Characterization of the specificity of O-GlcNAc reactive antibodies under conditions of starvation and stress.
Characterization of the specificity of O-GlcNAc reactive antibodies under conditions of starvation and stress.
复制标题
DOI：
10.1016/j.ab.2014.04.008
复制
发表时间：
2014-07-15
影响因子：
2.9
通讯作者：
Zachara NE
中科院分区：
文献类型：
--
作者：
Reeves RA;Lee A;Henry R;Zachara NE
O-连接的β-N-乙酰基-D-氨基葡萄糖（O-GlcNAc）对细胞核、细胞质和线粒体蛋白的动态修饰已显示以类似于蛋白磷酸化的方式调节超过3，000种蛋白。O-GlcNAc酰化调节细胞应激反应、细胞周期，并且与神经变性、II型糖尿病和癌症的病因学有关。抗体CTD 110.6通常用于检测O-GlcNAc修饰的变化。最近，已经证明CTD 110.6识别N-连接的N，N '-二乙酰基壳二糖，其被认为在经历严重葡萄糖剥夺的细胞中积累。在这项研究中，我们解决了两个问题：1）哪些其他抗体用于检测O-GlcNAc与N-连接的N，N '-二乙酰壳二糖交叉反应？2)N-连接的N，N '-二乙酰基壳二糖是否在对其他细胞应激源的反应中积累？为了在O-GlcNAc和N-连接的N，N '-二乙酰基壳二糖之间描绘，我们开发了一种工作流程，该工作流程已用于确认多种O-GlcNAc特异性抗体的特异性。使用该工作流程，我们证明了热休克、渗透压应激、内质网应激、氧化应激、DNA损伤、蛋白酶体抑制和ATP耗竭诱导O-GlcNAc酰化，但不诱导N-连接的N，N '-二乙酰壳二糖。此外，我们证明，虽然葡萄糖剥夺导致O-GlcNAc和N-连接的N，N '-二乙酰基壳二糖的诱导，但N-连接的N，N'-二乙酰基壳二糖的诱导因胎牛血清的去除而加剧。
The dynamic modification of nuclear, cytoplasmic, and mitochondrial proteins by O-linked β-N-acetyl-D-Glucosamine (O-GlcNAc) has been shown to regulate over 3,000 proteins in a manner analogous to protein phosphorylation. O-GlcNAcylation regulates the cellular stress response, the cell cycle, and is implicated in the etiology of neurodegeneration, type II diabetes, and cancer. The antibody CTD110.6 is often used to detect changes in the O-GlcNAc modification. Recently, it has been demonstrated that CTD110.6 recognizes N-linked N,N’-diacetylchitobiose, which is thought to accumulate in cells experiencing severe glucose deprivation. In this study, we have addressed two questions: 1) Which other antibodies used to detect O-GlcNAc cross-react with N-linked N,N’-diacetylchitobiose? 2) Does N-linked N,N’-diacetylchitobiose accumulate in response to other cellular stressors? To delineate between O-GlcNAc and N-linked N,N’-diacetylchitobiose, we developed a workflow that has been used to confirm the specificity of a variety of O-GlcNAc specific antibodies. Using this workflow we demonstrated that heat shock, osmotic stress, endoplasmic reticulum stress, oxidative stress, DNA damage, proteasomal inhibition, and ATP depletion induce O-GlcNAcylation but not N-linked N,N’-diacetylchitobiose. Moreover, we demonstrated that while glucose deprivation results in an induction in both O-GlcNAc and N-linked N,N’-diacetylchitobiose, the induction of N-linked N,N’-diacetylchitobiose is exacerbated by the removal of fetal bovine serum.
登录
查看更多内容
影响因子：
4.8
作者：
Taylor, Rodrick P.;Geisler, Taylor S.;McClain, Donald A.
通讯作者：
McClain, Donald A.
影响因子：
4.8
作者：
Kreppel, LK;Hart, GW
通讯作者：
Hart, GW
DOI：
10.1073/pnas.87.15.5608
发表时间：
1990-08-01
影响因子：
11.1
作者：
TURNER, JR;TARTAKOFF, AM;GREENSPAN, NS
通讯作者：
GREENSPAN, NS
影响因子：
3
作者：
McClain, DA
通讯作者：
McClain, DA
影响因子：
4.8
作者：
Zachara, NE;O'Donnell, N;Hart, GW
通讯作者：
Hart, GW