Proteasome

1
Proteasome other proteases
19-1
Proteasome - core complex and regulatory
cap Other proteases - HslUV, ClpAP, ClpXP, Lon,
FtsH
2
The proteasome
19-2

• 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)

• the evolution of the proteasomes 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?

3
Proteasomecomponents
19-3
4
The proteasome regulatory particle
19-4
5
Proteasome structure
19-5

• 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

active site
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 Ns). 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 a subunits, showing the
structure of the pore. (d) Cut-away view of the
Saccharomyces cerevisiae proteasome core. (e)
Ribbon diagram of two S. cerevisiae a 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.
6
Eukaryotic proteasome catalytic site
19-6

• 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)

• 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

7
11S proteasome regulator
19-7

• 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

Proteasome co-crystallized with 11S regulator
particle
Proteasome without 11S regulator
Proteasome with 11S regulator
8
Bacterial proteasome-like proteases?
19-8

• 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

HslUV
HslU
HslV (2 rings)
structures of 2 subunits superimposable to the
beta subunits of the archaeal proteasome
em picture
inside view of HslV protease (active site)

• 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

9
ClpAP, ClpXP proteases
19-9

• ClpAP and ClpXP are ATP-dependent proteases
• ClpA, ClpX are chaperones
• ClpP is the protease
• substrates soluble, abnormal proteins
• ClpAP 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 proteases
- ClpP (2 rings) has been crystallized
- ClpA, ClpX attach As single rings on Opposite
sides of ClpP

• 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 dont have this
  symmetry mismatch, so relevance is not clear)

10
ATP-dependent protease mechanisms
19-10

• unfolding, then degradation is a common
  mechanism to ATP-dependent proteases?
• work with ClpAP, ClpXP suggest that this is the
  case (presentation and assigned paper)

• 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

• 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

11
Compartmentalization
19-11

• 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

• 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