A simple methodology to assess endolysosomal protease activity involved in antigen processing in human primary cells.
BMC Cell Biol
; 14: 35, 2013 Aug 09.
Article
em En
| MEDLINE
| ID: mdl-23937268
ABSTRACT
BACKGROUND:
Endolysosomes play a key role in maintaining the homeostasis of the cell. They are made of a complex set of proteins that degrade lipids, proteins and sugars. Studies involving endolysosome contribution to cellular functions such as MHC class I and II epitope production have used recombinant endolysosomal proteins, knockout mice that lack one of the enzymes or purified organelles from human tissue. Each of these approaches has some caveats in analyzing endolysosomal enzyme functions.RESULTS:
In this study, we have developed a simple methodology to assess endolysosomal protease activity. By varying the pH in crude lysate from human peripheral blood mononuclear cells (PBMCs), we documented increased endolysosomal cathepsin activity in acidic conditions. Using this new method, we showed that the degradation of HIV peptides in low pH extracts analyzed by mass spectrometry followed similar kinetics and degradation patterns as those performed with purified endolysosomes.CONCLUSION:
By using crude lysate in the place of purified organelles this method will be a quick and useful tool to assess endolysosomal protease activities in primary cells of limited availability. This quick method will especially be useful to screen peptide susceptibility to degradation in endolysosomal compartments for antigen processing studies, following which detailed analysis using purified organelles may be used to study specific peptides.
Texto completo:
1
Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Peptídeo Hidrolases
/
Espectrometria de Massas
/
Leucócitos Mononucleares
/
Apresentação de Antígeno
/
Lisossomos
Tipo de estudo:
Evaluation_studies
Limite:
Humans
Idioma:
En
Revista:
BMC Cell Biol
Ano de publicação:
2013
Tipo de documento:
Article
País de afiliação:
Estados Unidos