Erythroid-specific 5-aminolevulinate synthase protein is stabilized by low oxygen and proteasomal inhibition
Erythroid-specific 5-aminolevulinate synthase protein is stabilized by low oxygen and proteasomal inhibition
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DOI：
10.1139/o05-045
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发表时间：
2005-10-01
影响因子：
2.9
通讯作者：
Willmore, WG
中科院分区：
文献类型：
--
作者：
Abu-Farha, M;Niles, J;Willmore, WG
5-氨基乙酰丙酸合酶（ALAS；E.C. 2.3.1.37）催化线粒体内血红素合成的第一步和限速步骤。 ALAS 存在两种同工酶，由不同的基因编码。 ALAS1 广泛表达，为细胞色素和其他血红素蛋白提供血红素。 ALAS2 仅在红细胞中表达，并专门合成血红蛋白的血红素。对可能受氧张力调节的蛋白质的数据库搜索显示，ALAS2 包含 ALAS1 中不存在的氨基酸序列（LXXLAP，其中 L 是亮氨酸，X 是任何氨基酸，A 是丙氨酸，P 是脯氨酸），该序列可能在常氧条件下 (21% O-2) 被羟基化，并靶向酶进行泛素化和被蛋白酶体降解。我们在 K562 细胞中检测了放线菌酮存在下 ALAS2 的蛋白质周转。常氧 ALAS2 的周转时间约为 36 小时。缺氧（10% O-2）和蛋白酶体抑制增加了 ALAS2 的稳定性和比活性（分别大于 2 倍和 7 倍，超过 72 It 处理）。 ALAS2 LXXLAP 序列中关键脯氨酸的突变也使该蛋白质在常氧条件下稳定超过 36 小时。 von Hippel-Lindau (vHL) 蛋白与常氧细胞中而非缺氧细胞中产生的 FLAG 表位标记的 ALAS2 进行免疫沉淀，表明 ALAS2 在常氧条件下被羟基化，并被 E3 泛素连接酶系统靶向泛素化。使用体外泛素化测定，ALAS2 也可以在常氧条件下被泛素化。本研究提供的证据表明，ALAS2 在常氧条件下被蛋白酶体分解，并且 protyl-4-羟化酶/vHL E3 泛素连接酶途径可能参与其中。
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% O-2) 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(10% O-2) and inhibition of the proteasome increased both the stability and the specific activity of ALAS2 (greater than 2- and 7-fold, respectively, over 72 It 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 protyl-4-hydroxylase/vHL E3 ubiquitin ligase pathway may be involved.