Metabolomics,Unknown,Transcriptomics,Genomics,Proteomics

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Transcription profiling of SH-SY5Y cells after treatment with soluble Abeta42_wt, Abeta42_G33A and Abeta40 for different timepoints

ABSTRACT: Although amyloid-beta42 (Abeta42) has long been implicated in the pathogenesis of Alzheimer disease (AD), the biological basis of its effects remains uncertain. Intraneuronal Abeta accumulation in brains of patients with AD or Down syndrome, in animal models, and in cultured cells has suggested an early pathophysiological role specific for intracellular Abeta40 and Abeta42. As a consequence of intraneuronal oligomerization, cell death can result from ER stress, endosomal/lysosomal leakage, and mitochondrial dysfunction. A possible nuclear role of intraneuronal Abeta has remained unexplored so far. We here study the role of intranuclear Abeta40 versus Abeta42 and Abeta42_G33A, and an untreated vehicle control in synchronized SH-SY5Y cells. RNA samples for microarray gene expression profiling were collected at t=0, 2, 6, 8 and 12 hours after incubation with Abeta40, Abeta42 and Abeta42_G33A peptides, respectively.'

ORGANISM(S): Homo sapiens

SUBMITTER: Georg Beckmann

PROVIDER: E-MTAB-1841 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:SUMO modification of proteins (sumoylation) is essential for mitotic progression from yeast to humans, but only a limited number of sumoylated proteins with functions in mitosis have been discovered. Vertebrates express three SUMO paralogs, SUMO-1, SUMO-2 and SUMO-3, but only SUMO-2 and -3 are chromosome-associated in mitosis. In this study, we used chromosome spreads to more precisely define the localization of endogenous SUMO-2 and -3 to the centromere as well as the chromosome protein scaffold. Furthermore, we developed methodologies for the immunopurification of endogenous SUMO-2 and -3 modified proteins from cell extracts. Using LC-MS/MS, we analyzed proteins immunopurified from mitotic chromosome fractions and G0 nuclear fractions. We identified 296 putative sumoylated proteins, with 138 being modified specifically in mitosis. We identified proteins known to localize to the centromere and kinetochore and the chromosome protein scaffold, consistent with SUMO-2/3 localization. Furthermore, we demonstrate that utilizing cell synchronization and fractionation allowed for the discovery of low abundance sumoylated proteins that are not detected in large asynchronous analyses. Our results provide a foundation for further characterization of the roles of sumoylation in regulating diverse aspects of chromosome segregation in mitosis. Thermo .raw files were uploaded to the ProHits (Liu et al, 2010) analytical suite and converted to .mzXML format using ReAdW software. Data were searched using X!Tandem (Craig & Beavis, 2004) against human ORFs (RefSeq v45). Search parameters specified a parent MS tolerance of +/- 15ppm, and an MS/MS tolerance of 0.4 Da, with up to two missed cleavages for trypsin. Oxidation of methionine and tryptophan, ubiquitylation of lysine, and alkylation of cysteine (by NEM) were allowed as variable modifications. Statistical validation of the results was performed using Peptide Prophet and Protein Prophet (Keller et al, 2002; Nesvizhskii et al, 2003) as part of the trans-proteomic pipeline. For each search, the Protein Prophet probability at a 1% false discovery rate was used as a cutoff value to generate SAINT-compatible input files. SAINT parameters were as follows: 5000 iterations, low mode Off (0), minFold 1 and normalization (1) (Choi et al, 2011). SAINT cutoff values of 0.75 were used to generate a high-confidence list of protein identifications.

Dataset Information

Transcription profiling of SH-SY5Y cells after treatment with soluble Abeta42_wt, Abeta42_G33A and Abeta40 for different timepoints

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