Suppression of p53 response by targeting p53-Mediator binding with a stapled peptide
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Abstract DNA-binding transcription factors (TFs) remain challenging to target with molecular probes. Many TFs function in part through interaction with Mediator; we sought to block p53 function by disrupting the p53-Mediator interaction. Through rational design and activity-based screening, we characterized a stapled peptide, with functional mimics of both p53 activation domains, that selectively inhibited p53- and Mediator-dependent transcription in vitro . This “bivalent peptide” also suppressed p53 transcriptional response in human cancer cells. Our strategy circumvents the TF and instead targets the TF-Mediator interface, with desired transcriptional outcomes. Different TFs target Mediator through different subunits, suggesting this strategy could be broadly applied.Keywords:
Mediator
Transcription
Transcriptional activity
Protein-protein interactions play an essential role in cellular processes. Certain proteins form stable complexes with their partner proteins, whereas others function by forming transient complexes. The conventional protein-protein interaction model describes an interaction between two proteins under the assumption that a protein binds to its partner protein through a single binding site. In this study, we improved the conventional interaction model by developing a Multiple-Site (MS) model in which a protein binds to its partner protein through closely located multiple binding sites on a surface of the partner protein by transiently docking at each binding site with individual binding free energies. To test this model, we used the protein-protein interaction mediated by Src homology 3 (SH3) domains. SH3 domains recognize their partners via a weak, transient interaction and are therefore promiscuous in nature. Because the MS model requires large amounts of data compared with the conventional interaction model, we used experimental data from the positionally addressable syntheses of peptides on cellulose membranes (SPOT-synthesis) technique. From the analysis of the experimental data, individual binding free energies for each binding site of peptides were extracted. A comparison of the individual binding free energies from the analysis with those from atomistic force fields gave a correlation coefficient of 0.66. Furthermore, application of the MS model to 10 SH3 domains lowers the prediction error by up to 9% compared with the conventional interaction model. This improvement in prediction originates from a more realistic description of complex formation than the conventional interaction model. The results suggested that, in many cases, SH3 domains increased the protein complex population through multiple binding sites of their partner proteins. Our study indicates that the consideration of general complex formation is important for the accurate description of protein complex formation, and especially for those of weak or transient protein complexes.
Docking (animal)
Threading (protein sequence)
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Abstract We present a possible molecular basis for the opposite activity of two homologues proteins that bind similar ligands and show that this is achieved by fine-tuning of the interaction interface. The highly homologous ASPP proteins have opposite roles in regulating apoptosis: ASPP2 induces apoptosis while iASPP inhibits it. The ASPP proteins are regulated by an autoinhibitory interaction between their Ank-SH3 and Pro domains. We performed a detailed biophysical and molecular study of the Pro – Ank-SH3 interaction in iASPP and compared it to the interaction in ASPP2. We found that iASPP Pro is disordered and that the interaction sites are entirely different: iASPP Ank-SH3 binds iASPP Pro via its fourth Ank repeat and RT loop while ASPP2 Ank-SH3 binds ASPP2 Pro via its first Ank repeat and the n-src loop. It is possible that by using different moieties in the same interface, the proteins can have distinct and specific interactions resulting in differential regulation and ultimately different biological activities.
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Abstract DNA-binding transcription factors (TFs) remain challenging to target with molecular probes. Many TFs function in part through interaction with Mediator; we sought to block p53 function by disrupting the p53-Mediator interaction. Through rational design and activity-based screening, we characterized a stapled peptide, with functional mimics of both p53 activation domains, that selectively inhibited p53- and Mediator-dependent transcription in vitro . This “bivalent peptide” also suppressed p53 transcriptional response in human cancer cells. Our strategy circumvents the TF and instead targets the TF-Mediator interface, with desired transcriptional outcomes. Different TFs target Mediator through different subunits, suggesting this strategy could be broadly applied.
Mediator
Transcription
Transcriptional activity
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Arf-GAP with Rho-GAP domain, ANK repeat and PH domain-containing protein 1 (ARAP1), Cbl-interacting protein of 85 kDa (CIN85), and casitas B-lineage lymphoma (Cbl) play important roles in epidermal growth factor receptor (EGFR) internalization and recycling. In previous studies, ARAP1 was found to interact with CIN85, and their interaction attenuated the ubiquitination of EGFR. However, the molecular mechanism was still unclear. In this study, we first biochemically and structurally characterized the interaction between ARAP1 and CIN85, and found that the CIN85 SH3B domain bound to the ARAP1 PXPXXRX (except P) XXR/H/K motif with high affinity and specificity. Based on this binding model, we further predicted other potential CIN85 binding partners and tested their interactions biochemically. Moreover, our swapping data and structure alignment analysis suggested that the β2-β3 loops of the CIN85 SH3 domains and the H87ARAP1/E132CIN85 interaction were critical for ARAP1 binding specificity. Finally, our competitive analytical gel-filtration chromatography and isothermal titration calorimetry (ITC) results showed that ARAP1 could compete with Cbl for CIN85 binding, which provides a biochemical basis for the regulatory roles of ARAP1 in the CIN85-mediated EGFR internalizing process.
Isothermal Titration Calorimetry
Internalization
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Protein-protein interactions (PPIs) and protein-metabolite interactions play a key role in many biochemical processes, yet they are often viewed as being independent. However, the fact that small molecule drugs have been successful in inhibiting PPIs suggests a deeper relationship between protein pockets that bind small molecules and PPIs. We demonstrate that 2/3 of PPI interfaces, including antibody-epitope interfaces, contain at least one significant small molecule ligand binding pocket. In a representative library of 50 distinct protein-protein interactions involving hundreds of mutations, >75% of hot spot residues overlap with small molecule ligand binding pockets. Hence, ligand binding pockets play an essential role in PPIs. In representative cases, evolutionary unrelated monomers that are involved in different multimeric interactions yet share the same pocket are predicted to bind the same metabolites/drugs; these results are confirmed by examples in the PDB. Thus, the binding of a metabolite can shift the equilibrium between monomers and multimers. This implicit coupling of PPI equilibria, termed "metabolic entanglement", was successfully employed to suggest novel functional relationships among protein multimers that do not directly interact. Thus, the current work provides an approach to unify metabolomics and protein interactomics.
Protein ligand
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During the development of the mammalian cardiovascular system, the formation of a mature and fully functional cardiovascular system needs the fine coordination of the morphogenesis of various molecules, cells, tissues, and organs. Abnormalities in these processes usually lead to serious congenital heart defects. The determination and maintenance of cell fate in multicellular organisms depend to a large extent on the precise timing and control of RNA polymerase II (Pol II) transcription, and the transcription Mediator complex plays an irreplaceable role in the Pol II transcription process. Mediator is an evolutionarily conserved multi-subunit protein complex, including four parts: head, middle, tail, and kinase. It is a functional bridge between transcription factors and basic transcription machines. In recent years, due to the key role of Mediator in the transcriptional regulation of gene expression, many of human heart diseases have been confirmed to be related to specific Mediator gene mutations, such as heart valve defects, translocation of the great arteries, DiGeorge syndrome and some cardiovascular diseases related to energy homeostasis. In this review, we summarize the role of Mediator in cardiovascular development and disease, focusing on the role of Mediator in the development of cardiovascular disease, and provides a broad idea for the research on Mediator-related cardiovascular system development and diseases.哺乳动物心血管系统发育过程中,各分子、细胞和组织器官形态发生过程的精细协调对于形成成熟且功能齐全的心血管系统是不可或缺的,这些过程出现异常通常会导致严重的先天性心血管发育缺陷。多细胞生物中细胞命运的决定和维持在很大程度上依赖于对RNA聚合酶II (Pol II)转录活性的时空精确调控,而转录中介体(Mediator)在Pol II转录过程中起着重要的协同作用。Mediator是一种进化上保守的多亚基蛋白质复合体,包括头部、中部、尾部和激酶部四个部分,是转录因子和基础转录机器之间的功能联系的桥梁。近年来,鉴于Mediator在基因表达中的关键作用,越来越多的人类心血管疾病被证实与特定的Mediator基因突变相关,如心脏瓣膜缺陷、大动脉转位、DiGeorge综合征及一些与能量稳态失衡相关的心血管疾病。本文就Mediator在心血管系统发育和疾病中的作用进行综述,重点讨论Mediator对转录调控的影响在心血管疾病发生发展中的作用,旨在为与Mediator相关的心血管系统发育和疾病的研究提供广阔的研究思路。.
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RNA polymerase II
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Identifying the contact regions between a protein and its binding partners is essential for creating therapies that block the interaction. Unfortunately, such contact regions are extremely difficult to characterize because they are hidden inside the binding interface. Here we introduce protein painting as a new tool that employs small molecules as molecular paints to tightly coat the surface of protein-protein complexes. The molecular paints, which block trypsin cleavage sites, are excluded from the binding interface. Following mass spectrometry, only peptides hidden in the interface emerge as positive hits, revealing the functional contact regions that are drug targets. We use protein painting to discover contact regions between the three-way interaction of IL1β ligand, the receptor IL1RI and the accessory protein IL1RAcP. We then use this information to create peptides and monoclonal antibodies that block the interaction and abolish IL1β cell signalling. The technology is broadly applicable to discover protein interaction drug targets.
Surface protein
Molecular Recognition
Protein Array Analysis
Protein ligand
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Abstract The pulsatile nature of transcription has recently emerged as an important property of gene expression. Here we report on the characterization of a RNA polymerase II transgene that is transcribed in the nucleolus. Using the MS2-GFP reporter system and live cell imaging, we found that the synthesis of a MS2-tagged transcript in the nucleolus was discontinuous in all of the cells that were observed, with periods of activity lasting from 15 minutes to 21 hours. The frequency of pulse lengths could be fitted with an exponential function, from which we determined that transcription occurs on average for periods of 20 minutes. These ON periods alternate with periods of inactivity which last on average 29 minutes. The post-mitotic reactivation of transcription was found to be asynchronous in daughter cell pairs. Our observation of discontinuous transcriptional activity in the nucleolus may reflect cycling in the assembly and disassembly of active chromatin structure in and/or around the rDNA genes.
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Transcriptional activity
RNA polymerase I
RNA polymerase II
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Mediator, originally discovered in yeast and composed of multi-subunits with a high molecular mass , is an essential component of the RNA polymerase Ⅱgeneral transcriptional machinery and plays a crucial part in the activation and repression of eukaryotic mRNA synthesis. Regulatory information could be conveyed through changes in Mediator conformation that would influence the transcription initiation process. Recent studies have defined the subunit composition and associated activities of mammalian Mediator, and revealed a striking evolutionary conservation of Mediator structure and function from yeast to man.
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Eukaryotic transcription
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