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: 10246944
• 负责人: Timothy Oliver Street
• 金额: $ 39.83万
• 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/10246944
• 关键词: ATP Hydrolysis; Antineoplastic Agents; Area; Attention; Behavior; Binding Sites; Biological; Biotechnology; Cells; Client; Clinical Trials; Collaborations; Complex; Cytosol; Detection; Disease; Dissociation; Disulfides; Drug Targeting; Endoplasmic Reticulum; Environment; Family; Goals; Golgi Apparatus; Growth Factor; Health; Heat-Shock Proteins 70; Hormones; Human; IGF1 gene; IGF2 gene; In Vitro; Individual; Insulin; Link; Malignant Neoplasms; Measurement; Mitochondria; Molecular Chaperones; Molecular Conformation; Molecular Structure; Non-Insulin-Dependent Diabetes Mellitus; Oncogenic; Pathology; Peptides; Process; Production; Proinsulin; Property; Proteins; Recording of previous events; Research; Role; Somatomedins; Structure of beta Cell of islet; System; Testing; Time; Universities; Work; Yeasts; analog; improved; inhibitor/antagonist; innovation; insulin secretion; member; novel; peptide hormone; protein folding; single-molecule FRET

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.

正确折叠和分泌需要内质网（ER）伴侣GRP94 胰岛素样生长因子（IGF），但尚不清楚潜在的机制。我们的目标是发现 GRP94有助于IGF蛋白的折叠。 GRP94是HSP90伴侣家族的成员，它需要ATP- 驱动的构型循环以伴侣的“客户蛋白”伴侣。许多致癌蛋白取决于HSP90 为了它们的功能。结果，HSP90的ATP竞争抑制剂已被广泛研究为抗 癌症药物。尽管胞质HSP90S是临床试验中抑制剂的第一个预期靶标，但GRP94为 作为药物靶标，人们受到越来越多的关注。 GRP94可以从BIP（HSP70家族链条）接收客户 在急诊室。通过机会发现，我们发现BIP和HSP90抑制剂锁的组合 GRP94在一种新颖的构象中，一个州显然被毒害的客户从BIP转移到GRP94。客户转移 从BIP到GRP94需要两个顺序的步骤：将客户端从BIP到GRP94进行交换，然后是BIP 与三元复合物解离。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Stall GRP94和BIP处于客户转移状态，因为ATP依赖性咨询变化 GRP94的of需要从BIP中移交客户，然后从GRP94中取代BIP。我们将测试这个 使用IGF2的假设，正如我们发现该GRP94客户端还具有明确定义的BIP结合位点。 AIM1：确定GRP94的依赖ATP的会议变化是否导致BIP移交界限 客户蛋白，以及HSP90抑制剂是否破坏了该客户端转移功能。 AIM2：确定是否ATP- 需要GRP94的依赖会议更改才能积极地从GRP94中取代BIP。目标1和2将测试 一种统一的机制，可以解释HSP90抑制剂如何破坏GRP94的功能，但不能解释什么 IGF蛋白提供了BIP和GRP94的必要伴侣功能。我们发现IGF2 形成动态低聚物。类似于其他已知的肽恐怖，这种自我关联 可以使IGF2在出口到高尔基体之前有效地浓缩和包装。尽管有合理 IGF2低聚的生物学作用，我们还观察到IGF2可以从动态可逆性转变 将异性骨料的寡聚物。这是我们的第二个假设是必需的伴侣功能 BIP和GRP94的含量是维持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对PROIGF2的寡聚特性的影响。