Abstract
α-Crystallin B (CRYAB or HspB5) is a chaperone member of the small heat-shock protein family that prevents aggregation of many cytosolic client proteins by means of its ATP-independent holdase activity. Surprisingly, several reports show that CRYAB exerts a protective role also extracellularly, and it has been recently demonstrated that CRYAB is secreted from human retinal pigment epithelial cells by an unconventional secretion pathway that involves multi-vesicular bodies. Here we show that autophagy is crucial for this unconventional secretion pathway and that phosphorylation at serine 59 residue regulates CRYAB secretion by inhibiting its recruitment to the autophagosomes. In addition, we found that autophagosomes containing CRYAB are not able to fuse with lysosomes. Therefore, CRYAB is capable to highjack and divert autophagosomes toward the exocytic pathway, inhibiting their canonical route leading to the lysosomal compartment. Potential implications of these findings in the context of disease-associated mutant proteins turn-over are discussed.
Highlights
Α-Crystallin B (CRYAB or HspB5) is a chaperone member of the small heat-shock protein family that prevents aggregation of many cytosolic client proteins by means of its ATP-independent holdase activity
To quantify and verify the secretion efficiency of both endogenous and transfected forms of CRYAB, COS-7 cells were transiently transfected with 3xFlag-CRYAB and after an over-night incubation at 37 °C the medium was replaced with Dulbecco’s modified essential medium (DMEM) supplemented with 1% foetal bovine serum (FBS) and 1% l-Glutamine (Gln)
After 6 hours, equal volumes of each medium and lysate were separated by SDS-PAGE and endogenous and over-expressed CRYAB were detected by using a mouse monoclonal anti-CRYAB and anti-FLAG antibodies, respectively
Summary
Α-Crystallin B (CRYAB or HspB5) is a chaperone member of the small heat-shock protein family that prevents aggregation of many cytosolic client proteins by means of its ATP-independent holdase activity. It has been shown that autophagy might be involved and contribute to UPS: the exosomes-mediated secretion requires first the fusion of autophagosomes with multi-vesicular bodies (MVBs) and the fusion with the plasma membrane[13,14]. Α-Crystallin B (CRYAB or HspB5) belongs to the group of small heat shock proteins (sHSPs, molecular mass 15–30 kDa) It forms functional oligomers (both homo- and hetero-oligomers), comprising up to 50 subunits and its chaperone activity consists in binding to either cytosolic or transmembrane proteins and preventing their aggregation through an ATP-independent holdase activity[16,17,18,19]. A recent report has shown that CRYAB can exert a protective function in the extracellular compartment, following to its exosome-dependent secretion from polarized human RPE cells, which is mediated by an UPS pathway that involves multi-vesicular-bodies (MVB)[27]. We highlight the phosphorylation on a key serine residue of the protein as a crucial negative regulator for its recruitment into autophagosome and consequent secretion
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