A simple methodology to assess endolysosomal protease activity involved in antigen processing in human primary cells

Area of Science:

• Cell Biology
• Biochemistry
• Immunology

Background:

• Endolysosomes are crucial for cellular homeostasis, degrading various biomolecules.
• Current methods for studying endolysosomal enzyme function have limitations.
• Endolysosomes are involved in antigen presentation pathways, including MHC class I and II epitope production.

Purpose of the Study:

• To develop a straightforward method for assessing endolysosomal protease activity.
• To validate the new method using human peripheral blood mononuclear cells (PBMCs).
• To compare the degradation kinetics of HIV peptides in crude lysates versus purified endolysosomes.

Main Methods:

• Utilizing crude cell lysates from PBMCs.
• Manipulating pH conditions to optimize endolysosomal protease activity.
• Employing mass spectrometry to analyze peptide degradation patterns.
• Comparing results with studies using purified endolysosomes.

Main Results:

• Endolysosomal cathepsin activity was enhanced under acidic conditions in crude lysates.
• The novel method demonstrated similar peptide degradation kinetics and patterns to purified endolysosome studies.
• HIV peptide degradation in low pH extracts mirrored that in purified endolysosomes.

Conclusions:

• The developed method offers a rapid and effective way to assess endolysosomal protease activity in primary cells.
• This approach is particularly valuable for screening peptide susceptibility to degradation in endolysosomal compartments for antigen processing.
• The method facilitates preliminary analysis, with detailed studies using purified organelles for specific peptides.