scholarly journals An evolutionarily distinct chaperone promotes 20S proteasome α-ring assembly in plants

2020 ◽  
Vol 133 (21) ◽  
pp. jcs249862
Author(s):  
Richard S. Marshall ◽  
David C. Gemperline ◽  
Fionn McLoughlin ◽  
Adam J. Book ◽  
Kay Hofmann ◽  
...  
Keyword(s):  
Active Sites ◽  
26S Proteasome ◽  
20S Proteasome ◽  
Β Subunit ◽  
The Core ◽  
Ring Assembly ◽  
Evolutionarily Conserved ◽  
Α And Β Subunits ◽  
Α Subunits

ABSTRACTThe core protease (CP) subcomplex of the 26S proteasome houses the proteolytic active sites and assumes a barrel shape comprised of four co-axially stacked heptameric rings formed by structurally related α- and β-subunits. CP biogenesis typically begins with the assembly of the α-ring, which then provides a template for β-subunit integration. In eukaryotes, α-ring assembly is partially mediated by two hetero-dimeric chaperones, termed Pba1–Pba2 (Add66) and Pba3–Pba4 (also known as Irc25–Poc4) in yeast. Pba1–Pba2 initially promotes orderly recruitment of the α-subunits through interactions between their C-terminal HbYX or HbF motifs and pockets at the α5–α6 and α6–α7 interfaces. Here, we identified PBAC5 as a fifth α-ring assembly chaperone in Arabidopsis that directly binds the Pba1 homolog PBAC1 to form a trimeric PBAC5–PBAC1–PBAC2 complex. PBAC5 harbors a HbYX motif that docks with a pocket between the α4 and α5 subunits during α-ring construction. Arabidopsis lacking PBAC5, PBAC1 and/or PBAC2 are hypersensitive to proteotoxic, salt and osmotic stresses, and display proteasome assembly defects. Remarkably, whereas PBAC5 is evolutionarily conserved among plants, sequence relatives are also dispersed within other kingdoms, including a scattered array of fungal, metazoan and oomycete species.

scholarly journals Structural Fluctuations of the Human Proteasome α7 Homo-Tetradecamer Double Ring Imply the Proteasomal α-Ring Assembly Mechanism

2021 ◽  
Vol 22 (9) ◽  
pp. 4519
Author(s):  
Chihong Song ◽  
Tadashi Satoh ◽  
Taichiro Sekiguchi ◽  
Koichi Kato ◽  
Kazuyoshi Murata
Keyword(s):  
Ring Structure ◽  
20S Proteasome ◽  
Core Complex ◽  
Double Ring ◽  
The Core ◽  
Ring Assembly ◽  
Cryo Electron Microscopy ◽  
Assembly Mechanism ◽  
Structural Fluctuations ◽  
Α Subunits

The 20S proteasome, which is composed of layered α and β heptameric rings, is the core complex of the eukaryotic proteasome involved in proteolysis. The α7 subunit is a component of the α ring, and it self-assembles into a homo-tetradecamer consisting of two layers of α7 heptameric rings. However, the structure of the α7 double ring in solution has not been fully elucidated. We applied cryo-electron microscopy to delineate the structure of the α7 double ring in solution, revealing a structure different from the previously reported crystallographic model. The D7-symmetrical double ring was stacked with a 15° clockwise twist and a separation of 3 Å between the two rings. Two more conformations, dislocated and fully open, were also identified. Our observations suggest that the α7 double-ring structure fluctuates considerably in solution, allowing for the insertion of homologous α subunits, finally converting to the hetero-heptameric α rings in the 20S proteasome.

scholarly journals The Contribution of the 20S Proteasome to Proteostasis

Biomolecules ◽  
2019 ◽  
Vol 9 (5) ◽  
pp. 190 ◽  
Author(s):  
Fanindra Kumar Deshmukh ◽  
Dana Yaffe ◽  
Maya Olshina ◽  
Gili Ben-Nissan ◽  
Michal Sharon
Keyword(s):  
Biological Process ◽  
Intrinsically Disordered Protein ◽  
26S Proteasome ◽  
20S Proteasome ◽  
Biological Processes ◽  
Neuronal Communication ◽  
Intrinsically Disordered ◽  
Disordered Protein ◽  
The Core ◽  
Functional Aspects

The last decade has seen accumulating evidence of various proteins being degraded by the core 20S proteasome, without its regulatory particle(s). Here, we will describe recent advances in our knowledge of the functional aspects of the 20S proteasome, exploring several different systems and processes. These include neuronal communication, post-translational processing, oxidative stress, intrinsically disordered protein regulation, and extracellular proteasomes. Taken together, these findings suggest that the 20S proteasome, like the well-studied 26S proteasome, is involved in multiple biological processes. Clarifying our understanding of its workings calls for a transformation in our perception of 20S proteasome-mediated degradation—no longer as a passive and marginal path, but rather as an independent, coordinated biological process. Nevertheless, in spite of impressive progress made thus far, the field still lags far behind the front lines of 26S proteasome research. Therefore, we also touch on the gaps in our knowledge of the 20S proteasome that remain to be bridged in the future.

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 Proteasome storage granules protect proteasomes from autophagic degradation upon carbon starvation

eLife ◽  
2018 ◽  
Vol 7 ◽  
Author(s):  
Richard S Marshall ◽  
Richard D Vierstra
Keyword(s):  
Nitrogen Starvation ◽  
Carbon Starvation ◽  
26S Proteasome ◽  
Energy Status ◽  
Cellular Energy ◽  
The Core ◽  
Evolutionarily Conserved ◽  
Autophagic Degradation ◽  
Storage Granules ◽  
Multiple Levels

26S proteasome abundance is tightly regulated at multiple levels, including the elimination of excess or inactive particles by autophagy. In yeast, this proteaphagy occurs upon nitrogen starvation but not carbon starvation, which instead stimulates the rapid sequestration of proteasomes into cytoplasmic puncta termed proteasome storage granules (PSGs). Here, we show that PSGs help protect proteasomes from autophagic degradation. Both the core protease and regulatory particle sub-complexes are sequestered separately into PSGs via pathways dependent on the accessory proteins Blm10 and Spg5, respectively. Modulating PSG formation, either by perturbing cellular energy status or pH, or by genetically eliminating factors required for granule assembly, not only influences the rate of proteasome degradation, but also impacts cell viability upon recovery from carbon starvation. PSG formation and concomitant protection against proteaphagy also occurs in Arabidopsis, suggesting that PSGs represent an evolutionarily conserved cache of proteasomes that can be rapidly re-mobilized based on energy availability.

scholarly journals Assembly of the elongated collagen prolyl 4-hydroxylase α2β2 heterotetramer around a central α2 dimer

2017 ◽  
Vol 474 (5) ◽  
pp. 751-769 ◽  
Author(s):  
M. Kristian Koski ◽  
Jothi Anantharajan ◽  
Petri Kursula ◽  
Prathusha Dhavala ◽  
Abhinandan V. Murthy ◽  
...  
Keyword(s):  
Three Dimensional ◽  
Coiled Coil ◽  
Β Subunit ◽  
Dimerization Domain ◽  
Α Subunit ◽  
Protein Dimer ◽  
Symmetric Molecule ◽  
Asymmetric Shape ◽  
Proline Rich Peptide ◽  
Α Subunits

Collagen prolyl 4-hydroxylase (C-P4H), an α2β2 heterotetramer, is a crucial enzyme for collagen synthesis. The α-subunit consists of an N-terminal dimerization domain, a central peptide substrate-binding (PSB) domain, and a C-terminal catalytic (CAT) domain. The β-subunit [also known as protein disulfide isomerase (PDI)] acts as a chaperone, stabilizing the functional conformation of C-P4H. C-P4H has been studied for decades, but its structure has remained elusive. Here, we present a three-dimensional small-angle X-ray scattering model of the entire human C-P4H-I heterotetramer. C-P4H is an elongated, bilobal, symmetric molecule with a length of 290 Å. The dimerization domains from the two α-subunits form a protein–protein dimer interface, assembled around the central antiparallel coiled-coil interface of their N-terminal α-helices. This region forms a thin waist in the bilobal tetramer. The two PSB/CAT units, each complexed with a PDI/β-subunit, form two bulky lobes pointing outward from this waist region, such that the PDI/β-subunits locate at the far ends of the βααβ complex. The PDI/β-subunit interacts extensively with the CAT domain. The asymmetric shape of two truncated C-P4H-I variants, also characterized in the present study, agrees with this assembly. Furthermore, data from these truncated variants show that dimerization between the α-subunits has an important role in achieving the correct PSB–CAT assembly competent for catalytic activity. Kinetic assays with various proline-rich peptide substrates and inhibitors suggest that, in the competent assembly, the PSB domain binds to the procollagen substrate downstream from the CAT domain.

scholarly journals Assembly of the Drosophila 26 S proteasome is accompanied by extensive subunit rearrangements

2002 ◽  
Vol 365 (2) ◽  
pp. 527-536 ◽  
Author(s):  
Éva KURUCZ ◽  
István ANDÓ ◽  
Máté SÜMEGI ◽  
Harald HÖLZL ◽  
Barbara KAPELARI ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
26S Proteasome ◽  
Cross Linking ◽  
20S Proteasome ◽  
Electron Microscopic ◽  
Immunoblot Assay ◽  
Optimal Conformation ◽  
Regulatory Complex ◽  
Atpase Subunits ◽  
The Cross

The subunit contacts in the regulatory complex of the Drosophila 26 S proteasome were studied through the cross-linking of closely spaced subunits of the complex, and analysis of the cross-linking pattern in an immunoblot assay with the use of subunit-specific monoclonal antibodies. The cross-linking pattern of the purified 26 S proteasome exhibits significant differences as compared with that of the purified free regulatory complex. It is shown that the observed differences are due to extensive rearrangement of the subunit contacts accompanying the assembly of the 26 S proteasome from the regulatory complex and the 20S proteasome. Cross-linking studies and electron microscopic examinations revealed that these changes are reversible and follow the assembly or the disassembly of the 26 S proteasome. Although the majority of the changes observed in the subunit contacts affected the hexameric ring of the ATPase subunits, the alterations extended over the whole of the regulatory complex, affecting subunit contacts even in the lid subcomplex. Changes in the subunit contacts, similar to those in the regulatory complex, were detected in the 20S proteasome. These observations indicate that the assembly of the 26 S proteasome is not simply a passive docking of two rigid subcomplexes. In the course of the assembly, the interacting subcomplexes mutually rearrange their structures so as to create the optimal conformation required for the assembly and the proper functioning of the 26S proteasome.

scholarly journals Phosphorylation of 20S proteasome alpha subunit C8 (alpha7) stabilizes the 26S proteasome and plays a role in the regulation of proteasome complexes by gamma-interferon

2004 ◽  
Vol 378 (1) ◽  
pp. 177-184 ◽  
Author(s):  
Suchira BOSE ◽  
Fiona L. L. STRATFORD ◽  
Kerry I. BROADFOOT ◽  
Grant G. F. MASON ◽  
A. Jennifer RIVETT
Keyword(s):  
Mammalian Cells ◽  
Substantial Effect ◽  
26S Proteasome ◽  
20S Proteasome ◽  
Phosphorylation Sites ◽  
Animal Cells ◽  
Catalytic Subunits ◽  
Kinase Ck2 ◽  
Γ Interferon

In animal cells there are several regulatory complexes which interact with 20S proteasomes and give rise to functionally distinct proteasome complexes. γ-Interferon upregulates three immuno beta catalytic subunits of the 20S proteasome and the PA28 regulator, and decreases the level of 26S proteasomes. It also decreases the level of phosphorylation of two proteasome alpha subunits, C8 (α7) and C9 (α3). In the present study we have investigated the role of phosphorylation of C8 by protein kinase CK2 in the formation and stability of 26S proteasomes. An epitope-tagged C8 subunit expressed in mammalian cells was efficiently incorporated into both 20S proteasomes and 26S proteasomes. Investigation of mutants of C8 at the two known CK2 phosphorylation sites demonstrated that these are the two phosphorylation sites of C8 in animal cells. Although phosphorylation of C8 was not absolutely essential for the formation of 26S proteasomes, it did have a substantial effect on their stability. Also, when cells were treated with γ-interferon, there was a marked decrease in phosphorylation of C8, a decrease in the level of 26S proteasomes, and an increase in immunoproteasomes and PA28 complexes. These results suggest that the down-regulation of 26S proteasomes after γ-interferon treatment results from the destabilization that occurs after dephosphorylation of the C8 subunit.

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