Discovery of ADDL--targeting small molecule drugs for Alzheimer's disease.

Amyloid beta-derived diffusible ligands (ADDLs) comprise the neurotoxic subset of soluble Abeta(1-42) oligomers, now widely considered to be the molecular cause of memory malfunction and neurodegeneration in Alzheimer's disease (AD). We have developed a screening cascade which identifies small molecule modulators of ADDL-mediated neurotoxicity. The primary screen involves a fluorescence resonance energy transfer (FRET)-based assay which selects inhibitors of Abeta1-42 oligomer assembly.

Localization of the rap1GAP catalytic domain and sites of phosphorylation by mutational analysis.

rap1GAP is a GTPase-activating protein that specifically stimulates the GTP hydrolytic rate of p21rap1. We have defined the catalytic domain of rap1GAP by constructing a series of cDNAs coding for mutant proteins progressively deleted at the amino- and carboxy-terminal ends. Analysis of the purified mutant proteins shows that of 663 amino acid residues, only amino acids 75 to 416 are necessary for full GAP activity.

Molecular cloning of a GTPase activating protein specific for the Krev-1 protein p21rap1.

The rap1/Krev-1 gene encodes a ras-related protein that suppresses transformation by ras oncogenes. We have purified an 88 kd GTPase activating protein (GAP), specific for the rap1/Krev-1 gene product, from bovine brain. Based on partial amino acid sequences obtained from this protein, a 3.

Purification, characterization, and western blot analysis of human GTPase-activating protein from native and recombinant sources.

Human ras GTPase-activating protein (GAP) is a cytoplasmic factor that stimulates the GTPase activity of normal N-ras p21 while having no stimulatory effect on the GTPase activity of oncogenic variants of N-ras p21. We have purified two forms of native ras GAP from human placental tissue. In addition to the Mr = 120,000 type I GAP reported previously (1), an equivalent amount of an Mr = 95,000 molecule with GAP activity was recovered and shown to have the N-terminal sequence expected for type II GAP.

The GAP-related domain of the neurofibromatosis type 1 gene product interacts with ras p21.

The neurofibromatosis type 1 (NF1) protein contains a region of significant sequence similarity to ras p21 GTPase-activating protein (GAP) and the yeast IRA1 gene product. A fragment of NF1 cDNA encoding the GAP-related domain (NF1 GRD) was expressed, immunoaffinity purified, and assayed for effects on N-ras p21 GTPase activity. The GTPase of wild-type ras p21 was stimulated by NF1 GRD, but oncogenic mutants of ras p21 (Asp-12 and Val-12) were unaffected, and the GTPase of an effector mutant (Ala-38) was only weakly stimulated.