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.

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 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 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 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 Structures of the substrate-engaged 26S proteasome reveal the mechanisms for ATP hydrolysis-driven translocation

2018 ◽  
Author(s):  
Andres H. de la Peña ◽  
Ellen A. Goodall ◽  
Stephanie N. Gates ◽  
Gabriel C. Lander ◽  
Andreas Martin
Keyword(s):  
Conformational Changes ◽  
Atp Hydrolysis ◽  
26S Proteasome ◽  
Atp Binding ◽  
Conformational States ◽  
Phosphate Release ◽  
Protein Substrates ◽  
Cellular Processes ◽  
Hexameric Atpase ◽  
Central Pore

AbstractThe 26S proteasome is the primary eukaryotic degradation machine and thus critically involved in numerous cellular processes. The hetero-hexameric 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-EM 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+ motor to induce conformational changes and propel the substrate through the central pore.

scholarly journals Omic-Sig: Utilizing Omics Data to Explore and Visualize Kinase-Substrate Interactions

2019 ◽  
Author(s):  
T. Mamie Lih ◽  
David J. Clark ◽  
Hui Zhang
Keyword(s):  
Protein Phosphorylation ◽  
Software Tool ◽  
Omics Data ◽  
Additional Insight ◽  
Post Translational Modifications ◽  
Kinase Substrate ◽  
Cellular Processes ◽  
Visualization Software ◽  
And Control ◽  
Insight Into

AbstractProtein phosphorylation is one of the most prevalent post-translational modifications, resulting from the activity of protein kinases phosphorylating specific substrates. Multiple cellular processes are regulated via protein phosphorylation, with aberrant signaling driven by dysregulation of phosphorylation events and associating with disease progression (e.g., cancer). Mass spectrometry-based phosphoprotomics approaches can be leveraged for studying alterations of kinase-substrate activity in clinical cohorts. However, the information gained via interrogation of global proteomes and transcriptomes can offer additional insight into the interaction of kinases and their respective substrates. Therefore, we have developed the bioinformatics, data visualization software tool, Omic-Sig, which can stratify prominent phospho-substrates and their associated kinases based on differential abundances between case and control samples (e.g., tumors and their normal adjacent tissues from a cancer cohort) in a multi-omics fashion. Omic-Sig is available at https://github.com/hzhangjhu/Omic-Sig.

scholarly journals Mechanistic insight into substrate processing and allosteric inhibition of human p97

2021 ◽  
Author(s):  
Man Pan ◽  
Yuanyuan Yu ◽  
Huasong Ai ◽  
Qingyun Zheng ◽  
Yuan Xie ◽  
...  
Keyword(s):  
Conformational Changes ◽  
Cellular Protein ◽  
Power Stroke ◽  
Cryo Electron Microscopy ◽  
D2 Domain ◽  
The Central Nervous System ◽  
Degenerative Disorder ◽  
Substrate Processing ◽  
Structural Insights ◽  
Insight Into

ABSTRACTp97, also known as valosin-containing protein (VCP), processes ubiquitinated substrates and plays a central role in cellular protein homeostasis. Mutations in human p97 are associated with multisystem proteinopathy (MSP), a dominantly inherited degenerative disorder that can affect muscle, bone and the central nervous system. It is also a drug target for cancer therapy with various inhibitors developed over the past decade. Despite significant structural insights into the fungal homologue of p97, Cdc48, little is known about how human p97 processes its substrates and how the activity is allosterically affected by inhibitors. Here, we report a series of cryo-electron microscopy (cryo-EM) structures of substrate-engaged human p97 complex with resolutions ranging from 2.9 to 3.8 Å that captured “power stroke”-like motions of both the D1 and D2 ATPase rings of p97. The structures elucidated how the unfolded substrate is engaged in the pore at atomic level. Critical conformational changes of the inter-subunit signaling (ISS) motifs were revealed, providing molecular insights into substrate translocation. Furthermore, we also determined cryo-EM structures of human p97 in complex with NMS-873, the most potent p97 inhibitor, at a resolution of 2.4 Å. The structures showed that NMS-873 binds at a cryptic groove in the D2 domain and interacts with the ISS motif, preventing its conformational change, thus blocking substrate translocation allosterically. Finally, using NMS-873 at a substoichiometric concentration, we captured a series of intermediate states, suggesting how the cofactor Npl4 coordinates with the D1 ring of p97 to initiate the translocation.

scholarly journals The proteasome cap RPT5/Rpt5p subunit prevents aggregation of unfolded ricin A chain

2013 ◽  
Vol 453 (3) ◽  
pp. 435-445 ◽  
Author(s):  
Paola Pietroni ◽  
Nishi Vasisht ◽  
Jonathan P. Cook ◽  
David M. Roberts ◽  
J. Michael Lord ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Proteasome Inhibitors ◽  
Retrograde Transport ◽  
26S Proteasome ◽  
Atpase Subunit ◽  
Aaa Atpase ◽  
A Chain ◽  
Ricin A

The plant cytotoxin ricin enters mammalian cells by receptor-mediated endocytosis, undergoing retrograde transport to the ER (endoplasmic reticulum) where its catalytic A chain (RTA) is reductively separated from the holotoxin to enter the cytosol and inactivate ribosomes. The currently accepted model is that the bulk of ER-dislocated RTA is degraded by proteasomes. We show in the present study that the proteasome has a more complex role in ricin intoxication than previously recognized, that the previously reported increase in sensitivity of mammalian cells to ricin in the presence of proteasome inhibitors simply reflects toxicity of the inhibitors themselves, and that RTA is a very poor substrate for proteasomal degradation. Denatured RTA and casein compete for a binding site on the regulatory particle of the 26S proteasome, but their fates differ. Casein is degraded, but the mammalian 26S proteasome AAA (ATPase associated with various cellular activities)-ATPase subunit RPT5 acts as a chaperone that prevents aggregation of denatured RTA and stimulates recovery of catalytic RTA activity in vitro. Furthermore, in vivo, the ATPase activity of Rpt5p is required for maximal toxicity of RTA dislocated from the Saccharomyces cerevisiae ER. The results of the present study implicate RPT5/Rpt5p in the triage of substrates in which either activation (folding) or inactivation (degradation) pathways may be initiated.

scholarly journals Conserved prolines in the coiled coil-OB domain linkers of proteasomal ATPases facilitate eukaryotic proteasome base assembly

2020 ◽  
Author(s):  
Chin Leng Cheng ◽  
Michael K Wong ◽  
Yanjie Li ◽  
Mark Hochstrasser
Keyword(s):  
Protein Quality ◽  
Coiled Coil ◽  
26S Proteasome ◽  
Single Type ◽  
Mutant Form ◽  
Peptide Bonds ◽  
Atpase Subunit ◽  
Aaa Atpase ◽  
Growth Defects ◽  
Atpase Subunits

AbstractThe proteasome is a large protease complex that degrades both misfolded and regulatory proteins. In eukaryotes, the 26S proteasome contains six different AAA+ ATPase subunits, Rpt1-Rpt6, which form a hexameric ring as part of the base subcomplex that drives unfolding and translocation of substrates into the proteasome core. Archaeal proteasomes contain only a single type of ATPase subunit, the proteasome-activating nucleotidase (PAN), which forms a trimer-of-dimers and is homologous to the eukaryotic Rpt subunits. A key PAN proline residue (P91) forms cis and trans peptide bonds in successive subunits around the ring, allowing efficient dimerization through upstream coiled coils. The importance of the equivalent Rpt prolines in eukaryotic proteasome assembly was unknown. We show an equivalent proline is strictly conserved in Rpt3 (in S. cerevisiae, P93) and Rpt5 (P76), well conserved in Rpt2 (P103), and loosely conserved in Rpt1 (P96) in deeply divergent eukaryotes, but in no case is its mutation strongly deleterious to yeast growth. However, the rpt2-P103A, rpt3-P93A, and rpt5-P76A mutations all cause synthetic defects with specific base assembly chaperone deletions. The Rpt5-P76A mutation decreases the levels of the protein and induces a mild proteasome assembly defect. The yeast rpt2-P103A rpt5-P76A double mutant has strong growth defects attributable to defects in proteasome base formation. Several Rpt subunits in this mutant form aggregates that are cleared, at least in part, by the Hsp42-mediated protein quality control (PQC) machinery. We propose that the conserved Rpt linker prolines promote efficient 26S proteasome base assembly by facilitating specific ATPase heterodimerization.

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.

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