代傳統上用來解析某個特定空間中的蛋白質體或是研究蛋白質相互作用的方法，像是共免疫沈澱法。在這篇研究中，我們利用BioID2去做鄰近標記來找尋PTPN9的潛在相互作用者。PTPN9和非專一性生物素連接酶BioID2融合之後，位在PTPN9周圍的蛋白就能被BioID2標記上生物素。並利用鏈霉親和素蛋白來分離被標記上生物素的蛋白，再用質譜法去辨認在PTPN9周圍的是哪些種類的蛋白。最重要的是，根據我們實驗結果的生物資訊分析進一步證明PTPN9在膜運輸中扮演著重要的角色。此外，我們還找到了幾種PTPN9的受質，例如TBC1D5，並指出PTPN9可能參與高基式體和胞內體系統間物質的運輸，纖毛和Retromer調控的胞內物質分選。

PTPN9, one of non-receptor type protein tyrosine phosphatases (PTPs), is distinct from other PTPs due to the CRAL-TRIO lipid binding domain at the N terminus. This tyrosine phosphatase was reported to be involved in insulin responsiveness and immune system.Despite these, the physiological roles of PTPN9 have remained poorly characterized because the substrates of PTPN9 have not yet been explored well. Proximity labeling has emerged as an alternative to traditional approaches for the investigation of spatially resolved proteomes or protein-protein interactions (PPIs) in living cells. Here we exploit BioID2-dependent proximity labeling to investigate the potential interactors of PTPN9. The proteins in the proximity of PTPN9 are labeled with biotin by fusing PTPN9 with the promiscuous biotin ligase, BioID2. These biotinylated proteins could be captured by streptavidin and identified using mass spectrometry. Most importantly, bioinformatic analysis based on our proximity labeling data further reveals that PTPN9 plays a pivotal role in membrane trafficking. Additionally, we also validated several PTPN9 substrates, such as TBC1D5 and indicated that PTPN9 may be involved in Golgi-endosomal system transport, cilia and retromer-dependent cargo sorting.