scholarly journals Visualizing ATP-dependent substrate-processing dynamics of the human 26S proteasome at near-atomic resolution

2018 ◽  
Author(s):  
Yuanchen Dong ◽  
Shuwen Zhang ◽  
Zhaolong Wu ◽  
Xuemei Li ◽  
Wei Li Wang ◽  
...  
Keyword(s):  
Protein Degradation ◽  
Atp Hydrolysis ◽  
26S Proteasome ◽  
Eukaryotic Cells ◽  
Power Stroke ◽  
The Third ◽  
Atpase Subunits ◽  
Adp Release ◽  
Substrate Processing ◽  
Ubiquitylated Protein

AbstractThe proteasome is an ATP-dependent 2.5-megadalton machine responsible for ubiquitylated protein degradation in all eukaryotic cells. Here we present cryo-EM structures of the substrate-engaged human 26S proteasome in seven conformational states at 2.8-3.6 Å resolution, captured during polyubiquitylated protein degradation. These structures visualize a continuum of dynamic substrate-proteasome interactions from ubiquitin recognition to processive substrate translocation, during which ATP hydrolysis sequentially navigate through all six ATPase subunits. Three principle modes of coordinated ATP hydrolysis are observed, featuring hydrolytic events in two oppositely positioned ATPases, in two consecutive ATPases, and in one ATPase at a time. They regulate deubiquitylation, translocation initiation and processive unfolding of substrates, respectively. A collective power stroke in the ATPase motor is generated by synchronized ATP binding and ADP release in the substrate-engaging and disengaging ATPases, respectively. It is amplified largely in the substrate-disengaging ATPase, and propagated unidirectionally by coordinated ATP hydrolysis in the third consecutive ATPase.

scholarly journals Unraveling a Force-Generating Allosteric Pathway of Actomyosin Communication Associated with ADP and Pi Release

2020 ◽  
Vol 22 (1) ◽  
pp. 104
Author(s):  
Peter Franz ◽  
Wiebke Ewert ◽  
Matthias Preller ◽  
Georgios Tsiavaliaris
Keyword(s):  
Structural Changes ◽  
Atp Hydrolysis ◽  
Power Stroke ◽  
Duty Ratio ◽  
Prolonged Duration ◽  
Adp Release ◽  
Strong Transition ◽  
Cleft Closure ◽  
Kinetic Investigations ◽  
Allosteric Pathway

The actomyosin system generates mechanical work with the execution of the power stroke, an ATP-driven, two-step rotational swing of the myosin-neck that occurs post ATP hydrolysis during the transition from weakly to strongly actin-bound myosin states concomitant with Pi release and prior to ADP dissociation. The activating role of actin on product release and force generation is well documented; however, the communication paths associated with weak-to-strong transitions are poorly characterized. With the aid of mutant analyses based on kinetic investigations and simulations, we identified the W-helix as an important hub coupling the structural changes of switch elements during ATP hydrolysis to temporally controlled interactions with actin that are passed to the central transducer and converter. Disturbing the W-helix/transducer pathway increased actin-activated ATP turnover and reduced motor performance as a consequence of prolonged duration of the strongly actin-attached states. Actin-triggered Pi release was accelerated, while ADP release considerably decelerated, both limiting maximum ATPase, thus transforming myosin-2 into a high-duty-ratio motor. This kinetic signature of the mutant allowed us to define the fractional occupancies of intermediate states during the ATPase cycle providing evidence that myosin populates a cleft-closure state of strong actin interaction during the weak-to-strong transition with bound hydrolysis products before accomplishing the power stroke.

scholarly journals Proteasomal conformation controls unfolding ability

2021 ◽  
Vol 118 (25) ◽  
pp. e2101004118
Author(s):  
Julianna R. Cresti ◽  
Abramo J. Manfredonia ◽  
Christopher E. Bragança ◽  
Joseph A. Boscia ◽  
Christina M. Hurley ◽  
...  
Keyword(s):  
Conformational Changes ◽  
Fluorescent Proteins ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Conformational State ◽  
26S Proteasome ◽  
Eukaryotic Cells ◽  
Ubiquitinated Protein ◽  
Equilibrium Conditions ◽  
Substrate Processing

The 26S proteasome is the macromolecular machine responsible for the bulk of protein degradation in eukaryotic cells. As it degrades a ubiquitinated protein, the proteasome transitions from a substrate-accepting conformation (s1) to a set of substrate-processing conformations (s3 like), each stabilized by different intramolecular contacts. Tools to study these conformational changes remain limited, and although several interactions have been proposed to be important for stabilizing the proteasome’s various conformations, it has been difficult to test these directly under equilibrium conditions. Here, we describe a conformationally sensitive Förster resonance energy transfer assay, in which fluorescent proteins are fused to Sem1 and Rpn6, which are nearer each other in substrate-processing conformations than in the substrate-accepting conformation. Using this assay, we find that two sets of interactions, one involving Rpn5 and another involving Rpn2, are both important for stabilizing substrate-processing conformations. Mutations that disrupt these interactions both destabilize substrate-processing conformations relative to the substrate-accepting conformation and diminish the proteasome’s ability to successfully unfold and degrade hard-to-unfold substrates, providing a link between the proteasome’s conformational state and its unfolding ability.

scholarly journals Structural and Computational Insights into a Blebbistatin-Bound Myosin•ADP Complex with Characteristics of an ADP-Release Conformation along the Two-Step Myosin Power Stoke

2020 ◽  
Vol 21 (19) ◽  
pp. 7417 ◽  
Author(s):  
Wiebke Ewert ◽  
Peter Franz ◽  
Georgios Tsiavaliaris ◽  
Matthias Preller
Keyword(s):  
Atp Hydrolysis ◽  
Hydrolysis Product ◽  
Force Production ◽  
Salt Bridge ◽  
Adenosine Diphosphate ◽  
Power Stroke ◽  
Directed Movement ◽  
Wide Range ◽  
Adp Release ◽  
Dynamics Simulations

The motor protein myosin drives a wide range of cellular and muscular functions by generating directed movement and force, fueled through adenosine triphosphate (ATP) hydrolysis. Release of the hydrolysis product adenosine diphosphate (ADP) is a fundamental and regulatory process during force production. However, details about the molecular mechanism accompanying ADP release are scarce due to the lack of representative structures. Here we solved a novel blebbistatin-bound myosin conformation with critical structural elements in positions between the myosin pre-power stroke and rigor states. ADP in this structure is repositioned towards the surface by the phosphate-sensing P-loop, and stabilized in a partially unbound conformation via a salt-bridge between Arg131 and Glu187. A 5 Å rotation separates the mechanical converter in this conformation from the rigor position. The crystallized myosin structure thus resembles a conformation towards the end of the two-step power stroke, associated with ADP release. Computationally reconstructing ADP release from myosin by means of molecular dynamics simulations further supported the existence of an equivalent conformation along the power stroke that shows the same major characteristics in the myosin motor domain as the resolved blebbistatin-bound myosin-II·ADP crystal structure, and identified a communication hub centered on Arg232 that mediates chemomechanical energy transduction.

scholarly journals Structure of the human 26S proteasome at a resolution of 3.9 Å

2016 ◽  
Vol 113 (28) ◽  
pp. 7816-7821 ◽  
Author(s):  
Andreas Schweitzer ◽  
Antje Aufderheide ◽  
Till Rudack ◽  
Florian Beck ◽  
Günter Pfeifer ◽  
...  
Keyword(s):  
Ubiquitin Proteasome System ◽  
Structural Features ◽  
26S Proteasome ◽  
The Other ◽  
Aaa Atpase ◽  
Loop Region ◽  
C Terminus ◽  
Proteasome Subunits ◽  
Atpase Subunits ◽  
Substrate Processing

Protein degradation in eukaryotic cells is performed by the Ubiquitin-Proteasome System (UPS). The 26S proteasome holocomplex consists of a core particle (CP) that proteolytically degrades polyubiquitylated proteins, and a regulatory particle (RP) containing the AAA-ATPase module. This module controls access to the proteolytic chamber inside the CP and is surrounded by non-ATPase subunits (Rpns) that recognize substrates and deubiquitylate them before unfolding and degradation. The architecture of the 26S holocomplex is highly conserved between yeast and humans. The structure of the human 26S holocomplex described here reveals previously unidentified features of the AAA-ATPase heterohexamer. One subunit, Rpt6, has ADP bound, whereas the other five have ATP in their binding pockets. Rpt6 is structurally distinct from the other five Rpt subunits, most notably in its pore loop region. For Rpns, the map reveals two main, previously undetected, features: the C terminus of Rpn3 protrudes into the mouth of the ATPase ring; and Rpn1 and Rpn2, the largest proteasome subunits, are linked by an extended connection. The structural features of the 26S proteasome observed in this study are likely to be important for coordinating the proteasomal subunits during substrate processing.

scholarly journals Deconvolution of substrate processing by the 26S proteasome reveals a selective kinetic gateway to degradation

2018 ◽  
Author(s):  
J.A.M. Bard ◽  
C. Bashore ◽  
K.C. Dong ◽  
A. Martin
Keyword(s):  
Amino Acid ◽  
Protein Degradation ◽  
Unnatural Amino Acid ◽  
Conformational State ◽  
26S Proteasome ◽  
The Individual ◽  
Detailed Kinetics ◽  
Substrate Processing ◽  
Kinetic Proofreading ◽  
Processing Steps

AbstractThe 26S proteasome is the principle macromolecular machine responsible for protein degradation in eukaryotes. However, little is known about the detailed kinetics and coordination of the underlying substrate-processing steps of the proteasome, and their correlation with observed conformational states. Here, we used reconstituted 26S proteasomes with unnatural amino acid-attached fluorophores in a series of FRET and anisotropy-based assays to probe substrate-proteasome interactions, the individual steps of the processing pathway, and the conformational state of the proteasome itself. We develop a complete kinetic picture of proteasomal degradation, which reveals that the engagement steps prior to substrate commitment are fast relative to subsequent deubiquitination, translocation and unfolding. Furthermore, we find that non-ideal substrates are rapidly rejected by the proteasome, which thus employs a kinetic proofreading mechanism to ensure degradation fidelity and substrate prioritization.

scholarly journals Ubiquitin-Independent Proteasomal Degradation Mediated by Antizyme

2020 ◽  
Author(s):  
Noriyuki Murai
Keyword(s):  
Protein Degradation ◽  
Ornithine Decarboxylase ◽  
Feedback Regulation ◽  
Proteasomal Degradation ◽  
Degradation Pathway ◽  
26S Proteasome ◽  
Eukaryotic Cells ◽  
Proteolytic Degradation ◽  
Dependent Manner ◽  
Independent Protein

Most of the proteins in eukaryotic cells are degraded by the proteasome in an ubiquitin-dependent manner. However, ubiquitin-independent protein degradation pathway by the 26S proteasome exists in the cells. Ornithine decarboxylase (ODC) is a well-known protein that is degraded by the 26S proteasome without ubiquitination. Degradation of ODC requires the protein, “antizyme (AZ),” that is induced by polyamine and binds to the ODC monomer to inhibit ODC activity and target it to the 26S proteasome for proteolytic degradation. Namely, AZ contributes the feedback regulation of intracellular polyamine level. ODC has been considered to be the only protein that AZ binds and accelerates its degradation. However, recently AZ-mediated proteasomal protein degradation will gradually increase. Most recently, we found that one of the antizyme families, AZ2, accelerates c-Myc degradation by the proteasome without ubiquitination. In this chapter, we introduce latest several ubiquitin-independent proteasomal degradation mediated by antizyme.

scholarly journals Assembly of the Drosophila 26 S proteasome is accompanied by extensive subunit rearrangements

2002 ◽  
Vol 365 (2) ◽  
pp. 527-536 ◽  
Author(s):  
Éva KURUCZ ◽  
István ANDÓ ◽  
Máté SÜMEGI ◽  
Harald HÖLZL ◽  
Barbara KAPELARI ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
26S Proteasome ◽  
Cross Linking ◽  
20S Proteasome ◽  
Electron Microscopic ◽  
Immunoblot Assay ◽  
Optimal Conformation ◽  
Regulatory Complex ◽  
Atpase Subunits ◽  
The Cross

The subunit contacts in the regulatory complex of the Drosophila 26 S proteasome were studied through the cross-linking of closely spaced subunits of the complex, and analysis of the cross-linking pattern in an immunoblot assay with the use of subunit-specific monoclonal antibodies. The cross-linking pattern of the purified 26 S proteasome exhibits significant differences as compared with that of the purified free regulatory complex. It is shown that the observed differences are due to extensive rearrangement of the subunit contacts accompanying the assembly of the 26 S proteasome from the regulatory complex and the 20S proteasome. Cross-linking studies and electron microscopic examinations revealed that these changes are reversible and follow the assembly or the disassembly of the 26 S proteasome. Although the majority of the changes observed in the subunit contacts affected the hexameric ring of the ATPase subunits, the alterations extended over the whole of the regulatory complex, affecting subunit contacts even in the lid subcomplex. Changes in the subunit contacts, similar to those in the regulatory complex, were detected in the 20S proteasome. These observations indicate that the assembly of the 26 S proteasome is not simply a passive docking of two rigid subcomplexes. In the course of the assembly, the interacting subcomplexes mutually rearrange their structures so as to create the optimal conformation required for the assembly and the proper functioning of the 26S proteasome.

Deafness mutation in the MYO3A motor domain impairs actin protrusion elongation mechanism

2021 ◽  
Author(s):  
Laura K. Gunther ◽  
Joseph A Cirilo ◽  
Rohini Desetty ◽  
Christopher M. Yengo
Keyword(s):  
Atp Hydrolysis ◽  
Kinetic Mechanism ◽  
Duty Ratio ◽  
Slight Reduction ◽  
Average Intensity ◽  
Class Iii ◽  
Length Regulation ◽  
Adp Release ◽  
Actin Protrusions

Class III myosins are actin-based motors proposed to transport cargo to the distal tips of stereocilia in the inner ear hairs cells and/or to participate in stereocilia length regulation, which is especially important during development. Mutations in the MYO3A gene are associated with delayed onset deafness. A previous study demonstrated that L697W, a dominant deafness mutation, disrupts MYO3A ATPase and motor properties but does not impair its ability to localize to the tips of actin protrusions. In the current study, we characterized the transient kinetic mechanism of the L697W motor ATPase cycle. Our kinetic analysis demonstrates that the mutation slows the ADP release and ATP hydrolysis steps, which results in a slight reduction in the duty ratio and slows detachment kinetics. Fluorescence recovery after photobleaching (FRAP) of filopodia tip localized L697W and WT MYO3A in COS-7 cells revealed that the mutant does not alter turnover or average intensity at the actin protrusion tips. We demonstrate that the mutation slows filopodia extension velocity in COS-7 cells which correlates with its 2-fold slower in vitro actin gliding velocity. Overall, this work allowed us to propose a model for how the motor properties of MYO3A are crucial for facilitating actin protrusion length regulation.

scholarly journals UBL domain of Usp14 and other proteins stimulates proteasome activities and protein degradation in cells

2018 ◽  
Vol 115 (50) ◽  
pp. E11642-E11650 ◽  
Author(s):  
Hyoung Tae Kim ◽  
Alfred L. Goldberg
Keyword(s):  
Protein Degradation ◽  
Atp Hydrolysis ◽  
Deubiquitinating Enzyme ◽  
Wild Type ◽  
Peptide Hydrolysis ◽  
Ubiquitin Chain ◽  
Important Regulator ◽  
General Response ◽  
Ubiquitin Conjugate ◽  
Stimulation Of

The best-known function of ubiquitin-like (UBL) domains in proteins is to enable their binding to 26S proteasomes. The proteasome-associated deubiquitinating enzyme Usp14/UBP6 contains an N-terminal UBL domain and is an important regulator of proteasomal activity. Usp14 by itself represses multiple proteasomal activities but, upon binding a ubiquitin chain, Usp14 stimulates these activities and promotes ubiquitin-conjugate degradation. Here, we demonstrate that Usp14’s UBL domain alone mimics this activation of proteasomes by ubiquitin chains. Addition of this UBL domain to purified 26S proteasomes stimulated the same activities inhibited by Usp14: peptide entry and hydrolysis, protein-dependent ATP hydrolysis, deubiquitination by Rpn11, and the degradation of ubiquitinated and nonubiquitinated proteins. Thus, the binding of Usp14’s UBL (apparently to Rpn1’s T2 site) seems to mediate the activation of proteasomes by ubiquitinated substrates. However, the stimulation of these various activities was greater in proteasomes lacking Usp14 than in wild-type particles and thus is a general response that does not involve some displacement of Usp14. Furthermore, the UBL domains from hHR23 and hPLIC1/UBQLN1 also stimulated peptide hydrolysis, and the expression of hHR23A’s UBL domain in HeLa cells stimulated overall protein degradation. Therefore, many UBL-containing proteins that bind to proteasomes may also enhance allosterically its proteolytic activity.

scholarly journals Ccr4 is a novel shuttle factor required for ubiquitin-dependent proteolysis by the 26S proteasome

2020 ◽  
Author(s):  
Ganapathi Kandasamy ◽  
Ashis Kumar Pradhan ◽  
R Palanimurugan
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Protein Degradation ◽  
Ubiquitin Proteasome System ◽  
Proteasomal Degradation ◽  
Rna Degradation ◽  
26S Proteasome ◽  
Cellular Proteins ◽  
Cellular Homeostasis ◽  
Substrate Degradation ◽  
Ubiquitin Proteasome

AbstractDegradation of short-lived and abnormal proteins are essential for normal cellular homeostasis. In eukaryotes, such unstable cellular proteins are selectively degraded by the ubiquitin proteasome system (UPS). Furthermore, abnormalities in protein degradation by the UPS have been linked to several human diseases. Ccr4 protein is a known component of the Ccr4-Not complex, which has established roles in transcription, mRNA de-adenylation and RNA degradation etc. Excitingly in this study, we show that Ccr4 protein has a novel function as a shuttle factor that promotes ubiquitin-dependent degradation of short-lived proteins by the 26S proteasome. Using a substrate of the well-studied ubiquitin fusion degradation (UFD) pathway, we found that its UPS-mediated degradation was severely impaired upon deletion of CCR4 in Saccharomyces cerevisiae. Additionally, we show that Ccr4 binds to cellular ubiquitin conjugates and the proteasome. In contrast to Ccr4, most other subunits of the Ccr4-Not complex proteins are dispensable for UFD substrate degradation. From our findings we conclude that Ccr4 functions in the UPS as a shuttle factor targeting ubiquitylated substrates for proteasomal degradation.

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