title: Consequences of impaired purine recycling on the proteome in a cellular model of Lesch-Nyhan disease.
abstract: The importance of specific pathways of purine metabolism for normal brain function is highlighted by several inherited disorders, such as Lesch-Nyhan disease (LND). In this disorder, deficiency of the purine recycling enzyme, hypoxanthine-guanine phosphoribosyltransferase (HGprt), causes severe neurological and behavioral abnormalities. Despite many years of research, the mechanisms linking the defect in purine recycling to the neurobehavioral abnormalities remain unclear. In the current studies, an unbiased approach to the identification of potential mechanisms was undertaken by examining changes in protein expression in a model of HGprt deficiency based on the dopaminergic rat PC6-3 line, before and after differentiation with nerve growth factor (NGF). Protein expression profiles of 5 mutant sublines carrying different mutations affecting HGprt enzyme activity were compared to the HGprt-competent parent line using the method of stable isotopic labeling by amino acids in cell culture (SILAC) followed by denaturing gel electrophoresis with liquid chromatography and tandem mass spectrometry (LC-MS/MS) of tryptic digests, and subsequent identification of affected biochemical pathways using the Database for Annotation, Visualization and Integrated Discovery (DAVID) functional annotation chart analysis. The results demonstrate that HGprt deficiency causes broad changes in protein expression that depend on whether the cells are differentiated or not. Several of the pathways identified reflect predictable consequences of defective purine recycling. Other pathways were not anticipated, disclosing previously unknown connections with purine metabolism and novel insights into the pathogenesis of LND.
authors.affiliation: Department of Biochemistry, Emory University, Atlanta, GA.
authors.forename: Eric B
authors.initials: EB
authors.lastname: Dammer
authors.affiliation: Department of Neurology, Emory University, Atlanta, GA.
authors.forename: Martin
authors.initials: M
authors.lastname: Göttle
authors.affiliation: Department of Biochemistry, Emory University, Atlanta, GA.
authors.forename: Duc M
authors.initials: DM
authors.lastname: Duong
authors.affiliation: Department of Biostatistics and Bioinformatics, Emory University, Atlanta, GA.
authors.forename: John
authors.initials: J
authors.lastname: Hanfelt
authors.affiliation: Department of Biochemistry, Emory University, Atlanta, GA.
authors.forename: Nicholas T
authors.initials: NT
authors.lastname: Seyfried
authors.affiliation: Department of Neurology, Emory University, Atlanta, GA.|Department of Human Genetics & Pediatrics, Emory University, Atlanta, GA.
authors.forename: H A
authors.initials: HA
authors.lastname: Jinnah
mesh_terms: D000818:Animals; D002454:Cell Differentiation / Q000235:genetics / Q000502:physiology; D002460:Cell Line; D004298:Dopamine / Q000378:metabolism; D059290:Dopaminergic Neurons / Q000502:physiology; D007041:Hypoxanthine Phosphoribosyltransferase / Q000172:deficiency / Q000235:genetics; D007926:Lesch-Nyhan Syndrome / Q000235:genetics* / Q000378:metabolism*; D020932:Nerve Growth Factor; D020543:Proteome; D011687:Purines / Q000378:metabolism*; D051381:Rats; D053719:Tandem Mass Spectrometry; D059467:Transcriptome
chemical_list: C502240:NGF protein, human; D020543:Proteome; D011687:Purines; D020932:Nerve Growth Factor; D007041:Hypoxanthine Phosphoribosyltransferase; D004298:Dopamine
keywords: Dopamine neurons; Hypoxanthine–guanine phosphoribosyltransferase; Lesch–Nyhan disease; Proteome