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 New insights into the human 26S proteasome function and regulation

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.

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 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 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 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 Altered phosphorylation of the proteasome subunit Rpt6 has minimal impact on synaptic plasticity and learning

2019 ◽  
Author(s):  
Samantha L. Scudder ◽  
Frankie R. Gonzales ◽  
Kristin K. Howell ◽  
Ivar S. Stein ◽  
Lara E. Dozier ◽  
...  
Keyword(s):  
Synaptic Plasticity ◽  
Long Term Potentiation ◽  
26S Proteasome ◽  
Compensatory Mechanisms ◽  
Atpase Subunit ◽  
Minimal Impact ◽  
Proteasome Subunit ◽  
Aaa Atpase

ABSTRACTDynamic control of protein degradation via the ubiquitin proteasome system is thought to play a crucial role in neuronal function and synaptic plasticity. The proteasome subunit Rpt6, an AAA ATPase subunit of the 19S regulatory particle, has emerged as an important site for regulation of 26S proteasome function in neurons. Phosphorylation of Rpt6 on serine 120 (S120) can stimulate the catalytic rate of substrate degradation by the 26S proteasome and this site is targeted by the plasticity-related kinase calcium/calmodulin-dependent kinase II (CaMKII), making it an attractive candidate for regulation of proteasome function in neurons. Several in vitro studies have shown that altered Rpt6 S120 phosphorylation can affect the structure and function of synapses. To evaluate the importance of Rpt6 S120 phosphorylation in vivo, we created two mouse models which feature mutations at S120 that block or mimic phosphorylation at this site. We find that peptidase and ATPase activities are upregulated in the phospho-mimetic mutant and downregulated in the phospho-dead mutant (S120 mutated to aspartic acid (S120D) or alanine (S120A), respectively). Surprisingly, these mutations had no effect on basal synaptic transmission, long-term potentiation, and dendritic spine dynamics and density in the hippocampus. Furthermore, these mutants displayed no deficits in cued and contextual fear memory. Thus, in a mouse model that blocks or mimics phosphorylation at this site, either compensatory mechanisms negate these effects, or small variations in proteasome activity are not enough to induce significant changes in synaptic structure, plasticity, or behavior.

Torbafylline (HWA 448) inhibits enhanced skeletal muscle ubiquitin–proteasome-dependent proteolysis in cancer and septic rats

2002 ◽  
Vol 361 (2) ◽  
pp. 185-192 ◽  
Author(s):  
Lydie COMBARET ◽  
Thomas TILIGNAC ◽  
Agnès CLAUSTRE ◽  
Laure VOISIN ◽  
Daniel TAILLANDIER ◽  
...  
Keyword(s):  
Muscle Wasting ◽  
26S Proteasome ◽  
Atpase Subunit ◽  
Xanthine Derivative ◽  
Xanthine Derivatives ◽  
Response To Chemotherapy ◽  
Inhibit Tumour Necrosis Factor ◽  
Regulatory Complex ◽  
Atpase Subunits ◽  
Factor Α

The development of new pharmacological approaches for preventing muscle wasting in cancer is an important goal because cachectic patients display a reduced response to chemotherapy and radiotherapy. Xanthine derivatives such as pentoxifylline inhibit tumour necrosis factor-α (TNF) production, which has been implicated in the signalling of muscle wasting. However, the effect of pentoxifylline has been inconclusive in clinical trials. We report here the first direct evidence that daily injections of torbafylline (also known as HWA 448), another xanthine derivative, had no effect by itself on muscle proteolysis in control healthy rats. In cancer rats, the drug blocked the lipopolysaccharide-induced hyperproduction of TNF and prevented muscle wasting. In these animals HWA 448 suppressed the enhanced proteasome-dependent proteolysis, which is sensitive to the proteasome inhibitor MG132, and the accumulation of high-molecular-mass ubiquitin (Ub) conjugates in the myofibrillar fraction. The drug also normalized the enhanced muscle expression of Ub, which prevails in the atrophying muscles from cancer rats. In contrast, HWA 448 did not reduce the increased expression of either the 14kDa Ub conjugating enzyme E2 or the ATPase and non-ATPase subunits of the 19S regulatory complex of the 26S proteasome, including the non-ATPase subunit S5a, which recognizes polyUb degradation signals. Finally, the drug also prevented muscle wasting in septic rats (which exhibit increased TNF production), and was much more potent than pentoxifylline or other xanthine derivatives. Taken together, the data indicate that HWA 448 is a powerful inhibitor of muscle wasting that blocks enhanced Ub—proteasome-dependent proteolysis in situations where TNF production rises, including cancer and sepsis.

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.

Sign in / Sign up

Export Citation Format

Share Document