Quantification of the HIV transcriptional activator complex in live cells by image‐based protein–protein interaction analysis

Asamitsu, Kaori; Omagari, Katsuhisa; Okuda, Tomoya; Hibi, Yurina; Okamoto, Takashi

doi:10.1111/gtc.12375

Cited by 7 publications

(8 citation statements)

References 47 publications

(58 reference statements)

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“…S1 Table summarizes the molecular details of the hydrogen bonding and hydrophobic interactions between CycT1 and CDK9 induced by Tat binding. Consistent with this finding from the MD simulation, we have previously reported that Tat induces increased binding affinity between CycT1 and CDK9 using image-based protein-protein interaction analysis in live cells [ 52 ].…”

Section: Resultssupporting

confidence: 84%

“…In order to clarify the structural arrangement of the Tat/P-TEFb complex, the phosphorylated Thr186 in CDK9 within the 3MI9 model was computationally substituted with an unphosphorylated Thr. The Tat sequence in the PDB data (3MI9) was substituted to match the Tat sequence used in our laboratory [ 25 , 26 , 52 ]: the substituted residues include R7N, K29R, T23N, Q35Y, V36A, I39T, T40R, and A42G (the activities of these Tat molecules are considered nearly identical). Missing side chains, loops, tails, and mutations were built using the homology modeling module in MOE ver.20140901 (Chemical Computing Group Inc.).…”

Section: Methodsmentioning

confidence: 99%

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MD simulation of the Tat/Cyclin T1/CDK9 complex revealing the hidden catalytic cavity within the CDK9 molecule upon Tat binding

2017

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In this study, we applied molecular dynamics (MD) simulation to analyze the dynamic behavior of the Tat/CycT1/CDK9 tri-molecular complex and revealed the structural changes of P-TEFb upon Tat binding. We found that Tat could deliberately change the local flexibility of CycT1. Although the structural coordinates of the H1 and H2 helices did not substantially change, H1ʹ, H2ʹ, and H3ʹ exhibited significant changes en masse. Consequently, the CycT1 residues involved in Tat binding, namely Tat-recognition residues (TRRs), lost their flexibility with the addition of Tat to P-TEFb. In addition, we clarified the structural variation of CDK9 in complex with CycT1 in the presence or absence of Tat. Interestingly, Tat addition significantly reduced the structural variability of the T-loop, thus consolidating the structural integrity of P-TEFb. Finally, we deciphered the formation of the hidden catalytic cavity of CDK9 upon Tat binding. MD simulation revealed that the PITALRE signature sequence of CDK9 flips the inactive kinase cavity of CDK9 into the active form by connecting with Thr186, which is crucial for its activity, thus presumably recruiting the substrate peptide such as the C-terminal domain of RNA pol II. These findings provide vital information for the development of effective novel anti-HIV drugs with CDK9 catalytic activity as the target.

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Section: Resultssupporting

confidence: 84%

Section: Methodsmentioning

confidence: 99%

MD simulation of the Tat/Cyclin T1/CDK9 complex revealing the hidden catalytic cavity within the CDK9 molecule upon Tat binding

2017

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“…The fluorescent protein tags in this droplet are detected as fluorescent puncta in a reversible manner. Although the Fluoppi system is useful for detecting PPIs in living cells 24 – 26 , it was recently extended to allow visualization of protein homodimerization by development of a single fusion construct containing PB1 and mAG1 (homoFluoppi) that is fused to a protein of interest 23 .…”

Section: Introductionmentioning

confidence: 99%

Visualization and quantification of dynamic STAT3 homodimerization in living cells using homoFluoppi

Okada

Watanabe

Shoji

et al. 2018

Sci Rep

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Dimerization in signal transduction is a dynamically regulated process and a key regulatory mechanism. Signal transducer and activator of transcription 3 (STAT3) dimerizes after tyrosine phosphorylation upon cytokine stimulation. Because only the STAT3 dimer possesses the trans-activation activity, dimerization is an indispensable process for cytokine signaling. Here we report the detection of dynamic STAT3 dimerization in living cells using the homoFluoppi system. This method allowed us to validate the presence of an intact Src homology 2 domain and STAT3 Tyr705 phosphorylation, which facilitate puncta formation and homodimerization. Puncta formation was reversible, as determined by a decreased punctate signal after washout of oncostatin M. We analyzed STAT3 mutants, which have been reported in patients with hyper IgE syndrome and inflammatory hepatocellular adenoma (IHCA). Analysis of the IHCA mutants using homoFluoppi revealed constitutive activity independent of cytokine stimulation and novel insight into kinetics of dimer dissociation process. Next, we used homoFluoppi to screen for inhibitors of STAT3 dimerization, and identified 3,4-methylenedioxy-β-nitrostyrene as a novel inhibitor. The results of this study show that homoFluoppi is a useful research tool for the analysis of proteins like STAT3 that dynamically dimerize, and is applicable for the screening of dimerization modulators.

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“…We termed this method Fluoppi for Fluo rescent ppi -visualization. Fluoppi was previously employed as a general method for PPI detection242526 before its operational principle was understood or studied.…”

Section: Resultsmentioning

confidence: 99%

Genetic visualization of protein interactions harnessing liquid phase transitions

Watanabe

Seki

Fukano

et al. 2017

Sci Rep

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Protein-protein interactions (PPIs) are essential components of cellular function. Current fluorescence-based technologies to measure PPIs have limited dynamic range and quantitative reproducibility. Here, we describe a genetically-encoded PPI visualization system that harnesses the dynamics of condensed liquid-phase transitions to analyze protein interactions in living cells. The fluorescent protein Azami-Green and p62-PB1 domain when fused to PPI partners triggered a rapid concatenation/oligomerization process that drove the condensation of liquid-phase droplets for real-time analysis of the interaction with unlimited dynamic range in the fluorescence signal. Proof-of-principle studies revealed novel insights on the live cell dynamics of XIAP-Smac and ERK2-dimer interactions. A photoconvertible variant allowed time-resolved optical highlighting for PPI kinetic analysis. Our system, called Fluoppi, demonstrates the unique signal amplification properties of liquid-phase condensation to detect PPIs. The findings introduce a general method for discovery of novel PPIs and modulators of established PPIs.

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Quantification of the HIV transcriptional activator complex in live cells by image‐based protein–protein interaction analysis

Cited by 7 publications

References 47 publications

MD simulation of the Tat/Cyclin T1/CDK9 complex revealing the hidden catalytic cavity within the CDK9 molecule upon Tat binding

MD simulation of the Tat/Cyclin T1/CDK9 complex revealing the hidden catalytic cavity within the CDK9 molecule upon Tat binding

Visualization and quantification of dynamic STAT3 homodimerization in living cells using homoFluoppi

Genetic visualization of protein interactions harnessing liquid phase transitions

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