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ATP-dependent proteases differ substantially in their unfolding ability. a, linear representation of the multidomain substrates targeted to proteases through the C termini. b, degradation of N -DHFR-barnase- ssrA- C substrate by ClpAP at 30 °C (see under “Experimental Procedures”), with and without 100 M methotrexate; data shown as autoradiograms of SDS-polyacrylamide gels. c, their quantification, amount of substrate proteins remaining ( Ϫ ligand, E ; ϩ ligand, F ) and the amount of degradation end products ( Ϫ ligand, XI ; ϩ ligand, f ) . d, observed unfolding ability of ClpAP is independent of protease concentration. Unfolding ability values were measured at two different protease concentrations (0.2 M ClpA 6 , 0.4 M Clp 14 and 0.8 M ClpA 6 , 1.6 M Clp 14 ). Values are means Ϯ S.E. from three experiments. e and f, degradation of N -DHFR-barnase-
Source publication
ATP-dependent proteases control the concentrations of hundreds of regulatory proteins and remove damaged or misfolded proteins
from cells. They select their substrates primarily by recognizing sequence motifs or covalent modifications. Once a substrate
is bound to the protease, it has to be unfolded and translocated into the proteolytic chamber to...
Contexts in source publication
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... Unfolding Abilities of AAA Proteases-To compare the unfolding abilities of AAA pro- teases, we again followed the deg- radation of model proteins com- posed of appropriate targeting signals followed by a barnase and then a DHFR domain (Fig. 2a). The barnase domain was destabi- lized by mutagenesis in some experiments so that all proteases were able to unfold and degrade the domain completely. The sta- bility of the DHFR domain was adjusted by changing assay tem- perature or by replacing mouse DHFR with the more stable E. coli DHFR (G unfolding 6.1 kcal mol 1 for E. coli DHFR ...
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... the DHFR domain, the targeting signals have already been degraded. Thus, the inter- mediate, once released, cannot be retargeted to the protease (i.e. k rel is negligible) and instead accumulates as a fragment. Indeed, the amount of fragment formed did not change with protease or substrate concentrations as expected in the absence of rebinding ( Fig. 2d and supplemental Fig. 2 and data not ...
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... signals have already been degraded. Thus, the inter- mediate, once released, cannot be retargeted to the protease (i.e. k rel is negligible) and instead accumulates as a fragment. Indeed, the amount of fragment formed did not change with protease or substrate concentrations as expected in the absence of rebinding ( Fig. 2d and supplemental Fig. 2 and data not ...
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... Unfold Model Proteins-We first compared protease unfold- ing abilities for substrates targeted through C-terminal signals ( Fig. 2 and Table 1). In these constructs, the DHFR domain is located at the N terminus of the substrate, followed by the bar- nase domain and the C-terminal targeting signal (Fig. 2a). Strikingly, the unfolding abilities of the proteases differed by more than 2 orders of magnitude (Fig. 2g). Among the bacterial proteases, ClpAP was the ...
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... Unfold Model Proteins-We first compared protease unfold- ing abilities for substrates targeted through C-terminal signals ( Fig. 2 and Table 1). In these constructs, the DHFR domain is located at the N terminus of the substrate, followed by the bar- nase domain and the C-terminal targeting signal (Fig. 2a). Strikingly, the unfolding abilities of the proteases differed by more than 2 orders of magnitude (Fig. 2g). Among the bacterial proteases, ClpAP was the strongest (U 1.3 0.1 for the more stable E. coli DHFR at 30 °C), followed by ClpXP (U 0.4 0.1) and HslUV (U 0.1 0.03). Lon exhibited a weak unfolding ability (U 0), and as expected ...
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... ing abilities for substrates targeted through C-terminal signals ( Fig. 2 and Table 1). In these constructs, the DHFR domain is located at the N terminus of the substrate, followed by the bar- nase domain and the C-terminal targeting signal (Fig. 2a). Strikingly, the unfolding abilities of the proteases differed by more than 2 orders of magnitude (Fig. 2g). Among the bacterial proteases, ClpAP was the strongest (U 1.3 0.1 for the more stable E. coli DHFR at 30 °C), followed by ClpXP (U 0.4 0.1) and HslUV (U 0.1 0.03). Lon exhibited a weak unfolding ability (U 0), and as expected (30), FtsH failed to degrade wild type DHFR even at elevated temperatures ( Table 1). The archaebacterial ...
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... Abilities and Substrate Selection-Despite the common degradation mechanism, the unfolding abilities of ATP-dependent proteases differ by more than 2 orders of mag- nitude. ATP-dependent proteases select proteins for destruc- Fig. 2a, and the details are given under "Experimental Procedures." b, unfolding ability values estimated for different proteases using barnase (clear bar graphs), barstar (light gray bar graphs), and CheY (dark gray bar graphs). Measurements are means S.E. from three repeat experiments. Asterisk indicates HslUV and Lon were unable to degrade ...
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Citations
... For example, Lon's reported weak unfolding ability is a property that might seem to limit widespread efficacy. 33 However, analysis of Lon mechanics has been hampered by lack of a high-affinity degron that converts model proteins into substrates. Our use of the M. florum Lon/ssrA pair solved this issue and allowed us to characterize unfoldase strength, translocation step size and rate, and processivity. ...
... MfLon is an unexpectedly strong and processive unfoldase E. coli Lon ranked as a "weak" unfoldase in experiments challenging AAA+ proteases with substrates of increasing thermodynamic stabilities, positioned well behind ClpXP, ClpAP, and the 26S proteasome. 33 However, MfLon unfolds and degrades degron-tagged GFP and titin I27 , which are difficult proteins to unfold. Moreover, MfLon is highly processive, and many of our single-molecule traces included a Halo unfolding event, indicating that the entire substrate was degraded ( Figures 5A and 5C), even though MfLon is a slower translocase and unfoldase than other AAA+ proteases. ...
Lon is a widely distributed AAA+ (ATPases associated with diverse cellular activities) protease known for degrading poorly folded and damaged proteins and is often classified as a weak protein unfoldase. Here, using a Lon-degron pair from Mesoplasma florum (MfLon and MfssrA, respectively), we perform ensemble and single-molecule experiments to elucidate the molecular mechanisms underpinning MfLon function. Notably, we find that MfLon unfolds and degrades stably folded substrates and that translocation of these unfolded polypeptides occurs with a ∼6-amino-acid step size. Moreover, the time required to hydrolyze one ATP corresponds to the dwell time between steps, indicating that one step occurs per ATP-hydrolysis-fueled "power stroke." Comparison of MfLon to related AAA+ enzymes now provides strong evidence that HCLR-clade enzymes function using a shared power-stroke mechanism and, surprisingly, that MfLon is more processive than ClpXP and ClpAP. We propose that ample unfoldase strength and substantial processivity are features that contribute to the Lon family's evolutionary success.
... Similarly, in the blue module, we identified the Os07g0424400 hub gene in chr7 that played an important role in cellulose synthase A catalytic subunit 3 [UDP-forming] that governs a major mechanism of cell wall formation (Taylor et al., 2000) and ultimately helps in supporting the plant growth against abiotic stress. In the brown module, the top potential hub gene, Os01g0743600, is located on chr1 with the reported function of ATP-dependent protease La domain containing protein (Koodathingal et al., 2009) and it is one of the key components in providing protection against the harmful effects of unfolded proteins. It is activated by stress conditions in the endoplasmic reticulum (ER) and it supports plant defense as well as response to abiotic stresses (Bao and Howell, 2017). ...
Rice is an important staple food grain consumed by most of the population around the world. With climate and environmental changes, rice has undergone a tremendous stress state which has impacted crop production and productivity. Plant growth hormones are essential component that controls the overall outcome of the growth and development of the plant. Cytokinin is a hormone that plays an important role in plant immunity and defense systems. Trans-zeatin is an active form of cytokinin that can affect plant growth which is mediated by a multi-step two-component phosphorelay system that has different roles in various developmental stages. Systems biology is an approach for pathway analysis to trans-zeatin treated rice that could provide a deep understanding of different molecules associated with them. In this study, we have used a weighted gene co-expression network analysis method to identify the functional modules and hub genes involved in the cytokinin pathway. We have identified nine functional modules comprising of different hub genes which contribute to the cytokinin signaling route. The biological significance of these identified hub genes has been tested by applying well-proven statistical techniques to establish the association with the experimentally validated QTLs and annotated by the DAVID server. The establishment of key genes in different pathways has been confirmed. These results will be useful to design new stress-resistant cultivars which can provide sustainable yield in stress-specific conditions.
... This weak activity seems to act as a regulatory mechanism to ensure these proteases do not aberrantly process correctly folded proteins. 114 Interestingly, both Lon and FtsH are further distinguished from the other ATP-dependent proteases as their ATPase domain and C-terminal proteolytic domain are covalently linked, and it is unclear whether this structural feature contributes to their comparatively weaker proteolytic activity. 111,115 The FtsH protease is responsible for degradation of misfolded or uncomplexed membrane proteins and shortlived transcription factors. ...
... All cleavage sites are sequestered on the inside of core particle and their entry is controlled by regulatory particles attach to the alpha ring on one or both of the sides. ATPase are present in the form of hexametric rings at the base of regulatory particle and function as a driving force in substrate unfolding and translocation (Koodathingal et al., 2009). Proteasome secondary structure and its active site residues are shown in Figure 1. ...
Tuberculosis (TB) has been recently declared as a health emergency because of sporadic increase in Multidrug-resistant Tuberculosis (MDR-TB) problem throughout the world. TB causing bacteria, Mycobacterium tuberculosis has become resistant to the first line of treatment along with second line of treatment and drugs, which are accessible to us. Thus, there is an urgent need of identification of key targets and development of potential therapeutic approach(s), which can overcome the Mycobacterium tuberculosis complications. In the present study, Mycobacterium tuberculosis proteasome has been taken as a potential target as it is one of the key regulatory proteins in Mycobacterium tuberculosis propagation. Further, a library of 400 compounds (small molecule) from Medicines for Malaria Venture (MMV) were screened against the target (proteasome) using molecular docking and simulation approach, and selected lead compounds were validated in in vitro model. In this study, we have identified two potent small molecules from the MMV Pathogen Box library, MMV019838 and MMV687146 with À9.8 kcal/mol and À8.7 kcal/mol binding energy respectively, which actively interact with the catalytic domain/active domain of Mycobacterium tuberculosis proteasome and inhibit the Mycobacterium tuberculosis growth in in vitro culture. Furthermore, the molecular docking and simulation study of MMV019838 and MMV687146 with proteasome show strong and stable interaction with Mycobacterium tuberculosis compared to human proteasome and show no cytotoxicity effect. A better understanding of proteasome inhibition in Mycobacterium tuberculosis in in vitro and in vivo model would eventually allow us to understand the molecular mechanism(s) and discover a novel and potent therapeutic agent against Tuberculosis. Active efflux of drugs mediated by efflux pumps that confer drug resistance is one of the mechanisms developed by bacteria to counter the adverse effects of antibiotics and chemicals. Efflux pump activity was tested for a specific compound MMV019838 which was showing good in silico results than MIC values. ARTICLE HISTORY
... One possible explanation is that the proteasome grips the partially unfolded substrate more tightly, preventing the backsliding that would be required for refolding to occur. In agreement with this, we have found that the proteasome has a much higher unfolding ability (the ratio between unfolding and release rates) than bacterial ATP-dependent proteases (21) and that mammalian proteasome has an even higher unfolding ability than yeast proteasome because of a slower release rate, despite a slower ATPase rate and unfolding rate (36). Given that the bacterial ATPases tend to run faster than the proteasome (ClpXP has an ATPase rate ;5-10-fold faster than that of the proteasome), it seems most likely that the higher unfolding ability of the proteasome, and its lower tendency to stall on GFP, is due to a slower release of partially degraded substrates. ...
The Ubiquitin-proteasome system (UPS) is the canonical pathway for protein degradation in eukaryotic cells. Green fluorescent protein (GFP) is frequently used as a reporter in proteasomal degradation assays. However, there are multiple variants of GFP in use, and these variants have different intrinsic stabilities. Further, there are multiple means by which substrates are targeted to the proteasome, and these differences could also affect the proteasome's ability to unfold and degrade substrates. Herein we investigate how the fate of GFP variants of differing intrinsic stabilities is determined by the mode of targeting to the proteasome. We compared two targeting systems: linear Ub4 degrons and the UBL domain from yeast Rad23, both of which are commonly used in degradation experiments. Surprisingly, the UBL degron allows for degradation of the most stable sGFP-containing substrates, while the Ub4 degron does not. Destabilizing the GFP by circular permutation allows degradation with either targeting signal, indicating that domain stability and mode of targeting combine to determine substrate fate. Difficult-to-unfold substrates are released and re-engaged multiple times, with removal of the degradation initiation region providing an alternative clipping pathway that precludes unfolding and degradation; the UBL degron favors degradation of even difficult-to-unfold substrates while the Ub4 degron favors clipping. Finally, we show that the ubiquitin receptor Rpn13 is primarily responsible for the enhanced ability of the proteasome to degrade stable UBL-tagged substrates. Our results indicate that the choice of targeting method and reporter protein are critical to the design of protein degradation experiments.
... These ATP-dependent proteases remove damaged, denatured and aberrantly folded proteins from the cell [43]. However, it is still poorly understood how these unfoldases discriminate between potential target proteins [44][45][46][47][48]. As our data suggested that Rac acetylates and influences the substrate specificity of these two unfoldases, we constructed single and double knockout P. aeruginosa strains of clpX and hslU. ...
In this study, we describe the biological function of the phage-encoded protein RNA polymerase alpha subunit cleavage protein (Rac), a predicted Gcn5-related acetyltransferase encoded by phiKMV-like viruses. These phages encode a single-subunit RNA polymerase for transcription of their late (structure- and lysis-associated) genes, whereas the bacterial RNA polymerase is used at the earlier stages of infection. Rac mediates the inactivation of bacterial transcription by introducing a specific cleavage in the α subunit of the bacterial RNA polymerase. This cleavage occurs within the flexible linker sequence and disconnects the C-terminal domain, required for transcription initiation from most highly active cellular promoters. To achieve this, Rac likely taps into a novel post-translational modification (PTM) mechanism within the host Pseudomonas aeruginosa. From an evolutionary perspective, this novel phage-encoded regulation mechanism confirms the importance of PTMs in the prokaryotic metabolism and represents a new way by which phages can hijack the bacterial host metabolism.
... On the other hand, circular permutants of GFP that unfold in a single step have been shown to be degraded by the bacterial ATP-dependent protease ClpXP without stalling (20). The proteasome has been suggested to be even more processive than ClpXP (21) and thus capable of degrading even difficult-to-unfold substrates like GFP without stalling (17). However, in some cases GFP unfolding and degradation in cells requires the unfoldase cdc48/p97 in addition to the proteasome's own unfoldase activity (22)(23)(24). ...
The Ubiquitin-proteasome system (UPS) is the canonical pathway for protein degradation in eukaryotic cells. Green fluorescent protein (GFP) is frequently used as a reporter in proteasomal degradation assays. However, there are multiple variants of GFP in use, and these variants have different stabilities. We previously found that the proteasomes ability to unfold and degrade substrates is enhanced by polyubiquitin chains on the substrate, and that proteasomal ubiquitin receptors mediate this activation. Herein we investigate how the fate of GFP variants of differing stabilities is determined by the mode of targeting to the proteasome. We compared two targeting systems: linear Ub 4 degrons and the UBL domain from yeast Rad23, both of which are commonly used in degradation experiments. Surprisingly, the UBL degron allows for degradation of the most stable sGFP-containing substrates, while the Ub 4 degron does not. Destabilizing the GFP by circular permutation allows degradation with either targeting signal, indicating that domain stability and mode of targeting combine to determine substrate fate. Finally, we show that the ubiquitin receptor Rpn13 is primarily responsible for the enhanced ability of the proteasome to degrade stable UBL-tagged substrates.
... The best-studied degron for E. coli ClpXP is the 11-residue ssrA tag, which is cotranslationally appended to the C-terminus of a protein by the ubiquitous tmRNA system when a bacterial ribosome stalls prior to completing synthesis (11,12). Recombinant addition of this tag to almost any polypeptide or protein makes it a substrate for ClpXP degradation (13)(14)(15)(16)(17)(18)(19). ATP or ATPγS must bind to the AAA+ ring of ClpX to support binding to the ssrA tag, but hydrolysis of these nucleotides is not required for ssrA-tag recognition (15,(20)(21)(22)(23)(24). ...
... The copyright holder for this preprint (which this version posted May 5, 2020. . https://doi.org/10.1101/2020.05.04.076331 doi: bioRxiv preprint domain is encountered, and single-domain substrates are also likely to be released multiple times prior to degradation (16,19,(25)(26)(27)(28). Single-chain ClpX was labeled at residue 170 of one subunit with a fluorescent TAMRA dye, and a cysteine between native titin and the ssrA tag was labeled with a black-hole quencher. ...
E. coli ClpXP is one of the most thoroughly studied AAA+ proteases, but relatively little is known about the reactions that allow it to bind and then engage specific protein substrates before the ATP-fueled mechanical unfolding and translocation steps that lead to processive degradation. Here, we employ a fluorescence-quenching assay to study the binding of ssrA-tagged substrates to ClpXP. Polyphasic stopped-flow association and dissociation kinetics support the existence of at least three distinct substrate-bound ClpXP complexes. These kinetic data fit well to a model in which ClpXP and substrate form an initial binding complex, followed by an intermediate complex, and then an engaged complex that is competent for substrate unfolding. The initial association and dissociation steps do not require ATP hydrolysis, but subsequent forward and reverse kinetic steps are accelerated by faster ATP hydrolysis. Our results, together with recent cryo-EM structures of ClpXP bound to substrates, support a model in which the ssrA degron initially binds in the top portion of the axial channel of the ClpX hexamer and then is translocated deeper into the channel in steps that eventually pull the native portion of the substrate against the channel opening. Reversible initial substrate binding allows ClpXP to check potential substrates for degrons, potentially increasing specificity. Subsequent substrateengagement steps allow ClpXP to grip a wide variety of sequences to ensure efficient unfolding and translocation of almost any native substrate.
Significance
AAA+ proteases play key regulatory and quality-control roles in all domains of life. These destructive enzymes recognize damaged, unneeded, or regulatory proteins via specific degrons and unfold them prior to processive degradation. Here, we show that E. coli ClpXP, a model AAA+ protease, recognizes ssrA-tagged substrates in a multistep binding and engagement reaction. Together with recent cryo-EM structures, our experiments reveal how ClpXP transitions from a machine that checks potential substrates for appropriate degrons to one that can unfold and translocate almost any protein. Other AAA+ proteases in organelles and bacteria are likely to use similar mechanisms to specifically identify and then destroy their target proteins.
... La protéase Lon est quant à elle capable de reconnaître largement les protéines mal repliées. Comparée aux autres protéases dépendantes de l'énergie, la protéase Lon a la plus faible capacité de dépliement 244 , mais un répertoire de substrat étonnamment diversifié. En effet, Lon semble reconnaître les résidus hydrophobes sur des substrats mal repliés qui sont généralement enfouis dans la structure native comme étant son principal déterminant de reconnaissance plutôt qu'un motif de séquence unique 245 . ...
... Dans ce contexte, il a été suggéré que l'adhérence au substrat protéique est réduite lorsque l'anneau ClpX n'est que partiellement saturé en ATP, ce qui affecte les protéines les plus stables qui nécessitent une adhérence forte pour être dépliées 323 . ClpXP peut même libérer des protéines partiellement dégradées 244,289,316,324 . La libération de protéines substrat stables (sur le plan mécanique) partiellement dégradées peut se produire en présence d'un autre substrat protéique moins stable. ...
... Une autre hypothèse est que les GFPssrA seraient partiellement prises en charge par le protéasome PAN-20S, qui les relâcherait sous forme partiellement dépliées et éventuellement partiellement dégradées. Cela a été décrit pour la prise en charge de protéines stables d'un point de vue mécanique par le système ClpXP244,289,316,324 . Il a également été suggéré que la libération de substrats protéiques stables au profit de la prise en charge de protéines substrat moins stables permettrait à ClpXP de dégrader préférentiellement les substrats protéiques les moins stables dans une population316 . ...
Les protéasomes sont de grands assemblages macromoléculaires ubiquitaires composés d’un complexe catalytique 20S et d’une particule régulatrice comprenant un module AAA-ATPase. Cette machine cellulaire est chargée de dégrader sélectivement les protéines intracellulaires pour permettre le renouvellement du protéome, éliminer les protéines défectueuses et contrôler de nombreuses fonctions biologiques. Le travail de cette thèse avait pour objectif de mettre à jour les mécanismes qui permettent aux complexes AAA-ATPase de déplier sélectivement les protéines substrat et de les transférer à la particule 20S, dans laquelle elles sont détruites. Pour cela, une approche novatrice a été utilisée en combinant la diffusion de neutrons aux petits angles résolue en temps (TR-SANS) avec la spectroscopie de fluorescence permettant de suivre l’activité biochimique. Le protéasome de l’archée hyperthermophile Methanocaldococcus jannaschii, a été utilisé comme système modèle. Il est composé de la protéase 20S et de la particule régulatrice AAA-ATPase PAN. Un variant de la protéine fluorescente GFP a été utilisé comme protéine substrat.Les données obtenues montrent que l’activité de dépliement de PAN génère des formes de GFP dénaturée formant des agrégats. En revanche, l’association avec la particule 20S prévient la formation de ces espèces et indique qu’une fois le dépliement d’une protéine par PAN engagé, les processus de transfert dans le complexe 20S et de dégradation sont étroitement couplés. L’analyse des spectres de diffusion neutronique du substrat GFP montrent que la population de GFP native disparait rapidement au profit des peptides générés par la protéase 20S, comme confirmé par une analyse en spectrométrie de masse. Cela démontre le caractère hautement processif du protéasome. Enfin, deux modes d'action de PAN ont été mis en évidence selon la quantité de protéines à dégrader par rapport au protéasome PAN-20S. Ces travaux permettent de valider expérimentalement un des modèles de fonctionnement du protéasome préalablement proposés et soulignent l’importance d’un contrôle de l’association des protéasomes in vivo. Cette étude met également en valeur l’intérêt de la technique TR-SANS pour étudier la dynamique fonctionnelle de grandes machines cellulaires.
... For simplicity, non-productive deubiquitination of the substrate, which can occur in competition with engagement and initial degradation, is not shown. Control experiments in which the order of the DHFR and barnase domains are reversed confirm that degradation proceeds from the N-terminus of the protein, as DHFR stabilized by methotrexate (MTX) is able to protect barnase from degradation ( Supplementary Fig. S1a-d) [25][26][27] . Capping the N-terminus of the protein with maltose-binding protein (MBP) slows degradation but doesn't affect the size of the fragment produced, further demonstrating the directionality of degradation ( Supplementary Fig. S1e-h). ...
The ubiquitin-proteasome system (UPS) is responsible for the bulk of protein degradation in eukaryotic cells, but the factors that cause different substrates to be unfolded and degraded to different extents are still poorly understood. We previously showed that polyubiquitinated substrates were degraded with greater processivity (with a higher tendency to be unfolded and degraded than released) than ubiquitin-independent substrates. Thus, even though ubiquitin chains are removed before unfolding and degradation occur, they affect the unfolding of a protein domain. How do ubiquitin chains activate the proteasome’s unfolding ability? We investigated the roles of the three intrinsic proteasomal ubiquitin receptors - Rpn1, Rpn10 and Rpn13 - in this activation. We find that these receptors are required for substrate-mediated activation of the proteasome’s unfolding ability. Rpn13 plays the largest role, but there is also partial redundancy between receptors. The architecture of substrate ubiquitination determines which receptors are needed for maximal unfolding ability, and, in some cases, simultaneous engagement of ubiquitin by multiple receptors may be required. Our results suggest physical models for how ubiquitin receptors communicate with the proteasomal motor proteins.
