水木未来·视界iss.10丨 冷冻电镜新突破，HHMI研究员成功解析Janus激酶的全长结构

经过二十多年的努力，一项新的研究揭示了一个关键信号分子的结构，为生产更好的抗癌药物打开了新的大门。这一突破性成果发表在分子生物学家Christopher Garcia的生日当天。

More than two decades of effort went into a project that has now revealed the structure of a crucial signaling molecule, opening the door to new and better drugs for some cancers. The breakthrough came on molecular biologist Christopher Garcia’s birthday.

20多年来，Garcia的团队和世界各地的其他科学家们一直在苦苦追踪一个难以捉摸的课题：细胞中一种关键信号蛋白的三维结构。在2021年末，他们的电子显微镜下的分子图像开始变得清晰了。12月8日，博士后Tsutsumi Naotaka和研究生Caleb Glassman给他发了一封电子邮件，其中有一张令人吃惊的清晰图片，显示该蛋白质与一个关键受体相连接。斯坦福大学霍华德·休斯医学研究所(HHMI)的研究员Garcia回忆说："当时我正坐在一个会议上，我意识到我们快成功了。我立即离开了会议，跑回了实验室"。

For more than 20 years, his team and others around the world had been chasing an elusive quarry – the 3D structure of a crucial signaling protein in cells. In late 2021, his electron microscope images of the molecule started to come into focus. On December 8, postdoc Naotaka Tsutsumi and graduate student Caleb Glassman sent him an email with a startlingly clear picture of the protein latched on to a key receptor. “I was sitting in a meeting, and I realized we had it,” recalls Garcia, a Howard Hughes Medical Institute Investigator at Stanford University. “I immediately left the meeting and ran back to the lab.”

刚搬到波士顿做哈佛大学博士后的Glassman取消了他的越野旅行计划，连夜赶回了斯坦福。然后，三位研究人员夜以继日地工作，确定了这种被称为Janus激酶的蛋白质的完整结构，并在这场竞速赛中击败了对手的实验室。Garcia说："这是全世界许多优秀科研团队之间的一场赛马比赛，而我们的团队全力冲刺，拿下了第一名。" 12月26日，他们匆匆向Science杂志提交了一份手稿，该杂志于2022年3月10日发表了这项工作。

Glassman, who had just moved to Boston for a Harvard postdoc, canceled his planned backcountry trip, and rushed back to Stanford. “I wanted to finish what Naotaka and I had started,” he explains. Then the three researchers worked around the clock to nail the complete structure of the protein, known as a Janus kinase, and beat competing labs to the discovery. “It was a big horse race between many great groups worldwide, and we were sprinting towards the finish line,” Garcia says. On December 26, they rushed a manuscript to the journal Science, which published the work on March 10, 2022.

Garcia的团队不仅获得了一个极其重要的信号分子的完整结构，而且还获得了这些激酶的工作机制。美国国立卫生研究院NIH的免疫学家John O’Shea说，弄清这些激酶的工作机制一直是 "一个决定性的基础生物学难题"。O’Shea帮助开发了第一批阻断Janus激酶功能的药物之一，但遗憾的是，他并没有参与这项由Garcia主导的新研究。这一新研究的结果可能会加速某些癌症的新型药物研发，对此，O'Shea盛赞道："这真是了不起的工作。 "

Garcia’s team has nabbed not just the full structure of a vitally important signaling molecule, but also the mechanism for how these kinases work, which had been “a fundamental question in biology,” says John O’Shea, an immunologist at the National Institutes of Health who helped to develop one of the first drugs to block Janus kinase function and was not involved with the new research. Because the proteins can go awry in disease, the results could lead to new and better drugs against certain cancers. “It’s amazing work,” O’Shea says. 

Janus激酶是动物界的通信专家之一。它们接收来自外部细胞的信号并将信息传递给内部分子。经过多年研究，科学家们已经知道Janus激酶的功能失调可以导致疾病。一些损害Janus激酶的突变会严重削弱身体抵抗感染的能力，导致一种与 "气泡男孩症"几乎相同的状况。而当基因出现故障，或频繁的信号使激酶过度激活时，其结果可能是血癌：如白血病，以及过敏性或自身免疫性疾病。

Janus kinases are one of the communication whizzes of the animal kingdom. They take signals that come from outside cells and pass the info along to molecules inside. Scientists have known for years that malfunctioning Janus kinases can cause disease. Some mutations that impair Janus kinases can severely curtail the body’s ability to fight off infection, causing a condition virtually identical to “bubble boy disease.” And when genetic glitches and exaggerated signals rev up the kinases too much, the result can be blood cancers like leukemia, and allergic or autoimmune diseases.

研究人员知道这些蛋白质部分的结构，包括相关的酶和分子末端的调节区域，这使它们被命名为Janus激酶，其中Janus指代罗马神话中的两面神。而此前繁杂的药物筛选已经发现了抑制这些蛋白质的分子，使医生有办法治疗一些癌症和类风湿性关节炎等疾病。但是科学家们在开发这些药物时并不知道这些分子的完整结构或它们是如何被激活的。因此，目前近十种药物中的大多数，加上更多处于临床实验阶段的药物，都是双刃剑：它们既能抑制突变的Janus激酶，也能抑制那些正常的Janus激酶。所以，尽管这些药物可以治疗从湿疹到新冠的许多疾病，它们也会引起一系列的副作用。

Researchers knew the shape of parts of the proteins, including related enzyme and regulatory regions at the end of the molecule, which earned them the name Janus kinases, after the two-faced mythological Roman god. And sophisticated drug screens have unearthed molecules that inhibit these proteins, giving doctors a way to treat some cancers and disorders like rheumatoid arthritis. But scientists developed the drugs without knowing the molecules’ full structure or how they become activated. So most of the current arsenal of nearly a dozen drugs, plus more in clinical trials, are relatively blunt instruments, blocking both healthy and mutated Janus kinases. They can still treat many diseases, from eczema to COVID-19, but also can cause a range of side effects.

Garcia希望更详细地了解这些蛋白质，但是，正如他在1995年的博士后阶段首次尝试对这些分子进行成像时所了解到的那样，这是一个令人生畏的挑战。众所周知，激酶很难在实验室里制作。而且它们不容易形成晶体，而科学家需要用X射线晶体学来捕捉三维结构。因此，多年来，Garcia和其他人每次只能获得关于激酶的一点点信息碎片。Garcia说："我们一直在努力，但成果却乏善可陈。"

Garcia wanted a more detailed view of the proteins but, as he learned when he first tried to image the molecules as a postdoc in 1995, it was a daunting challenge. The kinases are notoriously difficult to make in the lab. And they don’t easily form crystals, which scientists need to capture 3D structures using x-ray crystallography. So, for many years, Garcia and others could only view bits of the kinases at a time. “We kept chipping away without much to show for it,” he says.

在辛苦拼凑多年的信息碎片后，Garcia的团队逐渐迈向成功。一个极其关键的进展是冷冻电镜技术的发展：科学家们将样品冷冻起来，然后用电子显微镜观察它们，这个技术成功规避了激酶难以结晶的难题。另一个进展来自于Garcia团队的正确决策：他们选择研究小鼠的Janus激酶，而不是不太稳定的人类激酶。他们还在小鼠激酶中引入了一个常见的致癌突变，这使分子更加稳定。 

In the last few years, the pieces began to fall into place. One key advance was a method called cryo-EM, where scientists freeze samples and then view them using an electron microscope. Another was the choice by Garcia’s team to study a mouse Janus kinase rather than a less stable human one. They also introduced a common cancer-causing mutation into the mouse kinase, which stabilized the molecule even further. 

Garcia团队的工作揭示了一种叫做JAK1的Janus激酶的结构，并概述了它在细胞内发送信号的步骤：

Garcia’s team’s work reveals the structure of a Janus kinase called JAK1 and outlines the steps it uses to sends signals within cells.

首先，受体蛋白钉在细胞膜上，像牙签穿过三明治一样从细胞的内部和外部探出。然后，细胞内的一个Janus激酶附着在受体上，等待接收信号。接下来，被称为细胞因子的分子接近细胞的外部，每个都与两个受体结合。Garcia解释说，这些细胞因子就像一座桥梁，将两个受体拉得更近。这使Janus激酶的活性端聚集在一起，使它们开启。就像烽火台一样，激酶在中间传递了一个信号，告诉基因打开或关闭。

First, receptor proteins stud cell membranes, poking from the inner and outer surfaces of the cell like a toothpick through a sandwich. Then, a single Janus kinase inside the cell attaches to the receptors, waiting for a signal. Next, molecules called cytokines approach the cell’s exterior, each binding to two receptors. The cytokines act like a bridge that pulls the two receptors even closer, Garcia explains. That brings the active ends of the Janus kinase together, switching them on. Like a match lighting a fire, the kinase relays a signal that tells genes to turn on or off.

该结构还揭示了致癌突变是如何使信息传递过程发生故障的：致癌突变将Janus激酶的两个部分粘在了一起，这导致这两个活性区域即使在没有外部细胞因子的情况下也会一直保持开启状态，引发不受控制的活动，从而导致癌症。

The structure also reveals how the cancer-causing mutation short-circuits this messaging chain – by gluing two parts of the Janus kinase together. That causes the two active regions to stay switched on even when there are no outside cytokines, sparking uncontrolled activity that can trigger cancers.

Garcia希望新的结果能够帮助科学家设计出更好的药物：这些药物只会针对有缺陷的Janus激酶，但同时能让健康的激酶继续履行其职责。他说，这项研究是典型的 "学以致用"。    

Garcia hopes the new results could help scientists design better drugs that target only defective Janus kinases, allowing healthy versions to keep performing their normal duties. The work, he says, is an example of an “ideal situation in science, where solving a basic problem also has direct relevance for disease.”

相关文章

DOI: 10.1126/science.abn8933"Structure of a Janus kinase cytokine receptor complex reveals the basis for dimeric activation" 

细胞因子通过细胞表面受体二聚体发出信号，启动了细胞内Janus激酶（JAKs）的激活。我们报告了全长的JAK1与细胞因子受体的细胞内Box1/Box2结构域复合的3.6Å分辨率的冷冻电镜结构，该结构被捕获为带有在骨髓增生性肿瘤中普遍存在的Val→Phe（VF）突变的活化同源二聚体。JAK1的七个结构域形成了一个扩展的结构单元，其二聚体化是由紧密包装的假激酶（PK）结构域介导的。致癌性的VF突变位于JAK1 PK二聚体的核心，增强了包装的互补性以促进独立配体的激活。C端的酪氨酸激酶结构域准备对从悬垂的FERM-SH2结构域投射出来的受体STAT诱导图案进行磷酸化。持续活化的JAK突变体的分布佐证了两步别构激活机制，并揭示了选择性治疗致癌JAK信号的新机会。

Cytokines signal through cell surface receptor dimers to initiate activation of intracellular Janus Kinases (JAKs). We report the 3.6-Å resolution cryo-EM structure of full-length JAK1 complexed with a cytokine receptor intracellular Box1/Box2 domain, captured as an activated homodimer bearing the Val→Phe (VF) mutation prevalent in myeloproliferative neoplasms. The seven domains of JAK1 form an extended structural unit whose dimerization is mediated by close-packed pseudokinase (PK) domains. The oncogenic VF mutation lies within the core of the JAK1 PK dimer interface, enhancing packing complementarity to facilitate ligand-independent activation. The C-terminal tyrosine kinase domains are poised to phosphorylate the receptor STAT-recruiting motifs projecting from the overhanging FERM-SH2 domains. Mapping of constitutively active JAK mutants supports a two-step allosteric activation mechanism and reveals new opportunities for selective therapeutic targeting of oncogenic JAK signaling.

转载自HHMI News:
"After More Than 20 Years, Scientists Have Finally Solved the Full-Length Structure of a Janus Kinase"

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