Regulation of PPAR-alpha pathway by Dicer revealed through proteomic analysis

Nandini A. Sahasrabuddhe, Tai Chung Huang, Sartaj Ahmad, Min Sik Kim, Yi Yang, Bidyut Ghosh, Steven D. Leach, Harsha Gowda, Babu L. Somani, Raghothama Chaerkady, Akhilesh Pandey

Research output: Contribution to journalArticle

12 Citations (Scopus)

Abstract

Dicer is a crucial RNase III enzyme in miRNA biogenesis pathway. Although numerous studies have been carried out to investigate the role of miRNAs and Dicer in the regulation of biological processes, few studies have examined proteomic alterations upon knockout of Dicer. We employed a Cre-loxP-based inducible knockout mouse system to investigate the proteome regulated by Dicer-dependent miRNAs. We utilized spiked liver lysates from metabolically labeled mice to quantify the subtle changes in the liver proteome upon deletion of Dicer. We identified 2137 proteins using high resolution tandem mass spectrometry analysis. The upregulated proteins included several enzymes involved in peroxisomal β-oxidation of fatty acids and a large majority of the upregulated proteins involved in lipid metabolism were known PPARα targets. MRM-based assays were carried out to confirm the upregulation of enzymes including peroxisomal bifunctional enzyme, phosphoenolpyruvate carboxykinase 1, cytochrome P450 3A13, cytochrome P450 3A41 and myristoylated alanine-rich protein kinase C substrate. Further, miRNA-124 which is predicted to regulate expression of peroxisomal bifunctional enzyme was confirmed to be downregulated in the Dicer knockout mice. Our study demonstrates the strength of coupling knockout mouse models and quantitative proteomic strategies to investigate functions of individual proteins in vivo. Biological significance: Dicer dependent miRNA biogenesis is the major pathway for generation of mature miRNAs. We developed SILAC mouse-based proteomics screen to identify protein targets of Dicer-dependent miRNAs in liver of Dicer knockout mice. We spiked liver lysates of induced and uninduced Dicer knockout mice with liver lysate of SILAC labeled mice for identification of dysregulated proteome. We quantitated 1217 proteins of which 257 were upregulated in induced Dicer knockout mice. We observed enrichment of PPAR-α targets and proteins involved in lipid metabolism among upregulated proteins. We further carried out MRM-based validation of peroxisomal bifunctional enzyme, phosphoenolpyruvate carboxykinase 1, Cyp3A13, Cyp3A41 and myristoylated alanine-rich protein kinase C substrate. We further validated upregulation of peroxisomal bifunctional enzyme using Western blot analysis and downregulation of its predicted upstream miRNA, miR-124 using qRT-PCR. Our study demonstrates that upon ablation of Dicer, certain Dicer-dependent miRNAs are dysregulated which result in dysregulation of their target proteins such as proteins associated with lipid metabolism. Our study illustrates the use of SILAC strategy for quantitative proteomic investigations of animal model systems.

Original languageEnglish (US)
Pages (from-to)306-315
Number of pages10
JournalJournal of Proteomics
Volume108
DOIs
StatePublished - Aug 28 2014
Externally publishedYes

Fingerprint

PPAR alpha
Proteomics
MicroRNAs
Peroxisomal Bifunctional Enzyme
Knockout Mice
Liver
Proteins
Proteome
Lipid Metabolism
Peroxisome Proliferator-Activated Receptors
Phosphoenolpyruvate
Alanine
Cytochrome P-450 Enzyme System
Protein Kinase C
Up-Regulation
Enzymes
Down-Regulation
Ribonuclease III
Biological Phenomena
Substrates

Keywords

  • Dicer
  • Peroxisomal β-oxidation
  • Quantitative proteomics
  • SILAC mouse

ASJC Scopus subject areas

  • Biophysics
  • Biochemistry

Cite this

Sahasrabuddhe, N. A., Huang, T. C., Ahmad, S., Kim, M. S., Yang, Y., Ghosh, B., ... Pandey, A. (2014). Regulation of PPAR-alpha pathway by Dicer revealed through proteomic analysis. Journal of Proteomics, 108, 306-315. https://doi.org/10.1016/j.jprot.2014.04.027

Regulation of PPAR-alpha pathway by Dicer revealed through proteomic analysis. / Sahasrabuddhe, Nandini A.; Huang, Tai Chung; Ahmad, Sartaj; Kim, Min Sik; Yang, Yi; Ghosh, Bidyut; Leach, Steven D.; Gowda, Harsha; Somani, Babu L.; Chaerkady, Raghothama; Pandey, Akhilesh.

In: Journal of Proteomics, Vol. 108, 28.08.2014, p. 306-315.

Research output: Contribution to journalArticle

Sahasrabuddhe, NA, Huang, TC, Ahmad, S, Kim, MS, Yang, Y, Ghosh, B, Leach, SD, Gowda, H, Somani, BL, Chaerkady, R & Pandey, A 2014, 'Regulation of PPAR-alpha pathway by Dicer revealed through proteomic analysis', Journal of Proteomics, vol. 108, pp. 306-315. https://doi.org/10.1016/j.jprot.2014.04.027
Sahasrabuddhe NA, Huang TC, Ahmad S, Kim MS, Yang Y, Ghosh B et al. Regulation of PPAR-alpha pathway by Dicer revealed through proteomic analysis. Journal of Proteomics. 2014 Aug 28;108:306-315. https://doi.org/10.1016/j.jprot.2014.04.027
Sahasrabuddhe, Nandini A. ; Huang, Tai Chung ; Ahmad, Sartaj ; Kim, Min Sik ; Yang, Yi ; Ghosh, Bidyut ; Leach, Steven D. ; Gowda, Harsha ; Somani, Babu L. ; Chaerkady, Raghothama ; Pandey, Akhilesh. / Regulation of PPAR-alpha pathway by Dicer revealed through proteomic analysis. In: Journal of Proteomics. 2014 ; Vol. 108. pp. 306-315.
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