Sterol Regulatory Element-binding Protein Cleavage Research Articles

Three mutations in sterol-sensing domain of SCAP block interaction with insig and render SREBP cleavage insensitive to sterols.

We report the isolation and characterization of a new line of mutant Chinese hamster ovary cells, designated SRD-5, that are resistant to 25HC, a potent suppressor of cleavage of sterol regulatory element-binding proteins (SREBPs) in mammalian cells. In SRD-5 cells, SREBPs are cleaved constitutively, generating transcriptionally active nuclear SREBP even in the presence of sterols. Sequence analysis of SREBP cleavage-activating protein (SCAP) transcripts from SRD-5 cells revealed the presence of a mutation in one SCAP allele that results in substitution of a conserved Leu by Phe at amino acid 315 within the sterol-sensing domain. Sterols fail to inhibit the packaging of SREBPSCAP(L315F) complexes into budding vesicles in vitro. Sterols also fail to induce binding of SCAP(L315F) to insig-1 or insig-2, two proteins that function in the sterol-mediated retention of SREBPSCAP complexes in the endoplasmic reticulum. Similar findings were observed for SCAP(D443N) and SCAP(Y298C), both of which cause a sterol-resistant phenotype. Thus, three different point mutations, each within the sterol-sensing domain of SCAP, prevent sterol-induced binding of SCAP to insig proteins and abolish feedback regulation of SREBP processing by sterols.

Sterol regulatory element-binding proteins (SREBPs) as regulators of lipid metabolism: polyunsaturated fatty acids oppose cholesterol-mediated induction of SREBP-1 maturation.

Cellular cholesterol and fatty acid metabolism in mammals is controlled by a family of transcription factors called sterol regulatory element-binding protein isoforms, three of which (SREBP-1a, 1c, and 2) are well characterized. These proteins, which are synthesized as precursors, are inserted into the endoplasmic reticulum (ER) membrane with both the amino and carboxylic acid domains facing the cytosolic face of the membrane. In sterol-deficient cells, proteolytic cleavage of SREBPs occurs, thereby releasing their N-terminal mature and active forms and enabling them to enter the nucleus, where they bind to the sterol regulatory response element (SRE) and/or E-box sequences and activate genes involved in cholesterol, triglyceride, and fatty acid biosynthesis. Of the three SREBP isoforms, SREBP-1c gene expression is induced by cholesterol and repressed by polyunsaturated fatty acids (PUFA). We have examined the changes in SREBP-1c mRNA and protein levels as well as the mRNA levels of several SREBP-1c target genes when a high-cholesterol diet is combined with diets rich in PUFA of the n-6 series. Our studies show that PUFA oppose the cholesterol-mediated SREBP-1 maturation without affecting the cholesterol-mediated increase of SREBP-1c mRNA and precursor protein. The decrease in SREBP-1 mature protein paralleled the decrease in mRNAs for genes of fatty acid and cholesterol biosynthesis, such as HMG-CoA synthase and fatty acid synthase, but interestingly gene expression of stearoyl-CoA desaturase 1 (SCD1) was instead induced. These studies suggest that the main point of control of PUFA-mediated suppression of lipogenic gene expression is the inhibition of SREBP-1 maturation. The studies also reveal that the induction of SCD1 gene expression by cholesterol occurs through a mechanism independent of SREBP-1 maturation.

Androgens stimulate lipogenic gene expression in prostate cancer cells by activation of the sterol regulatory element-binding protein cleavage activating protein/sterol regulatory element-binding protein pathway.

Using two independent prostate cancer cell lines (LNCaP and MDA-PCa-2a), we demonstrate that coordinated stimulation of lipogenic gene expression by androgens is a common phenomenon in androgen-responsive prostate tumor lines and involves activation of the sterol regulatory element-binding protein (SREBP) pathway. We show 1) that in both cell lines, androgens stimulate the expression of fatty acid synthase and hydroxymethylglutaryl-coenzyme A synthase, two key lipogenic genes representative for the fatty acid and the cholesterol synthesis pathway, respectively; 2) that treatment with androgens results in increased nuclear levels of active SREBP; 3) that the effects of androgens on promoter-reporter constructs derived from both lipogenic genes (fatty acid synthase and hydroxymethylglutaryl-coenzyme A synthase) depend on the presence of intact SREBP-binding sites; and 4) that cotransfection with dominant-negative forms of SREBPs abolishes the effects of androgens. Related to the mechanism underlying androgen activation of the SREBP pathway, we show that in addition to minor effects on SREBP precursor levels, androgens induce a major increase in the expression of sterol regulatory element-binding protein cleavage-activating protein (SCAP), an escort protein that transports SREBPs from their site of synthesis in the endoplasmic reticulum to their site of proteolytical activation in the Golgi. Both time course studies and overexpression experiments showing that increasing levels of SCAP enhance the production of mature SREBP and stimulate lipogenic gene expression support the contention that SCAP plays a pivotal role in the lipogenic effects of androgens in tumor cells.

Androgens Stimulate Lipogenic Gene Expression in Prostate Cancer Cells by Activation of the Sterol Regulatory Element-Binding Protein Cleavage Activating Protein/Sterol Regulatory Element-Binding Protein Pathway

Androgens Stimulate Lipogenic Gene Expression in Prostate Cancer Cells by Activation of the Sterol Regulatory Element-Binding Protein Cleavage Activating Protein/Sterol Regulatory Element-Binding Protein Pathway

CREating a SCAP-less liver keeps SREBPs pinned in the ER membrane and prevents increased lipid synthesis in response to low cholesterol and high insulin.

Cellular cholesterol homeostasis in mammals is governed by a small subfamily of bHLHLZ transcription factors called the sterol regulatory element binding proteins (SREBPs). When they were first identified in 1993 (Hua et al. 1993; Tontonoz et al. 1993; Yokoyama et al. 1993), the significance of the amino-terminal bHLHLZ domain as a transcriptional regulatory protein was obvious because of its similarity with other proteins such as Myc and Max. However, because the remaining 50% of the predicted protein sequence was not similar to any other known proteins, its significance was not appreciated. Elegant studies over the ensuing years have shown that the SREBPs are synthesized as precursor proteins that are threaded into the endoplasmic reticulum (ER) membrane and anchored there through a two-pass membrane-spanning domain (Brown and Goldstein 1999). The twomembrane domains pin the inactive precursor in a rigid hairpin orienting both the aminoand carboxy-terminal domains to the cytosolic face of the membrane. SREBPs are maintained in this inactive form until a low sterol level is sensed and they are needed to increase lipid accumulation. The low sterol level sets a proteolytic cascade in motion that results in nuclear accumulation of an amino-terminal fragment that corresponds to the mature SREBP transcriptional regulatory protein. Key steps in the mechanism for the sterol-regulated cleavage process have been revealed through a classic combination of somatic cell-molecular genetics and biochemistry (Brown and Goldstein 1999). Three additional membrane-bound proteins are involved in the multistep regulation in addition to the SREBPs themselves. These include two proteases (Rawson et al. 1997; Sakai et al. 1998) and a polytopic ER membrane protein with a putative sterol-sensing domain (Hua et al. 1996a), which is involved in modulating access of the substrate SREBPs with the first protease. Although the need for proteolysis was obvious, the role of the regulatory protein, called SREBP cleavage activation protein or SCAP, was not apparent. It is now clear that SCAP is the key to sterol regulation because the identical proteases, but not SCAP, are involved in releasing the ATF 6 protein from ER membranes in response to cellular stress (Ye et al. 2000). In addition, a complex between SCAP and SREBPs moves from the ER to the golgi in a sterol-regulated manner (Nohturfft et al. 1999, 2000). This places the substrate SREBPs into the same cellular compartment as the proteases (Fig. 1). Thus, the role of SCAP is to escort the SREBPs from one cellular compartment to another in a sterol-regulated fashion Sheng et al. (1995) showed that processing of SREBP-1 and SREBP-2 is regulated differently in animals, whereas their processing is coordinately regulated in cultured cells in response to sterol depletion (Wang et al. 1994; Hua et al. 1996b). SCAP is required for maturation of both SREBP-1 and SREBP-2 isoforms in cultured cells, but because of the results of Sheng et al. (1995) there was a question as to whether SCAP was really involved in processing both SREBP-1 and SREBP-2 in animals. Recently, Matsuda et al. (2001) used Cre–loxP gene knockout technology combined with animal feeding studies to evaluate SCAP’s role in processing precursor SREBPs in livers of adult mammals. Loss of liver SCAP resulted in severely reduced levels of mRNAs and precursor proteins for both SREBP-1 and SREBP-2 in livers of animals fed a standard rodent balanced chow diet. Thus, SCAP is definitely involved in maturation of both SREBPs, but there is surely more to be uncovered in this system because of the differential regulation noted by Sheng et al. (1995). In the first part of this article, I provide a brief overview to the SREBP pathway and refer the reader to other recent reviews of this more general topic (Brown and Goldstein 1999; Edwards and Ericsson 1999; Edwards et al. 2000; Osborne 2000). The second part of the 1E-MAIL tfosborn@uci.edu; FAX (949) 824-8551. Article and publication are at http://www.genesdev.org/cgi/doi/10.1101/ gad.916601.

Cholesterol Signaling at the Endoplasmic Reticulum Occurs in npc1−/− but Not in npc1−/−, LDLR−/− Mice

It remains controversial whether deficiency of the Niemann–Pick C1 (npc1) protein results in altered cholesterol signaling at the endoplasmic reticulum (ER). In this report, we have measured the processed, nuclear form of sterol regulatory element binding protein (SREBP)-1 in livers of npc1 wild-type, heterozygous, and homozygous deficient mice, alone, and in combination with deficiencies of the low density lipoprotein receptor (LDLR) or the multiple drug resistant (mdr)1a, P-glycoprotein. Cleavage of SREBPs to activated forms normally occurs when the ER is deficient in cholesterol. A large decrease in processed SREBP-1 was evident in fasted npc1−/− mice and npc1−/−, mdr1a−/− mice, with no decrease evident in npc1−/−, LDLR−/− mice. These results suggest that the increase in cellular cholesterol which occurs in npc1−/− and in npc1−/−, mdr1a−/− mice includes the sites responsible for cholesterol signaling, while the similar increase in cholesterol found in npc1−/−, LDLR−/− mice does not.

Proteolytic Activation of SREBPs during Adipocyte Differentiation

A member of sterol regulatory element-binding protein (SREBP) family, SREBP-1, is a key regulator of adipocyte differentiation. Expression of the SREBP-1 gene is induced during adipocyte differentiation, but proteolytic activation of the synthesized precursor form of SREBP-1 has not been well analyzed. The proteolytic processing of SREBPs is severely suppressed in sterol loaded culture cells. Here we report that a splicing isoform, SREBP-1a, is predominantly expressed in 3T3-L1 preadipocytes and adipocytes, and that the nuclear active form of SREBP-1 protein increases in adipocyte differentiation. We further show that the amount of nuclear SREBP-2 protein also increases despite no increase in SREBP-2 mRNA, suggesting that proteolytic cleavage of SREBPs is induced in lipid loaded adipocytes. Northern blot analyses reveal that mRNA levels for SREBP cleavage-activating protein (SCAP), Site-1 protease (S1P), and Site-2 protease (S2P), which participate in the proteolytic processing of SREBPs, are relatively unaffected in adipogenesis. These results demonstrate that SREBP-2 appears to promote adipocyte differentiation as well as SREBP-1 and that the proteolytic activation of SREBPs may be induced by an as-yet unidentified mechanism in lipid loaded adipocytes.

Lipogenic enzymes fatty acid synthase and acetyl-coenzyme A carboxylase are coexpressed with sterol regulatory element binding protein and Ki-67 in fetal tissues.

Endogenous fatty acid synthesis has been observed in some rapidly proliferating cells and tissues, both normal and neoplastic, and probably supports membrane synthesis. Sterol regulatory element binding proteins (SREBPs) are transcription factors that regulate the expression of genes for both cholesterol and fatty acid synthesis. The inactive precursor form resides in cytoplasmic membranes. Intracellular lipid depletion triggers proteolytic cleavage of SREBP, allowing the amino terminus to enter the nucleus and activate the expression of enzymes, including acetyl-CoA carboxylase (ACC) and fatty acid synthase (FAS), major biosynthetic enzymes for fatty acid synthesis. The expression patterns of ACC, FAS, SREBP, and Ki-67 in fetal tissues were compared to determine whether SREBP is likely to participate in the regulation of proliferation-associated fatty acid synthesis during fetal growth. Tissues from 22 fetuses, 12 first-trimester and 10 second-trimester (range 7.0 to 21.6 weeks), were studied. Serial 5-microm sections were stained with antibodies to ACC, FAS, SREBP, and Ki-67 and were compared. ACC, FAS, SREBP, and Ki-67 were coexpressed in the proliferative compartments of the intestines, skin, and kidney. ACC, FAS, and Ki-67 were coexpressed with little SREBP in lung and cytotrophoblast. SREBP, ACC, and FAS were coexpressed without Ki-67 in hepatocytes, ganglion cells, and intermediate trophoblast. The close linkage of SREBP, ACC, FAS, and Ki-67 in some proliferating fetal tissues suggests that in these tissues SREBP participates in the transcriptional regulation of lipogenic genes during proliferation. SREBP, ACC, and FAS coexpression without Ki-67 occurs in differentiated tissues that may synthesize fatty acids for other functions.

Sterols Promote Retention

Lipid biosynthesis and uptake is regulated by the intracellular sterol level, indirectly by sterol-regulatory element binding proteins (SREBPs), which control the synthesis of lipogenic genes. The intracellular sterol level is sensed by SREBP cleavage-activating proteins (SCAPs) in the endoplasmic reticulum (ER), which then control the movement and subsequent activation of the SREBP transcription factors. The transcription factors arise from cleavage of SREBPs after transport to the Golgi. SCAP and SREBP form a complex that moves from the ER to the Golgi, and SCAPs have a sterol-sensing domain that allows increased intracellular sterols to block this ER-to-Golgi transport of the SCAP/SREBP complex. Yang et al . transfected cells with cDNAs encoding the sterol-sensing domain of SCAP or encoding a SCAP unable to interact with SREBPs. These SCAP fragments promoted Golgi delivery and blocked the suppression of SREBP cleavage and nuclear accumulation of active SREBP in response to increasing sterol concentration. When the sterol-sensing domain of the SCAP fragments was mutated, then this inhibition did not occur. These data suggest that the SCAP fragments compete for binding to a retention molecule in the presence of increased sterols. This supports a model whereby SCAP/SREBP is constitutively transported to the Golgi and increased sterols promote ER retention of the complex. Yang, T., Goldstein, J.L., and Brown, M.S. (2000) Overexpression of membrane domain of SCAP prevent sterols from inhibiting SCAP-SREBP exit from endoplasmic reticulum. J. Biol. Chem. 275 : 29881-29886. [Abstract] [Full Text]

Inhibition of sterol regulatory element-binding protein cleavage by the prodomain of human SKI-1 protease

Inhibition of sterol regulatory element-binding protein cleavage by the prodomain of human SKI-1 protease

Failure to Cleave Sterol Regulatory Element-binding Proteins (SREBPs) Causes Cholesterol Auxotrophy in Chinese Hamster Ovary Cells with Genetic Absence of SREBP Cleavage-activating Protein

We describe a line of mutant Chinese hamster ovary cells, designated SRD-13A, that cannot cleave sterol regulatory element-binding proteins (SREBPs) at site 1, due to mutations in the gene encoding SREBP cleavage-activating protein (SCAP). The SRD-13A cells were obtained by two rounds of gamma-irradiation followed first by selection for a deficiency of low density lipoprotein receptors and second for cholesterol auxotrophy. In the SRD-13A cells, the only detectable SCAP allele encodes a truncated nonfunctional protein. In the absence of SCAP, the site 1 protease fails to cleave SREBPs, and their transcriptionally active NH(2)-terminal fragments cannot enter the nucleus. As a result, the cells manifest a marked reduction in the synthesis of cholesterol and its uptake from low density lipoproteins. The SRD-13A cells grow only when cholesterol is added to the culture medium. SREBP cleavage is restored and the cholesterol requirement is abolished when SRD-13A cells are transfected with expression vectors encoding SCAP. These results provide formal proof that SCAP is essential for the cleavage of SREBPs at site 1.

Sterols regulate cycling of SREBP cleavage-activating protein (SCAP) between endoplasmic reticulum and Golgi.

The proteolytic cleavage of sterol regulatory element-binding proteins (SREBPs) is regulated by SREBP cleavage-activating protein (SCAP), which forms complexes with SREBPs in membranes of the endoplasmic reticulum (ER). In sterol-depleted cells, SCAP facilitates cleavage of SREBPs by Site-1 protease, thereby initiating release of active NH(2)-terminal fragments from the ER membrane so that they can enter the nucleus and activate gene expression. In sterol-overloaded cells, the activity of SCAP is blocked, SREBPs remain bound to membranes, and transcription of sterol-regulated genes declines. Here, we provide evidence that sterols act by inhibiting the cycling of SCAP between the ER and Golgi. We use glycosidases, glycosidase inhibitors, and a glycosylation-defective mutant cell line to demonstrate that the N-linked carbohydrates of SCAP are modified by Golgi enzymes in sterol-depleted cells. After modification, SCAP returns to the ER, as indicated by experiments that show that the Golgi-modified forms of SCAP cofractionate with ER membranes on density gradients. In sterol-overloaded cells, the Golgi modifications of SCAP do not occur, apparently because SCAP fails to leave the ER. Golgi modifications of SCAP are restored when sterol-overloaded cells are treated with brefeldin A, which causes Golgi enzymes to translocate to the ER. These studies suggest that sterols regulate the cleavage of SREBPs by modulating the ability of SCAP to transport SREBPs to a post-ER compartment that houses active Site-1 protease.

Blunted feedback suppression of SREBP processing by dietary cholesterol in transgenic mice expressing sterol-resistant SCAP(D443N).

Feedback regulation of cholesterol biosynthesis is mediated by membrane-bound transcription factors designated sterol regulatory element-binding proteins (SREBP)-1 and -2. In sterol-deprived cultured cells, SREBPs are released from membranes by a proteolytic process that is stimulated by SREBP cleavage-activating protein (SCAP), a membrane protein containing a sterol-sensing domain. Sterols suppress SREBP cleavage by blocking the action of SCAP, thereby decreasing cholesterol synthesis. A point mutation in SCAP(D443N) causes resistance to sterol suppression. In this article, we produced transgenic mice that express mutant SCAP(D443N) in liver. In these livers the nuclear content of SREBP-1 and -2 was increased, mRNAs encoding proteins involved in uptake and synthesis of cholesterol and fatty acids were elevated, and the livers were engorged with cholesteryl esters and triglycerides enriched in monounsaturated fatty acids. When the mice were challenged with a high cholesterol diet, cleavage of SREBP-1 and -2 was reduced in wild-type livers and less so in transgenic livers. We conclude that SCAP(D443N) stimulates proteolytic processing of native SREBPs in liver and decreases the normal sterol-mediated feedback regulation of SREBP cleavage, suggesting a central role for SCAP as a sterol sensor in liver.

Sterols regulate processing of carbohydrate chains of wild-type SREBP cleavage-activating protein (SCAP), but not sterol-resistant mutants Y298C or D443N.

SREBP cleavage activating protein (SCAP), a membrane-bound glycoprotein, regulates the proteolytic activation of sterol regulatory element binding proteins (SREBPs), which are membrane-bound transcription factors that control lipid synthesis in animal cells. SCAP-stimulated proteolysis releases active fragments of SREBPs from membranes of the endoplasmic reticulum and allows them to enter the nucleus where they activate transcription. Sterols such as 25-hydroxycholesterol inactivate SCAP, suppressing SREBP proteolysis and turning off cholesterol synthesis. We here report the isolation of Chinese hamster ovary cells with a point mutation in SCAP (Y298C) that renders the protein resistant to inhibition by 25-hydroxycholesterol. Like the previously described D443N mutation, the Y298C mutation occurs within the putative sterol-sensing domain, which is part of the polytopic membrane attachment region of SCAP. Cells that express SCAP(Y298C) continued to process SREBPs in the presence of 25-hydroxycholesterol and hence they resisted killing by this sterol. In wild-type Chinese hamster ovary cells the N-linked carbohydrate chains of SCAP were mostly in the endoglycosidase H-sensitive form when cells were grown in medium containing 25-hydroxycholesterol. In contrast, when cells were grown in sterol-depleted medium, these chains were converted to an endoglycosidase H-resistant form. 25-Hydroxycholesterol had virtually no effect in cells expressing SCAP(D443N) or SCAP(Y298C). The relation between this regulated carbohydrate processing to the SCAP-regulated proteolysis of SREBP remains to be explored.

BetaAPP gamma-secretase and SREBP site 2 protease are two different enzymes.

The cleavage process of sterol regulatory element binding proteins (SREBPs) shares several characteristics with that of beta-amyloid precursor protein (betaAPP). We studied the secretion of amyloid beta protein (Abeta) from mutant M19 Chinese hamster ovary (CHO) cells that are defective in the site 2 cleavage of SREBPs. The levels as well as the C-terminal properties of Abeta secreted from M19 cells were similar to those from wild-type CHO cells, demonstrating that gamma-cleavage of betaAPP occurs normally in M19 cells. The present data indicated that gamma-secretase of betaAPP and SREBP site 2 protease are distinct enzymes.

Characterization of two Chinese hamster ovary cell lines expressing the COOH-terminal domains of sterol regulatory element-binding protein (SREBP)-1.

Sterol regulatory element-binding proteins (SREBPs) regulate transcription of genes encoding enzymes in the cholesterol biosynthetic pathway and the LDL receptor. These proteins are synthesized as membrane-bound precursors and processed to generate the NH2-terminal domains, mature transcription factors. We established two Chinese hamster ovary (CHO) cell lines, CHO-421 expressing the truncated hamster SREBP-1 (amino acids 421-1133) with two transmenbrane domains and CHO-557 expressing another truncated SREBP-1 (amino acids 557-1133) without any transmembrane domains, to investigate the fate of the COOH terminus after cleavage of the NH2-terminal mature SREBP. The cell fractionation experiments revealed that the two proteins, regardless of the absence of transmembrane domains in the SREBP (557-1133), similarly localized in the nuclear envelope and the microsomal membrane fractions, suggesting that these proteins appear to be tightly bound to a membrane protein(s) localizing on the nuclear and endoplasmic reticulum (ER) membranes. Although we predicted that overexpression of the COOH-terminal domains, which were thought to be involved in the regulation of SREBP processing, would result in disruption of the SREBP-dependent transcriptional regulation of several genes, the mRNA levels for 3-hydroxy-3-methylglutaryl coenzyme A (HMG CoA) synthase in these two cell lines were regulated in a sterol-dependent manner. Pulse-chase experiments revealed that the SREBP (421-1133) was relatively rapidly degraded (t1/2 = 4-6 hr) and that the intracellular cholesterol level did not affect the half-life time. The degradation of the SREBP (421-1133) was not suppressed by the treatment of a calpain inhibitor, N-acetyl-leucyl-leucyl-norleucynal (ALLN), which blocks the proteolysis of some proteins within or near the ER. In CHO-557 cells the SREBP (557-1133) was much more rapidly degraded (t1/2 = 1-2 hr), suggesting that the cytosolic COOH-terminal domain is accessible to the enzymatic attacks from the cytoplasm. Taken together, overexpression of the COOH-terminal domains does not affect the regulation of SREBP processing and the domains are rapidly turned over by the cytosolic proteolytic process distinct from the ALLN-sensitive ER degradative pathway.

Complementation Cloning of S2P, a Gene Encoding a Putative Metalloprotease Required for Intramembrane Cleavage of SREBPs

We report the cloning of a gene, S2P, that encodes a putative metalloprotease required for intramembrane proteolysis of sterol-regulatory element–binding proteins (SREBPs) at Site-2. SREBPs are membrane-bound transcription factors that activate genes regulating cholesterol metabolism. The active NH 2-terminal domains of SREBPs are released from membranes by sequential cleavage at two sites: Site-1, within the lumen of the endoplasmic reticulum; and Site-2, within a transmembrane segment. The human S2P gene was cloned by complementation of mutant CHO cells that cannot cleave SREBPs at Site-2 and are cholesterol auxotrophs. S2P defines a new family of polytopic membrane proteins that contain an HE XX H sequence characteristic of zinc metalloproteases. Mutation of the putative zinc-binding residues abolishes S2P activity. S2P encodes an unusual metalloprotease that cleaves proteins within transmembrane segments.

Identification of Complexes between the COOH-terminal Domains of Sterol Regulatory Element-binding Proteins (SREBPs) and SREBP Cleavage-Activating Protein

SREBP cleavage-activating protein (SCAP) stimulates the proteolytic cleavage of membrane-bound SREBPs, thereby initiating the release of NH2-terminal fragments from cell membranes. The liberated fragments enter the nucleus and stimulate transcription of genes involved in synthesis and uptake of cholesterol and fatty acids. Sterols repress cleavage of SREBPs, apparently by interacting with the membrane attachment domain of SCAP. In the present studies we show that SCAP, like the SREBPs, is located in membranes of the endoplasmic reticulum and nuclear envelope. The COOH-terminal domain of SCAP, like that of the SREBPs, is located on the cytosolic face of the membranes. Co-immunoprecipitation experiments show that SCAP and SREBP-2 form a complex that can be precipitated with antibodies to either component. Complex formation occurs when cells express only the COOH-terminal domain of either SREBP-2 or SCAP, indicating that the complex forms between the two COOH-terminal domains. Truncation of SREBP-2 at its COOH terminus prevents the formation of complexes with SCAP and simultaneously reduces proteolytic cleavage. We conclude that proteolytic cleavage of SREBPs requires the formation of a complex with the COOH-terminal domain of SCAP and that SCAP is therefore a required element in the regulation of sterol and fatty acid metabolism in animal cells.

Purification and Characterization of an Interleukin-1β-converting Enzyme Family Protease That Activates Cysteine Protease P32 (CPP32)

CPP32, a member of the interleukin-1beta-converting enzyme (ICE) family of cysteine proteases, cleaves poly(ADP-ribose) polymerase and sterol regulatory element binding proteins during apoptosis. CPP32 normally exists in the cytosol as a 32-kDa inactive precursor and only becomes activated when cells are undergoing apoptosis. The activation is a proteolytic event that generates a p20/p11 heterodimer. We report here the identification, purification, and characterization of a hamster CPP32-activating protease (CAP) that cleaves and activates CPP32. The biochemical properties of CAP suggest that it is another member of the ICE family of proteases. Purified CAP consists of two prominent polypeptides of 19 and 13 kDa. Protein sequencing revealed that CAP is derived from the hamster homolog of Mch2alpha, a member of the ICE family recently identified based on the sequence conservation among the ICE family members. CAP activity is inhibited by CrmA, a cowpox virus protein that prevents host cell apoptosis. CAP itself is also activated through proteolytic cleavage. These data are consistent with the idea that the activation of the ICE family of proteases during apoptosis proceeds through a cascade of proteolytic events.

Cleavage of sterol regulatory element binding proteins (SREBPs) by CPP32 during apoptosis.

Cellular cholesterol homeostasis is controlled by sterol-regulated proteolysis of membrane-bound transcription factors called sterol-regulatory element binding proteins (SREBPs). CPP32, a cysteine protease, was shown previously to cleave SREBP-1 and SREBP-2 in vitro at an aspartic acid between the basic helix-loop-helix leucine zipper domain and the first trans-membrane domain, liberating a transcriptionally active fragment. Here, we show that CPP32 exists in an inactive 32 kDa form in Chinese hamster ovary (CHO) cells. When apoptosis was induced with the protein kinase inhibitor staurosporine, CPP32 was cleaved to subunits of 20 and 10 kDa to form the active protease. Under these conditions membrane-bound SREBP-1 and SREBP-2 were both cleaved, and the transcriptionally active N-terminal fragments were found in nuclear extracts. Similar results were obtained in human U937 cells induced to undergo apoptosis by anti-Fas and etoposide. The apoptosis-induced cleavage of SREBPs was not suppressed by sterols, indicating that apoptosis-induced cleavage and sterol-regulated cleavage are mediated by different proteases. CHO cells expressing a mutant SREBP-2 with an Asp--> Ala mutation at the CPP32 cleavage site showed sterol-regulated cleavage but no apoptosis-induced cleavage. These data are consistent with the emerging concept that CPP32 is a central mediator in apoptosis. They also indicate that SREBPs, like poly (ADP) ribose polymerase, are cleaved by CPP32 during programmed cell death.