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Differential regulation of human ALAS1 mRNA and protein levels by heme and cobalt protoporphyrin

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Abstract

5-Aminolevulinic acid synthase 1 (ALAS1) is the first and rate-controlling enzyme of heme biosynthesis. This study was to determine the effects of heme and selected nonheme metalloporphyrins on human ALAS1 gene expression in hepatocytes. We found that, upon heme and cobalt protoporphyrin (CoPP) treatments, ALAS1 mRNA levels were down-regulated significantly by ca. 50% or more. Measurement of mRNA in the presence of actinomycin D showed that these down-regulations were due to the decreases in mRNA half-lives. Furthermore, the levels of mitochondrial mature ALAS1 protein were down-regulated by 60–70%, but those of the cytosolic precursor protein were up-regulated by 2–5-fold. Measurement of protein in the presence of cycloheximide (CHX) suggests that elevation of the precursor form is due to the increase in protein half-lives. These results provide novel insights into the mechanisms of heme repressional effects on ALAS1 and provide a rationale for further investigation of CoPP as a therapeutic agent for acute porphyric syndromes.

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Abbreviations

ALAS1:

5-Aminolevulinic acid synthase 1

ALAS2:

5-Aminolevulinic acid synthase 2

CHX:

Cycloheximide

CoPP:

Cobalt protoporphyrin

CrMP:

Chromium mesoporphyrin

GAPDH:

Glyceraldehyde phosphate dehydrogenase

HO-1:

Heme oxygenase 1

LMH:

Leghorn male hepatoma

DMSO:

Dimethyl sulfoxide

MnPP:

Manganese protoporphyrin

PCR:

Polymerase chain reaction

qRT-PCR:

Quantitative real-time polymerase chain reaction

SDS-PAGE:

Sodium dodecyl sulfate polyacrylamide gel electrophoresis

PBST:

Phosphate-buffered saline Tween-20

PVDF:

Polyvinylidene fluoride

SnMP:

Tin mesoporphyrin

ZnMP:

Zinc mesoporphyrin

ZnPP:

Zinc protoporphyrin

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Acknowledgments

This work was supported by United States Public Health Service Grants RO1-DK38825 and contracts NO-1 DK92326 and UO-1 DK065193. Authors thank Dr. Henry M. Furneaux (Center for Vascular Biology, University of Connecticut Health Center, Farmington, CT) for helpful comments and discussion, Dr. John A. Watts (Carolinas Medical Center, Charlotte, NC) for suggestions, and Dr. Rixin Wang (Yale Center for Medical Informatics, New Haven, CT) for statistical assistance in the data analysis.

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  1. Herbert L. Bonkovsky

    Present address: Cannon Research Center, Suite 201, Carolinas Medical Center, 1000 Blythe Blvd, Charlotte, NC, 28203, USA

Authors and Affiliations

  1. The Liver, Digestive Disease, & Metabolism Laboratory, Carolinas Medical Center, Charlotte, NC, 28203, USA

    Jianyu Zheng & Herbert L. Bonkovsky

  2. Department of Biology, The University of North Carolina at Charlotte, Charlotte, NC, 28223, USA

    Jianyu Zheng & Herbert L. Bonkovsky

  3. Department of Medicine, The University of Massachusetts Medical School, Worcester, MA, 01655, USA

    Ying Shan

  4. Department of Medicine, The University of Connecticut Health Center, Farmington, CT, 06030, USA

    Richard W. Lambrecht, Susan E. Donohue & Herbert L. Bonkovsky

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  1. Jianyu Zheng
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  2. Ying Shan
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  3. Richard W. Lambrecht
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  5. Herbert L. Bonkovsky
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Correspondence to Herbert L. Bonkovsky.

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Zheng, J., Shan, Y., Lambrecht, R.W. et al. Differential regulation of human ALAS1 mRNA and protein levels by heme and cobalt protoporphyrin. Mol Cell Biochem 319, 153–161 (2008). https://doi.org/10.1007/s11010-008-9888-0

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  • DOI: https://doi.org/10.1007/s11010-008-9888-0

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