br Expression and Purification of Atg and
Expression and Purification of Atg4 and Atg8 Proteins for In Vitro Assays
Measurement of Atg4 Activity In Vitro and Ex Vivo
Kinetics Analysis of Atg4 Enzymes
Summary Atg4 proteases play a crucial role in preparing Atg8 for conjugation to lipid membranes and for the deconjugation of Atg8 from the autophagosomes (Till & Subramani, 2010). Of the four proteases, Atg4B GNE-317 1500-fold more catalytically efficient for LC3B activation than the other Atg4s, whereas Atg4A is most selective toward GATE-16 (Li et al., 2011). Atg4B and LC3B are best studied in autophagy and Atg4B seems to be most potent (Nakatogawa et al., 2007, Tanida et al., 2004). These findings suggest that Atg4B could be a potential target for inhibiting autophagy. HTS assays for Atg4B inhibitors have been conducted (Li et al., 2012, Shu et al., 2012, Xu et al., 2016), but it is still a long journey to the availability of practically useful agents with sufficient potency and specificity. We hope that the methods discussed in this chapter will further stimulate studies of Atg4 enzymes and the development of Atg4 modulators.
The Conflict-of-Interest and Financial Disclosure Statements: There were no potential conflicts of interest and no significant financial support for this work to disclose.