Browse
Submit Data
Databases
API
Help

Dataset Information

0 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

The proteasome under the microscope: the regulatory particle in focus.

ABSTRACT: Since first imaged by electron microscopy, much effort has been placed into determining the structure and mechanism of the 26S proteasome. While the proteolytic core is understood in atomic detail, how substrates are engaged and transported to this core remains elusive. Substrate delivery is accomplished by a 19-subunit regulatory particle that binds to ubiquitinated substrates, detaches ubiquitin tags, unfolds the substrate, and translocates it into the peptidase in an ATP-dependent fashion. Recently, several labs have determined subnanometer cryoEM structures of the 26S proteasome, shedding light on the architecture of the regulatory complex. We discuss the biological insights into substrate processing provided by these structures, and the technical hurdles ahead to achieve an atomic resolution structure of the 26 proteasome.

SUBMITTER: Lander GC

PROVIDER: S-EPMC3676703 | biostudies-literature | 2013 Apr

REPOSITORIES: biostudies-literature

ACCESS DATA

Json Xml

Publications

The proteasome under the microscope: the regulatory particle in focus.

Lander Gabriel C GC Martin Andreas A Nogales Eva E

Current opinion in structural biology 20130313 2

Since first imaged by electron microscopy, much effort has been placed into determining the structure and mechanism of the 26S proteasome. While the proteolytic core is understood in atomic detail, how substrates are engaged and transported to this core remains elusive. Substrate delivery is accomplished by a 19-subunit regulatory particle that binds to ubiquitinated substrates, detaches ubiquitin tags, unfolds the substrate, and translocates it into the peptidase in an ATP-dependent fashion. Re ...[more]

PMID: 23498601

Similar Datasets

Complete subunit architecture of the proteasome regulatory particle.

Project description:The proteasome is the major ATP-dependent protease in eukaryotic cells, but limited structural information restricts a mechanistic understanding of its activities. The proteasome regulatory particle, consisting of the lid and base subcomplexes, recognizes and processes polyubiquitinated substrates. Here we used electron microscopy and a new heterologous expression system for the lid to delineate the complete subunit architecture of the regulatory particle from yeast. Our studies reveal the spatial arrangement of ubiquitin receptors, deubiquitinating enzymes and the protein unfolding machinery at subnanometre resolution, outlining the substrate's path to degradation. Unexpectedly, the ATPase subunits within the base unfoldase are arranged in a spiral staircase, providing insight into potential mechanisms for substrate translocation through the central pore. Large conformational rearrangements of the lid upon holoenzyme formation suggest allosteric regulation of deubiquitination. We provide a structural basis for the ability of the proteasome to degrade a diverse set of substrates and thus regulate vital cellular processes.

| S-EPMC3285539 | biostudies-literature

Chaperone-mediated pathway of proteasome regulatory particle assembly.

Project description:The proteasome is a protease that controls diverse processes in eukaryotic cells. Its regulatory particle (RP) initiates the degradation of ubiquitin-protein conjugates by unfolding the substrate and translocating it into the proteasome core particle (CP) to be degraded. The RP has 19 subunits, and their pathway of assembly is not understood. Here we show that in the yeast Saccharomyces cerevisiae three proteins are found associated with RP but not with the RP-CP holoenzyme: Nas6, Rpn14 and Hsm3. Mutations in the corresponding genes confer proteasome loss-of-function phenotypes, despite their virtual absence from the holoenzyme. These effects result from deficient RP assembly. Thus, Nas6, Rpn14 and Hsm3 are RP chaperones. The RP contains six ATPases-the Rpt proteins-and each RP chaperone binds to the carboxy-terminal domain of a specific Rpt. We show in an accompanying study that RP assembly is templated through the Rpt C termini, apparently by their insertion into binding pockets in the CP. Thus, RP chaperones may regulate proteasome assembly by directly restricting the accessibility of Rpt C termini to the CP. In addition, competition between the RP chaperones and the CP for Rpt engagement may explain the release of RP chaperones as proteasomes mature.

| S-EPMC2727592 | biostudies-literature

The regulatory particle of the Saccharomyces cerevisiae proteasome.

Project description:The proteasome is a multisubunit protease responsible for degrading proteins conjugated to ubiquitin. The 670-kDa core particle of the proteasome contains the proteolytic active sites, which face an interior chamber within the particle and are thus protected from the cytoplasm. The entry of substrates into this chamber is thought to be governed by the regulatory particle of the proteasome, which covers the presumed channels leading into the interior of the core particle. We have resolved native yeast proteasomes into two electrophoretic variants and have shown that these represent core particles capped with one or two regulatory particles. To determine the subunit composition of the regulatory particle, yeast proteasomes were purified and analyzed by gradient sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Resolution of the individual polypeptides revealed 17 distinct proteins, whose identities were determined by amino acid sequence analysis. Six of the subunits have sequence features of ATPases (Rpt1 to Rpt6). Affinity chromatography was used to purify regulatory particles from various strains, each of which expressed one of the ATPases tagged with hexahistidine. In all cases, multiple untagged ATPases copurified, indicating that the ATPases assembled together into a heteromeric complex. Of the remaining 11 subunits that we have identified (Rpn1 to Rpn3 and Rpn5 to Rpn12), 8 are encoded by previously described genes and 3 are encoded by genes not previously characterized for yeasts. One of the previously unidentified subunits exhibits limited sequence similarity with deubiquitinating enzymes. Overall, regulatory particles from yeasts and mammals are remarkably similar, suggesting that the specific mechanistic features of the proteasome have been closely conserved over the course of evolution.

| S-EPMC108897 | biostudies-literature

Structural defects in the regulatory particle-core particle interface of the proteasome induce a novel proteasome stress response.

Project description:Proteasomes consist of a 19-subunit regulatory particle (RP) and 28-subunit core particle (CP), an ?(7)?(7)?(7)?(7) structure. The RP recognizes substrates and translocates them into the CP for degradation. At the RP-CP interface, a heterohexameric Rpt ring joins to a heteroheptameric CP ? ring. Rpt C termini insert individually into the ? ring pockets to form a salt bridge with a pocket lysine residue. We report that substitutions of ? pocket lysine residues produce an unexpected block to CP assembly, arising from a late stage defect in ? ring assembly. Substitutions ?5(K66A) and ?6(K62A) resulted in abundant incorporation of immature CP ? subunits, associated with a complete ? ring, into proteasome holoenzymes. Incorporation of immature CP into the proteasome depended on a proteasome-associated protein, Ecm29. Using ump1 mutants, we identified Ecm29 as a potent negative regulator of RP assembly and confirmed our previous findings that proper RP assembly requires the CP. Ecm29 was enriched on proteasomes of pocket lysine mutants, as well as those of rpt4-?1 and rpt6-?1 mutants, in which the C-terminal residue, thought to contact the pocket lysine, is deleted. In both rpt6-?1 and ?6(K62A) proteasomes, Ecm29 suppressed opening of the CP substrate translocation channel, which is gated through interactions between Rpt C termini and the ? pockets. The ubiquitin ligase Hul5 was recruited to these proteasomes together with Ecm29. Proteasome remodeling through the addition of Ecm29 and Hul5 suggests a new layer of the proteasome stress response and may be a common response to structurally aberrant proteasomes or deficient proteasome function.

| S-EPMC3196138 | biostudies-literature

Structural insights into proteasome activation by the 19S regulatory particle.

Project description:Since its discovery in the late 1970s, the ubiquitin-proteasome system (UPS) has become recognized as the major pathway for regulated cellular proteolysis. Processes such as cell cycle control, pathogen resistance, and protein quality control rely on selective protein degradation at the proteasome for homeostatic function. Perhaps as a consequence of the importance of this pathway, and the genesis of severe diseases upon its dysregulation, protein degradation by the UPS is highly controlled from the level of substrate recognition to proteolysis. Technological advances over the past decade have created an explosion of structural and mechanistic information that has underscored the complexity of the proteasome and its upstream regulatory factors. Significant insights have come from the study of the 19S proteasome regulatory particle (RP) responsible for recognition and processing of ubiquitinated substrates destined for proteolysis. Established as a highly dynamic proteasome activator, the RP has a large number of both permanent and transient components with specialized functional roles that are critical for proteasome function. In this review, we highlight recent mechanistic developments in the study of proteasome activation by the RP and how they provide context to our current understanding of the UPS.

| S-EPMC3864829 | biostudies-literature

Conformational Landscape of the p28-Bound Human Proteasome Regulatory Particle.

Project description:The proteasome holoenzyme is activated by its regulatory particle (RP) consisting of two subcomplexes, the lid and the base. A key event in base assembly is the formation of a heterohexameric ring of AAA-ATPases, which is guided by at least four RP assembly chaperones in mammals: PAAF1, p28/gankyrin, p27/PSMD9, and S5b. Using cryogenic electron microscopy, we analyzed the non-AAA structure of the p28-bound human RP at 4.5 Å resolution and determined seven distinct conformations of the Rpn1-p28-AAA subcomplex within the p28-bound RP at subnanometer resolutions. Remarkably, the p28-bound AAA ring does not form a channel in the free RP and spontaneously samples multiple "open" and "closed" topologies at the Rpt2-Rpt6 and Rpt3-Rpt4 interfaces. Our analysis suggests that p28 assists the proteolytic core particle to select a specific conformation of the ATPase ring for RP engagement and is released in a shoehorn-like fashion in the last step of the chaperone-mediated proteasome assembly.

| S-EPMC5580496 | biostudies-literature

Maturation of the proteasome core particle induces an affinity switch that controls regulatory particle association.

Project description:Proteasome assembly is a complex process, requiring 66 subunits distributed over several subcomplexes to associate in a coordinated fashion. Ten proteasome-specific chaperones have been identified that assist in this process. For two of these, the Pba1-Pba2 dimer, it is well established that they only bind immature core particles (CPs) in vivo. In contrast, the regulatory particle (RP) utilizes the same binding surface but only interacts with the mature CP in vivo. It is unclear how these binding events are regulated. Here, we show that Pba1-Pba2 binds tightly to the immature CP, preventing RP binding. Changes in the CP that occur on maturation significantly reduce its affinity for Pba1-Pba2, enabling the RP to displace the chaperone. Mathematical modelling indicates that this 'affinity switch' mechanism has likely evolved to improve assembly efficiency by preventing the accumulation of stable, non-productive intermediates. Our work thus provides mechanistic insights into a crucial step in proteasome biogenesis.

| S-EPMC4380239 | biostudies-literature

Multiple assembly chaperones govern biogenesis of the proteasome regulatory particle base.

Project description:The central protease of eukaryotes, the 26S proteasome, has a 20S proteolytic core particle (CP) and an attached 19S regulatory particle (RP). The RP is further subdivided into lid and base subcomplexes. Little is known about RP assembly. Here, we show that four conserved assembly factors govern biogenesis of the yeast RP base. Nas2 forms a complex with the Rpt4 and Rpt5 ATPases and enhances 26S proteasome formation in vivo and in vitro. Other RP subcomplexes contain Hsm3, which is related to mammalian proteasome subunit S5b. Hsm3 also contributes to base assembly. Larger Hsm3-containing complexes include two additional proteins, Nas6 and Rpn14, which function as assembly chaperones as well. Specific deletion combinations affecting these four factors cause severe perturbations to RP assembly. Our results demonstrate that proteasomal RP biogenesis requires multiple, functionally overlapping chaperones and suggest a model in which subunits form specific subcomplexes that then assemble into the base.

| S-EPMC2718848 | biostudies-literature

The central unit within the 19S regulatory particle of the proteasome.

Project description:The 26S proteasome is a multisubunit enzyme composed of a cylindrical catalytic core (20S) and a regulatory particle (19S) that together perform the essential degradation of cellular proteins tagged by ubiquitin. To date, however, substrate trajectory within the complex remains elusive. Here we describe a previously unknown functional unit within the 19S, comprising two subunits, Rpn1 and Rpn2. These toroids physically link the site of substrate recruitment with the site of proteolysis. Rpn2 interfaces with the 20S, whereas Rpn1 sits atop Rpn2, serving as a docking site for a substrate-recruitment factor. The 19S ATPases encircle the Rpn1-Rpn2 stack, covering the remainder of the 20S surface. Both Rpn1-Rpn2 and the ATPases are required for substrate translocation and gating of the proteolytic channel. Similar pairing of units is found in unfoldases and nuclear transporters, exposing common features of these protein nanomachines.

| S-EPMC2481239 | biostudies-literature

Structural insights into the regulatory particle of the proteasome from Methanocaldococcus jannaschii.

Project description:Eukaryotic proteasome consists of a core particle (CP), which degrades unfolded protein, and a regulatory particle (RP), which is responsible for recognition, ATP-dependent unfolding, and translocation of polyubiquitinated substrate protein. In the archaea Methanocaldococcus jannaschii, the RP is a homohexameric complex of proteasome-activating nucleotidase (PAN). Here, we report the crystal structures of essential elements of the archaeal proteasome: the CP, the ATPase domain of PAN, and a distal subcomplex that is likely the first to encounter substrate. The distal subcomplex contains a coiled-coil segment and an OB-fold domain, both of which appear to be conserved in the eukaryotic proteasome. The OB domains of PAN form a hexameric ring with a 13 A pore, which likely constitutes the outermost constriction of the substrate translocation channel. These studies reveal structural codes and architecture of the complete proteasome, identify potential substrate-binding sites, and uncover unexpected asymmetry in the RP of archaea and eukaryotes.

| S-EPMC3268689 | biostudies-literature

OmicsDI is part of the ELIXIR infrastructure

OmicsDI is an Elixir interoperability service. Learn more ›

Tweets

OmicsDI Databases

PRIDE
PeptideAtlas
MassIVE
JPOST Repository
Physiome Model Repository

EGA
EVA
ENA
LINCS
PAXDB
Cell Collective

MetaboLights
Metabolomics Workbench
MetabolomeExpress
GNPS
BioModels
FAIRDOMHub

ArrayExpress
dbGaP
ExpressionAtlas
GEO
NODE

Information

Databases
Help
API
Contact us
Code on GitHub
Terms of use
Submit Data