Understanding the impact of Fbw7 mutations on substrate function and cancer cell biology

FBXW 7肿瘤抑制基因编码SCFFbw 7泛素连接酶的底物识别亚基，并靶向降解许多致癌蛋白，如c-Myc、c-Jun、Notch和细胞周期蛋白E。Fbw 7底物结合区的杂合错义突变是Fbw 7最常见的遗传变异，常见于T细胞白血病、卵巢癌、子宫内膜癌和胃肠癌。这些突变体在底物降解中部分受损，被认为是“无效”等位基因，但它们对底物功能和细胞生物学的影响仍不清楚。引人注目的是，我们发现，尽管肿瘤衍生的Fbw 7突变体不能有效地指导蛋白质降解，但它们保留了结合和泛素化至少一些底物的能力。此外，Fbw 7底物的某些功能在Fbw 7缺失细胞中受损，认为Fbw 7促进了它们的一些活性。基于初步数据，我们认为杂合Fbw 7突变的影响超出了损害底物周转，并可能对底物功能产生额外的非蛋白水解作用。我们建议严格分析癌症来源的Fbw 7突变体的泛素化如何影响多种底物的活性，其下游途径和癌细胞生理学。我们将使用体外试验直接测试突变对底物识别和Fbw 7催化活性的影响，并使用组织培养和小鼠异种移植模型分析其对底物功能和细胞生物学的影响。

The FBXW7 tumor suppressor gene encodes a substrate-recognition subunit of the SCFFbw7 ubiqui-tin ligase and targets for degradation a number of oncogenic proteins, such as c-Myc, c-Jun, Notch, and cyclin E. Heterozygous missense mutations in the substrate-binding domain are the most com-mon genetic alteration in Fbw7 and are found in T-cell leukemias, ovarian, endometrial and gastroin-testinal cancers. These mutants are partially compromised in substrate degradation and are consid-ered to be “null” alleles, but their impact on substrate function and cell biology remains largely unclear. Strikingly, we find that although tumor-derived Fbw7 mutants do not efficiently direct protein degradation, they retain the ability to bind and ubiquitinate at least some substrates. Moreover, certain functions of Fbw7 substrates are impaired in Fbw7-null cells, arguing that Fbw7 promotes some of their activities. Based on preliminary data, we suggest that the impact of heterozygous Fbw7 mutations extends beyond compromising substrate turnover and can have additional non-proteolytic effects on substrate function. We propose to rigorously analyze how ubiquitination by the cancer-derived Fbw7 mutants affects the activity of multiple substrates, their downstream pathways and cancer cell physiology. We will directly test the impact of mutations on substrate recognition and the catalytic activity of Fbw7 using in vitro assays and analyze their impact on substrate function and cell biology using tissue culture and mouse xenograft models.