Calcineurin Targets Involved in Stress Survival and Fungal Virulence.
Calcineurin Targets Involved in Stress Survival and Fungal Virulence.
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钙调神经素靶标参与应力存活和真菌毒力。
DOI：
10.1371/journal.ppat.1005873
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发表时间：
2016-09
期刊：
影响因子：
6.7
通讯作者：
Cardenas ME
中科院分区：
文献类型：
--
作者：
Park HS;Chow EW;Fu C;Soderblom EJ;Moseley MA;Heitman J;Cardenas ME
钙调神经磷酸酶控制人类真菌病原体新型隐球菌的应激生存、性分化和毒力。钙调神经磷酸酶被应激引起的 Ca2+ 水平升高所激活，并通过去磷酸化蛋白质底物来转导信号。在此，我们通过采用磷酸蛋白质组 TiO2 富集和定量质谱法鉴定并表征了新型隐球菌中的钙调神经磷酸酶底物。已确定的靶标包括反式激活因子 Crz1 以及功能与 P 体/应激颗粒 (PB/SG) 以及 mRNA 翻译和衰变相关的新型底物，例如 Pbp1 和 Puf4。我们证明 Crz1 是一种真正的钙调神经磷酸酶底物，并且 Crz1 定位和转录活性受钙调神经磷酸酶控制。我们之前证明热应力和其他应力会触发钙调神经磷酸酶定位到 PBs/SGs。 PBs/SG 中的多个钙调神经磷酸酶靶标（包括 Puf4 和 Pbp1）单独或与 Crz1 一起有助于抗应激和毒力。此外，Pbp1也是性发育所必需的。遗传上位分析表明，Crz1 和新靶点 Lhp1、Puf4 和 Pbp1 在分支钙调磷酸酶通路中发挥作用，协调应激生存和毒力。这些发现支持了一种模型，即钙调神经磷酸酶在转录水平上通过 Crz1 控制应激和毒力，并在转录后通过定位到 PB/SG 并作用于 mRNA 代谢相关的靶标来控制应激和毒力。除 Crz1 外，本研究中确定的钙调神经磷酸酶靶标与已知的钙调神经磷酸酶底物几乎没有重叠。特别是，mRNA 结合蛋白和 PBs/SGs 驻留物包含一组新的钙调神经磷酸酶靶标，这些靶标之前尚未与哺乳动物或酿酒酵母中的钙调神经磷酸酶联系起来。这项研究表明，要么是钙调磷酸酶途径的广泛进化重新布线，要么是这些新的钙调磷酸酶靶标尚未被表征为其他生物体中的钙调磷酸酶靶标。这些发现进一步强调了新型隐球菌作为将钙调磷酸酶响应毒力网络定义为抗真菌治疗靶点的杰出模型。钙调神经磷酸酶是一种 Ca2+/钙调蛋白依赖性蛋白磷酸酶，对于人类真菌病原体新型隐球菌和其他与全球人类健康相关的主要病原真菌的应激生存、性发育和毒力至关重要。然而，病原真菌中尚无已知的钙调神经磷酸酶底物。采用最先进的磷酸蛋白质组学方法，我们鉴定了钙调磷酸酶底物，包括钙调磷酸酶本身和已知在钙信号传导和应激生存中发挥作用的保守 Crz1 转录激活剂。值得注意的是，我们的研究还发现了参与 RNA 加工、稳定性和翻译的新钙调神经磷酸酶靶标，这些靶标在热应激时与应激颗粒/P 体中的钙调磷酸酶一起共定位。这些发现支持了一个模型，即钙调神经磷酸酶通过 Crz1 和几个已确定的新靶点在分支途径中发挥作用，控制转录和转录后回路，以驱动应激生存、性发育和真菌毒力。我们的研究强调新型隐球菌作为一种实验模型，可以定义全球热应激反应毒力网络中钙调神经磷酸酶信号传导的基本范式，该网络可以作为真菌治疗的目标。
Calcineurin governs stress survival, sexual differentiation, and virulence of the human fungal pathogen Cryptococcus neoformans. Calcineurin is activated by increased Ca2+ levels caused by stress, and transduces signals by dephosphorylating protein substrates. Herein, we identified and characterized calcineurin substrates in C. neoformans by employing phosphoproteomic TiO2 enrichment and quantitative mass spectrometry. The identified targets include the transactivator Crz1 as well as novel substrates whose functions are linked to P-bodies/stress granules (PBs/SGs) and mRNA translation and decay, such as Pbp1 and Puf4. We show that Crz1 is a bona fide calcineurin substrate, and Crz1 localization and transcriptional activity are controlled by calcineurin. We previously demonstrated that thermal and other stresses trigger calcineurin localization to PBs/SGs. Several calcineurin targets localized to PBs/SGs, including Puf4 and Pbp1, contribute to stress resistance and virulence individually or in conjunction with Crz1. Moreover, Pbp1 is also required for sexual development. Genetic epistasis analysis revealed that Crz1 and the novel targets Lhp1, Puf4, and Pbp1 function in a branched calcineurin pathway that orchestrates stress survival and virulence. These findings support a model whereby calcineurin controls stress and virulence, at the transcriptional level via Crz1, and post-transcriptionally by localizing to PBs/SGs and acting on targets involved in mRNA metabolism. The calcineurin targets identified in this study share little overlap with known calcineurin substrates, with the exception of Crz1. In particular, the mRNA binding proteins and PBs/SGs residents comprise a cohort of novel calcineurin targets that have not been previously linked to calcineurin in mammals or in Saccharomyces cerevisiae. This study suggests either extensive evolutionary rewiring of the calcineurin pathway, or alternatively that these novel calcineurin targets have yet to be characterized as calcineurin targets in other organisms. These findings further highlight C. neoformans as an outstanding model to define calcineurin-responsive virulence networks as targets for antifungal therapy. Calcineurin is a Ca2+/calmodulin-dependent protein phosphatase essential for stress survival, sexual development, and virulence of the human fungal pathogen Cryptococcus neoformans and other major pathogenic fungi of global human health relevance. However, no calcineurin substrates are known in pathogenic fungi. Employing state-of-the-art phosphoproteomic approaches we identified calcineurin substrates, including calcineurin itself and the conserved Crz1 transcriptional activator known to function in calcium signaling and stress survival. Remarkably, our study also identified novel calcineurin targets involved in RNA processing, stability, and translation, which colocalize together with calcineurin in stress granules/P-bodies upon thermal stress. These findings support a model whereby calcineurin functions in a branched pathway, via Crz1 and several of the identified novel targets, that governs transcriptional and posttranscriptional circuits to drive stress survival, sexual development, and fungal virulence. Our study underscores C. neoformans as an experimental model to define basic paradigms of calcineurin signaling in global thermostress responsive virulence networks that can be targeted for fungal therapy.
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