Cathepsin D links TNF-induced acid sphingomyelinase to Bid-mediated caspase-9 and -3 activation

Area of Science:

• Cell Biology
• Molecular Biology
• Biochemistry

Background:

• Acidic noncaspase proteases, such as cathepsins, are implicated in apoptosis.
• The precise role of these proteases and the endolysosomal compartment in apoptotic signaling requires further elucidation.
• Understanding the interplay between noncaspases and the caspase pathway is crucial for defining their role in cell death.

Purpose of the Study:

• To investigate the role of cathepsin D (CTSD) in tumor necrosis factor (TNF)-mediated apoptosis.
• To determine the relationship between CTSD, acid sphingomyelinase (A-SMase), and the mitochondrial apoptotic pathway.
• To identify downstream targets of CTSD in the apoptotic cascade.

Main Methods:

• Utilized CTSD-deficient fibroblasts and ectopic CTSD expression to assess apoptotic responses.
• Investigated the colocalization of CTSD with the proapoptotic protein Bid using immunofluorescence microscopy.
• Performed in vitro cleavage assays to confirm CTSD's enzymatic activity on Bid.
• Analyzed caspase-9 and caspase-3 activation in A-SMase and CTSD deficiency models.

Main Results:

• TNF-induced CTSD activation was dependent on functional A-SMase expression.
• Ectopic CTSD expression enhanced TNF-mediated apoptosis in deficient cells.
• CTSD directly cleaved Bid in vitro, and Bid activation was impaired in cathepsin-deficient cells, identifying Bid as a downstream target.
• CTSD and Bid colocalized within endosomal compartments (Rab5-positive).
• Caspase-9 and caspase-3 activation were partially dependent on A-SMase and CTSD.

Conclusions:

• Novel endosomal intermediates, A-SMase and CTSD, are linked to the mitochondrial apoptotic TNF pathway.
• CTSD acts as a key mediator in TNF-induced apoptosis, primarily through the direct cleavage of Bid.
• The findings establish a critical connection between the endolysosomal system and extrinsic apoptotic signaling cascades.