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

项目摘要 内质网（ER）伴侣Grp94是胰岛素样多肽的正确折叠和分泌所必需的。 生长因子（IGF），但其潜在机制尚不清楚。我们的目标是发现Grp94如何帮助 IGF蛋白的折叠Grp94是Hsp90分子伴侣家族的成员，其需要ATP驱动 构象循环以陪伴它们的“客户蛋白”。许多致癌蛋白依赖于Hsp90进行其功能。 功能因此，Hsp90的ATP竞争性抑制剂作为抗癌药物已被广泛研究。 毒品虽然细胞溶质Hsp90是临床试验中抑制剂的第一个预期靶点，但Grp94正在接受 作为药物靶点越来越受到关注。Grp94可以接收来自BiP的客户，BiP是ER中的Hsp70家族伴侣。 通过一次偶然的发现，我们发现BiP和Hsp90抑制剂的组合将Grp94锁定在 新的构象，一个国家显然准备从BiP客户端转移到Grp94。客户端从BiP转移到 Grp94需要两个连续的步骤：将客户端从BiP切换到Grp94，然后将BiP从 三元复合体我们的第一个假设是Hsp90抑制剂阻断了这两个步骤： 和BiP处于客户端转移状态，因为需要Grp94的ATP依赖性构象变化 从BiP切换客户端，然后从Grp94替换BiP。我们将用IGF2来检验这一假设， 因为我们已经发现该Grp94客户端也具有明确的BiP结合位点。目标1：确定是否 Grp94的ATP依赖性构象变化导致BiP移交结合的客户蛋白，以及 Hsp90抑制剂破坏了这种客户转移功能。目的2：确定是否存在ATP依赖性构象 需要Grp94的变化来主动地从Grp94置换BiP。目标1和2将测试一个统一的机制， 可以解释Hsp90抑制剂如何破坏Grp94功能，但不能解释什么是必要的伴侣 BiP和Grp94为IGF蛋白提供功能。我们已经发现IGF2形成动态寡聚体。 类似于已知寡聚化的其他肽激素，这种自缔合可能使IGF2能够被寡聚化。 在出口到高尔基体之前有效地浓缩和包装。尽管IGF2具有合理的生物学作用， 我们还观察到IGF2可以从动态可逆寡聚体转变为不可逆寡聚体， 集料.因此，我们的第二个假设是BiP和Grp94的基本伴侣功能是 维持IGF2的动态寡聚化状态。这个想法在目标3中得到了验证：确定 Grp94和BiP对ProIGF 2的寡聚化性质的影响。

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.