Role of HSP90 Family Chaperone Proteins in Cellular Sign

HSP90和GRP94是分别在胞浆和内质网中发现的同源细胞伴侣。几年前，我们发现苯醌类抗生素的成员，包括赫比霉素A和格尔达那霉素(GA)，与HSP90和GRP94结合，并破坏这些蛋白质所在的某些多分子复合体。我们利用对HSP90和GRP94活性的药物干扰来研究这些伴侣蛋白在细胞信号转导中的功能。多种信号转导蛋白与这些蛋白相互作用，包括src、erbB2、c-raf-1、Akt、Kit、Met、bcr-Ab1、转录因子HIF-1α和突变型(但不是野生型)p53。伴侣/信号蛋白复合体的药物破坏的一般后果是导致信号蛋白的显著不稳定和不正确的亚细胞定位。这种不稳定性是由于伴侣蛋白解离后26S蛋白酶体蛋白分解复合体刺激信号蛋白的靶向性降解所致。我们发现HSP90与胞浆蛋白RIP有关，RIP是导致NFkB激活的肿瘤坏死因子信号通路的关键成分。我们已经确定，格尔达那霉素破坏RIP的稳定性可以阻止肿瘤坏死因子诱导的NFkB，但不能阻止肿瘤坏死因子对JNK的信号转导，从而使细胞对肿瘤坏死因子的凋亡特性敏感。此外，我们还观察到另一种与细胞存活相关的激酶Akt对格尔达霉素敏感。格尔达那霉素可阻断除肿瘤坏死因子外的多种刺激对NFkB的诱导，包括化疗药物和IL-1。它这样做的能力可能与它对Akt的不稳定有关。苯醌阿霉素类药物(格尔达霉素)是唯一能特异性干扰HSP90/GRP94功能的药物。最近，我们发现自由基代表了一类能够与HSP90结合的新型天然产物。自由基和阿萨米星都在氨基末端的核苷酸口袋与HSP90结合。最近，我们已经确定了第三类天然产物，新生物素，它也与HSP90结合，尽管亲和力低于苯醌、阿萨米星或自由基。尽管如此，新诺贝菌素似乎对“客户蛋白”产生了与阿萨霉素和自由基相同的生物效应。令人惊讶的是，novobiocin似乎与HSP90上的羧基末端区域相互作用，这实际上是以前未被识别的第二核苷酸结合部位。初步的动物试验显示，在一个多月的时间里，每天两次服用新诺贝菌素没有毒性。该方案在erbB2驱动的转基因小鼠乳腺癌模型中显示出显著的抗肿瘤活性。我们已经观察到，格尔达霉素逆转了黑色素瘤细胞中的β-连环素酪氨酸磷酸化，可能是由于这些细胞迅速失去了erbB2。在未经处理的细胞中，erbB2和β-连环蛋白可以很容易地共沉淀。在体外，β-连环素酪氨酸磷酸化缺失导致与E-钙粘附素的相关性增加，并降低细胞的运动性。这是第一个表明调节黑色素瘤细胞中β-连环素的酪氨酸磷酸化状态与运动性降低有关的迹象。事实是，在3/3的黑色素瘤细胞系中，β-连环素酪氨酸磷酸化似乎是由erbB2-一种对格尔达霉素敏感的酪氨酸激酶-介导的，这一事实表明格尔达霉素治疗可能是抗转移的。这一假说目前正在体内转移模型中进行测试。在堪萨斯州，我们已经鉴定了一系列新生物素衍生物，与亲本化合物相比，它们表现出更好的结合亲和力和抗Hsp90活性，并且我们已经证明了这些衍生物中的几个能够耗尽肿瘤细胞中的Hsp90客户蛋白。ErbB受体酪氨酸激酶家族包括四个成员。

HSP90 and GRP94 are homologous cellular chaperones found in cytosol and endoplasmic reticulum, respectively. Several years ago, we discovered that members of the benzoquinone ansamycin class of antibiotics, including herbimycin A and geldanamycin (GA) bound to HSP90 and GRP94 and disrupted certain multi-molecular complexes of which these proteins were a part. We have utilized pharmacologic disruption of HSP90 and GRP94 activity to study the function of these chaperones in cellular signal transduction. Multiple signal transduction proteins interact with these charperones, including the kinases src, erbB2, c-raf-1, Akt, Kit, Met, Bcr-Abl, the transcription factor HIF-1alpha, and mutated (but not wild type) p53. A general consequence of pharmacologic disruption of the chaperone/signal protein complex is the resultant marked instability and incorrect subcellular localization of the signalling protein. The instability is due to stimulation of targeted degradation of the signalling protein by the 26S proteasome proteolytic complex following chaperone dissociation. We made the novel observation that HSP90 associates with the cytosolic kinase RIP, a key component of the TNF signalling pathway which leads to NFkB activation. We have determined that disruption of RIP stability by geldanamycin prevents NFkB induction by TNF, but not TNF signalling to Jnk, thus sensitizing cells to the apoptotic properties of TNF. We have additionally observed that another kinase associated with cell survival, Akt, is sensitive to geldanamycin. Geldanamycin blocks NFkB induction by a wide variety of stimuli other than TNF, including chemotherapeutic drugs and IL-1. Its ability to do this may relate to its destabilization of Akt. Benzoquinone ansamycins (geldanamycin) had been the only agents capable of specifically interfering in HSP90/GRP94 function. Recently, we identified radicicol as representing a novel class of natural product capable of binding to HSP90. Both radicicol and the ansamycins bind to HSP90 at an amino terminal nucleotide pocket. Most recently, we have identified a third class of natural product, novobiocin, which also binds to HSP90, although at a lower affinity than either benzoquinone ansamycins or radicicol. Nonetheless, novobiocin appears to cause the same biologic effects on "client proteins" as ansamycins and radicicol. Surprisingly, novobiocin appears to interact with a carboxyl terminal region on HSP90, which is in fact a previously unrecognized second nucleotide binding site. Preliminary animal testing has revealed no toxicity after twice daily administration of novobiocin for more than one month. This regimen demonstrates significant anti-tumor activity in a transgenic murine model of erbB2-driven breast cancer. We have observed that geldanamycin reverses beta-catenin tyrosine phosphorylation in melanoma cells, probably due to the rapid loss of erbB2 from these cells. In untreated cells, erbB2 and beta-catenin can be readily co-precipitated. Loss of beta-catenin tyrosine phosphorylation leads to an increased association with E-cadherin and decreased cell motility in vitro. This is the first indication that modulation of the tyrosine phosphorylation status of beta catenin in melanoma cells is associated with decreased motility. The fact that beta-catenin tyrosine phosphorylation seems to be mediated, in 3/3 melanoma cell lines examined, by erbB2 - a geldanamycin-sensitive tyrosine kinase - suggests that geldanamycin treatment may be anti-metastatic. This hypothesis is currently being tested in an in vivo metastasis model.In collaboration with Brian Blagg of the Univ. of Kansas, we have identified a series of novobiocin derivatives that demonstrate improved binding affinity and anti-Hsp90 activity compared to the parental compound, and we have demonstrated the ability of several of these derivatives to deplete Hsp90 client proteins in tumor cells.The ErbB family of receptor tyrosine kinases contains four members.