Background of Chaperone-Mediated Autophagy Activity
Chaperone-mediated autophagy (CMA) is a very selective form. Most cells show some basal level of CMA activity, but several types of cellular stressors upregulate CMA, such as starvation, oxidative stress, hypoxia, lipotoxicity, or DNA damage. Researchers have found that CMA activity is reduced in almost all tissues during aging, which may exacerbate the course of age-related diseases associated with protein toxicity. reduced CMA activity has been described in neurodegenerative diseases such as Parkinson's disease and bullous pemphigoid, as well as metabolic disorders such as diabetes and diet-induced obesity. In contrast, upregulation of CMA activity is common in many types of cancer. this link between CMA and different human diseases has stimulated interest in understanding this fundamental cellular process and using it for therapeutic purposes.
Fig. 1. Assays to measure CMA activity. (Patel B, et al., 2015)
Chaperone-Mediated Autophagy Activity Assessment Services
Our scientists have a keen interest in measuring changes in CMA activity under different physiological and pathological conditions. At CD BioSciences, we are committed to providing comprehensive services for the assessment of chaperone-mediated autophagic activity to our clients worldwide. We develop a variety of experimental model platforms for CMA research.
Mammalian Cell Model Platforms
- Bird and above animals.
- LAMP-2 knockout mice.
- Double transgenic mouse model.
In Vitro Cell Model Platforms
- Many different types of transformed cells, such as mouse fibroblasts, human renal epithelial cells, Chinese hamster ovary cells, rat renal epithelial cells, rat hepatoblastoma, human hepatoblastoma, astrocytome, several human lung cancer cell lines and several primary cells in culture.
- Different tissues of rodents, such as liver, kidney, heart, spleen, and lung.
In addition, we offer several strategies for assessing CMA activity in different organs of animals or in vitro cell cultures, including:
(1) Measurement of Protein Degradation Rates
Proteins degraded in lysosomes generally have a long half-life. We offer the measurement of degradation rates of long-lived proteins in cultured cells by metabolic markers in pulse/chase experiments to assess lysosomal function.
(2) Measurement of Levels of Key CMA Components
The levels of hsc70 remain constant for the most part. However, lysosomal levels of LAMP-2A and lysosomal-hsc70 increase with increasing CMA activity. We provide monitoring of CMA activity of LAMP-2A and lys-hsc70 in lysosomes isolated from tissues/cells of interest by immunoblotting.
(3) Analysis of the Subcellular Location of CMA-Active Lysosomes
Only lysosomes containing LAMP-2A are competent for CMA. We performed immunofluorescence co-localization of LAMP-2A and hsc70 to monitor CMA in cultured cells and tissue sections.
(4) In Vitro Assay to Measure Translocation of CMA Substrates
Both the purity of the lysosomal fraction and the integrity of the lysosomal membrane are critical for proper assessment of CMA substrate translocation. We measured radiolabeled CMA substrate by isolated lysosomes as well. Parallel experiments with lysosomal disruption by hypotonic shock were performed to determine possible changes in protein hydrolysis unrelated to binding/uptake.
Why Choose Us
- Our dedicated team has unique insights into chaperone-mediated autophagy.
- Accurate and reliable combination of a range of methods.
- Can be used to gather information on CMA failure in different disease models and physiological states.
- Various experimental models to quantify changes in CMA activity under different conditions.
CD BioSciences can meet any reasonable needs of our clients, taking time and budget into consideration for you. Our aim is to be customer-centric and to provide the highest quality services to customers. Our customer service representatives are enthusiastic and trustworthy 24 hours a day, 7 days a week. If you are interested in our services, please feel free to contact us for more information or a detailed discussion.
Reference
- Patel B, Cuervo AM. (2015) Methods to study chaperone-mediated autophagy. Methods. 75:133-40.