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ALA Induced Heme Synthesis: Fine Tuning Mechanisms of PBG Deaminase and ALA Dehydratase

Malik Zvi, Berkovitch-Luria G, Nudelman A and Rephaeli A

During erythroid differentiation and maturation, three critical components α-globin, β-globin, and heme are critical for the formation of stable α2β2 hemoglobin (Hb) complexes. Heme synthesis enhances globin formation, not only because it is the precursor of Hb but also by modulating the machinery of gene expression and globin synthesis and coordinating erythroid cell maturation. Exogenous 5-aminolevulinic acid (ALA), the first precursor in the heme biosynthesis pathway, circumvents the rate limiting enzyme ALA synthase, and accelerates the heme synthesis pathway. ALA induced erythroid differentiation of human myelogenous leukemia K562 and murine erythroleukemia MEL cells, leads to hemoglobinization and cellular typical maturation. Only two key enzymes where known as regulator of the accumulation of PpIx: Porphobilinogen deaminase and Ferrochelatase. Recently we demonstrated that downregulation or over expression of ALA dehydratase (ALAD) and porphobilinogen deaminase (PBGD), the second and the third enzymes of the pathway, by specific shRNAs caused a marked alteration in PpIX synthesis in K562 erythroleukemic cells. PBGD down regulation induced an elevation in ALAD activity, while over expression of PBGD reduced ALAD activity, indicating a novel regulation feedback of PBGD on ALAD activity. This feedback mechanism enabled partial PpIX synthesis under PBGD silencing, whereas ALAD silencing reduced PpIX production to a minimum, only ALAD loss resulted in reduced PpIX and Hb synthesis.

Hemoglobin synthesis and assembly is largely dependent on coordinated gene expression control, it is well established that the histone deacetylase inhibitor butyric acid activates transcription of heme synthesis an globin mRNA expression, leading to changes in cell morphology and induction of erythroid differentiation of erythroleukemic cell lines. We have demonstrated that the activity of the multitargeting ALA prodrug, AlaAcBu in the induction of protoporphyrin IX (PpIX) and anti-cancer activity following light irradiation. The prodrug AlaAcBu undergos enzymatic intracellular hydrolysis releasing ALA, butyric acid and acetaldehyde and these active components induce accelerated heme and hemoglobin synthesis. Erythroid differentiation was characterized by cellular maturation characterized by cytoplasm hemoglobinization, expression of the marker glycophorin A and polar arrangement of mitochondria and a developed central vacuolar system preceding nuclear extrusion. The ability of AlaAcBu to promote differentiation along the erythroid lineage and to dramatically induce hemoglobin synthesis is presented in this report.