scholarly journals The proteasome: a macromolecular assembly designed for controlled proteolysis

1999 ◽  
Vol 354 (1389) ◽  
pp. 1501-1511 ◽  
Author(s):  
P. Zwickl ◽  
D. Voges ◽  
W. Baumeister
Keyword(s):  
Functional Organization ◽  
26S Proteasome ◽  
Eukaryotic Cells ◽  
Core Complex ◽  
Ubiquitin Pathway ◽  
Enzymatic Mechanism ◽  
Macromolecular Assembly ◽  
Regulatory Complex ◽  
Structure Assembly ◽  
Made In

In eukaryotic cells, the vast majority of proteins in the cytosol and nucleus are degraded via the proteasome–ubiquitin pathway. The 26S proteasome is a huge protein degradation machine of 2.5 MDa, built of approximately 35 different subunits. It contains a proteolytic core complex, the 20S proteasome and one or two 19S regulatory complexes which associate with the termini of the barrel–shaped 20S core. The 19S regulatory complex serves to recognize ubiquitylated target proteins and is implicated to have a role in their unfolding and translocation into the interior of the 20S complex where they are degraded into oligopeptides. While much progress has been made in recent years in elucidating the structure, assembly and enzymatic mechanism of the 20S complex, our knowledge of the functional organization of the 19S regulator is rather limited. Most of its subunits have been identified, but specific functions can be assigned to only a few of them.

Formation of Proteasome-PA700 Complexes Directly Correlates with Activation of Peptidase Activity

1998 ◽  
Vol 4 (S2) ◽  
pp. 952-953
Author(s):  
G. Adams ◽  
B. Crotchett ◽  
C. Slaughter ◽  
G. DeMartino ◽  
E. Gogol
Keyword(s):  
26S Proteasome ◽  
Cavity Surface ◽  
Detectable Effect ◽  
Peptidase Activity ◽  
Core Complex ◽  
Central Cavity ◽  
Protein Subunits ◽  
Catalytic Sites ◽  
Regulatory Complex ◽  
Dependent Pathway

The eukaryotic 20S proteasome (700 kDa) serves as the core complex for the breakdown of most cytosolic proteins by the ubiquitin-dependent pathway. This cylinder-shaped proteolytic assembly (17 nm long, 11 nm diameter) consists of four stacked heptameric rings that help to form a central cavity where degradation occurs. The catalytic sites are located on the cavity surface of the two middle β rings while the outer α rings serve as docking sites for various regulatory complexes.PA700 (700 kDa, 20 protein subunits) is the major regulatory complex of the proteasome, forming a “cap” that attaches to either or both proteasome ends. The resulting superassembly structurally resembles the larger, more active 26S proteasome and exhibits both a higher intrinsic peptidase activity as well as a wider range of substrate specificity, including ubiquitin-tagged proteins. A second regulatory complex, the modulator (300 kDa), further stimulates the effects of PA700 at subsaturating concentrations of the activator with no detectable effect on the proteasome in the absence of PA700.

Formation of Proteasome-PA700 Complexes Directly Correlates with Activation of Peptidase Activity

1998 ◽  
Vol 4 (S2) ◽  
pp. 982-983
Author(s):  
E. Gogol ◽  
G. Adams ◽  
B. Crotchett ◽  
C. Slaughter ◽  
G. DeMartino
Keyword(s):  
26S Proteasome ◽  
Cavity Surface ◽  
Detectable Effect ◽  
Peptidase Activity ◽  
Core Complex ◽  
Central Cavity ◽  
Protein Subunits ◽  
Catalytic Sites ◽  
Regulatory Complex ◽  
Dependent Pathway

The eukaryotic 20S proteasome (700 kDa) serves as the core complex for the breakdown of most cytosolic proteins by the ubiquitin-dependent pathway. This cylinder-shaped proteolytic assembly (17 nm long, 11 nm diameter) consists of four stacked heptameric rings that help to form a central cavity where degradation occurs. The catalytic sites are located on the cavity surface of the two middle (3 rings while the outer a rings serve as docking sites for various regulatory complexes.PA700 (700 kDa, 20 protein subunits) is the major regulatory complex of the proteasome, forming a “cap” that attaches to either or both proteasome ends. The resulting superassembly structurally resembles the larger, more active 26S proteasome and exhibits both a higher intrinsic peptidase activity as well as a wider range of substrate specificity, including ubiquitin-tagged proteins. A second regulatory complex, the modulator (300 kDa), further stimulates the effects of PA700 at subsaturating concentrations of the activator with no detectable effect on the proteasome in the absence of PA700.

scholarly journals The 26S proteasome of the fission yeast Schizosaccharomyces pombe

1999 ◽  
Vol 354 (1389) ◽  
pp. 1523-1532 ◽  
Author(s):  
C. R. M. Wilkinson ◽  
M. Penny ◽  
G. McGurk ◽  
M. Wallace ◽  
C. Gordon
Keyword(s):  
Fission Yeast ◽  
Nuclear Periphery ◽  
26S Proteasome ◽  
Live Cells ◽  
Multiprotein Complex ◽  
Major Site ◽  
Catalytic Core ◽  
Ubiquitin Pathway ◽  
Dynamic Distribution ◽  
Regulatory Complex

The 26S proteasome is the multiprotein complex that degrades proteins that have been marked for destruction by the ubiquitin pathway. It is made up of two multisubunit complexes, the 20S catalytic core and the 19S regulatory complex. We describe the isolation and characterisation of conditional mutants in the regulatory complex and their use to investigate interactions between different subunits. In addition we have investigated the localisation of the 26S proteasome in fission yeast, by immunofluoresence in fixed cells and live cells using a GFP tagged subunit. Surprisingly we find that in mitotic cells the 26S proteasome occupies a discrete intracellular compartment, the nuclear periphery. EM analysis demonstrates that the complex resides inside the nuclear envelope. During meiosis the localisation showed a more dynamic distribution. In meiosis I the proteasome remained around the nuclear periphery. However, during meiosis II there was a dramatic relocalisation wherebye initially the signal occupied the area between the dividing nuclei. At the end of mitosis the signal dispersed returning to the nuclear periphery upon ascospore formation. This observation implies that the nuclear periphery is a major site of proteolysis in yeast during mitotic growth and raises important questions about the function of the 26S proteasome in protein degradation.

High resolution spot scan imaging of frozen, hydrated actin bundles with 400kV electrons

1992 ◽  
Vol 50 (1) ◽  
pp. 512-513
Author(s):  
J. Jakana ◽  
M.F. Schmid ◽  
P. Matsudaira ◽  
W. Chiu
Keyword(s):  
High Resolution ◽  
Binding Proteins ◽  
Actin Binding ◽  
Eukaryotic Cells ◽  
Dimensional Structure ◽  
Structural Basis ◽  
Actin Bundle ◽  
Actin Bundles ◽  
3 Dimensional ◽  
Macromolecular Assembly

Actin is a protein found in all eukaryotic cells. In its polymerized form, the cells use it for motility, cytokinesis and for cytoskeletal support. An example of this latter class is the actin bundle in the acrosomal process from the Limulus sperm. The different functions actin performs seem to arise from its interaction with the actin binding proteins. A 3-dimensional structure of this macromolecular assembly is essential to provide a structural basis for understanding this interaction in relationship to its development and functions.

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 Proteasomal conformation controls unfolding ability

2021 ◽  
Vol 118 (25) ◽  
pp. e2101004118
Author(s):  
Julianna R. Cresti ◽  
Abramo J. Manfredonia ◽  
Christopher E. Bragança ◽  
Joseph A. Boscia ◽  
Christina M. Hurley ◽  
...  
Keyword(s):  
Conformational Changes ◽  
Fluorescent Proteins ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Conformational State ◽  
26S Proteasome ◽  
Eukaryotic Cells ◽  
Ubiquitinated Protein ◽  
Equilibrium Conditions ◽  
Substrate Processing

The 26S proteasome is the macromolecular machine responsible for the bulk of protein degradation in eukaryotic cells. As it degrades a ubiquitinated protein, the proteasome transitions from a substrate-accepting conformation (s1) to a set of substrate-processing conformations (s3 like), each stabilized by different intramolecular contacts. Tools to study these conformational changes remain limited, and although several interactions have been proposed to be important for stabilizing the proteasome’s various conformations, it has been difficult to test these directly under equilibrium conditions. Here, we describe a conformationally sensitive Förster resonance energy transfer assay, in which fluorescent proteins are fused to Sem1 and Rpn6, which are nearer each other in substrate-processing conformations than in the substrate-accepting conformation. Using this assay, we find that two sets of interactions, one involving Rpn5 and another involving Rpn2, are both important for stabilizing substrate-processing conformations. Mutations that disrupt these interactions both destabilize substrate-processing conformations relative to the substrate-accepting conformation and diminish the proteasome’s ability to successfully unfold and degrade hard-to-unfold substrates, providing a link between the proteasome’s conformational state and its unfolding ability.

scholarly journals Therapeutic targeting of MLL

Blood ◽  
2009 ◽  
Vol 113 (24) ◽  
pp. 6061-6068 ◽  
Author(s):  
Michaela Liedtke ◽  
Michael L. Cleary
Keyword(s):  
Hematologic Malignancies ◽  
Therapeutic Strategies ◽  
Detailed Structure ◽  
Core Complex ◽  
Acute Leukemias ◽  
Therapeutic Targeting ◽  
Molecular Abnormalities ◽  
Novel Therapeutic Strategies ◽  
Genetic Rearrangements ◽  
Made In

AbstractTreatment of hematologic malignancies is evolving from a uniform approach to targeted therapies directed at the underlying molecular abnormalities of disease. The mixed lineage leukemia (MLL) proto-oncogene is a recurrent site of genetic rearrangements in acute leukemias; and since its discovery in 1992, many advances have been made in understanding its role in leukemogenesis. A variety of MLL translocation partners have been described, and detailed structure/function studies have identified functional domains that are required for transformation. Proteins associated with the MLL core complex or its fusion partners have been isolated and characterized for their critical roles in leukemia pathogenesis. Downstream mediators of MLL transcriptional regulation and multiple collaborating signaling pathways have been described and characterized. These advances in our understanding of MLL-related leukemogenesis provide a foundation for ongoing and future efforts to develop novel therapeutic strategies that will hopefully result in better treatment outcomes.

scholarly journals Characterization of the DNA target site for the yeast ARGR regulatory complex, a sequence able to mediate repression or induction by arginine.

1992 ◽  
Vol 12 (1) ◽  
pp. 68-81 ◽  
Author(s):  
M De Rijcke ◽  
S Seneca ◽  
B Punyammalee ◽  
N Glansdorff ◽  
M Crabeel
Keyword(s):  
Binding Site ◽  
Transcription Initiation ◽  
Functional Organization ◽  
Tata Box ◽  
Saturation Mutagenesis ◽  
Published Data ◽  
Zinc Cluster ◽  
Regulatory Complex ◽  
Specific Control

We have determined the sequences and positions of the cis elements required for proper functioning of the ARG3 promoter and proper arginine-specific control. A TATA box located 100 nucleotides upstream of the transcription start was shown to be essential for ARG3 transcription. Two sequences involved in normal arginine-mediated repression lie immediately downstream of the TATA box: an essential one (arginine box 1 [AB1]) and a secondary one (arginine box 2 [AB2]). AB1 was defined by saturation mutagenesis and is an asymmetrical sequence. A stringently required CGPu motif in AB1 is conserved in all known target sites of C6 zinc cluster DNA-binding proteins, leading us to propose that AB1 is the binding site of ARGRII, another member of the C6 family. The palindromic AB2 sequence is suggested, on the basis of published data, to be the binding site of ARGRI, possibly in heterodimerization with MCM1. AB2 and AB1 correspond respectively to the 5' and 3' halves of two adjacent similar sequences of 29 bp that appear to constitute tandem operators. Indeed, mutations increasing the similarity of the other halves with AB1 and AB2 cause hyperrepression. To mediate repression, the operator must be located close to the transcription initiation region. It remains functional if the TATA box is moved downstream of it but becomes inoperative in repression when displaced to a far-upstream position where it mediates an arginine and ARGR-dependent induction of gene expression. The ability of the ARG3 operator to act either as an operator or as an upstream activator sequence, depending on its location, and the functional organization of the anabolic and catabolic arginine genes suggest a simple model for arginine regulation in which an activator complex can turn into a repressor when able to interfere sterically with the process of transcription initiation.

scholarly journals ATP-Dependent Inactivation and Sequestration of Ornithine Decarboxylase by the 26S Proteasome Are Prerequisites for Degradation

1999 ◽  
Vol 19 (10) ◽  
pp. 7216-7227 ◽  
Author(s):  
Yasuko Murakami ◽  
Senya Matsufuji ◽  
Shin-Ichi Hayashi ◽  
Nobuyuki Tanahashi ◽  
Keiji Tanaka
Keyword(s):  
Proteolytic Activity ◽  
Ornithine Decarboxylase ◽  
Molecular Basis ◽  
Energy Requirement ◽  
26S Proteasome ◽  
20S Proteasome ◽  
Catalytic Core ◽  
Dependent Inactivation ◽  
Regulatory Complex

ABSTRACT The 26S proteasome is a eukaryotic ATP-dependent protease, but the molecular basis of its energy requirement is largely unknown. Ornithine decarboxylase (ODC) is the only known enzyme to be degraded by the 26S proteasome without ubiquitinylation. We report here that the 26S proteasome is responsible for the irreversible inactivation coupled to sequestration of ODC, a process requiring ATP and antizyme (AZ) but not proteolytic activity. Neither the 20S proteasome (catalytic core) nor PA700 (the regulatory complex) by itself contributed to this ODC inactivation. Analysis with a C-terminal mutant ODC revealed that the 26S proteasome recognizes the C-terminal degradation signal of ODC exposed by attachment of AZ, and subsequent ATP-dependent sequestration of ODC in the 26S proteasome causes irreversible inactivation, possibly unfolding, of ODC and dissociation of AZ. These processes may be linked to the translocation of ODC into the 20S proteasomal inner cavity, centralized within the 26S proteasome, for degradation.

Sign in / Sign up

Export Citation Format

Share Document