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是一种多功能细胞因子，具有有效的免疫调节特性。 白细胞谱系的几乎所有细胞表达了细胞因子。 尽管尚未完全理解，但TGF-β的作用已经在许多过程中确立，包括炎症反应，致癌作用和口服耐受性作为主要的抑制性细胞因子。 通过表达细胞周期蛋白依赖性激酶抑制剂p27分子，TGF-β治疗在G1后期导致细胞周期停滞。 TGF-β受体由两个链I型和II链组成。两者都是受体丝氨酸/苏氨酸激酶。 TGF-β与II型受体募集的结合并激活I型受体，这又激活了SMAD信号通路，从而导致基因表达的调节。由于缺乏结构性工作，受体和TGF-β的配体结合区域尚不清楚。我们的目标是确定TGF-β受体的晶体结构及其与TGF-的复合物。 我们希望获得对受体激活的功能性洞察力。我们的首次尝试是与TGF-BETA1复合物中表达的杆状病毒II型TGF-β受体（TBRII）。 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。随后的退化糖基化过程产生了部分退化的受体，当与TGF-BETA1复合时，该受体产生了小晶体。但是，这些晶体在X射线束中衍射较差，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率，我们试图在几种细菌表达载体系统中表达可溶性II型受体。 由于该受体中存在的大量二硫键键（12个半胱氨酸），其他组以前试图重新构建表达受体的细菌都失败了。 我们制作的第一个细菌是将II型TGF-β受体表示为GST融合蛋白。 从TGF-β结合ELISA分析中可以明显看出，这导致了功能受体的产生。 但是，GST融合构建体的TBRII产量仍然很低。 这主要是由于GST和受体之间接头区域的降解，这导致大多数从谷胱甘肽亲和力柱中洗脱样品是游离GST，而不是GST-TBRII融合蛋白。 典型的10升细菌表达实验产生的纯化受体蛋白小于0.5 mg。为了克服低表达产量的问题，尽管其他人以前没有成功，但我们开始通过重新折叠方法来调查纯化方案。 结果，我们将ecto-tbrii（没有任何融合伙伴）归为两个Novagen的宠物矢量PET14和PET30，它们都是由T7聚合酶驱动的。 该构建体的表达导致大量包含体。我们的初步重构实验表明，可以轻松获得毫克量的TBRII，并且在TGF-beta ELISA测定中似乎有活性。最近，我们使用该受体的结晶实验产生了TBRII的小晶体。一旦我们使用TBRII证明了细菌包容体表达系统的适用性，就采用了相同的方法来表达受体配体TGF-BETA1，TGF-BETA2和TGF？beta3。 我们的初步结果表明，TGF-β的所有三种同工型都可以使用此方法表示并重构为活动形式。