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

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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.