Synthetic substrates for measuring activity of autophagy proteases: autophagins (Atg4)

Autophagy. 2010 Oct;6(7):936-47. doi: 10.4161/auto.6.7.13075. Epub 2010 Oct 19.

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.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Adaptor Proteins, Signal Transducing / genetics
  • Adaptor Proteins, Signal Transducing / metabolism
  • Amino Acid Sequence
  • Autophagy / physiology*
  • Autophagy-Related Protein 8 Family
  • Autophagy-Related Proteins
  • Cysteine Endopeptidases / genetics
  • Cysteine Endopeptidases / metabolism*
  • HeLa Cells
  • Humans
  • Microfilament Proteins / genetics
  • Microfilament Proteins / metabolism
  • Molecular Sequence Data
  • Molecular Structure
  • Peptide Hydrolases / metabolism*
  • Peptide Library
  • Peptides / chemistry
  • Peptides / genetics
  • Peptides / metabolism
  • Protein Isoforms / genetics
  • Protein Isoforms / metabolism*
  • Recombinant Fusion Proteins / genetics
  • Recombinant Fusion Proteins / metabolism
  • Substrate Specificity

Substances

  • Adaptor Proteins, Signal Transducing
  • Autophagy-Related Protein 8 Family
  • Autophagy-Related Proteins
  • GABARAPL2 protein, human
  • Microfilament Proteins
  • Peptide Library
  • Peptides
  • Protein Isoforms
  • Recombinant Fusion Proteins
  • Peptide Hydrolases
  • ATG4B protein, human
  • Cysteine Endopeptidases