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PPARα Activation Induces N ε-Lys-Acetylation of Rat Liver Peroxisomal Multifunctional Enzyme Type 1

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Lipids

Abstract

Peroxisomes are ubiquitous subcellular organelles that participate in metabolic and disease processes, with few of its proteins undergoing posttranslational modifications. As the role of lysine-acetylation has expanded into the cellular intermediary metabolism, we used a combination of differential centrifugation, organelle isolation by linear density gradient centrifugation, western blot analysis, and peptide fingerprinting and amino acid sequencing by mass spectrometry to investigate protein acetylation in control and ciprofibrate-treated rat liver peroxisomes. Organelle protein samples isolated by density gradient centrifugation from PPARα-agonist treated rat liver screened with an anti-N ε-acetyl lysine antibody revealed a single protein band of 75 kDa. Immunoprecipitation with this antibody resulted in the precipitation of a protein from the protein pool of ciprofibrate-induced peroxisomes, but not from the protein pool of non-induced peroxisomes. Peptide mass fingerprinting analysis identified the protein as the peroxisomal multifunctional enzyme type 1. In addition, mass spectrometry-based amino acid sequencing resulted in the identification of unique peptides containing 4 acetylated-Lys residues (K155, K173, K190, and K583). This is the first report that demonstrates posttranslational acetylation of a peroxisomal enzyme in PPARα-dependent proliferation of peroxisomes in rat liver.

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Abbreviations

ABC:

ATP-binding cassette

ACN:

Acetonitrile

ACOX:

Acyl-CoA oxidase

CIP:

Ciprofibrate

CoA:

Coenzyme A

HAD:

Hydroxyacyl-CoA dehydrogenase

LC/ESI–MS/MS:

Liquid chromatography–electrospray ionization–tandem mass spectrometry

MALDI-TOF/TOF:

Matrix-assisted laser desorption ionization-time of flight/time of flight

MFE-2:

Multifunctional enzyme type 2

MS:

Mass spectrometry

N ε-AcK:

N ε-acetyl-lysine

PAA:

Polyacrylamide

Pex:

Peroxin

PI:

Protein immunoprecipitation

PPARα:

Peroxisome proliferator-activated receptor alpha

PTM:

Posttranslational modifications

rpMFE-1:

Rat peroxisomal multifunctional enzyme type 1

SCPx:

Sterol carrier protein x

TFA:

Trifluoroacetic acid

VLCFA:

Very long chain fatty acids

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Acknowledgments

The authors greatly appreciate the technical assistance of Ms. Joyce Bryan and Ms. Desiree Vonkollmar, and the help of Dr. Osamu Morinaga for raising the polyclonal antibodies against very long chain acyl-CoA synthetase. They also thank Antonio Contreras for reading the manuscript. This work was supported by Grants from the Extramural Research Facilities Program of the National Center for Research Resources [C06 RR018823 and C06 RR015455] and from the National Institutes of Health [NS-22576, NS-34741, NS-37766, and NS-64195].

Conflict of interest

The authors declare that they do not have conflicts of interest.

Author information

Author notes

  1. Miguel A. Contreras

    Present address: Office of Research Infrastructure Programs, Division of Comparative Medicine, National Institutes of Health, 6701 Democracy Boulevard, Room 945, Bethesda, MD, 20892, USA

  2. Oscar Alzate

    Present address: Paul L. Foster School of Medicine, Texas Tech University Health Sciences Center, 5001 El Paso Dr., El Paso, TX, 79905, USA

Authors and Affiliations

  1. Department of Pediatrics, The Darby Children’s Research Institute, Medical University of South Carolina, Charleston, SC, 29425, USA

    Miguel A. Contreras, Avtar K. Singh & Inderjit Singh

  2. Department of Pathology, Ralph H. Johnson VA Medical Center, Charleston, SC, 29401, USA

    Avtar K. Singh

  3. Systems Proteomics Core Laboratory, Department of Cell and Developmental Biology, University of North Carolina, Chapel Hill, NC, 27599, USA

    Oscar Alzate

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  1. Miguel A. Contreras
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Correspondence to Inderjit Singh.

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Contreras, M.A., Alzate, O., Singh, A.K. et al. PPARα Activation Induces N ε-Lys-Acetylation of Rat Liver Peroxisomal Multifunctional Enzyme Type 1. Lipids 49, 119–131 (2014). https://doi.org/10.1007/s11745-013-3843-x

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