Cholesterol feedback: from Schoenheimer's bottle to Scap's MELADL

Abstract

Cholesterol biosynthesis is among the most intensely regulated processes in biology. Synthetic rates vary over hundreds of fold depending on the availability of an external source of cholesterol. Studies of this feedback regulatory process have a rich history. The field began 75 years ago when Rudolf Schoenheimer measured cholesterol balance in mice in a bottle. He found that cholesterol feeding led to decreased cholesterol synthesis, thereby introducing the general phenomenon by which end products of biosynthetic pathways inhibit their own synthesis. Recently, cholesterol feedback has been explained at a molecular level with the discovery of membrane-bound transcription factors called sterol regulatory element-binding proteins (SREBPs), and an appreciation of the sterol-sensing role of their partner, an escort protein called Scap. The key element in Scap is a hexapeptide sequence designated MELADL (rhymes with bottle). Thus, over 75 years, Schoenheimer's bottle led to Scap's MELADL. In addition to their basic importance in membrane biology, these studies have implications for the regulation of plasma cholesterol levels and consequently for the development of atherosclerotic plaques, myocardial infarctions, and strokes. In this article we review the major milestones in the cholesterol feedback story.

Fig. 1.

Two classic papers on cholesterol homeostasis in animals. (A) In 1933, Schoenheimer and Breusch (1) published the first example of a biological feedback system. They used balance techniques to study cholesterol synthesis and degradation in mice on low and high cholesterol diets. Their major conclusion is shown in quotation marks. (B) In 1953, Gould et al. (4) performed one of the first studies to use radioisotopes to measure the synthesis of cholesterol. They showed a reduction in the incorporation of [¹⁴C]acetate into [¹⁴C]cholesterol in liver slices from dogs fed a high cholesterol diet (1% cholesterol). The animals were fed the diets for 7 days, and the incubation time was 3 hours.

Fig. 2.

Feedback regulation of HMG-CoA reductase activity in fibroblasts from a normal subject (•) and from an FH homozygote (○). A: Monolayers of cells were grown in dishes containing 10% fetal calf serum. At zero time, the medium was replaced with fresh medium containing 5% human serum from which the lipoproteins had been removed. At the indicated time, extracts were prepared, and HMG-CoA reductase activity was measured. B: After a 24-h incubation with 5% human lipoprotein-deficient serum, human LDL was added to give the indicated cholesterol concentration. HMG-CoA reductase activity was measured in cell-free extracts at the indicated time. [Reprinted with permission from Goldstein and Brown (7)].

Fig. 3.

The SREBP pathway. When cells are depleted of sterols, Scap transports SREBPs from the ER to the Golgi apparatus. Release of SREBPs from the membrane is initiated by Site-1 Protease (S1P), a Golgi-located protease that cleaves SREBPs in the luminal loop between the two membrane-spanning sequences. Once the two halves of the SREBP are separated, a second Golgi protease, Site-2 Protease (S2P), cleaves the NH₂-terminal bHLH-Zip domain of SREBP at a site located within the membrane-spanning region. After the second cleavage, the NH₂-terminal bHLH-Zip domain leaves the membrane, carrying three hydrophobic residues at its COOH-terminus. The cleaved SREBP enters the nucleus, where it activates genes controlling lipid synthesis and uptake.

Fig. 4.

Mechanisms by which sterols alter Scap's conformation and inhibit transport of SREBPs from ER to Golgi. A: Amino acid sequence and topology of the membrane domain of hamster Scap. The sterol-sensing domain of Scap (transmembranes 2-5) is shown in blue. Amino acids 447-452 (shown in red) denote the hexapeptide MELADL sequence that targets Scap to CopII coat proteins. Arginine-503 (shown in purple) denotes the amino acid residue that is subject to trypsin-induced proteolysis in ER membrane from cholesterol-treated cells but not sterol-depleted cells. B: Differential binding of cholesterol and oxysterols to Scap and Insig, respectively. When the concentration of cholesterol in the ER membrane is low, the hexapeptide MELADL sorting signal in loop 6 of Scap binds to Sec24, a component of the Sar1/Sec23/Sec24 complex of CopII coat proteins. This binding mediates the sequestration of Scap/SREBP complexes in CopII-coated vesicles that leave the ER. Cholesterol and oxysterols promote Scap binding to Insigs, but by different membranes. Cholesterol binds directly to Scap, triggering Scap to bind to Insig, an ER retention protein. Oxysterols bind directly to Insig, triggering Insig to bind to Scap. In both cases, the end result is a conformational change in the cytoplasmic loop 6 of Scap that prevents CopII proteins from gaining access to the MELADL sequence (C). The diagrams in A–C are likely to be oversimplifications because the protein complexes can potentially form large oligomeric complexes.

Fig. 5.

Alterations in hepatic cholesterol synthesis in gene-manipulated mice. A: Increased cholesterol synthesis occurs in livers of the following gene-manipulated mice: transgenic mice expressing a truncated form of SREBP-2 (100) that lacks the transmembrane and C-terminal regulatory domains; transgenic mice expressing a mutant version of Scap (D443N) that does not bind Insig in the presence of sterols and thus undergoes constitutive ER-to-Golgi transport (62, 101); and knockout mice lacking both Insig-1 and Insig-2 (102). B: Decreased cholesterol synthesis occurs in livers of the following gene-manipulated mice: transgenic mice that overexpress Insig-1 (87); knockout mice lacking Scap (103); and knockout mice lacking site-1 protease (104).