Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UMLS:C0033036 (APC)
 10,214 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Nonsense or frameshift mutations, which result in a truncated gene product, are prevalent in a variety of disease-related genes, including APC (implicated in colorectal cancer), BRCA1 and BRCA2 (breast and ovarian cancer), PKD1 (polycystic kidney disease), NF1 and NF2 (neurofibromatosis), and DMD (Duchenne muscular dystrophy). Such chain-truncating mutations can be detected using the protein truncation test (PTT). This test is based on cell-free transcription and translation of either PCR-amplified portions of the target gene or RT-PCR amplified target mRNA, followed by analysis of the product(s) for shortened polypeptide fragments. However, conventional PTT is not easily adapted to high-throughput applications because it involves SDS-PAGE followed by autoradiography or western blotting. It is also subject to human error, as it relies on visual inspection to detect the mobility of shifted bands. To overcome these limitations, we have developed a high-throughput solid-phase protein truncation test (HTS-PTT). HTS-PTT uses a combination of misaminoacylated tRNAs, which incorporate affinity tags for surface capture of the cell-free expressed protein fragments, and specially designed PCR primers, which introduce N- and C-terminal markers for measuring the relative level of shortened polypeptides produced by the chain-truncation mutation. After cell-free translation of the protein fragments, capture and detection are accomplished in a single well using a standard 96-well microtiter plate enzyme-linked immunosorbent assay (ELISA) format and chemiluminescence readout. We demonstrate the use of the technique to detect chain-truncation mutations in the APC gene using DNA or RNA from cancer cell lines as well as DNA of individuals diagnosed with familial adenomatous polyposis (FAP). HTS-PTT can also provide a high-throughput method for noninvasive colorectal cancer screening when used in conjunction with methods of enriching and amplifying low-abundance mutant DNA.
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PMID:A high-throughput nonisotopic protein truncation test. 1252 52

Interleukin 1 receptor activation innervates a cascade of signal transduction events that ultimately lead to the activation of inflammatory and immune response genes. TRAF6 is a Ub ligase (E3) involved in this pathway, and inhibition of this critical enzyme may provide a means for treating inflammatory and immune diseases. A TR-FRET assay has been developed and evaluated for HTS for TRAF6 inhibitors. Bio-Ub and Eu-Ub were polymerized in the presence of Ub activating enzyme E1, conjugating enzyme E2, and TRAF6. Following a 2-h incubation, the reaction was stopped with a buffer containing 10 m M EDTA and the fluorescence donor SA-APC. Fluorescence energy transfer from Eu to APC was measured as a ratio of fluorescence intensity at 655 nm to that at 615 nm (excitation at 340 nm). This homogeneous assay has been optimized and validated in a 384-well format. A window of five- to eightfold and Z' factor of 0.6-0.8 suggests that this assay can be applied to screen for inhibitors of the polyubiquitination activity of TRAF6.
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PMID:Development of a high throughput time-resolved fluorescence resonance energy transfer assay for TRAF6 ubiquitin polymerization. 1509 Jan 44