A proteolytic pathway that controls the cholesterol content of membranes, cells, and blood (sterol regulatory element-binding proteinsytranscriptionySite-1 proteaseySite-2 proteaseysterol-sensing domain)

The integrity of cell membranes is maintained by a balance between the amount of cholesterol and the amounts of unsaturated and saturated fatty acids in phospholipids. This balance is maintained by membrane-bound transcription factors called sterol regulatory element-binding proteins (SREBPs) that activate genes encoding enzymes of cholesterol and fatty acid biosynthesis. To enhance transcription, the active NH2-terminal domains of SREBPs are released from endoplasmic reticulum membranes by two sequential cleavages. The first is catalyzed by Site-1 protease (S1P), a membrane-bound subtilisin-related serine protease that cleaves the hydrophilic loop of SREBP that projects into the endoplasmic reticulum lumen. The second cleavage, at Site-2, requires the action of S2P, a hydrophobic protein that appears to be a zinc metalloprotease. This cleavage is unusual because it occurs within a membrane-spanning domain of SREBP. Sterols block SREBP processing by inhibiting S1P. This response is mediated by SREBP cleavage-activating protein (SCAP), a regulatory protein that activates S1P and also serves as a sterol sensor, losing its activity when sterols overaccumulate in cells. These regulated proteolytic cleavage reactions are ultimately responsible for controlling the level of cholesterol in membranes, cells, and blood. Cholesterol has long been known to play an important role in modulating fluidity and phase transitions in the plasma membranes of animal cells (1). Recently, a new role for cholesterol has been appreciated. Cholesterol, together with sphingomyelin, forms plasma membrane rafts or caveolae that are sites where signaling molecules are concentrated (2, 3). To perform these functions, membrane cholesterol must be maintained at a constant level. This homeostasis is achieved by a feedback regulatory system that senses the level of cholesterol in cell membranes and modulates the transcription of genes encoding enzymes of cholesterol biosynthesis and uptake from plasma lipoproteins. The modulators are a family of membrane-bound transcription factors called sterol regulatory element-binding proteins (SREBPs), which must be released proteolytically from membranes to act (4). This article summarizes recent progress in understanding the SREBPs and the sterolregulated proteases that release them. Three SREBPs are currently recognized. Two are produced from a single gene through the use of alternate promoters that produce transcripts with different first exons (5). The cDNAs for these proteins, designated as SREBP-1a and SREBP-1c, were cloned from human and mouse cells (6–8). SREBP-1c was cloned independently from rat adipocytes and was designated ADD-1 (9). The third isoform, SREBP-2 is produced from a separate gene (5, 10). The SREBPs are three-domain proteins of '1,150 amino acids that are bound to membranes of the endoplasmic reticulum (ER) and nuclear envelope in a hairpin orientation (4) (see Fig. 1). The NH2-terminal domain of '480 amino acids and the COOH-terminal domain of '590 amino acids project into the cytosol. They are anchored to membranes by a central domain of '80 amino acids that comprises two membrane-spanning sequences separated by a short 31-aa loop that projects into the lumen of the ER and nuclear envelope. The NH2-terminal domains of SREBPs are transcription factors of the basic-loop-helix-leucine zipper (bHLH-Zip) family (4, 11). The extreme NH2 terminus contains a stretch of acidic amino acids that recruits transcriptional coactivators, including CBP (12). In SREBP-1a and SREBP-2, these acidic sequences are relatively long. In SREBP-1c, the acidic sequence is shorter, and this protein is a much weaker activator than the other two SREBPs (7, 8, 13). The NH2-terminal domains of all three SREBPs also contain a bHLH-Zip motif that mediates dimerization, nuclear entry, and DNA binding. Within the basic region of this motif, the SREBPs contain a tyrosine in place of an arginine that is conserved in nearly all of the other bHLH family members (11, 14). This substitution allows SREBPs to recognize decanucleotide segments of DNA called sterol regulatory elements (SREs) (14). In contrast to the usual binding sites for bHLH proteins, which are palindromic, SREs are nonpalindromic, and they usually contain one or two copies of the sequence CAC (6, 11). When tested for binding activity against random sequences of DNA (14), the SREBPs show a strong preference for the SRE sequence that was originally defined in the enhancers of the genes encoding the low density lipoprotein (LDL) receptor and 3-hydroxy-3-methylglutaryl CoA (HMG-CoA) synthase, namely, TCACCCCACT (15, 16). In other promoters, the SREBPs recognize different sequences, and a clear consensus has not been defined (17). In sterol-depleted cells, the NH2-terminal domains of the SREBPs are released from membranes by two sequential proteolytic cleavages that must occur in the proper order (18). The NH2-terminal domain then travels to the nucleus, where it binds to SREs in the enhancers of multiple genes encoding enzymes of cholesterol biosynthesis, unsaturated fatty acid biosynthesis, triglyceride biosynthesis, and lipid uptake (reviewed in ref. 19). In the cholesterol biosynthetic pathway, well defined target genes include HMG-CoA synthase, HMG-CoA PNAS is available online at www.pnas.org. Abbreviations: bHLH-Zip, basic-helix-loop-helix-leucine zipper; CHO, Chinese hamster ovary; ER, endoplasmic reticulum; HMGCoA, 3-hydroxy-3-methylglutaryl CoA; LDL, low density lipoprotein; PLAP, placental alkaline phosphatase; SRE, sterol regulatory element; SREBP, sterol regulatory element-binding protein; SCAP, SREBP cleavage-activating protein; S1P; Site-1 protease; S2P; Site-2 protease. *E-mail: [email protected] or [email protected].