CHIP: A Co-chaperone for Degradation by the Proteasome - The Networking of Chaperones by Co-chaperones - page 218

proteasome. The 19S regulatory subunit is a protein complex that functions to acti-
vate the 20S particle for protein degradation (Unverdorben et al. 2014 ). The size of
the central pore of the proteasome ranges between 50-13 ￅ and entry is restricted
by the alpha subunit gate. The 19S proteasome is required to open the gate in the
20S proteasome, thereby permitting entry of substrate proteins into the catalytic
sites within the proteasome core (da Fonseca and Morris 2008 ; Sledz et al. 2013 ;
Unverdorben et al. 2014 ). Polyubiquitinated proteins targeted for degradation
need to be deubiquitinated and delivered to the proteolytic active site of the pro-
teasome that is buried within the 20S core particle. The substrate is thought to
be partially unfolded during translocation into the cavity. Within the proteasome
core, substrate peptide bonds are hydrolysed by nucleophilic attack dependent on
catalytic threonine residues that extend into the cavity from the beta subunits. The
resultant peptides released from the proteasome range between 4 and 25 residues
(Babbitt et al. 2005 ; Ortega et al. 2005 ; Goldberg et al. 1997 ).
Degradation of proteins by the proteasome is preceded by the conjugation of
ubiquitin to the substrate via a series of sequential enzyme catalysed reactions
(Fig. 11.2 ) (Hershko and Ciechanover 1998 ). Ubiquitin is a small, abundant pro-
tein (~ 8 kDa) found in all eukaryotic cells which, when added to proteins in a
polyubiquitin chain, functions as the degradation signal (Smith 1988 ; Johnson
et al. 1992 ; Johnson et al. 1995 ). Ubiquitin is initially activated by conjugation to
an ubiquitin activating enzyme (E1) in an ATP dependent manner via a thioester
linkage (Lee and Schindelin 2008 ). Ubiquitin is subsequently transferred via an
ubiquitin conjugating enzyme (E2) intermediate to the substrate protein targeted
for degradation (Olsen and Lima 2013 ). This reaction is catalysed by an ubiq-
uitin ligase enzyme (E3) and results in the formation of a peptide bond between
a glycine residue in the C-terminus of ubiquitin and lysine residues within the
substrate protein (Scheffner et al. 1995 ; Wilkinson 2000 ). There are a range of
different E2 and E3 isoforms that may combine for different substrate proteins,
suggesting a diverse and discriminatory recognition system for ubiquitin con-
jugation (Spratt et al. 2012 ). This process may be repeated a number of times,
often involving the conjugation of subsequent ubiquitin molecules to lysines
within ubiquitin itself, leading to the formation of covalently linked polyubiq-
uitin chains. A fourth enzyme may also be involved in this cascade. Known as
E4, this protein acts as an ubiquitin chain elongation enzyme to catalyse the as-
sembly of polyubiquitin chains on protein substrates (Koegl et al. 1999 ). While
monoubiquitination may induce changes in activity or subcellular localisation
of proteins, the conjugation of a polyubiquitin chain to a substrate protein is
required for degradation by the proteasome (Johnson et al. 1992 ). Ubiquitin con-
tains 7 lysines residues (K6, K11, K27, K29, K33, K48, and K63), in addition
to its N-terminus, which act as potential sites of conjugation. The lysine residue
involved in the bond can also impact on the outcome of ubiquitination (Hershko
and Ciechanover 1998 ). K48 linked ubiquitin chains, where the covalent linkage
of the ubiquitin chain is via the K48 residue of ubiquitin, is the canonical signal
for proteasomal degradation (Jacobson et al. 2009 ). K48 linkage is regulated