Kehon omasta
kolesterolin muodostustien feedback mekanismista, josa tuoteet,
sterolit vaiuttavat HMGCoA reduktaasin joutumista
ERAD-hajoitukseen. Tämä säätely toimii luonnon eläimillä.
Volume
19, Issue 6, 16 September 2005, Pages 829-840
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 NH2-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.
https://ars.els-cdn.com/content/image/1-s2.0-S1097276505015273-gr6.jpg
Inga kommentarer:
Skicka en kommentar