Bunney Tom D, Kampyli Charis, Gregory Ashley, Katan Matilda
Institute of Structural and Molecular Biology, Division of Biosciences, University College London, London, UK.
Institute of Structural and Molecular Biology, Division of Biosciences, University College London, London, UK.
Adv Biol Regul. 2024 Dec;94:101053. doi: 10.1016/j.jbior.2024.101053. Epub 2024 Sep 19.
The phospholipase C enzyme PLCγ2 is best characterised in the context of immune cell regulation. Furthermore, many mutations discovered in PLCγ2 have been linked to the development of complex immune disorders as well as resistance to ibrutinib treatment in chronic lymphocytic leukaemia. Importantly, it has also been found that a rare variant of PLCγ2 (P522R) has a protective role in Alzheimer's disease (AD). Despite initial characterisation of these disease-linked variants, a comprehensive understanding of their differences and underpinning molecular mechanisms, needed to facilitate therapeutic efforts, is lacking. Here, we used available structural insights for PLCγ enzymes to further analyse PLCγ2 M1141K mutation, representative for mutations in immune disorders and cancer resistance, and the AD-protective variant, PLCγ2 P522R. Together with several other mutations in the autoinhibitory interface, the PLCγ2 M1141K mutation was strongly activating in a cell-based assay, under basal and stimulated conditions. Measurements of PLC activity in various in vitro assays demonstrated enhanced activity of PLCγ2 M1141K while the activity of PLCγ2 P522R was not significantly different from the WT. Similar trends were observed in several other assays, including direct liposome binding. However, an enhanced rate of phosphorylation of a functionally important tyrosine by Btk in vitro was observed for PLCγ2 P522R variants. To further assess implications of these in vitro findings in a cellular context relevant for the PLCγ2 P522R variant, microglia (BV2) stable cell lines were generated and analysed under growth conditions. The PLC activity in cells expressing PLCγ2 P522R at physiologically relevant levels was clearly enhanced compared to the WT, and differences in cell morphology observed. These data, combined with the structural insights, suggest that the PLCγ2 P522R variant has subtle, localised structural changes that do not directly affect the PLC activity by compromising autoinhibition, as determined for PLCγ2 M1141K. It is also likely that in contrast to the PLCγ2 M1141K, the functional impact of the P522R substitution completely depends on further interactions with upstream kinases and other regulatory proteins in a relevant cellular context, where changes in the PLCγ2 P522R variant could facilitate processes such as phosphorylation and protein-protein interactions.
磷脂酶C酶PLCγ2在免疫细胞调节方面的特征最为明显。此外，在PLCγ2中发现的许多突变与复杂免疫疾病的发展以及慢性淋巴细胞白血病对伊布替尼治疗的耐药性有关。重要的是，还发现PLCγ2的一种罕见变体（P522R）在阿尔茨海默病（AD）中具有保护作用。尽管对这些与疾病相关的变体进行了初步表征，但仍缺乏对它们的差异和潜在分子机制的全面理解，而这对于促进治疗研究是必要的。在这里，我们利用PLCγ酶的现有结构见解，进一步分析了PLCγ2 M1141K突变（代表免疫疾病和癌症耐药性中的突变）以及AD保护性变体PLCγ2 P522R。与自抑制界面中的其他几个突变一起，PLCγ2 M1141K突变在基于细胞的测定中，在基础和刺激条件下均具有强烈的激活作用。在各种体外测定中对PLC活性的测量表明，PLCγ2 M1141K的活性增强，而PLCγ2 P522R的活性与野生型无显著差异。在包括直接脂质体结合在内的其他几种测定中也观察到了类似的趋势。然而，在体外观察到，PLCγ2 P522R变体中Btk对功能重要酪氨酸的磷酸化速率有所提高。为了在与PLCγ2 P522R变体相关的细胞环境中进一步评估这些体外研究结果的影响，我们构建并分析了小胶质细胞（BV2）稳定细胞系在生长条件下的情况。与野生型相比，在生理相关水平表达PLCγ2 P522R的细胞中的PLC活性明显增强，并且观察到细胞形态存在差异。这些数据与结构见解相结合，表明PLCγ2 P522R变体具有细微的局部结构变化，这些变化不会像PLCγ2 M1141K那样通过损害自抑制直接影响PLC活性。与PLCγ2 M1141K相比，P522R替代的功能影响也很可能完全取决于在相关细胞环境中与上游激酶和其他调节蛋白的进一步相互作用，在这种环境中，PLCγ2 P522R变体的变化可能促进磷酸化和蛋白质 - 蛋白质相互作用等过程。
