title: N-terminally truncated forms of human cathepsin F accumulate in aggresome-like inclusions.
abstract: The contribution of individual cysteine cathepsins as positive mediators of programmed cell death is dependent on several factors, such as the type of stimuli, intensity and duration of the stimulus, and cell type involved. Of the eleven human cysteine cathepsins, cathepsin F is the only cathepsin that exhibits an extended N-terminal proregion, which contains a cystatin-like domain. We predicted that the wild-type human cathepsin F contains three natively disordered regions within the enzyme's propeptide and various amino acid stretches with high fibrillation propensity. Wild-type human cathepsin F and its N-terminally truncated forms, Ala(20)-Asp(484) (Δ(19)CatF), Pro(126)-Asp(484) (Δ(125)CatF), and Met(147)-Asp(484) (Δ(146)CatF) were cloned into the pcDNA3 vector and overexpressed in HEK 293T cells. Wild-type human cathepsin F displayed a clear vesicular labeling and colocalized with the LAMP2 protein, a lysosomal marker. However, all three N-terminally truncated forms of human cathepsin F were recovered as insoluble proteins, suggesting that the deletion of at least the signal peptides (Δ(19)CatF), results in protein aggregation. Noteworthy, they concentrated large perinuclear-juxtanuclear aggregates that accumulated within aggresome-like inclusions. These inclusions showed p62-positive immunoreactivity and were colocalized with the autophagy marker LC3B, but not with the LAMP2 protein. In addition, an approximately 2-3 fold increase in DEVDase activity was not sufficient to induce apoptotic cell death. These results suggested the clearance of the N-terminally truncated forms of human cathepsin F via the autophagy pathway, underlying its protective and prosurvival mechanisms.
authors.affiliation: Department of Biochemistry and Molecular and Structural Biology, J. Stefan Institute, Ljubljana, Slovenia.
authors.forename: Barbara
authors.initials: B
authors.lastname: Jerič
authors.forename: Iztok
authors.initials: I
authors.lastname: Dolenc
authors.forename: Marko
authors.initials: M
authors.lastname: Mihelič
authors.forename: Martina
authors.initials: M
authors.lastname: Klarić
authors.forename: Tina
authors.initials: T
authors.lastname: Zavašnik-Bergant
authors.forename: Gregor
authors.initials: G
authors.lastname: Gunčar
authors.forename: Boris
authors.initials: B
authors.lastname: Turk
authors.forename: Vito
authors.initials: V
authors.lastname: Turk
authors.forename: Veronika
authors.initials: V
authors.lastname: Stoka
mesh_terms: D048868:Adaptor Proteins, Signal Transducing / Q000378:metabolism*; D000595:Amino Acid Sequence; D017209:Apoptosis; D001343:Autophagy; D015153:Blotting, Western; D020169:Caspases / Q000378:metabolism*; D056646:Cathepsin F / Q000235:genetics / Q000378:metabolism*; D002478:Cells, Cultured; D004789:Enzyme Activation; D005455:Fluorescent Antibody Technique; D006031:Glycosylation; D006801:Humans; D007124:Immunoenzyme Techniques; D052119:Lysosomal-Associated Membrane Protein 2; D051907:Lysosomal Membrane Proteins / Q000378:metabolism*; D008869:Microtubule-Associated Proteins / Q000378:metabolism*; D008969:Molecular Sequence Data; D010957:Plasmids; D055503:Protein Multimerization; D000071456:Sequestosome-1 Protein; D013347:Subcellular Fractions
chemical_list: D048868:Adaptor Proteins, Signal Transducing; C497811:LAMP2 protein, human; D052119:Lysosomal-Associated Membrane Protein 2; D051907:Lysosomal Membrane Proteins; C502070:MAP1LC3B protein, human; D008869:Microtubule-Associated Proteins; C099718:SQSTM1 protein, human; D000071456:Sequestosome-1 Protein; D020169:Caspases; D056646:Cathepsin F
keywords: Aggregation-prone; Aggresome; Aggresome-like inclusion; Autophagy; Caspase activation; Cathepsin F; HRP; Mw; horseradish peroxidase; molecular weight; truncated form of human cathepsin F (Ala(20)–Asp(484)); truncated form of human cathepsin F (Met(147)–Asp(484)); truncated form of human cathepsin F (Pro(126)–Asp(484)); wild-type cathepsin F; wtCatF; Δ(125)CatF; Δ(146)CatF; Δ(19)CatF