Protein inhibitor of neuronal nitric oxide synthase

Details Protein inhibitor of neuronal nitric oxide synthase Protein inhibitor of neuronal nitric oxide synthase

1998-12-23

2001-01-02

Bui, Phuong T. (Department: 1645)

Chemistry: molecular biology and microbiology

Measuring or testing process involving enzymes or...

Involving antigen-antibody binding, specific binding protein...

C435S004000, C435S006120, C435S007200, C435S007210, C435S007310, C435S007800, C435S041000, C435S069100, C435S069700, C435S070400, C435S070300, C435S320100, C530S300000, C530S402000, C530S827000, C530S839000

Utility Patent

active

06168926

ABSTRACT:

TECHNICAL AREA OF THE INVENTION
The invention relates to the area of neurotransmitter regulation. More particularly, the invention relates to the regulation of neuronal nitric oxide synthase.
BACKGROUND OF THE INVENTION
Nitric oxide (NO) is a major messenger molecule in the cardiovascular, immune and nervous systems. In the brain, NO is responsible for the glutamate-linked enhancement of 3′, 5′ cyclic guanosine monophosphate (cGMP) levels (S. R. Jaffrey and S. H. Snyder,
Annu. Rev. Cell Dev. Biol.
11, 417, 1995) and may be involved in apoptosis (E. Bonofoco et al.,
Proc. Natl. Acad Sci. U.S.A.
92, 7162, 1995; J. B. Mannick et al.,
Cell
79, 1137, 1994), synaptogenesis (Jaffrey and Snyder, ibid.; T. Wang, Z. Xie, and B. Lu,
Nature
374, 262, 1995) and neuronal development (Jaffrey and Snyder, ibid.).
Since NO cannot be stored in vesicles like other neurotransmitters, its release is regulated by the activity of the enzyme which makes it, NO synthase (NOS). Although a number of substances are known to regulate transcription of NOS, it is possible that regulation occurs at other levels as well. For example, several enzymes are influenced by physiologically associated proteins that serve as enzyme inhibitors. Examples include cyclin-dependent kinase inhibitors (A. Kamb,
Trends Genet.,
11, 136, 1995; S. J. Elledge and J. W. Harper,
Curr. Opin. Cell Biol.
6, 847, 1994), the FoF1 ATPase inhibitor (J. E. Walker,
Curr. Opin. Struct. Biol.
4, 912, 1994), and the ornithine decarboxylase inhibitor antizyme (J. S. Heller, W. F. Fong, E. S. Canellakis,
Proc. Natl. Acad. Sci. U.S.A.
73, 1858, 1976). Such regulation of NOS by protein inhibitors was not known.
NO mediates glutamate neurotoxicity, which has been implicated in debilitating and lethal neurodegenerative disorders such as Alzheimer's and Huntington's diseases (D. W. Choi,
J. Neurosci
10, 2493-2501; B. Meldrum and J. Garthwaite,
Trends Phannacol. Sci.
11, 379-387, 1990). Thus, there is a continuing need in the art of neurotransmitter regulation for methods of affecting the activity of neuronal NOS, so that one can manipulate NO levels when required for therapeutic effect in such disorders.
SUMMARY OF THE INVENTION
It is an object of the invention to provide an isolated mammalian PIN-1 (Protein Inhibitor of nNOS) protein.
It is another object of the invention to provide a fusion protein comprising at least eight contiguous amino acids selected from the PIN-1 amino acid sequence shown in SEQ ID NO:2.
It is yet another object of the invention to provide an isolated polypeptide consisting of at least eight contiguous amino acids of PIN-1 as shown in SEQ ID NO:2 and capable of binding a rat nNOS domain within amino acids 163-245 as shown in SEQ ID NO:3.
It is still another object of the invention to provide a preparation of antibodies which specifically bind to a PIN-1 protein as shown in SEQ ID NO:2.
It is even another object of the invention to provide a subgenomic polynucleotide which encodes a PIN-1 protein as shown in SEQ ID NO:2.
It is yet another object of the invention to provide a recombinant DNA construct for expressing PIN-1 antisense nucleic acids.
It is still another object of the invention to provide a method of inhibiting a mammalian neuronal nitric oxide synthase (nNOS).
It is even another object of the invention to provide methods of screening test compounds for the ability to decrease or augment the activity of nNOS.
These and other objects of the invention are provided by one or more of the embodiments described below. One embodiment of the invention provides an isolated mammalian PIN-1 protein which has the sequence shown in SEQ ID NO:2 and naturally occurring biologically active variants thereof.
Another embodiment of the invention provides a mammalian PIN-1 fusion protein which comprises two protein segments fused to each other by means of a peptide bond, wherein one of the protein segments consists of at least eight contiguous amino acids selected from the amino acid sequence shown in SEQ ID NO:2.
Yet another embodiment of the invention provides an isolated polypeptide which consists of at least eight contiguous amino acids of PIN-1 as shown in SEQ ID NO:2, wherein the polypeptide binds to a rat nNOS domain within amino acids 163-245 as shown in SEQ ID NO:3.
Still another embodiment of the invention provides a preparation of antibodies which specifically bind to a mammalian PIN-1 protein as shown in SEQ ID NO:2.
Even another embodiment of the invention provides a subgenomic polynucleotide which encodes a PIN-1 protein as shown in SEQ ID NO:2.
Yet another embodiment of the invention provides a recombinant DNA construct for expressing PIN-1 antisense nucleic acids, comprising a promoter and a coding sequence for PIN-1 consisting of at least 12 contiguous base pairs selected from SEQ ID NO: 1, wherein the coding sequence is in an inverted orientation with respect to the promoter, such that upon transcription from the promoter an RNA is produced that is complementary to native mRNA encoding PIN-1.
Still another embodiment of the invention provides a method of decreasing a mammalian nNOS, comprising the step of contacting a nNOS with a PIN-1 protein having an amino acid sequence as shown in SEQ ID NO:2.
Even another embodiment of the invention provides a method of screening test compounds for the ability to decrease or augment nNOS activity comprising the steps of: (a) contacting a test compound with a mixture of a mammalian PIN-1 protein and two molecules which bind to each other by virtue of a nNOS dimerization domain or naturally occurring biologically active variants thereof, and (b) measuring the amount of PIN-1 or of at least one of the two molecules that is bound or unbound in the presence of the test compound. A test compound that increases the amount of PIN-1 or decreases the amount of the two molecules that are bound is a potential drug for decreasing nNOS activity. A test compound that decreases the amount of PIN-1 or increases the amount of the two molecules that are bound is a potential drug for augmenting nNOS activity.
In one embodiment of the invention the test compound is contacted with a cell lysate containing nNOS and PIN-1 or naturally occurring biologically active variants thereof. The proteins in the lysate are separated by electrophoresis in an SDS-polyacrylamide gel under non-reducing conditions, and the amount of NNOS monomers or dimers in the gel is measured by immunoblotting. A test compound that decreases the amount of nNOS monomers or increases the amount of nNOS dimers is a potential drug for augmenting nNOS activity. A test compound that increases the amount of nNOS monomers or decreases the amount of nNOS dimers is a potential drug for decreasing nNOS activity.
Yet another embodiment of the invention provides a method of screening test compounds for the ability to decrease or augment nNOS activity comprising the steps of: (a) contacting a cell with a test compound, wherein the cell comprises i) a first fusion protein comprising (1) a DNA binding domain and (2) all or a portion of a mammalian PIN-1 protein, wherein the portion consists of a contiguous sequence of amino acids selected from the amino acid sequence shown in SEQ ID NO:2, wherein the portion is capable of binding to nNOS; ii) a second fusion protein comprising (1) a transcriptional activating domain and 2) all or a portion of nNOS, wherein the portion consists of a contiguous sequence of amino acids selected from amino acids 163-245 as shown in SEQ ID NO:3, or naturally occurring biologically active variants thereof, and wherein the interaction of the portion of the PIN-1 protein with the portion of nNOS reconstitutes a sequence specific transcriptional activating factor; and iii) a reporter gene comprising a DNA sequence to which the DNA binding domain of the first fusion protein specifically binds; and (b) measuring the expression of the reporter gene. A test compound that increases the expression of the reporter gene is a potential drug for decreasing nNOS activity. A test compound tha

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