Exon skipping of cathepsin B: mitochondrial targeting of a lysosomal peptidase provokes cell death.
The alternatively spliced messenger RNA of the human cysteine peptidase cathepsin B missing exons 2 and 3 encodes a truncated form of the enzyme lacking the signal peptide and part of the inhibitory propeptide. This deletion results in a new N-terminal leader sequence characteristic of proteins predestined for transport into mitochondria. We determined enzyme targeting to intracellular organelles by transfecting HeLa cells with constructs containing segments of variable length of the N terminus of truncated cathepsin B fused to green fluorescent protein. Co-localization of the constructs with mitochondria and the endoplasmic reticulum was probed with specific markers. None of the chimeric products were found in the endoplasmic reticulum, showing that truncated cathepsin B is misrouted from its regular biosynthetic pathway and forced to enter the mitochondria instead of lysosomes as its final destination. The first 20 amino acids of the new N terminus were necessary and sufficient for mitochondrial targeting, but only cells expressing the complete truncated cathepsin B sequence died by nuclear fragmentation. This new and unexpected behavior draws attention to an additional extralysosomal role for a cysteine peptidase with several recognized important pathophysiological functions. Mitochondrial targeting of cathepsin B may have significant consequences on cell life in pathological or physiological situations characterized by excessive transcription of the cathepsin B message lacking exons 2 and 3, as observed for instance in osteoarthritic cartilage.
Müntener
Kathrin
K
Department of Biochemistry, University of Zurich, Winterthurerstrasse 190, CH-8057 Zurich, Switzerland.
Zwicky
Roman
R
Csucs
Gabor
G
Rohrer
Jack
J
Baici
Antonio
A
D017398:Alternative Splicing; D015153:Blotting, Western; D002401:Cathepsin B / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D016923:Cell Death; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D002498:Centrifugation; D003600:Cytosol / Q000378:metabolism; D004721:Endoplasmic Reticulum / Q000378:metabolism; D005091:Exons; D017930:Genes, Reporter; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins; D006367:HeLa Cells; D006801:Humans; D008164:Luminescent Proteins / Q000378:metabolism; D008247:Lysosomes / Q000201:enzymology*; D008856:Microscopy, Fluorescence; D008928:Mitochondria / Q000201:enzymology* / Q000378:metabolism; D008958:Models, Molecular; D010447:Peptide Hydrolases / Q000378:metabolism*; D010455:Peptides / Q000737:chemistry; D010957:Plasmids / Q000378:metabolism; D011485:Protein Binding; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D012333:RNA, Messenger / Q000378:metabolism; D014162:Transfection
D008164:Luminescent Proteins; D010455:Peptides; D012333:RNA, Messenger; D049452:Green Fluorescent Proteins; D010447:Peptide Hydrolases; D002401:Cathepsin B