Biotin-Based Strategies to Explore the World of Ubiquitin and Ubiquitin-Like Modifiers.

A complex code of cellular signals is mediated by ubiquitin and ubiquitin-like (Ub/UbL) modifications on substrate proteins. The so-called Ubiquitin Code specifies protein fates, such as stability, subcellular localization, functional activation or suppression, and interactions. Hundreds of enzymes are involved in placing and removing Ub/UbL on thousands of substrates, while the consequences of modifications and the mechanisms of specificity are still poorly defined. Challenges include rapid and transient engagement of enzymes and Ub/UbL interactors, low stoichiometry of modified versus non-modified cellular substrates, and protease-mediated loss of Ub/UbL in lysates. To decipher this complexity and confront the challenges, many tools have been created to trap and identify substrates and interactors linked to Ub/UbL modification. This review focuses on an assortment of biotin-based tools developed for this purpose (for example BioUbLs, UbL-ID, BioE3, BioID), taking advantage of the strong affinity of biotin-streptavidin and the stringent lysis/washing approach allowed by it, paired with sensitive mass-spectrometry-based proteomic methods. Knowing how substrates change during development and disease, the consequences of substrate modification, and matching substrates to particular UbL-ligating enzymes will contribute new insights into how Ub/UbL signaling works and how it can be exploited for therapies.

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