CMT2N-causing aminoacylation domain mutants enable Nrp1 interaction with AlaRS.
CMT2N-causing aminoacylation domain mutants enable Nrp1 interaction with AlaRS.
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DOI：
10.1073/pnas.2012898118
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发表时间：
2021-03-30
影响因子：
11.1
通讯作者：
Yang XL
中科院分区：
文献类型：
--
作者：
Sun L;Wei N;Kuhle B;Blocquel D;Novick S;Matuszek Z;Zhou H;He W;Zhang J;Weber T;Horvath R;Latour P;Pan T;Schimmel P;Griffin PR;Yang XL
Charcot-Marie-Tooth病(CMT)是一种毁灭性的运动和感觉神经病，在美国估计有10万名患者。出乎意料的是，氨基酰-tRNA合成酶是最大的疾病相关蛋白家族。一种自然的解释是，这种疾病与翻译不力或误译(由编辑缺陷引起)有关。然而，我们对AlaRS中六个不同致病突变的结果排除了氨基酰化或编辑缺陷是原因因素。相反，特定的突变蛋白获得了神经粘连蛋白1(Nrp1)-AlaRS的相互作用。以前，Nrp1与另一种致病tRNA合成酶相互作用的增加被认为与CMT的病理机制有关。因此，我们的结果提出了这样一种可能性，即Nrp1的病理参与至少在tRNA合成酶相关的CMT病例中是共同的。通过显性突变，氨基酰-tRNA合成酶构成了与Charcot-Marie-Tooth病(CMT)相关的最大蛋白质家族。一个例子是CMT亚型2N(CMT2N)，由AlaRS中分布的单个突变引起，包括氨基酰化结构域中的三个突变，从而表明tRNA荷电缺陷的作用。然而，我们在这里发现有两个是氨基酰化缺陷的，但在体外和未分离的患者细胞样本中，分布最广泛的R329H作为纯化蛋白是正常的。值得注意的是，与野生型(WT)AlaRS相比，所有三个突变蛋白都获得了与神经毛细蛋白1(Nrp1)相互作用的能力，Nrp1是GlyRS中与CMT发病相关的受体。携带R329H突变的患者样本进一步证实了AlaRS-Nrp1的异常相互作用。然而，CMT2N在氨基酰化结构域之外的突变不会诱导Nrp1相互作用。详细的生化和生物物理研究，包括X射线结晶学、小角X射线散射、氢-氚交换(HDX)、SwitchSENSE流体动力学直径测定和蛋白酶消化，揭示了突变导致的氨基酰化结构域的结构松动，这与Nrp1相互作用有关。Nrp1的B1B2结构域负责与R329H AlaRs的相互作用。结果表明，Nrp1与tRNA合成酶家族中与CMT相关的成员有更广泛的联系。此外，我们还发现编辑结构域的突变导致了明显的结构松动效应，而C-ALA结构域的突变对构象没有影响，这表明同一蛋白的突变可能通过不同的机制引起神经病变。我们的结果表明，与其他CMT相关的tRNA合成酶一样，氨基酰化本身与病理无关。
Charcot-Marie-Tooth disease (CMT) is a devastating motor and sensory neuropathy with an estimated 100,000 afflicted individuals in the US. Unexpectedly, aminoacyl-tRNA synthetases are the largest disease-associated protein family. A natural explanation is that the disease is associated with weak translation or mistranslation (caused by editing defects). However, our results with six different disease-causing mutants in AlaRS ruled out defects in aminoacylation or editing as causal factors. Instead, specific mutant proteins gained a neuropilin 1 (Nrp1)-AlaRS interaction. Previously a gain of Nrp1 interaction with a different disease-causing tRNA synthetase was mechanistically linked to the pathology of CMT. Thus, our results raise the possibility that pathological engagement of Nrp1 is common to at least a subset of tRNA synthetase-associated cases of CMT. Through dominant mutations, aminoacyl-tRNA synthetases constitute the largest protein family linked to Charcot-Marie-Tooth disease (CMT). An example is CMT subtype 2N (CMT2N), caused by individual mutations spread out in AlaRS, including three in the aminoacylation domain, thereby suggesting a role for a tRNA-charging defect. However, here we found that two are aminoacylation defective but that the most widely distributed R329H is normal as a purified protein in vitro and in unfractionated patient cell samples. Remarkably, in contrast to wild-type (WT) AlaRS, all three mutant proteins gained the ability to interact with neuropilin 1 (Nrp1), the receptor previously linked to CMT pathogenesis in GlyRS. The aberrant AlaRS-Nrp1 interaction is further confirmed in patient samples carrying the R329H mutation. However, CMT2N mutations outside the aminoacylation domain do not induce the Nrp1 interaction. Detailed biochemical and biophysical investigations, including X-ray crystallography, small-angle X-ray scattering, hydrogen-deuterium exchange (HDX), switchSENSE hydrodynamic diameter determinations, and protease digestions reveal a mutation-induced structural loosening of the aminoacylation domain that correlates with the Nrp1 interaction. The b1b2 domains of Nrp1 are responsible for the interaction with R329H AlaRS. The results suggest Nrp1 is more broadly associated with CMT-associated members of the tRNA synthetase family. Moreover, we revealed a distinct structural loosening effect induced by a mutation in the editing domain and a lack of conformational impact with C-Ala domain mutations, indicating mutations in the same protein may cause neuropathy through different mechanisms. Our results show that, as with other CMT-associated tRNA synthetases, aminoacylation per se is not relevant to the pathology.
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