Native mass spectrometry analyses of chaperonin complex TRiC/CCT reveal subunit N-terminal processing and re-association patterns.
Native mass spectrometry analyses of chaperonin complex TRiC/CCT reveal subunit N-terminal processing and re-association patterns.
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DOI：
10.1038/s41598-021-91086-6
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发表时间：
2021-06-22
影响因子：
4.6
通讯作者：
Frydman J
中科院分区：
文献类型：
--
作者：
Collier MP;Moreira KB;Li KH;Chen YC;Itzhak D;Samant R;Leitner A;Burlingame A;Frydman J
真核伴侣蛋白TRiC/CCT是细胞蛋白质折叠所必需的大型atp依赖性复合体。它的亚基排列成两个堆叠的八元异聚物环，从酵母到人类是保守的。最近的一项突破使从昆虫细胞中生产功能性人类TRiC （hTRiC）成为可能。在这里，我们应用一套质谱技术来表征重组hTRiC。我们发现所有的CCT1-8亚基都是通过甲硫氨酸切除和乙酰化的组合进行n端加工的。有机溶剂解离产生的主要是单体亚基和少量的CCT二聚体。值得注意的是，一些二聚体具有初始hTRiC中不存在的非规范亚基间接触。这表明单个CCT单体可以混杂地重新组装成二聚体，并且缺乏在整体配合物中承担特定界面配对的信息。CCT5一直是最稳定的亚基，参与最多的非规范二聚体配对。这些发现证实了重组hTRiC在生理上的翻译后加工和功能，并为定量了解TRiC亚基和界面的相对稳定性提供了依据，这是重建其组装机制的关键一步。我们的研究结果还强调了在研究多聚体组装时，分配由溶液解离后的天然质谱鉴定的接触作为规范或非规范的重要性。
The eukaryotic chaperonin TRiC/CCT is a large ATP-dependent complex essential for cellular protein folding. Its subunit arrangement into two stacked eight-membered hetero-oligomeric rings is conserved from yeast to man. A recent breakthrough enables production of functional human TRiC (hTRiC) from insect cells. Here, we apply a suite of mass spectrometry techniques to characterize recombinant hTRiC. We find all subunits CCT1-8 are N-terminally processed by combinations of methionine excision and acetylation observed in native human TRiC. Dissociation by organic solvents yields primarily monomeric subunits with a small population of CCT dimers. Notably, some dimers feature non-canonical inter-subunit contacts absent in the initial hTRiC. This indicates individual CCT monomers can promiscuously re-assemble into dimers, and lack the information to assume the specific interface pairings in the holocomplex. CCT5 is consistently the most stable subunit and engages in the greatest number of non-canonical dimer pairings. These findings confirm physiologically relevant post-translational processing and function of recombinant hTRiC and offer quantitative insight into the relative stabilities of TRiC subunits and interfaces, a key step toward reconstructing its assembly mechanism. Our results also highlight the importance of assigning contacts identified by native mass spectrometry after solution dissociation as canonical or non-canonical when investigating multimeric assemblies.
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影响因子：
7.4
作者：
Landreh, Michael;Alvelius, Gunvor;Jornvall, Hans
通讯作者：
Jornvall, Hans
影响因子：
3.3
作者：
Elliott KL;Svanström A;Spiess M;Karlsson R;Grantham J
通讯作者：
Grantham J
影响因子：
48
作者：
Gibson, Daniel G.;Young, Lei;Smith, Hamilton O.
通讯作者：
Smith, Hamilton O.
DOI：
10.1073/pnas.91.19.9111
发表时间：
1994-09-13
影响因子：
11.1
作者：
CHEN, XY;SULLIVAN, DS;HUFFAKER, TC
通讯作者：
HUFFAKER, TC
DOI：
10.1073/pnas.1320298111
发表时间：
2014-07-01
影响因子：
11.1
作者：
Leitner, Alexander;Joachimiak, Lukasz A.;Aebersold, Ruedi
通讯作者：
Aebersold, Ruedi