Expression of murine 5-aminolevulinate synthase variants causes protoporphyrin IX accumulation and light-induced mammalian cell death.
Expression of murine 5-aminolevulinate synthase variants causes protoporphyrin IX accumulation and light-induced mammalian cell death.
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DOI：
10.1371/journal.pone.0093078
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发表时间：
2014
期刊：
影响因子：
3.7
通讯作者：
Ferreira GC
中科院分区：
文献类型：
--
作者：
Fratz EJ;Hunter GA;Ferreira GC
5-氨基乙酰丙酸合成酶(ALAS；EC 2.3.1.37)催化动物体内血红素生物合成的第一步。红系特异性ALAS同工酶(ALAS2)在线粒体输入水平上受到血红素的负调控，在其成熟形式中，小鼠ALAS2活性部位环的某些突变导致血红素前体原卟啉IX(PPIX)的产生增加。重要的是，PPIX的产生是广泛应用于癌症和其他异型增生的光动力疗法(PDT)的关键组成部分。导致高水平PPIX积累的ALAS2变体提供了一种新的靶向和潜在增强的光敏化手段。为了评估ALAS2变异体在PDT生产PPIX中的应用前景，将ALAS2变异体的表达载体导入K562人红白血病细胞和HeLa人宫颈癌细胞，获得了比野生型酶更高的酶活性。流式细胞仪结合荧光检测分析ALAS2表达细胞中PPIX的积聚水平。此外，表达ALAS2变异体的细胞被白光(21-22Klux)处理10分钟，然后测定细胞存活率。用小鼠ALAS2变异体的表达载体转染HeLa细胞，特别是那些线粒体前序列突变和活性位点环突变的细胞，导致PPIX在细胞内显著积累，特别是在膜上。光处理显示，与产生类似数量的PPIX的氨基酮丙酸(ALA)处理相比，ALAS2的表达导致细胞死亡增加。稳定和高活性的ALAS2变异体的交付具有扩展和改进当前光动力疗法的潜力。
5-Aminolevulinate synthase (ALAS; EC 2.3.1.37) catalyzes the first committed step of heme biosynthesis in animals. The erythroid-specific ALAS isozyme (ALAS2) is negatively regulated by heme at the level of mitochondrial import and, in its mature form, certain mutations of the murine ALAS2 active site loop result in increased production of protoporphyrin IX (PPIX), the precursor for heme. Importantly, generation of PPIX is a crucial component in the widely used photodynamic therapies (PDT) of cancer and other dysplasias. ALAS2 variants that cause high levels of PPIX accumulation provide a new means of targeted, and potentially enhanced, photosensitization. In order to assess the prospective utility of ALAS2 variants in PPIX production for PDT, K562 human erythroleukemia cells and HeLa human cervical carcinoma cells were transfected with expression plasmids for ALAS2 variants with greater enzymatic activity than the wild-type enzyme. The levels of accumulated PPIX in ALAS2-expressing cells were analyzed using flow cytometry with fluorescence detection. Further, cells expressing ALAS2 variants were subjected to white light treatments (21–22 kLux) for 10 minutes after which cell viability was determined. Transfection of HeLa cells with expression plasmids for murine ALAS2 variants, specifically for those with mutated mitochondrial presequences and a mutation in the active site loop, caused significant cellular accumulation of PPIX, particularly in the membrane. Light treatments revealed that ALAS2 expression results in an increase in cell death in comparison to aminolevulinic acid (ALA) treatment producing a similar amount of PPIX. The delivery of stable and highly active ALAS2 variants has the potential to expand and improve upon current PDT regimes.
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DOI：
10.3322/caac.20114
发表时间：
2011-07
期刊：
CA: a cancer journal for clinicians
影响因子：
--
作者：
Agostinis P;Berg K;Cengel KA;Foster TH;Girotti AW;Gollnick SO;Hahn SM;Hamblin MR;Juzeniene A;Kessel D;Korbelik M;Moan J;Mroz P;Nowis D;Piette J;Wilson BC;Golab J
通讯作者：
Golab J
影响因子：
4.7
作者：
Felitsyn N;McLeod C;Shroads AL;Stacpoole PW;Notterpek L
通讯作者：
Notterpek L
影响因子：
11.4
作者：
Astner, I;Schulze, JO;Heinz, DW
通讯作者：
Heinz, DW
影响因子：
64.5
作者：
BOSHART, M;WEBER, F;SCHAFFNER, W
通讯作者：
SCHAFFNER, W
影响因子：
2.3
作者：
CANTI, G;LATTUADA, D;CUBEDDU, R
通讯作者：
CUBEDDU, R