Biomedical Engineering Reference
In-Depth Information
E2
Ub
RING
Substrate
4
E3
3
1
2
Ub
E2
E1
Ub
Ub
E1
E2
HECT
Ub
Substrate
E3
Ub
Ub
Ub
Substrate proteolysis
3
ATP
ADP + Pi
E2
E1
HECT
4
E3
26S
proteasome
Ub
Substrate
Ub
FIGURE 4.1
Schematic diagram of the ubiquitination pathway. The ubiquitin-proteasome is
composed of four major enzyme types — E1, E2, E3, and the 26S proteasome — which
function in a hierarchical fashion: (1) Ubiquitin is activated in an adenosine triphosphate
(ATP)-dependent manner by a single ubiquitin-activating enzyme, E1. (2) The activated
ubiquitin moiety is transferred to a number of different E2s. (3) The activated ubiquitin is
attached to a Cys residue on homologous to the E6-AP carboxyl terminus (HECT) E3s and
is then transferred to substrate or to a substrate-bound polyubiquitin chain. For RING E3s,
ubiquitin is transferred directly from E2 to a substrate. Each E2 can interact with several E3s.
Each E3 may target several different substrates, and certain substrates may be targeted by
more than one E3. (4) The 26S proteasome recognizes polyubiquitin-modified substrates and
proteolyzes them into short peptides, releasing ubiquitin for reuse.
extracts in 1986 and, shortly after its cytoplasmic inhibitor, I
κ
B (inhibitor of NF-
κ
B)
[1,2,3]. This work suggested a model of NF-
κ
B activation that required its liberation
from the inhibitory effects of I
B and ubiquitin
became apparent from research into the physiological mechanism of this release
following agonist stimulation.
Using a simple detergent treatment of cell extracts, Baeuerle and Baltimore
provided the first proof of concept for this model, showing that while inducing the
dissociation from its inhibitor, NF-
κ
B. The connection between NF-
κ
B was activated [3]. Subsequent
in vivo
exper-
iments using cell lines suggested that stimulus-induced I
κ
κ
B phosphorylation trig-
gered release of associated NF-
κ
B and could therefore be the critical event for the
physiological activation of NF-
κ
B [4]. However, it was later demonstrated that I
κ
B
phosphorylation was in fact insufficient for NF-
B activation [5,6]. In 1993, several
groups (Siebenlist's, Greene's, Baldwin's, and Goodbourn's) [7,8,9,10] made the
seminal observation that NF-
κ
B degradation.
Furthermore, Baeuerle, Ben-Neriah, and colleagues showed that blockade of I
κ
B activation was accompanied by I
κ
κ
B
degradation prevented NF-
κ
B activation [11]. Elucidation of the mechanism of
I
B proteolysis followed. Maniatis, Goldberg, and colleagues first showed that
proteasome inhibitors inhibited NF-
κ
B degradation,
implicating the proteasome in the activation process [12]. Soon after, the groups
of Maniatis, Ben-Neriah, and Ciechanover demonstrated that signal-induced ubiq-
uitination was required for the elimination of I
κ
B activation by blocking I
κ
κ
B from the NF-
κ
B complex by the
proteasome [13,14].
