Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving antigen-antibody binding – specific binding protein...
Reexamination Certificate
2001-03-13
2002-10-15
Kemmerer, Elizabeth (Department: 1646)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving antigen-antibody binding, specific binding protein...
C435S006120, C436S501000
Reexamination Certificate
active
06465196
ABSTRACT:
FIELD OF THE INVENTION
The field of this invention is cholesterol regulation.
BACKGROUND OF THE INVENTION
Atherogenic low density lipoproteins are cleared from the circulation by low density lipoprotein receptors (LDLR) in the liver. Two inherited forms of hypercholesterolemia result from loss of hepatic LDLR activity: the autosomal dominant disorder, familial hypercholesterolemia (FH), caused by mutations in the LDLR gene, and autosomal recessive hypercholesterolemia (ARH) in which the LDLR gene is normal. We mapped the ARH locus to a ~1 centimorgan interval on chromosome 1p35 and identified six mutations in a gene encoding a new adaptor protein, LDLR adaptin, in 32 alleles from 16 unrelated ARH patients. LDLR adaptin contains a phosphotyrosine binding (PTB) domain, which binds NPXY (SEQ ID NO:7) motifs in the cytoplasmic tails of cell surface receptors, including the LDLR. Defects in LDLR adaptin result in selective impairment of LDLR function in the liver, whereas correction of LDLR adaptin defects and stabilization of LDLR adaptin function reduces associated hypercholesterolemia.
SUMMARY OF THE INVENTION
The invention provides methods and compositions relating to hypercholesterolemia. The inventors disclose a novel, key component of human cholesterol regulation, which provides a target for rational drug design and screening, therapeutic intervention, and diagnosis.
In particular embodiments, the invention provides a variety of LDLR adaptin and LDLR adaptin PTB and CC domain compositions, including compositions comprising a recombined natural human LDLR adaptin PTB or CC domain, and in vitro compositions comprising a determined amount of a natural human LDLR adaptin PTB domain and a ligand such as an NPXY (SEQ ID NO:7) peptide, or a determined amount of a natural human LDLR adaptin CC domain and ligand such as an LDL Receptor (SEQ ID NO:8) or a fragment thereof sufficient to selectively bind said domain.
The LDLR adaptin reagents are used, inter alia, in rational drug screening methods. An exemplary method comprises the steps of combining a disclosed LDLR adaptin reagent with a ligand and an agent under conditions wherein but for the presence of the agent, the domain and ligand engage in a first binding; and detecting a second binding of the domain and ligand, wherein a difference between the first and second bindings indicates the agent modulates the binding of the domain and the ligand. In other embodiments, the assay is a NMR-based assay wherein the detecting step comprises detecting a binding-dependent NMR shift in the mixture.
The invention also provides polynucleotides encoding the subject LDLR adaptin polypeptides, including natural coding sequences. Natural coding sequence polypeptides may be used as probes or primers for detecting or amplifying LDLR adaptin genes and transcripts. Accordingly, the invention also provides methods for detecting a mutation or polymorphism in a natural human LDLR adaptin gene in a patient predetermined to have or be predisposed to hypercholesterolemia, comprising the steps of detecting a natural, endogenous human LDLR adaptin gene or protein in the patient; and determining if the gene or protein structure or expression is associated with hypercholesterolemia or a predisposition to hypercholesterolemia. In this method, the detecting step may comprise, for example, detecting a natural, endogenous LDLR adaptin gene by specific probe hybridization, or detecting a natural, endogenous LDLR adaptin protein by specific antibody binding.
In a particular embodiment, the method is extended to therapeutic intervention by further comprising the steps of modulating the gene or protein structure or expression, and confirming a resultant change in cholesterol level of the patient, particularly wherein the modulating step comprises enhancing or stabilizing binding of the protein to an NPXY peptide. A wide variety of methods are disclosed for modulating the LDLR adaptin protein, including protein modulated with antisense polynucleotides, intrabodies, a dominant negative LDLR adaptin mutant, or a product of a disclosed rational drug screen.
REFERENCES:
patent: 6280964 (2001-08-01), Kavanaugh et al.
patent: 6323177 (2001-11-01), Curran et al.
Gotthardt et al, Interactions of the low density lipoprotein receptor gene family with cytosolic adaptor and scaffold proteins suggest diverse biological functions in cellular communication and signal transduction, J. Biol. Chem. 275:25616-25624, 2000.*
Morris, S. M. and Cooper, J. A. Disabled-2 colocalizes with the LDLR in clathrin-coated pits and interacts with AP-2 Traffic 2:111-123, 2001.*
Trommsdorff et al, Interaction of cytosolic adaptor proteins with neuronal apolipoprotein E receptors and the amyloid precursor protein. J. Biol. Chem. 273: 33556-33560, 1998.
Barnes Robert I.
Cohen Jonathan
Garcia Christine K.
Hobbs Helen H.
Board of Regents , The University of Texas System
Kemmerer Elizabeth
Li Ruixiang
Osman Richard Aron
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