Endoplasmic reticulum stress-independent activation of unfolded protein response kinases by a small molecule ATP-mimic.
Endoplasmic reticulum stress-independent activation of unfolded protein response kinases by a small molecule ATP-mimic.
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DOI：
10.7554/elife.05434
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发表时间：
2015-05-19
期刊：
影响因子：
7.7
通讯作者：
Walter P
中科院分区：
文献类型：
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作者：
Mendez AS;Alfaro J;Morales-Soto MA;Dar AC;McCullagh E;Gotthardt K;Li H;Acosta-Alvear D;Sidrauski C;Korennykh AV;Bernales S;Shokat KM;Walter P
两个ER膜驻留跨膜激酶，IRE 1和PERK，在未折叠的蛋白质反应中起应激传感器的作用。IRE 1还具有核糖核酸内切酶活性，可启动非常规的mRNA剪接反应，而PERK可磷酸化eIF 2 α。我们设计了一种有效的小分子IPA，它与IRE 1的ATP结合口袋结合，使激酶结构域易于寡聚化，激活其RNA酶。IPA也抑制PERK，但矛盾的是，在低浓度下激活它，导致钟形激活曲线。我们从纯化的组分中重建了PERK介导的eIF 2 α磷酸化的IPA激活。我们估计，在最大活化的条件下，反应中少于15%的PERK分子被IPA占据。我们认为IPA结合使PERK激酶偏向其活性构象，从而反式激活apo-PERK分子。抑制剂的部分占据可以激活激酶的机制可能是广泛的，并且对激酶抑制剂的设计和治疗应用具有重要意义。DOI：http://dx.doi.org/10.7554/eLife.05434.001细胞含有数千种蛋白质，它们执行生存所需的基本任务。在它们能够工作之前，蛋白质必须首先折叠成特定的三维形状。内质网是一个专门将新产生的蛋白质正确折叠成天然状态的细胞隔室，对于蛋白质成熟过程至关重要。如果内质网中的折叠酶不能与它们必须折叠的蛋白质负荷适当平衡，内质网可能会被积累的未折叠蛋白质淹没，导致“内质网应激”。细胞通过触发“未折叠蛋白反应”（UPR）来应对内质网应激。这种反应有助于通过增加内质网的大小和其中折叠酶的浓度以及通过减少新产生的蛋白质流入内质网来清除未折叠的蛋白质。UPR与内质网膜中的信号分子结合，其中有两种信号酶，称为IRE 1和PERK。激活这些信号酶的药物可以帮助细胞处理未折叠的蛋白质，防止内质网应激引起的毒性，并抵御由此引起的疾病。Mendez，Alfaro，Morales-Soto等人开发了一种称为IPA（IRE 1/PERK Activator的缩写）的小分子，旨在结合并激活IRE 1。无意中，IPA不仅激活了IRE 1，还激活了PERK。令人惊讶的是，仅在低IPA浓度下观察到PERK活化，其中IPA仅在少数PERK分子中占据活性位点，而在较高浓度和完全占据下，IPA完全抑制PERK。Mendez、Alfaro、Morales-Soto等人提出，在部分IPA占据的条件下，少数IPA结合的PERK分子呈现活化状态，其传播到没有IPA结合的相邻PERK分子，并活化它们。先前在临床使用的药物中观察到类似的剂量依赖性激活，该药物旨在抑制在癌症进展中重要的类似信号传导酶。结合门德斯、阿尔法罗、莫拉莱斯-索托等人的观察，这些结果表明，对类似治疗方法的研究必须考虑到存在一个“最小剂量”，低于该剂量的药物可能会产生与预期相反的效果。仍需要进一步的工作来充分理解产生这种行为的机制。DOI：http://dx.doi.org/10.7554/eLife.05434.002网站
Two ER membrane-resident transmembrane kinases, IRE1 and PERK, function as stress sensors in the unfolded protein response. IRE1 also has an endoribonuclease activity, which initiates a non-conventional mRNA splicing reaction, while PERK phosphorylates eIF2α. We engineered a potent small molecule, IPA, that binds to IRE1's ATP-binding pocket and predisposes the kinase domain to oligomerization, activating its RNase. IPA also inhibits PERK but, paradoxically, activates it at low concentrations, resulting in a bell-shaped activation profile. We reconstituted IPA-activation of PERK-mediated eIF2α phosphorylation from purified components. We estimate that under conditions of maximal activation less than 15% of PERK molecules in the reaction are occupied by IPA. We propose that IPA binding biases the PERK kinase towards its active conformation, which trans-activates apo-PERK molecules. The mechanism by which partial occupancy with an inhibitor can activate kinases may be wide-spread and carries major implications for design and therapeutic application of kinase inhibitors. DOI: http://dx.doi.org/10.7554/eLife.05434.001 Cells contain thousands of proteins that carry out the essential tasks needed for survival. Before they can work, proteins must first fold into specific three-dimensional shapes. The endoplasmic reticulum, a cellular compartment that specializes in properly folding newly made proteins into their native states, is critical for this protein maturation process. If folding-enzymes in the endoplasmic reticulum are not properly balanced with the load of proteins they must fold, the endoplasmic reticulum can be overwhelmed with unfolded proteins that accumulate, leading to ‘endoplasmic reticulum stress’. The cell copes with endoplasmic reticulum stress by triggering the ‘unfolded protein response’ (UPR). This response helps to clear the unfolded proteins by increasing the size of the endoplasmic reticulum and the concentration of folding enzymes within it, and by decreasing the influx of newly made protein into the endoplasmic reticulum. The UPR engages signaling molecules in the endoplasmic reticulum membrane, among them two signaling enzymes called IRE1 and PERK. Drugs that activate these signaling enzymes could help the cell to deal with unfolded proteins, prevent toxicity resulting from endoplasmic reticulum stress, and ward off the diseases that result from it. Mendez, Alfaro, Morales-Soto et al. developed a small molecule, called IPA (short for IRE1/PERK Activator), that was designed to bind to and activate IRE1. Serendipitously, IPA not only activated IRE1 but also activated PERK. Surprisingly, PERK activation was only observed at low IPA concentrations in which IPA occupied the active sites in only a few PERK molecules, whereas at higher concentrations and full occupancy IPA completely inhibited PERK. Mendez, Alfaro, Morales-Soto et al. proposed that, under conditions of partial IPA occupancy, a minority of IPA-bound PERK molecules assume an activated state that propagates to adjacent PERK molecules that have no IPA bound to them, and activates them. Similar dose-dependent activation was previously observed for a clinically used drug designed to inhibit a similar signaling enzyme that is important in cancer progression. Together with the observations of Mendez, Alfaro, Morales-Soto et al., these results suggest that research into similar treatments must consider that a ‘minimal dose’ can exist, below which drugs may have the opposite effect to what is desired. Further work is still needed to fully understand the mechanisms that produce such behavior. DOI: http://dx.doi.org/10.7554/eLife.05434.002