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

Biomolecules ◽  
2020 ◽  
Vol 10 (4) ◽  
pp. 629 ◽  
Author(s):  
Shuwen Zhang ◽  
Youdong Mao
Keyword(s):  
Protein Degradation ◽  
Conformational Changes ◽  
Dynamic Models ◽  
Three Dimensional ◽  
26S Proteasome ◽  
Aaa Atpase ◽  
Substrate Protein ◽  
Function Relationship ◽  
Atp Binding And Hydrolysis ◽  
Aaa Atpases

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.

scholarly journals Structural basis for dynamic regulation of the human 26S proteasome

2016 ◽  
Vol 113 (46) ◽  
pp. 12991-12996 ◽  
Author(s):  
Shuobing Chen ◽  
Jiayi Wu ◽  
Ying Lu ◽  
Yong-Bei Ma ◽  
Byung-Hoon Lee ◽  
...  
Keyword(s):  
Conformational Changes ◽  
Ground State ◽  
Atp Hydrolysis ◽  
26S Proteasome ◽  
Structural Basis ◽  
Core Particle ◽  
Dynamic Regulation ◽  
Aaa Atpase ◽  
The Core ◽  
Aaa Atpases

The proteasome is the major engine of protein degradation in all eukaryotic cells. At the heart of this machine is a heterohexameric ring of AAA (ATPases associated with diverse cellular activities) proteins that unfolds ubiquitylated target proteins that are concurrently translocated into a proteolytic chamber and degraded into peptides. Using cryoelectron microscopy, we determined a near–atomic-resolution structure of the 2.5-MDa human proteasome in its ground state, as well as subnanometer-resolution structures of the holoenzyme in three alternative conformational states. The substrate-unfolding AAA-ATPase channel is narrowed by 10 inward-facing pore loops arranged into two helices that run in parallel with each other, one hydrophobic in character and the other highly charged. The gate of the core particle was unexpectedly found closed in the ground state and open in only one of the alternative states. Coordinated, stepwise conformational changes of the regulatory particle couple ATP hydrolysis to substrate translocation and regulate gating of the core particle, leading to processive degradation.

scholarly journals Specific lid-base contacts in the 26s proteasome control the conformational switching required for substrate degradation

eLife ◽  
2019 ◽  
Vol 8 ◽  
Author(s):  
Eric R Greene ◽  
Ellen A Goodall ◽  
Andres H de la Peña ◽  
Mary E Matyskiela ◽  
Gabriel C Lander ◽  
...  
Keyword(s):  
Conformational Changes ◽  
Degradation Process ◽  
26S Proteasome ◽  
Conformational Switching ◽  
Conformational Switch ◽  
Atpase Subunit ◽  
Aaa Atpase ◽  
Early Degradation ◽  
Processing Steps ◽  
Insight Into

The 26S proteasome is essential for proteostasis and the regulation of vital processes through ATP-dependent degradation of ubiquitinated substrates. To accomplish the multi-step degradation process, the proteasome’s regulatory particle, consisting of lid and base subcomplexes, undergoes major conformational changes whose origin is unknown. Investigating the Saccharomyces cerevisiae proteasome, we found that peripheral interactions between the lid subunit Rpn5 and the base AAA+ ATPase ring are important for stabilizing the substrate-engagement-competent state and coordinating the conformational switch to processing states upon substrate engagement. Disrupting these interactions perturbs the conformational equilibrium and interferes with degradation initiation, while later processing steps remain unaffected. Similar defects in early degradation steps are observed when eliminating hydrolysis in the ATPase subunit Rpt6, whose nucleotide state seems to control proteasome conformational transitions. These results provide important insight into interaction networks that coordinate conformational changes with various stages of degradation, and how modulators of conformational equilibria may influence substrate turnover.

scholarly journals Structure, Dynamics and Function of the 26S Proteasome

2020 ◽  
pp. 1-151
Author(s):  
Youdong Mao
Keyword(s):  
Atp Hydrolysis ◽  
Three Dimensional ◽  
Ubiquitin Proteasome System ◽  
26S Proteasome ◽  
Large Network ◽  
Processing Efficiency ◽  
Aaa Atpase ◽  
Substrate Degradation ◽  
Dynamics And Function ◽  
Almost All

AbstractThe 26S proteasome is the most complex ATP-dependent protease machinery, of ~2.5 MDa mass, ubiquitously found in all eukaryotes. It selectively degrades ubiquitin-conjugated proteins and plays fundamentally indispensable roles in regulating almost all major aspects of cellular activities. To serve as the sole terminal “processor” for myriad ubiquitylation pathways, the proteasome evolved exceptional adaptability in dynamically organizing a large network of proteins, including ubiquitin receptors, shuttle factors, deubiquitinases, AAA-ATPase unfoldases, and ubiquitin ligases, to enable substrate selectivity and processing efficiency and to achieve regulation precision of a vast diversity of substrates. The inner working of the 26S proteasome is among the most sophisticated, enigmatic mechanisms of enzyme machinery in eukaryotic cells. Recent breakthroughs in three-dimensional atomic-level visualization of the 26S proteasome dynamics during polyubiquitylated substrate degradation elucidated an extensively detailed picture of its functional mechanisms, owing to progressive methodological advances associated with cryogenic electron microscopy (cryo-EM). Multiple sites of ubiquitin binding in the proteasome revealed a canonical mode of ubiquitin-dependent substrate engagement. The proteasome conformation in the act of substrate deubiquitylation provided insights into how the deubiquitylating activity of RPN11 is enhanced in the holoenzyme and is coupled to substrate translocation. Intriguingly, three principal modes of coordinated ATP hydrolysis in the heterohexameric AAA-ATPase motor  were discovered to regulate intermediate functional steps of the proteasome, including ubiquitin-substrate engagement, deubiquitylation, initiation of substrate translocation and processive substrate degradation. The atomic dissection of the innermost working of the 26S proteasome opens up a new era in our understanding of the ubiquitin-proteasome system and has far-reaching implications in health and disease.

scholarly journals Cryo-EM structures of the archaeal PAN-proteasome reveal an around-the-ring ATPase cycle

2018 ◽  
Vol 116 (2) ◽  
pp. 534-539 ◽  
Author(s):  
Parijat Majumder ◽  
Till Rudack ◽  
Florian Beck ◽  
Radostin Danev ◽  
Günter Pfeifer ◽  
...  
Keyword(s):  
26S Proteasome ◽  
Structural Basis ◽  
Particle Analysis ◽  
Aaa Atpase ◽  
Cellular Processes ◽  
Domains Of Life ◽  
Atpase Subunits ◽  
Domain Motions ◽  
And Control ◽  
Aaa Atpases

Proteasomes occur in all three domains of life, and are the principal molecular machines for the regulated degradation of intracellular proteins. They play key roles in the maintenance of protein homeostasis, and control vital cellular processes. While the eukaryotic 26S proteasome is extensively characterized, its putative evolutionary precursor, the archaeal proteasome, remains poorly understood. The primordial archaeal proteasome consists of a 20S proteolytic core particle (CP), and an AAA-ATPase module. This minimal complex degrades protein unassisted by non-ATPase subunits that are present in a 26S proteasome regulatory particle (RP). Using cryo-EM single-particle analysis, we determined structures of the archaeal CP in complex with the AAA-ATPase PAN (proteasome-activating nucleotidase). Five conformational states were identified, elucidating the functional cycle of PAN, and its interaction with the CP. Coexisting nucleotide states, and correlated intersubunit signaling features, coordinate rotation of the PAN-ATPase staircase, and allosterically regulate N-domain motions and CP gate opening. These findings reveal the structural basis for a sequential around-the-ring ATPase cycle, which is likely conserved in AAA-ATPases.

scholarly journals Ubiquitin-dependent chloroplast-associated protein degradation in plants

Science ◽  
2019 ◽  
Vol 363 (6429) ◽  
pp. eaav4467 ◽  
Author(s):  
Qihua Ling ◽  
William Broad ◽  
Raphael Trösch ◽  
Mats Töpel ◽  
Tijen Demiral Sert ◽  
...  
Keyword(s):  
Protein Degradation ◽  
Forward Genetics ◽  
26S Proteasome ◽  
Chloroplast Envelope ◽  
Outer Envelope ◽  
Aaa Atpase ◽  
Ubiquitin E3 Ligase ◽  
Outer Envelope Membrane ◽  
Underlying Mechanisms ◽  
Envelope Membranes

Chloroplasts contain thousands of nucleus-encoded proteins that are imported from the cytosol by translocases in the chloroplast envelope membranes. Proteolytic regulation of the translocases is critically important, but little is known about the underlying mechanisms. We applied forward genetics and proteomics in Arabidopsis to identify factors required for chloroplast outer envelope membrane (OEM) protein degradation. We identified SP2, an Omp85-type β-barrel channel of the OEM, and CDC48, a cytosolic AAA+ (ATPase associated with diverse cellular activities) chaperone. Both proteins acted in the same pathway as the ubiquitin E3 ligase SP1, which regulates OEM translocase components. SP2 and CDC48 cooperated to bring about retrotranslocation of ubiquitinated substrates from the OEM (fulfilling conductance and motor functions, respectively), enabling degradation of the substrates by the 26S proteasome in the cytosol. Such chloroplast-associated protein degradation (CHLORAD) is vital for organellar functions and plant development.

scholarly journals Deep manifold learning reveals hidden dynamics of proteasome autoregulation

2020 ◽  
Author(s):  
Zhaolong Wu ◽  
Shuwen Zhang ◽  
Wei Li Wang ◽  
Yinping Ma ◽  
Yuanchen Dong ◽  
...  
Keyword(s):  
Manifold Learning ◽  
Conformational Changes ◽  
26S Proteasome ◽  
Nucleophilic Attack ◽  
Nucleotide Exchange ◽  
Polyubiquitin Chains ◽  
Aaa Atpase ◽  
Cellular Processes ◽  
Systemic Analysis ◽  
Manifold Embedding

AbstractThe 2.5-MDa 26S proteasome maintains proteostasis and regulates myriad cellular processes1. How polyubiquitylated substrate interactions regulate proteasome activity is not understood1,2. Here we introduce a deep manifold learning framework, named AlphaCryo4D, which enables atomic-level cryogenic electron microscopy (cryo-EM) reconstructions of nonequilibrium conformational continuum and reconstitutes ‘hidden’ dynamics of proteasome autoregulation in the act of substrate degradation. AlphaCryo4D integrates 3D deep residual learning3 with manifold embedding4 of free-energy landscapes5, which directs 3D clustering via an energy-based particle-voting algorithm. In blind assessments using simulated heterogeneous cryo-EM datasets, AlphaCryo4D achieved 3D classification accuracy three times that of conventional methods6–9 and reconstructed continuous conformational changes of a 130-kDa protein at sub-3-Å resolution. By using AlphaCryo4D to analyze a single experimental cryo-EM dataset2, we identified 64 conformers of the substrate-bound human 26S proteasome, revealing conformational entanglement of two regulatory particles in the doubly capped holoenzymes and their energetic differences with singly capped ones. Novel ubiquitin-binding sites are discovered on the RPN2, RPN10 and α5 subunits to remodel polyubiquitin chains for deubiquitylation and recycle. Importantly, AlphaCryo4D choreographs single-nucleotide-exchange dynamics of proteasomal AAA-ATPase motor during translocation initiation, which upregulates proteolytic activity by allosterically promoting nucleophilic attack. Our systemic analysis illuminates a grand hierarchical allostery for proteasome autoregulation.

scholarly journals Nucleotide-Driven Triple-State Remodeling of the AAA-ATPase Channel in the Activated Human 26S Proteasome

2017 ◽  
Author(s):  
Yanan Zhu ◽  
Wei Li Wang ◽  
Daqi Yu ◽  
Qi Ouyang ◽  
Ying Lu ◽  
...  
Keyword(s):  
Conformational Changes ◽  
26S Proteasome ◽  
Eukaryotic Cells ◽  
Alternative States ◽  
Nucleotide Exchange ◽  
Core Particle ◽  
Aaa Atpase ◽  
The Core ◽  
Gate Opening ◽  
Proteolytic Chamber

SUMMARYThe proteasome is a sophisticated ATP-dependent molecular machine responsible for protein degradation in all eukaryotic cells. It remains elusive how conformational changes of the AAA-ATPase unfoldase in the regulatory particle (RP) control the gating of substrate-translocation channel to the proteolytic chamber of the core particle (CP). Here we report three alternative states of the ATP-γS-bound human proteasome, in which the CP gate is asymmetrically open, visualized by cryo-EM at near-atomic resolutions. Only four nucleotides are stably bound to the AAA-ATPase ring in the open-gate states. Concerted nucleotide exchange gives rise to a back-and-forth wobbling motion of the AAA-ATPase channel, coincident with remarkable transitions of their pore loops between the spiral staircase and saddle-shaped circle topologies. Gate opening in the CP is thus controlled with nucleotide-driven remodeling of the AAA-ATPase unfoldase. These findings demonstrate an elegant mechanism of allosteric coordination among sub-machines within the holoenzyme that is crucial for substrate translocation.

scholarly journals Substrate-engaged 26S proteasome structures reveal mechanisms for ATP-hydrolysis–driven translocation

Science ◽  
2018 ◽  
Vol 362 (6418) ◽  
pp. eaav0725 ◽  
Author(s):  
Andres H. de la Peña ◽  
Ellen A. Goodall ◽  
Stephanie N. Gates ◽  
Gabriel C. Lander ◽  
Andreas Martin
Keyword(s):  
Electron Microscopy ◽  
Conformational Changes ◽  
Adenosine Triphosphatase ◽  
Atp Hydrolysis ◽  
26S Proteasome ◽  
Phosphate Release ◽  
Protein Substrates ◽  
Cellular Processes ◽  
Cryo Electron Microscopy ◽  
Aaa Atpases

The 26S proteasome is the primary eukaryotic degradation machine and thus is critically involved in numerous cellular processes. The heterohexameric adenosine triphosphatase (ATPase) motor of the proteasome unfolds and translocates targeted protein substrates into the open gate of a proteolytic core while a proteasomal deubiquitinase concomitantly removes substrate-attached ubiquitin chains. However, the mechanisms by which ATP hydrolysis drives the conformational changes responsible for these processes have remained elusive. Here we present the cryo–electron microscopy structures of four distinct conformational states of the actively ATP-hydrolyzing, substrate-engaged 26S proteasome. These structures reveal how mechanical substrate translocation accelerates deubiquitination and how ATP-binding, -hydrolysis, and phosphate-release events are coordinated within the AAA+ (ATPases associated with diverse cellular activities) motor to induce conformational changes and propel the substrate through the central pore.

scholarly journals Specific lid-base contacts in the 26S proteasome control the conformational switching required for substrate engagement and degradation

2019 ◽  
Author(s):  
Eric R. Greene ◽  
Ellen A. Goodall ◽  
Andres H. de la Peña ◽  
Mary E. Matyskiela ◽  
Gabriel C. Lander ◽  
...  
Keyword(s):  
Conformational Changes ◽  
26S Proteasome ◽  
Conformational Switching ◽  
Atpase Subunit ◽  
Aaa Atpase ◽  
Cellular Processes ◽  
Substrate Processing ◽  
And Control ◽  
Early Degradation ◽  
Insight Into

AbstractThe 26S proteasome is essential for protein homeostasis and the regulation of vital cellular processes through ATP-dependent degradation of ubiquitinated substrates. To accomplish the multi-step reaction of protein degradation, the proteasome’s regulatory particle, consisting of the lid and base subcomplexes, undergoes major conformational changes whose origin and control are largely unknown. Investigating the Saccharomyces cerevisiae 26S proteasome, we found that peripheral interactions between the lid subunit Rpn5 and the base AAA+ ATPase ring play critical roles in stabilizing the substrate-engagement-competent state and coordinating the conformational switch to processing states after a substrate has been engaged. Disrupting these interactions perturbs the conformational equilibrium and interferes with degradation initiation, while later steps of substrate processing remain unaffected. Similar defects in the early degradation steps are also observed when eliminating hydrolysis in the ATPase subunit Rpt6, whose nucleotide state seems to control conformational transitions of the proteasome. These results provide important insight into the network of interactions that coordinate conformational changes with various stages of proteasomal degradation, and how modulators of conformational equilibria may influence substrate turnover.

Electron Microscopy and image reconstruction reveal the structural basis for spectrin's elastic properties

1992 ◽  
Vol 50 (1) ◽  
pp. 510-511
Author(s):  
Amy M. McGough ◽  
Robert Josephs
Keyword(s):  
Electron Microscopy ◽  
Elastic Properties ◽  
Conformational Changes ◽  
Quaternary Structure ◽  
Three Dimensional ◽  
Membrane Skeleton ◽  
Projection Algorithm ◽  
Structural Basis ◽  
Iterative Approximation ◽  
Membrane Skeletons

The remarkable deformability of the erythrocyte derives in large part from the elastic properties of spectrin, the major component of the membrane skeleton. It is generally accepted that spectrin's elasticity arises from marked conformational changes which include variations in its overall length (1). In this work the structure of spectrin in partially expanded membrane skeletons was studied by electron microscopy to determine the molecular basis for spectrin's elastic properties. Spectrin molecules were analysed with respect to three features: length, conformation, and quaternary structure. The results of these studies lead to a model of how spectrin mediates the elastic deformation of the erythrocyte.Membrane skeletons were isolated from erythrocyte membrane ghosts, negatively stained, and examined by transmission electron microscopy (2). Particle lengths and end-to-end distances were measured from enlarged prints using the computer program MACMEASURE. Spectrin conformation (straightness) was assessed by calculating the particles’ correlation length by iterative approximation (3). Digitised spectrin images were correlation averaged or Fourier filtered to improve their signal-to-noise ratios. Three-dimensional reconstructions were performed using a suite of programs which were based on the filtered back-projection algorithm and executed on a cluster of Microvax 3200 workstations (4).

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