Processing of human cathepsin D in lysosomes in vitro

Area of Science:

• Cell Biology
• Biochemistry
• Molecular Biology

Background:

• Cathepsin D is a key lysosomal protease involved in protein degradation.
• Its proper maturation is essential for lysosomal function and cellular homeostasis.
• Understanding the regulatory mechanisms of cathepsin D processing is crucial for cell biology research.

Purpose of the Study:

• To investigate the proteolytic maturation of cathepsin D polypeptides within isolated lysosomes.
• To elucidate the role of lysosomal environment and energy sources in this process.
• To identify key factors and conditions influencing cathepsin D processing.

Main Methods:

• Utilized metabolically labeled fibroblasts and isolated lysosomes for in vitro studies.
• Assessed cathepsin D polypeptide processing using radiolabeling and chase experiments.
• Manipulated lysosomal pH and utilized various inhibitors to study processing dependency.

Main Results:

• Identified a Mr = 47,000 polypeptide as a precursor to mature cathepsin D.
• Demonstrated that lysosomal integrity, optimal pH (6-7), and ATP are critical for processing.
• Showed ATP-dependent stimulation linked to lysosomal acidification, but also a distinct ATP effect at pH 5.5.
• Confirmed the involvement of cysteine proteinases and N-ethylmaleimide-sensitive pathways.

Conclusions:

• Cathepsin D maturation in lysosomes is dependent on cysteine proteinases.
• ATP-driven lysosomal acidification significantly stimulates cathepsin D processing.
• ATP also enhances maturation at pH 5.5 through a mechanism independent of proton pumping.