Atomic Force Microscopy Reveals Two Conformations of the 20 S Proteasome from Fission Yeast

Osmulski, Paweł A.; Gaczyńska, Maria

doi:10.1074/jbc.c901035199

Cited by 59 publications

(66 citation statements)

References 19 publications

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“…If these sites are located inside the particle, then the spontaneous opening of the channel (10,29) presumably enables the peptides to bind to these sites in our experiments, and thereby prevents the return of the channel to the closed conformation. The observation that potassium ions, which decrease spontaneous opening of the channel (10), delay (but do not prevent) the onset of the activation by the hydrophobic peptides (Fig.…”

Section: Discussionmentioning

confidence: 93%

“…However, we cannot exclude the possibility that additional effects are required for maintaining the channel in the open conformation. Osmulski and Gaczynska (29) reported that blocking Suc-LLVY-amc cleavage at the chymotrypsin-like site by the inhibitor abolishes the effect of these peptides on the channel in the ␣-ring and suggested that a catalytic event is required to promote channel opening. Although the present results demonstrate that hydrophobic peptides need to bind to the non-catalytic sites to stimulate peptide hydrolysis, we cannot exclude the possibility that peptides may also have to bind to any of the catalytic sites to trigger channel opening, because in all of our experiments peptide substrates had to be present for us to assay the activities of the catalytic sites.…”

Section: Discussionmentioning

confidence: 99%

See 1 more Smart Citation

Binding of Hydrophobic Peptides to Several Non-catalytic Sites Promotes Peptide Hydrolysis by All Active Sites of 20 S Proteasomes

Kisselev

Kaganovich

Goldberg

2002

Journal of Biological Chemistry

171

156

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The eukaryotic 20 S proteasome contains the following 6 active sites: 2 chymotrypsin-like, 2 trypsin-like, and 2 caspase-like. We previously showed that hydrophobic peptide substrates of the chymotrypsin-like sites allosterically stimulate peptide hydrolysis by the caspase-like sites and their own cleavage. More thorough analysis revealed that these peptides also stimulate peptide hydrolysis by the trypsin-like site. This general activation by hydrophobic peptides occurred even if the chymotrypsin-like sites were occupied by a covalent inhibitor and was highly cooperative, with an average Hill coefficient of 7. Therefore, this stimulation of peptide hydrolysis at all active sites occurs upon binding of hydrophobic peptides to several non-catalytic sites. The stimulation by hydrophobic peptides was not observed in the yeast ⌬N␣3 mutant 20 S proteasomes, in 20 S-PA26 complexes, or SDS-activated proteasomes and was significantly lower in 26 S proteasomes, all of which appear to have the gated channel in the ␣-rings in an open conformation and hydrolyze peptides at much faster rates than 20 S proteasomes. Also the hydrophobic peptides altered K m , V max of active sites in a similar fashion as PA26 and the ⌬N␣3 mutation. The activation by hydrophobic peptides was decreased in K ؉ -containing buffer, which favors the closed state of the channels. Therefore, hydrophobic peptides stimulate peptide hydrolysis most likely by promoting the opening of the channels in the ␣-rings. During protein breakdown, this peptide-induced channel opening may function to facilitate the release of products from the proteasome.The majority of proteins in mammalian cells is degraded by 26 S proteasomes (1). This 2.4-MDa proteolytic enzyme consists of the 20 S proteasome and one or two 19 S regulatory complexes (2, 3). The 20 S proteasome, which also exists in mammalian cells as a free 700-kDa particle, is a hollow cylinder composed of two outer ␣-and two inner ␤-rings (3). Each ring contains seven different subunits, and each ␤-ring contains three proteolytic sites, which differ in their substrate specificities. The "chymotrypsin-like" (␤5) site cleaves peptide bonds preferentially after hydrophobic residues; the "trypsinlike" (␤2) site cuts mainly after basic residues, and the third site (␤1) cuts preferentially after acidic residues (4 -7). This latter site has been traditionally termed "post-glutamyl peptide hydrolase" site. However, because it hydrolyzes standard fluorogenic substrates of caspases and cleaves after aspartate residues better than after glutamates, we prefer the more accurate and simpler term "caspase-like" site (8).When isolated under gentle conditions (e.g. in the presence of glycerol), 20 S proteasomes are in a latent state (9) in which they are unable to degrade proteins and hydrolyze model peptide substrates only at low rates. This low peptidase activity is suppressed further by physiological concentrations of potassium ions (10), but the activity of such preparations increases dramatically upon a variety of treatme...

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Section: Discussionmentioning

confidence: 93%

Section: Discussionmentioning

confidence: 99%

Binding of Hydrophobic Peptides to Several Non-catalytic Sites Promotes Peptide Hydrolysis by All Active Sites of 20 S Proteasomes

Kisselev

Kaganovich

Goldberg

2002

Journal of Biological Chemistry

171

156

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“…The appearance of the proteasomes in the crystals did not change with time during repeated scanning. Based on AFM recordings, yeast 20 S proteasomes have been reported to switch between 2 distinct conformations (47). In contrast, the AFM images of proteasomes in two-dimensional arrays did not reveal the existence of different conformational states.…”

Section: Binding Of His-tagged Proteins To Metal-chelating Lipidmentioning

confidence: 84%

Specific Orientation and Two-dimensional Crystallization of the Proteasome at Metal-chelating Lipid Interfaces

Theß¹,

Hutschenreiter²,

Hofmann³

et al. 2002

Journal of Biological Chemistry

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The potential of a protein-engineered His tag to immobilize macromolecules in a predictable orientation at metal-chelating lipid interfaces was investigated using recombinant 20 S proteasomes His-tagged in various positions. Electron micrographs demonstrated that the orientation of proteasomes bound to chelating lipid films could be controlled via the location of their His tags: proteasomes His-tagged at their sides displayed exclusively side-on views, while proteasomes His-tagged at their ends displayed exclusively end-on views. The activity of proteasomes immobilized at chelating lipid interfaces was well preserved. In solution, His-tagged proteasomes hydrolyzed casein at rates comparable with wild-type proteasomes, unless the His tags were located in the vicinity of the N termini of ␣-subunits. The N termini of ␣-subunits might partly occlude the entrance channel in ␣-rings through which substrates enter the proteasome for subsequent degradation. A combination of electron micrographs and atomic force microscope topographs revealed a propensity of vertically oriented proteasomes to crystallize in two dimensions on fluid lipid films. The oriented immobilization of His-tagged proteins at biocompatible lipid interfaces will assist structural studies as well as the investigation of biomolecular interaction via a wide variety of surface-sensitive techniques including single-molecule analysis.

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“…As outlined earlier, substrates and certain competitive inhibitors binding to orthosteric sites may double as allosteric ligands, promoting opening of the gate (99,100,103). Gate opening can be detected by AFM imaging of the 20S core and confirmed by functional studies of the latent or allosterically open core decorated with 11S activator (103).…”

Section: Allosteric Route: From the Catalytic Centers To The Grooves mentioning

confidence: 99%

“…There is one more intriguing aspect of the conformational dynamics related to effects of rapamycin. Treatment of the 20S core with the saturating concentration of a model peptide substrate induces maximum 75% of the open particles (99,100,103). A saturating concentration of rapamycin pushes the equilibrium to about 60% of the open proteasomes.…”

Section: Allosteric Route: From the Grooves On A Face To The Gatementioning

confidence: 99%

Harnessing Proteasome Dynamics and Allostery in Drug Design

Gaczyńska

Osmulski

2014

Antioxidants & Redox Signaling

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Significance: The proteasome is the essential protease that is responsible for regulated cleavage of the bulk of intracellular proteins. Its central role in cellular physiology has been exploited in therapies against aggressive cancers where proteasome-specific competitive inhibitors that block proteasome active centers are very effectively used. However, drugs regulating this essential protease are likely to have broader clinical usefulness.

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Atomic Force Microscopy Reveals Two Conformations of the 20 S Proteasome from Fission Yeast

Cited by 59 publications

References 19 publications

Binding of Hydrophobic Peptides to Several Non-catalytic Sites Promotes Peptide Hydrolysis by All Active Sites of 20 S Proteasomes

Binding of Hydrophobic Peptides to Several Non-catalytic Sites Promotes Peptide Hydrolysis by All Active Sites of 20 S Proteasomes

Specific Orientation and Two-dimensional Crystallization of the Proteasome at Metal-chelating Lipid Interfaces

Harnessing Proteasome Dynamics and Allostery in Drug Design

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