title: Synthetic substrates for measuring activity of autophagy proteases: autophagins (Atg4).
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.
authors.affiliation: Sanford-Burnham Medical Research Institute, Program on Apoptosis and Cell Death Research, La Jolla, CA, USA.
authors.forename: Chih-Wen
authors.initials: CW
authors.lastname: Shu
authors.forename: Marcin
authors.initials: M
authors.lastname: Drag
authors.forename: Miklos
authors.initials: M
authors.lastname: Bekes
authors.forename: Dayong
authors.initials: D
authors.lastname: Zhai
authors.forename: Guy S
authors.initials: GS
authors.lastname: Salvesen
authors.forename: John C
authors.initials: JC
authors.lastname: Reed
mesh_terms: D048868:Adaptor Proteins, Signal Transducing / Q000235:genetics / Q000378:metabolism; D000595:Amino Acid Sequence; D001343:Autophagy / Q000502:physiology*; D000071190:Autophagy-Related Protein 8 Family; D000071183:Autophagy-Related Proteins; D003546:Cysteine Endopeptidases / Q000235:genetics / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D008840:Microfilament Proteins / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D015394:Molecular Structure; D010447:Peptide Hydrolases / Q000378:metabolism*; D019151:Peptide Library; D010455:Peptides / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D020033:Protein Isoforms / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity
chemical_list: D048868:Adaptor Proteins, Signal Transducing; D000071190:Autophagy-Related Protein 8 Family; D000071183:Autophagy-Related Proteins; C498698:GABARAPL2 protein, human; D008840:Microfilament Proteins; D019151:Peptide Library; D010455:Peptides; D020033:Protein Isoforms; D011993:Recombinant Fusion Proteins; D010447:Peptide Hydrolases; C507524:ATG4B protein, human; D003546:Cysteine Endopeptidases
references.citation: J Cell Biol. 1999 Oct 18;147(2):435-46
references.pmid: 10525546
references.citation: Science. 2004 Jun 4;304(5676):1500-2
references.pmid: 15131264
references.citation: Biochem J. 1997 Jun 1;324 ( Pt 2):361-4
references.pmid: 9182691
references.citation: Cell. 2008 Jan 11;132(1):27-42
references.pmid: 18191218
references.citation: Protein Sci. 2008 Jun;17(6):1035-43
references.pmid: 18424514
references.citation: J Biol Chem. 2007 Jun 22;282(25):18573-18583
references.pmid: 17442669
references.citation: J Biol Chem. 2003 Dec 19;278(51):51841-50
references.pmid: 14530254
references.citation: Cell. 2005 Sep 23;122(6):927-39
references.pmid: 16179260
references.citation: Science. 2006 May 26;312(5777):1160-1
references.pmid: 16728626
references.citation: EMBO J. 2007 Apr 4;26(7):1749-60
references.pmid: 17347651
references.citation: Methods Enzymol. 2000;322:91-100
references.pmid: 10914007
references.citation: J Cell Biol. 2001 Feb 19;152(4):657-68
references.pmid: 11266458
references.citation: J Cell Sci. 2004 Jun 1;117(Pt 13):2805-12
references.pmid: 15169837
references.citation: FEBS Lett. 1993 Oct 25;333(1-2):169-74
references.pmid: 8224160
references.citation: Nat Rev Mol Cell Biol. 2009 Jul;10(7):458-67
references.pmid: 19491929
references.citation: Nature. 1998 Sep 24;395(6700):395-8
references.pmid: 9759731
references.citation: FEBS Lett. 1994 Aug 1;349(2):275-80
references.pmid: 8050581
references.citation: Cell. 2007 Jul 13;130(1):165-78
references.pmid: 17632063
references.citation: J Biol Chem. 1997 Jul 18;272(29):17907-11
references.pmid: 9218414
references.citation: Nature. 2008 Feb 28;451(7182):1069-75
references.pmid: 18305538
references.citation: J Biol Chem. 2004 Aug 27;279(35):36268-76
references.pmid: 15187094
references.citation: Nature. 1981 Feb 12;289(5798):604-6
references.pmid: 7464926
references.citation: J Biol Chem. 2004 Nov 12;279(46):47704-10
references.pmid: 15355958
references.citation: J Biol Chem. 2003 Feb 7;278(6):3671-8
references.pmid: 12446702
references.citation: EMBO J. 2009 May 6;28(9):1341-50
references.pmid: 19322194
references.citation: Biochem J. 2008 Jan 15;409(2):461-9
references.pmid: 17916063
references.citation: J Cell Sci. 2009 Jul 15;122(Pt 14):2554-66
references.pmid: 19549685
references.citation: J Biol Chem. 2001 Jan 19;276(3):1701-6
references.pmid: 11096062
references.citation: J Biol Chem. 2007 Sep 7;282(36):26217-24
references.pmid: 17591783
references.citation: J Cell Biol. 2000 Oct 16;151(2):263-76
references.pmid: 11038174
references.citation: Biochem J. 2008 Nov 1;415(3):367-75
references.pmid: 18601651