title: Linking a compound-heterozygous Parkin mutant (Q311R and A371T) to Parkinson's disease by using proteomic and molecular approaches.
abstract: Parkin is an E3-protein ubiquitin ligase, which plays an important role as a scavenger in cell metabolism. Since the discovery of the link between Parkin and Parkinson's disease, Parkin was placed in the center of Parkinson's disease research. Previously, we isolated a mutant form of the Parkin protein (Q311R and A371T) from a Parkinson's disease patient. In this study, we aimed at characterizing this mutant Parkin protein by using biochemical and proteomic approaches. We used neuroblastoma cells (SH-SY5Y) as our model and created two inducible cell lines that expressed the wild type and the mutant Parkin proteins. We first investigated the effect of expressing both the wild type and the mutant Parkin proteins on the overall proteome by using 2D-DIGE approach. The experiments yielded the identification of 22 differentially regulated proteins, of which 13 were regulated in the mutant Parkin expressing cells. Classification of the identified proteins based on biological process and molecular function revealed that the majority of the regulated proteins belonged to protein folding and energy metabolism. Ingenuity Pathway Analysis predicted the presence of a link between the regulated proteins of the mutant Parkin expressing cells and Parkinson's disease. We also performed biochemical characterization studies on the wild type and the mutant Parkin proteins to make sense out of the differences observed at the proteome level. Both proteins displayed biological activity, had similar stabilities and localized similarly to the cytoplasm and the nucleus in SH-SY5Y cells. The mutant protein, however, was cut by a protease and subjected to a post-translational modification. The observed differences at the proteome level might be due to the differences in processing of the mutant Parkin protein. Overall, we were able to create a possible link between a pair of Parkin mutations to its pertinent disease by using 2D-DIGE in combination with biochemical and molecular approaches.
authors: Department of Medical Biology, Kocaeli University School of Medicine, 41380 Kocaeli, Turkey.
authors: Sinem
authors: S
authors: Ozgul
authors: Department of Medical Biology, Kocaeli University School of Medicine, 41380 Kocaeli, Turkey; DEKART Proteomics Laboratory, Kocaeli University School of Medicine, 41380 Kocaeli, Turkey. Electronic address: mkasap2008@gmail.com.
authors: Murat
authors: M
authors: Kasap
authors: Department of Medical Biology, Kocaeli University School of Medicine, 41380 Kocaeli, Turkey; DEKART Proteomics Laboratory, Kocaeli University School of Medicine, 41380 Kocaeli, Turkey.
authors: Gurler
authors: G
authors: Akpinar
authors: Department of Medical Biology, Kocaeli University School of Medicine, 41380 Kocaeli, Turkey; DEKART Proteomics Laboratory, Kocaeli University School of Medicine, 41380 Kocaeli, Turkey.
authors: Aylin
authors: A
authors: Kanli
authors: Department of Medical Biology, Kocaeli University School of Medicine, 41380 Kocaeli, Turkey; DEKART Proteomics Laboratory, Kocaeli University School of Medicine, 41380 Kocaeli, Turkey.
authors: Nil
authors: N
authors: Güzel
authors: DEKART Proteomics Laboratory, Kocaeli University School of Medicine, 41380 Kocaeli, Turkey; Department of Biomedical Engineering, Kocaeli University School of Technology, 41380 Kocaeli, Turkey.
authors: Kübra
authors: K
authors: Karaosmanoglu
authors: TUBITAK Marmara Research Center, Genetic Engineering and Biotechnology Institute (GEBI), Gebze, 41470 Kocaeli, Turkey.
authors: Ahmet Tarik
authors: AT
authors: Baykal
authors: Department of Neurology, Kocaeli University School of Medicine, 41380 Kocaeli, Turkey.
authors: Pervin
authors: P
authors: Iseri
mesh_terms: D045744:Cell Line, Tumor; D018076:DNA, Complementary; D015180:Electrophoresis, Gel, Two-Dimensional; D006579:Heterozygote; D006801:Humans; D008958:Models, Molecular; D009154:Mutation; D010300:Parkinson Disease / Q000235:genetics*; D040901:Proteomics; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D044767:Ubiquitin-Protein Ligases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism
chemical_list: D018076:DNA, Complementary; D044767:Ubiquitin-Protein Ligases; C111567:parkin protein
keywords: 2D-DIGE; LC-MS/MS; MALDI-TOF/TOF; Neuroproteomics; Parkin; Parkinson's disease