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

实现细胞中内源性目的蛋白（POI）的快速有效降解是理解蛋白质功能和蛋白质治疗靶向的研究工具包所需的。通过基因敲除（例如通过CRISPR/Cas9基因组编辑）实现的蛋白质沉默是不可逆的，耗时的，并且通常不可行。同样，RNA干扰方法需要长期治疗，可能导致蛋白质不完全耗尽，并且通常与脱靶效应相关。P0 I的直接蛋白水解可以潜在地克服这些限制。Sapkota实验室已经开发了亲和定向蛋白导弹（AdPROM）系统，其允许快速有效地破坏许多细胞系中的内源性靶蛋白（1，2）。蛋白水解AdPROM系统融合特定蛋白质和von Hippel-Lindau（VHL）蛋白质的小的、高亲和力多肽结合剂，例如纳米抗体和单体抗体，以募集CUL 2-RING E3连接酶复合物。当通过逆转录病毒感染在任何细胞中递送时，所得的AdPROM系统通过蛋白酶体导致内源性靶蛋白的有效降解。Ciulli实验室致力于开发小分子降解剂，也称为蛋白水解靶向嵌合体（PROTAC），其诱导细胞中的特定蛋白质通过泛素蛋白酶体系统降解（3）。Ciulli实验室已经开发出PROTAC，通过将它们招募到VHL-CUL 2 E3连接酶复合物中，用于HALO标记蛋白的蛋白酶体破坏，选择性地快速降解细胞中的HALO标记蛋白。这种方法的关键要求是内源性靶蛋白需要首先用HALO标签标记，以便HaloPROTAC起作用。在内源性蛋白质上引入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/单抗体与HALO标签融合，并使用HaloPROTAC诱导降解纳米/单抗体靶向的目的蛋白质，从而开发有效的诱导型AdPROM系统。该系统的优点是，它将适用于表达靶蛋白的任何细胞系，因此可以在任何细胞环境中快速评估蛋白质的功能。在缺乏有效靶向纳米/单体抗体的情况下，CRISPR/Cas9基因组编辑可用于首先在靶蛋白上快速引入GFP标签，并使用融合至HALO标签的抗GFP纳米抗体作为用于降解POI的诱导型AdPROM。在开发出强大的AdPROM技术后，将测试其降解许多不同细胞类型中多种靶蛋白的功效。新型HaloPROTAC将在Ciulli实验室合成，并进行测试，以优化系统的降解效率。这种方法有可能在快速评估蛋白水解作为一种药物的方法，许多所谓的'undruggable'的目标蛋白参与许多人类疾病。学生将接受多学科领域的优秀培训：Sapkota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.富尔彻湖J.，Macartney，T. J.，Turnbull，C.，哈钦森湖，和Sapkota，G. P.（2017）通过亲和力导向的蛋白质导弹系统靶向内源性蛋白质降解。开放生物学7：1700662。Fulcher，A. J.，Macartney，T.，Bozatzi，P.，Hornberger，A.，Rojas-Fernandez，A.，和Sapkota，G. P.（2016）一种用于靶向蛋白水解的亲和定向蛋白质导弹（AdPROM）系统。开放生物学6：1602551.3。Gadd，M.美国，Testa，A.，卢卡斯，X，Chan，K.- H、陈威拉蒙，D. J.，Zengerle，M.和Ciulli，A.（2017）选择性蛋白质降解的PROTAC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.