The chaperone Hsp70 is a BH3 receptor activated by the pro-apoptotic Bim to stabilize anti-apoptotic clients

Zongwei Guo,Ting Song,Ziqian Wang,Donghai Lin,Keke Cao,Peng Liu,Yingang Feng,Xiaodong Zhang,Peiran Wang,Fangkui Yin,Jian Dai,Sheng Zhou,Zhichao Zhang

doi:10.1074/jbc.ra120.013364

Abstract

The chaperone heat shock protein 70 (Hsp70) is crucial for avoiding protein misfolding under stress, but is also up-regulated in many kinds of cancers, where its ability to buffer cellular stress prevents apoptosis. Previous research has suggested Hsp70 interacts with pro-apoptotic Bcl-2 family proteins, including Bim and Bax. However, a definitive demonstration of this interaction awaits, and insights into the structural basis and molecular mechanism remain unclear. Earlier studies have identified a Bcl-2 homology 3 (BH3) domain present in Bcl-2 family members that engages receptors to stimulate apoptosis. We now show that Hsp70 physically interacts with pro-apoptotic multidomain and BH3-only proteins via a BH3 domain, thereby serving as a novel BH3 receptor, using in vitro fluorescent polarization (FP), isothermal titration calorimetry (ITC), and cell-based co-immunoprecipitation (co-IP) experiments, 1H-15N-transverse relaxation optimized spectroscopy (TROSY-HSQC), trypsin proteolysis, ATPase activity, and denatured rhodanese aggregation measurements further demonstrated that BimBH3 binds to a novel allosteric site in the nucleotide-binding domain (NBD) of Hsp70, by which Bim acts as a positive co-chaperone to promote the ATPase activity and chaperone functions. A dual role of Hsp70's anti-apoptotic function was revealed that when it keeps Bim in check to inhibit apoptosis, it simultaneously stabilizes oncogenic clients including AKT and Raf-1 with the aid of Bim. Two faces of Bim in cell fate regulation were revealed that in opposite to its well-established pro-apoptotic activator role, Bim could help the folding of oncogenic proteins.

Highlights

heat shock protein 70 (Hsp70) is an ATP-dependent molecular chaperone that is abundantly expressed in most cancer cells to facilitate the maturation, activation, and stabilization of many oncogenic clients to buffer cellular stress [1,2,3]
The protein-protein interactions (PPIs) of the Bcl-2 family proteins dictate apoptosis, which is mediated by a Bcl-2 homology 3 (BH3) domain of the pro-apoptotic protein that inserts into a BH3 receptor groove on the surface of anti-apoptotic proteins [12]
Bim acts as a co-chaperone to positively regulate Hsp70

Summary

Results and discussion

We characterized the binding ability of Hsp proteins to BH3 domains of Bid, Bim, Noxa, Bax, and Bak by using in vitro biochemical assays and cell-based assays. The combination of Hsp (1 mM) and BimBH3 (0.1 mM), which resembled the ratio of Bim-bound Hsp in total Hsp as determined by semi-quantification assay in cells (Fig. S8) resulted in a 3.8-fold increase of the rate of ATP consumption to the addition of J domain (4.2fold), and both of their stimulations of ATPase activity were blunted by MKT-077. It reminds us of a growing emergence of new partners of BH3-only protein, which endows BH3-only proteins with new roles on directly regulating the partners’ activity and biological process beyond their canonical pro-apoptotic function [12, 13, 31]. The solid physical complex of Hsp70/Bim as well as the biological relevance between them as evidenced by bioinformatics analysis highlights the further exploration of the consequent biological events of Hsp70/Bim dimer in cell death and proliferation

Cell lines

Protein expression and purification

NMR titrations

Partial proteolysis

Rhodanese aggregation assay

Data availability statements