Structure Determination of TGF-beta Receptor

TGF-β受体的结构测定

• 批准号: 6431697
• 负责人: PETER D. SUN
• 金额: --
• 依托单位: NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/6431697
• 关键词: Baculoviridae; crystallization; disulfide bond; enzyme linked immunosorbent assay; glycosylation; growth factor receptors; molecular cloning; protein folding; protein reconstitution; protein structure; receptor expression; structural biology; transfection /expression vector; transforming growth factors

TGF-beta is a pleiotropic, multifunctional cytokine with potent immunoregulatory properties. Nearly all cells of leukocyte lineage express the cytokine. Although not completely understood, the role of TGF-beta has been established in many processes including inflammatory response, carcinogenesis and oral tolerance as the primary inhibitory cytokine. TGF-beta treatment leads to cell-cycle arrest at the late G1 phase through the expression of a cyclin-dependent kinase inhibitor p27 molecule. The TGF-beta receptor consists of two chains, type I and II; both are receptor serine/threonine kinases. The binding of TGF-beta to the type II receptor recruits and activates the type I receptor, which in turn activates the SMAD signaling pathway, leading to the regulation in gene expression. Due to the lack of structural work, the ligand-binding region of both the receptor and TGF-beta remains unclear. Our objective is to determine the crystal structures of the TGF-beta receptor and its complex with TGF-. We hope to gain functional insight into the receptor activation. Our first attempt was to crystallize a baculovirus expressed human type II TGF-beta receptor (TBRII) in complex with TGF-beta1. Due to extensive glycosylation on the receptor, the crystallization trials with this receptor did not yield any crystals. Subsequent deglycosylation procedure yielded a partially deglycosylated receptor that produced small crystals when complexed with TGF-beta1. However, these crystals diffracted poorly in the X-ray beam and were not suitable for structure determination. To overcome the carbohydrate heterogeneity and low expression yield of the baculovirus expression system, we have attempted to express the soluble type II receptor in several bacteria expression vector systems. Due to a large number of disulfide bonds (12 cysteines) present in this receptor, previous attempts by other groups to reconstitute the bacteria expressed receptor have all failed. The first bacteria construct we made was to express the type II TGF-beta receptor as a GST fusion protein. This resulted in the production of a functional receptor, as evident from the TGF-beta binding ELISA assays, in soluble fraction of cell lysates. However, the yield of TBRII from the GST fusion construct remained low. This was primarily due to the degradation of the linker region between GST and the receptor, which resulted in the majority eluted sample from a glutathione affinity column being free GST rather than GST-TBRII fusion protein. A typical 10 liter bacteria expression experiment yielded less than 0.5 mg of purified receptor protein. To overcome the problem of low expression yield, we began to investigate the purification schemes by refolding methods, despite previous attempts by others not being successful. As a result, we subcloned the ecto-TBRII (without any fusion partner) into two Novagen's pET vectors, pET14 and pET30, both of which are driven by T7 polymerase. The expression of this construct resulted in large quantities of inclusion bodies. Our preliminary reconstitution experiment suggests that milligram quantities of TBRII can be obtained readily and the reconstituted protein appears to be active in a TGF-beta ELISA assay. Recently, our crystallization experiments using this receptor have produced small crystals of TBRII. Once we demonstrated the suitability of the bacterial inclusion body expression system using TBRII, the same method was applied to express the receptor ligands, TGF-beta1, TGF-beta2, and TGF?beta3. Our preliminary results indicate that all three isoforms of TGF-beta can be expressed and reconstituted into active form using this method.

TGF-β 是一种多效性、多功能细胞因子，具有有效的免疫调节特性。 几乎所有白细胞谱系的细胞都表达细胞因子。 尽管尚未完全了解，但 TGF-β 在许多过程中的作用已被确定为主要抑制性细胞因子，包括炎症反应、致癌和口服耐受。 TGF-β 处理通过细胞周期蛋白依赖性激酶抑制剂 p27 分子的表达导致细胞周期停滞在 G1 期晚期。 TGF-β受体由两条链组成，I型和II型；两者都是受体丝氨酸/苏氨酸激酶。 TGF-β 与 II 型受体的结合会募集并激活 I 型受体，进而激活 SMAD 信号通路，从而调节基因表达。由于缺乏结构研究，受体和 TGF-β 的配体结合区域仍不清楚。我们的目标是确定 TGF-β 受体及其与 TGF-β 复合物的晶体结构。 我们希望深入了解受体激活的功能。我们的第一次尝试是结晶表达人 II 型 TGF-β 受体 (TBRII) 与 TGF-β1 复合物的杆状病毒。 由于受体上广泛的糖基化，该受体的结晶试验没有产生任何晶体。随后的去糖基化过程产生了部分去糖基化的受体，当与 TGF-β1 复合时，该受体产生小晶体。然而，这些晶体在 X 射线束中的衍射性能很差，不适合结构测定。 为了克服杆状病毒表达系统的碳水化合物异质性和低表达产量，我们尝试在几种细菌表达载体系统中表达可溶性II型受体。 由于该受体中存在大量二硫键（12个半胱氨酸），之前其他小组重建细菌表达受体的尝试均失败了。 我们制作的第一个细菌构建体是将 II 型 TGF-β 受体表达为 GST 融合蛋白。 这导致在细胞裂解物的可溶部分中产生功能性受体，如 TGF-β 结合 ELISA 测定所证明的那样。 然而，GST 融合构建体的 TBRII 产量仍然很低。 这主要是由于 GST 和受体之间的连接区降解，导致从谷胱甘肽亲和柱中洗脱的大部分样品是游离 GST，而不是 GST-TBRII 融合蛋白。 典型的 10 升细菌表达实验产生少于 0.5 毫克的纯化受体蛋白。为了克服表达产量低的问题，我们开始研究重折叠方法的纯化方案，尽管其他人之前的尝试没有成功。 因此，我们将 ecto-TBRII（没有任何融合伴侣）亚克隆到 Novagen 的两个 pET 载体 pET14 和 pET30 中，这两个载体均由 T7 聚合酶驱动。 该构建体的表达产生了大量的包涵体。我们的初步重构实验表明，可以轻松获得毫克量的 TBRII，并且重构的蛋白质在 TGF-β ELISA 测定中似乎具有活性。最近，我们使用该受体的结晶实验产生了 TBRII 的小晶体。一旦我们证明了使用 TBRII 的细菌包涵体表达系统的适用性，就应用相同的方法来表达受体配体 TGF-β1、TGF-β2 和 TGFβ3。 我们的初步结果表明，使用这种方法可以表达 TGF-β 的所有三种亚型并重构为活性形式。