Mechanisms of Nonstop Extension Mutations in Tumor Suppressor Genes

癌症是一种遗传疾病，错义和无义点突变或拷贝数改变已被研究作为致病的遗传改变。在之前的项目中，我们报告了在癌症中从未研究过的一类不间断突变的功能相关性的发现。这些突变将终止密码子转化为常规密码子，并在蛋白质上添加c端延伸。我们已经建立了一个包含3412个癌症不间断突变的广泛数据库（NonStopDB）。在癌症相关基因中，肿瘤抑制基因SMAD4最常受到不间断突变的影响。引人注目的是，在外源性和内源性精确基因组编辑模型中，不间断突变降低了SMAD4蛋白的表达。我们发现了一个可转移的十个氨基酸的降解基序，其中包含疏水性大块残基，可诱导SMAD4泛素化和蛋白酶体降解（Dhamija, Nat Cell Biol 2020）。此外，我们的初步数据发现肿瘤抑制因子VHL和BAP1的复发性不间断突变与肾癌有关。这些也降低了蛋白质表达，但与SMAD4有显著差异：VHL突变仅影响内源性环境下的表达，而不影响过表达，而其扩展也丰富了大块残留物。BAP1的延伸很长（205aa），不含大量残基。因此，我们假设不间断突变在癌症基因中具有迄今未被探索的作用，并且c端延伸和序列基序通常可以控制蛋白质丰度。在该项目中，我们在三个工作包（WP）中追求三个目标：在WP1中，我们探索VHL中不间断突变的功能相关性和分子机制，特别强调外源性和内源性引入突变之间的差异。在WP2中，我们阐明了不间断突变对BAP1的机制影响，并在长扩展中绘制了序列基序。在最大的WP3中，我们系统地分析了在癌症中发现的所有不间断突变对报告蛋白表达的影响。这不仅可以识别对表达有强烈影响的单个不间断延伸，还可以分析与蛋白质丰度相关的c端序列基序，这将进一步研究。值得注意的是，我们已经发现不间断延伸的氨基酸组成与硅蛋白质组的氨基酸组成存在显著差异，其中大块氨基酸富集而酸性残基耗尽。综上所述，本项目将详细阐明重要肿瘤抑制基因VHL和BAP1个体不间断突变的分子机制，并系统分析所有癌症特异性c端延伸控制蛋白表达水平的影响和基序。重要的是，WPs可以协同实现，并从共享的方法和模型系统中受益，但所有目标也可以独立实现。

Cancer is a genetic disease for which missense and nonsense point mutations or copy number alterations have been studied as causative genetic changes.For the preceding project, we report the discovery of the functional relevance of the class nonstop mutations, which had never been studied in cancer. These mutations convert a stop codon into a regular codon adding a C-terminal extension to the protein. We have generated an extensive database of 3412 nonstop mutations in cancer (NonStopDB). Among cancer-related genes, the tumor suppressor SMAD4 was most frequently affected by nonstop mutations. Strikingly, nonstop mutations diminished SMAD4 protein expression in exogenous and endogenous precision genome editing models. We identified a transferable ten amino acid degron motif containing hydrophobic bulky residues that induces SMAD4 ubiquitination and proteasomal degradation (Dhamija, Nat Cell Biol 2020).Moreover, our preliminary data identified recurrent nonstop mutations in the tumor suppressors VHL and BAP1 linked to kidney cancer. These also diminished protein expression - but with notable differences to SMAD4: the VHL mutation only affected expression in the endogenous setting, but not upon overexpression, while its extension was also enriched for bulky residues. The extension of BAP1 was very long (205 aa) and did not contain a stretch of bulky residues.Thus, we hypothesize that nonstop mutations have a so far unexplored role in cancer genes and that C-terminal extensions and sequence motifs can generally control protein abundance.In the proposed project, we pursue three aims in three work packages (WP): In WP1, we explore the functional relevance and the molecular mechanism of the nonstop mutations in VHL with a special emphasis on the discrepancy between exogenously and endogenously introduced mutations. In WP2, we elucidate the mechanistic impact of nonstop mutations on BAP1 and also map sequence motifs in the long extension. In the largest WP3, we systematically analyze all nonstop mutations found in cancer for their impact on reporter protein expression. This will not only identify individual nonstop extensions with a strong impact on expression, but also enable analyses of C-terminal sequence motifs correlating with protein abundance, which will be further studied. Notably, we already found significant differences in the amino acid composition of the nonstop extensions versus the proteome in silico with an enrichment of bulky amino acids and a depletion of acidic residues.In summary, the proposed project will elucidate in detail the molecular mechanisms of individual nonstop mutations in the important tumor suppressor genes VHL and BAP1, and also systematically analyze the impact and motifs in all cancer-specific C-terminal extensions controlling protein expression levels. Importantly, the WPs can be pursued synergistically and benefit from shared methodologies and model systems, but all aims can also be achieved independently.