中血红素的合成水平在哺乳动物细胞中测量
Summary
Altered intracellular heme levels are associated with common diseases such as cancer. Thus, there is a need to measure heme biosynthesis levels in diverse cells. The goal of this protocol is to provide a fast and sensitive method to measure and compare the levels of heme synthesis in different cells.
Abstract
血红素作为辅基为多种已知作为血红素蛋白，例如血红蛋白，肌红蛋白和细胞色素蛋白。它参与各种分子和细胞过程，如基因转录，翻译，细胞分化和细胞增殖。血红素的生物合成水平在不同组织和细胞类型而变化，并改变在疾病状况如贫血，神经病变和癌症。这种技术使用[4- 14 C] 5-氨基乙酰丙酸（[14 C] 5-ALA），早期前体中的血红素生物合成途径来测量哺乳动物细胞血红素合成的级别中的一个。该测定涉及细胞与[14 C] 5-ALA进行萃取血红素结合到血红素的放射性的和测量的孵化。这个过程是准确，快速。该方法测量血红素生物合成的相对水平，而不是总血红素含量。为了证明使用该TECHN的神游血红素生物合成的水平测定几种哺乳动物细胞系。
Introduction
血红素，亚铁离子，原卟啉IX的络合物是中央分子用于运输和利用在几乎所有的活生物1-3氧。血红素的独特结构使其能够用作双原子气体和电子的载流子，以及执行各种其它功能1-5。例如，血红素结合氧血红蛋白和肌红蛋白对氧6,7的传输和存储。它也可以作为在细胞色素电子载体呼吸期间和充当用于通过细胞色素P450酶8,9-催化氧化还原反应的电子供体。之一的血红素的最显著特征是，它可以在细胞和分子过程，如基因转录，蛋白合成和微RNA的生物发生4发挥调节作用。例如，它通过控制哺乳动物转录阻遏BACH1的活性和哺乳动物核REC影响许多基因的转录eptor REV-erbα10-15。血红素调节血红素活化蛋白（HAP）1，它起着参与呼吸和控制氧化损伤的基因的活化中起重要作用，响应于血红素或氧气16的激活。血红素也调节基因转录在通 过神经生长因子（NGF）信令3,17-20神经元细胞。它还通过调节血红素调节eIF2α的激酶（HRI）21-24活性调节蛋白质合成在哺乳动物红系细胞。此外，血红素影响关键信号传导蛋白如酪氨酸激酶Jak2的和Src的，这对于适当的细胞功能和细胞生长4,20,25必需的活性。结果发现，在HeLa细胞中的血红素抑制导致细胞周期阻滞与衰老和凋亡26相关的标记物和活化。既血红素不足或血红素水平增加都与人类27严重的健康影响有关。最近的一个分子第二流行病学研究显示高血红素摄入正相关和疾病，如2型糖尿病，冠状动脉心脏疾病和多种癌症，包括肺癌，结肠直肠癌和胰腺癌27,28的风险增加。使用配对正常和癌症肺细胞作者的实验室已经发现，癌细胞已经耗氧量增加，血红素合成和参与血红素摄取和利用氧的蛋白质28的水平。有趣的是，抑制血红素合成减少耗氧量，增殖，迁移和集落形成的癌细胞28。因此，内源性血红素水平波动起着分子和细胞过程3,4,28,29的调节中起重要作用。
在哺乳动物中，血红素的生物合成发生在八个步骤，涉及位于线粒体和胞液4（ 图1）的酶。血红素生物合成论文中线粒体的基质开始与甘氨酸和琥珀酰辅酶A的缩合，以形成5-氨基乙酰丙酸（5-ALA）的通过ALA合成酶（ALAS）4,31催化。这是速率限制步骤中nonerythroid细胞血红素生物合成。 5-ALA然后出口出来到接下来的四个步骤发生，以形成粪卟啉原III（CPgenIII），然后将其导入回线粒体，在那里它被转换成原卟啉IX（PPIX）的胞质溶胶中。最后，铁的一个分子掺入到原卟啉IX（PPIX）以产生血红素，通过铁螯合（FECH）2,4-催化的反应。
血红素生物合成的水平主要取决于ALAS酶被紧紧通过细胞内的铁和血红素4控制的水平。血红素的生物合成可受遗传缺陷，某些矿物质和维生素（ 例如，核黄素，锌），暴露于毒素（ 例如，铝，铅的可用性），某些类固醇（缺氧的，发烧和等级，例如雌激素）32-35。血红素合成的电平被改变的各种疾病状况。血红素下降合成可引起贫血和神经系统疾病3,36。可替换地，增加的血红素生物合成起着某些癌症28,37的发展中起重要作用。血红素已被证明是用于哺乳动物脂肪，红系和神经元细胞4,38-41的生长，分化和存活的关键。例如，血红素缺乏导致通过谷氨酸NMDA（N-甲基-D-天冬氨酸）受体17的抑制轴突损伤在原代小鼠皮质神经元。此外，抑制血红素合成引起程序性细胞死亡的上皮宫颈癌HeLa细胞26,41。因此，测量在不同条件下在各种细胞血红素生物合成水平是研究病因和progressi重要对许多疾病的。
在这里，我们描述了一种快速，灵敏的方法，使用[4- 14 C〕-5- aminolevulic酸来测量细胞内血红素合成的水平。这是一种替代方法，使用55 Fe或59的Fe的其他方法。我们喜欢使用14 C，因为它的辐射非常微弱。相反，需要用铁同位素工作有力的保障。此外，这种方法的目的是测量并以快速的方式在不同的细胞进行比较并行血红素合成。为了测量绝对血红素水平，人们可以使用的涉及使用的HPLC 42,43的先前建立的方法。
Protocol
Representative Results
Discussion
血红素起着经由呼吸26在细胞能量的产生的关键作用。改变血红素代谢是已知的各种疾病，包括癌症28,41相关联。血红素合成的抑制已知会导致细胞周期阻滞和凋亡的Hela细胞26,41。它已经表明，高血红素合成水平与肺癌细胞28的进展有关。因此，这将是非常重要的测量在不同条件下的细胞血红素生物合成的水平。这里讨论不量化血红素的总量因此该方法，但它不提?…
Divulgations
The authors have nothing to disclose.
Acknowledgements
在HCC4017和HBEC30KT细胞系被好心医生约翰·明纳的实验室提供。这项工作是由塞西尔H.和艾达绿色基金李章博士的支持。
Materials
| Acetone | Sigma | 650501 | |
| Diethy ether | Sigma | 296082 | |
| HCl (Hydrochloric acid) | Fisher | A481-212 | |
| Liquid Scintillation cocktail | MP Biomedicals | 882470 | |
| Trypan blue | Gibco | 15250 | |
| Radiolabeled 4-14C aminolevulinic acid | Perkin-Elmer life sciences | Store @ -80 °C | |
| CelLytic M | Sigma | C2978 | Mammalian cell lysis reagent |
| Pierce BCA Protein Assay Kit | Thermo Scientific | 23227 | |
| Specific reagent | |||
| Component | Dispense | ||
| Heme extraction buffer- Acetone: HCl:Water (25:1.3:5) | Acetone | 25ml | |
| Concentrated HCl | 1.3ml | ||
| Water | 5ml |
References
- Furuyama, K., Kaneko, K., Vargas, P. D. Heme as a magnificent molecule with multiple missions: heme determines its own fate and governs cellular homeostasis. Tohoku J Exp Med. 213, 1-16 (2007).
- Hamza, I., Dailey, H. A. One ring to rule them all: trafficking of heme and heme synthesis intermediates in the metazoans. Biochim Biophys Acta. 1823, 1617-1632 (2012).
- Zhu, Y., Hon, T., Ye, W., Zhang, L. Heme deficiency interferes with the Ras-mitogen-activated protein kinase signaling pathway and expression of a subset of neuronal genes. Cell Growth Differ. 13, 431-439 (2002).
- Zhang, L. HEME BIOLOGY: The Secret Life of Heme in Regulating Diverse Biological Processes. Singapore: World Scientific Publishing Company. , (2011).
- Mense, S. M., Zhang, L. Heme: a versatile signaling molecule controlling the activities of diverse regulators ranging from transcription factors to MAP kinases. Cell Res. 16, 681-692 (2006).
- Ingram, D. J., Kendrew, J. C. Orientation of the haem group in myoglobin and its relation to the polypeptide chain direction. Nature. 178, 905-906 (1956).
- Perutz, M. F. X-ray analysis of hemoglobin. Science. 140, 863-869 (1963).
- Chance, B. The nature of electron transfer and energy coupling reactions. FEBS Lett. 23, 3-20 (1972).
- Guengerich, F. P., MacDonald, T. L. Mechanisms of cytochrome P-450 catalysis. Faseb J. 4, 2453-2459 (1990).
- Igarashi, K., et al. Multivalent DNA binding complex generated by small Maf and Bach1 as a possible biochemical basis for beta-globin locus control region complex. J Biol Chem. 273, 11783-11790 (1998).
- Ogawa, K., et al. Heme mediates derepression of Maf recognition element through direct binding to transcription repressor Bach1. Embo J. 20, 2835-2843 (2001).
- Oyake, T., et al. Bach proteins belong to a novel family of BTB-basic leucine zipper transcription factors that interact with MafK and regulate transcription through the NF-E2 site. Mol Cell Biol. 16, 6083-6095 (1996).
- Snyder, S. H., Jaffrey, S. R., Zakhary, R. Nitric oxide and carbon monoxide: parallel roles as neural messengers. Brain Res Brain Res Rev. 26, 167-175 (1998).
- Sun, J., et al. Hemoprotein Bach1 regulates enhancer availability of heme oxygenase-1 gene. Embo J. 21, 5216-5224 (2002).
- Zhang, L., Guarente, L. Heme binds to a short sequence that serves a regulatory function in diverse proteins. Embo J. 14, 313-320 (1995).
- Hon, T., Lee, H. C., Hu, Z., Iyer, V. R., Zhang, L. The heme activator protein Hap1 represses transcription by a heme-independent mechanism in Saccharomyces cerevisiae. Génétique. 169, 1343-1352 (2005).
- Chernova, T., et al. Neurite degeneration induced by heme deficiency mediated via inhibition of NMDA receptor-dependent extracellular signal-regulated kinase 1/2 activation. J Neurosci. 27, 8475-8485 (2007).
- Chernova, T., et al. Early failure of N-methyl-D-aspartate receptors and deficient spine formation induced by reduction of regulatory heme in neurons. Mol Pharmacol. 79, 844-854 (2011).
- Sengupta, A., Hon, T., Zhang, L. Heme deficiency suppresses the expression of key neuronal genes and causes neuronal cell death. Brain Res Mol Brain Res. 137, 23-30 (2005).
- Smith, A. G., Raven, E. L., Chernova, T. The regulatory role of heme in neurons. Metallomics. 3, 955-962 (2011).
- Raghuram, S., et al. Identification of heme as the ligand for the orphan nuclear receptors REV-ERBalpha and REV-ERBbeta. Nat Struct Mol Biol. 14, 1207-1213 (2007).
- Wu, N., Yin, L., Hanniman, E. A., Joshi, S., Lazar, M. A. Negative feedback maintenance of heme homeostasis by its receptor Rev-erbalpha. Genes Dev. 23, 2201-2209 (2009).
- Yin, L., et al. Rev-erbalpha, a heme sensor that coordinates metabolic and circadian pathways. Science. 318, 1786-1789 (2007).
- Zhu, Y., Hon, T., Zhang, L. Heme initiates changes in the expression of a wide array of genes during the early erythroid differentiation stage. Biochemical and biophysical research communications. 258, 87-93 (1999).
- Yao, X., Balamurugan, P., Arvey, A., Leslie, C., Zhang, L. Heme controls the regulation of protein tyrosine kinases Jak2 and Src. Biochemical and biophysical research communications. 402, 30-35 (2010).
- Ye, W., Zhang, L. Heme controls the expression of cell cycle regulators and cell growth in HeLa cells. Biochem and biophys res comm. 315, 546-554 (2004).
- Hooda, J., Shah, A., Zhang, L. Heme, an essential nutrient from dietary proteins, critically impacts diverse physiological and pathological processes. Nutrients. 6, 1080-1102 (2014).
- Hooda, J., et al. Enhanced heme function and mitochondrial respiration promote the progression of lung cancer cells. PloS one. 8, e63402 (2013).
- Atamna, H., Walter, P. B., Ames, B. N. The role of heme and iron-sulfur clusters in mitochondrial biogenesis, maintenance, and decay with age. Arch Biochem Biophys. 397, 345-353 (2002).
- Atamna, H., Killilea, D. W., Killilea, A. N., Ames, B. N. Heme deficiency may be a factor in the mitochondrial and neuronal decay of aging. Proc Natl Acad Sci U S A. 99, 14807-14812 (2002).
- Ponka, P. Cell biology of heme. Am J Med Sci. 318, 241-256 (1999).
- Brawer, J. R., Naftolin, F., Martin, J., Sonnenschein, C. Effects of a single injection of estradiol valerate on the hypothalamic arcuate nucleus and on reproductive function in the female rat. Endocrinol. 103, 501-512 (1978).
- Daniell, W. E., et al. Environmental chemical exposures and disturbances of heme synthesis. Environ Health Perspect. 105, 37-53 (1997).
- Kihara, T., et al. Hepatic heme metabolism in rats with fever induced by interleukin 1beta. Res Commun Mol Pathol Pharmacol. 104, 115-126 (1999).
- Vijayasarathy, C., Damle, S., Prabu, S. K., Otto, C. M., Avadhani, N. G. Adaptive changes in the expression of nuclear and mitochondrial encoded subunits of cytochrome c oxidase and the catalytic activity during hypoxia. Eur J Biochem. 270, 871-879 (2003).
- Anderson, K. E. S. S., Bishop, D. F., Desnick, R. J. Disorders of heme biosynthesis: X-linked sideroblastic anemia and the porphyrias. The Metabolic and Molecular Bases of Inherited Disease. , 1-53 (2009).
- Salvo, M. L., Contestabile, R., Paiardini, A., Maras, B. Glycine consumption and mitochondrial serine hydroxymethyltransferase in cancer cells: the heme connection. Med Hypotheses. 80, 633-636 (2013).
- Ishii, D. N., Maniatis, G. M. Haemin promotes rapid neurite outgrowth in cultured mouse neuroblastoma cells. Nature. 274, 372-374 (1978).
- Padmanaban, G., Venkateswar, V., Rangarajan, P. N. Haem as a multifunctional regulator. Trends Biochem Sci. 14, 492-496 (1989).
- Rutherford, T. R., Clegg, J. B., Weatherall, D. J. K562 human leukaemic cells synthesise embryonic haemoglobin in response to haemin. Nature. 280, 164-165 (1979).
- Ye, W., Zhang, L. Heme deficiency causes apoptosis but does not increase ROS generation in HeLa cells. Biochemical and biophysical research communications. 319, 1065-1071 (2004).
- Bonkovsky, H. L., et al. High-performance liquid chromatographic separation and quantitation of tetrapyrroles from biological materials. Anal Biochem. 155, 56-64 (1986).
- Sinclair, P. R., Gorman, N., Jacobs, J. M. Measurement of heme concentration. Curr Protoc Toxicol. 8, Unit 8.3 (2001).
- Barros, M. H., Carlson, C. G., Glerum, D. M., Tzagoloff, A. Involvement of mitochondrial ferredoxin and Cox15p in hydroxylation of heme O. FEBS Lett. 492, 133-138 (2001).
- Shinjyo, N., Kita, K. Up-regulation of heme biosynthesis during differentiation of Neuro2a cells. J Biochem. 139, 373-381 (2006).
- Israels, L. G., Yoda, B., Schacter, B. A. Heme binding and its possible significance in heme movement and availability in the cell. Ann N Y Acad Sci. 244, 651-661 (1975).
- Yannoni, C. Z., Robinson, S. H. Early-labelled haem in erythroid and hepatic cells. Nature. 258, 330-331 (1975).
- Robinson, S. H. Formation of bilirubin from erythroid and nonerythroid sources. Semin Hematol. 9, 43-53 (1972).
- Granick, S., Granick, D. Nucleolar necklaces in chick embryo myoblasts formed by lack of arginine. J Cell Biol. 51, 636-642 (1971).
- Morell, D. B., Barrett, J., Clezy, P. S. The prosthetic group of cytochrome oxidase. 1. Purification as porphyrin alpha and conversion into haemin alpha. Biochem J. 78, 793-797 (1961).
- Sinclair, P., Gibbs, A. H., Sinclair, J. F., de Matteis, F. Formation of cobalt protoporphyrin in the liver of rats. A mechanism for the inhibition of liver haem biosynthesis by inorganic cobalt. Biochem J. 178, 529-538 (1979).
- Chung, J., Haile, D. J., Wessling-Resnick, M. Copper-induced ferroportin-1 expression in J774 macrophages is associated with increased iron efflux. Proc Natl Acad Sci U S A. 101, 2700-2705 (2004).
