NSF/MCB-BSF: Systematic analysis of degrons recognized for nuclear protein quality control

细胞生理和活力依赖于蛋白质采用精确的三维（3D）结构，为蛋白质的功能和活性提供正确的结构。蛋白质经常会受损，失去正确的3D结构，从而使它们更容易聚集和形成有毒聚集体。错误折叠的蛋白质聚集是所有细胞都会遇到的一个基本问题，要么是由于蛋白质合成错误而随机产生的，要么是由于蛋白质暴露于有害物质而引起的应激诱导现象。为了对抗错误折叠的蛋白质聚集，真核细胞已经进化出强大的细胞器特异性蛋白质质量控制（PQC）途径来管理这些蛋白质。本项目旨在通过高通量筛选和计算分析，研究模式生物Saccharomyces cerevisiae（出芽酵母）中错误折叠和聚集蛋白靶向不同PQC途径的机制。它还将揭示细胞核中错误折叠蛋白毒性的关键特征。更广泛地说，这项研究将为从高中生和本科生到研究生和研究科学家的各种经验水平的科学家提供大量的培训机会。将根据生成的数据创建公开可用的数据集。还将设立国际讲习班，为教育和信息传播提供全球手段。本研究的重点是通过多种泛素-蛋白酶体系统蛋白质量控制（UPS PQC）途径研究靶向错误折叠蛋白的机制，了解真核细胞中的核细胞器如何保护自己免受错误折叠蛋白的侵害并形成有毒聚集体。该实验系统旨在最大限度地确定控制UPS PQC降解途径的降解信号谱的概率。为此，将构建酵母cDNA片段文库并将其附加到核报告系统中。由此产生的核蛋白文库将能够测量每个单个片段对蛋白质稳定性的贡献。具体来说，为了对细胞核中不同的UPS PQC途径进行分类，报告文库将在酵母菌株中表达特定核PQC泛素连接酶的单个或组合缺失。数据分析将依赖于深入的生物信息学，将不稳定因素与其影响途径联系起来。随后，将不稳定序列（degrons）与互补酵母2杂交分析鉴定的序列进行比较，这些序列是核蛋白与其同源泛素连接酶的相互作用序列。一旦根据其同源降解途径对降解进行分类，进一步的计算分析将识别结构特征，特别是降解序列，结构，生化和生物物理性质。不同的特征将通过实验进行测试。为了更好地理解降解物聚集倾向与其对细胞毒性的贡献之间的关系，将采用细胞生长和荧光测定相结合的方法。总之，新的UPS PQC度的鉴定将使细胞核中错误折叠蛋白质量控制的系统研究成为可能。反过来，这将为错误折叠的蛋白质如何聚集及其后果提供新的生物学假设。这个美国/以色列合作项目由美国国家科学基金会和以色列两国科学基金会支持。

Cell physiology and viability depend on proteins adopting precise 3-dimensional (3D) structures that provide the correct architecture for protein function and activity. Proteins can often become damaged and lose their correct 3D structures thus making them more prone to aggregation and the formation of toxic aggregates. Misfolded protein aggregation is a fundamental problem that all cells encounter, either randomly due to errors in protein synthesis or as a stress-induced phenomenon caused by exposure of proteins to damaging agents. To counteract misfolded protein aggregation, eukaryotic cells have evolved robust organelle-specific protein quality control (PQC) pathways that manage these proteins. This project seeks to examine the mechanisms of targeting of misfolded and aggregated proteins to different PQC pathways in the model organism, Saccharomyces cerevisiae (budding yeast), using high throughput screening and computational analysis. It will also reveal the key features of misfolded protein toxicity in the nucleus. More broadly, the research will provide numerous training opportunities for scientists at all levels of experience, from high school and undergraduate students to graduate students and research scientists. Publically available datasets will be created from the data generated. International workshops will also be created to provide a global means for education and information dissemination. The focus of this research is to understand how the nuclear organelle in eukaryotic cells protects itself from proteins that have become incorrectly folded and form toxic aggregates by examining the mechanisms of targeting misfolded proteins through the multiple ubiquitin-proteasome system protein quality control (UPS PQC) pathways. The experimental system is designed to maximize the probability of identifying the spectrum of degradation signals that govern the UPS PQC degradation pathways in the nucleus. To this end, a library of yeast cDNA fragments will be constructed and appended to a nuclear reporter system. The resulting nuclear protein library will enable measurement of the contribution of each individual fragment to protein stability. Specifically, to sort the different UPS PQC pathways in the nucleus, the reporter library will be expressed in yeast strains with single or combined deletions of specific nuclear PQC ubiquitin ligases. Data analysis will rely on in-depth bioinformatics that will correlate the destabilizing elements with their affecting pathway(s). Subsequently, the destabilizing sequences (degrons) will be compared to sequences identified by a complementary yeast 2-hybrid analysis as interaction sequences of nuclear proteins with their cognate ubiquitin ligases. Once degrons are classified by their cognate degradation pathways, further computational analyses will discern structural features particularly degron sequence, structure, biochemical and biophysical properties. Distinct features will be tested experimentally. To better understand the relationship between a tendency of a degron to aggregate and its contribution to cell toxicity, a combination of cell-growth and fluorescent assays will be employed. Altogether, the identification of novel UPS PQC degrons will enable systematic investigation of misfolded protein quality control in the nucleus. This, in turn, will provide new biological hypotheses about how misfolded proteins aggregates and its consequences. This collaborative US/Israel project is supported by the US National Science Foundation and the Israeli Binational Science Foundation.