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

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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Inhibition of Ocular Aldose Reductase by a New Benzofuroxane Derivative Ameliorates Rat Endotoxic Uveitis

Mediators of Inflammation ◽

10.1155/2014/857958 ◽

2014 ◽

Vol 2014 ◽

pp. 1-9 ◽

Cited By ~ 6

Author(s):

C. Di Filippo ◽

M. V. Zippo ◽

R. Maisto ◽

M. C. Trotta ◽

D. Siniscalco ◽

...

Keyword(s):

Aldose Reductase ◽

Manganese Superoxide Dismutase ◽

Clinical Score ◽

Ubiquitin Proteasome System ◽

26S Proteasome ◽

Dependent Manner ◽

Proteasome Subunits ◽

The Right ◽

Antioxidant Markers ◽

Tissue Alterations

The study investigated the effects of the aldose reductase (AR) inhibitor benzofuroxane derivative 5(6)-(benzo[d]thiazol-2-ylmethoxy) benzofuroxane (herein referred to as BF-5m) on the biochemical and tissue alterations induced by endotoxic uveitis in rats. BF-5m has been administered directly into the vitreous, in order to assess the expression and levels of (i) inflammatory markers such as the ocular ubiquitin-proteasome system, NF-κB, TNF-α, and MCP-1; (ii) prooxidant and antioxidant markers such as nitrotyrosine, manganese superoxide dismutase (MnSOD), and glutathione peroxidase (GPX); (iii) apoptotic/antiapoptotic factors caspases and Bcl-xl; (iv) markers of endothelial progenitor cells (EPCs) recruitment such as CD34 and CD117. 5 μL of BF-5m (0.01; 0.05; and 0.1 μM) into the right eye decreased in a dose-dependent manner the LPS-induced inflammation of the eye, reporting a clinical score 1. It reduced the ocular levels of ubiquitin, 20S and 26S proteasome subunits, NF-κB subunits, TNF-α, MCP-1, and nitrotyrosine. BF-5m ameliorated LPS-induced decrease in levels of MnSOD and GPX. Antiapoptotic effects were seen from BF-5m by monitoring the expression of Bcl-xl, an antiapoptotic protein. Similarly, BF-5m increased recruitment of the EPCs within the eye, as evidenced by CD34 and CD117 antibodies.

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New insights into the human 26S proteasome function and regulation

10.1101/2021.10.03.462214 ◽

2021 ◽

Author(s):

Miglė Kišonaitė ◽

Pavel Afanasyev ◽

Jonida Tafilaku ◽

Ana Toste Rêgo ◽

Paula C. A. da Fonseca

Keyword(s):

High Resolution ◽

Structural Data ◽

26S Proteasome ◽

Aaa Atpase ◽

Physiological Processes ◽

Allosteric Communication ◽

Atpase Subunits ◽

Strict Regulation ◽

Major Conformation

SummaryThe 26S proteasome is a protease complex essential for proteostasis and strict regulation of diverse critical physiological processes, the mechanisms of which are still not fully described. The human 26S proteasome purification was optimized without exogenous nucleotides, to preserve the endogenous nucleotide occupancy and conformation of its AAA-ATPase subunits. This unveiled important effects on the proteasome function and structure resulting from exposure to Ca2+ or Mg2+, with important physiological implications. This sample, with an added model degron designed to mimic the minimum canonical ubiquitin signal for proteasomal recognition, was analysed by high-resolution cryo-EM. Two proteasome conformations were resolved, with only one capable of degron binding. The structural data show that this occurs without major conformation rearrangements and allows to infer into the allosteric communication between ubiquitin degron binding and the peptidase activities. These results revise existing concepts on the 26S proteasome function and regulation, opening important opportunities for further research.

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The Saccharomyces cerevisiae ubiquitin–proteasome system

Philosophical Transactions of the Royal Society B Biological Sciences ◽

10.1098/rstb.1999.0495 ◽

1999 ◽

Vol 354 (1389) ◽

pp. 1513-1522 ◽

Cited By ~ 18

Author(s):

M. Hochstrasser ◽

P. R. Johnson ◽

C. S. Arendt ◽

A. Y. Amerik ◽

S. Swaminathan ◽

...

Keyword(s):

Ubiquitin Proteasome System ◽

26S Proteasome ◽

Site Formation ◽

Protein Protein Interaction ◽

Proteasome Subunits ◽

Ubiquitin Proteasome ◽

Proteasome Pathway ◽

Diploid Cells ◽

Ubiquitin Conjugating Enzymes

Our studies of the yeast ubiquitin-proteasome pathway have uncovered a number of general principles that govern substrate selectivity and proteolysis in this complex system. Much of the work has focused on the destruction of a yeast transcription factor, MATα2. The α2 protein is polyubiquitinated and rapidly degraded in α–haploid cells. One pathway of proteolytic targeting, which depends on two distinct endoplasmic reticulum–localized ubiquitin–conjugating enzymes, recognizes the hydrophobic face of an amphipathic helix in α2. Interestingly, degradation of α2 is blocked in a /α–diploid cells by heterodimer formation between the α2 and a 1 homeodomain proteins. The data suggest that degradation signals may overlap protein–protein interaction surfaces, allowing a straightforward steric mechanism for regulated degradation. Analysis of α2 degradation led to the identification of both 20S and 26S proteasome subunits, and several key features of proteasome assembly and active–site formation were subsequently uncovered. Finally, it has become clear that protein (poly)ubiquitination is highly dynamic in vivo , and our studies of yeast de–ubiquitinating enzymes illustrate how such enzymes can facilitate the proteolysis of diverse substrates.

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

10.1101/2020.11.13.381962 ◽

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.

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Structure, Dynamics and Function of the 26S Proteasome

Subcellular Biochemistry - Macromolecular Protein Complexes III: Structure and Function ◽

10.1007/978-3-030-58971-4_1 ◽

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.

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Structure and Function of the 26S Proteasome

Annual Review of Biochemistry ◽

10.1146/annurev-biochem-062917-011931 ◽

2018 ◽

Vol 87 (1) ◽

pp. 697-724 ◽

Cited By ~ 158

Author(s):

Jared A.M. Bard ◽

Ellen A. Goodall ◽

Eric R. Greene ◽

Erik Jonsson ◽

Ken C. Dong ◽

...

Keyword(s):

Ubiquitin Proteasome System ◽

26S Proteasome ◽

Substrate Selection ◽

Cellular Functions ◽

Cellular Environment ◽

Ubiquitin Proteasome ◽

General Protein ◽

The Many ◽

And Function ◽

Substrate Processing

As the endpoint for the ubiquitin-proteasome system, the 26S proteasome is the principal proteolytic machine responsible for regulated protein degradation in eukaryotic cells. The proteasome's cellular functions range from general protein homeostasis and stress response to the control of vital processes such as cell division and signal transduction. To reliably process all the proteins presented to it in the complex cellular environment, the proteasome must combine high promiscuity with exceptional substrate selectivity. Recent structural and biochemical studies have shed new light on the many steps involved in proteasomal substrate processing, including recognition, deubiquitination, and ATP-driven translocation and unfolding. In addition, these studies revealed a complex conformational landscape that ensures proper substrate selection before the proteasome commits to processive degradation. These advances in our understanding of the proteasome's intricate machinery set the stage for future studies on how the proteasome functions as a major regulator of the eukaryotic proteome.

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

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1817752116 ◽

2018 ◽

Vol 116 (2) ◽

pp. 534-539 ◽

Cited By ~ 35

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.

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

10.1101/419051 ◽

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.

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Structure of the PUB Domain from Ubiquitin Regulatory X Domain Protein 1 (UBXD1) and Its Interaction with the p97 AAA+ ATPase

Biomolecules ◽

10.3390/biom9120876 ◽

2019 ◽

Vol 9 (12) ◽

pp. 876

Author(s):

Mike Blueggel ◽

Johannes van den Boom ◽

Hemmo Meyer ◽

Peter Bayer ◽

Christine Beuck

Keyword(s):

Stress Responses ◽

Mutational Analysis ◽

Binding Pocket ◽

Binding Studies ◽

Aaa Atpase ◽

Cellular Processes ◽

C Terminus ◽

Cellular Stress Responses ◽

Domain Protein ◽

Substrate Processing

AAA+ ATPase p97/valosin-containing protein (VCP)/Cdc48 is a key player in various cellular stress responses in which it unfolds ubiquitinated proteins to facilitate their degradation by the proteasome. P97 works in different cellular processes using alternative sets of cofactors and is implicated in multiple degenerative diseases. Ubiquitin regulatory X domain protein 1 (UBXD1) has been linked to pathogenesis and is unique amongst p97 cofactors because it interacts with both termini of p97. Its N-domain binds to the N-domain and N/D1 interface of p97 and regulates its ATPase activity. The PUB (peptide:N-glycanase and UBA or UBX-containing proteins) domain binds the p97 C-terminus, but how it controls p97 function is still unknown. Here we present the NMR structure of UBXD1-PUB together with binding studies, mutational analysis, and a model of UBXD1-PUB in complex with the p97 C-terminus. While the binding pocket is conserved among PUB domains, UBXD1-PUB features a unique loop and turn regions suggesting a role in coordinating interaction with downstream regulators and substrate processing

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Proteasome in action: substrate degradation by the 26S proteasome

Biochemical Society Transactions ◽

10.1042/bst20200382 ◽

2021 ◽

Author(s):

Indrajit Sahu ◽

Michael H. Glickman

Keyword(s):

Structural Features ◽

26S Proteasome ◽

Catalytic Core ◽

Substrate Protein ◽

Gate Opening ◽

Ubiquitin Receptors ◽

Substrate Processing ◽

Ubiquitin Conjugate ◽

Insight Into ◽

Recent Insight

Ubiquitination is the major criteria for the recognition of a substrate-protein by the 26S proteasome. Additionally, a disordered segment on the substrate — either intrinsic or induced — is critical for proteasome engagement. The proteasome is geared to interact with both of these substrate features and prepare it for degradation. To facilitate substrate accessibility, resting proteasomes are characterised by a peripheral distribution of ubiquitin receptors on the 19S regulatory particle (RP) and a wide-open lateral surface on the ATPase ring. In this substrate accepting state, the internal channel through the ATPase ring is discontinuous, thereby obstructing translocation of potential substrates. The binding of the conjugated ubiquitin to the ubiquitin receptors leads to contraction of the 19S RP. Next, the ATPases engage the substrate at a disordered segment, energetically unravel the polypeptide and translocate it towards the 20S catalytic core (CP). In this substrate engaged state, Rpn11 is repositioned at the pore of the ATPase channel to remove remaining ubiquitin modifications and accelerate translocation. C-termini of five of the six ATPases insert into corresponding lysine-pockets on the 20S α-ring to complete 20S CP gate opening. In the resulting substrate processing state, the ATPase channel is fully contiguous with the translocation channel into the 20S CP, where the substrate is proteolyzed. Complete degradation of a typical ubiquitin-conjugate takes place over a few tens of seconds while hydrolysing tens of ATP molecules in the process (50 kDa/∼50 s/∼80ATP). This article reviews recent insight into biochemical and structural features that underlie substrate recognition and processing by the 26S proteasome.

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