Quantitative proteomic analysis of cultured skin fibroblast cells derived from patients with triglyceride deposit cardiomyovasculopathyReportar como inadecuado

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Orphanet Journal of Rare Diseases

, 8:197

First Online: 21 December 2013Received: 24 September 2013Accepted: 12 December 2013


BackgroundTriglyceride deposit cardiomyovasculopathy TGCV is a rare disease, characterized by the massive accumulation of triglyceride TG in multiple tissues, especially skeletal muscle, heart muscle and the coronary artery. TGCV is caused by mutation of adipose triglyceride lipase, which is an essential molecule for the hydrolysis of TG. TGCV is at high risk for skeletal myopathy and heart dysfunction, and therefore premature death. Development of therapeutic methods for TGCV is highly desirable. This study aims to discover specific molecules responsible for TGCV pathogenesis.

MethodsTo identify differentially expressed proteins in TGCV patient cells, the stable isotope labeling with amino acids in cell culture SILAC method coupled with LC-MS-MS was performed using skin fibroblast cells derived from two TGCV patients and three healthy volunteers. Altered protein expression in TGCV cells was confirmed using the selected reaction monitoring SRM method. Microarray-based transcriptome analysis was simultaneously performed to identify changes in gene expression in TGCV cells.

ResultsUsing SILAC proteomics, 4033 proteins were quantified, 53 of which showed significantly altered expression in both TGCV patient cells. Twenty altered proteins were chosen and confirmed using SRM. SRM analysis successfully quantified 14 proteins, 13 of which showed the same trend as SILAC proteomics. The altered protein expression data set was used in Ingenuity Pathway Analysis IPA, and significant networks were identified. Several of these proteins have been previously implicated in lipid metabolism, while others represent new therapeutic targets or markers for TGCV. Microarray analysis quantified 20743 transcripts, and 252 genes showed significantly altered expression in both TGCV patient cells. Ten altered genes were chosen, 9 of which were successfully confirmed using quantitative RT-PCR. Biological networks of altered genes were analyzed using an IPA search.

ConclusionsWe performed the SILAC- and SRM-based identification-through-confirmation study using skin fibroblast cells derived from TGCV patients, and first identified altered proteins specific for TGCV. Microarray analysis also identified changes in gene expression. The functional networks of the altered proteins and genes are discussed. Our findings will be exploited to elucidate the pathogenesis of TGCV and discover clinically relevant molecules for TGCV in the near future.

KeywordsProteome Triglyceride deposit cardiomyovasculopathy SILAC SRM-MRM ATGL Rare disease AbbreviationsPBSPhosphate-buffered saline


LC-MS-MSLiquid chromatography-tandem mass spectrometry

SI-peptideStable isotope labeled peptide

PCRPolymerase chain reaction

GAPDHGlyceraldehyde-3-phosphate dehydrogenase

ACTBActin, beta

PPIAPeptidylprolyl isomerase A

RPLP1Ribosomal protein, large, P1

RPLP2Ribosomal protein, large, P2

RPS18Ribosomal protein S18

ACOX2Acyl-CoA oxidase 2, branched chain

AKR1B1Aldo-keto reductase family 1, member B1

DHCR2424-dehydrocholesterol reductase

DPP4Dipeptidyl-peptidase 4

FABP3Fatty acid binding protein 3

FADS2Fatty acid desaturase 2

FDFT1Farnesyl-diphosphate farnesyltransferase 1

ITGA6Integrin, alpha 6

PLIN2Perilipin 2

PON2Paraoxonase 2

PTGS1Prostaglandin-endoperoxide synthase 1

TRPV2Transient receptor potential cation channel, subfamily V, member 2

COL18A1Collagen, type XVIII, alpha 1

COL6A3Collagen, type VI, alpha 3

COL8A1Collagen, type VIII, alpha 1

CTHRC1Collagen triple helix repeat containing 1

TBPTATA box binding protein


FBN2Fibrillin 2

FGL2Fibrinogen-like 2


ALDH1B1Aldehyde dehydrogenase 1 family, member B1

CSPG4Chondroitin sulfate proteoglycan 4

FBLN1Fibulin 1

GATA6GATA binding protein 6

LAMA1Laminin, alpha 1

LAMA2Laminin, alpha 2

MMP1Matrix metallopeptidase 1

OLR1Oxidized low density lipoprotein lectin-like receptor 1

PLA2G4APhospholipase A2, group IVA

TGFB2Transforming growth factor, beta 2

PTGS2Prostaglandin-endoperoxide synthase 2.

Electronic supplementary materialThe online version of this article doi:10.1186-1750-1172-8-197 contains supplementary material, which is available to authorized users.

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Autor: Yasuhiro Hara - Naoko Kawasaki - Ken-ichi Hirano - Yuuki Hashimoto - Jun Adachi - Shio Watanabe - Takeshi Tomonaga

Fuente: https://link.springer.com/

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