ARD1-mediated Hsp70 acetylation balances stress-induced protein refolding and degradation

Heat shock protein (Hsp)70 is a molecular chaperone that maintains protein homoeostasis during cellular stress through two opposing mechanisms: protein refolding and degradation. However, the mechanisms by which Hsp70 balances these opposing functions under stress conditions remain unknown. Here, we demonstrate that Hsp70 preferentially facilitates protein refolding after stress, gradually switching to protein degradation via a mechanism dependent on ARD1-mediated Hsp70 acetylation. During the early stress response, Hsp70 is immediately acetylated by ARD1 at K77, and the acetylated Hsp70 binds to the co-chaperone Hop to allow protein refolding. Thereafter, Hsp70 is deacetylated and binds to the ubiquitin ligase protein CHIP to complete protein degradation during later stages. This switch is required for the maintenance of protein homoeostasis and ultimately rescues cells from stress-induced cell death in vitro and in vivo. Therefore, ARD1-mediated Hsp70 acetylation is a regulatory mechanism that temporally balances protein refolding/degradation in response to stress.

(a) Etoposide treatment gradually changes acetylation level and co-chaperone complexes of Hsp70. After treating HEK293T cells with 100 mM etoposide, endogenous Hsp70 was precipitated, and its co-chaperone binding partners were analyzed by western blotting.
Acetylation of endogenous Hsp70 was accessed by immunoprecipitation using an anti-Lys-Ac antibody.
(b) MPP + treatment gradually changes acetylation level and co-chaperone bindings of Hsp70.
After SH-SY5Y cells stably expressing GFP-Hsp70 were treated with 1 mM MPP + , GFP-Hsp70 was precipitated using an anti-GFP antibody, and its co-chaperone complexes were determined by western blotting. Acetylation of GFP-Hsp70 was analyzed by immunoprecipitation using an anti-Lys-Ac antibody.
(c) NaCl treatment changes acetylation level and co-chaperone complexes of Hsp70.
HEK293T cells were treated with 100 mM NaCl for the indicated time. Acetylation of endogenous Hsp70 was accessed by immunoprecipitation using an anti-Lys-Ac antibody. For co-chaperone binding, endogenous Hsp70 was immunoprecipitated using an anti-Hsp70 antibody and its binding to Hop and CHIP was analyzed by western blotting.
(d) EtOH treatment changes acetylation level and co-chaperone complexes of Hsp70. After treating HEK293T cells with 150 mM EtOH, endogenous Hsp70 was precipitated, and its cochaperone binding partners were analyzed by western blotting. Acetylation of endogenous Hsp70 was accessed by immunoprecipitation using an anti-Lys-Ac antibody.
(e) After brief expose of cellular stress, Hsp70 gradually alters its acetylation level and cochaperone binding partners. HEK293T cells were treated with 1 mM H 2 O 2 for 1 h. During the recovery time, the temporal changes in acetylation and co-chaperone binding of endogenous Hsp70 were assessed by immunoprecipitation and western blotting. Hsp70 binding to FLAG-HA-ARD1 was identified by mass spectrometry. The Hsp70 band is indicated by an asterisk (*), and Hsp70 peptide sequences identified by mass spectrometry are indicated in red.   (b) HDAC4 deacetylates HSP70 in vitro. After GST-Hsp70 was acetylated by GST-ARD1 in vitro, FLAG-HDAC4 precipitated from HEK293T cells was subjected to in vitro deacetylation assays. Acetylation levels of GST-Hsp70 were analyzed by western blotting using an anti-Lys-Ac antibody.
(c) HDAC4 changes the co-chaperone binding pattern of Hsp70. HEK293T cells expressing HDAC4 were treated with 1 mM H 2 O 2 for 1 h with or without TSA treatment for 4 h.
Endogenous Hsp70 was precipitated, and its acetylation and co-chaperone bindings were assessed by western blotting.
(d) HDAC4 is a relevant deacetylase to K77 acetylation of Hsp70. HEK293T cells transfected with HDAC4 siRNA were treated with 1 mM H 2 O 2 for the indicated times.
Endogenous Hsp70 was precipitated, and its acetylation and co-chaperone binding was assessed by western blotting. HEK293T cells were treated with 0.3 mM H 2 O 2 for 30 min and then the cells were recovered for 2 h. Cell lysates were extracted and the total amount of soluble luciferase was analyzed by 11 western blotting. (d) Hsp70 is required for protein refolding in the early phase after H 2 O 2induced stress. HEK293T cells were pretreated with Hsp70 inhibitor, VER-155008, for 30 min before H 2 O 2 stimulation. After treatment with 0.3 mM H 2 O 2 for 30 min with or without VER-155008, the cells were recovered for 2 h and protein refolding was measured.
(e) K77 acetylation of Hsp70 is necessary for protein refolding in the early phase after H 2 O 2induced stress. HEK293T cells were transfected with the indicated Hsp70 plasmids. After treatment with 0.3 mM H 2 O 2 for 30 min, the cells were recovered for 2 h and protein refolding was measured.

Stress-induced protein aggregation was enhanced by both the K77R and K77Q mutations in
Hsp70. HEK293T cells were treated with 1 mM H 2 O 2 for 1 h, and then washed with phosphate-buffered saline to remove H 2 O 2 . After 24 h, the accumulation of protein aggregation was analyzed using protein aggregation detection dye.