Promoter sequences

Details Promoter sequences Promoter sequences

2001-06-26

2004-04-20

Prouty, Rebecca E. (Department: 1652)

Chemistry: molecular biology and microbiology

Micro-organism, per se ; compositions thereof; proces of...

Bacteria or actinomycetales; media therefor

C435S320100, C435S325000, C536S024100

Reexamination Certificate

active

06723553

ABSTRACT:

TECHNICAL FIELD
The present invention relates an isolated human Site-1 Protease promoter region. The invention also relates to screening methods for agents decreasing the expression of Site-1 protease and thereby being potentially useful for the treatment of medical conditions related to obesity and/or diabetes.
BACKGROUND ART
Sterol Regulatory Element-Binding Proteins (SREBPs)
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 partly maintained by membrane-bound transcription factors called Sterol Regulatory Element-Binding Proteins (SREBPs; for reviews, see Brown & Goldstein (1997) Cell 89, 331-340; Brown & Goldstein (1999) Proc. Natl. Acad. Sci. U.S.A. 96, 11041-11048) that activate genes encoding enzymes of cholesterol and fatty acid biosynthesis. To enhance transcription, the active NH
2
-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. These regulated proteolytic cleavage reactions are ultimately responsible for controlling the level of cholesterol in membranes, cells, and blood.
Three isoforms of SREBPs have been identified. SREBP-1a and SREBP-1c are encoded by a single gene and differ in their N-terminal acid transcription activation domains. The N-terminus of SREBP-1a is longer and includes additional acidic amino acids, consistent with the observation that it is a more powerful transcription factor (Pai et al. (1998) Proc. Natl. Acad. Sci. U.S.A. 40, 26138-26148). SREBP-2 is produced by a different gene and contains a long activation domain resembling that of SREBP-1a. Recent evidence suggests that the main function of SREBP-2 is to regulate cholesterol synthesis whilst that of SREBP-1 is to regulate fatty acid synthesis (Pai et al., supra).
Inhibition of SREBP transcription factor function will lead to reduced cellular synthesis of free fatty acids and cholesterol, the clinical benefits of which are expected to include increased cellular insulin sensitivity and reduced coronary artery disease (CAD). Furthermore, SREBP-1 represents a cellular mechanism for increasing both fat cell size and number (Kim et al. (1998) J. Clin. Invest. 101, 1-9). Since most obesity generally involves an increase in both cell size and cell number, inhibition of SREBP-1 might be expected to have a positive effect on obesity. The hypolipidemic effects of dietary polyunsaturated fatty acids are believed to derive from a direct inhibitory effect on SREBP-1 expression (Xu et al. (1999) J. Biol. Chem. 274, 23577-23583).
There is data indicating independent regulation of SREBP-1 and SREBP-2 in hamster liver, suggesting the possibility for specific targeting of SREBP-1 or -2 (Sheng et al. (1995) Proc. Natl. Acad. Sci. U.S.A. 92, 935-938).
Transgenic mice over-expressing a dominant-positive form of SREBP-2 in the liver and adipose tissue showed greatly increased levels of mRNAs encoding multiple enzymes of cholesterol synthesis. Enzymes involved in fatty acid synthesis were also increased, however, to a lesser extent (Horton et al. (1998) J. Clin. Invest. 101, 2331-2339). Transgenic mice over-expressing a constitutively active SREBP-1a in the liver and adipose tissue showed greatly increased mRNA levels for enzymes involved in fatty acid and cholesterol (Shimano et al. (1996) J. Clin. Invest. 98, 1575-1584). Their livers were enlarged about 4-fold due to a massive accumulation of free fatty acids and cholesterol. Over-expression of a corresponding version of SREBP-1c in adipocytes of transgenic mice yielded insulin resistance and diabetes (Shimomura et al. (1999) Genes Dev. 12, 3182-3194). In cell culture such overexpression was previously shown to promote adipocyte differentiation. It has further been shown that overnutrition increases SREBP-1c expression in liver and islets of obese fa/fa Zucker diabetic fatty rats (Kakuma, T. et al. (2000) Proc. Natl. Acad. Sci. U.S.A. 97: 8536-8541).
S-1 Protease
As discussed above, SREBPs are activated by proteolysis, which releases the active transcription factor. The luminal subtilisin-like protease Site-1 Protease (S1P) is responsible for the first of the two proteolytic steps. Cleavage by S1P enables further cleavage by a Site-2 protease. S1P is the target for feedback inhibition by cholesterol.
S1P from hamster has been cloned (Sakai et al. (1998) Molecular Cell 2, 505-514). (GenBank accession no. AF078105; SEQ ID NOS: 5 and 6). The corresponding sequence of the (then unidentified) human gene was disclosed by Nagase et al. (1995) DNA Research 2, 37-43 (GenBank Accession no. D420453; SEQ ID NOS: 3 and 4)
SREBP and S1P are co-localized with a third protein: SREBP Cleavage-Activating Protein (SCAP), which is required for Site-1 cleavage in vivo. SCAP contains a site for sterol regulation, conserved in a small number of proteins, e.g. HMG-CoA reductase.
Only one S1P has been identified among the human expressed sequence tags (ESTs). Thus, S1P may be the only member of a subfamily among the subtilisin-like proteases.
Consequently, SREBPs are important regulators of fat and sugar metabolism in mammals and direct or indirect down-regulation of SREBPs may be of therapeutic value in type II diabetes; obesity, hypercholesterolemia, and other cardiovascular diseases or dyslipidemias.
Site-1 Protease represents a molecular target for therapeutic intervention which is expected to interfere with the SREBP pathway. Two principally distinct concepts for inhibition of the site-1-protease activity may be postulated; (i) by inactivation of the proteolytic activity (classical inhibitors) or (ii) by modulation of the site-1-protease gene expression level. In order to modulate the expression of the site-1-protease gene, there is a need for identification of regulatory regions responsible for the regulation of Site-1 protease promoter. Such regulatory regions in the promoter could be used for the identification of agents that inhibit expression of Site-1 protease, and thereby for the inhibition of the SREBP pathway.
DISCLOSURE OF THE INVENTION
The 5′-flanking region (promoter region) of the human Site-1 Protease (S1P) gene has been cloned and sequenced. This promoter region is useful in biological assays for the identification of compounds that inhibit the transcription of the Site-1 Protease. Inhibition of the SREBP pathway is expected to have therapeutic value in type II diabetes; obesity, hypercholesterolemia, and other cardiovascular diseases or dyslipidemias.
Consequently, in a first aspect this invention provides an isolated human site-1 protease promoter region comprising a sequence selected from:
(a) the nucleotide sequence set forth as SEQ ID) NO: 2, or a fragment thereof exhibiting site-1 protease promoter activity;
(b) the complementary strand of (a); and
(c) nucleotide sequences capable of hybridizing, under stringent hybridization conditions, to a nucleotide sequence as defined in (a) or (b).
The term “promoter region” refers to a region of DNA that functions to control the transcription of one or more genes, and is structurally identified by the presence of a binding site for DNA-dependent RNA polymerase and of other DNA sequences on the same molecule which interact to regulate promoter function.
The nucleic acid molecules according to the present invention includes cDNA, chemically synthesized DNA, DNA isolated by PCR, genomic DNA, and combinations thereof. Genomic DNA may be obtained by screening a genomic library with the cDNA described herein, using methods that are well known in the art.
In a preferred form of the invention, the said nucleic acid molecule has a nucleotide sequence identical with SEQ ID NO: 2 of the Sequence Listing. However, the nucl

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