Antisense glutaminase inhibition modifies the O-GlcNAc pattern and flux through the hexosamine pathway in breast cancer cells
Antisense glutaminase inhibition modifies the O-GlcNAc pattern and flux through the hexosamine pathway in breast cancer cells
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DOI：
10.1002/jcb.21449
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发表时间：
2008-02-15
影响因子：
4
通讯作者：
Segura, Juan A.
中科院分区：
文献类型：
--
作者：
Donadio, Ana C.;Lobo, Carolina;Segura, Juan A.
谷氨酰胺是肿瘤和快速分裂细胞的关键营养素。谷氨酰胺酶是这些细胞中主要的谷氨酰胺利用酶，其活性与谷氨酰胺消耗和生长速率相关。我们在人MCF 7乳腺癌细胞中进行了反义L-型转氨酶抑制，以研究其对己糖胺途径和蛋白质O-糖基化模式的影响。表达反义mRNA精氨酸酶的细胞，命名为ORF 19，呈现出比亲本细胞低50%的增殖率，显示出更分化的表型。ORF 19细胞的谷氨酰胺：果糖-6-P酰胺转移酶活性降低了80%，这是己糖胺途径的限速步骤。虽然总体细胞蛋白O-糖基化没有改变，但几个关键蛋白的O-糖基化状态发生了改变。O-GlcNAc转移酶（OGT），将N-乙酰葡糖胺连接到蛋白质上的酶，在ORF 19中的O-糖基化比野生型细胞低5倍。抑制谷氨酰胺酶也引起Sp1表达增加10倍，并且Sp1和Rpt 2蛋白酶体组分的O-糖基化与总蛋白的比率显著降低。这些变化伴随着更高的Sp1转录活性。O-糖基化蛋白的蛋白质组分析允许检测两个新的OGT靶蛋白：伴侣蛋白TCP-1 θ和癌基因Ets相关蛋白亚型7。两者合计，我们的研究结果支持己糖胺途径和蛋白质的O-糖基化是细胞的营养和能量状态的传感器机制。
Glutamine behaves as a key nutrient for tumors and rapidly dividing cells. Glutaminase is the main glutamine-utilizing enzyme in these cells, and its activity correlates with glutamine consumption and growth rate. We have carried out the antisense L-type glutaminase inhibition in human MCF7 breast cancer cells, in order to study its effect on the hexosamine pathway and the pattern of protein O-glycosylation. The antisense mRNA glutaminase expressing cells, named ORF19, presented a 50% lower proliferation rate than parental cells, showing a more differentiated phenotype. ORF19 cells had an 80% reduction in glutamine:fructose-6-P amidotransferase activity, which is the rate-limiting step of the hexosamine pathway. Although the overall cellular protein O-glycosylation did not change, the O-glycosylation status of several key proteins was altered. O-glycosylation of O-GlcNAc transferase (OGT), theenzymethat links N-acetylglucosamineto proteins, was fivefold lower in ORF19 than in wild type cells. Inhibition of glutaminase also provoked a 10-fold increase in Sp1 expression, and a significant decrease in the ratio of O-glycosylated to total protein for both Sp1 and the Rpt2 proteasome component. These changes were accompanied by a higher Sp1 transcriptional activity. Proteome analysis of O-glycosylated proteins permitted the detection of two new OGT target proteins: the chaperonin TCP-1 theta and the oncogene Ets-related protein isoform 7. Taken together, our results support the hexosamine pathway and the O-glycosylation of proteins being a sensor mechanism of the nutritional and energetic states of the cell.