Mechanism by which the Grp94 molecular chaperone folds insulin-like growth factors

Grp94分子伴侣折叠胰岛素样生长因子的机制

• 批准号: 10612146
• 负责人: Timothy Oliver Street
• 金额: $ 3.74万
• 依托单位: BRANDEIS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-09-01 至 2024-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10612146
• 关键词: Antineoplastic Agents; Attention; Binding Sites; Biological; Client; Clinical Trials; Complex; Dissociation; Drug Targeting; Endoplasmic Reticulum; Family; Goals; Golgi Apparatus; Growth Factor; Heat-Shock Proteins 70; IGF2 gene; Molecular Chaperones; Molecular Conformation; Oncogenic; Property; Proteins; Role; Somatomedins; Testing; inhibitor; insulin secretion; member; novel; peptide hormone

Project Summary The endoplasmic reticulum (ER) chaperone Grp94 is required for the correct folding and secretion of insulin-like growth factors (IGF), but the underlying mechanism is not understood. Our goal is to discover how Grp94 assists the folding of IGF proteins. Grp94 is a member of the Hsp90 chaperone family, which require ATP-driven conformational cycling to chaperone their “client proteins”. Many oncogenic proteins depend on Hsp90 for their function. As a result, ATP-competitive inhibitors of Hsp90 have been extensively investigated as anti-cancer drugs. Although cytosolic Hsp90s were the first intended targets of inhibitors in clinical trials, Grp94 is receiving increasing attention as a drug target. Grp94 can receive clients from BiP, the Hsp70-family chaperone in the ER. Through a chance discovery, we have found that the combination of BiP and Hsp90 inhibitors locks Grp94 in a novel conformation, a state apparently poised for client transfer from BiP to Grp94. Client transfer from BiP to Grp94 requires two sequential steps: handover of the client from BiP to Grp94, followed by BiP dissociation from the ternary complex. Our first hypothesis is that Hsp90 inhibitors block both steps: Hsp90 inhibitors stall Grp94 and BiP in a client transfer state because ATP-dependent conformational changes of Grp94 are required to handover the client from BiP and then displace BiP from Grp94. We will test this hypothesis with IGF2, as we have discovered that this Grp94 client also has a well-defined BiP binding site. Aim1: Determine whether ATP-dependent conformational changes of Grp94 cause BiP to handover a bound client protein, and whether Hsp90 inhibitors disrupt this client-transfer function. Aim2: Determine whether ATP-dependent conformational changes of Grp94 are required to actively displace BiP from Grp94. Aims 1&2 will test a unified mechanism that can explain how Hsp90 inhibitors disrupt Grp94 function, but does not explain what essential chaperoning function is provided by BiP and Grp94 for IGF proteins. We have discovered that IGF2 forms dynamic oligomers. Similar to other peptide hormones that are known to oligomerize, this self-association may enable IGF2 to be effectively concentrated and packaged prior to its export to the Golgi. Despite a plausible biological role for IGF2 oligomerization, we have also observed that IGF2 can transition from dynamic reversible oligomers to irreversible aggregates. Thus, our second hypothesis is that the essential chaperoning function of BiP and Grp94 is to maintain a dynamic oligomerization state of IGF2. This idea is tested in Aim3: Determine the influence of Grp94 and BiP on the oligomerization properties of ProIGF2.

项目摘要 内质网（ER）伴侣GRP94是正确折叠和分泌胰岛素样需要的 生长因子（IGF），但尚不理解潜在的机制。我们的目标是发现GRP94如何辅助 IGF蛋白的折叠。 GRP94是HSP90伴侣家族的成员，该家族需要ATP驱动 构型循环以伴侣的“客户蛋白”。许多致癌蛋白都取决于HSP90 功能。结果，HSP90的ATP竞争力抑制剂已被广泛研究为抗癌 毒品。尽管胞质HSP90S是临床试验中抑制剂的第一个预期靶标，但GRP94正在接受 作为药物靶标的注意力越来越多。 GRP94可以从ER中的HSP70家族伴侣BIP接收客户。 通过偶然发现，我们发现BIP和HSP90抑制剂的组合将GRP94锁定在A 新颖的构象，一个显然被中毒的国家从BIP转移到GRP94。客户从BIP转移到 GRP94需要两个顺序的步骤：将客户从BIP转移到Grp94，然后从BIP解离 三元建筑群。我们的第一个假设是HSP90抑制剂阻止了这两个步骤：HSP90抑制剂失速GRP94 和BIP处于客户转移状态，因为需要ATP的咨询更改GRP94 要从BIP中移交客户，然后将BIP从GRP94移位。我们将用IGF2检验该假设， 正如我们发现该GRP94客户端还具有明确定义的BIP绑定位点。 AIM1：确定是否 GRP94的ATP依赖性咨询变化导致BIP移交有限的客户蛋白，以及是否是否 HSP90抑制剂破坏了此客户端转移功能。 AIM2：确定是否依赖ATP的构象 需要GRP94的变化才能积极地从GRP94中取代BIP。目标1和2将测试统一的机制 可以解释HSP90抑制剂如何破坏GRP94的功能，但不能解释什么必需的链 功能由BIP和GRP94用于IGF蛋白。我们已经发现IGF2形成动态低聚物。 类似于其他已知的肽激素，这种自我关联可能使IGF2成为 尽管IGF2具有合理的生物学作用 低聚，我们还观察到IGF2可以从动态可逆性低聚物过渡到不可逆 聚合。这是我们的第二个假设是，BIP和GRP94的基本伴侣功能是 保持IGF2的动态寡聚状态。这个想法在AIM3中进行了测试：确定 grp94和bip on proigf2的寡聚特性。