Insig-dependent Ubiquitination and Degradation of Mammalian 3-Hydroxy-3-methylglutaryl-CoA Reductase Stimulated by Sterols and Geranylgeraniol

Abstract

The endoplasmic reticulum enzyme 3-hydroxy-3-methylglutaryl-CoA reductase produces mevalonate, which is converted to sterols and to other products, including geranylgeraniol groups attached to proteins. The enzyme is known to be ubiquitinated and rapidly degraded when sterols and nonsterol end products of mevalonate metabolism accumulate in cells. Here, we use RNA interference to show that sterol-accelerated ubiquitination of reductase requires Insig-1 and Insig-2, membrane-bound proteins of the endoplasmic reticulum that were shown previously to accelerate degradation of reductase when overexpressed by transfection. Alanine substitution experiments reveal that binding of reductase to Insigs and subsequent ubiquitination require the tetrapeptide sequence YIYF in the second membrane-spanning helix of reductase. The YIYF peptide is also found in the sterol-sensing domain of SCAP, another protein that binds to Insigs in a sterolstimulated fashion. When lysine 248 of reductase is substituted with arginine, Insig binding persists, but the reductase is no longer ubiquitinated and degradation is markedly slowed. Lysine 248 is predicted to lie immediately adjacent to a membrane-spanning helix, suggesting that a membrane-bound ubiquitin transferase is responsible. Finally, we show that Insig-dependent, sterolstimulated degradation of reductase is further accelerated when cells are also supplied with the 20-carbon isoprenoid geranylgeraniol, but not the 15-carbon farnesol, raising the possibility that the nonsterol potentiator of reductase regulation is a geranylgeranylated protein.The enzyme 3-hydroxy-3-methylglutaryl-CoA reductase (HMG-CoA reductase) 1 catalyzes the two-step reduction of HMG-CoA to mevalonate, a crucial intermediate in the synthesis of cholesterol and nonsterol isoprenoids (1). Mevalonatederived products include ubiquinone, heme, and the farnesyl and geranylgeranyl groups that become attached to many proteins, directing them to membranes. HMG-CoA reductase is subject to tight regulation by a multivalent feedback mechanism mediated by nonsterol and sterol end products of mevalonate metabolism (2). These end products decrease reductase activity by inhibiting transcription of the reductase gene, blocking translation of the reductase mRNA, and accelerating degradation of the reductase protein. The transcriptional effects are mediated through the action of sterol regulatory elementbinding proteins (SREBPs), membrane-bound transcription factors that enhance transcription of genes encoding cholesterol biosynthetic enzymes and the low density lipoprotein receptor (3). The translational effects are mediated by nonsterol mevalonate-derived isoprenoids, which act by an undefined mechanism (4). The accelerated degradation of reductase is mediated both by sterols and nonsterol end products of mevalonate metabolism (4, 5) and appears to be carried out by the 26 S proteasome (6, 7). Hamster HMG-CoA reductase is an endoplasmic reticulum (ER) resident glycoprotein of 887 amino acids and consists of two distinct domains ...

Dislocation of HMG-CoA Reductase and Insig-1, Two Polytopic Endoplasmic Reticulum Proteins, En Route to Proteasomal Degradation

“…Despite its poor release and deglycosylation (Figure 5Ai, lanes 3 and 4), the dislocated HMG 350 -HA (YIYF3 AIAF) that was associated with Insig-1-Myc was predominantly un/deglycosylated, similar to the wild-type protein (Figure 5Bi, lanes 1-4). Radioactive pulse-chase experiments confirmed that the turnover of HMG 350 -HA (YIYF3 AIAF) mutant was not regulated by sterols (data not shown).A mutant HMG 350 -HA in which lysines 89 and 248 were replaced by arginines does not undergo sterol-regulated polyubiquitination and degradation (Sever et al, 2003a;Doolman et al, 2004). In response to sterols, the amounts of HMG 350 -HA (K89ϩ248R) that were released to the supernatant and pulled down along with Insig-1-Myc did not increase but were even slightly reduced ( Figure 5, Ai and Bi, lanes 5 and 6).…”

“…A mutant HMG 350 -HA in which lysines 89 and 248 were replaced by arginines does not undergo sterol-regulated polyubiquitination and degradation (Sever et al, 2003a;Doolman et al, 2004). In response to sterols, the amounts of HMG 350 -HA (K89ϩ248R) that were released to the supernatant and pulled down along with Insig-1-Myc did not increase but were even slightly reduced ( Figure 5, Ai and Bi, lanes 5 and 6).…”

ERAD‐dependent control of the Wnt secretory factor Evi

“…Besides its role in ensuring protein quality, ERAD also has the capability to respond to the physiological requirement of mature proteins that transit or reside within the ER to regulate their homeostasis accordingly. This is well described for the negative feedback regulation of sterol biosynthesis, where abundant sterol metabolites induce ERAD‐mediated degradation of sterol biosynthetic enzymes like 3‐hydroxy‐3‐methylglutaryl‐coenzyme‐A reductase (HMGCR) (Gil et al , 1985; Sever et al , 2003) and squalene monooxygenase (SQLE) (Gill et al , 2011; Foresti et al , 2013). Beyond this pathway, only a few other substrates have been identified for regulatory ERAD.…”

“…Since ERAD‐dependent quality control has been linked to a range of cellular processes and human diseases (Zettl et al , 2011; Guerriero & Brodsky, 2012; Perrody et al , 2016), understanding its molecular mechanisms is an important step to develop potential treatment strategies (Tsai & Weissman, 2010; Hetz et al , 2013). In addition to its role in protein quality control of nascent polypeptides, ERAD has been implicated in regulating the abundance of mature proteins in response to changes in physiological conditions (Wiertz et al , 1996a,b; Sever et al , 2003; Brodsky & Fisher, 2008; Adle et al , 2009; Foresti et al , 2013; Avci et al , 2014; van den Boomen et al , 2014). However, only a few regulatory ERAD substrates have been described so far.…”

Multifaceted role of tocotrienols in cardioprotection supports their structure: function relation

“…This pathway is activated when sterols and nonsterol end products of mevalonate metabolism accumulate in cells. Sterol-accelerated ubiquitination of HMG-CoA reductase requires Insig-1 and Insig-2; membrane-bound proteins of the ER (Sever et al 2003). Recently, it has been elegantly demonstrated that dtocotrienol stimulates the ubiquitination and degradation of HMG-CoA reductase and blocks processing of SREBPs, another sterol-mediated action of Insigs.…”