Secreted Site-1 Protease Cleaves Peptides Corresponding to Luminal Loop of Sterol Regulatory Element-binding Proteins

We describe a permanent line of Chinese hamster ovary cells transfected with a cDNA encoding a truncated form of Site-1 protease (S1P) that is secreted into the culture medium in an enzymatically active form. S1P, a subtilisin-like protease, normally cleaves the luminal loop of sterol regulatory element-binding proteins (SREBPs). This cleavage initiates the two-step proteolytic process by which the NH2-terminal domains of SREBPs are released from cell membranes for translocation to the nucleus, where they activate transcription of genes involved in the biosynthesis and uptake of cholesterol and fatty acids. Truncated S1P (amino acids 1–983), produced by the transfected Chinese hamster ovary cells, lacks the COOH-terminal membrane anchor. Like native S1P, this truncated protein undergoes normal autocatalytic processing after residue 137 to release an NH2-terminal propeptide, thereby generating an active form, designated S1P-B. Prior to secretion, truncated S1P-B, like native S1P-B, is cleaved further after residue 186 to generate S1P-C, which is the only form that appears in the culture medium. The secreted enzyme, designated S1P(983)-C, cleaves a synthetic peptide that terminates in a 7-amino-4-methyl-coumarin fluorochrome. This peptide, RSLK-MCA, corresponds to the internal propeptide cleavage site that generates S1P-B as described in the accompanying paper (Espenshade, P. J., Cheng, D., Goldstein, J. L., and Brown, M. S. (1999), J. Biol. Chem. 274, 22795–22804). The secreted enzyme does not cleave RSVL-MCA, a peptide corresponding to the physiologic cleavage site in SREBP-2. However, S1P(983)-C does cleave after this leucine when the RSVL sequence is contained within a 16-residue peptide corresponding to the central portion of the SREBP-2 luminal loop. The catalytic activity of S1P(983)-C differs from that of furin/prohormone convertases, two related proteases, in its more alkaline pH optimum (pH 7–8), its relative resistance to calcium chelating agents, and its ability to cleave after lysine or leucine rather than arginine. These data provide direct biochemical evidence that S1P is the protease that cleaves SREBPs and thereby functions to control lipid biosynthesis and uptake in animal cells. We describe a permanent line of Chinese hamster ovary cells transfected with a cDNA encoding a truncated form of Site-1 protease (S1P) that is secreted into the culture medium in an enzymatically active form. S1P, a subtilisin-like protease, normally cleaves the luminal loop of sterol regulatory element-binding proteins (SREBPs). This cleavage initiates the two-step proteolytic process by which the NH2-terminal domains of SREBPs are released from cell membranes for translocation to the nucleus, where they activate transcription of genes involved in the biosynthesis and uptake of cholesterol and fatty acids. Truncated S1P (amino acids 1–983), produced by the transfected Chinese hamster ovary cells, lacks the COOH-terminal membrane anchor. Like native S1P, this truncated protein undergoes normal autocatalytic processing after residue 137 to release an NH2-terminal propeptide, thereby generating an active form, designated S1P-B. Prior to secretion, truncated S1P-B, like native S1P-B, is cleaved further after residue 186 to generate S1P-C, which is the only form that appears in the culture medium. The secreted enzyme, designated S1P(983)-C, cleaves a synthetic peptide that terminates in a 7-amino-4-methyl-coumarin fluorochrome. This peptide, RSLK-MCA, corresponds to the internal propeptide cleavage site that generates S1P-B as described in the accompanying paper (Espenshade, P. J., Cheng, D., Goldstein, J. L., and Brown, M. S. (1999), J. Biol. Chem. 274, 22795–22804). The secreted enzyme does not cleave RSVL-MCA, a peptide corresponding to the physiologic cleavage site in SREBP-2. However, S1P(983)-C does cleave after this leucine when the RSVL sequence is contained within a 16-residue peptide corresponding to the central portion of the SREBP-2 luminal loop. The catalytic activity of S1P(983)-C differs from that of furin/prohormone convertases, two related proteases, in its more alkaline pH optimum (pH 7–8), its relative resistance to calcium chelating agents, and its ability to cleave after lysine or leucine rather than arginine. These data provide direct biochemical evidence that S1P is the protease that cleaves SREBPs and thereby functions to control lipid biosynthesis and uptake in animal cells. Site-1 protease (S1P) 1The abbreviations used are: S1P, Site-1 protease; S2P, Site-2 protease; AMC, 7-amino-4-methyl-coumarin; CHO, Chinese hamster ovary; CMV, cytomegalovirus; endo H, endoglycosidase H; MCA, 4-methyl-coumaryl-7-amide; PAGE, polyacrylamide gel electrophoresis; SREBP, sterol regulatory element-binding protein; SCAP, SREBP cleavage-activating protein; ER, endoplasmic reticulum; HPLC, high performance liquid chromatography; kb, kilobase; Mes, 4-morpholineethanesulfonic acid is a mammalian subtilisin-related serine protease that cleaves sterol regulatory element-binding proteins (SREBPs) and initiates the release of their NH2-terminal domains from membranes (1Sakai J. Rawson R.B. Espenshade P.J. Cheng D. Seegmiller A.C. Goldstein J.L. Brown M.S. Mol. Cell. 1998; 2: 505-514Abstract Full Text Full Text PDF PubMed Scopus (328) Google Scholar). The NH2-terminal domains enter the nucleus, where they enhance transcription of multiple genes encoding enzymes of cholesterol and fatty acid biosynthesis and the low density lipoprotein receptor. Excess cholesterol inhibits its own synthesis by blocking the activity of S1P, thereby preventing the NH2-terminal domains of SREBPs from entering the nucleus. This feedback inhibition controls the cholesterol content of cells and plasma (2Brown M.S. Goldstein J.L. Cell. 1997; 89: 331-340Abstract Full Text Full Text PDF PubMed Scopus (3029) Google Scholar). The RSVLS target sequence for S1P is located in a luminal loop between two membrane-spanning helices of SREBP-2 (3Hua X. Sakai J. Ho Y.K. Goldstein J.L. Brown M.S. J. Biol. Chem. 1995; 270: 29422-29427Abstract Full Text Full Text PDF PubMed Scopus (148) Google Scholar). The NH2-terminal and COOH-terminal domains of SREBPs extend into the cytosol, and the luminal loop projects into the endoplasmic reticulum (ER) and nuclear envelope. Cleavage by S1P separates SREBP-2 into two halves. This allows a second protease to cleave at Site-2, which is within the first transmembrane helix, thereby liberating the NH2-terminal domain from the membrane (4Sakai J. Duncan E.A. Rawson R.B. Hua X. Brown M.S. Goldstein J.L. Cell. 1996; 85: 1037-1046Abstract Full Text Full Text PDF PubMed Scopus (435) Google Scholar, 5Duncan E.A. Davé U.P. Sakai J. Goldstein J.L. Brown M.S. J. Biol. Chem. 1998; 273: 17801-17809Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar). SREBPs are complexed with a polytopic membrane protein called SREBP cleavage-activating protein (SCAP), the presence of which is required for S1P to act within cells (6Sakai J. Nohturfft A. Cheng D. Ho Y.K. Brown M.S. Goldstein J.L. J. Biol. Chem. 1997; 272: 20213-20221Abstract Full Text Full Text PDF PubMed Scopus (199) Google Scholar, 7Sakai J. Nohturfft A. Goldstein J.L. Brown M.S. J. Biol. Chem. 1998; 273: 5785-5793Abstract Full Text Full Text PDF PubMed Scopus (191) Google Scholar). Sterols inhibit Site-1 cleavage by abolishing the activity of SCAP. The S1P cleavage reaction has been studied in intact cells that were transfected with cDNAs encoding wild-type or mutant forms of SREBP-2. S1P cleaves SREBP-2 between the leucine and serine of the sequence RSVLS (8Duncan E.A. Brown M.S. Goldstein J.L. Sakai J. J. Biol. Chem. 1997; 272: 12778-12785Abstract Full Text Full Text PDF PubMed Scopus (154) Google Scholar). Cleavage absolutely requires a basic residue at the P4 position. Either of the serines or the valine can be replaced with alanine without affecting cleavage, but a leucine to valine replacement abolishes cleavage. In an accompanying paper (9Espenshade P.J. Cheng D. Goldstein J.L. Brown M.S. J. Biol. Chem. 1999; 274: 22795-22804Abstract Full Text Full Text PDF PubMed Scopus (146) Google Scholar), we show that the S1P precursor is inserted into the ER lumen by virtue of a cleaved NH2-terminal signal sequence. The protein remains attached to ER membranes through a hydrophobic transmembrane segment near the COOH terminus. This segment is followed by a short positively charged sequence that extends into the cytosol. Signal peptidase generates the pro form of S1P, which is designated S1P-A. This form is inactive. S1P-A is activated by intramolecular cleavage at Site-B, which follows lysine-137. The hamster sequence at this site is RSLKF. This releases a propeptide of 115 amino acids and activates the enzyme. This propeptide is secreted from cells, apparently in an intact form. Subsequently, S1P undergoes cleavage after the second leucine of the sequence RRLLR, which removes an additional 49 amino acids and generates S1P-C (amino acids 187–1052). The carbohydrates of S1P-A and S1P-B remain endo H-sensitive, indicating that these proteins are located in pre-Golgi compartments. S1P-C is endo H-resistant, suggesting that it resides in the Golgi or more distal compartments. Mutation of the active site serine of S1P (S414A) inactivates the enzyme and prevents generation of either S1P-B or S1P-C (9Espenshade P.J. Cheng D. Goldstein J.L. Brown M.S. J. Biol. Chem. 1999; 274: 22795-22804Abstract Full Text Full Text PDF PubMed Scopus (146) Google Scholar), indicating that these cleavages are autocatalytic. Studies of S1P are facilitated by the availability of SRD-12B cells, a line of mutant Chinese hamster ovary (CHO) cells that fails to produce S1P mRNA and protein, owing to mutations in both copies of the S1P gene (1Sakai J. Rawson R.B. Espenshade P.J. Cheng D. Seegmiller A.C. Goldstein J.L. Brown M.S. Mol. Cell. 1998; 2: 505-514Abstract Full Text Full Text PDF PubMed Scopus (328) Google Scholar, 10Rawson R.B. Cheng D. Brown M.S. Goldstein J.L. J. Biol. Chem. 1998; 273: 28261-28269Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar). As a result of this defect, SRD-12B cells are unable to release SREBPs from membranes, and they fail to transcribe multiple genes encoding the low density lipoprotein receptor and the enzymes of cholesterol and unsaturated fatty acid biosynthesis. These cells can grow only if they are supplied with exogenous sources of cholesterol and oleate. The activity of S1P in cleaving SREBPs has been studied so far only in intact cells. To understand this reaction more completely and to show directly that S1P is able to cleave the SREBP-2 recognition sequence, in the current studies, we prepared a permanent line of SRD-12B cells that expresses a truncated form of S1P that lacks the COOH-terminal membrane anchor. Despite the lack of membrane attachment, this truncated form of S1P processes SREBPs normally and alleviates the lipid growth requirements of the cells. Truncated S1P is secreted from the cells and can be isolated from the culture medium. This secreted enzyme cleaves peptide substrates corresponding to the recognition site in SREBP-2 and to the internal recognition site within S1P that releases the propeptide. These developments to the catalytic activity of S1P in We medium from and from from and and from and were from and and were by by of the peptide with or followed by and were in synthesis a peptide a the NH2-terminal domain of hamster SREBP-2 (amino acids prepared as described J. Brown M.S. Ho Y.K. Goldstein J.L. J. Biol. Chem. 1995; 270: Full Text Full Text PDF PubMed Scopus Google Scholar). were from sources described in the accompanying (9Espenshade P.J. Cheng D. Goldstein J.L. Brown M.S. J. Biol. Chem. 1999; 274: 22795-22804Abstract Full Text Full Text PDF PubMed Scopus (146) Google Scholar). an acid protein that of an amino acids of hamster S1P (1Sakai J. Rawson R.B. Espenshade P.J. Cheng D. Seegmiller A.C. Goldstein J.L. Brown M.S. Mol. Cell. 1998; 2: 505-514Abstract Full Text Full Text PDF PubMed Scopus (328) Google Scholar), amino acids by the sequence of copies of the from the protein amino acids by and is by the in as an by of a released from the and of a released from and (1Sakai J. Rawson R.B. Espenshade P.J. Cheng D. Seegmiller A.C. Goldstein J.L. Brown M.S. Mol. Cell. 1998; 2: 505-514Abstract Full Text Full Text PDF PubMed Scopus (328) Google Scholar), and a released from the of a from the the and an by of the a released from a and of a released from a and of and a released from the of a from the the and a of were These to two additional copies of the The were into the released from the and of The from this is designated the protein as does that the encoding the amino acids of S1P so as to the membrane and COOH-terminal The for the of this to that for for the second in which a from the and as described of the and in of the were by cholesterol SRD-12B cells were at a density of in (9Espenshade P.J. Cheng D. Goldstein J.L. Brown M.S. J. Biol. Chem. 1999; 274: 22795-22804Abstract Full Text Full Text PDF PubMed Scopus (146) Google with and oleate. cells were transfected with of either or an and in with the medium to with without The medium second were of the growth of SRD-12B cells in the of (1Sakai J. Rawson R.B. Espenshade P.J. Cheng D. Seegmiller A.C. Goldstein J.L. Brown M.S. Mol. Cell. 1998; 2: 505-514Abstract Full Text Full Text PDF PubMed Scopus (328) Google Scholar). cell that S1P were isolated by and for S1P by with the The cell and are designated and cells, of cells were in in (9Espenshade P.J. Cheng D. Goldstein J.L. Brown M.S. J. Biol. Chem. 1999; 274: 22795-22804Abstract Full Text Full Text PDF PubMed Scopus (146) Google with and of cells were at a density of the medium replaced with of medium The medium from from to The medium through low protein membrane and at for the medium to pH by of at a of and with of at pH The at at The with of with followed by of without with of pH The with a followed by a The with an of and in multiple at without of activity for at of cells of S1P activity with reaction in of Mes, and to pH with contained in and of for at reaction by of of The 7-amino-4-methyl-coumarin with a of with of reaction in a of of a acid synthetic peptide and of for at the reaction were by a a in acid at with a of were by a from with acid as the the of the S1P that were used in these The S1P signal sequence at residue The propeptide cleavage site is after residue Cleavage at this site generates S1P-B. This is followed by a second cleavage after residue 186 to generate The active site serine is at residue and the transmembrane sequence at residue The wild-type protein terminates at residue We prepared a cDNA encoding a truncated form of S1P that terminates after residue the transmembrane sequence and the COOH-terminal copies of a and are at the COOH of both cDNAs encoding the proteins in were into SRD-12B cells, which lack S1P R.B. Cheng D. Brown M.S. Goldstein J.L. J. Biol. Chem. 1998; 273: 28261-28269Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar). cell were in the of cholesterol and a that for cells that The permanent that and truncated protein are called and To of the transfected S1P, the cells were and a membrane to and with an the In to the of S1P we the cells in the of with or without the S1P is to be active both We in the presence of a of and cholesterol that is to S1P activity (1Sakai J. Rawson R.B. Espenshade P.J. Cheng D. Seegmiller A.C. Goldstein J.L. Brown M.S. Mol. Cell. 1998; 2: 505-514Abstract Full Text Full Text PDF PubMed Scopus (328) Google Scholar). As membranes from wild-type cells and mutant SRD-12B cells not a protein that with the from the cells of protein that to the from and from the cells two that were than the in the cells We that these and These proteins were in the membrane they lack a transmembrane anchor. The of the S1P were not when the cells were in the presence of or As in nuclear from cells contained of the NH2-terminal segment of as by when the cells were in the of The when the cells were further of by with The when the cells were with The SRD-12B cells to nuclear SREBP-2 of these In the of nuclear SREBP-2 in both the cells and the cells in either the or presence of In both nuclear SREBP-2 by of and These data that the truncated S1P and the S1P were active and normally by in the transfected cells. of that membranes from of the cells contained the precursor form of SREBP-2. an to the transfected cells secreted S1P and to the form of the secreted this the cells were in medium for after which the medium and the cells were in a of the cell corresponding to of cells and of medium corresponding to of cells were to and with In the cells, protein in the and in the medium The contained corresponding to and In the cells, the contained two corresponding to the for and The medium contained of a protein corresponding to the of S1P(983)-C the the of S1P(983)-C in the medium to the of in the with the in the accompanying paper (9Espenshade P.J. Cheng D. Goldstein J.L. Brown M.S. J. Biol. Chem. 1999; 274: 22795-22804Abstract Full Text Full Text PDF PubMed Scopus (146) Google Scholar), in the cells, and after with peptide indicating that forms the only of S1P-A and S1P-B. S1P-B with S1P-C, which by endo In from the cells, the and forms were both to endo as as to peptide form and The medium from these cells contained a protein that in to S1P(983)-C This protein to peptide but to endo To the of this we the cells in in and the protein from the medium by of the a of an gel this corresponding from an gel and to the NH2-terminal sequence in This sequence corresponds to the from cleavage at of P.J. Cheng D. Goldstein J.L. Brown M.S. J. Biol. Chem. 1999; 274: 22795-22804Abstract Full Text Full Text PDF PubMed Scopus (146) Google Scholar). To the catalytic activity of the secreted enzyme, we prepared a of that a COOH-terminal of at the COOH terminus. does not show at it has been released from the peptide through substrates been used to convertases, and subtilisin-related enzymes J. 1997; PubMed Scopus Google Scholar). the when the peptide with secreted The sequence of this peptide corresponds to the sequence at of S1P, which is to be cleaved by S1P in an autocatalytic reaction (9Espenshade P.J. Cheng D. Goldstein J.L. Brown M.S. J. Biol. Chem. 1999; 274: 22795-22804Abstract Full Text Full Text PDF PubMed Scopus (146) Google Scholar). with for a with the at the The a of these which of and of of only of enzyme, which is but is for Cleavage absolutely the at the P4 cleavage of the peptide the enzyme not cleave the corresponding to Site-1 in SREBP-2 The of cleavage of at pH and cleavage at pH This differs from the with and the convertases, the pH of which are in the of J. 1997; PubMed Scopus Google Scholar). To further the recognition requirements for the we prepared a of of sequence and with the peptide by two its to cleavage by in from to were not S1P not cleave that to the recognition sequence for the convertases, or The peptide has been used for in of J. Biol. Chem. Full Text PDF PubMed Google cleavage of synthetic substrates by with of S1P(983)-C at for as described The S1P activity of of at and the activity of the substrates relative to this the of which from by in a The with of S1P(983)-C at for as described The S1P activity of of at and the activity of the substrates relative to this the of which from by Like J. 1997; PubMed Scopus Google Scholar), S1P of serine proteases, as and by high of which is more than The enzyme to by and but only at high The of In not we that the enzyme reaction not by of calcium the and in the we that the reaction by calcium cleavage of of protease of the peptide were with of S1P(983)-C in the or presence of the at for as described the of which from by in a of the peptide were with of S1P(983)-C in the or presence of the at for as described the of which from by The low activity of the S1P the that the activity be to a of a more active enzyme that not be the To this we an S1P(983)-C with a control or the and were to and with As in but not the control the The and were for the ability to cleave the with a control the enzyme activity in the the activity in the These data that the cleavage of the peptide in this is to the activity of The of S1P(983)-C to cleave the been to an of the enzyme to the RSVL sequence, or it be to the resistance of the to cleavage. To between these we prepared 16-residue of the amino acids the internal cleavage in S1P or Site-1 in SREBP-2 The were with or without S1P(983)-C, and the were by In the of S1P, both the with S1P(983)-C, these were cleaved to generate two These were by and the were to to from cleavage after and S1P(983)-C has the to cleave Site-1 in SREBP-2. The current that a truncated form of S1P is able to cleave peptide substrates corresponding to Site-1 in SREBP-2 and to the internal propeptide cleavage site within S1P The of these in from the for and the convertases, which are the only subtilisin-like enzymes in animal cells. The of secreted S1P were facilitated by the of SRD-12B cells, the growth requirements of which be by of S1P, when the protein truncated so as to the membrane-spanning and SRD-12B cells with encoding truncated S1P, we were able to for high by the cells in the of and of the cells for in the of truncated S1P in the The protein contained a that by cleavage by signal the S1P designated undergoes two further cleavages to generate S1P-B and S1P-C (9Espenshade P.J. Cheng D. Goldstein J.L. Brown M.S. J. Biol. Chem. 1999; 274: 22795-22804Abstract Full Text Full Text PDF PubMed Scopus (146) Google Scholar). with mutant forms of S1P that S1P-A is and that S1P-B is for of the cleavage of S1P-C, not cleave SREBPs within cells. This follows from with cells transfected with cDNAs encoding forms of SREBP-2 that in the luminal loop (8Duncan E.A. Brown M.S. Goldstein J.L. Sakai J. J. Biol. Chem. 1997; 272: 12778-12785Abstract Full Text Full Text PDF PubMed Scopus (154) Google Scholar). These show that SREBP-2 is cleaved when are in the endo the protein has the Golgi The carbohydrates S1P-A and S1P-B are in the endo form, indicating that these enzymes are in the as the In the carbohydrates S1P-C are in the endo form, indicating that S1P-C is either in the or it is to the Golgi after it is In either of the S1P-C is not in the where SREBP cleavage In the current studies, we that S1P(983)-C the only form of truncated S1P that secreted from transfected cells. Truncated and were only within the cells These proteins were to membranes, they membrane These data that and a sequence that allows to to a membrane protein that in a pre-Golgi S1P to the Golgi only after cleavage at for this is in the of in which the precursor is in a pre-Golgi by a which has been to of to the ER protein in J. 1997; PubMed Scopus Google Scholar). be for the of S1P-A and S1P-B that from cell by an S1P (9Espenshade P.J. Cheng D. Goldstein J.L. Brown M.S. J. Biol. Chem. 1999; 274: 22795-22804Abstract Full Text Full Text PDF PubMed Scopus (146) Google Scholar). after the propeptide cleavage reaction has remains the propeptide remains attached to the enzyme in a active only after it an where a further cleavage within the propeptide, its release from the enzyme J. 1997; PubMed Scopus Google Scholar). We not the propeptide from S1P after cleavage at or it remains attached secreted S1P(983)-C catalytic activity the of cleavage The only of enzyme. This is the by for in a of enzyme J. Biol. Chem. Full Text PDF PubMed Google for this low activity be the of in to SREBPs and is required for Site-1 cleavage in intact cells J. Nohturfft A. Goldstein J.L. Brown M.S. J. Biol. Chem. 1998; 273: 5785-5793Abstract Full Text Full Text PDF PubMed Scopus (191) Google Scholar). S1P differs from and the in its for (1Sakai J. Rawson R.B. Espenshade P.J. Cheng D. Seegmiller A.C. Goldstein J.L. Brown M.S. Mol. Cell. 1998; 2: 505-514Abstract Full Text Full Text PDF PubMed Scopus (328) Google Scholar, 7Sakai J. Nohturfft A. Goldstein J.L. Brown M.S. J. Biol. Chem. 1998; 273: 5785-5793Abstract Full Text Full Text PDF PubMed Scopus (191) Google Scholar). We not directly activates S1P or its only is to SREBPs to the that does activate the enzyme, we not be able to we to into the with SREBPs the of the membrane through the of the segment of to the COOH-terminal regulatory segment of SREBPs (6Sakai J. Nohturfft A. Cheng D. Ho Y.K. Brown M.S. Goldstein J.L. J. Biol. Chem. 1997; 272: 20213-20221Abstract Full Text Full Text PDF PubMed Scopus (199) Google Scholar). In to this in it be to SREBP as The in peptide cleavage additional in which S1P differs from and the In to the in pH optimum that has an for and it is by J. 1997; PubMed Scopus Google Scholar, J. Biol. Chem. Full Text PDF PubMed Google Scholar). S1P not calcium in the and it only by high of calcium This to the in the in which the two enzymes in the distal Golgi and in where the pH is and the calcium is S1P is to act in a pre-Golgi that is to a pH and a low calcium S1P is in its ability to cleave with lysine at the position. and the an for at the and lysine for this J. 1997; PubMed Scopus Google Scholar). In S1P cleaves after the lysine of the sequence to generate the propeptide. S1P cleaves peptide in but it does not cleave which is a of J. 1997; PubMed Scopus Google Scholar). S1P not cleave the peptide, which corresponds to Site-1 in but it cleave this sequence when it located at the of a 16-residue peptide the SREBP-2 sequence. is that the presence of the the of S1P for when in to is at the position. These are with the by J. S. A. J. M. M. M. S. A. 1999; PubMed Scopus Google Scholar), described the of a subtilisin-related called that is to These that they data that culture medium from cells with a encoding unable to cleave the peptide but able to cleave between the sequence when it contained in a acid synthetic peptide corresponding to the luminal loop sequence of SREBP-2. We and for for protein for and for and and for culture