Ubiquitin Profiling in Liver Using a Transgenic Mouse with Biotinylated Ubiquitin

Ubiquitination
is behind most cellular processes, with ubiquitin
substrates being regulated variously according to the number of covalently
conjugated ubiquitin molecules and type of chain formed. Here we report
the first mammalian system for ubiquitin proteomics allowing direct
validation of the MS-identified proteins. We created a transgenic
mouse expressing biotinylated ubiquitin and demonstrate its use for
the isolation of ubiquitinated proteins from liver and other tissues.
The specificity and strength of the biotin–avidin interaction
allow very stringent washes, so only proteins conjugated to ubiquitin
are isolated. In contrast with recently available antibody-based approaches,
our strategy allows direct validation by immunoblotting, therefore
revealing
the type of ubiquitin chains (mono or poly) formed in vivo. We also
identify the conjugating E2 enzymes that are ubiquitin-loaded in the
mouse tissue. Furthermore, our strategy allows the identification
of candidate cysteine-ubiquitinated proteins, providing a strategy
to identify those on a proteomic scale. The novel in vivo system described
here allows broad access to tissue-specific ubiquitomes and can be
combined with established mouse disease models to investigate ubiquitin-dependent
therapeutical approaches.

泛素化（Ubiquitination）是绝大多数细胞过程的核心调控基础，泛素底物的调控模式会依据共价结合的泛素分子数量以及所形成的泛素链类型而呈现多样化差异。本研究首次构建了可直接验证质谱（Mass Spectrometry，MS）鉴定蛋白的哺乳动物泛素蛋白质组学研究体系。我们构建了一株表达生物素标记泛素的转基因小鼠，并验证了该模型可用于分离肝脏及其他组织中的泛素化蛋白。生物素-亲和素相互作用的高特异性与强结合力允许使用严苛的洗涤条件，从而仅能分离得到与泛素共价结合的蛋白。与当前已有的基于抗体的研究方法相比，本策略可通过免疫印迹（immunoblotting）实现直接验证，进而揭示体内形成的泛素链类型（单泛素链或多泛素链）。此外，本研究还鉴定了小鼠组织中负载泛素的泛素结合酶E2。进一步而言，本策略可用于筛选半胱氨酸泛素化候选蛋白，为在蛋白质组规模上鉴定此类蛋白提供了可行方案。本研究描述的新型体内研究体系可广泛获取组织特异性泛素组（ubiquitome），且可与已建立的小鼠疾病模型相结合，用于探究依赖泛素化通路的治疗策略。