Lysosomal Serine Protease CLN2 Regulates Tumor Necrosis Factor-α-mediated Apoptosis in a Bid-dependent Manner

doi:10.1074/jbc.M807151200

Lysosomal Serine Protease CLN2 Regulates Tumor Necrosis Factor-α-mediated Apoptosis in a Bid-dependent Manner^*

^{^‡}INSERM U858, 31432 Toulouse, ^{^§}Université Toulouse III Paul Sabatier, Institut de Médecine Moléculaire de Rangueil, IFR31, 31432 Toulouse, ^{^¶}INSERM U567, Institut Cochin, Université Paris Descartes, CNRS (UMR 8104), 75014 Paris, and ^{^∥}Laboratoire de Biochimie Métabolique, CHU de Toulouse, 31432 Toulouse, France

1 To whom correspondence should be addressed: U858 INSERM, Institut de Médecine Moléculaire de Rangueil BP84225, 31432 Toulouse Cedex 4, France. Tel.: 33-561-32-35-31; Fax: 33-561-32-20-84; E-mail: nathalie.andrieu{at}inserm.fr.

Abstract

Apoptosis is a highly organized, energy-dependent program by which multicellular organisms eliminate damaged, superfluous, and potentially harmful cells. Although caspases are the most prominent group of proteases involved in the apoptotic process, the role of lysosomes has only recently been unmasked. This study investigated the role of the lysosomal serine protease CLN2 in apoptosis. We report that cells isolated from patients affected with late infantile neuronal ceroid lipofuscinosis (LINCL) having a deficient activity of CLN2 are resistant to the toxic effect of death ligands such as tumor necrosis factor (TNF), CD95 ligand, or tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) but not to receptor-independent stress agents. CLN2-deficient cells exhibited a defect in TNF-induced Bid cleavage, release of cytochrome c, and caspase-9 and -3 activation. Moreover, extracts from CLN2-overexpressing cells or a CLN2 recombinant protein were able to catalyze the in vitro cleavage of Bid. Noteworthy, correction of the lysosomal enzyme defect of LINCL fibroblasts using a medium enriched in CLN2 protein enabled restoration of TNF-induced Bid and caspase-3 processing and toxicity. Conversely, transfection of CLN2-corrected cells with small interfering RNA targeting Bid abrogated TNF-induced cell death. Altogether, our study demonstrates that genetic deletion of the lysosomal serine protease CLN2 and the subsequent loss of its catalytic function confer resistance to TNF in non-neuronal somatic cells, indicating that CLN2 plays a yet unsuspected role in TNF-induced cell death.

Footnotes

↵² The abbreviations used are: TNF, tumor necrosis factor-α; TNFR, TNF receptor; AAF-AMC, H-Ala-Ala-Phe amino methylcoumarin; AAF-cmk, H-Ala-Ala-Phe-fluoromethyl ketone; Ac-DEVD-AMC, Ac-Asp-Glu-Val-Asp-aminomethylcoumarin; CLN, ceroid lipofuscinosis; FCS, fetal calf serum; LINCL, late-infantile neuronal ceroid lipofuscinosis; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; AEBSF, 4-(2-aminoethyl)-benzenesulfonyl fluoride hydrochloride; TRAIL, tumor necrosis factor-related apoptosis-inducing ligand; CHO, Chinese hamster ovary; PBS, phosphate-buffered saline; PMSF, phenylmethanesulfonyl fluoride; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; DMEM, Dulbecco's modified Eagle's medium; mAb, monoclonal antibody; siRNA, small interfering RNA.
↵³ Upon incubation with TNF, CLN2 redistributed from lysosomes to the cytoplasm as indicated by a diffuse staining observed by immunocytochemical studies (V. Albinet, T. Levade, and N. Andrieu-Abadie, unpublished observation).
↵^* This work was supported by grants from INSERM, Université Paul Sabatier, the Association Vaincre les Maladies Lysosomales, the Fédération pour la Recherche sur le Cerveau, and Groupement d'Intérêt Scientifique Institut des Maladies Rares.
- Received September 16, 2008.
- Revision received February 12, 2009.
The American Society for Biochemistry and Molecular Biology, Inc.