Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving hydrolase
Reexamination Certificate
2000-03-10
2001-12-25
Achutamurthy, Ponnathapu (Department: 1652)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving hydrolase
C435S023000, C435S219000, C435S350000, C435S395000, C435S320100, C435S254200, C435S325000, C435S348000, C435S349000, C530S350000, C536S023400
Reexamination Certificate
active
06333167
ABSTRACT:
CROSS REFERENCE TO RELATED APPLICATIONS
not applicable
ACKNOWLEDGEMENT OF FEDERAL RESEARCH SUPPORT
not applicable
BACKGROUND OF THE INVENTION
The present invention relates to methods and reagents for identifying compounds which inhibit proteolysis of polypeptides, especially membrane-associated polypeptides.
The amyloid precursor protein (APP) is an integral membrane protein located predominantly within intracellular vesicles. One of the well-characterized proteolytic processing pathways of APP results in the generation of a 40 or 42 amino acid peptide (A&bgr;40-42). Extracellular deposition and plaque formation nucleated by these amyloid A&bgr; peptides form a hallmark lesion in the brains of Alzheimer's disease patients [Selkoe, D. J. (1996)
J. Biol. Chem
. 271:18295-18298]. A&bgr;40-42 peptides are generated from the specific and sequential cleavage of the membrane-bound APP by the &bgr; and &ggr;-secretases [Selkoe, D. J. (1997)
Science
275:630-631; Lamb, B. T. (1997)
Nature Med
. 3:28-29]. &bgr;-secretase cleaves on the lumenal side of the ER and, together with the subsequent intrinsic membrane cleavage by &ggr;-secretase, determines the rate of A&bgr;40-42 generation. Notably, the &ggr;-secretase cleavage of APP bears a striking similarity to that of the SREBP S2P cleavage and, except for Notch processing (discussed below), is the only other known example of specific proteolysis occurring within a membrane-spanning segment [Brown and Goldstein (1997)
Cell
89:331-340; Sakai et al. (1996)
Cell
85:1037-1046]. Recently, the APP &ggr;-secretase and SREBP S2P have been shown to be two distinct enzymes [Tomita et al. (1998)
NeuroReport
9:911-913].
The Notch receptor family includes Notch in Drosophila, LIN-12 and GLP-1 in C. elegans, and mNotch1 and mNotch2 in mouse, among others [Artavanis-Tsakonas et al. (1995)
Science
268:225-232]. During development, Notch mediates cell-cell communications required for a variety of cell fate decisions and for axon guidance. Notch family members are large, multidomain proteins that consist of a single transmembrane domain and large extracellular and intracellular domains. Proteolytic cleavage of Notch is believed to result in release of the intracellular domain, which translocates to the nucleus and associates with a DNA-binding subunit [Li, X, and Greenwald, I. (1998)
Proc. Natl. Acad. Sci. USA
95:7109-7114; Thinakaran et al. (1996)
Neuron
17:181-190; Podlisny et al. (1997)
Neurobiol. Dis
. 3:325-337; Capell et al. (1998)
J. Biol. Chem
. 273:3205-3211]. A processing step responsible for releasing the intracellular domain takes place in or near the transmembrane domain [Li and Greenwald (1998) supra]. Thus, Notch appears to undergo proteolytic events that resemble those involved in cleavage of APP, i.e., sequential hydrolysis by &bgr; and &ggr;-secretases.
The regulation of hepatic cholesterol biosynthesis is central to the understanding of hypercholesterolemia as a risk factor for cardiovascular disease [Goldstein and Brown (1997)
Nature
343:425-430]. A sterol-activated transcription factor, the sterol regulatory element binding protein (SREBP), specifically binds sterol regulatory elements (SREs) in the regulatory region of a number of coordinately regulated genes, including the promoters of the low density lipoprotein (LDL) receptor and 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase [Briggs et al. (1993)
Cell
. 77:53-62; Wang et al. (1993)
J. Biol. Chem
. 268:14497-14504]. SREBP is maintained in an inactive state by its targeted localization to the endoplasmic reticulum (ER) membrane. Upon cholesterol deprivation, the amino-terminal portion of SREBP is specifically proteolyzed and liberated from its ER anchor in a two-step process [Wang et al. (1994)
J. Biol. Chem
. 271:10379-10384; Hua et al. (1996)
J. Biol. Chem
. 271:10379-10384; Sakai et al. (1996) supra]. This cleavage releases the amino-terminal segment of SREBP, allowing it to enter the nucleus, where it binds to enhancers and activates transcription of genes encoding the LDL receptor and multiple enzymes of cholesterol and fatty acid biosynthesis [Brown and Goldstein (1997) supra]. Sterols regulate initial proteolysis at site 1, which appears to be a prerequisite for site 2 hydrolysis, which occurs within the ER bilayer. The cDNA encoding the protease responsible for site 2 cleavage of the SREBPs (site 2 protease (S2P)) was isolated by complementation cloning and encodes a putative zinc metalloprotease with multiple transmembrane domains [Rawson et al. (1997)
Mol. Cell
. 1:47-57]. The site 1 protease (S1P), a membrane-bound subtilisin-related serine protease, has also recently been cloned and characterized [Espenshade et al. (1999)
J. Biol. Chem
. 274:22795-22804].
Due to the importance of membrane-associated proteolysis in gene expression, cellular differentiation and disease, considerable research effort has focused on membrane proteins, and particularly on identifying enzymes involved in their proteolytic pathways. Despite these efforts, until recently, only one of the several inferred proteolytic enzymes responsible for APP, SREBP and Notch maturation had been identified. Although at least three such proteases have now been cloned, specific and nontoxic inhibitors of these enzymes are not yet available. Thus, a need exists for an accurate, sensitive and economical high throughput screen to identify novel nontoxic inhibitors of proteolysis, especially membrane-associated proteolysis. In addition, due to the difficulty in isolating enzymes involved in lumenal and intrinsic membrane proteolysis, there exists a need for a simple and reliable technique which will enable tho large-scale screening of compounds for antiproteolytic activity without the need to first clone the target protease. The present invention fulfills these and other needs.
SUMMARY OF THE INVENTION
The present invention provides methods and compositions for identifying compounds which inhibit proteolysis of cellular proteins, particularly intrinsic membrane proteins and membrane-associated proteins. More specifically, the invention relates to methods and compositions for identifying compounds which prevent the functional interaction between a protease and its corresponding recognition site on a proteolytic substrate. Compounds that disrupt or prevent the functional interaction between these two proteins have significant modulatory effects on receptor maturation and signal transduction, and thus are useful for treating and/or preventing a wide variety of diseases and pathological conditions associated with transmembrane proteolytic events. Such conditions include, for example, neurological disorders, cardiac diseases, and metabolic diseases. Many of these compounds have potent neuroprotective properties which prove especially usefull for the prevention and/or treatment of neurodegenerative disorders such as Alzheimer's disease, as well as for the prevention and/or treatment of cardiovascular diseases and diabetes.
In one aspect, the invention provides methods of evaluating and screening candidate compounds for the ability to inhibit proteolysis of a proteolytic substrate. The methods comprise preparing a genetically modified (recombinant) host cell which expresses (i) a chimeric polypeptide comprising a transcription repressor and a protease recognition site, wherein the chimeric polypeptide is desirably attached to a non-nuclear membrane, and (ii) a reporter gene operably linked to transcription regulatory sequences which are responsive to the transcription repressor and which include a promoter. The candidate compound is then added to the modified host cell, and expression of the reporter gene is monitored. An increase in reporter gene expression over expression in the absence of the test compound is an indication that the compound inhibits proteolysis of the proteolytic substrate.
Reporter genes desirably give rise to gene products which
Quinet Elaine M.
Shuey David J.
Achutamurthy Ponnathapu
American Home Products Corp.
Fronda Christian L.
Greenlee Winner and Sullican PC
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