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

内质网（ER）伴侣Grp 94是正确折叠和分泌 胰岛素样生长因子（IGF），但其潜在机制尚不清楚。我们的目标是发现 Grp 94有助于IGF蛋白的折叠。Grp 94是Hsp 90伴侣蛋白家族的成员，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Hsp 90 因为它们的功能。因此，Hsp 90的ATP竞争性抑制剂已被广泛研究作为抗Hsp 90的药物。 抗癌药虽然细胞溶质Hsp 90是临床试验中抑制剂的第一个预期靶点，但Grp 94是一种靶点。 作为药物靶点越来越受到关注。Grp 94可以从BiP（Hsp 70家族伴侣）接收客户 在急诊室通过一次偶然的发现，我们发现BiP和Hsp 90抑制剂的组合锁定了 Grp 94在一个新的构象，一个国家显然准备从BiP客户端转移到Grp 94。客户机传送 从BiP到Grp 94需要两个连续的步骤：将客户端从BiP切换到Grp 94，然后是BiP 从三元复合物中解离。我们的第一个假设是Hsp 90抑制剂阻断了这两个步骤： 抑制剂使Grp 94和BiP停滞在客户端转移状态，因为ATP依赖性构象变化 需要从BiP切换客户端，然后从Grp 94替换BiP。我们将测试这个 这与IGF 2的假设是一致的，因为我们已经发现该Grp 94客户端也具有明确的BiP结合位点。 目的1：确定Grp 94的ATP依赖性构象变化是否导致BiP切换结合 客户蛋白，以及Hsp 90抑制剂是否破坏这种客户转移功能。目标2：确定ATP是否- Grp 94的依赖性构象变化需要从Grp 94主动置换BiP。目标1&2将测试 一个统一的机制，可以解释如何热休克蛋白90抑制剂破坏Grp 94功能，但没有解释什么， BiP和Grp 94为IGF蛋白提供了必需的伴侣功能。我们发现IGF 2 形成动态低聚物。类似于已知的寡聚化的其他肽类激素， 可以使IGF 2在输出到高尔基体之前被有效地浓缩和包装。尽管一个看似合理的 对于IGF 2寡聚化的生物学作用，我们还观察到IGF 2可以从动态可逆的 低聚物转化为不可逆聚集体。因此，我们的第二个假设是， BiP和Grp 94的作用是维持IGF 2的动态寡聚化状态。这个想法在目标3中得到了验证： 确定Grp 94和BiP对ProIGF 2的寡聚性质的影响。

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.