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. Versatile biotin-based tools are highlighted here for studying ubiquitin- and ubiquitin-like modifications in cells and organisms. These powerful proteomic methods can eventually be coupled with other chemical biology concepts (e. g. induced proximity pharmacology, targeted protein degradation, PROTACs, molecular glues, etc.) for addressing specificity and unraveling mechanisms in the complex ubiquitin signaling system. Created with BioRender.com.+image