11/24/2023 0 Comments Chaperone mediated autophagy assayThey constitute a very heterogeneous pool of intracellular proteins including among others some glycolytic enzymes (glyceraldehyde-3-phosphate dehydrogenase, aldolase, phosphoglyceromutase), particular transcription factors and inhibitors of transcription factors (c-fos, the inhibitor of NFκB (IκB)), calcium-binding proteins (Annexins I, II, IV and VI), vesicular trafficking proteins (α-synuclein), cytosolic forms of secretory proteins (α-2-microglobulin) and even some of the catalytic and regulatory subunits of the proteasome, the major cytosolic protease (reviewed in Dice, 2007 Majeski and Dice, 2004 Massey et al., 2004 Massey et al., 2006b). These two features, the selectivity towards substrate proteins and their direct translocation across the lysosomal membrane, make CMA distinct from the other types of autophagy in mammalian cells, namely macroautophagy and microautophagy, where cargo is typically delivered in bulk to lysosomes through processes involving vesicular fusion (macroautophagy) and/or membrane excision (microautophagy) ( Cuervo, 2004a Levine and Klionsky, 2004 Shintani and Klionsky, 2004).Īpproximately 30% of soluble cytosolic proteins contain the CMA-targeting motif ( Dice, 1990). Once in the lysosomal lumen, CMA substrates are rapidly degraded (in 5–10 min) by the broad array of lysosomal proteases. After unfolding, the substrate protein is translocated across the lysosomal membrane in an ATP-dependent manner, assisted by a resident lysosomal chaperone (lys-hsc70) ( Agarraberes et al., 1997). The substrate/chaperone complex is delivered to the surface of the lysosomes where it binds to a CMA receptor, the lysosome-associated membrane protein type–2A (LAMP-2A) ( Cuervo and Dice, 1996). This motif is recognized by a cytosolic chaperone, the heat shock cognate protein of 70 kDa (hsc70), in complex with its cochaperones ( Chiang et al., 1989). In this chapter we review the different experimental approaches utilized to assess CMA activity both in cells in culture and in different organs from animals.Ĭhaperone-mediated autophagy (CMA) is a type of autophagy responsible for the degradation of a subset of cytosolic proteins bearing in their amino acid sequence a consensus motif, biochemically related to KFERQ, that targets them for lysosomal degradation ( Dice, 1990). These findings have drawn renewed attention to CMA and a growing interest in the measurement of changes in CMA activity under different physiological and pathological conditions. Furthermore, impairment of CMA underlies the pathogenesis of certain human pathologies such as neurodegenerative disorders. CMA activity decreases with age, likely contributing to the accumulation of altered proteins characteristic in tissues from old organisms. CMA is part of the cellular quality control systems and as such, essential for the cellular response to stress. In contrast to other forms of autophagy where cargo is sequestered and delivered to lysosomes through membrane fusion/excision, CMA substrates reach the lysosomal lumen after direct translocation across the lysosomal membrane. Chaperone-mediated autophagy (CMA) is a selective type of autophagy responsible for the lysosomal degradation of soluble cytosolic proteins.
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