Dataset Information

Capture of candidate calpain5 interacting proteins from lysates of the human neuroblastoma SH-SY5Y cell line with analysis of LC-MSMS data using SWATH

ABSTRACT: Calpain5 (CAPN5) is a nonclassical calpain in that it lacks a pentaEF hand domain. Although widely expressed dominantly inherited mutations of CAPN5 display phenotypes primarily localized to the eye. These experiments aimed to identify proteins from the neuroblastoma cell line SH-SY5Y that may interact with the catalytic core domain of CAPN5 (phe2-leu348) and to complement co-immunoprecipitation studies using SH-SY5Y cell overexpressing full length CAPN5. Given that domains are independent folding units within the intact protein and interacting surfaces are in general localized some valid interacting proteins are expected to be captured. It is also clear that some interacting partners may be lost due to a decreased number of, or affinity of, interactions..

INSTRUMENT(S):

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Cell Culture, Neuroblast

DISEASE(S): Neuroblastoma

SUBMITTER: Carlos Gartner

LAB HEAD: Calvin Vary

PROVIDER: PXD071887 | Pride | 2026-02-02

REPOSITORIES: Pride

ACCESS DATA

Dataset's files

Source:

			Action	DRS
	DCroall_IPsOnlyLib_20200925.group	Other
	DDA-90m-DCroall-S1-019.wiff	Wiff
	DDA-90m-DCroall-S1-019.wiff.scan	Wiff
	DDA-90m-DCroall-S2-0020.wiff	Wiff
	DDA-90m-DCroall-S2-0020.wiff.scan	Wiff

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Publications

Candidate Interaction Partners of Calpain-5 Suggest Clues to Its Involvement in Neovascular Inflammatory Vitreoretinopathy.

Gal Jozsef J Bondada Vimala V Crasta Rachel R Croall Dorothy E DE Vary Calvin P CP Geddes James W JW

Cells 20260113 2

Although calpain-5/CAPN5 is widely expressed in mammals, little is known regarding its functions. Pathogenic mutations of CAPN5 are causal for a devastating autoimmune eye disease, neovascular inflammatory vitreoretinopathy (NIV). To provide insight into both the physiological and pathological roles of CAPN5, it is essential to identify candidate interaction partners and possible substrates. Human SH-SY5Y neuroblastoma cells, transfected with full-length catalytically dead (Cys81Ala) CAPN5-3×FLA ...[more]

PMID: 41597217

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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.