T URNOVER OF AB ERR ANT PROT EINS IN E. COLI . . . . . . . . . . . . . . . . . 445 The Lon Protease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 446 DnaK. DnaJ. GrpE. GroEL and GroES . . . . . . . . . . . . . . . . . . . . . . . . . . 447 The Clp Protease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 448 The DegP Protease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 452

The Function of Heat-Shock Proteins in Stress Tolerance: Degradation and Reactivation of Damaged Proteins

Using a combination of computer methods for iterative database searches and multiple sequence alignment, we show that protein sequences related to the AAA family of ATPases are far more prevalent than reported previously. Among these are regulatory components of Lon and Clp proteases, proteins involved in DNA replication, recombination, and restriction (including subunits of the origin recognition complex, replication factor C proteins, MCM DNA-licensing factors and the bacterial DnaA, RuvB, and McrB proteins), prokaryotic NtrC-related transcription regulators, the Bacillus sporulation protein SpoVJ, Mg2+, and Co2+ chelatases, the Halobacterium GvpN gas vesicle synthesis protein, dynein motor proteins, TorsinA, and Rubisco activase. Alignment of these sequences, in light of the structures of the clamp loader delta' subunit of Escherichia coli DNA polymerase III and the hexamerization component of N-ethylmaleimide-sensitive fusion protein, provides structural and mechanistic insights into these proteins, collectively designated the AAA+ class. Whole-genome analysis indicates that this class is ancient and has undergone considerable functional divergence prior to the emergence of the major divisions of life. These proteins often perform chaperone-like functions that assist in the assembly, operation, or disassembly of protein complexes. The hexameric architecture often associated with this class can provide a hole through which DNA or RNA can be thread; this may be important for assembly or remodeling of DNA-protein complexes.

AAA+: A Class of Chaperone-Like ATPases Associated with the Assembly, Operation, and Disassembly of Protein Complexes

▪ Abstract A growing number of cellular regulatory mechanisms are being linked to protein modification by the polypeptide ubiquitin. These include key transitions in the cell cycle, class I antigen processing, signal transduction pathways, and receptor-mediated endocytosis. In most, but not all, of these examples, ubiquitination of a protein leads to its degradation by the 26S proteasome. Following attachment of ubiquitin to a substrate and binding of the ubiquitinated protein to the proteasome, the bound substrate must be unfolded (and eventually deubiquitinated) and translocated through a narrow set of channels that leads to the proteasome interior, where the polypeptide is cleaved into short peptides. Protein ubiquitination and deubiquitination are both mediated by large enzyme families, and the proteasome itself comprises a family of related but functionally distinct particles. This diversity underlies both the high substrate specificity of the ubiquitin system and the variety of regulatory mechanisms...

Ubiquitin-dependent protein degradation

Heat shock protein 90 (HSP90) is a highly conserved molecular chaperone that facilitates the maturation of a wide range of proteins (known as clients). Clients are enriched in signal transducers, including kinases and transcription factors. Therefore, HSP90 regulates diverse cellular functions and exerts marked effects on normal biology, disease and evolutionary processes. Recent structural and functional analyses have provided new insights on the transcriptional and biochemical regulation of HSP90 and the structural dynamics it uses to act on a diverse client repertoire. Comprehensive understanding of how HSP90 functions promises not only to provide new avenues for therapeutic intervention, but to shed light on fundamental biological questions.

HSP90 at the hub of protein homeostasis: emerging mechanistic insights

The proteasome is an intricate molecular machine, which serves to degrade proteins following their conjugation to ubiquitin. Substrates dock onto the proteasome at its 19-subunit regulatory particle via a diverse set of ubiquitin receptors and are then translocated into an internal chamber within the 28-subunit proteolytic core particle (CP), where they are hydrolyzed. Substrate is threaded into the CP through a narrow gated channel, and thus translocation requires unfolding of the substrate. Six distinct ATPases in the regulatory particle appear to form a ring complex and to drive unfolding as well as translocation. ATP-dependent, degradation-coupled deubiquitination of the substrate is required both for efficient substrate degradation and for preventing the degradation of the ubiquitin tag. However, the proteasome also contains deubiquitinating enzymes (DUBs) that can remove ubiquitin before substrate degradation initiates, thus allowing some substrates to dissociate from the proteasome and escape degradation. Here we examine the key elements of this molecular machine and how they cooperate in the processing of proteolytic substrates.

Recognition and Processing of Ubiquitin-Protein Conjugates by the Proteasome

The two major molecular chaperone families that mediate ATP-dependent protein folding and refolding are the heat shock proteins Hsp60s (GroEL) and Hsp70s (DnaK). Clp proteins, like chaperones, are highly conserved, present in all organisms, and contain ATP and polypeptide binding sites. We discovered that ClpA, the ATPase component of the ATP-dependent ClpAP protease, is a molecular chaperone. ClpA performs the ATP-dependent chaperone function of DnaK and DnaJ in the in vitro activation of the plasmid P1 RepA replication initiator protein. RepA is activated by the conversion of dimers to monomers. We show that ClpA targets RepA for degradation by ClpP, demonstrating a direct link between the protein unfolding function of chaperones and proteolysis. In another chaperone assay, ClpA protects luciferase from irreversible heat inactivation but is unable to reactivate luciferase.

https://www.pnas.org/content/pnas/91/25/12218.full.pdf

A molecular chaperone, ClpA, functions like DnaK and DnaJ

All organisms use rapid degradation of specific proteins as one way to tightly control important regulatory factors and developmental switches. Regulation of biological functions by proteolysis requires accurate substrate recognition at a precise time. In eukaryotes, fidelity of protein degradation depends on ubiquitin-tagging, a process that occurs in multicomponent complexes involving regulatory factors and various ubiquitin pathway enzymes. The ubiquitinated proteins are then targeted to the proteasome for degradation (for review, see Voges et al. 1999). In Escherichia coli, an example of substrate tagging as a means for regulating proteolysis is the degradation of incomplete proteins made from truncated mRNAs. In this case, short peptides coded by the SsrA RNA are added cotranslationally to polypeptides that have become stalled on ribosomes, and the tagged proteins are then degraded (Keiler et al. 1996; Gottesman et al. 1998; Roche and Sauer 1999). In general, E. coli proteins regulated by degradation interact directly with the proteases themselves, with each of the five known E. coli ATP-dependent proteases degrading a different but somewhat overlapping set of substrates (Gottesman 1996; Wickner et al. 1999). For many of the highly unstable E. coli proteins, degradation is rapid under all conditions, and synthesis is tightly controlled.

However, the activity of some proteins in E. coli is controlled by regulated degradation. For example, σ32 (RpoH), the heat shock sigma factor, is rapidly degraded under normal growth conditions by the AAA protease, FtsH, in a reaction modulated by the DnaJ/DnaK/GrpE chaperone system. During heat shock, σ32 is transiently stabilized, and this stabilization results in the rapid increase in the synthesis of the heat shock proteins (Yura and Nakahigashi 1999).

The stationary phase sigma factor, σS (RpoS), is another example of a protein whose activity is controlled by regulated proteolysis. σS promotes expression of more than 50 genes involved in responses to many stresses, including starvation, osmotic stress, acid shock, cold shock, heat shock, and oxidative damage, as well as the transition to stationary phase (Loewen and Hengge-Aronis 1994; Hengge-Aronis 2000). Although σS is present at very low levels during exponential cell growth, owing largely to its rapid degradation (half-life of ∼2 min), its stability increases ∼10-fold following transition to stationary phase or other stress treatments. Regulated degradation plays a major role in determining the amount of σS in the cell, but σS accumulation is also regulated at the transcriptional and translational levels (Lange and Hengge-Aronis 1994). The protease responsible for σS turnover in exponentially growing cells is ClpXP (Schweder et al. 1996), an ATP-dependent protease consisting of a regulatory component, ClpX, and a proteolytic component, ClpP (Gottesman et al. 1993; Wojtkowiak et al. 1993).

Degradation of σS requires an additional protein, RssB (Regulator of Sigma S; also referred to as SprE in E. coli, MviA in Salmonella, and ExpM in Erwinia) that is homologous to response regulator proteins (Bearson et al. 1996; Muffler et al. 1996b; Pratt and Silhavy 1996; Andersson et al. 1999). Genetic evidence shows that RssB is required for σS degradation but not for another ClpXP substrate, λ O, which indicates that RssB specifically targets σS for degradation (Zhou and Gottesman 1998). One of the hallmarks of the response regulator component of two-component signal transduction systems in prokaryotes is the presence of a conserved aspartate that is phosphorylated by the cognate sensor component. The N-terminal domain of RssB contains this conserved aspartate, although a cognate sensor protein has not been identified. RssB, like many other response regulators, is phosphorylated by acetyl phosphate in vitro (Bouche et al. 1998). In addition, phosphorylated RssB forms a stable complex with σS in vitro (Becker et al. 1999).

In this report we investigate RssB-regulated degradation of σS by reconstituting the pathway of σS degradation in vitro with purified RssB and ClpXP. We discovered that RssB acts directly and catalytically in stimulating degradation of σS by ClpXP in a reaction requiring acetyl phosphate and ATP. We have isolated and characterized subassemblies of the degradation machinery including σS–RssB, σS–RssB–ClpX, and σS–RssB–ClpXP complexes, and suggest a probable pathway for the degradation of σS.

/pdf/the-rssb-response-regulator-directly-targets-ss-for-3nnmc418wm.pdf

The RssB response regulator directly targets ςS for degradation by ClpXP

ClpX and ClpA are molecular chaperones that interact with specific proteins and, together with ClpP, activate their ATP-dependent degradation. The chaperone activity is thought to convert proteins into an extended conformation that can access the sequestered active sites of ClpP. We now show that ClpX can catalyze unfolding of a green fluorescent protein fused to a ClpX recognition motif (GFP-SsrA). Unfolding of GFP-SsrA depends on ATP hydrolysis. GFP-SsrA unfolded either by ClpX or by treatment with denaturants binds to ClpX in the presence of adenosine 5′-O-(3-thiotriphosphate) and is released slowly (t1/2 ≈ 15 min). Unlike ClpA, ClpX cannot trap unfolded proteins in stable complexes unless they also have a high-affinity binding motif. Addition of ATP or ADP accelerates release (t1/2 ≈ 1 min), consistent with a model in which ATP hydrolysis induces a conformation of ClpX with low affinity for unfolded substrates. Proteolytically inactive complexes of ClpXP and ClpAP unfold GFP-SsrA and translocate the protein to ClpP, where it remains unfolded. Complexes of ClpXP with translocated substrate within the ClpP chamber retain the ability to unfold GFP-SsrA. Our results suggest a bipartite mode of interaction between ClpX and substrates. ClpX preferentially targets motifs exposed in specific proteins. As the protein is unfolded by ClpX, additional motifs are exposed that facilitate its retention and favor its translocation to ClpP for degradation.

https://www.pnas.org/content/pnas/97/16/8898.full.pdf

Unfolding and internalization of proteins by the ATP-dependent proteases ClpXP and ClpAP.

DnaK is a major heat shock protein of Escherichia coli and the homolog of hsp70 in eukaryotes. We demonstrate the mechanism by which DnaK and another heat shock protein, DnaJ, render the plasmid P1 initiator RepA 100-fold more active for binding to the P1 origin of replication. Activation is the conversion of RepA dimers into monomers in an ATP-dependent reaction and the monomer form binds with high affinity to oriP1 DNA. Reversible chemical denaturants also convert RepA dimers to monomers and simultaneously activate oriP1 DNA binding. Increasing protein concentration converts monomers to dimers and deactivates RepA. Based on our data and previous work, we present a model for heat shock protein action under normal and stress conditions.

Monomerization of RepA dimers by heat shock proteins activates binding to DNA replication origin

ClpA, a bacterial member of the Clp/Hsp100 chaperone family, is an ATP-dependent molecular chaperone and the regulatory component of the ATP-dependent ClpAP protease. To study the mechanism of binding and unfolding of proteins by ClpA and translocation to ClpP, we used as a model substrate a fusion protein that joined the ClpA recognition signal from RepA to green fluorescent protein (GFP). ClpAP degrades the fusion protein in vivo and in vitro. The substrate binds specifically to ClpA in a reaction requiring ATP binding but not hydrolysis. Binding alone is not sufficient to destabilize the native structure of the GFP portion of the fusion protein. Upon ATP hydrolysis the GFP fusion protein is unfolded, and the unfolded intermediate can be sequestered by ClpA if a nonhydrolyzable analog is added to displace ATP. ATP is required for release. We found that although ClpA is unable to recognize native proteins lacking recognition signals, including GFP and rhodanese, it interacts with those same proteins when they are unfolded. Unfolded GFP is held in a nonnative conformation while associated with ClpA and its release requires ATP hydrolysis. Degradation of unfolded untagged proteins by ClpAP requires ATP even though the initial ATP-dependent unfolding reaction is bypassed. These results suggest that there are two ATP-requiring steps: an initial protein unfolding step followed by translocation of the unfolded protein to ClpP or in some cases release from the complex.

https://www.pnas.org/content/pnas/97/16/8892.full.pdf

Joel R. Hoskins

Papers

A molecular chaperone, ClpA, functions like DnaK and DnaJ

The RssB response regulator directly targets ςS for degradation by ClpXP

Unfolding and internalization of proteins by the ATP-dependent proteases ClpXP and ClpAP.

Monomerization of RepA dimers by heat shock proteins activates binding to DNA replication origin

Protein binding and unfolding by the chaperone ClpA and degradation by the protease ClpAP.