Proteasome & other proteasesProteasome & other proteases

Proteasome- core complex and regulatory cap

Other proteases- HslUV, ClpAP, ClpXP, Lon, FtsH

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The proteasomeThe proteasome the proteasome is a cylindrical proteasome consisting of four stacked, seven-membered rings

the two outer rings are alpha subunits (inactive) the two inner rings are beta subunits; these are proteolytically-active

archaeal proteasome one type of alpha subunit one type of beta subunit

core particle (20S)

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core particle (20S)

regulatory particle (19S)

26S

eukaryotic proteasome seven types of alpha subunits seven types of beta subunits regulatory cap (at least 17 subunits)

the evolution of the proteasome’s subunit complexity therefore parallels that of archaeal/eukaryal prefoldin/chaperonin

archaeal prefoldin, 2 subunit types; archaeal chaperonin, 1-3 types eukaryotic prefoldin, 6 subunit types; eukaryotic CCT, 8 types co-evolution with substrates?

ProteasomeProteasomecomponentscomponents

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The The proteasome proteasome regulatory regulatory

particleparticle

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Proteasome structureProteasome structure 19S cap (regulatory complex) is only present in eukaryotic proteasome and its crystal structure is unknown

the circled ‘N’ in Fig. (c) and (e) represent the N-termini of archaeal and yeast proteasome

archaea has only alpha and beta subunits whereas eukarya has different homologous alpha and beta subunits

Proteasomes from eukaryotes and archaea, showing the cap complex (magenta), core complex (blue, where alpha and beta subunits are shown), slice surface (green), active sites (white circles) and N-termini (circled ‘N’s). In (c) and (f), cyan indicates the residues visualized that are closest to the N-termini (threonine 13 and serine 11 respectively). (a) Electron micrograph of proteasome holoenzyme from a representative eukaryote (Xenopus laevis). (b) Medial cut-away view of the Thermoplasma acidophilum proteasome core. The lumen is divided into three chambers, and the central chamber contains the peptidase active sites (red). (c) Ribbon diagram of two Thermoplasma acidophilum subunits, showing the structure of the pore. (d) Cut-away view of the Saccharomyces cerevisiae proteasome core. (e) Ribbon diagram of two S. cerevisiae subunits (left: Pre9/Y13; right: Pre10/Prs1). The N-termini of these subunits are shown to occlude the channel. Adapted from Dan Finley, Encylopedia of Life Sciences.

active site

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NN

N

N

Eukaryotic proteasome catalytic siteEukaryotic proteasome catalytic site

active sites (shown with white circles) are on three separate beta subunits; threonine residues are critical during catalysis

the proteasome contains three separate proteolytic activities: - trypsin-like (arg, lys) - chymotrypsin-like (tyr, phe) - post-glutamyl (glu)

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controversial: the distance between the active site thr residues is 28A, which may determine the length of the proteolytic fragments, i.e., ~ 8 amino acids

11S proteasome regulator11S proteasome regulator proteasome 11S regulator also consist of heptameric rings and bind the 20S core much as the 19S regulatory cap does

also called PA26, PA28 and REG

binding of the 11S particle stimulates proteasomal activity

may facilitate product release by opening proteasome ‘gate’

reduction in processivity expected for an open conformation of the exit gate may explain the role of 11S regulators in the production of ligands for MHC class I molecules

11S carboxy-terminal tails provide binding affinity by inserting into pockets on the 20S proteasome, and 11S activation loops induce conformational changes in alpha-subunits that open the gate separating the proteasome interior from the intracellular environment

Proteasomewithout 11S

regulator

Proteasomewith 11Sregulator

Proteasome co-crystallizedwith 11S regulator particle

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smallerpeptides

largerpeptides

Bacterial proteasome-like proteases?Bacterial proteasome-like proteases? MOST Bacteria do not possess proteins that are closely related to the proteasome, but:

HslV is is structurally-related to proteasome HslU is the regulatory particle HslVU is responsible for the degradation of the cell division inhibitor SulA;its repertoire of substrates likely includes other cellular proteins

em picture inside view ofHslV protease(active site)

structures of 2 subunits;superimposable to the betasubunits of the archaealproteasome

HslV(2 rings)

HslU

HslUV

Lon and FtsH have combined regulatory and protease domains into one single polypeptide that assembles into a ring structure

shown to have chaperone-like activity, can disassemble aggregates, and can mediate protein degradation

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Bacterial proteasomes - comparisonBacterial proteasomes - comparisonFigure 1 Comparison of different classes of ATP-dependent pro- teases, shown as a side-on cross-section. (A) The eukaryotic 26S proteasome, composed of a 20S core particle (blue a and b subunits) flanked by 19S regulatory subunits (magenta and orange). The 19S subunits bind to the substrate through the covalent ubiquitin modification (yellow) and unfold it by pulling on the unstructured initiation site (bright green). Ubiquitin is removed to be recycled during the degradation process. (B) The bacterial protease ClpXP, composed of rings of the protease ClpP (dark green) and the ATPase motor ClpX (red). ClpX binds to the degradation signal, in this case the ssrA peptide sequence (green), which also serves as the site for the initiation of degradation. (C) The actinobacterial proteasome, consisting of a 20S core particle similar to that of the 26S proteasome, and a single ring of the ATPase Mpa (purple). Mpa binds to the substrate through the covalent Pup modification (light green). Pup has an N-terminal unstructured region, which serves as the site for the initiation of degradation, leading to complete degradation of Pup. (D) Archaeal proteasome is most closely related to the eukaryotic proteasome, with core alpha and beta subunits and Rpt-like AAA ATPase subunits (termed PAN for Proteasome Activating Nucleotidase) involved in protein unfolding.

Kraut and Matouschek EMBO J. 2009

eukaryotic bacterial

archaeal

Dactino-bacteria

only

allbacteria

*

*

ClpAP, ClpXP proteasesClpAP, ClpXP proteases ClpAP and ClpXP are ATP-dependent proteases

ClpA, ClpX are chaperones ClpP is the protease substrates: soluble, abnormal proteinsClpAP and ClpXP can also degrade any protein tagged with SsrA, an 11-residue peptide that is added to arrested chains in bacteria

ClpA and ClpX (in the absence of ClpP) can also disassemble protein complexes(similar to how Hsp104 from yeast can disentangle protein aggregates)

ClpAP, ClpXP are ‘active’ proteasesClpP by itself not active as protease

symmetry mismatch ClpA and ClpX have six subunits per ring; ClpP is a homo-heptameric ring symmetry mismatch may have implications for activity (but, other proteases don’t have this symmetry mismatch, so relevance is not clear)

- ClpP (2 rings) hasbeen crystallized

- ClpA, ClpX attachAs single rings onOpposite sides of ClpP

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ATP-dependent protease mechanismsATP-dependent protease mechanisms unfolding, then degradation is a common mechanism to ATP-dependent proteases?

work with ClpAP, ClpXP suggest that this is the case

PAN (Proteasome Activating Nucleotidase) associated with archaeal proteasome; stimulates its activity AAA ATPase (as with base of 19S proteasome cap); hexameric ring work with PAN also suggests unfolding then degradation mechanism

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the ATPase subunits of the proteasome regulatory particle

shown to have chaperone activity Braun et al. (1999) Nat. Cell Biol. 1, 221-226.

likely also involved in unfolding substrates just before translocation into the core particle

next lecture: evidence for ClpA unfolding

CompartmentalizationCompartmentalization compartmentalization, with respect to protein folding and degradation, refers to the encapsulation of substrates within a cavity, or a shielded environment

chaperones chaperonins possess a cavity that is capped by a cofactor (in the case of GroEL/GroES) or with protrusions (in the case of Group II chaperonins CCT and thermosome) AAA ATPases are also ring-shaped structures that possess a cavity prefoldin may partially envelopes substrates this encapsulation provides shielding of substrate hydrophobic residues in the case of chaperonins it provides ‘infinite dilution’ for substrates

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proteases most oligomeric proteases have a cavity that is shielded fromthe bulk cytosol

- e.g., proteasome, HslUV, ClpAP/XP, Tricorn protease, etc. shielding the active site is necessary to preventunregulated proteolysis encapsulation may assist processivity of protease

folding newly-made, non-native proteins are shielded from the bulk cytosol by chaperones