Dataset Information

Protein expression in MOCK and neuronal nitric oxide synthase 1 (NOS1) transduced SH-SY5Y human neuroblastoma cells on stimulation with rapamycin or serum free starvation

ABSTRACT: Neuronal nitric oxide synthase (nNOS/NOS1) is upregulated in the aging brain and contributes to age-and stress-associated neuronal dysfunctions. To mimic this situation for the study of underlying mechanisms we generated a cell model in which human SH-SH5Y neuroblastoma cells constitutively express NOS1 at a level comparable with the rodent brain, whereas the control cells have only low levels. SH-SY5Y cells are the most widely used cell model for neurodegenerative and aging research. They were stably transduced with a lentiviral vector for NOS1 and FACS sorted. Control cells were transduced with the backbone vector, referred to as MOCK or "WT-LV205". Neuronal aging of SH-SY5Y can be imposed with a number of pharmacological, genetic or stress stimuli including retinoic acid and repeated cycles of starvation. Hence, to further challenge the cells we imposed proteostasis stress by treatment with 1 µM rapamycin for 24h or culture in serum-free medium for 24h. Control cells were cultured in full medium, which was RPMI 1640 medium supplemented with heat-inactivated 10% fetal bovine serum, 100 U/ml penicillin/streptomycin, and 2 mM glutamine. All cells were kept at 37°C in 5% CO2 atmosphere in a humidified tissue culture incubator. Rapamycin elicits mTOR dependent autophagy, whereas starvation is a stimulus for chaperone-mediated autophagy, but also affects proteolysis by the proteasome and lysosome and eventually, ER stress. During stimulation, all cells were supplemented with NOS cofactors including 10 µM NAD, 40 µM NADPH, and 100 µM tetrahydrobiopterin. The proteome data set shows nNOS/NOS1-dependent alterations of protein expression in SH-SY5Y neuroblastoma cells in full medium, on stimulation with rapamycin or exposure to serum-free starvation.

INSTRUMENT(S): Q Exactive

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Cell Culture, Neuroblast

SUBMITTER: Irmgard Tegeder

LAB HEAD: Ilka Wittig

PROVIDER: PXD010538 | Pride | 2018-10-17

REPOSITORIES: Pride

ACCESS DATA

Dataset's files

Source:

			Action	DRS
	160923_A12_FullProteome2_NOS_19_pellet_SF_B.raw	Raw
	160923_A12_FullProteome2_NOS_19_pellet_SF_C.raw	Raw
	160923_A12_FullProteome2_NOS_19_pellet_SF_DE.raw	Raw
	160923_A12_FullProteome2_NOS_19_supernatant_SF_SCX12.raw	Raw
	160923_A12_FullProteome2_NOS_19_supernatant_SF_SCX3.raw	Raw

Items per page:

1 - 5 of 289

Publications

Nitric oxide contributes to protein homeostasis by S-nitrosylations of the chaperone HSPA8 and the ubiquitin ligase UBE2D.

Valek Lucie L Heidler Juliana J Scheving Reynir R Wittig Ilka I Tegeder Irmgard I

Redox biology 20181016

Upregulations of neuronal nitric oxide synthase (nNOS) in the rodent brain have been associated with neuronal aging. To address underlying mechanisms we generated SH-SY5Y neuronal cells constitutively expressing nNOS at a level similar to mouse brain (nNOS+ versus MOCK). Initial experiments revealed S-nitrosylations (SNO) of key players of protein homeostasis: heat shock cognate HSC70/HSPA8 within its nucleotide-binding site, and UBE2D ubiquitin conjugating enzymes at the catalytic site cysteine ...[more]

PMID: 30368041

Publication: 1/2

Similar Datasets

Project description:Background: SH-SY5Y cells exhibit a neuronal phenotype when treated with all-trans retinoic acid (RA), but the molecular mechanism of activation in the signaling pathway mediated by phosphatidylinositol 3-kinase (PI3K) is not sufficiently understood. To shed new light on the mechanism, we comprehensively compared the gene expression profiles between SK-N-SH cells and two subtypes of SH-SY5Y cells (SH-SY5Y-A and SH-SY5Y-E), each of which showed a different phenotype during RA-mediated differentiation. Results: SH-SY5Y-A cells differentiated in the presence of RA, whereas RA-treated SH-SY5Y-E cells required additional treatment with brain-derived neurotrophic factor (BDNF) for full differentiation. In combination with perturbation using a PI3K inhibitor, LY294002, we identified 386 genes and categorized them into two clusters dependent on the PI3K signaling pathway during RA-mediated differentiation in SH-SY5Y-A cells. Transcriptional regulation of the gene cluster was greatly reduced in SK-N-SH cells or partially impaired in SH-SY5Y-E cells in coincidence with a defect in the neuronal phenotype of these cell lines. Additional stimulation with BDNF induced a set of neural genes which were down-regulated in RA-treated SH-SY5Y-E cells but were abundant in the differentiated SH-SY5Y-A cells. Conclusions: We identified the gene clusters controlled by PI3K- and TRKB-mediated signaling pathways during differentiation in two subtypes of SH-SY5Y cells. TRKB-mediated bypass pathway compensates for the impaired neural functions generated by defects in several signaling pathways including PI3K in SH-SY5Y-E cells. The expression profiling data are useful for further studies to elucidate the signal transduction-transcriptional network including PI3K and/or TRKB. Experiment Overall Design: Human neuroblastomas, SK-N-SH (HTB-11) and SH-SY5Y-A cells (CRL-2266) were obtained from the American Type Culture Collection (ATCC). We also obtained SH-SY5Y-E cells (EC94030304) from the European Collection of Cell Cultures (ECACC). Tissue culture cells were maintained in D-MEM/F12 1:1 mixture supplemented with 15% FBS (Fetal Bovine Serum) and 1% NEAA (Non-essential amino acid) in a 5% CO2 humidified incubator at 37oC. The culture medium was changed twice a week. For the RA-inducible experiment, random culture cells from two clone subtypes of SH-SY5Y and SK-N-SH were seeded in laminin coated culture dishes (BioCoat Laminin Cellware; BD Biosciences, Billerica, MA, USA) for 1 day and then transferred to a medium containing 10 μM of RA in the presence or the absence of LY294002 (10μM) for five days. For BDNF-induced sequential differentiation of the SH-SY5Y-E strain, cells were washed with D-MEM/F12 twice after five days in the presence of RA and then incubated with 50 ng/ml of BDNF in D-MEM/F12 without serum for three days.