Sue Wickner

Overlapping recognition determinants within the ssrA degradation tag allow modulation of proteolysis

The ssrA tag, an 11-aa peptide added to the C terminus of proteins stalled during translation, targets proteins for degradation by ClpXP and ClpAP. Mutational analysis of the ssrA tag reveals independent, but overlapping determinants for its interactions with ClpX, ClpA, and SspB, a specificity-enhancing factor for ClpX. ClpX interacts with residues 9–11 at the C terminus of the tag, whereas ClpA recognizes positions 8–10 in addition to residues 1–2 at the N terminus. SspB interacts with residues 1–4 and 7, N-terminal to the ClpX-binding determinants, but overlapping the ClpA determinants. As a result, SspB and ClpX work together to recognize ssrA-tagged substrates efficiently, whereas SspB inhibits recognition of these substrates by ClpA. Thus, dissection of the recognition signals within the ssrA tag provides insight into how multiple proteins function in concert to modulate proteolysis.

Mini-P1 plasmid replication: The autoregulation-sequestration paradox

It has been proposed that the initiator protein RepA is rate limiting for mini-P1 plasmid replication, and that the role of the plasmid copy number control locus is to sequester the initiator and thus reduce replication. This proposal appears inconsistent with the observation that RepA is autoregulated, since the protein lost by sequestration should be replenished. A resolution of this autoregulation-sequestration paradox is possible if the sequestered RepA, unavailable for replication, is still available for promoter repression. We demonstrate that RepA binds to the control locus and to the promoter region simultaneously, causing the intervening DNA to loop. DNA looping could provide the requisite mechanism by which RepA bound to the control locus might exert repression.

E. coli chaperones DnaK, Hsp33 and Spy inhibit bacterial functional amyloid assembly

Amyloid formation is an ordered aggregation process, where β-sheet rich polymers are assembled from unstructured or partially folded monomers. We examined how two Escherichia coli cytosolic chaperones, DnaK and Hsp33, and a more recently characterized periplasmic chaperone, Spy, modulate the aggregation of a functional amyloid protein, CsgA. We found that DnaK, the Hsp70 homolog in E. coli, and Hsp33, a redox-regulated holdase, potently inhibited CsgA amyloidogenesis. The Hsp33 anti-amyloidogenesis activity was oxidation dependent, as oxidized Hsp33 was significantly more efficient than reduced Hsp33 at preventing CsgA aggregation. When soluble CsgA was seeded with preformed amyloid fibers, neither Hsp33 nor DnaK were able to efficiently prevent soluble CsgA from adopting the amyloid conformation. Moreover, both DnaK and Hsp33 increased the time that CsgA was reactive with the amyloid oligomer conformation-specific A11 antibody. Since CsgA must also pass through the periplasm during secretion, we assessed the ability of the periplasmic chaperone Spy to inhibit CsgA polymerization. Like DnaK and Hsp33, Spy also inhibited CsgA polymerization in vitro. Overexpression of Spy resulted in increased chaperone activity in periplasmic extracts and in reduced curli biogenesis in vivo. We propose that DnaK, Hsp33 and Spy exert their effects during the nucleation stages of CsgA fibrillation. Thus, both housekeeping and stress induced cytosolic and periplasmic chaperones may be involved in discouraging premature CsgA interactions during curli biogenesis.

Functional and physical interaction between yeast Hsp90 and Hsp70

Significance The Hsp90 molecular chaperone remodels and activates a large variety of client proteins. Its activity requires collaboration with the more than 25 Hsp90 cochaperone proteins, including the Hsp70 chaperone. In higher eukaryotes, Hsp90 collaborates with Hsp70 through a bridging protein, the Hop cochaperone. We show that yeast Hsp90 (Hsp82) and Hsp70 (Ssa1) directly interact in vitro in the absence of yeast Hop (Sti1) and identify a region in the middle domain of yeast Hsp90 that is important for interaction with Hsp70. This region of Hsp90 is also important for interactions with several cochaperones and client proteins, suggesting that collaboration between Hsp70 and Hsp90 in protein remodeling may be modulated through competition between Hsp70 and Hsp90 cochaperones for the interaction surface. Heat shock protein 90 (Hsp90) is a highly conserved ATP-dependent molecular chaperone that is essential in eukaryotes. It is required for the activation and stabilization of more than 200 client proteins, including many kinases and steroid hormone receptors involved in cell-signaling pathways. Hsp90 chaperone activity requires collaboration with a subset of the many Hsp90 cochaperones, including the Hsp70 chaperone. In higher eukaryotes, the collaboration between Hsp90 and Hsp70 is indirect and involves Hop, a cochaperone that interacts with both Hsp90 and Hsp70. Here we show that yeast Hsp90 (Hsp82) and yeast Hsp70 (Ssa1), directly interact in vitro in the absence of the yeast Hop homolog (Sti1), and identify a region in the middle domain of yeast Hsp90 that is required for the interaction. In vivo results using Hsp90 substitution mutants showed that several residues in this region were important or essential for growth at high temperature. Moreover, mutants in this region were defective in interaction with Hsp70 in cell lysates. In vitro, the purified Hsp82 mutant proteins were defective in direct physical interaction with Ssa1 and in protein remodeling in collaboration with Ssa1 and cochaperones. This region of Hsp90 is also important for interactions with several Hsp90 cochaperones and client proteins, suggesting that collaboration between Hsp70 and Hsp90 in protein remodeling may be modulated through competition between Hsp70 and Hsp90 cochaperones for the interaction surface.

Bacterial Hsp90 ATPase Assays

Bacterial Hsp90 is an ATP-dependent molecular chaperone involved in protein remodeling and activation. The E. coli Hsp90, Hsp90Ec, collaborates in protein remodeling with another ATP-dependent chaperone, DnaK, the E. coli Hsp70. Both Hsp90Ec and DnaK hydrolyze ATP and client (substrate) proteins stimulate the hydrolysis. Additionally, ATP hydrolysis by the combination of Hsp90Ec and DnaK is synergistically stimulated in the presence of client (substrate). Here, we describe two steady-state ATPase assays used to monitor ATP hydrolysis by Hsp90Ec and DnaK as well as the synergistic stimulation of ATP hydrolysis by the combination of Hsp90Ec and DnaK in the presence of a client (substrate). The first assay is a spectrophotometric assay based on enzyme-coupled reactions that utilize the ADP formed during ATP hydrolysis to oxidize NADH. The second assay is a more sensitive method that directly quantifies the radioactive inorganic phosphate released following the hydrolysis of [γ-33P] ATP or [γ-32P] ATP.

Abstract 1180: Targeting the HSP40/HSP70 chaperone axis as a novel strategy to treat castration-resistant prostate cancer

Castration-resistant prostate cancer (CRPC) is frequently characterized by elevated expression of nuclear receptors able to at least partially maintain the androgen receptor (AR) transcriptional program. Elevated expression of a number of constitutively active AR splice variants lacking the ligand binding domain (LBD) (e.g., ARv7, which is ligand-independent and correlates with poor prognosis, reduced survival, and resistance to existing LBD-targeted standard of care therapy) is a frequent occurrence in CRPC. Thus, alternative approaches to disrupt AR signaling in CRPC are of great clinical importance, and a single strategy able to target AR and ARv7 remains a critical unmet need. As a steroid hormone nuclear receptor, the AR exists in an interactive and dynamic cycle with the molecular chaperones (heat shock proteins, HSPs) HSP40/HSP70/HSP90 for proper folding and remodeling of the AR LBD to bind ligand. Notably, HSP90 inhibitors promote AR degradation and display efficacy in prostate cancer xenograft models. Although it has been shown that ARv7 functions independently of HSP90, additional chaperone requirements of LBD-deficient ARv7 are not known. Thus, we tested the hypothesis that both AR and ARv7 are dependent on HSP40/HSP70 and that targeting these chaperones with specific inhibitors (C86 and JG98, respectively) will lead to AR/ARv7 destabilization and loss of transcriptional activity in models of CRPC. To determine if AR proteins associate with HSP40/HSP70, 22Rv1 CRPC cells (expressing endogenous AR and ARv7) were first transfected with FLAG-HSP40 or FLAG-HSP70. Immunoprecipitation with FLAG beads revealed AR and ARv7 associated with both chaperones, indicating potential functional dependence of these nuclear receptors on HSP40/HSP70. To further characterize these interactions, 22Rv1 lysate was probed with biotinylated-C86 and subjected to IP with streptavidin beads. C86 bound a significant fraction of HSP40 complexed with HSP70, AR, and ARv7. Excess unlabeled C86 or JG98 effectively competed away binding of HSP40/HSP70 to biotinylated-C86 with concomitant loss of associated AR and ARv7. Treatment of 22Rv1 cells with C86 or JG98 led to a time and dose-dependent decrease in AR and ARv7 protein, concomitant with a significant loss of viability. We also observed that HSP40/HSP70 inhibition markedly reduced AR and ARv7 transcriptional activity, as indicated by decreased AR (KLK3, TMPRSS2) and ARv7 (UBE2C) target gene expression. Finally, treatment of mice bearing 22Rv1 xenografts with JG231 (an analog of JG98 with enhanced PK properties) led to significantly smaller tumors relative to vehicle treated mice. Together, these data confirm the continued dependence of AR and ARv7 on HSP40/HSP70 molecular chaperones and they demonstrate the feasibility of targeting the HSP40/HSP70 axis to abrogate sustained AR-mediated signaling in CRPC. Citation Format: Michael A. Moses, Yeong Sang Kim, Genesis Rivera-Marquez, Matthew J. Watson, Sunmin Lee, Andrea Kravats, Sue Wickner, Jason Gestwicki, Jane Trepel, Len Neckers. Targeting the HSP40/HSP70 chaperone axis as a novel strategy to treat castration-resistant prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1180. doi:10.1158/1538-7445.AM2017-1180

Pathways of Protein Remodeling by Escherichia Coli Molecular Chaperones

A new concept in molecular biology that has evolved over the past ten years is that proteins fold with assistance from other proteins, collectively referred to as molecular chaperones. All organisms, from bacteria to humans, possess several classes of highly conserved molecular chaperones, defined generally as proteins that bind to non-native conformations of proteins and facilitate correct folding and to native proteins and promote refolding. Extensive work has shown that the function as well as the structure of chaperones are highly conserved and the results obtained from studies of one organism are usually applicable to other organisms (1–3). This review will focus on several ATP-dependent molecular chaperone systems in Escherichia coli, including (i) DnaK, and its co-chaperones, DnaJ and GrpE, (ii) Clp proteins and (iii) GroEL and its co-chaperone, GroES (summarized in Table 1).

The Replication Initiator Protein of P1 Is Activated by two E. coli Heat Shock Proteins, DnaJ and DnaK

Two E. coli heat shock proteins, DnaK (the hsp70 homolog) and DnaJ activate the specific DNA binding function of the replication initiator protein, RepA, of plasmid P1 by about 100-fold (Wickner et al., 1991). The activation is ATP-dependent and DNA-independent. The mechanism of activation is the conversion of RepA dimers to monomers and the monomer form binds with high affinity to P1 origin DNA. Treatment of RepA with reversible chemical denaturants also converts dimers to monomers and simultaneously activates P1 origin binding. Increasing protein concentration converts monomers to dimers and deactivates RepA.

DnaJ and DnaK Heat Shock Proteins Activate Sequence Specific DNA Binding by RepA

Two families of heat shock proteins, HSP60s and HSP70s, have been implicated in mediating the folding and unfolding of proteins and the assembly and disassembly of oligomeric protein structures (for review see Rothman, 1989). The mechanisms by which heat shock proteins (HSPs) catalyze these reactions have not been elucidated and are the object of great interest, particularly in view of their ubiquity and high conservation during evolution (for reviews see Neidhardt and Van Bogelen, 1987; Lindquist and Craig, 1988; Munro and Pelham, 1986). DnaK is the E. coli HSP70 homolog. It is involved in chromosome segregation at normal temperatures (Bukau and Walker, 1989) and is essential for growth (Itikawa and Ryu, 1979; Sato and Uchida, 1978) and DNA replication (Sakakibara, 1988) at high temperatures. It is also involved in replication of phage 1 and plasmids mini-F and mini- P1. Two other heat shock proteins, DnaJ and GrpE are also involved in the replication of these three replicons (Georgopoulos, 1977; Sunshine etal., 1977; Saito and Uchida, 1977; Zylicz etal., 1989; Mensa-Wilmot etal., 1989; Tilly and Yarmolinsky, 1989; Bukau and Walker, 1989; Wickner, 1990; Kawasaki etal., 1990; Ezaki etal., 1989).