Although the crystal structures of Atg C and
Although the crystal structures of Atg4C and Atg4D are not available, their three dimensional structures can be acquired by homology modeling, using the structure of Atg4B as a template (Zhang, Li, Ouyang, Liu, & Cheng, 2016). In that model, the catalytic triad is conserved (Cys110/Asp345/His347 for Atg4C and Cys134/Asp356/His358 for Atg4D). It is of note that Atg4C and Atg4D have longer sequences than Atg4A and Atg4B, and the catalytic triad may be spatially positioned differently, rendering access to the substrate difficult. Interestingly, deletion of the N-terminal 63 amino acids of Atg4D stimulated its catalytic activity toward Atg8L (Betin & Lane, 2009), indicating that structural changes of Atg4D could improve substrate access to the active sites.
Overview of the Methods to Detect the Atg4 Activity In Vitro and Ex Vivo The classical method to measure Atg4 activity is based on electrophoretic separation of cleaved Atg8 substrates with a C-terminal tag after the cleavage site (Kabeya et al., 2000, Kirisako et al., 2000, Sugawara et al., 2005). This method is described in details later. By measuring the amount of cleaved band of Atg8 migrating at the expected position on SDS-PAGE, one can determine the activity of Atg4. Compared with the SDS-PAGE-based assay, adoption of specific fluorogenic substrates can make the process more automated and convenient. One example is the use of a site-specific fluorogenic tetrapeptide (Shu et al., 2010), such as acetyl-Gly-Thr-Phe-Gly-AFC (Ac-GTFG-AFC), which is close to the cleavage site (Fig. 1). Release of the fluorogenic AFC following Atg4 cleavage can be detected with a fluorescence spectrometer. A concern on the use of the short 4-amino PF-562271 sale peptide is that the cleavage efficiency by Atg4 is quite low (Shu et al., 2010, Vezenkov et al., 2015) (M. Li and X.-M. Yin, unpublished observations). Although different peptide substrates have been optimized with self-immolative linker, which might be convenient for single point read assays, the KM of such peptides are still 10-fold higher than that measured with full-length LC3 (Vezenkov et al., 2015). Another in vitro system uses the enzyme phospholipase A2, which is inactivated as the result of fusion to the C-terminus of LC3B adjacent to the cleavage site (Ketteler and Seed, 2008, Shu et al., 2010). When the fusion protein is cleaved by Atg4B, PLA2 is released to regain its activity, which can be determined using a separate substrate for PLA2. Although this method is sensitive, quantitative, and suitable for high-throughput screening (HTS), the Atg4 activity is indirectly measured based on PLA2 activity and thus could be affected by any agents that interfere with the PLA2 reaction. Methods to measure ex vivo Atg4 activity have been developed. One early study fused Gaussia princeps luciferase (GLUC) and β-actin to the N- and C-terminus of LC3, respectively (Tannous, Kim, Fernandez, Weissleder, & Breakefield, 2005). When expressed in cells, the fusion protein is anchored to the cytoskeleton via β-actin and the soluble cytosol contains few GLUC activity. The increased GLUC activity in the cytosol represents that of Atg4B, which cleaves the fusion protein, releasing it from the cytoskeleton. This method does not interrupt normal metabolism in cells with the overexpression of the substrate. As this is an indirect method, any factors that affect β-actin interaction with the substrates or affect GLUC activity can lead to assay instability, and require multiple parallel controls. Simple one-reaction designs for ex vivo measurement have been reported, which considerably reduce the complexity and interferences. In one assay (Choi et al., 2011), FITC-labeled Atg4-substrate peptides are conjugated to polymeric nanoparticles, which are highly permeable to cells. Once inside the cells, the substrates are cleaved by Atg4, releasing and dequenching the labeled fluorescent dye. The fluorescence is measured and the intensity reflects the Atg4 activity. In another assay, two cell penetrating peptides are used (Ni et al., 2015). One peptide named as AU4S contains the Atg4-recognizable motif “GTFG” and the fluorophore FITC. The other peptide named as AU4R is used as a control, and is not recognized and cleaved by Atg4. The fluorescence ratio defined as “F–D value” between AU4S and AU4R changes as the Atg4 activity. The concern on the ex vivo use of the short 4-amino acid peptide is the same as in the in vitro case, that is, the cleavage efficiency of Atg4 on peptides is quite low (Shu et al., 2010, Vezenkov et al., 2015) (M. Li and X.-M. Yin, unpublished observations). Thus, the use of full-length substrate would be preferred to better reflect Atg4 activity. Toward that end a fluorescence resonance energy transfer (FRET)-based ex vivo assay has been developed (Li, Chen, Ye, Vogt, & Yin, 2012), which is detailed in the following section.