BAG2 Interferes with CHIP-Mediated Ubiquitination of HSP72

Abstract

The maintenance of cellular proteostasis is dependent on molecular chaperones and protein degradation pathways. Chaperones facilitate protein folding, maturation, and degradation, and the particular fate of a misfolded protein is determined by the interaction of chaperones with co-chaperones. The co-factor CHIP (C-terminus of HSP70-inteacting protein, STUB1) ubiquitinates chaperone substrates and directs proteins to the cellular degradation systems. The activity of CHIP is regulated by two co-chaperones, BAG2 and HSPBP1, which are potent inhibitors of the E3 ubiquitin ligase activity. Here, we examined the functional correlation of HSP72, CHIP, and BAG2, employing human primary fibroblasts. We showed that HSP72 is a substrate of CHIP and that BAG2 efficiently prevented the ubiquitination of HSP72 in young cells as well as aged cells. Aging is associated with a decline in proteostasis and we observed increased protein levels of CHIP as well as BAG2 in senescent cells. Interestingly, the ubiquitination of HSP72 was strongly reduced during aging, which revealed that BAG2 functionally counteracted the increased levels of CHIP. Interestingly, HSPBP1 protein levels were down-regulated during aging. The data presented here demonstrates that the co-chaperone BAG2 influences HSP72 protein levels and is an important modulator of the ubiquitination activity of CHIP in young as well as aged cells.

Keywords: BAG2; CHIP; HSP72; aging; proteostasis; ubiquitination.

Figure 1

BAG2 modulates HSP72 protein levels. (a) Immunoblot analysis of cells that were transfected with the indicated plasmids. Actin served as control for equal loading. Statistics are depicted as mean ± SD. *** p < 0.001, n = 6; (b) Immunoblot analysis of cells that were manipulated with nonsense or BAG2 siRNA. Actin served as loading control. Statistics are depicted as mean ± SD. * p < 0.05, n = 4; (c) Real-time PCR analyses of HSP72 mRNA levels in cells that were manipulated as indicated. Statistics are depicted as mean values (±SD) relative to HSP72 mRNA levels in cells that are treated with empty vector or nonsense siRNA, respectively. n = 3.

Figure 2

(a) Fibroblasts were transiently transfected with HA-tagged ubiquitin and either EGFP as control, or BAG2 or myc-CHIP, or BAG2 and Myc-CHIP. Endogenous HSP72 was immunoprecipitated and the level of ubiquitination was monitored by anti-HA antibody. Purified mouse IgG was used as negative control (not shown). The lower panel shows protein levels in total cell lysates used for Co-IP (Input); (b) Cells were transfected with HA-tagged ubiquitin together with either BAG2, Myc-CHIP or EGFP as control. Endogenous HSP72 was immunoprecipitated and BAG2 or myc-CHIP were detected by specific antibodies. The lower panels show protein levels of the analyzed proteins in total cell lysates used for Co-IP (Input). CHIP: C-terminus of HSP70-inteacting protein, STUB1; OE: overexpression; HA: hemagglutinin.

Figure 3

(a) Immunoblot analysis of aging marker proteins in young (PDL 20) and aged (PDL + 50) fibroblasts. Actin served as control of equal loading. One representative blot is shown. n = 3; (b) Immunoblot analysis of BAG2, HSPBP1, CHIP, and HSP72 in young and aged fibroblasts. Actin served as loading control. Statistics are depicted as mean ± SD. * p < 0.05, *** p < 0.001, n = 4; (c) Immunoblot analysis of indicated proteins in young fibroblasts, that were treated repeatedly with 200 µM (SIPS I) and 500 µM (SIPS II) hydrogen peroxide (H₂O₂) to induce premature senescence (SIPS). Tubulin served as loading control. Statistics are depicted as mean ± SD. * p < 0.05, *** p < 0.001, n = 3; (d) Immunoblot analysis of BAG2 and HSPBP1 levels in fibroblasts that were treated with H₂O₂ or 4-hydroxy-nonenal (4-HNE). Poly-Ubiquitin and CDKN1A indicate proteostatic stress. Tubulin served as loading control. Statistics are depicted as mean ± SD. n = 3.

Figure 4

(a) Co-IP analysis of young and aged fibroblasts that were transfected with HA-tagged ubiquitin and Flag-tagged HSP72. The accumulation of poly-ubiquitinated proteins was induced by MG132 treatment (4 µM, 4 h) prior to cell lysis. The levels of HSP72 ubiquitination were visualized by immunoblot analysis (left panel). The input fraction is shown on the right panel; (b) Aged fibroblasts were transiently transfected with HA-tagged ubiquitin and FLAG-tagged HSP72 and either EGFP (control), BAG2, CHIP or with BAG2 and CHIP. The accumulation of poly-ubiquitinated proteins was induced by treatment with MG132 (4 µM, 4 h) prior to cell lysis. HSP72 was immunoprecipitated and levels of ubiquitination proteins were verified by immunoblot analysis (left panel). Purified mouse IgG was used as control (not shown). The right panel shows relative protein levels in cell lysates used for Co-IP (Input).