scholarly journals A cell-based high-content screen identifies isocotoin as a small molecule inhibitor of the meiosis-specific MEIOB–SPATA22 complex†

2020 ◽  
Vol 103 (2) ◽  
pp. 333-342 ◽  
Author(s):  
Yang Xu ◽  
Rong Liu ◽  
N Adrian Leu ◽  
Lei Zhang ◽  
Ilsiya Ibragmova ◽  
...  
Keyword(s):  
Small Molecule ◽  
Protein Interactions ◽  
Cultured Cells ◽  
Bimolecular Fluorescence Complementation ◽  
Hek293 Cells ◽  
Dependent Manner ◽  
Bifc Assay ◽  
Small Molecule Libraries ◽  
A Cell ◽  
Specific Proteins

Abstract MEIOB and SPATA22 are meiosis-specific proteins, interact with each other, and are essential for meiotic recombination and fertility. Aspartic acid 383 (D383) in MEIOB is critical for its interaction with SPATA22 in biochemical studies. Here we report that genetic studies validate the requirement of D383 for the function of MEIOB in mice. The MeiobD383A/D383A mice display meiotic arrest due to depletion of both MEIOB and SPATA22 proteins in the testes. We developed a cell-based bimolecular fluorescence complementation (BiFC) assay, in which MEIOB and SPATA22 are fused to split YFP moieties and their co-expression in cultured cells leads to the MEIOB–SPATA22 dimerization and reconstitution of the fluorophore. As expected, the interaction-disrupting D383A substitution results in the absence of YFP fluorescence in the BiFC assay. A high-throughput screen of small molecule libraries identified candidate hit compounds at a rate of 0.7%. Isocotoin, a hit compound from the natural product library, inhibits the MEIOB–SPATA22 interaction and promotes their degradation in HEK293 cells in a dose-dependent manner. Therefore, the BiFC assay can be employed to screen for small molecule inhibitors that disrupt protein–protein interactions or promote degradation of meiosis-specific proteins.

scholarly journals MKL1 promotes endothelial-to-mesenchymal transition and liver fibrosis by activating TWIST1 transcription

2019 ◽  
Vol 10 (12) ◽  
Author(s):  
Zilong Li ◽  
Baoyu Chen ◽  
Wenhui Dong ◽  
Ming Kong ◽  
Zhiwen Fan ◽  
...  
Keyword(s):  
Liver Fibrosis ◽  
Small Molecule ◽  
Cultured Cells ◽  
Underlying Mechanism ◽  
Dependent Manner ◽  
Mesenchymal Transition ◽  
Duct Ligation ◽  
Small Molecule Compound ◽  
Liver Fibrogenesis ◽  
Endothelial To Mesenchymal Transition

AbstractExcessive fibrogenic response in the liver disrupts normal hepatic anatomy and function heralding such end-stage liver diseases as hepatocellular carcinoma and cirrhosis. Sinusoidal endothelial cells contribute to myofibroblast activation and liver fibrosis by undergoing endothelial-mesenchymal transition (EndMT). The underlying mechanism remains poorly defined. Here we report that inhibition or endothelial-specific deletion of MKL1, a transcriptional modulator, attenuated liver fibrosis in mice. MKL1 inhibition or deletion suppressed EndMT induced by TGF-β. Mechanistically, MKL1 was recruited to the promoter region of TWIST1, a master regulator of EndMT, and activated TWIST1 transcription in a STAT3-dependent manner. A small-molecule STAT3 inhibitor (C188-9) alleviated EndMT in cultured cells and bile duct ligation (BDL) induced liver fibrosis in mice. Finally, direct inhibition of TWIST1 by a small-molecule compound harmine was paralleled by blockade of EndMT in cultured cells and liver fibrosis in mice. In conclusion, our data unveil a novel mechanism underlying EndMT and liver fibrosis and highlight the possibility of targeting the STAT3-MKL1-TWIST1 axis in the intervention of aberrant liver fibrogenesis.

scholarly journals A conformation-specific ON-switch for controlling CAR T cells with an orally available drug

2020 ◽  
Vol 117 (26) ◽  
pp. 14926-14935 ◽  
Author(s):  
Charlotte U. Zajc ◽  
Markus Dobersberger ◽  
Irene Schaffner ◽  
Georg Mlynek ◽  
Dominic Pühringer ◽  
...  
Keyword(s):  
T Cells ◽  
Small Molecule ◽  
Conformational Changes ◽  
Protein Interactions ◽  
Fold Increase ◽  
Retinol Binding Protein ◽  
Dependent Manner ◽  
Car T Cells ◽  
Car T

Molecular ON-switches in which a chemical compound induces protein–protein interactions can allow cellular function to be controlled with small molecules. ON-switches based on clinically applicable compounds and human proteins would greatly facilitate their therapeutic use. Here, we developed an ON-switch system in which the human retinol binding protein 4 (hRBP4) of the lipocalin family interacts with engineered hRBP4 binders in a small molecule-dependent manner. Two different protein scaffolds were engineered to bind to hRBP4 when loaded with the orally available small molecule A1120. The crystal structure of an assembled ON-switch shows that the engineered binder specifically recognizes the conformational changes induced by A1120 in two loop regions of hRBP4. We demonstrate that this conformation-specific ON-switch is highly dependent on the presence of A1120, as demonstrated by an ∼500-fold increase in affinity upon addition of the small molecule drug. Furthermore, the ON-switch successfully regulated the activity of primary human CAR T cells in vitro. We anticipate that lipocalin-based ON-switches have the potential to be broadly applied for the safe pharmacological control of cellular therapeutics.

scholarly journals ATAD5 promotes replication restart by regulating RAD51 and PCNA in response to replication stress

2019 ◽  
Vol 10 (1) ◽  
Author(s):  
Su Hyung Park ◽  
Nalae Kang ◽  
Eunho Song ◽  
Minwoo Wie ◽  
Eun A. Lee ◽  
...  
Keyword(s):  
Single Molecule ◽  
Protein Interactions ◽  
Cultured Cells ◽  
Replication Stress ◽  
Dependent Manner ◽  
Dna Breaks ◽  
Single Molecule Fret ◽  
Hydroxyurea Treatment ◽  
Fork Regression

AbstractMaintaining stability of replication forks is important for genomic integrity. However, it is not clear how replisome proteins contribute to fork stability under replication stress. Here, we report that ATAD5, a PCNA unloader, plays multiple functions at stalled forks including promoting its restart. ATAD5 depletion increases genomic instability upon hydroxyurea treatment in cultured cells and mice. ATAD5 recruits RAD51 to stalled forks in an ATR kinase-dependent manner by hydroxyurea-enhanced protein-protein interactions and timely removes PCNA from stalled forks for RAD51 recruitment. Consistent with the role of RAD51 in fork regression, ATAD5 depletion inhibits slowdown of fork progression and native 5-bromo-2ʹ-deoxyuridine signal induced by hydroxyurea. Single-molecule FRET showed that PCNA itself acts as a mechanical barrier to fork regression. Consequently, DNA breaks required for fork restart are reduced by ATAD5 depletion. Collectively, our results suggest an important role of ATAD5 in maintaining genome integrity during replication stress.

scholarly journals Strategies to improve the fluorescent signal of the tripartite sfGFP system

2020 ◽  
Vol 52 (9) ◽  
pp. 998-1006
Author(s):  
Jing Shen ◽  
Wenlu Zhang ◽  
Chunyang Gan ◽  
Xiafei Wei ◽  
Jie Li ◽  
...  
Keyword(s):  
Protein Interactions ◽  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Bimolecular Fluorescence Complementation ◽  
Fluorescence Signal ◽  
Recovery Efficiency ◽  
Good Recovery ◽  
Protein Protein Interactions ◽  
Bifc Assay ◽  
Green Fluorescent

Abstract Bimolecular fluorescence complementation (BiFC) is a popular method used to detect protein–protein interactions. For a BiFC assay, a fluorescent protein is usually split into two parts, and the fluorescence is recovered upon the interaction between the fused proteins of interest. As an elegant extension of BiFC, a tripartite superfold green fluorescent protein (sfGFP) system that has the advantages of low background fluorescence and small fusion tag size has been developed. However, the tripartite system exhibits a low fluorescence signal in some cases. To address this problem, we proposed to increase the affinity between the two parts, G1–9 and G11, of the tripartite system by adding affinity pairs. Among the three affinity pairs tested, LgBiT-HiBiT improved both the signal and signal-to-noise (S/N) ratio to the greatest extent. More strikingly, the direct covalent fusion of G11 to G1–9, which converted the tripartite system into a new bipartite system, enhanced the S/N ratio from 20 to 146, which is superior to the bipartite sfGFP system split at 157/158 or 173/174. Our results implied that the 10th β-strand of sfGFP has a low affinity and a good recovery efficiency to construct a robust BiFC system, and this concept might be applied to other fluorescent proteins with similar structure to construct new BiFC systems.

scholarly journals Development of a Split Esterase 1 for Protein-Protein 2 Interaction Dependent Small Molecule Activation

2019 ◽  
Author(s):  
Krysten A. Jones ◽  
Kaitlin Kentala ◽  
Michael Beck ◽  
Weiwei An ◽  
Alexander Lippert ◽  
...  
Keyword(s):  
Synthetic Biology ◽  
Small Molecule ◽  
Protein Interactions ◽  
Simultaneous Detection ◽  
Cell Killing ◽  
Dependent Manner ◽  
Protein Protein Interactions ◽  
Biological Studies ◽  
Cytotoxic Molecule ◽  
Measured Input

Split reporters based on fluorescent proteins and luciferases have emerged as valuable tools for measuring interactions in biological systems. Relatedly, biosensors that transduce measured input signals into outputs that influence the host system are key components of engineered gene circuits for synthetic biology applications. While small molecule-based imaging agents are widely used in biological studies, and small molecule-based drugs and chemical probes can target a range of biological processes, a general method for generating a target small molecule in a biological system based on a measured input signal is lacking. Here, we develop a proximity-dependent split esterase that selectively unmasks ester-protected small molecules in an interaction-dependent manner. Exploiting the versatility of an ester-protected small molecule output, we demonstrate fluorescent, chemiluminescent, and pharmacological probe generation, each created by masking key alcohol functional groups on a target small molecule. We show the split esterase system can be used in combination with ester-masked fluorescent or luminescent probes to measure a protein-protein interactions and protein-protein interaction inhibitor engagement. We demonstrate the esterase-based reporter system is compatible with other commonly-used split reporter imaging systems for the simultaneous detection of multiple protein-protein interactions. Finally, we develop a system for selective small molecule-dependent cell killing by unmasking a cytotoxic molecule using an inducible split esterase. Presaging utility in future synthetic biology-based therapeutic applications, we also show the system can be used for intercellular cell killing via a bystander effect, where one activated cell unmasks a cytotoxic molecule and kills cells physically adjacent to the activated cells. Collectively, this work illustrates that the split esterase system is a valuable new addition to the split protein toolbox, with particularly exciting potential in synthetic biology applications.

scholarly journals Development of a Split Esterase for Protein-Protein Interaction Dependent Small Molecule Activation

2019 ◽  
Author(s):  
Krysten A. Jones ◽  
Kaitlin Kentala ◽  
Michael Beck ◽  
Weiwei An ◽  
Alexander Lippert ◽  
...  
Keyword(s):  
Synthetic Biology ◽  
Small Molecule ◽  
Protein Interaction ◽  
Protein Interactions ◽  
Cell Killing ◽  
Dependent Manner ◽  
Protein Protein Interactions ◽  
Protein Protein Interaction ◽  
Cytotoxic Molecule ◽  
Measured Input

Split reporters based on fluorescent proteins and luciferases have emerged as valuable tools for measuring interactions in biological systems. Relatedly, biosensors that transduce measured input signals into outputs that influence the host system are key components of engineered gene circuits for synthetic biology applications. While small molecule-based imaging agents are widely used in biological studies, and small molecule-based drugs and chemical probes can target a range of biological processes, a general method for generating a target small molecule in a biological system based on a measured input signal is lacking. Here, we develop a proximity-dependent split esterase that selectively unmasks ester-protected small molecules in an interaction-dependent manner. Exploiting the versatility of an ester-protected small molecule output, we demonstrate fluorescent, chemiluminescent, and pharmacological probe generation, each created by masking key alcohol functional groups on a target small molecule. We show the split esterase system can be used in combination with ester-masked fluorescent or luminescent probes to measure a protein-protein interactions and protein-protein interaction inhibitor engagement. We demonstrate the esterase-based reporter system is compatible with other commonly-used split reporter imaging systems for the simultaneous detection of multiple protein-protein interactions. Finally, we develop a system for selective small molecule-dependent cell killing by unmasking a cytotoxic molecule using an inducible split esterase. Presaging utility in future synthetic biology-based therapeutic applications, we also show the system can be used for intercellular cell killing via a bystander effect, where one activated cell unmasks a cytotoxic molecule and kills cells physically adjacent to the activated cells. Collectively, this work illustrates that the split esterase system is a valuable new addition to the split protein toolbox, with particularly exciting potential in synthetic biology applications.

scholarly journals Transcription of Repeats Activates INterferon (TRAIN) in response to chromatin destabilization induced with anti-cancer small molecule

2017 ◽  
Author(s):  
Katerina Leonova ◽  
Alfiya Safina ◽  
Elimelech Nesher ◽  
Poorva Sandlesh ◽  
Rachel Pratt ◽  
...  
Keyword(s):  
Small Molecule ◽  
Interferon Response ◽  
Drug Candidate ◽  
Type I ◽  
Dependent Manner ◽  
Dna Hypomethylation ◽  
Deacetylase Inhibitor ◽  
Dna Elements ◽  
A Cell ◽  
Nf Kappab

AbstractThe anticancer activity of genotoxic agents has been intensively studied, while the mechanisms of action of drugs destabilizing the epigenome are far less understood. We previously found that DNA hypomethylation in the absence of p53 leads to transcriptional desilencing of repetitive DNA elements, such as pericentromeric repeats and endogenous retroelements, which is associated with an interferon type I response, a phenomenon we named TRAIN (Transcription ofRepeatsActivatesINterferon). Here, we report that curaxin, a small molecule anticancer drug candidate, which destabilizes nucleosomes via disruption of histone/DNA interactions, can induce TRAIN independently of the p53 status of a cell. Furthermore, curaxin inhibits oncogene-induced transformation in an interferon-dependent manner, suggesting that cancer prevention by curaxin, previously attributed to its p53-activating and NF-kappaB-inhibiting activities, may also involve the induction of the interferon response to epigenetic derepression of the cellular “repeatome.” Moreover, we observed that another type of drugs decondensing chromatin, histone deacetylase inhibitor, also induces TRAIN. Thus, we proposed that TRAIN may be one of the mechanisms ensuring epigenetic integrity of cells via elimination of cells with desilenced chromatin.

A calcineurin B-like protein participates in low oxygen signalling in rice

2017 ◽  
Vol 44 (9) ◽  
pp. 917 ◽  
Author(s):  
Viet The Ho ◽  
Anh Nguyet Tran ◽  
Francesco Cardarelli ◽  
Pierdomenico Perata ◽  
Chiara Pucciariello
Keyword(s):  
Fluorescence Recovery After Photobleaching ◽  
Ruthenium Red ◽  
Bimolecular Fluorescence Complementation ◽  
Starch Degradation ◽  
Dependent Manner ◽  
Low Oxygen ◽  
Interacting Protein ◽  
Bifc Assay ◽  
Calcineurin B ◽  
Downstream Effectors

Following the identification of the calcineurin B-like interacting protein kinase 15 (CIPK15), which is a regulator of starch degradation, the low O2 signal elicited during rice germination under submergence has been linked to the sugar sensing cascade and calcium (Ca2+) signalling. CIPK proteins are downstream effectors of calcineurin B-like proteins (CBLs), which act as Ca2+ sensors, whose role under low O2 has yet to be established. In the present study we describe CBL4 as a putative CIPK15 partner, transcriptionally activated under low O2 in rice coleoptiles. The transactivation of the rice embryo CBL4 transcript and CBL4 promoter was influenced by the Ca2+ blocker ruthenium red (RR). The bimolecular fluorescence complementation (BiFC) assay associated to fluorescence recovery after photobleaching (FRAP) analysis confirmed that CBL4 interacts with CIPK15. The CBL4-CIPK15 complex is localised in the cytoplasm and the plasma-membrane. Experiments in protoplasts showed a dampening of α-amylase 3 (RAMY3D) expression after CBL4 silencing by artificial miRNA. Our results suggest that under low O2, the Ca2+ sensor CBL4 interacts with CIPK15 to regulate RAMY3D expression in a Ca2+-dependent manner.

scholarly journals Two dynamin-like proteins stabilize FtsZ rings duringStreptomycessporulation

2017 ◽  
Vol 114 (30) ◽  
pp. E6176-E6183 ◽  
Author(s):  
Susan Schlimpert ◽  
Sebastian Wasserstrom ◽  
Govind Chandra ◽  
Maureen J. Bibb ◽  
Kim C. Findlay ◽  
...  
Keyword(s):  
Cell Division ◽  
Protein Interactions ◽  
Time Lapse ◽  
Dependent Manner ◽  
Protein Protein Interactions ◽  
Cellular Processes ◽  
Organelle Division ◽  
Z Ring ◽  
A Cell ◽  
Massive Cell

During sporulation, the filamentous bacteriaStreptomycesundergo a massive cell division event in which the synthesis of ladders of sporulation septa convert multigenomic hyphae into chains of unigenomic spores. This process requires cytokinetic Z-rings formed by the bacterial tubulin homolog FtsZ, and the stabilization of the newly formed Z-rings is crucial for completion of septum synthesis. Here we show that two dynamin-like proteins, DynA and DynB, play critical roles in this process. Dynamins are a family of large, multidomain GTPases involved in key cellular processes in eukaryotes, including vesicle trafficking and organelle division. Many bacterial genomes encode dynamin-like proteins, but the biological function of these proteins has remained largely enigmatic. Using a cell biological approach, we show that the twoStreptomycesdynamins specifically localize to sporulation septa in an FtsZ-dependent manner. Moreover, dynamin mutants have a cell division defect due to the decreased stability of sporulation-specific Z-rings, as demonstrated by kymographs derived from time-lapse images of FtsZ ladder formation. This defect causes the premature disassembly of individual Z-rings, leading to the frequent abortion of septum synthesis, which in turn results in the production of long spore-like compartments with multiple chromosomes. Two-hybrid analysis revealed that the dynamins are part of the cell division machinery and that they mediate their effects on Z-ring stability during developmentally controlled cell division via a network of protein–protein interactions involving DynA, DynB, FtsZ, SepF, SepF2, and the FtsZ-positioning protein SsgB.

scholarly journals S-Nitrosylation in Tumor Microenvironment

2021 ◽  
Vol 22 (9) ◽  
pp. 4600
Author(s):  
Vandana Sharma ◽  
Veani Fernando ◽  
Joshua Letson ◽  
Yashna Walia ◽  
Xunzhen Zheng ◽  
...  
Keyword(s):  
Tumor Microenvironment ◽  
Cancer Progression ◽  
Protein Interactions ◽  
Dependent Manner ◽  
Protein Protein Interactions ◽  
Post Translational Modification ◽  
Tissue Microenvironment ◽  
Cell Tissue ◽  
Protein Thiols ◽  
A Cell

S-nitrosylation is a selective and reversible post-translational modification of protein thiols by nitric oxide (NO), which is a bioactive signaling molecule, to exert a variety of effects. These effects include the modulation of protein conformation, activity, stability, and protein-protein interactions. S-nitrosylation plays a central role in propagating NO signals within a cell, tissue, and tissue microenvironment, as the nitrosyl moiety can rapidly be transferred from one protein to another upon contact. This modification has also been reported to confer either tumor-suppressing or tumor-promoting effects and is portrayed as a process involved in every stage of cancer progression. In particular, S-nitrosylation has recently been found as an essential regulator of the tumor microenvironment (TME), the environment around a tumor governing the disease pathogenesis. This review aims to outline the effects of S-nitrosylation on different resident cells in the TME and the diverse outcomes in a context-dependent manner. Furthermore, we will discuss the therapeutic potentials of modulating S-nitrosylation levels in tumors.

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