Erythroid-specific 5-aminolevulinate synthase protein is stabilized by low oxygen and proteasomal inhibition

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Abstract
5-aminolevulinate synthase (ALAS; E.C. 2.3.1.37) catalyzes the first and rate-limiting step of heme synthesis within the mitochondria. Two isozymes of ALAS, encoded by separate genes, exist. ALAS1 is ubiquitously expressed and provides heme for cytochromes and other hemoproteins. ALAS2 is expressed exclusively in erythroid cells and synthesizes heme specifically for haemoglobin. A database search for proteins potentially regulated by oxygen tension revealed that ALAS2 contained a sequence of amino acids (LXXLAP where L is leucine, X is any amino acid, A is alanine, and P is proline) not occurring in ALAS1, which may be hydroxylated under normoxic conditions (21% O2) and target the enzyme for ubiquitination and degradation by the proteasome. We examined protein turnover of ALAS2 in the presence of cycloheximide in K562 cells. Normoxic ALAS2 had a turnover time of approximately 36 h. Hypoxia (1% O2) and inhibition of the proteasome increased both the stability and the specific activity of ALAS2 (greater than 2- and 7-fold, respectively, over 72 h of treatment). Mutation of a key proline within the LXXLAP sequence of ALAS2 also stabilized the protein beyond 36 h under normoxic conditions. The von Hippel-Lindau (vHL) protein was immunoprecipitated with FLAG epitope-tagged ALAS2 produced in normoxic cells but not in hypoxic cells, suggesting that the ALAS2 is hydroxylated under normoxic conditions and targeted for ubiquitination by the E3 ubiquitin ligase system. ALAS2 could also be ubiquitinated under normoxia using an in vitro ubiquitination assay. The present study provides evidence that ALAS2 is broken down under normoxic conditions by the proteasome and that the prolyl-4-hydroxylase/vHL E3 ubiquitin ligase pathway may be involved.Key words: erythroid-specific 5-aminolevulinate synthase, hypoxia, hydroxylation, prolyl-4-hydroxylases, E3 ubiquitin ligases, von Hippel-Lindau protein, proteasome.