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首页> 外文期刊>Infection and immunity >Pertussis toxin-catalyzed ADP-ribosylation of Gi-2 and Gi-3 in CHO cells is modulated by inhibitors of intracellular trafficking.
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Pertussis toxin-catalyzed ADP-ribosylation of Gi-2 and Gi-3 in CHO cells is modulated by inhibitors of intracellular trafficking.

机译：CHO-2中GI-2和GI-3的PERTUSSIS毒素催化的ADP-核糖基化由细胞内运输的抑制剂调节。

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In previous studies, an in vitro ADP-ribosylation assay was developed to quantitatively evaluate the in vivo ADP-ribosylation of eukaryotic target proteins in intact Chinese hamster ovary (CHO) cells by pertussis toxin (PT). Immunoblot analysis identified the two PT-sensitive target proteins in CHO cells as Gi-2 and Gi-3. In this in vitro ADP-ribosylation assay, the ability of PT and ADP-ribosylate Gi-2 and Gi-3 intact CHO cells was not inhibited by cytochalasin D but was inhibited by chloroquine, monensin, and nocodazole. These data implicated the involvement of a cytochalasin D-independent endocytic mechanism, a pH-sensitive step, and microtubules in the ADP-ribosylation of Gi-2 and Gi-3 by PT in intact CHO cells. Preincubation of CHO cells with cycloheximide, at concentrations that reduced protein synthesis by > 95%, did not inhibit the ability of PT to ADP-ribosylate Gi-2 and Gi-3. Control experiments showed that these agents did not affect either the ability of PT to directly ADP-ribosylate the heterotrimeric G protein, Gt, or the binding of PT to CHO cells, except that monensin slightly inhibited the binding of PT to CHO cells. These results are consistent with a model in which PT is internalized by receptor-mediated endocytosis, probably via a cytochalasin D-independent pathway, which involves intracellular trafficking through late endosomes and the Golgi apparatus. An alternative model predicts the presence of a eukaryotic factor that traffics within cells via this pathway and is required by PT to ADP-ribosylate Gi proteins.
机译：在先前的研究中，开发了体外ADP-核糖基化测定以通过Pertussis毒素（PT）定量评价真核毒物卵巢（CHO）细胞的真核靶蛋白的体内ADP-核糖基化。免疫印迹分析将CHO细胞中的两个Pt敏感靶蛋白鉴定为GI-2和GI-3。在该体外ADP-核糖基化测定中，CTOCOALASIN D不抑制PT和ADP-核糖基酯GI-2和GI-3完整CHO细胞的能力，但受氯喹，莫素和Nocodazole抑制。这些数据涉及通过PT在完整的CHO细胞中通过PT在GI-2和Gi-3的ADP-核糖化中的促进核糖化中的细胞蛋白D-独立的内核化机制，pH敏感性步骤和微管的累积。 CHO细胞与环己酰亚胺预孵育，在减少蛋白质合成的浓度> 95％的浓度下，不抑制Pt至Adp-核糖酸酯Gi-2和Gi-3的能力。对照实验表明，这些试剂不影响Pt直接Adp-核糖基酯的能力，除了宫内蛋白略微抑制Pt至Cho细胞的结合之外，除了Pt至Cho细胞的结合的能力。这些结果与受体介导的内吞作用的PT内化的模型一致，可能通过细胞蛋白D-独立途径，这涉及通过晚期内体和高尔基装置的细胞内运输。另一种模型预测通过该途径通过该途径在细胞内运输的真核因子的存在，并且由Pt至Adp-核糖基酯Gi蛋白。

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