Control of cholesterol synthesis through regulated ER-associated degradation of HMG CoA reductase

Abstract

Multiple mechanisms for feedback control of cholesterol synthesis converge on the rate-limiting enzyme in the pathway, 3-hydroxy-3-methylglutaryl coenzyme A reductase. This complex feedback regulatory system is mediated by sterol and nonsterol metabolites of mevalonate, the immediate product of reductase activity. One mechanism for feedback control of reductase involves rapid degradation of the enzyme from membranes of the endoplasmic reticulum (ER). This degradation results from the accumulation of sterols in ER membranes, which triggers binding of reductase to ER membrane proteins called Insig-1 and Insig-2. Insig binding leads to the recruitment of a membrane-associated ubiquitin ligase called gp78 that initiates ubiquitination of reductase. Ubiquitinated reductase then becomes extracted from ER membranes and is delivered to cytosolic 26S proteasomes through an unknown mechanism that is mediated by the gp78-associated ATPase Valosin-containing protein/p97 and appears to be augmented by nonsterol isoprenoids. Here, we will highlight several advances that have led to the current view of mechanisms for sterol-accelerated, ER-associated degradation of reductase. In addition, we will discuss potential mechanisms for other aspects of the pathway such as selection of reductase for gp78-mediated ubiquitination, extraction of the ubiquitinated enzyme from ER membranes, and the contribution of Insig-mediated degradation to overall regulation of reductase in whole animals.

Figure 1

Schematic representation of the mevalonate pathway in animal cells. Statins, competitive inhibitors of HMG CoA reductase, are highlighted in red. The abbreviation “PP” (i.g., isopentyl-PP) designates pyrophosphate.

Figure 2

Domain structure of HMG CoA reductase. (A) As discussed in the text, HMG CoA reductase consists of two distinct domains: a hydrophobic N-terminal domain with eight membrane-spanning segments that plays a key role in sterol-accelerated degradation of the enzyme and a hydrophilic C-terminal domain that directs enzymatic activity. (B) Amino acid sequence and topology of the membrane domain of HMG CoA reductase. The lysine residues that are required for Insig-mediated, sterol-induced ubiquitination of HMG CoA reductase are enlarged, highlighted in red, and denoted by arrows. Sequences required for sterol-regulated binding of HMG CoA reductase to Insigs (YIYF, Ser-60, Gly-87, and Ala-333) are enlarged and highlighted in yellow.

Figure 3

Current model for sterol-accelerated ERAD of HMG CoA reductase. Accumulation of certain sterols (e.g., oxysterols such as 25-hydroxycholesterol and the cholesterol synthesis intermediate 24,25-dihydrolanosterol) stimulates binding of Insigs to the membrane domain of HMG CoA reductase. Some of the Insig molecules are associated with gp78, a membrane-anchored ubiquitin ligase that associates with the ubiquitin conjugating enzyme Ubc7 and the AAA-ATPase VCP/p97. Ubc7 and gp78 combine to initiate the polyubiquitination of two cytosolic lysine residues in the membrane domain of HMG CoA reductase. This ubiquitination triggers extraction of HMG CoA reductase from ER membranes through the action of VCP/p97 and its associated cofactors; this step appears to be enhanced by the 20-carbon nonsterol isoprenoid geranylgeraniol through an undefined mechanism. Once extracted, HMG CoA reductase is delivered to proteasomes for degradation.

Figure 4

The S. cerevisiae Hrd1 ubiquitin ligase complex. Schematic representation of the Hrd1 complex in yeast that includes factors involved in substrate selection (Kar2 and Yos9), ubiquitination (Ubc7 and Cue1), and recruitment of cdc48 (Ubx2) and Der1 (Usa1). Yeast proteins are shown in black and their mammalian homologs are shown in magenta. Hrd1 complex components required for Insig-mediated degradation of HMG CoA reductase in Drosophila S2 cells are denoted by asterisks.

Figure 5

Proposed model for Insig-mediated selection of mammalian HMG CoA reductase for ubiquitination/degradation. (A) As discussed in the text, Hrd1-mediated degradation of proteins with misfolded lumenal domain (ERAD-L substrates) in yeast begins with their recognition by the chaperone Kar2, which associates with the lectin-like protein Yos9. These substrates are then transferred to the Hrd1 complex through a mechanism that is mediated by interactions between Yos9 and Hrd3. In subsequent steps, ERAD-L substrates become dislocated into the cytosol, ubiquitinated, and presented to proteasomes for degradation through the actions of other Hrd1 complex components shown in Figure 4. (B) Reconstitution experiments reveal that Drosophila Hrd1 and Sel1 (the Hrd3 homolog) are required for Insig-mediated, sterol-accelerated degradation of mammalian HMG CoA reductase in S2 cells. By analogy to the model present in A, this degradation may involve a mechanism whereby Insigs bridge HMG CoA reductase to the dHrd1 complex through interactions with an unknown intermediary protein(s) that plays a role similar to that of Yos9 in degradation of ERAD-L substrates in yeast.