背景:谷氨酸能突触功能障碍被认为是许多个体自闭症谱系障碍(ASD)和智力障碍(ID)发展的基础。然而,众所周知,鉴定导致这些个体突触功能障碍的遗传标记是非常困难的。基于基因组分析,结构建模,和功能数据,我们最近确定TRIO-RAC1通路参与ASD和ID.此外,我们在TRIO的GEF1域中发现了一个病理性从头错义突变热点。该结构域中与ASD/ID相关的错义突变会损害谷氨酸能突触功能,并可能有助于ASD/ID的发展。在TRIO的GEF1域内鉴定出突变的ASD/ID病例数量正在增加。然而,缺乏准确预测这种突变是否对蛋白质功能有害的工具。
方法:在这里,我们部署了先进的蛋白质结构建模技术来预测TRIO的GEF1域内潜在的从头致病性和良性突变。在器官型培养的海马切片的CA1锥体神经元中产生并表达突变体TRIO-9构建体。使用双全细胞膜片钳电生理学检查了这些神经元中AMPA受体介导的突触后电流。我们还使用正交免疫共沉淀和荧光寿命成像(FLIM-FRET)实验验证了这些发现,以测定TRIO突变体过表达对TRIO-RAC1结合和HEK293/T细胞中RAC1活性的影响。
结果:在TRIO的GEF1结构域中预测会破坏TRIO-RAC1结合或稳定性的错义突变进行了实验测试,发现极大地削弱了TRIO-9对谷氨酸能突触功能的影响。相比之下,在我们的实验测定中,TRIO-9对谷氨酸能突触功能的影响不大,预计TRIO-的GEF1结构域错义突变对TRIO-RAC1结合或稳定性影响最小。在正交试验中,我们发现大多数预测会破坏结合显示功能丧失的突变,但预测会破坏稳定性的突变并不能反映我们从神经元电生理数据中得到的结果。
结论:我们提出了一种方法来预测TRIO的GEF1域中可能损害TRIO功能的错义突变,并在有限数量的测定中测试效果。这里使用的模型系统可能产生的限制可以在未来的研究中解决。我们的方法没有提供这些突变是否赋予ASD/ID风险或此类突变将导致ASD/ID发展的可能性的证据。
结论:在这里,我们表明,基于结构的计算预测和实验验证的组合可以用于可靠地预测人类TRIO基因中的错义突变是否会阻碍TRIO蛋白功能并损害TRIO在谷氨酸能突触调节中的作用。随着基因组测序的普及,在病理突变的准确鉴定中使用这些工具将有助于ASD/ID的诊断.
Glutamatergic synapse dysfunction is believed to underlie the development of Autism Spectrum Disorder (ASD) and Intellectual Disability (ID) in many individuals. However, identification of genetic markers that contribute to synaptic dysfunction in these individuals is notoriously difficult. Based on genomic analysis, structural modeling, and functional data, we recently established the involvement of the TRIO-RAC1 pathway in ASD and ID. Furthermore, we identified a pathological de novo missense mutation hotspot in TRIO\'s GEF1 domain. ASD/ID-related missense mutations within this domain compromise glutamatergic synapse function and likely contribute to the development of ASD/ID. The number of ASD/ID cases with mutations identified within TRIO\'s GEF1 domain is increasing. However, tools for accurately predicting whether such mutations are detrimental to protein function are lacking.
Here we deployed advanced protein structural modeling techniques to predict potential de novo pathogenic and benign mutations within TRIO\'s GEF1 domain. Mutant TRIO-9 constructs were generated and expressed in CA1 pyramidal neurons of organotypic cultured hippocampal slices. AMPA receptor-mediated postsynaptic currents were examined in these neurons using dual whole-cell patch clamp electrophysiology. We also validated these findings using orthogonal co-immunoprecipitation and fluorescence lifetime imaging (FLIM-FRET) experiments to assay TRIO mutant overexpression effects on TRIO-RAC1 binding and on RAC1 activity in HEK293/T cells.
Missense mutations in TRIO\'s GEF1 domain that were predicted to disrupt TRIO-RAC1 binding or stability were tested experimentally and found to greatly impair TRIO-9\'s influence on glutamatergic synapse function. In contrast, missense mutations in TRIO\'s GEF1 domain that were predicted to have minimal effect on TRIO-RAC1 binding or stability did not impair TRIO-9\'s influence on glutamatergic synapse function in our experimental assays. In orthogonal assays, we find most of the mutations predicted to disrupt binding display loss of function but mutants predicted to disrupt stability do not reflect our results from neuronal electrophysiological data.
We present a method to predict missense mutations in TRIO\'s GEF1 domain that may compromise TRIO function and test for effects in a limited number of assays. Possible limitations arising from the model systems employed here can be addressed in future studies. Our method does not provide evidence for whether these mutations confer ASD/ID risk or the likelihood that such mutations will result in the development of ASD/ID.
Here we show that a combination of structure-based computational predictions and experimental validation can be employed to reliably predict whether missense mutations in the human TRIO gene impede TRIO protein function and compromise TRIO\'s role in glutamatergic synapse regulation. With the growing accessibility of genome sequencing, the use of such tools in the accurate identification of pathological mutations will be instrumental in diagnostics of ASD/ID.