Department of General and Medical Biochemistry, Faculty of Biology, University of Gdansk, Poland.
Laboratory of Virus Molecular Biology, Intercollegiate Faculty of Biotechnology University of Gdansk - Medical University of Gdansk, Gdansk, Poland.
J Proteomics. 2018 Apr 15;177:88-111. doi: 10.1016/j.jprot.2018.02.022. Epub 2018 Mar 2.
The human HtrA3 protease is involved in placentation, mitochondrial homeostasis, stimulation of apoptosis and proposed to be a tumor suppressor. Molecular mechanisms of the HtrA3 functions are poorly understood and knowledge concerning its cellular targets is very limited. There are two HtrA3 isoforms, the long (HtrA3L) and short (HtrA3S). Upon stress, their N-terminal domains are removed, resulting in the more active ΔN-HtrA3. By pull down and mass spectrometry techniques, we identified a panel of putative ΔN-HtrA3L/S substrates. We confirmed that ΔN-HtrA3L/S formed complexes with actin, β-tubulin, vimentin and TCP1α in vitro and in a cell and partially co-localized with the actin and vimentin filaments, microtubules and TCP1α in a cell. In vitro, both isoforms cleaved the cytoskeleton proteins, promoted tubulin polymerization and displayed chaperone-like activity, with ΔN-HtrA3S being more efficient in proteolysis and ΔN-HtrA3L - in polymerization. TCP1α, essential for the actin and tubulin folding, was directly bound by the ΔN-HtrA3L/S but not cleaved. These results indicate that actin, β-tubulin, vimentin, and TCP1α are HtrA3 cellular partners and suggest that HtrA3 may influence cytoskeleton dynamics. They also suggest different roles of the HtrA3 isoforms and a possibility that HtrA3 protease may also function as a co-chaperone.
The HtrA3 protease stimulates apoptosis and is proposed to be a tumor suppressor and a therapeutic target, however little is known about its function at the molecular level and very few HtrA3 physiological substrates have been identified so far. Furthermore, HtrA3 is the only member of the HtrA family of proteins which, apart from the long isoform possessing the PD and PDZ domains (HtrA3L), has a short isoform (HtrA3S) lacking the PDZ domain. In this work we identified a large panel (about 150) of the tentative HtrA3L/S cellular partners which provides a good basis for further research concerning the HtrA3 function. We have shown that the cytoskeleton proteins actin, β-tubulin and vimentin, and the TCP1α chaperonin are cellular partners of both HtrA3 isoforms. Our findings indicate that HtrA3 may promote destabilization of the actin and vimentin cytoskeleton and suggest that it may influence the dynamics of the microtubule network, with the HtrA3S being more efficient in cytoskeleton protein cleavage and HtrA3L - in tubulin polymerization. Also, we have shown for the first time that HtrA3 has a chaperone-like, holdase activity in vitro - activity typical for co-chaperone proteins. The proposed HtrA3 influence on the cytoskeleton dynamics may be one of the ways in which HtrA3 promotes cell death and affects cancerogenesis. We believe that the results of this study provide a new insight into the role of HtrA3 in a cell and further confirm the notion that HtrA3 should be considered as a target of new anti-cancer therapies.
人 HtrA3 蛋白酶参与胎盘形成、线粒体动态平衡、凋亡刺激，并被提议作为肿瘤抑制因子。HtrA3 功能的分子机制了解甚少，关于其细胞靶标的知识非常有限。有两种 HtrA3 同种型，长（HtrA3L）和短（HtrA3S）。在应激下，它们的 N 端结构域被去除，导致更活跃的 ΔN-HtrA3。通过下拉和质谱技术，我们鉴定了一组推定的 ΔN-HtrA3L/S 底物。我们证实 ΔN-HtrA3L/S 在体外和细胞中与肌动蛋白、β-微管蛋白、波形蛋白和 TCP1α 形成复合物，并部分与肌动蛋白和波形蛋白丝、微管和 TCP1α 在细胞中共定位。在体外，两种同工酶都能切割细胞骨架蛋白，促进微管蛋白聚合，并表现出伴侣样活性，其中 ΔN-HtrA3S 在蛋白水解方面更有效，ΔN-HtrA3L 在聚合方面更有效。TCP1α 是肌动蛋白和微管蛋白折叠所必需的，直接与 ΔN-HtrA3L/S 结合，但不被切割。这些结果表明，肌动蛋白、β-微管蛋白、波形蛋白和 TCP1α 是 HtrA3 的细胞伴侣，并表明 HtrA3 可能影响细胞骨架动力学。它们还表明 HtrA3 同工型的不同作用以及 HtrA3 蛋白酶可能也作为共伴侣发挥作用的可能性。
HtrA3 蛋白酶刺激细胞凋亡，被提议作为肿瘤抑制因子和治疗靶点，然而，目前对其分子水平的功能知之甚少，迄今为止很少有鉴定出 HtrA3 的生理底物。此外，HtrA3 是 HtrA 家族蛋白中唯一的成员，除了具有 PD 和 PDZ 结构域的长同工型（HtrA3L）外，还有一个缺乏 PDZ 结构域的短同工型（HtrA3S）。在这项工作中，我们鉴定了一个约 150 个的 HtrA3L/S 细胞伴侣的暂定面板，为进一步研究 HtrA3 功能提供了良好的基础。我们已经表明，细胞骨架蛋白肌动蛋白、β-微管蛋白和波形蛋白，以及 TCP1α 伴侣蛋白是两种 HtrA3 同工型的细胞伴侣。我们的发现表明，HtrA3 可能促进肌动蛋白和波形蛋白细胞骨架的不稳定性，并表明它可能影响微管网络的动态，其中 HtrA3S 在细胞骨架蛋白切割方面更有效，HtrA3L 在微管蛋白聚合方面更有效。此外，我们首次表明 HtrA3 在体外具有伴侣样、持留酶活性 - 这是共伴侣蛋白的典型活性。HtrA3 对细胞骨架动力学的影响可能是 HtrA3 促进细胞死亡和影响癌症发生的方式之一。我们相信，这项研究的结果为 HtrA3 在细胞中的作用提供了新的见解，并进一步证实了 HtrA3 应该被视为新的抗癌治疗靶点的观点。
