Engineering an Affinity-directed Protein Missile System (AdPROM) for inducible degradation of target proteins

在细胞中实现内源性感兴趣蛋白(POI)的快速有效降解是了解蛋白质功能和治疗靶向蛋白质的理想工具。通过基因敲除来沉默蛋白质，例如通过CRISPR/Cas9基因组编辑，是不可逆转的、耗时的，而且往往是不可行的。同样，RNA干扰方法需要长时间的治疗，可能导致蛋白质不完全耗尽，并且经常与靶外效应有关。POI的直接蛋白分解有可能克服这些限制。Sapkota实验室开发了一种亲和定向蛋白导弹(AdPROM)系统，允许快速有效地破坏许多细胞系(1，2)中的内源性目标蛋白。蛋白水解型AdPROM系统将特定蛋白质和von Hippel-Lindau(VHL)蛋白的小而高亲和力的多肽结合物(如纳米体和单体)与von Hippel-Lindau(VHL)蛋白融合在一起，以招募CUL2环E3连接酶复合体。由此产生的AdPROM系统，当通过逆转录病毒感染在任何细胞中传递时，通过蛋白酶体导致内源性目标蛋白的有效降解。Ciulli实验室致力于开发小分子降解剂，也称为针对嵌合体的蛋白分解(PROTACs)，它通过泛素蛋白酶体系统诱导细胞中的特定蛋白质降解(3)。Ciulli实验室已经开发出PROTAC，通过将细胞中的HALO标记的蛋白质重新招募到VHL-CUL2 E3连接酶复合体上，选择性地快速降解HALO标记的蛋白质，从而对HALO标记的蛋白质进行蛋白酶体破坏。这种方法的一个关键要求是，为了使HaloPROTAC发挥作用，需要首先用Halo-tag标记内源目标蛋白。在内源蛋白质上引入Halo标签不仅具有挑战性，而且往往会损害蛋白质的功能。本项目旨在通过将纳米/单体与Halo标签融合来开发一种有效的可诱导AdPROM系统，并使用HaloPROTAC诱导降解纳米/单体靶向的目标蛋白质。该系统的优点是，它将适用于任何表达目标蛋白的细胞系，因此可以在任何细胞环境中快速评估该蛋白的功能。在缺乏有效靶向纳米/单体的情况下，CRISPR/Cas9基因组编辑可以首先在靶蛋白上快速引入GFP-Tag，然后使用融合到Halo-Tag上的抗GFP纳米体作为可诱导的AdPROM来降解POI。在开发出强大的AdPROM技术后，将测试其在许多不同细胞类型中降解多种目标蛋白的有效性。新型卤代烃将在Ciulli实验室合成并进行测试，目的是优化系统的降解效率。这种方法有可能快速评估蛋白质分解作为一种可用药的方法，因为许多所谓的“不可用药”的靶蛋白与许多人类疾病有关。学生将接受多学科领域的优秀培训：萨普科塔实验室的尖端细胞生物学和生物化学技术，以及Ciulli实验室的最先进的化学生物学技术。参考文献：1.Fulcher，L.J.，Macartney，T.J.，Turnbull，C.，Hutchinson，L.和Sapkota，G.P.(2017)通过亲和力定向的蛋白质导弹系统瞄准内源性蛋白质进行降解。开放生物学7：1700662。Fulcher，A.J.，Macartney，T.，Bozatzi，P.，Hornberger，A.，Rojas-Fernandez，A.和Sapkota，G.P.(2016)一种用于靶向蛋白质降解的亲和定向蛋白质导弹(AdPROM)系统。开放生物学6：1602551.3。GADD，M.S.，Testa，A.，Lucas，X.，Chan，K.-H.，Chen，W.Lamont，D.J.，Zengerle，M.和Ciulli，A.(2017)PROTAC选择性蛋白质降解合作识别的结构基础。NAT Chem Biol 13,514-521。

Achieving a rapid and efficient degradation of endogenous proteins of interest (POIs) in cells is desirable as a research toolkit to understand protein function, and for therapeutic targeting of proteins. Protein silencing through gene knockouts, for example by CRISPR/Cas9 genome editing, are irreversible, time consuming and often not feasible. Similarly, RNA interference approaches require prolonged treatments, can lead to incomplete protein depletion and are often associated with off-target effects. Direct proteolysis of POIs can potentially overcome these limitations.The Sapkota lab has developed an affinity directed protein missile (AdPROM) system that allows for rapid and efficient destruction of endogenous target proteins in many cell lines (1, 2). The proteolytic AdPROM system fuses small, high-affinity polypeptide binders, such as nanobodies and monobodies, of specific proteins and the von Hippel-Lindau (VHL) protein to recruit the CUL2-RING E3 ligase complex. The resulting AdPROM system, when delivered in any cell, through retroviral infections, leads to efficient degradation of endogenous target proteins through the proteasome. The Ciulli lab works on developing small molecule degraders, also known as proteolysis targeting chimeras (PROTACs), that induce specific proteins in cells to be degraded via the ubiquitin proteasome system (3). The Ciulli lab has developed PROTACs that selectively and rapidly degrade the HALO-tagged proteins from cells by recruiting them to VHL-CUL2 E3 ligase complex for proteasomal destruction of the HALO-tagged proteins. A key requirement for this approach is that the endogenous target proteins need to be tagged with a HALO-tag first in order for the HaloPROTACs to work. Introducing a HALO-tag on an endogenous protein is not only challenging but can often compromise the function of the protein as well.This project aims to develop an effective inducible AdPROM system by fusing nano/monobodies with a HALO-tag and inducibly degrade the proteins of interest targeted by the nano/monobodies using HaloPROTACs. The advantage of this system is that it will be applicable to any cell lines where the target protein is expressed and so the function of the protein can be rapidly assessed in any cellular context. In the absence of an effective targeting nano/monobodies, CRISPR/Cas9 genome editing can be used to rapidly introduce a GFP-tag on the target protein first and use anti-GFP nanobody fused to HALO-tag as the inducible AdPROM for the degradation of the POI. After developing a robust AdPROM technology, its efficacy to degrade a wide array of target proteins in many different cell types will be tested. Novel HaloPROTACs will be synthesized in the Ciulli Lab and tested with the goal to optimize the degradation efficiency of the system. This approach has the potential in rapidly evaluating proteolysis as a druggable approach for many so called 'undruggable' target proteins involved in many human diseases. The student will receive excellent training in multidisciplinary areas: cutting-edge cell biology and biochemistry technologies from the Sapkota lab and state-of-the-art chemical biology techniques from the Ciulli lab. References:1. Fulcher, L. J., Macartney, T. J., Turnbull, C., Hutchinson, L., and Sapkota, G. P. (2017) Targeting endogenous proteins for degradation through the affinity-directed protein missile system. Open biology 7: 1700662. Fulcher, A. J., Macartney, T., Bozatzi, P., Hornberger, A., Rojas-Fernandez, A., and Sapkota, G. P. (2016) An Affinity-directed PROtein Missile (AdPROM) system for targeted proteolysis. Open biology 6:1602551.3. Gadd, M. S., Testa, A., Lucas, X., Chan, K.-H., Chen, W. Lamont, D.J., Zengerle, M. and Ciulli, A. (2017) Structural basis of PROTAC cooperative recognition for selective protein degradation. Nat Chem Biol 13, 514-521.