Crystal Structures of the Rhodococcus Proteasome with and without its Pro-peptides: Implications for the Role of the Pro-peptide in Proteasome Assembly

Kwon, Young Do; Nagy, István; Adams, Paul D.; Baumeister, Wolfgang; Jap, Bing K.

doi:10.1016/j.jmb.2003.08.029

Cited by 76 publications

(97 citation statements)

References 38 publications

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“…C, pulse-chase analysis of pre␤5 processing in the H74A mutant. 35 S-Labeled proteins were immunoprecipitated with anti-LMP7-His 6 serum. Asterisks, cleavage products produced when autoprocessing at the normal G-T cleavage site is inhibited.…”

Section: Rowsmentioning

confidence: 99%

“…Subunit-specific appendages that are part of the mature subunits as well as ␤-subunit propeptides contribute to assembly by supplementing specific subunit-subunit interfaces within the assembling proteasome and possibly by recruiting or positioning extrinsic assembly chaperones (25,26,35). The most prominent "intramolecular chaperone" is the ␤5 propeptide (26,36).…”

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confidence: 99%

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Distinct Elements in the Proteasomal β5 Subunit Propeptide Required for Autocatalytic Processing and Proteasome Assembly

Arendt

et al. 2016

Journal of Biological Chemistry

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Eukaryotic 20S proteasome assembly remains poorly understood. The subunits stack into four heteroheptameric rings; three inner-ring subunits (␤1, ␤2, and ␤5) bear the protease catalytic residues and are synthesized with N-terminal propeptides. These propeptides are removed autocatalytically late in assembly. In Saccharomyces cerevisiae, ␤5 (Doa3/Pre2) has a 75-residue propeptide, ␤5pro, that is essential for proteasome assembly and can work in trans. We show that deletion of the poorly conserved N-terminal half of the ␤5 propeptide nonetheless causes substantial defects in proteasome maturation. Sequences closer to the cleavage site have critical but redundant roles in both assembly and self-cleavage. A conserved histidine two residues upstream of the autocleavage site strongly promotes processing. Surprisingly, although ␤5pro is functionally linked to the Ump1 assembly factor, trans-expressed ␤5pro associates only weakly with Ump1-containing precursors. Several genes were identified as dosage suppressors of trans-expressed ␤5pro mutants; the strongest encoded the ␤7 proteasome subunit. Previous data suggested that ␤7 and ␤5pro have overlapping roles in bringing together two half-proteasomes, but the timing of ␤7 addition relative to half-mer joining was unclear. Here we report conditions where dimerization lags behind ␤7 incorporation into the half-mer. Our results suggest that ␤7 insertion precedes half-mer dimerization, and the ␤7 tail and ␤5 propeptide have unequal roles in half-mer joining.

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Section: Rowsmentioning

confidence: 99%

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confidence: 99%

Distinct Elements in the Proteasomal β5 Subunit Propeptide Required for Autocatalytic Processing and Proteasome Assembly

Arendt

et al. 2016

Journal of Biological Chemistry

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“…Plasmids and Mutagenesis-For reasons of simplicity and the availability of the crystal structure of a simplified ␣1␤1 20 S proteasome from R. erythropolis (24), only one type of ␣-subunit and one type of ␤-subunit was used for the in vitro and in vivo assembly experiments. The single gene constructs pT7-7 ␣1His 6 and pT7-7 ␤1His 6 encoding the C-terminal His-tagged ␣-and ␤-subunit, respectively, where used for the in vitro assembly experiments.…”

Section: Methodsmentioning

confidence: 99%

“…It is established, however, that the assembly proceeds via the formation of halfproteasomes composed of one ␣-subunit ring and one unprocessed ␤-subunit ring (␣ 7 ␤ 7 ). Two half-proteasomes assemble further to form a preholoproteasome, a species that still contains the unprocessed ␤-subunits (13,23,24). Finally, processing of the ␤-subunits occurs, and the mature holoproteasome (␣ 7 ␤ 7 ␤ 7 ␣ 7 ) is formed (9,20).…”

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confidence: 99%

Mass Spectrometry Reveals the Missing Links in the Assembly Pathway of the Bacterial 20 S Proteasome

Sharon

Witt

Glasmacher

et al. 2007

Journal of Biological Chemistry

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The 20 S proteasome is an essential proteolytic particle, responsible for degrading short-lived and abnormal intracellular proteins. The 700-kDa assembly is comprised of 14 ␣-type and 14 ␤-type subunits, which form a cylindrical architecture composed of four stacked heptameric rings (␣ 7 ␤ 7 ␤ 7 ␣ 7 ). The formation of the 20 S proteasome is a complex process that involves a cascade of folding, assembly, and processing events. To date, the understanding of the assembly pathway is incomplete due to the experimental challenges of capturing shortlived intermediates. In this study, we have applied a real-time mass spectrometry approach to capture transient species along the assembly pathway of the 20 S proteasome from Rhodococcus erythropolis. In the course of assembly, we observed formation of an early ␣/␤-heterodimer as well as an unprocessed half-proteasome particle. Formation of mature holoproteasomes occurred in concert with the disappearance of half-proteasomes. We also analyzed the ␤-subunits before and during assembly and reveal that those with longer propeptides are incorporated into half-and full proteasomes more rapidly than those that are heavily truncated. To characterize the preholoproteasome, formed by docking of two unprocessed half-proteasomes and not observed during assembly of wild type subunits, we trapped this intermediate using a ␤-subunit mutational variant. In summary, this study provides evidence for transient intermediates in the assembly pathway and reveals detailed insight into the cleavage sites of the propeptide.

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“…These propeptides appear to prevent premature enzymatic activation, but they are dispensable for assembly [6]. In contrast, Rhodococcus β subunit propeptides are much longer (around 60 amino acid residues), contain conserved residues that interact with adjacent α subunits, and they are required for normal proteasome assembly [7]. Assembly of eukaryotic proteasomes also requires catalytic β subunit propeptides.…”

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confidence: 99%

Differential intra-proteasome interactions involving standard and immunosubunits

Jayarapu

Griffin

2007

Biochemical and Biophysical Research Communications

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Animals with immune systems have two types of proteasomes, "standard proteasomes" and "immunoproteasomes" that respectively contain constitutively expressed catalytic subunits or interferon-γ-inducible catalytic subunits. Interestingly, proteasome assembly is biased against formation of most mixed proteasomes containing combinations of standard subunits and immunosubunits. We previously demonstrated that catalytic subunit propeptide differences contribute to this assembly specificity. In the current study, we investigated the contributions of catalytic subunit propeptides and C-terminal extensions to intra-proteasome protein-protein interactions that are potentially involved in mediating biased assembly of human proteasomes, and we found a number of interactions that differentially depended on these structures. For example, the C-terminal extension of standard subunit β2 is required for β2's interaction with adjacent β3, whereas the C-terminal extension of immunosubunit β2i is dispensable for β2i's interaction with β3. Taken together, our results suggest mechanisms whereby differential intra-proteasome interactions could contribute to proteasome assembly specificity. KeywordsProteasome; Immunoproteasome; Proteasome assembly; Propeptide; Protein-protein interaction; Yeast two-hybrid interaction Proteasomes are multi-subunit multi-catalytic intracellular proteases that are responsible for most non-lysosomal protein degradation in eukaryotic cells. Proteasomes are an integral component of ubiquitin-mediated protein degradation, and as such they are involved in many cellular processes, including cell cycle control, cellular stress responses, intra-cellular signaling, and MHC class 1 antigen processing [1][2][3]. The 20S proteasome core contains the proteolytic active sites and consists of four heptameric stacked rings configured α7β7β7α7. This quaternary structure is conserved from archeae to humans. Proteasomes in the archebacterium Thermoplasma acidophilum are composed of only two different subunits, with all α subunits and β subunits identical. Conversely, eukaryotic proteasomes are composed of seven different α subunits and seven different β subunits. In animals with immune systems, there is increased complexity with two proteasome subtypes, "standard proteasomes" and Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. As proteasome structural complexity increases, so too does complexity of proteasome assembly. In Thermoplasma, β subunits are synthesized as inactive precursor proteins with very short N-terminal propeptides that are removed at the end of the assembly p...

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Crystal Structures of the Rhodococcus Proteasome with and without its Pro-peptides: Implications for the Role of the Pro-peptide in Proteasome Assembly

Cited by 76 publications

References 38 publications

Distinct Elements in the Proteasomal β5 Subunit Propeptide Required for Autocatalytic Processing and Proteasome Assembly

Distinct Elements in the Proteasomal β5 Subunit Propeptide Required for Autocatalytic Processing and Proteasome Assembly

Mass Spectrometry Reveals the Missing Links in the Assembly Pathway of the Bacterial 20 S Proteasome

Differential intra-proteasome interactions involving standard and immunosubunits

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