Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or...
Reexamination Certificate
1999-07-23
2001-11-27
Martin, Jill D. (Department: 1632)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
C435S006120, C435S320100, C435S325000, C435S455000, C530S350000, C536S023100, C536S023400
Reexamination Certificate
active
06322962
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates generally to the fields of medicine. More particularly, it concerns assays for the identification of modulators of cholesterol and fatty acid metabolism.
2. Description of Related Art
Cholesterol and fatty acids are the hydrophobic building blocks of cell membranes. Their synthesis and uptake must be coordinated so as to supply sufficient amounts for new membrane synthesis while avoiding over accumulation. Coordination is achieved by a family of transcription factors designated sterol regulatory element binding proteins (SREBPs) that are bound to membranes of the endoplasmic reticulum (ER) and nuclear envelope (Brown and Goldstein, 1997). When cells are deprived of sterols, a two-step proteolytic process releases the active portions of the SREBPs from cell membranes, allowing them to translocate to the nucleus where they activate transcription of more than a dozen genes encoding enzymes required for biosynthesis and uptake of cholesterol and unsaturated fatty acids. When sterols build up in cells, the proteolytic release process is blocked, the SREBPs remain membrane-bound, and transcription of the target genes declines. A crucial component in this regulatory pathway is the Site-1 protease (S1P), which makes the first cut in the SREBPs, thereby initiating release (Sakai et al., 1996; Duncan et al.,1997). S1P is the target of feedback regulation: its activity is extinguished in sterol-overloaded cells. So far, nothing is known about the structure or properties of S1P.
SREBPs are bound to the ER membrane and nuclear envelope in a hairpin orientation. The NH
2
-terminal and COOH-terminal domains, each about 500 amino acids in length, project into the cytosol. They are linked by a pair of membrane-spanning sequences that flank a short 31-amino acid hydrophilic loop that projects into the lumen of the ER and nuclear envelope (Brown and Goldstein, 1997). When cells are depleted of sterols, S1P cleaves the SREBPs at a leucine-serine bond in the luminal loop, thereby separating the proteins into halves, each with a single membrane spanning sequence (Duncan et al., 1997). The NH
2
-terminal half is called the “intermediate” form of SREBP. Next, a second protease, designated Site-2 protease (S2P), cleaves the NH
2
-terminal intermediate at a leucine-cysteine bond that is located just within the first membrane spanning segment (Duncan et al., 1998). This liberates the NH
2
-terminal fragment, which dissociates from the membrane with three hydrophobic residues at its COOH-terminus. This fragment, designated nuclear SREBP (nSREBP) enters the nucleus and activates gene transcription.
The Site-1 cleavage reaction requires the participation of a membrane-bound regulatory protein designated SREBP cleavage-activating protein (SCAP) (Hua et al., 1996a). SCAP has two domains: a hydrophobic NH
2
-terminal membrane domain consisting of eight membrane spanning sequences and a hydrophilic COOH-terminal domain containing five “WD” repeats that projects into the cytosol (Nohturfft et al., 1998). The COOH-terminal domain of SCAP forms a tight complex with the COOH-terminal domain of the SREBPs (Sakai et al., 1997; Sakai et al., 1998a). Disruption of this complex by overexpression of truncated dominant negative versions of SCAP or SREBP blocks Site-1 cleavage of SREBPs, indicating that the SCAP/SREBP complex is absolutely required for cleavage. Moreover, truncated versions of SREBPs, which lack the COOH-terminal domain, fail to form complexes with SCAP and fail to undergo Site-1 cleavage (Sakai et al., 1998a).
Although the SCAP/SREBP complex is created by interactions on the cytosolic side of the membrane, the complex activates S1P. which cuts the SREBPs on the opposite (luminal) side (Sakai et al., 1998a). The protease cuts between the leucine and serine of the sequence RSVLS (SEQ ID NO:10). Recognition requires only the arginine and leucine: the other residues can be replaced with alanines without reducing cleavage (Duncan et al., 1997).
The Site-1 processing reaction is the target for feedback regulation of lipid biosynthesis and uptake in animal cells. When sterols accumulate in cells, the Site-1 cleavage reaction is blocked (Brown and Goldstein, 1997). The Site-2 cleavage reaction is blocked secondarily since it requires prior cleavage by S1P. The sterol effect appears to be mediated by five of the eight membrane spanning sequences of SCAP, which are designated as the sterol sensor (Hua et al., 1996a). Point mutations at two positions within the sterol sensor render SCAP constitutively active and prevent sterol-mediated suppression of Site-1 cleavage (Nohturfft et al., 1996). Sequences that resemble the sterol-sensing domain are found in three other proteins that are postulated to interact with sterols (Loftus et all., 1997; Nohturfft et al., 1998).
The human gene for S2P has recently been cloned by complementation of the growth defect in a mutant line of Chinese hamster ovary (CHO) cells that fails to synthesize cholesterol owing to a deletion of the S2P gene (Rawson et al., 1997). This gene encodes a unique hydrophobic zinc metalloprotease that cleaves the intermediate forms of SREBPs within their transmembrane sequences.
A similar approach to complementation cloning of S1P has been unavailable up to now because of the difficulty which has been associated with isolating a mutant cell line that fails to carry out Site-1 cleavage. This inability has represented a significant obstacle in the pursuit of further knowledge regarding the regulation of cholesterol and fatty acid metabolism. There is, therefore, a great need in the art for obtaining further information regarding S1P and more specifically, for cloning S1P. Once S1P is cloned, there will be a great need to identify efficient assays which may be employed for the identification of inhibitors of S1P, the inhibitors potentially having great therapeutic value for the treatment of hypercholsterolemia or other lipid metabolism associated conditions.
SUMMARY OF THE INVENTION
In one aspect, the invention provides a method of preparing a candidate modulator of cholesterol biosynthesis in a target cell comprising identifying an agent capable of down regulating Site-1 protease activity in said cell and formulating a composition comprising said agent. In particular embodiments, the modulator is identified by a method comprising the steps of: (a) providing a cell comprising a Site-1 protease; (b) contacting said cell with a candidate modulator; and (c) monitoring said cell for an effect that is not present in the absence of said candidate modulator. The cell may be a mammalian cell, and may be a human cell a hamster cell, a cow cell, a goat cell, a sheep cell, a rat cell or a mouse cell. In one embodiment the mammalian cell is a hamster cell and said Site-1 protease comprises the amino acid sequence of SEQ ID NO:1, or is a human cell and said Site-1 protease comprises the amino acid sequence of SEQ ID NO:3. In another embodiment, the Site-1 protease is encoded by a transgene comprising the nucleotide sequence of SEQ ID NO:2.
In the method, the mammalian cell may be a human cell and said Site-1 protease may comprise the amino acid sequence of SEQ ID NO:3. The Site-1 protease may be encoded by a transgene comprising the nucleotide sequence of SEQ ID NO:4. In particular embodiments of the method, the effect is cholesterol or fatty acid biosynthesis or lipid uptake.
In another aspect, the invention provides a method for preparing a modulator of Site-1 protease comprising the steps of (a) preparing a solution comprising a candidate modulator, Site-1 protease, and a polypeptide target sequence capable of being cleaved by said Site-1 protease; (b) monitoring the rate cleavage of said target sequence in the presence of the candidate modulator relative to the rate of cleavage in the absence of the candidate: and (c) preparing a composition comprising said identified modulator. In other embodiments, the solution further comprises SREBP cleavage activating protein. In one embodiment
Brown Michael S.
Cheng Dong
Espenshade Peter J.
Goldstein Joseph L.
Rawson Robert B.
Board of Regents , The University of Texas System
Fulbright & Jaworski
Martin Jill D.
Paras, Jr. Peter
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