Quantitative Proteomics Combined with Two Genetic Strategies for Screening Substrates of Ubiquitin Ligase Hrt3

Ubiquitin ligases (E3s) serve as key regulators for the
ubiquitylation-mediated
pathway. The identification of the corresponding relationship between
E3 and its substrates is challenging but required for understanding
the regulatory network of ubiquitylation. The low abundance of ubiquitinated
conjugates and high redundancy of E3 substrate regulation made the
screening pretty hard. Herein, we combined SILAC-based quantitative
proteomics with two contrary genetic methods (overexpression and knockout)
in theory for E3 (Hrt3, the F-box subunit of the SCF complex) substrate
screening. The knockout method could not overcome the constraint mentioned
above, while the overexpression approach turned on the access to the
potential substrates of E3. Subsequently, we obtained 77 candidates,
which are involved in many critical biological processes and need
to be verified in the future. Within these candidates, we confirmed
the relationship between one of the candidates Nce103 and Hrt3 and
linked Hrt3 with oxygen sensitivity and oxidative stress response
in which Nce103 took part as well. This research is also beneficial
for understanding the impact of oxygen supply on regulation of yeast
growth through the ubiquitination of Nce103.

泛素连接酶（E3s）是泛素介导通路的关键调控因子。鉴定E3与其底物之间的对应关联极具挑战性，但却是解析泛素化调控网络的必要前提。泛素化结合物丰度极低，且E3底物调控存在高度冗余性，这使得筛选工作难度极大。本研究将基于SILAC（细胞培养稳定同位素标记氨基酸）的定量蛋白质组学技术，与两种截然相反的遗传学手段（过表达与基因敲除）相结合，理论上可用于E3（Hrt3，SCF复合物的F-box亚基）的底物筛选。其中基因敲除方法无法克服前述限制，而过表达策略则成功获取了E3的潜在底物。随后我们共获得77个候选底物，这些底物参与多项关键生物学过程，有待后续实验验证。在这些候选底物中，我们验证了候选蛋白Nce103与Hrt3之间的相互作用关系，并将Hrt3与Nce103共同参与的氧敏感性及氧化应激应答通路建立了关联。本研究还有助于解析通过Nce103泛素化调控氧气供给对酵母生长的影响机制。