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, 6 (7), 936-47

Synthetic Substrates for Measuring Activity of Autophagy Proteases: Autophagins (Atg4)

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Synthetic Substrates for Measuring Activity of Autophagy Proteases: Autophagins (Atg4)

Chih-Wen Shu et al. Autophagy.

Abstract

Atg4 cysteine proteases (autophagins) play crucial roles in autophagy by proteolytic activation of Atg8 paralogs for targeting to autophagic vesicles by lipid conjugation, as well as in subsequent deconjugation reactions. However, the means to measure the activity of autophagins is limited. Herein, we describe two novel substrates for autophagins suitable for a diversity of in vitro assays, including (i) fluorogenic tetrapeptide acetyl-Gly-L-Thr-L-Phe-Gly-AFC (Ac-GTFG-AFC) and (ii) a fusion protein comprised of the natural substrate LC3B appended to the N-terminus of phospholipase A(2) (LC3B-PLA(2)), which upon cleavage releases active PLA(2) for fluorogenic assay. To generate the synthetic tetrapeptide substrate, the preferred tetrapeptide sequence recognized by autophagin-1/Atg4B was determined using a positional scanning combinatorial fluorogenic tetrapeptide library. With the LC3B-PLA(2) substrate, we show that mutation of the glycine proximal to the scissile bond in LC3B abolishes activity. Both substrates showed high specificity for recombinant purified autophagin-1/Atg4B compared to closely related proteases and the LC3B-PLA(2) substrate afforded substantially higher catalytic rates (k(cat)/K(m) 5.26 x 10(5) M(-1)/sec(-1)) than Ac-GTFG-AFC peptide (0.92 M(-1)/sec(-1)), consistent with substrate-induced activation. Studies of autophagin-1 mutants were also performed, including the protease lacking a predicted autoinhibitory domain at residues 1 to 24 and lacking a regulatory loop at residues 259 to 262. The peptide and fusion protein substrates were also employed for measuring autophagin activity in cell lysates, showing a decrease in cells treated with autophagin-1/Atg4B siRNA or transfected with a plasmid encoding Atg4B (Cys74Ala) dominantnegative. Therefore, the synthetic substrates for autophagins reported here provide new research tools for studying autophagy.

Figures

Figure 1
Figure 1
Recombinant Atg4B protease cleaves Atg8 substrates in vitro. (A) Schematic of recombinant wild-type Atg4B protease, catalytic mutant Atg4B C74A and its substrate Atg8 paralogs are depicted. Atg4B and a mutant C74A were expressed in bacteria fused with His6-tag at N-terminus. Atg8 paralogs were expressed in bacteria with fused His6-tag and S-tag at N- and C-terminus, respectively. (B) His6-tagged Atg4B, catalytic mutant Atg4B C74A and His- and S-tagged Atg8 paralogs including LC3B, LC3C, GABARAP and GATE-16 were expressed in E. coli BL21 DE3 cells. Proteins were purified by Ni-NTA resin. The purity of proteins was assessed by SDS-PAGE analysis and staining with GelCode Blue. Stars indicate partially degraded bands in preparations of recombinant LC3B and GABARAP (C), 100 nM recombinant Atg4B wild-type (WT) or C74A mutant was incubated with 400 nM of the various substrates in reaction buffer (50 mM Tris, pH 8.0, 150 mM NaCl and 1 mM DTT) with or without 10 mM iodoacetic acid (IAA) at 37°C for 1 h. Cleavage of protein substrates was visualized by SDS-PAGE analysis with GelCode Blue staining (GB, top part) and by immunoblotting using anti-S-tag antibody (WB, bottom part). Molecular weight markers are indicated in kilodaltons (kDa). Stars indicate partially degraded bands in preparations of recombinant LC3B and GABARAP.
Figure 2
Figure 2
Estimating activity of recombinant Atg4B using full-length Atg8 paralog substrates. (A) Atg4B WT or C74A mutant (1 nM) was incubated with 400 nM Atg8 paralogs at 37°C for various times as indicated. Cleavage of substrates was determined by immunoblotting using anti-S-tag antibody. (B) Atg8 paralogs (400 nM) were incubated with two-fold serial dilutions of wild-type Atg4B in reaction buffer (50 mM Tris, pH 8.0, 150 mM NaCl and 1 µM DTT) at 37°C for 1 h (where maximum final concentration of Atg4B is either 1 or 10 nM). Proteins were subjected to SDS-PAGE and stained with GelCode Blue (GB, top part for each pair). Arrows indicate dilution at which cleavage is detectable. The cleavage of Atg8 paralogs was further analyzed by immunoblotting using anti-S-tag antibody (WB, bottom part for each pair). EC50 for different substrates was indicated with arrowhead. (C), EC50 values for Atg4B cleavage of LC3B were used for calculating catalytic efficiency kcat/Km. Molecular weight markers are indicated in kilodaltons (kDa). Data represent the mean ± S.D. of three experiments.
Figure 3
Figure 3
Synthetic substrate specificity of Atg4B. (A) The positional scanning strategy is depicted for using combinatorial peptide library to define optimal substrate sequence for Atg4B. (B) Summary of cleavage of fluorogenic tetrapeptides is presented. The enzyme concentration was 6–8 µM. ACC production was monitored with assay times varying from 15–60 min. Standard deviation for each measurement shown is <20%. The x-axis indicates the amino-acid tested at the P2, P3 or P4 positions (standard single letter code for natural L-amino acids; O, nor-leucine, hC, cyclohexylalanine, hP, homo-phenylalanine). The y-axis represents the average relative activity expressed as a percent of the best amino acid. ACC fluorescence was monitored using an fmax multiwell fluorescence plate reader at excitation wavelength of 355 nm and an emission wavelength of 460 nm for 15–60 mins.
Figure 4
Figure 4
Characterization of Atg4B activity using synthetic peptide substrates. (A) Various concentrations of recombinant Atg4B were incubated with 100 µM synthetic tetrapeptides conjugated with AFC in 50 mM Tris-HCl, pH 8.0, 5 mM DTT at 37°C for 30 min. Data are expressed at RFU per sec (mean ± S.D.; n = 3). (B) 1 µM Atg4B was incubated with two-fold serial dilutions of 100 µM synthetic tetrapeptide substrates at 37°C for 30 min. kcat/Km was calculated using AFC standard curve. (C) Activity of Atg4B was assayed in buffers (50 mM) titrated to various pH values as indicated in the presence of 150 mM NaCl and 5 mM DTT. The concentrations of Atg4B and Ac-GTFG-AFC were 1 µM and 50 µM, respectively. (D) Activity of Atg4B in 50 mM Tris-HCl buffer, pH 8.0, 5 mM DTT with different Hofmeister salts was assayed at 37°C for 30 min. The non-Hofmeister salt NaCl was used as a control to rule out effects from alteration of ionic strength. (E) 1 µM Atg4B was assayed with 50 µM Ac-GTFG-AFC in 50 µM Tris-HCl buffer, pH 8.0, 5 mM DTT in the presence of Na2Citrate (solid line) or (NH4)2Citrate (dashed line). (F) 1 µM Atg4B was assayed with 50 µM Ac-GTFG-AFC in 50 mM Tris-HCl buffer, pH 8.0, 5 mM DTT in the presence of various amounts of glycerol. Effect of different Hofmeister salts or glycerol on activity was expressed as fold increase. AFC production was measured with an excitation wavelength of 400 nm and an emission wavelength of 505 nm. Data represent the mean ± S.D. of four experiments.
Figure 5
Figure 5
Generation and characterization of a novel LC3B-PLA2 reporter substrate for Atg4B. (A) Scheme depicting basis for the LC3B-PLA2 reporter assay for monitoring Atg4B activity. Atg4B cleaves Gly117 site of proLC3B to release catalytically active PLA2, which hydrolyzes NBD-C6-HPC, generating fluorescence. LC3B-PLA2 fusion proteins were generated with various cleavage sites, containing the natural ETFG, altered GTFG or noncleavable ETFA sequences. (B) two-fold serial dilutions of Atg4B (maximum final concentration 1 nM) were incubated with 400 nM wild-type (ETFG) or mutant (GTFG, ETFA) LC3B-PLA2 fusion proteins at 37°C for 1 h. Cleavage of LC3B-PLA2 proteins was determined by SDS-PAGE with GelCode Blue staining. (C) Atg4B (0.05 nM) was mixed with two-fold dilutions of substrates to give a concentration range from ∼1.5–100 nM LC3B-PLA2 wild-type (ETFG) fusion protein in PLA2 reaction buffer at 37°C. PLA2 activity was monitored by hydrolysis of NBD-C6-HPC, measuring fluorescence with excitation and emission filters of 485 nm and 530 nm, respectively and reporting results as RFU (left part). Data points obtained with 25, 50 or 100 nM substrate are represented by the symbols of square, triangle and circle, respectively. Computer-generated curves were fit to the data for kcat/Km calculation of Atg4B. The deduced catalytic efficiency kcat/Km of Atg4B as measured with the various LC3B-PLA2 reporter substrates is shown in the right part. Data represent the mean ± S.D. of three experiments.
Figure 6
Figure 6
Structure-function analysis of Atg4B. (A) His6-tagged recombinant wild-type Atg4B (WT), N-terminal deletion (dN), regulatory loop deletion (dL) or catalytic mutant (C74A) proteins were expressed in bacteria and purified by Ni-NTA resin. Purified Atg4B proteins (1 nM) were incubated with 400 nM LC3B at 37°C for 1 h. The purity of recombinant proteins (left) and cleavage of LC3B (right) were accessed by SDS-PAGE analysis and staining with GelCode Blue (GB). In addition, LC3B cleavage was assessed by immunoblotting using anti-S-tag antibody as described for Figure 1 (WB). (B) Activity of wild-type and mutant Atg4B proteins (1 µM) was assayed using 100 µM Ac-GTFG-AFC in 50 mM Tris-HCl, pH 8.0, 5 mM DTT, with 0, 0.8 M or 1.2 M sodium citrate, at 37°C. The activity of Atg4B mutants was normalized relative to activity of wild-type Atg4B for each buffer condition and expressed as % control. (C), WT (diamonds), dN (circles), dL (triangles) and C74A mutant (squares) Atg4B proteins (0.1 nM) were assayed using various concentrations of LC3B-PLA2 in PLA2 reaction buffer at room temperature for 30 min. Data are expressed as % relative to WT Atg4B and represent mean ± S.D. of four experiments.
Figure 7
Figure 7
Specificity of tetrapeptide and LC3B-PLA2 substrates. (A) 1 µM of recombinant Atg4 enzymes (Atg4A or Atg4B), desumolating enzymes (SENP-1 or -2) or deubiquitinating enzyme (UCH-L3) was mixed with 50 µM Ac-GTFG-AFC at 37°C for 45 min to determine the specificity of the synthetic tetrapeptide (left part). Data represent percentage activity relative to Atg4B (mean ± std dev, n = 4). In addition, 1 µM Atg4B was combined with various concentrations of 100 nM Atg4B inhibitor LC3-vinyl sulfone (right part), measuring Atg4B activity using Ac-GTFG-AFC peptide as above. Data represent % activity relative to reactions without LC3-vinyl sulfone (mean ± std dev; n = 4). (B) Various enzymes (0.1 nM) were assayed using 20 nM LC3B-PLA2 as the substrate (left part). Atg4B was further assayed with Atg4B inhibitor LC3-vinyl sulfone (right part). (C) HeLa S3 cells were lysed by sonication to extract soluble proteins (1 [LC3B-PLA2] or 50 [Ac-GTFG-AFC] µg per reaction) for assaying Ac-GTFG-AFC (1 µM) or LC3B-PLA2 (100 nM) hydrolytic activity in the presence (+) or absence (−) of 1 µM (Ac-GTFG-AFC) or 100 nM (LC3B-PLA2) LC3-Vinyl Sulfone. (D) siRNA against Atg4B or Flag tagged dominant-negative Atg4B C74A was transfected into HeLa S3 cells. Efficiency of Atg4B siRNA knockdown or Atg4B C74A overexpression was determined by immunoblotting (middle part) using 20 µg total protein per lane and probing blots with antibodies to Atg4B (top) and b-actin (bottom). Cellular proteins extracted as above were incubated with 50 µM Ac-GTFG-AFC in 50 mM Tris-HCl, pH 8.0, 0.8 M sodium citrate, 5 mM DTT buffer at 37°C to measure Atg4B activity (left part). Extracted cellular proteins were also assayed with 200 nM LC3B-PLA2 fusion protein in PLA2 assay buffer in the presence of 20 µM NBD C6-HPC at room temperature for 30 min (right part). Data represent mean ± S.D. of three experiments.

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