Kehon omasta
kolesterolin muodostustien feedback mekanismista, josa tuoteet,
sterolit vaiuttavat HMGCoA reduktaasin joutumista
ERAD-hajoitukseen. Tämä säätely toimii luonnon eläimillä.
Article
Gp78, a
Membrane-Anchored Ubiquitin Ligase, Associates with Insig-1 and
Couples Sterol-Regulated Ubiquitination to Degradation of HMG CoA
Reductase
Summary
Sterol-regulated
ubiquitination
is an obligatory step in ER-associated degradation (ERAD) of
HMG
CoA reductase, a rate-limiting enzyme in
cholesterol
synthesis. Accelerated degradation of
reductase,
one of several strategies animal cells use to limit production of
cholesterol,
requires sterol-induced binding of the enzyme to ER
membrane
proteins called Insigs.
Once formed, the reductase-Insig complex is recognized by a
putative membrane-associated ubiquitin ligase (E3) that mediates the
reductase ubiquitination reaction. Here, we show that gp78, a
membrane bound E3, binds to Insig-1 and is required for
sterol-regulated ubiquitination of reductase. In addition, gp78
couples regulated ubiquitination to degradation of reductase by
binding to VCP, an
ATPase
that plays a key role in recognition and degradation of
ERAD
substrates. The current results identify gp78 as the E3 that
initiates sterol-accelerated degradation of reductase, and Insig-1 as
a bridge between gp78/VCP and the reductase substrate.
Introduction
An effective strategy in lowering plasma
low-density
lipoprotein cholesterol, a major risk factor in development of
coronary artery disease, is to block reduction of
3-hydroxy-3-methylglutaryl
coenzyme
A (HMG CoA) to mevalonate (
Illingworth
and Tobert, 2001). This rate-limiting reaction in
cholesterol
synthesis is catalyzed by the enzyme
HMG
CoA reductase,
the inhibition of which triggers regulatory responses that lead to a
decrease in the concentration of
blood
cholesterol (
Goldstein
and Brown, 1990). Aggressive cholesterol-lowering therapies using
competitive reductase inhibitors called
statins
reduce the incidence of heart attacks and prolong lives of patients
with preexisting coronary artery disease (
Heart
Protection Study Collaborative Group, 2002,
Scandinavian
Simvastatin Study Group, 1994). However, the action of statins
blocks production of mevalonate-derived regulatory products that
normally govern the levels and activity of reductase through a
multivalent feedback mechanism (
Brown
and Goldstein, 1980). In livers of animals and in cultured cells,
the absence of these regulatory molecules leads to a major increase
in the amount of active reductase protein, invoking the need for
higher doses of statins to maintain their cholesterol-lowering
effects (
Kita
et al., 1980,
Brown
et al., 1978,
Lee
et al., 2005). Thus, understanding how disruption of mevalonate
metabolism leads to a compensatory increase in reductase could prove
useful toward developing novel agents that block the increase and
could thereby improve the effectiveness of statins.
HMG
CoA reductase is anchored to membranes of the endoplasmic
reticulum (ER) through its hydrophobic NH
2-terminal
domain, which consists of eight membrane-spanning regions separated
by short loops (
Roitelman
et al., 1992). The membrane domain of reductase precedes a large
COOH-terminal domain that projects into the cytosol and exerts all of
the
catalytic
activity of the enzyme (
Gil
et al., 1985,
Liscum
et al., 1985).
Part of the underlying mechanism for feedback
inhibition of reductase involves its sterol-accelerated degradation,
an action achieved through sterol-induced binding of reductase to one
of a pair of ER
membrane
proteins called Insig-1 and
Insig-2
(
Sever
et al., 2003a). Formation of the reductase-Insig complex is
mediated by the reductase membrane domain and results in the
ubiquitination
of the enzyme, an obligate reaction for recognition and degradation
of reductase by 26S
proteasomes.
The mechanism for sterol-regulated ubiquitination of reductase was
examined in a permeabilized cell system (
Song
and DeBose-Boyd, 2004). After permeabilization of cells, nearly
all cytosolic proteins were released into the supernatant upon
centrifugation, while membrane proteins such as reductase remained
associated with the pellet fraction. This pellet fraction fully
supported Insig-dependent ubiquitination of reductase stimulated by
the in vitro addition of
sterols
and rat liver cytosol. Expanding on this observation, we recently
reconstituted ubiquitination of reductase by simply incubating
sterol-depleted membranes with sterols and purified
ubiquitin-activating
enzyme (E1) in vitro (
Song
et al., 2005). These results indicate that reductase
ubiquitination must be mediated by membrane-associated
ubiquitin-conjugating (E2) and -ligating (E3) enzymes.
In the current study, we utilized
affinity
purification, coupled with
tandem
mass spectrometry, to identify membrane proteins that associate
with the reductase-Insig-1 complex. Through this approach, we
identified gp78, a membrane bound E3, as an Insig-1-associated
protein. Insig-1 binds to the membrane domain of gp78 in the absence
or presence of sterols, and upon the addition of sterols, reductase
is recruited to the complex.
Knockdown of gp78 by
RNA
interference (RNAi) prevents sterol-dependent ubiquitination and
degradation of endogenous reductase.
Valosin-containing
protein (VCP), an
ATPase
that participates in postubiquitination steps of
ERAD,
indirectly associates with Insig-1 by directly binding gp78 and is
required for reductase degradation.
The current results identify gp78
as the
ubiquitin
ligase that initiates sterol-dependent degradation of reductase,
and Insig-1 as a bridge between gp78/VCP and the reductase substrate.
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