A Chemical Proteomics Approach to Reveal Direct Protein-Protein Interactions in Living Cells.
A Chemical Proteomics Approach to Reveal Direct Protein-Protein Interactions in Living Cells.
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一种化学蛋白质组学方法揭示了活细胞中直接蛋白质蛋白质相互作用。
DOI：
10.1016/j.chembiol.2017.10.001
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发表时间：
2018-01-18
影响因子：
8.6
通讯作者：
Kapoor TM
中科院分区：
文献类型：
--
作者：
Kleiner RE;Hang LE;Molloy KR;Chait BT;Kapoor TM
蛋白质-蛋白质相互作用介导重要的细胞过程，然而天然相互作用的检测是具有挑战性的，因为它们通常是低亲和力和环境依赖性的。在这里，我们开发了一种化学蛋白质组学方法在体内CLASPI [iCLASPI]（在体内交联辅助和稳定同位素标记的氨基酸在细胞培养[SILAC}为基础的蛋白质鉴定）依赖于光交联，琥珀抑制，和SILAC为基础的定量蛋白质组学的轮廓在活细胞中的上下文依赖性蛋白质-蛋白质相互作用。首先，我们使用iCLASPI来分析可溶性组蛋白H3或H4的N-末端尾部的体内结合伴侣。我们确定了已知的组蛋白伴侣和修饰蛋白，从而验证了我们的方法，并发现可溶性组蛋白H3和UBR 7，E3泛素连接酶，介导的UBR 7的PHD结构域之间的相互作用。此外，我们应用iCLASPI分析了不同细胞周期阶段染色质相关组蛋白H3的上下文依赖性蛋白质-蛋白质相互作用，并确定ANP 32A为有丝分裂特异性相互作用因子。我们的研究结果表明，iCLASPI方法可以提供一个通用的策略，用于识别本地的，上下文依赖的直接蛋白质-蛋白质相互作用，使用光交联和定量蛋白质组学。蛋白质-蛋白质相互作用介导重要的生物学过程，但在细胞中表征这些相互作用提出了一个重大挑战。Kleiner等人描述了一种结合光交联和基于SILAC的蛋白质组学的化学蛋白质组学方法，以表征活细胞中依赖于上下文的直接蛋白质-蛋白质相互作用。他们将他们的方法应用于涉及核心组蛋白H3和H4的相互作用。
Protein-protein interactions mediate essential cellular processes, however the detection of native interactions is challenging since they are often low affinity and context dependent. Here, we develop a chemical proteomics approach in vivo CLASPI [iCLASPI] (in vivo crosslinking-assisted and stable isotope labeling by amino acids in cell culture [SILAC}-based protein identification) relying upon photo-crosslinking, amber suppression, and SILAC-based quantitative proteomics to profile context-dependent protein-protein interactions in living cells. First, we use iCLASPI to profile in vivo binding partners of the N-terminal tails of soluble histone H3 or H4. We identify known histone chaperones and modifying proteins, thereby validating our approach, and find an interaction between soluble histone H3 and UBR7, an E3 ubiquitin ligase, mediated by UBR7’s PHD domain. Furthermore, we apply iCLASPI to profile the context-dependent protein-protein interactions of chromatin-associated histone H3 at different cell cycle stages, and identify ANP32A as a mitosis-specific interactor. Our results demonstrate that the iCLASPI approach can provide a general strategy for identifying native, context-dependent direct protein-protein interactions using photo-crosslinking and quantitative proteomics. Protein-protein interactions mediate essential biological processes, but characterizing these interactions in cells presents a major challenge. Kleiner et al. describe a chemical proteomics approach combining photocrosslinking and SILAC-based proteomics to characterize context-dependent direct protein-protein interactions in live cells. They apply their approach to profile interactions involving core histones H3 and H4.
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影响因子：
64.8
作者：
Kim, Min-Sik;Pinto, Sneha M.;Getnet, Derese;Nirujogi, Raja Sekhar;Manda, Srikanth S.;Chaerkady, Raghothama;Madugundu, Anil K.;Kelkar, Dhanashree S.;Isserlin, Ruth;Jain, Shobhit;Thomas, Joji K.;Muthusamy, Babylakshmi;Leal-Rojas, Pamela;Kumar, Praveen;Sahasrabuddhe, Nandini A.;Balakrishnan, Lavanya;Advani, Jayshree;George, Bijesh;Renuse, Santosh;Selvan, Lakshmi Dhevi N.;Patil, Arun H.;Nanjappa, Vishalakshi;Radhakrishnan, Aneesha;Prasad, Samarjeet;Subbannayya, Tejaswini;Raju, Rajesh;Kumar, Manish;Sreenivasamurthy, Sreelakshmi K.;Marimuthu, Arivusudar;Sathe, Gajanan J.;Chavan, Sandip;Datta, Keshava K.;Subbannayya, Yashwanth;Sahu, Apeksha;Yelamanchi, Soujanya D.;Jayaram, Savita;Rajagopalan, Pavithra;Sharma, Jyoti;Murthy, Krishna R.;Syed, Nazia;Goel, Renu;Khan, Aafaque A.;Ahmad, Sartaj;Dey, Gourav;Mudgal, Keshav;Chatterjee, Aditi;Huang, Tai-Chung;Zhong, Jun;Wu, Xinyan;Shaw, Patrick G.;Freed, Donald;Zahari, Muhammad S.;Mukherjee, Kanchan K.;Shankar, Subramanian;Mahadevan, Anita;Lam, Henry;Mitchell, Christopher J.;Shankar, Susarla Krishna;Satishchandra, Parthasarathy;Schroeder, John T.;Sirdeshmukh, Ravi;Maitra, Anirban;Leach, Steven D.;Drake, Charles G.;Halushka, Marc K.;Prasad, T. S. Keshava;Hruban, Ralph H.;Kerr, Candace L.;Bader, Gary D.;Iacobuzio-Donahue, Christine A.;Gowda, Harsha;Pandey, Akhilesh
通讯作者：
Pandey, Akhilesh
影响因子：
14.8
作者：
Kleiner RE;Verma P;Molloy KR;Chait BT;Kapoor TM
通讯作者：
Kapoor TM
影响因子：
2.9
作者：
Garcia, BA;Barber, CM;Hunt, DF
通讯作者：
Hunt, DF
DOI：
10.1038/nrm.2016.159
发表时间：
2017-03
期刊：
Nature reviews. Molecular cell biology
影响因子：
--
作者：
Hammond CM;Strømme CB;Huang H;Patel DJ;Groth A
通讯作者：
Groth A
DOI：
10.1083/jcb.110.5.1475
发表时间：
1990-05
期刊：
The Journal of cell biology
影响因子：
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作者：
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通讯作者：
Earnshaw WC