Showing papers by "David S. Bredt published in 1997"

PDZ domain of neuronal nitric oxide synthase recognizes novel C-terminal peptide sequences

Abstract: PDZ domains are multifunctional protein-interaction motifs that often bind to the C-terminus of protein targets. Nitric oxide (NO), an endogenous signaling molecule, plays critical roles in nervous, immune, and cardiovascular function. Although there are numerous physiological functions for neuron-derived NO, produced primarily by the neuronal NO synthase (nNOS), excess nNOS activity mediates brain injury in cerebral ischemia and in animal models of Parkinson's disease. Subcellular localization of nNOS activity must therefore be tightly regulated. To determine ligands for the PDZ domain of nNOS, we screened 13 billion distinct peptides and found that the nNOS-PDZ domain binds tightly to peptides ending Asp-X-Val. This differs from the only known (Thr/Ser)-X-Val consensus that interacts with PDZ domains from PSD-95. Preference for Asp at the -2 peptide position is mediated by Tyr-77 of nNOS. A Y77D78 to H77E78 substitution changes the binding specificity from Asp-X-Val to Thr-X-Val. Guided by the Asp-X-Val consensus, candidate nNOS interacting proteins have been identified including glutamate and melatonin receptors. Our results demonstrate that PDZ domains have distinct peptide binding specificity.

Actinin-associated LIM Protein: Identification of a Domain Interaction between PDZ and Spectrin-like Repeat Motifs

Abstract: PDZ motifs are protein–protein interaction domains that often bind to COOH-terminal peptide sequences. The two PDZ proteins characterized in skeletal muscle, syntrophin and neuronal nitric oxide synthase, occur in the dystrophin complex, suggesting a role for PDZ proteins in muscular dystrophy. Here, we identify actinin-associated LIM protein (ALP), a novel protein in skeletal muscle that contains an NH2-terminal PDZ domain and a COOH-terminal LIM motif. ALP is expressed at high levels only in differentiated skeletal muscle, while an alternatively spliced form occurs at low levels in the heart. ALP is not a component of the dystrophin complex, but occurs in association with α-actinin-2 at the Z lines of myofibers. Biochemical and yeast two-hybrid analyses demonstrate that the PDZ domain of ALP binds to the spectrin-like motifs of α-actinin-2, defining a new mode for PDZ domain interactions. Fine genetic mapping studies demonstrate that ALP occurs on chromosome 4q35, near the heterochromatic locus that is mutated in fascioscapulohumeral muscular dystrophy.

Regulation of neuronal nitric oxide synthase through alternative transcripts

Abstract: Nitric oxide (NO) participates in diverse physiological processes ranging from neurotransmission to muscle relaxation. Neuronal-derived NO can be either beneficial or detrimental depending on the cell

Muscular dystrophy, stroke, and neurodegenerative disease diagnosis and treatment

Abstract: Nitric oxide, neuronal nitric oxide synthase, neuronal nitric oxide synthase binding proteins, their inhibitors and a method of use of neuronal nitric oxide synthase, its binding proteins and their inhibitors for diagnosis and treatment of muscular dystrophy, stroke and other neurodegenerative diseases. A diagnostic assay for detection of absence of dystrophin or its mutated forms, neuronal nitric oxide synthase or its binding proteins. A method for treatment of muscular dystrophies by restoration of a functional dystrophin molecule in dystrophic muscles using gene therapy. Neuronal nitric oxide binding proteins PSD-95 and PSD-93 involved in management of stroke and other neurodegenerative diseases. The cloning and expression of the neuronal nitric oxide synthase binding proteins.

Ligand detection system and methods of use thereof

Abstract: The present invention relates to novel ligand detection systems and methods of using the systems to identify ligands capable of specifically binding orphan protein domains. The invention also relates to peptide ligands capable of specifically binding an orphan domain of interest such as the PDZ domain of neuronal nitric oxide synthase (nNOS). Further provided are methods of detecting the peptide ligands and those orphan protein domains capable of specifically binding the peptide ligands. The present invention is useful for a variety of applications including detecting peptide ligands with therapeutic capacity to treat human diseases.

Detection of Mycoplasma Infection of Mammalian Cells

Abstract: Mycoplasma infection was detected in cultures of COS cells with a novel, simple assay that detects the conversion of arginine to citrulline by the enzyme, arginine deiminase, specific to all species of mycoplasma. Transfection of COS cells was inhibited in mycoplasma-infected cells, a phenomenon that was readily reversed by treatment with a mycoplasma removal agent. Cultures of cells used for transfection should be regularly monitored for evidence of mycoplasma by assay of arginine deiminase activity or by other methods.