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

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.

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

Biomeditsinskaya Khimiya ◽

10.18097/pbmc20196504306 ◽

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.

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Structural basis for dynamic regulation of the human 26S proteasome

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1614614113 ◽

2016 ◽

Vol 113 (46) ◽

pp. 12991-12996 ◽

Cited By ~ 96

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.

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Exploring long-range cooperativity in the 20S proteasome core particle from Thermoplasma acidophilum using methyl-TROSY–based NMR

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1920770117 ◽

2020 ◽

Vol 117 (10) ◽

pp. 5298-5309

Author(s):

Enrico Rennella ◽

Rui Huang ◽

Zanlin Yu ◽

Lewis E. Kay

Keyword(s):

Molecular Machines ◽

Molecular Assembly ◽

20S Proteasome ◽

Core Particle ◽

Thermoplasma Acidophilum ◽

Transverse Relaxation ◽

Catalytic Sites ◽

Methyl Trosy ◽

Transverse Relaxation Optimized Spectroscopy ◽

Allosteric Pathway

The 20S core particle (CP) proteasome is a molecular assembly catalyzing the degradation of misfolded proteins or proteins no longer required for function. It is composed of four stacked heptameric rings that form a barrel-like structure, sequestering proteolytic sites inside its lumen. Proteasome function is regulated by gates derived from the termini of α-rings and through binding of regulatory particles (RPs) to one or both ends of the barrel. The CP is dynamic, with an extensive allosteric pathway extending from one end of the molecule to catalytic sites in its center. Here, using methyl-transverse relaxation optimized spectroscopy (TROSY)-based NMR optimized for studies of high–molecular-weight complexes, we evaluate whether the pathway extends over the entire 150-Å length of the molecule. By exploiting a number of different labeling schemes, the two halves of the molecule can be distinguished, so that the effects of 11S RP binding, or the introduction of gate or allosteric pathway mutations at one end of the barrel can be evaluated at the distal end. Our results establish that while 11S binding and the introduction of key mutations affect each half of the CP allosterically, they do not further couple opposite ends of the molecule. This may have implications for the function of so-called “hybrid” proteasomes where each end of the CP is bound with a different regulator, allowing the CP to be responsive to both RPs simultaneously. The methodology presented introduces a general NMR strategy for dissecting pathways of communication in homo-oligomeric molecular machines.

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

Biomolecules ◽

10.3390/biom9060213 ◽

2019 ◽

Vol 9 (6) ◽

pp. 213 ◽

Cited By ~ 3

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.

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

10.1101/132613 ◽

2017 ◽

Cited By ~ 1

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.

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