R1441G but not G2019S mutation enhances LRRK2 mediated Rab10 phosphorylation in human peripheral blood neutrophils.
R1441G but not G2019S mutation enhances LRRK2 mediated Rab10 phosphorylation in human peripheral blood neutrophils.
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R1441G而非G2019S突变增强人外周血中性粒细胞中LRRK2介导的Rab10磷酸化。
DOI：
10.1007/s00401-021-02325-z
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发表时间：
2021-09
影响因子：
12.7
通讯作者：
Sammler EM
中科院分区：
文献类型：
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作者：
Fan Y;Nirujogi RS;Garrido A;Ruiz-Martínez J;Bergareche-Yarza A;Mondragón-Rezola E;Vinagre-Aragón A;Croitoru I;Gorostidi Pagola A;Paternain Markinez L;Alcalay R;Hickman RA;Düring J;Gomes S;Pratuseviciute N;Padmanabhan S;Valldeoriola F;Pérez Sisqués L;Malagelada C;Ximelis T;Molina Porcel L;Martí MJ;Tolosa E;Alessi DR;Sammler EM
LRRK2（富含亮氨酸的重复激酶2）的杂合激酶功能变异导致1-2%的帕金森病（PD）病例，尽管具有不完全和年龄依赖的外显率。所有致病性LRRK2突变都位于LRRK2的两个催化结构域内——要么位于其激酶结构域（如G2019S），作用不大，要么位于其ROC-COR GTPase结构域（如R1441G/H），对LRRK2激酶活性有较大影响。我们之前报道了在人类生物样品中测量其内源性底物Rab10磷酸化的LRRK2激酶途径活性的实验，这反映了LRRK2激酶的激活状态。在这里，我们从101名参与者的新鲜外周血中分离出中性粒细胞，其中包括42名LRRK2突变携带者（21名G2019S突变携带者和21名R1441G突变携带者），27名特发性PD患者和32名对照组。采用双重方法，通过定量复用免疫印迹法测定pRab10Thr73/总Rab10，以及靶向质谱法测定pRab10Thr73的绝对占用率，测定LRRK2依赖性Rab10在苏氨酸73位点的磷酸化（pRab10Thr73）。我们发现，与临床疾病状态无关，LRRK2 R1441G突变携带者的pRab10Thr73磷酸化显著增加了四倍以上。LRRK2 G2019S突变的影响没有达到统计学意义。此外，我们发现LRRK2丝氨酸935位点的磷酸化并不是人类中性粒细胞中LRRK2激酶活性的标志。在分析死后脑样本中的pRab10Thr73磷酸化时，我们观察到与临床和LRRK2突变状态无关的总体高变异性，并将其归因于死前和死后时期对翻译后修饰（如蛋白质磷酸化）稳定性的不利影响。总体而言，人外周血中性粒细胞中LRRK2依赖性pRab10Thr73磷酸化是一种特异性的、稳健的、有前景的LRRK2激酶过度激活的生物标志物，与LRRK2 R1441G突变一样。可能需要额外的读数和/或检测来提高检测适度LRRK2激酶激活的灵敏度，如LRRK2 G2019S突变。我们的分析可能对LRRK2激酶抑制剂的患者分层和靶标参与研究有用。在线版本包含补充材料，可在10.1007/s00401-021-02325-z获得。
Heterozygous gain-of-kinase function variants in LRRK2 (leucine-rich repeat kinase 2) cause 1–2% of all cases of Parkinson’s disease (PD) albeit with incomplete and age-dependent penetrance. All pathogenic LRRK2 mutations reside within the two catalytic domains of LRRK2—either in its kinase domain (e.g. G2019S) with modest effect or its ROC-COR GTPase domain (e.g. R1441G/H) with large effect on LRRK2 kinase activity. We have previously reported assays to interrogate LRRK2 kinase pathway activity in human bio-samples measuring phosphorylation of its endogenous substrate Rab10, that mirrors LRRK2 kinase activation status. Here, we isolated neutrophils from fresh peripheral blood from 101 participants including 42 LRRK2 mutation carriers (21 with the G2019S and 21 with the R1441G mutations), 27 patients with idiopathic PD, and 32 controls. Using a dual approach, LRRK2 dependent Rab10 phosphorylation at Threonine 73 (pRab10Thr73) was measured by quantitative multiplexed immunoblotting for pRab10Thr73/total Rab10 as well as targeted mass-spectrometry for absolute pRab10Thr73 occupancy. We found a significant over fourfold increase in pRab10Thr73 phosphorylation in carriers of the LRRK2 R1441G mutation irrespective of clinical disease status. The effect of the LRRK2 G2019S mutation did not reach statistical significance. Furthermore, we show that LRRK2 phosphorylation at Serine 935 is not a marker for LRRK2 kinase activity in human neutrophils. When analysing pRab10Thr73 phosphorylation in post-mortem brain samples, we observed overall high variability irrespective of clinical and LRRK2 mutation status and attributed this mainly to the adverse effect of the peri- and post-mortem period on the stability of posttranslational modifications such as protein phosphorylation. Overall, in vivo LRRK2 dependent pRab10Thr73 phosphorylation in human peripheral blood neutrophils is a specific, robust and promising biomarker for significant LRRK2 kinase hyperactivation, as with the LRRK2 R1441G mutation. Additional readouts and/or assays may be needed to increase sensitivity to detect modest LRRK2 kinase activation, as with the LRRK2 G2019S mutation. Our assays could be useful for patient stratification and target engagement studies for LRRK2 kinase inhibitors. The online version contains supplementary material available at 10.1007/s00401-021-02325-z.
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DOI：
10.1042/bcj20200937
发表时间：
2021-02-12
期刊：
The Biochemical journal
影响因子：
--
作者：
Malik AU;Karapetsas A;Nirujogi RS;Mathea S;Chatterjee D;Pal P;Lis P;Taylor M;Purlyte E;Gourlay R;Dorward M;Weidlich S;Toth R;Polinski NK;Knapp S;Tonelli F;Alessi DR
通讯作者：
Alessi DR
影响因子：
11
作者：
Jankovic, Joseph;Tan, Eng King
通讯作者：
Tan, Eng King
DOI：
10.1042/bcj20180248
发表时间：
2018-06-06
期刊：
The Biochemical journal
影响因子：
--
作者：
Mir R;Tonelli F;Lis P;Macartney T;Polinski NK;Martinez TN;Chou MY;Howden AJM;König T;Hotzy C;Milenkovic I;Brücke T;Zimprich A;Sammler E;Alessi DR
通讯作者：
Alessi DR
影响因子：
11
作者：
HUGHES, AJ;DANIEL, SE;LEES, AJ
通讯作者：
LEES, AJ
影响因子：
168.9
作者：
Kalia, Lorraine V.;Lang, Anthony E.
通讯作者：
Lang, Anthony E.