Cover Image

Not for Sale

View/Hide Left Panel

reductase, farnesyl diphosphate synthase, and squalene synthase (20). The targets in the fatty acid and triglyceride biosynthetic pathways include acetyl CoA carboxylase, fatty acid synthase, stearoyl CoA desaturase, and glycerol-3-phosphate acyltransferase (4, 17, 20). The SREBPs also enhance transcription of the LDL receptor, which mediates cholesterol uptake from plasma lipoproteins. Overexpression of the NH2-terminal nuclear domains of SREBPs also elevates mRNAs encoding many other enzymes required for lipid synthesis, including enzymes that generate acetyl CoA and reduced pyridine nucleotides (21).

When sterols build up within cells, the proteolytic release of SREBPs from membranes is blocked. The NH2-terminal domains that have already entered the nucleus are rapidly degraded in a process that is blocked by inhibitors of proteasomes (22). As a result of these events, transcription ofallof the target genes declines. This decline is complete for the cholesterol biosynthetic enzymes, whose transcription is entirely dependent on SREBPs. The decline is less complete for the fatty acid biosynthetic enzymes whose basal transcription can be maintained by other factors (13, 23).

Two-Step Proteolytic Release of SREBPs

The two-step proteolytic release of the NH2-terminal domains is illustrated schematically in Fig. 1. The process begins when a protease, termed Site-1 protease (S1P), cleaves the SREBPs

FIG. 1. Model for the sterol-mediated proteolytic release of SREBPs from membranes. (Top) Release is initiated by Site-1 protease (S1P), a sterol-regulated protease that recognizes the SCAP/ SREBP complex and cleaves SREBP in the luminal loop between two membrane-spanning sequences. SCAP allows Site-1 cleavage to be activated when cells are deprived of sterols, and it inhibits this process when sterols are abundant. (Middle) Once the two halves of SREBP are separated, a second protease, Site-2 protease (S2P), cleaves the NH2-terminal bHLH-Zip domain of SREBP at a site located within the membrane-spanning region. (Bottom) After the second cleavage, the NH2-terminal bHLH-Zip domain leaves the membrane, carrying three hydrophobic residues at its COOH-terminus. The protein enters the nucleus, where it activates target genes controlling lipid synthesis and uptake.

at a site within the hydrophilic loop that projects into the lumen of the ER (Fig. 1 Top). In SREBP-2, this cleavage occurs between the leucine and serine of the sequence RSVLS (24). S1P absolutely requires a basic residue at the P4 position, and it strongly prefers a leucine at the P1 position. The residues at the P2, P3, and P1′ positions can be substituted freely without affecting cleavage (24).

Cleavage by S1P separates the SREBPs into two halves, both of which remain membrane-bound (Fig. 1 Middle). The separation can be detected by immunoprecipitation experiments; after cleavage, an antibody against the COOH-terminal domain no longer precipitates the membranebound NH2-terminal domain. The membrane-bound NH2-terminal domain is termed the intermediate fragment of SREBP (18).

After the two halves of the SREBP have separated, a second protease, designated Site-2 protease (S2P), cleaves the NH2-terminal intermediate fragment at a site that is just within its membrane-spanning domain (Fig. 1 Middle). In SREBP-2, this cleavage occurs between the leucine and cysteine of the sequence DRSRILLC (25). The second arginine of this sequence is believed to represent the boundary between the hydrophilic NH2-terminal domain and the hydrophobic membrane-spanning segment. Thus, the cleavage occurs three residues within the membrane-spanning segment. When the NH2-terminal fragment leaves the membrane to enter the nucleus, it carries the three hydrophobic ILL residues at its COOH-terminus (Fig. 1 Bottom). Studies of intact cells showed that recognition by S2P requiresallor part of the DRSR sequence. The exact recognition sequence has not been defined. Each of the ILLC residues can be replaced singly with alanines without affecting cleavage (25).

Sterols block the proteolytic release process by selectively inhibiting cleavage by S1P (Fig. 1 Top). Current evidence indicates that S2P is not regulated directly by sterols, but it is regulated indirectly because the enzyme cannot act until the two halves of SREBP have been separated through the action of S1P (18).

SREBP Cleavage-Activating Protein (SCAP)

The first advance in understanding SREBP regulation came with the isolation of a cDNA encoding SREBP cleavage-activating protein (SCAP), a regulatory protein that is required for cleavage at Site-1 (26). SCAP is an integral membrane protein of 1,276 amino acids with two distinct domains. The NH2-terminal domain of ≈730 amino acids consists of alternating hydrophilic and hydrophobic sequences that appear to form eight membrane-spanning helices (27). This domain anchors SREBP to membranes of the ER. The COOH-terminal domain of ≈550 amino acids projects into the cytosol. It contains five WD-repeats. Similar repeats, each ≈40 residues in length, are found in many intracellular proteins, where they often mediate protein-protein interactions (28). The crystal structure of one such protein, the β-subunit of heterotrimeric G proteins, revealed that the WD-repeats form the blades of a propeller-like structure that bridges the α- and γ-subunits (29, 30).

Within cells, SCAP is found in a tight complex with SREBPs (31, 32). The association is mediated by an interaction between the COOH-terminal regulatory domain of the SREBP and the WD-repeat domain of SCAP. Formation of this complex is required for Site-1 cleavage, as revealed by the following experiments (31, 32): (i) truncation of the COOH-terminal domain of SREBP-2 prevents interaction with SCAP and abolishes susceptibility to cleavage by S1P; (ii) Overexpression of a cDNA encoding the membrane-anchored COOH-terminal domain of either SCAP or SREBP-2 competitively disrupts the formation of the complex between endogenous

The National Academies of Sciences, Engineering, and Medicine
500 Fifth St. N.W. | Washington, D.C. 20001

Copyright © National Academy of Sciences. All rights reserved.
Terms of Use and Privacy Statement