Mechanisms of Nonstop Extension Mutations in Tumor Suppressor Genes

抑癌基因不间断延伸突变的机制

• 批准号: 397982491
• 负责人: Professor Dr. Sven Diederichs
• 金额: --
• 依托单位: Klinik für Thoraxchirurgie
• 依托单位国家: 德国
• 项目类别: Research Grants
• 资助国家: 德国
• 项目状态: 未结题
• 来源: https://gepris.dfg.de/gepris/projekt/397982491?language=en
• 关键词: Mechanisms; Nonstop; Extension; Mutations; Tumor

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.

癌症是一种遗传性疾病，错义和无义点突变或拷贝数改变被认为是导致癌症的遗传变化。在前一个项目中，我们报告了这类从未在癌症中研究过的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，向蛋白质添加C末端延伸。我们已经生成了一个包含3412个癌症非停止突变的广泛数据库（NonStopDB）。在癌症相关基因中，肿瘤抑制基因SMAD 4最常受到不停止突变的影响。引人注目的是，在外源性和内源性精确基因组编辑模型中，不间断突变减少了SMAD 4蛋白的表达。我们鉴定了一个可转移的10个氨基酸降解决定子基序，其含有诱导SMAD 4泛素化和蛋白酶体降解的疏水大残基（Dhamija，Nat Cell Biol 2020）。此外，我们的初步数据鉴定了与肾癌相关的肿瘤抑制因子VHL和BAP 1中的复发性不停止突变。这些也减少了蛋白质表达-但与SMAD 4有显著差异：VHL突变仅影响内源性环境中的表达，但不影响过表达，而其延伸也富集了大的残基。BAP 1的延伸非常长（205 aa），并且不包含一段庞大的残基。因此，我们假设不间断突变在癌症基因中具有迄今为止尚未探索的作用，并且C末端延伸和序列基序通常可以控制蛋白质丰度。在拟议的项目中，我们在三个工作包（WP）中追求三个目标：在WP 1中，我们探讨了VHL中不间断突变的功能相关性和分子机制，特别强调了外源性和内源性引入突变之间的差异。在WP 2中，我们阐明了BAP 1上的不停止突变的机制影响，并在长延伸中绘制了序列基序。在最大的WP 3中，我们系统地分析了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个体不间断延伸，还可以分析与蛋白质丰度相关的C-末端序列基序，这将进一步研究。值得注意的是，我们已经发现，与计算机蛋白质组相比，不间断延伸的氨基酸组成存在显着差异，大体积氨基酸富集，酸性残基耗尽。总之，拟议的项目将详细阐明重要肿瘤抑制基因VHL和BAP 1中单个不间断突变的分子机制，并且还系统地分析控制蛋白质表达水平的所有癌症特异性C-末端延伸中的影响和基序。重要的是，工作方案可以协同进行，并受益于共享的方法和模型系统，但所有目标也可以独立实现。