Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving antigen-antibody binding – specific binding protein...
Reexamination Certificate
2000-09-15
2003-05-06
Huff, Sheela (Department: 1642)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving antigen-antibody binding, specific binding protein...
C435S007100, C435S007210
Reexamination Certificate
active
06558912
ABSTRACT:
BACKGROUND OF THE INVENTION
The invention relates to proteins involved in neurotrophin signaling.
The regulation of cell growth and survival is believed to be under the control of a wide variety of signaling cascades. One such cascade is that which transduces the presence of a neurotrophin ligand. Early in vivo and in vitro experiments demonstrated that nerve growth factor (NGF) plays critical roles in the development of the nervous system. The cloning of brain-derived neurotrophic factor (BDNF) subsequently revealed a homology with NGF that spurred the cloning and characterization of neurotrophin-3 (NT-3), neurotrophin-4/5 (NT-4/5) and neurotrophin-6 (NT-6). Each of these proteins promotes survival of specific populations of neurons and affects aspects of the neuronal phenotype.
The mammalian neurotrophins interact with two types of cell surface receptors. The trk receptors (e.g., trkA, trkB, and trkC) are highly-related transmembrane receptor tyrosine kinases, each of which preferentially binds one or a subset of the neurotrophin family members trk receptors play a critical role in mediating the effects of the neurotrophins, and their activation results in the effects typically associated with neurotrophin action.
The p75NTR receptor (p75NTR), which binds all neurotrophins with approximately equal affinity, is a member of the tumor necrosis factor (TNF) receptor superfamily. In contrast to the rapid progress made in elucidating the mechanism of action of the trk receptors, the physiological roles of the p75NTR have been more difficult to discern. At present, the actions of p75NTR fall into two categories. First, p75NTR appears to functionally collaborate with trk receptors to either enhance or reduce neurotrophin-mediated trk receptor activation. Second, p75NTR acts autonomously to activate signaling cascades that may be involved in apoptosis and inflammation.
A number of proteins which directly interact with the intracellular domains of members of the TNF receptor superfamily have been identified, including TRADD, FADD, and members of the TRAF family. Generally, the intracellular domain of p75NTR is highly conserved across species, but not conserved with other members of the TNF receptor superfamily. The exception to the general lack of homology between p75NTR and the TNF receptor superfamily is the presence of a 90 amino acid stretch termed the “death domain.” In TNF receptor-1 (TNFR-1), fas, DR3, and other related receptors, the death domain is required to mediate interactions with either FADD or TRADD. We have previously found, however, that the p75NTR does not bind TRADD or FADD proteins. The failure of p75NTR to bind FADD or TRADD is likely due to the fact that the tertiary structure of the p75NTR death domain differs considerably from these other, receptors, suggesting that p75NTR must facilitate apoptosis through another mechanism. Thus, the mechanism by which neurotrophins signal via p75NTR to modulate apoptosis remains unknown.
There is a need to identify additional components of the neurotrophin signal transduction pathway. These components would be useful as targets for pharmacologic intervention in patients diagnosed with an apoptotic disease.
SUMMARY OF THE INVENTION
In a first aspect, the invention features a substantially pure NRAGE polypeptide (previously referred to as PRI-MAGE). In a preferred embodiment, the polypeptide is from a mammal (e.g., a human). In a second preferred embodiment, the polypeptide binds to p75NTR.
In a second aspect, the invention features a substantially pure polypeptide having 50% or greater amino acid sequence identity to the amino acid sequence of the protein of the first aspect and binds to p75NTR. Preferably, the polypeptide has 70% or greater amino acid sequence identity to the amino acid sequence of the protein of the first aspect and binds to p75NTR. More preferably, the polypeptide has 85% or greater amino acid sequence identity to the amino acid sequence of the protein of the first aspect and binds to p75NTR.
In a third aspect, the invention features a substantially pure polypeptide that modulates apoptosis, wherein the polypeptide has 50% or greater amino acid sequence identity to an amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2.
Preferably, the polypeptide has 70% or greater amino acid sequence identity to the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2. More preferably, the polypeptide has 80% or greater amino acid sequence identity to the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2.
In a fourth aspect, the invention features a substantially pure nucleic acid molecule encoding an NRAGE polypeptide. In one embodiment, the nucleic acid molecule is from a mammal (e.g., a human). In another embodiment, the nucleic acid is cDNA.
In a fifth aspect, the invention features substantially pure DNA having a sequence of
FIG. 1C
(SEQ ID NO: 3) or
FIG. 1D
(SEQ ID NO: 4), or degenerate variants thereof, and encoding an amino, acid sequence of
FIG. 1A
(SEQ ID NO: 1) or
FIG. 1B
(SEQ ID NO: 2).
In a sixth aspect, the invention features substantially pure DNA that binds at high stringency to a DNA sequence of
FIG. 1C
(SEQ ID NO: 3) or
FIG. 1D
(SEQ ID NO: 4).
In a seventh aspect, the invention features purified DNA sequence substantially identical to a DNA sequence shown in
FIG. 1C
(SEQ ID NO: 3) or
FIG. 1D
(SEQ ID NO: 4).
In one embodiment, the nucleic acid is operably linked to regulatory sequences for expression of the polypeptide encoded by the nucleic acid and wherein the regulatory sequences comprise a promoter. Preferably, the promoter is a constitutive promoter, is inducible by one or more external agents, or is cell-type specific.
In an eighth aspect, the invention features a vector that includes the nucleic acid of the seventh aspect, the vector being capable of directing expression of the polypeptide encoded by the nucleic acid in a vector-containing cell.
In a ninth aspect, the invention features a cell expressing the nucleic acid of the seventh aspect.
In a tenth aspect, the invention features a method for identifying a compound that modulates binding of NRAGE to p75NTR. The method includes: (a) providing a cell expressing an NRAGE polypeptide; (b) contacting the cell with a candidate compound; and (c) monitoring the level of binding of the NRAGE polypeptide to p75NTR, wherein a change in the level of the binding in response to the candidate compound relative to a level of binding in a cell not contacted with the candidate compound indicating the presence of a compound that modulates binding of NRAGE to p75NTR. Preferably, the cell is from a mammal (e.g., a human or a rodent).
In an eleventh aspect, the invention features a kit for determining the amount of NRAGE polypeptide in a sample, the kit including a substantially pure antibody that specifically binds an NRAGE polypeptide. Preferably, the kit further includes a means for detecting the binding of the antibody to the NRAGE polypeptide.
In a twelfth aspect, the invention feature a substantially pure antibody that specifically binds to an NRAGE polypeptide. In one embodiment, the antibody is selected from a group consisting of a polyclonal antibody, a monoclonal antibody, and a neutralizing antibody.
In a thirteenth aspect, the invention features a method for modulating cell apoptosis, the method includes administering an NRAGE polypeptide to the cell.
In a fourteenth aspect, the invention features a method for modulating apoptosis. The method includes administering to the cell a compound that modulates binding of NRAGE to p75NTR. In various embodiments, the compound is a chemical, a drug, or an antibody that specifically binds to an NRAGE polypeptide. A preferred antibody is a neutralizing antibody. In one preferred embodiment, the compound is an NRAGE antisense nucleic acid molecule.
In preferred embodiments of the thirteenth or fourteenth aspect, the cell is in a mammal (e.g., a human or a rodent).
In another embodiment of the thirteenth or fourteenth aspects, apoptosis is decreased in a mammal diagnosed as being HIV-positive, or as
Barker Philip
Salehi Amir
Verdi Joseph
Bieker-Brady Kristina
Clark & Elbing LLP
Huff Sheela
McGill University
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