Lysosomal Protein Degradation and Aging
The main focus of our laboratory is on understanding how proteins are transported into lysosomes for their degradation and how malfunctioning of the lysosomal system contributes to aging and age-related diseases.
A common feature of senescent cells is the accumulation of abnormal or damaged proteins in their cytosol that, undoubtedly, impairs cellular function. Protein accumulation results, at least in part, from impaired protein degradation with age. Among the different systems that participate in the intracellular degradation of proteins, lysosomes are the most affected by age. We have previously identified in many tissues of aged animals a decrease with age in the activity of a selective pathway for the degradation of cytosolic proteins in lysosomes known as chaperone-mediated autophagy. The main goal of our research is to identify the defect(s) that lead to the decreased activity of chaperone-mediated autophagy with age, and to analyze if the correction of those defects and recovery of normal proteolytic activity in old cells leads to an improvement in cellular function.
Chaperone-mediated autophagy is responsible for the degradation of as much as 30% of cytosolic proteins. This pathway is especially active in organs such as liver, kidney and heart, and it is mainly activated under conditions of stress, such as nutrient deprivation, oxidative stress and exposure to toxic compounds. Under those conditions, substrate proteins are selectively recognized by cytosolic chaperones (hsc73 and cochaperones) that stimulate their binding to a glycoprotein receptor in the lysosomal membrane (lamp2a). The transport of the cytosolic proteins into lysosomes for their degradation requires also the presence of another chaperone in the lysosomal matrix (lys-hsc73).
Our efforts are currently directed to the:
- Characterization of the different components involved in chaperone-mediated autophagy and identification of new players for this pathway.- We use an animal model that requires the isolation of intact lysosomes from several tissues (liver, kidney and spleen) of mice and rats. Using an in-vitro system we are able to separately analyze each step involved in the degradation of substrate proteins: binding to the lysosomal membrane, uptake into the lysosome, and complete degradation once at the lysosomal matrix. For the identification of the new components of the systems we are currently using different immunochemical approaches and the yeast two-hybrid system. We are currently tracking down two proteases, coimmunoprecipitated with the lysosomal receptor, that modulate chaperone-mediated autophagy activity by regulating the levels of the receptor.
- Correction of the age-related defect in chaperone-mediated autophagy.- We have recently found that the binding of substrates to the lysosomal membrane dramatically decreases with age. This defect seems to be associated with a decrease in the levels of the receptor protein in the lysosomal membrane. We are currently generating a bitransgenic mouse in which the levels of the lysosomal receptor can be regulated by administration of tetracycline, a common antibiotic. This animal model will allow us to test if chaperone-mediated autophagy can be restored in old animals by increasing the levels of the lysosomal receptor toward normal values. If normal activity is achieved we could then value the benefits, at the organ level, of correcting the impaired lysosomal function.
Kiffin R, Kaushik S, Zeng M, Bandyopadhyay U, Zhang C, Massey AC, Martinez-Vicente M, Cuervo AM. Altered dynamics of the lysosomal receptor for chaperone-mediated autophagy with age. J Cell Sci. 2007 120(Pt 5):782-91
Kaushik S, Massey AC, Cuervo AM. Lysosome membrane lipid microdomains: novel regulators of chaperone-mediated autophagy. EMBO J. 2006 25(17):3921-33
Massey AC, Kaushik S, Sovak G, Kiffin R, Cuervo AM. Consequences of the selective blockage of chaperone-mediated autophagy. Proc Natl Acad Sci U S A. 2006 103(15):5805-10.
Yu WH, Cuervo AM, Kumar A, Schmidt SD., Grbovi O., Terio NB, Jiang Y., Peterhoff C, Mathews P., Cataldo A, Nixon RA. Autophagy-dependent generation of the Amyloid-B (AB) peptide. J Cell Biol 2005; 171:87-98
Nixon RA, Wegiel J, Kumar A, Yu WH, Peterhoff C, Cataldo A, Cuervo AM. Extensive involvement of autophagy in Alzheimer disease: an immuno-electron microscopy study. J Neuropathol Exp Neurol. 2005 64(2):113-22.