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

内质网(ER)伴侣Grp94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 Grp94有助于IGF蛋白的折叠。Grp94是Hsp90伴侣家族的成员，需要ATP- 驱动构象循环来陪伴它们的“客户蛋白”。许多致癌蛋白依赖于Hsp90 因为他们的功能。因此，Hsp90的ATP竞争性抑制剂作为抗肿瘤药物被广泛研究。 抗癌药物。尽管胞浆Hsp90是临床试验中抑制剂的第一个预定目标，但Grp94是 作为毒品目标受到越来越多的关注。Grp94可以接收来自Hsp70系列监护人Bip的客户 在急诊室。通过一个偶然的发现，我们发现Bip和Hsp90抑制剂的组合会锁定 Grp94在一种新的构象中，一种状态显然准备将客户从BIP转移到Grp94。客户端转接 从Bip到Grp94需要两个连续步骤：客户端从Bip切换到Grp94，然后是Bip 从三元络合物中解离。我们的第一个假设是，Hsp90抑制剂阻断了这两个步骤： 由于依赖于ATP的构象变化，抑制剂使Grp94和Bip在客户转移状态中停滞 需要GRP94来从BIP切换客户端，然后从GRP94移位BIP。我们将对此进行测试 假设与IGF2，因为我们已经发现，这个Grp94客户端也有一个明确的BiP结合位点。 目的1：确定Grp94依赖于ATP的构象变化是否导致Bip交接结合 客户蛋白，以及Hsp90抑制剂是否破坏这一客户转移功能。AIM2：确定ATP是否- Grp94需要依赖的构象变化才能主动取代Grp94中的Bip。AIMS 1和2将进行测试 可以解释Hsp90抑制剂如何破坏Grp94功能的统一机制，但不能解释什么 Bip和Grp94为IGF蛋白提供了必需的伴侣功能。我们发现IGF2 形成动态低聚物。与已知的其他寡聚化的多肽激素类似，这种自我关联 可以使IGF2在出口到高尔基山脉之前得到有效的浓缩和包装。尽管有一个看似合理的 IGF2齐聚的生物学作用，我们还观察到IGF2可以从动态可逆转变 低聚物转变为不可逆的聚集体。因此，我们的第二个假设是，基本的陪伴功能 Bip和Grp94的目的是保持IGF2的动态齐聚状态。这一想法在Aim3中得到了验证： 确定Grp94和Bip对ProIGF2齐聚性能的影响。

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.