title: Biochemical Characterization and Substrate Specificity of Autophagin-2 from the Parasite Trypanosoma cruzi.
abstract: The genome of the parasite Trypanosoma cruzi encodes two copies of autophagy-related cysteine proteases, Atg4.1 and Atg4.2. T. cruzi autophagin-2 (TcAtg4.2) carries the majority of proteolytic activity and is responsible for processing Atg8 proteins near the carboxyl terminus, exposing a conserved glycine. This enables progression of autophagy and differentiation of the parasite, which is required for successful colonization of humans. The mechanism of substrate hydrolysis by Atg4 was found to be highly conserved among the species as critical mutations in the TcAtg4.2, including mutation of the conserved Gly-244 residue in the hinge region enabling flexibility of the regulatory loop, and deletion of the regulatory loop, completely abolished processing capacity of the mutants. Using the positional scanning-substrate combinatorial library (PS-SCL) we determined that TcAtg4.2 tolerates a broad spectrum of amino acids in the P4 and P3 positions, similar to the human orthologue autophagin-1 (HsAtg4B). In contrast, both human and trypanosome Atg4 orthologues exhibited exclusive preference for aromatic amino acid residues in the P2 position, and for Gly in the P1 position, which is absolutely conserved in the natural Atg8 substrates. Using an extended P2 substrate library, which also included the unnatural amino acid cyclohexylalanine (Cha) derivative of Phe, we generated highly selective tetrapeptide substrates acetyl-Lys-Lys-Cha-Gly-AFC (Ac-KKChaG-AFC) and acetyl-Lys-Thr-Cha-Gly-AFC (Ac-KTChaG-AFC). Althoughthese substrates were cleaved by cathepsins, making them unsuitable for analysis of complex cellular systems, they were recognized exclusively by TcAtg4.2, but not by HsAtg4B nor by the structurally related human proteases SENP1, SENP2, and UCH-L3.
authors.affiliation: Department of Biochemistry and Molecular and Structural Biology, Jožef Stefan Institute, Jamova Cesta 39, SI-1000 Ljubljana, Slovenia; Jožef Stefan International Postgraduate School, Jamova Cesta 39, SI-1000 Ljubljana, Slovenia.
authors.forename: Jelena
authors.initials: J
authors.lastname: Rajković
authors.affiliation: Department of Bioorganic Chemistry, Faculty of Chemistry, Wroclaw University of Technology, Wyb. Wyspianskiego 27, 50-370 Wroclaw, Poland.
authors.forename: Marcin
authors.initials: M
authors.lastname: Poreba
authors.affiliation: Department of Biochemistry and Molecular and Structural Biology, Jožef Stefan Institute, Jamova Cesta 39, SI-1000 Ljubljana, Slovenia; Department of Chemistry.
authors.forename: Dejan
authors.initials: D
authors.lastname: Caglič
authors.affiliation: Department of Biochemistry and Molecular and Structural Biology, Jožef Stefan Institute, Jamova Cesta 39, SI-1000 Ljubljana, Slovenia; Jožef Stefan International Postgraduate School, Jamova Cesta 39, SI-1000 Ljubljana, Slovenia.
authors.forename: Robert
authors.initials: R
authors.lastname: Vidmar
authors.affiliation: Department of Bioorganic Chemistry, Faculty of Chemistry, Wroclaw University of Technology, Wyb. Wyspianskiego 27, 50-370 Wroclaw, Poland.
authors.forename: Aleksandra
authors.initials: A
authors.lastname: Wilk
authors.affiliation: Department of Bioorganic Chemistry, Faculty of Chemistry, Wroclaw University of Technology, Wyb. Wyspianskiego 27, 50-370 Wroclaw, Poland.
authors.forename: Agata
authors.initials: A
authors.lastname: Borowik
authors.affiliation: Sanford-Burnham Medical Research Institute, Program in Cell Death and Survival Networks, The Scripps Research Institute, La Jolla, California 92037.
authors.forename: Guy
authors.initials: G
authors.lastname: Salvesen
authors.affiliation: Department of Biochemistry and Molecular and Structural Biology, Jožef Stefan Institute, Jamova Cesta 39, SI-1000 Ljubljana, Slovenia.
authors.forename: Vito
authors.initials: V
authors.lastname: Turk
authors.affiliation: Department of Bioorganic Chemistry, Faculty of Chemistry, Wroclaw University of Technology, Wyb. Wyspianskiego 27, 50-370 Wroclaw, Poland.
authors.forename: Marcin
authors.initials: M
authors.lastname: Drag
authors.affiliation: Department of Biochemistry and Molecular and Structural Biology, Jožef Stefan Institute, Jamova Cesta 39, SI-1000 Ljubljana, Slovenia; Centre of Excellence for Integrated Approaches in Chemistry and Biology of Proteins, Jamova Cesta 39, SI-1000 Ljubljana, Slovenia; Faculty of Chemistry and Chemical Technology, University of Ljubljana, Jamova Cesta 39, SI-1000 Ljubljana, Slovenia. Electronic address: boris.turk@ijs.si.
authors.forename: Boris
authors.initials: B
authors.lastname: Turk
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001343:Autophagy; D003546:Cysteine Endopeptidases / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D059748:Proteolysis; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D014349:Trypanosoma cruzi / Q000201:enzymology*
chemical_list: D003546:Cysteine Endopeptidases
keywords: Trypanosoma cruzi; autophagin (Atg4); autophagy; autophagy-related protein 8 (ATG8); cysteine protease; positional-scanning substrate combinatorial library (PS-SCL); proteolysis; substrate specificity