scholarly journals In-depth Analysis of the Lid Subunits Assembly Mechanism in Mammals

Biomolecules ◽  
2019 ◽  
Vol 9 (6) ◽  
pp. 213 ◽  
Author(s):  
Minghui Bai ◽  
Xian Zhao ◽  
Kazutaka Sahara ◽  
Yuki Ohte ◽  
Yuko Hirano ◽  
...  
Keyword(s):  
26S Proteasome ◽  
Core Particle ◽  
Proteasome Subunit ◽  
Ubiquitinated Proteins ◽  
Assembly Pathway ◽  
Assembly Mechanism ◽  
Depth Analysis ◽  
Regulatory Particle ◽  
Assembly Intermediate ◽  
Assembly Pathways

The 26S proteasome is a key player in the degradation of ubiquitinated proteins, comprising a 20S core particle (CP) and a 19S regulatory particle (RP). The RP is further divided into base and lid subcomplexes, which are assembled independently from each other. We have previously demonstrated the assembly pathway of the CP and the base by observing assembly intermediates resulting from knockdowns of each proteasome subunit and the assembly chaperones. In this study, we examine the assembly pathway of the mammalian lid, which remains to be elucidated. We show that the lid assembly pathway is conserved between humans and yeast. The final step is the incorporation of Rpn12 into the assembly intermediate consisting of two modular complexes, Rpn3-7-15 and Rpn5-6-8-9-11, in both humans and yeast. Furthermore, we dissect the assembly pathways of the two modular complexes by the knockdown of each lid subunit.

scholarly journals The Assembly Pathway of the 19S Regulatory Particle of the Yeast 26S Proteasome

2007 ◽  
Vol 18 (2) ◽  
pp. 569-580 ◽  
Author(s):  
Erika Isono ◽  
Kiyoshi Nishihara ◽  
Yasushi Saeki ◽  
Hideki Yashiroda ◽  
Naoko Kamata ◽  
...  
Keyword(s):  
26S Proteasome ◽  
Restrictive Temperature ◽  
Enzyme Complex ◽  
Core Particle ◽  
Yeast Saccharomyces Cerevisiae ◽  
The Core ◽  
Assembly Pathway ◽  
Regulatory Particle ◽  
Mutant Cells

The 26S proteasome consists of the 20S proteasome (core particle) and the 19S regulatory particle made of the base and lid substructures, and it is mainly localized in the nucleus in yeast. To examine how and where this huge enzyme complex is assembled, we performed biochemical and microscopic characterization of proteasomes produced in two lid mutants, rpn5-1 and rpn7-3, and a base mutant ΔN rpn2, of the yeast Saccharomyces cerevisiae. We found that, although lid formation was abolished in rpn5-1 mutant cells at the restrictive temperature, an apparently intact base was produced and localized in the nucleus. In contrast, in ΔN rpn2 cells, a free lid was formed and localized in the nucleus even at the restrictive temperature. These results indicate that the modules of the 26S proteasome, namely, the core particle, base, and lid, can be formed and imported into the nucleus independently of each other. Based on these observations, we propose a model for the assembly process of the yeast 26S proteasome.

scholarly journals Ubiquitinated proteins promote the association of proteasomes with the deubiquitinating enzyme Usp14 and the ubiquitin ligase Ube3c

2017 ◽  
Vol 114 (17) ◽  
pp. E3404-E3413 ◽  
Author(s):  
Chueh-Ling Kuo ◽  
Alfred Lewis Goldberg
Keyword(s):  
Ubiquitin Ligase ◽  
Mammalian Cells ◽  
Proteasome Activity ◽  
Deubiquitinating Enzyme ◽  
Core Particle ◽  
Ubiquitinated Proteins ◽  
Protein Ubiquitination ◽  
Cell Extracts ◽  
Regulatory Particle ◽  
In Cells

In mammalian cells, the 26S proteasomes vary in composition. In addition to the standard 28 subunits in the 20S core particle and 19 subunits in each 19S regulatory particle, a small fraction (about 10–20% in our preparations) also contains the deubiquitinating enzyme Usp14/Ubp6, which regulates proteasome activity, and the ubiquitin ligase, Ube3c/Hul5, which enhances proteasomal processivity. When degradation of ubiquitinated proteins in cells was inhibited, levels of Usp14 and Ube3c on proteasomes increased within minutes. Conversely, when protein ubiquitination was prevented, or when purified proteasomes hydrolyzed the associated ubiquitin conjugates, Usp14 and Ube3c dissociated rapidly (unlike other 26S subunits), but the inhibitor ubiquitin aldehyde slowed their dissociation. Recombinant Usp14 associated with purified proteasomes preferentially if they contained ubiquitin conjugates. In cells or extracts, adding Usp14 inhibitors (IU-1 or ubiquitin aldehyde) enhanced Usp14 and Ube3c binding further. Thus, in the substrate- or the inhibitor-bound conformations, Usp14 showed higher affinity for proteasomes and surprisingly enhanced Ube3c binding. Moreover, adding ubiquitinated proteins to cell extracts stimulated proteasome binding of both enzymes. Thus, Usp14 and Ube3c cycle together on and off proteasomes, and the presence of ubiquitinated substrates promotes their association. This mechanism enables proteasome activity to adapt to the supply of substrates.

Reverse Genetic Analysis of the Caenorhabditis elegans 26S Proteasome Subunits by RNA Interference

2002 ◽  
Vol 383 (7-8) ◽  
pp. 1263-1266 ◽  
Author(s):  
M. Takahashi ◽  
H. Iwasaki ◽  
H. Inoue ◽  
K. Takahashi
Keyword(s):  
Rna Interference ◽  
Caenorhabditis Elegans ◽  
Genetic Analysis ◽  
Postembryonic Development ◽  
26S Proteasome ◽  
Reverse Genetic ◽  
Proteasome Subunit ◽  
Proteasome Subunits ◽  
Regulatory Particle

Abstract Reverse genetic analysis was performed on the Caenorhabditis elegans 26S proteasome subunit genes by doublestranded RNAmediated interference (RNAi). Embryonic and postembryonic lethality was caused by interference of all of the eight tested 20S core subunits and all of the 19S regulatory particle subunits except for CeRpn9, CeRpn10, and Ce Rpn12, where RNAi caused no abnormality. However, synthetic suppression of CeRpn10 and CeRpn12 was lethal, whereas neither the combination of Ce Rpn9 with CeRpn10 nor with CeRpn12 resulted in abnormalities in RNAi. These results indicate that the 26S proteasome is indispensable for embryogenesis and postembryonic development, although Ce Rpn9, CeRpn10, and CeRpn12 are not essential, at least under the conditions used. CeRpn10 and Ce Rpn12 are considered to compensate for the suppression of each other.

Chaperone-driven proteasome assembly

2008 ◽  
Vol 36 (5) ◽  
pp. 807-812 ◽  
Author(s):  
Rina Rosenzweig ◽  
Michael H. Glickman
Keyword(s):  
26S Proteasome ◽  
Biologically Active ◽  
Core Particle ◽  
Regulatory Particle ◽  
Particle Attachment

Assembly of the 34-subunit, 2.5 MDa 26S proteasome is a carefully choreographed intricate process. It starts with formation of a seven-membered α-ring that serves as a template for assembly of the complementary β-ring-forming ‘half-proteasomes’. Dimerization results in a latent 20S core particle that can serve further as a platform for 19S regulatory particle attachment and formation of the biologically active 26S proteasome for ubiquitin-dependent proteolysis. Both general and dedicated proteasome assembly chaperones regulate the efficiency and outcome of critical steps in proteasome biogenesis, and in complex association.

scholarly journals Assembly manual for the proteasome regulatory particle: the first draft

2010 ◽  
Vol 38 (1) ◽  
pp. 6-13 ◽  
Author(s):  
Soyeon Park ◽  
Geng Tian ◽  
Jeroen Roelofs ◽  
Daniel Finley
Keyword(s):  
Biochemical Study ◽  
Release Mechanism ◽  
Substrate Selection ◽  
Core Particle ◽  
Genetic Studies ◽  
Macromolecular Assembly ◽  
Assembly Pathway ◽  
Ongoing Work ◽  
The Common ◽  
Regulatory Particle

The proteasome is the most complex protease known, with a molecular mass of approx. 3 MDa and 33 distinct subunits. Recent studies reported the discovery of four chaperones that promote the assembly of a 19-subunit subcomplex of the proteasome known as the regulatory particle, or RP. These and other findings define a new and highly unusual macromolecular assembly pathway. The RP mediates substrate selection by the proteasome and injects substrates into the CP (core particle) to be degraded. A heterohexameric ring of ATPases, the Rpt proteins, is critical for RP function. These ATPases abut the CP and their C-terminal tails help to stabilize the RP–CP interface. ATPase heterodimers bound to the chaperone proteins are early intermediates in assembly of the ATPase ring. The four chaperones have the common feature of binding the C-domains of Rpt proteins, apparently a remarkable example of convergent evolution; each chaperone binds a specific Rpt subunit. The C-domains are distinct from the C-terminal tails, but are proximal to them. Some, but probably not all, of the RP chaperones appear to compete with CP for binding of the Rpt proteins, as a result of the proximity of the tails to the C-domain. This competition may underlie the release mechanism for these chaperones. Genetic studies in yeast point to the importance of the interaction between the CP and the Rpt tails in assembly, and a recent biochemical study in mammals suggests that RP assembly takes place on pre-assembled CP. These results do not exclude a parallel CP-independent pathway of assembly. Ongoing work should soon clarify the roles of both the CP and the four chaperones in RP assembly.

Chaperone-assisted assembly of the proteasome core particle

2010 ◽  
Vol 38 (1) ◽  
pp. 29-33 ◽  
Author(s):  
Ana C. Matias ◽  
Paula C. Ramos ◽  
R. Jürgen Dohmen
Keyword(s):  
Active Sites ◽  
26S Proteasome ◽  
Eukaryotic Cells ◽  
Main Function ◽  
Core Particle ◽  
Lysosomal Protease ◽  
Assembly Pathway ◽  
Multimeric Complex ◽  
Autocatalytic Processing ◽  
Α Subunits

The 26S proteasome is a non-lysosomal protease in the cytosol and nucleus of eukaryotic cells. Its main function is to mediate ubiquitin-dependent proteolysis. The 26S proteasome is a multimeric complex composed by the 20S proteasome CP (core particle) and the 19S RPs (regulatory particles). Although the atomic structure of the 26S proteasome has not yet been determined, high-resolution structures are available for its CP. Studies on the complicated assembly pathway of the proteasome have revealed that it involves an unprecedented number of dedicated chaperones. Assembly of the CP alone involves three conserved proteasome-assembly chaperones [PAC1–PAC2, PAC3–PAC4 and UMP1 (ubiquitin-mediated proteolysis 1)]. Whereas the two heterodimeric PACs have been implicated in the formation of rings of the seven distinct α subunits, UMP1 is important for the formation and dimerization of proteasome precursor complexes containing β subunits. Dimerization coincides with the incorporation of the last β subunit (β7). Additional modules important for the assembly of precursor complexes and their dimerization reside in the β subunits themselves, either as transient or as permanent extensions. Particularly important domains are the propeptide of β5 and the C-terminal extensions of β2 and β7. Upon maturation of the active sites by autocatalytic processing, UMP1 is degraded by the native proteasome.

scholarly journals A biosensor study of protein interaction with the 20S proteasome core particle

2019 ◽  
Vol 65 (4) ◽  
pp. 306-310
Author(s):  
O.A. Buneeva ◽  
O.V. Gnedenko ◽  
M.V. Medvedeva ◽  
V.G. Zgoda ◽  
A.S. Ivanov ◽  
...  
Keyword(s):  
Pyruvate Kinase ◽  
Direct Interaction ◽  
26S Proteasome ◽  
Spectrometric Analysis ◽  
20S Proteasome ◽  
Core Particle ◽  
Ubiquitinated Proteins ◽  
Moonlighting Proteins ◽  
Biosensor Chip ◽  
Related Proteins

It becomes increasingly clear that ubiquitination of cellular proteins is not an indispensable prerequisite of their degradation in proteasomes. There are a number of proteins to be eliminated which are not pre-ubiquitinated for their recognition by regulatory subcomplex of 26S proteasome, but which directly interact with the 20S proteasome core particle (20S proteasome). The obligatory precondition for such interaction consists in existence of disordered (hydrophobic) fragments in the target protein. In this study we have investigated the interaction of a number of multifunctional (moonlighting) proteins (glyceraldehyde-3-phosphate dehydrogenase (GAPDH), aldolase, pyruvate kinase) and neurodegeneration-related proteins (a-synuclein, myelin basic protein) with 20S proteasome immobilized on the SPR-biosensor chip and stabilized by means of a bifunctional agent dimethyl pimelimidate (in order to prevent possible dissociation of this subcomplex). Only two of all investigated proteins (aldolase and pyruvate kinase) interacted with the immobilized 20S proteasome (Kd of 8.17´10-7 M and 5.56´10-7 M, respectively). In addition to earlier detected GAPDH ubiquitination, mass spectrometric analysis of the studied proteins revealed the presence of the ubiquitin signature (Lys-e-Gly-Gly) only in aldolase. Oxidation of aldolase and pyruvate kinase, which promotes elimination of proteins via their direct interaction with 20S proteasome, caused a 2-3-fold decrease in their Kd values as comparison with this parameter obtained for the intact proteins. The results of this study provide further evidence for direct interaction of both ubiquitinated proteins (aldolase), and non-ubiquitinated proteins (pyruvate kinase) with the 20S proteasome core particle (20S proteasome). The effectiveness of this interaction is basically equal for the ubiquitinated proteins and non-ubiquitinated proteins.

scholarly journals Signature activities of 20S proteasome include degradation of the ubiquitin-tag with the protein under hypoxia

2019 ◽  
Author(s):  
Indrajit Sahu ◽  
Sachitanand M. Mali ◽  
Prasad Sulkshane ◽  
Andrey Rozenberg ◽  
Cong Xu ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Conformational Changes ◽  
Unique Property ◽  
26S Proteasome ◽  
20S Proteasome ◽  
Core Particle ◽  
Ubiquitinated Proteins ◽  
Catalytic Sites ◽  
Intracellular Proteolysis ◽  
Ubiquitinated Substrate

AbstractCareful removal of unwanted proteins is necessary for cell survival. The primary constitutive intracellular protease is the 26S proteasome complex, often found in equilibrium with its free catalytic subcomplex– the 20S core particle. Protein degradation by 26S is tightly regulated by prior ubiquitination of substrates, whereas 20S is amenable to substrates with an unstructured segment. Differentiating their contributions to intracellular proteolysis is challenging due to their common catalytic sites. Here, by chemically synthesizing a synoptic set of homogenous ubiquitinated proteins, we ascribe signature features to 20S function and demonstrate a unique property: degrading the ubiquitin-tag along with the target protein. Cryo-EM confirms that a ubiquitinated substrate can induce asymmetric conformational changes to 20S. Mass-spectrometry of intracellular peptidome under hypoxia and in human failing heart identifies the signature properties of 20S in cells. Moreover, the ability of 20S proteasome to clear toxic proteins rapidly, contributes to better survival under these conditions.

scholarly journals Phosphatase UBLCP1 controls proteasome assembly

Open Biology ◽  
2017 ◽  
Vol 7 (5) ◽  
pp. 170042 ◽  
Author(s):  
Shuangwu Sun ◽  
Sisi Liu ◽  
Zhengmao Zhang ◽  
Wang Zeng ◽  
Chuang Sun ◽  
...  
Keyword(s):  
Atpase Activity ◽  
Essential Role ◽  
26S Proteasome ◽  
Core Particle ◽  
Proteasome Subunit ◽  
Bona Fide ◽  
Proteasome Regulation

Ubiquitin-like domain-containing C-terminal domain phosphatase 1 (UBLCP1), an FCP/SCP phosphatase family member, was identified as the first proteasome phosphatase. UBLCP1 binds to proteasome subunit Rpn1 and dephosphorylates the proteasome in vitro . However, it is still unclear which proteasome subunit(s) are the bona fide substrate(s) of UBLCP1 and the precise mechanism for proteasome regulation remains elusive. Here, we show that UBLCP1 selectively binds to the 19S regulatory particle (RP) through its interaction with Rpn1, but not the 20S core particle (CP) or the 26S proteasome holoenzyme. In the RP, UBLCP1 dephosphorylates the subunit Rpt1, impairs its ATPase activity, and consequently disrupts the 26S proteasome assembly, yet it has no effects on the RP assembly from precursor complexes. The Rpn1-binding and phosphatase activities of UBLCP1 are essential for its function on Rpt1 dephosphorylation and proteasome activity both in vivo and in vitro . Our study establishes the essential role of the UBLCP1/Rpn1/Rpt1 complex in regulating proteasome assembly.

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