The carboxyl-terminal region of erythroid-specific 5-aminolevulinate synthase acts as an intrinsic modifier for its catalytic activity and protein stability

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• 作者：Senkottuvelan Kadirvel^a ; Kazumichi Furuyama^a ; ^{furuyama@med.tohoku.ac.jp} ; Hideo Harigae^b ; Kiriko Kaneko^c ; Yoshiko Tamai^d ; Yoji Ishida^e ; Shigeki Shibahara^a
• 刊名：Experimental Hematology
• 出版年：2012
• 出版时间：June, 2012
• 年：2012
• 卷：40
• 期：6
• 页码：477-486.e1
• 全文大小：830 K

文摘

Erythroid-specific 5-aminolevulinate synthase (ALAS2) is essential for hemoglobin production, and a loss-of-function mutation of ALAS2 gene causes X-linked sideroblastic anemia. Human ALAS2 protein consists of 587 amino acids and its carboxyl(C)-terminal region of 33 amino acids is conserved in higher eukaryotes, but is not present in prokaryotic ALAS. We explored the role of this C-terminal region in the pathogenesis of X-linked sideroblastic anemia. In?vitro enzymatic activity was measured using bacterially expressed recombinant proteins. In?vivo catalytic activity was evaluated by comparing the accumulation of porphyrins in eukaryotic cells stably expressing each mutant ALAS2 tagged with FLAG, and the half-life of each FLAG-tagged ALAS2 protein was determined by Western blot analysis. Two novel mutations (Val562Ala and Met567Ile) were identified in patients with X-linked sideroblastic anemia. Val562Ala showed the higher catalytic activity in?vitro, but a shorter half-life in?vivo compared to those of wild-type ALAS2 (WT). In contrast, the in?vitro activity of Met567Ile mutant was about 25 % of WT, while its half-life was longer than that of WT. However, in?vivo catalytic activity of each mutant was lower than that of WT. In addition, the deletion of 33 amino acids at C-terminal end resulted in higher catalytic activity both in?vitro and in?vivo with the longer half-life compared to WT. In conclusion, the C-terminal region of ALAS2 protein may function as an intrinsic modifier that suppresses catalytic activity and increases the degradation of its protein, each function of which is enhanced by the Met567Ile mutation and the Val562Ala mutation, respectively.