Dataset Information

Identification of dystrophin Dp71dΔ71 associated proteins in PC12 cells by quantitative proteomics

ABSTRACT: Dystrophin Dp71 is the main product of the DMD (Duchenne muscular dystrophy) gene in the brain. Dystrophin Dp71 is essential for the development of the nervous system and its alteration is associated with intellectual disability. Different isoforms of Dp71, with different subcellular locations, are generated by alternative splicing; however, their functions have not been fully described. Here, we identified Dp71dΔ71-associated proteins to understand their functions. PC12 cells stably transfected with pTRE2pur-Myc/Dp71dΔ71 or the empty vector (EV, pTRE2pur-Myc) were analyzed by immunoprecipitation coupled with quantitative proteomics. Samples were resolved and digested in gel, and peptides were separated using a UHPLC ACQUITY M-Class. Spectral data were acquired in an MS with electrospray ionization and ion mobility separation Synapt G2-Si operated with data-independent acquisition and ion mobility spectrometry using the high-definition multiplexed MS/MS mode. We used the Hi3 method to quantify absolutely every protein detected. A total of 121 proteins were identified and absolutely quantified using the internal standard and the intensity of the most abundant peptides per protein with Progenesis QI software and the database UP000002494. Seven new proteins associated with Dp71dΔ71 were selected with at least 2-fold quantity between immunoprecipitated proteins of PC12 Myc/Dp71dΔ71 versus PC12 empty vector cells. Proteins included β-tubulin, S-adenosylmethionine synthase isoform type-2, adapter molecule crk, helicase with zinc finger 2, WD repeat domain 93, cyclin-L2 and myosin-10. Immunoprecipitation followed by quantitative proteomics revealed new proteins that interact with Dp71dΔ71. These proteins are related to cell migration or cell growth, suggesting that Dp71dΔ71 and its associated proteins are involved in these cell functions. The results lay the foundation for future research on the relationship between these proteins and dystrophin Dp71 isoforms

INSTRUMENT(S): SYNAPT G2-Si

ORGANISM(S): Rattus Norvegicus (rat)

TISSUE(S): Cell Culture

SUBMITTER: Emmanuel Rios-Castro

LAB HEAD: Silvia Cecilia Irene Montañez Ojeda

PROVIDER: PXD021886 | Pride | 2024-11-01

REPOSITORIES: Pride

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Dataset's files

Source:

			Action	DRS
	20200120_1_pos_R001.raw.zip	Raw
	20200120_1_pos_R001_IA_final_peptide.csv	Csv
	20200120_1_pos_R001_IA_final_protein.csv	Csv
	20200120_1_pos_R002.raw.zip	Raw
	20200120_1_pos_R002_IA_final_peptide.csv	Csv

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Publications

Identification of dystrophin Dp71dΔ71-associated proteins in PC12 cells by quantitative proteomics.

Azotla-Vilchis Coztli C Merino-Jiménez Candelaria C Ríos-Castro Emmanuel E Aragón Jorge J Ceja Víctor V Montanez Cecilia C

Biochimica et biophysica acta. Proteins and proteomics 20240928 1

Dystrophin Dp71 is essential for the development of the nervous system. Its alteration is associated with intellectual disability. Different Dp71 isoforms are generated by alternative splicing; however, their functions have not been fully described. Here, we identified Dp71dΔ71-associated proteins to understand the complex functions. PC12 cells, stably transfected with pTRE2pur-Myc/Dp71dΔ71 or pTRE2pur-Myc empty vector (EV), were analyzed by immunoprecipitation followed wit ...[more]

PMID: 39349271

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Project description:Obstructive nephropathy (ON) is a common clinical entity caused by different etiologies such as urolithiasis, prostatic hyperplasia in males, tumors, congenital stenosis, and others. Unilateral ureteral obstruction (UUO) in rodents is an experimental model widely used to explore the pathophysiology of ON, replicating vascular alterations, tubular atrophy, inflammation, and fibrosis development. On the other hand, mitochondria have been the subject of great attention, as morphological and functional alterations have been demonstrated in kidney diseases. Therefore, in this study, we explored the differences of the renal mitochondrial proteome during a time-course of 7, 14, and 21 days after the UUO in a rat model; and subsequently performed an overrepresentation analysis of biological processes to gain insight into the most relevant mitochondrial processes during UUO. We first isolated mitochondria from each UUO group and the control (sham) group, then the mitochondria were lysed in lysis buffer (8 M Urea, 20 mM HEPES, 1mM EDTA, pH 8.0) and digested using iST Sample Preparation Kit®. Peptides were separated using a UHPLC ACQUITY M-Class. Spectral data were acquired in an MS with electrospray ionization and ion mobility separation Synapt G2-Si operated with data-independent acquisition and ion mobility spectrometry in HDMSE mode. A total of 954 proteins were identified and quantified by label-free with Progenesis QI software and the database UP000002494 . Subsequently, after cross-checking with the MitoMiner database, we were left with 379 mitochondrial or mitochondrial transit proteins. Then, only proteins that had abundance values in at least 50% of the samples per study group and did not show atypical abundance values were selected, thus conserving 308 mitochondrial proteins for principal component analysis (PCA). PCA showed that 11 components can explain the total variation in protein abundance among samples and that the first two components can differentiate between the control group (sham) and the UUO groups. Considering the results obtained from the PCA analysis, a heat map was constructed with 243 proteins that strongly correlated (r ≤ -0.5 or r ≥ 0.5) with the first two principal components. The results show the abundance patterns for each UUO day and for the sham group and that these proteins are mainly involved in three metabolic pathways, oxidative phosphorylation (OXPHOS), the tricarboxylic acid cycle (TCA), and fatty acid (FA) metabolism.

Dataset Information

Identification of dystrophin Dp71dΔ71 associated proteins in PC12 cells by quantitative proteomics

Dataset's files

Publications

Identification of dystrophin Dp71d<sub>Δ71</sub>-associated proteins in PC12 cells by quantitative proteomics.

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Dataset Information

Identification of dystrophin Dp71dΔ71 associated proteins in PC12 cells by quantitative proteomics

Dataset's files

Publications

Identification of dystrophin Dp71d&lt;sub&gt;Δ71&lt;/sub&gt;-associated proteins in PC12 cells by quantitative proteomics.

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OmicsDI is part of the ELIXIR infrastructure

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Identification of dystrophin Dp71d<sub>Δ71</sub>-associated proteins in PC12 cells by quantitative proteomics.