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    • Heme Colorimetric Assay Kit br The Structure and Regulatory

    2020-08-06


    The Structure and Regulatory Machinery of Atg4 Atg4 proteases range from 393 to 474 Heme Colorimetric Assay Kit in size and possess several structural features of cysteine proteases. The crystal structure of Atg4A (PDB ID: 2P82) has been resolved (Fig. 2A). Atg4A shares a similar catalytic triad (Cys77/Asp279/His281) with Atg4B (Marino et al., 2003). Structure studies have shown that all the residues of Atg4B that interact with LC3B are conserved in Atg4A except Leu232. Atg4A possesses Ile233 instead of Leu232 at the corresponding position. When Ile233 is changed to Leu, the mutated Atg4A acquires a notable ability to cleave LC3B (Satoo et al., 2009). The crystal structure of human Atg4B (PDB ID: 2CY7) has also been resolved (Fig. 2B). The structure of Atg4B contains a papain-like fold and a small α/β-fold domain, which is thought to be the binding sites for Atg8 homologues. The active site of Atg4B is composed of Cys74, Asp278, and His280. Mutation of these sites is associated with the complete loss of its catalytic activity (Sugawara et al., 2005). The active site of free Atg4B is masked by a regulatory loop (residues 259–262) (Satoo et al., 2009). A large conformational change of Atg4B is induced in the regulatory loop and the N-terminal tail (residues 1–24) when Atg4B interacts with LC3 (Fig. 2C and D). In this process, the regulatory loop masking the entrance of the active site of Atg4B is lifted by LC3 Phe119, resulting in the formation of a groove, into which the LC3 tail can enter, gaining the access to the active site. Besides, the N-terminal tail is originally positioned at the back of the active sites and undergoes a large conformational change as well upon interaction with LC3, which could affect the exit of the cleaved substrates. Thus, deletion of this N-terminal region has been found to increase Atg4B activity (Satoo et al., 2009). 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 acid 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).