Biomolecules

Research

Jump to: Review

25 pages, 4067 KiB

Open AccessArticle

Insight into the RssB-Mediated Recognition and Delivery of σ^s to the AAA+ Protease, ClpXP

by Dimce Micevski, Kornelius Zeth, Terrence D. Mulhern, Verena J. Schuenemann, Jessica E. Zammit, Kaye N. Truscott and David A. Dougan

Biomolecules 2020, 10(4), 615; https://doi.org/10.3390/biom10040615 - 16 Apr 2020

Cited by 10 | Viewed by 3282

Abstract

In Escherichia coli, SigmaS (σ^S) is the master regulator of the general stress response. The cellular levels of σ^S are controlled by transcription, translation and protein stability. The turnover of σ^S, by the AAA+ protease (ClpXP), is [...] Read more.

In Escherichia coli, SigmaS (σ^S) is the master regulator of the general stress response. The cellular levels of σ^S are controlled by transcription, translation and protein stability. The turnover of σ^S, by the AAA+ protease (ClpXP), is tightly regulated by a dedicated adaptor protein, termed RssB (Regulator of Sigma S protein B)—which is an atypical member of the response regulator (RR) family. Currently however, the molecular mechanism of σ^S recognition and delivery by RssB is only poorly understood. Here we describe the crystal structures of both RssB domains (RssB_N and RssB_C) and the SAXS analysis of full-length RssB (both free and in complex with σ^S). Together with our biochemical analysis we propose a model for the recognition and delivery of σ^S by this essential adaptor protein. Similar to most bacterial RRs, the N-terminal domain of RssB (RssB_N) comprises a typical mixed (βα)₅-fold. Although phosphorylation of RssB_N (at Asp58) is essential for high affinity binding of σ^S, much of the direct binding to σ^S occurs via the C-terminal effector domain of RssB (RssB_C). In contrast to most RRs the effector domain of RssB forms a β-sandwich fold composed of two sheets surrounded by α-helical protrusions and as such, shares structural homology with serine/threonine phosphatases that exhibit a PPM/PP2C fold. Our biochemical data demonstrate that this domain plays a key role in both substrate interaction and docking to the zinc binding domain (ZBD) of ClpX. We propose that RssB docking to the ZBD of ClpX overlaps with the docking site of another regulator of RssB, the anti-adaptor IraD. Hence, we speculate that docking to ClpX may trigger release of its substrate through activation of a “closed” state (as seen in the RssB-IraD complex), thereby coupling adaptor docking (to ClpX) with substrate release. This competitive docking to RssB would prevent futile interaction of ClpX with the IraD-RssB complex (which lacks a substrate). Finally, substrate recognition by RssB appears to be regulated by a key residue (Arg117) within the α5 helix of the N-terminal domain. Importantly, this residue is not directly involved in σ^S interaction, as σ^S binding to the R117A mutant can be restored by phosphorylation. Likewise, R117A retains the ability to interact with and activate ClpX for degradation of σ^S, both in the presence and absence of acetyl phosphate. Therefore, we propose that this region of RssB (the α5 helix) plays a critical role in driving interaction with σ^S at a distal site. Full article

(This article belongs to the Special Issue AAA+ Proteins in Health and Disease: Structure, Physiological Function, and Mechanisms of Action)

► Show Figures

Figure 1

15 pages, 9650 KiB

Open AccessFeature PaperArticle

Sorafenib as an Inhibitor of RUVBL2

by Nardin Nano, Francisca Ugwu, Thiago V. Seraphim, Tangzhi Li, Gina Azer, Methvin Isaac, Michael Prakesch, Leandro R. S. Barbosa, Carlos H. I. Ramos, Alessandro Datti and Walid A. Houry

Biomolecules 2020, 10(4), 605; https://doi.org/10.3390/biom10040605 - 14 Apr 2020

Cited by 12 | Viewed by 4427

Abstract

RUVBL1 and RUVBL2 are highly conserved ATPases that belong to the AAA+ (ATPases Associated with various cellular Activities) superfamily and are involved in various complexes and cellular processes, several of which are closely linked to oncogenesis. The proteins were implicated in DNA damage [...] Read more.

RUVBL1 and RUVBL2 are highly conserved ATPases that belong to the AAA+ (ATPases Associated with various cellular Activities) superfamily and are involved in various complexes and cellular processes, several of which are closely linked to oncogenesis. The proteins were implicated in DNA damage signaling and repair, chromatin remodeling, telomerase activity, and in modulating the transcriptional activities of proto-oncogenes such as c-Myc and β-catenin. Moreover, both proteins were found to be overexpressed in several different types of cancers such as breast, lung, kidney, bladder, and leukemia. Given their various roles and strong involvement in carcinogenesis, the RUVBL proteins are considered to be novel targets for the discovery and development of therapeutic cancer drugs. Here, we describe the identification of sorafenib as a novel inhibitor of the ATPase activity of human RUVBL2. Enzyme kinetics and surface plasmon resonance experiments revealed that sorafenib is a weak, mixed non-competitive inhibitor of the protein’s ATPase activity. Size exclusion chromatography and small angle X-ray scattering data indicated that the interaction of sorafenib with RUVBL2 does not cause a significant effect on the solution conformation of the protein; however, the data suggested that the effect of sorafenib on RUVBL2 activity is mediated by the insertion domain in the protein. Sorafenib also inhibited the ATPase activity of the RUVBL1/2 complex. Hence, we propose that sorafenib could be further optimized to be a potent inhibitor of the RUVBL proteins. Full article

(This article belongs to the Special Issue AAA+ Proteins in Health and Disease: Structure, Physiological Function, and Mechanisms of Action)

► Show Figures

Figure 1

18 pages, 5624 KiB

Open AccessArticle

ClpG Provides Increased Heat Resistance by Acting as Superior Disaggregase

by Panagiotis Katikaridis, Lena Meins, Shady Mansour Kamal, Ute Römling and Axel Mogk

Biomolecules 2019, 9(12), 815; https://doi.org/10.3390/biom9120815 - 2 Dec 2019

Cited by 12 | Viewed by 3419

Abstract

Elevation of temperature within and above the physiological limit causes the unfolding and aggregation of cellular proteins, which can ultimately lead to cell death. Bacteria are therefore equipped with Hsp100 disaggregation machines that revert the aggregation process and reactivate proteins otherwise lost by [...] Read more.

Elevation of temperature within and above the physiological limit causes the unfolding and aggregation of cellular proteins, which can ultimately lead to cell death. Bacteria are therefore equipped with Hsp100 disaggregation machines that revert the aggregation process and reactivate proteins otherwise lost by aggregation. In Gram-negative bacteria, two disaggregation systems have been described: the widespread ClpB disaggregase, which requires cooperation with an Hsp70 chaperone, and the standalone ClpG disaggregase. ClpG co-exists with ClpB in selected bacteria and provides superior heat resistance. Here, we compared the activities of both disaggregases towards diverse model substrates aggregated in vitro and in vivo at different temperatures. We show that ClpG exhibits robust activity towards all disordered aggregates, whereas ClpB acts poorly on the protein aggregates formed at very high temperatures. Extreme temperatures are expected not only to cause extended protein unfolding, but also to result in an accelerated formation of protein aggregates with potentially altered chemical and physical parameters, including increased stability. We show that ClpG exerts higher threading forces as compared to ClpB, likely enabling ClpG to process “tight” aggregates formed during severe heat stress. This defines ClpG as a more powerful disaggregase and mechanistically explains how ClpG provides increased heat resistance. Full article

(This article belongs to the Special Issue AAA+ Proteins in Health and Disease: Structure, Physiological Function, and Mechanisms of Action)

► Show Figures

Figure 1

Review

Jump to: Research

22 pages, 4698 KiB

Open AccessReview

AAA+ ATPases in Protein Degradation: Structures, Functions and Mechanisms

by Shuwen Zhang and Youdong Mao

Biomolecules 2020, 10(4), 629; https://doi.org/10.3390/biom10040629 - 18 Apr 2020

Cited by 29 | Viewed by 7040

Abstract

Adenosine triphosphatases (ATPases) associated with a variety of cellular activities (AAA+), the hexameric ring-shaped motor complexes located in all ATP-driven proteolytic machines, are involved in many cellular processes. Powered by cycles of ATP binding and hydrolysis, conformational changes in AAA+ ATPases can generate [...] Read more.

Adenosine triphosphatases (ATPases) associated with a variety of cellular activities (AAA+), the hexameric ring-shaped motor complexes located in all ATP-driven proteolytic machines, are involved in many cellular processes. Powered by cycles of ATP binding and hydrolysis, conformational changes in AAA+ ATPases can generate mechanical work that unfolds a substrate protein inside the central axial channel of ATPase ring for degradation. Three-dimensional visualizations of several AAA+ ATPase complexes in the act of substrate processing for protein degradation have been resolved at the atomic level thanks to recent technical advances in cryogenic electron microscopy (cryo-EM). Here, we summarize the resulting advances in structural and biochemical studies of AAA+ proteases in the process of proteolysis reactions, with an emphasis on cryo-EM structural analyses of the 26S proteasome, Cdc48/p97 and FtsH-like mitochondrial proteases. These studies reveal three highly conserved patterns in the structure–function relationship of AAA+ ATPase hexamers that were observed in the human 26S proteasome, thus suggesting common dynamic models of mechanochemical coupling during force generation and substrate translocation. Full article

(This article belongs to the Special Issue AAA+ Proteins in Health and Disease: Structure, Physiological Function, and Mechanisms of Action)

► Show Figures

Figure 1

17 pages, 1482 KiB

Open AccessFeature PaperReview

The Role of Torsin AAA+ Proteins in Preserving Nuclear Envelope Integrity and Safeguarding Against Disease

by Anthony J. Rampello, Sarah M. Prophet and Christian Schlieker

Biomolecules 2020, 10(3), 468; https://doi.org/10.3390/biom10030468 - 19 Mar 2020

Cited by 18 | Viewed by 4766

Abstract

Torsin ATPases are members of the AAA+ (ATPases associated with various cellular activities) superfamily of proteins, which participate in essential cellular processes. While AAA+ proteins are ubiquitously expressed and demonstrate distinct subcellular localizations, Torsins are the only AAA+ to reside within the nuclear [...] Read more.

Torsin ATPases are members of the AAA+ (ATPases associated with various cellular activities) superfamily of proteins, which participate in essential cellular processes. While AAA+ proteins are ubiquitously expressed and demonstrate distinct subcellular localizations, Torsins are the only AAA+ to reside within the nuclear envelope (NE) and endoplasmic reticulum (ER) network. Moreover, due to the absence of integral catalytic features, Torsins require the NE- and ER-specific regulatory cofactors, lamina-associated polypeptide 1 (LAP1) and luminal domain like LAP1 (LULL1), to efficiently trigger their atypical mode of ATP hydrolysis. Despite their implication in an ever-growing list of diverse processes, the specific contributions of Torsin/cofactor assemblies in maintaining normal cellular physiology remain largely enigmatic. Resolving gaps in the functional and mechanistic principles of Torsins and their cofactors are of considerable medical importance, as aberrant Torsin behavior is the principal cause of the movement disorder DYT1 early-onset dystonia. In this review, we examine recent findings regarding the phenotypic consequences of compromised Torsin and cofactor activities. In particular, we focus on the molecular features underlying NE defects and the contributions of Torsins to nuclear pore complex biogenesis, as well as the growing implications of Torsins in cellular lipid metabolism. Additionally, we discuss how understanding Torsins may facilitate the study of essential but poorly understood processes at the NE and ER, and aid in the development of therapeutic strategies for dystonia. Full article

(This article belongs to the Special Issue AAA+ Proteins in Health and Disease: Structure, Physiological Function, and Mechanisms of Action)

► Show Figures

Figure 1

12 pages, 2372 KiB

Open AccessReview

Bacterial Enhancer Binding Proteins—AAA⁺ Proteins in Transcription Activation

by Forson Gao, Amy E. Danson, Fuzhou Ye, Milija Jovanovic, Martin Buck and Xiaodong Zhang

Biomolecules 2020, 10(3), 351; https://doi.org/10.3390/biom10030351 - 25 Feb 2020

Cited by 25 | Viewed by 4686

Abstract

Bacterial enhancer-binding proteins (bEBPs) are specialised transcriptional activators. bEBPs are hexameric AAA⁺ ATPases and use ATPase activities to remodel RNA polymerase (RNAP) complexes that contain the major variant sigma factor, σ⁵⁴ to convert the initial closed complex to the transcription competent [...] Read more.

Bacterial enhancer-binding proteins (bEBPs) are specialised transcriptional activators. bEBPs are hexameric AAA⁺ ATPases and use ATPase activities to remodel RNA polymerase (RNAP) complexes that contain the major variant sigma factor, σ⁵⁴ to convert the initial closed complex to the transcription competent open complex. Earlier crystal structures of AAA⁺ domains alone have led to proposals of how nucleotide-bound states are sensed and propagated to substrate interactions. Recently, the structure of the AAA⁺ domain of a bEBP bound to RNAP-σ⁵⁴-promoter DNA was revealed. Together with structures of the closed complex, an intermediate state where DNA is partially loaded into the RNAP cleft and the open promoter complex, a mechanistic understanding of how bEBPs use ATP to activate transcription can now be proposed. This review summarises current structural models and the emerging understanding of how this special class of AAA⁺ proteins utilises ATPase activities to allow σ⁵⁴-dependent transcription initiation. Full article

(This article belongs to the Special Issue AAA+ Proteins in Health and Disease: Structure, Physiological Function, and Mechanisms of Action)

► Show Figures

Figure 1

17 pages, 2067 KiB

Open AccessReview

The Mitochondrial Lon Protease: Novel Functions off the Beaten Track?

by Wolfgang Voos and Karen Pollecker

Biomolecules 2020, 10(2), 253; https://doi.org/10.3390/biom10020253 - 7 Feb 2020

Cited by 27 | Viewed by 4839

Abstract

To maintain organellar function, mitochondria contain an elaborate endogenous protein quality control system. As one of the two soluble energy-dependent proteolytic enzymes in the matrix compartment, the protease Lon is a major component of this system, responsible for the degradation of misfolded proteins, [...] Read more.

To maintain organellar function, mitochondria contain an elaborate endogenous protein quality control system. As one of the two soluble energy-dependent proteolytic enzymes in the matrix compartment, the protease Lon is a major component of this system, responsible for the degradation of misfolded proteins, in particular under oxidative stress conditions. Lon defects have been shown to negatively affect energy production by oxidative phosphorylation but also mitochondrial gene expression. In this review, recent studies on the role of Lon in mammalian cells, in particular on its protective action under diverse stress conditions and its relationship to important human diseases are summarized and commented. Full article

(This article belongs to the Special Issue AAA+ Proteins in Health and Disease: Structure, Physiological Function, and Mechanisms of Action)

► Show Figures

Figure 1

34 pages, 6462 KiB

Open AccessReview

Shaping the Nascent Ribosome: AAA-ATPases in Eukaryotic Ribosome Biogenesis

by Michael Prattes, Yu-Hua Lo, Helmut Bergler and Robin E. Stanley

Biomolecules 2019, 9(11), 715; https://doi.org/10.3390/biom9110715 - 7 Nov 2019

Cited by 27 | Viewed by 6892

Abstract

AAA-ATPases are molecular engines evolutionarily optimized for the remodeling of proteins and macromolecular assemblies. Three AAA-ATPases are currently known to be involved in the remodeling of the eukaryotic ribosome, a megadalton range ribonucleoprotein complex responsible for the translation of mRNAs into proteins. The [...] Read more.

AAA-ATPases are molecular engines evolutionarily optimized for the remodeling of proteins and macromolecular assemblies. Three AAA-ATPases are currently known to be involved in the remodeling of the eukaryotic ribosome, a megadalton range ribonucleoprotein complex responsible for the translation of mRNAs into proteins. The correct assembly of the ribosome is performed by a plethora of additional and transiently acting pre-ribosome maturation factors that act in a timely and spatially orchestrated manner. Minimal disorder of the assembly cascade prohibits the formation of functional ribosomes and results in defects in proliferation and growth. Rix7, Rea1, and Drg1, which are well conserved across eukaryotes, are involved in different maturation steps of pre-60S ribosomal particles. These AAA-ATPases provide energy for the efficient removal of specific assembly factors from pre-60S particles after they have fulfilled their function in the maturation cascade. Recent structural and functional insights have provided the first glimpse into the molecular mechanism of target recognition and remodeling by Rix7, Rea1, and Drg1. Here we summarize current knowledge on the AAA-ATPases involved in eukaryotic ribosome biogenesis. We highlight the latest insights into their mechanism of mechano-chemical complex remodeling driven by advanced cryo-EM structures and the use of highly specific AAA inhibitors. Full article

(This article belongs to the Special Issue AAA+ Proteins in Health and Disease: Structure, Physiological Function, and Mechanisms of Action)

► Show Figures