Non-canonical Interactions between Heat Shock Cognate Protein 70 (Hsc70) and Bcl2-associated Anthanogene (BAG) Co-Chaperones Are Important for Client Release

Jennifer N Rauch,Erik R.P Zuiderweg,Jason E Gestwicki

doi:10.1074/jbc.m116.742502

Abstract

Heat shock cognate protein 70 (Hsc70) regulates protein homeostasis through its reversible interactions with client proteins. Hsc70 has two major domains: a nucleotide-binding domain (NBD), that hydrolyzes ATP, and a substrate-binding domain (SBD), where clients are bound. Members of the BAG family of co-chaperones, including Bag1 and Bag3, are known to accelerate release of both ADP and client from Hsc70. The release of nucleotide is known to be mediated by interactions between the conserved BAG domain and the Hsc70 NBD. However, less is known about the regions required for client release, and it is often assumed that this activity also requires the BAG domain. It is important to better understand this step because it determines how long clients remain in the inactive, bound state. Here, we report the surprising observation that truncated versions of either human Bag1 or Bag3, comprised only the BAG domain, promoted rapid release of nucleotide, but not client, in vitro Rather, we found that a non-canonical interaction between Bag1/3 and the Hsc70 SBD is sufficient for accelerating this step. Moreover, client release did not seem to require the BAG domain or Hsc70 NBD. These results suggest that Bag1 and Bag3 control the stability of the Hsc70-client complex using at least two distinct protein-protein contacts, providing a previously under-appreciated layer of molecular regulation in the human Hsc70 system.

Highlights

Heat shock cognate protein 70 (Hsc70, HSPA8)2 is a molecular chaperone that regulates the folding, trafficking, function, stability, and turnover of its client proteins [1, 2]
Isolated BAG Domains Promote Release of Nucleotide, but Not Model Client—In this study, we focused on the interaction of human Hsc70 (HSPA8) with Bag1 and Bag3
These systems were chosen because the Hsc70-Bag1 complex has been implicated in directing clients to the ubiquitin-proteasome system (UPS) [40], while the Hsc70-Bag3 pair links some clients to the macroautophagy pathway [41]

Summary

Introduction

Heat shock cognate protein 70 (Hsc, HSPA8) is a molecular chaperone that regulates the folding, trafficking, function, stability, and turnover of its client proteins [1, 2]. Hydrolysis of nucleotide reorganizes lobes I and II, which “closes” the lid subdomain and strengthens the affinity for clients In this ADPbound state, the NBD and SBD are no longer docked; rather, they move relatively independently in solution [23]. Recent studies have shown that members of the Hsc family inhibit ligand binding to glucocorticoid receptor [28] This interaction keeps aggregation-prone clients, such as human telomerase repeat binding factor (hTRF1), on the folding trajectory by allowing proper secondary structures to form [29]. Hsc70-client complexes seem to represent a “timing” mechanism for client folding, function and turnover, possibly helping to ensure proper quality control These observations have focused attention on the protein-protein interaction between Hsc and BAG family proteins as potential targets for drug discovery [3]. Allosteric inhibitors of this interaction trigger degradation of a subset of Dengue viral proteins [30] and some oncogenes [31]

Methods

Results

Conclusion