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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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