Mechanism of polyubiquitin chain assembly by an ER-associated ubiquitin ligase

内质网相关泛素连接酶组装多聚泛素链的机制

• 批准号: 8939553
• 负责人: Yihong Ye
• 金额: $ 19.02万
• 依托单位: NATIONAL INSTITUTE OF DIABETES AND DIGESTIVE AND KIDNEY 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/8939553
• 关键词: Active Sites; Arginine; Aspartate; Binding Sites; Biological Assay; Cell physiology; Cells; Charge; Collaborations; Cysteine; Data; Dimerization; Electrostatics; Enzymes; Genes; Goals; Homologous Gene; In Vitro; Ligase; Ligase Gene; Link; Mediating; Modification; Molecular; Mutagenesis; Polyubiquitin; Polyubiquitination; Positioning Attribute; Protein C; Proteins; Reaction; Role; Site; Specific qualifier value; Surface; System; Testing; Ubiquitin; Ubiquitin-Conjugating Enzymes; Ubiquitination; Yeasts; carboxyl group; human UBE3A protein; interest; novel; research study; thioester; ubiquitin ligase; ubiquitin-protein ligase

Covalent modification of a protein with the 76-residue protein ubiquitin (Ub), thereby changing the stability, localization, or activity of the target protein, regulates almost all aspects of eukaryotic cellular function. This reaction requires the sequential actions of three types of enzymes; an activating enzyme (E1) forms a thioester linkage between its catalytic cysteine and the carboxyl group of Gly76 in ubiquitin to activate it; a conjugating enzyme (E2) that receives ubiquitin from the E1; a ubiquitin ligase (E3) that transfers the ubiquitin molecule from the E2 to a substrate. For every eukaryotic species, there are two E1 enzymes, whereas approximately dozens of E2 enzymes (Table 1) and thousands of E3 ligases are found. The E3 ligases can be classified into two major catagories, the HECT domain (homologous to E6-associated protein C-Terminus)-containing ligases and those carrying a catalytic RING (really interesting new gene) domain. We have established an in vitro ubiquitination system using purified gp78c, a cytosolic domain of an ER-associated RING ligase and ube2g2. The assay allows us to assemble Lys48 linked polyubiquitin chains. Using this system, we have demonstrated that polyubiquitin chains can be preassembled on the catalytic cysteine of Ube2g2 before being transferred to a substrate. Active site-linked polyubiquitin chains are detected in cells on Ube2g2 and its yeast homolog Ubc7p, but how these chains are assembled on an E2 active site is unclear. We recently discovered that gp78 forms an oligomer via two oligomerization sites, one of which being a hydrophobic segment in the gp78 cytosolic domain (gp78C). We further demonstrate that a gp78 oligomer can simultaneously associate with multiple Ube2g2 molecules using a novel interaction that is primarily mediated by an Ube2g2 surface distinct from the predicted RING binding site. Our data suggest that the formation of such a gp78-Ube2g2 hetero-oligomer brings multiple Ube2g2 molecules into close proximity, allowing ubiquitin moieties to be transferred between neighboring Ube2g2 to form active site-linked polyubiquitin chains. More recently, in collaboration with Dr. Wei Li, we demonstrate that Ube2g2 synthesizes linkage specific ubiquitin chains by forming an unprecedented homodimer: The dimerization of Ube2g2, mediated primarily by electrostatic interactions between two Ube2g2s, is also facilitated by the charged ubiquitin molecules. Mutagenesis studies show that Ube2g2 dimerization is required for ER-associated degradation (ERAD). In addition to E2 dimerization, we show that a highly conserved arginine residue in the donor Ube2g2 senses the presence of an aspartate in the acceptor ubiquitin to position only Lys48 of ubiquitin in proximity to the donor E2 active site. These results reveal an unanticipated mode of E2 self-association that allows the E2 to effectively engage two ubiquitins to specifically synthesize Lys48-linked ubiquitin chains.

用 76 个残基蛋白泛素 (Ub) 共价修饰蛋白质，从而改变靶蛋白的稳定性、定位或活性，调节真核细胞功能的几乎所有方面。 该反应需要三种酶的连续作用；激活酶(E1)在其催化的半胱氨酸与泛素中Gly76的羧基之间形成硫酯键以将其激活；结合酶 (E2)，从 E1 接收泛素；泛素连接酶 (E3)，可将泛素分子从 E2 转移至底物。对于每个真核物种，都有两种 E1 酶，而大约有数十种 E2 酶（表 1）和数千种 E3 连接酶。 E3 连接酶可分为两大类：含有 HECT 结构域（与 E6 相关蛋白 C 末端同源）的连接酶和携带催化 RING（非常有趣的新基因）结构域的连接酶。 我们使用纯化的 gp78c（ER 相关 RING 连接酶的胞质结构域）和 ube2g2 建立了体外泛素化系统。该测定允许我们组装 Lys48 连接的多聚泛素链。使用该系统，我们证明多聚泛素链可以在转移到底物之前预先组装在 Ube2g2 的催化半胱氨酸上。在 Ube2g2 及其酵母同源物 Ubc7p 的细胞中检测到活性位点连接的多聚泛素链，但这些链如何在 E2 活性位点上组装尚不清楚。我们最近发现 gp78 通过两个寡聚位点形成寡聚体，其中一个是 gp78 胞质结构域 (gp78C) 中的疏水片段。我们进一步证明，gp78 寡聚物可以使用一种新的相互作用同时与多个 Ube2g2 分子结合，该相互作用主要由与预测的 RING 结合位点不同的 Ube2g2 表面介导。我们的数据表明，这种 gp78-Ube2g2 异源寡聚体的形成使多个 Ube2g2 分子紧密相连，从而允许泛素部分在相邻的 Ube2g2 之间转移，形成活性位点连接的多泛素链。 最近，我们与李伟博士合作，证明Ube2g2通过形成前所未有的同型二聚体来合成连接特异性泛素链：Ube2g2的二聚化主要由两个Ube2g2之间的静电相互作用介导，带电的泛素分子也促进了这种二聚化。诱变研究表明，Ube2g2 二聚化是 ER 相关降解 (ERAD) 所必需的。除了E2二聚化之外，我们还发现供体Ube2g2中高度保守的精氨酸残基感知受体泛素中天冬氨酸的存在，从而仅将泛素的Lys48定位在供体E2活性位点附近。这些结果揭示了一种意想不到的 E2 自缔合模式，该模式允许 E2 有效地与两个泛素结合，特异性合成 Lys48 连接的泛素链。

项目成果

The E3 ubiquitin ligases Hrd1 and gp78 bind to and promote cholera toxin retro-translocation.

E3 泛素连接酶 Hrd1 和 gp78 结合并促进霍乱毒素逆向易位。

• 发表时间: 2010-01-01
• 影响因子: 3.3
• 作者: Bernardi, Kaleena M;Williams, Jeffrey M;Kikkert, Marjolein;van Voorden, Sjaak;Wiertz, Emmanuel J;Ye, Yihong;Tsai, Billy
• 通讯作者: Tsai, Billy

Polyubiquitin chains: functions, structures, and mechanisms.

多聚泛素链：功能、结构和机制。

• 发表时间: 2008-08
• 作者: Li, W;Ye, Y
• 通讯作者: Ye, Y