scholarly journals Chemical profiling of DNA G-quadruplex-interacting proteins in live cells

2021 ◽  
Author(s):  
Xiaoyun Zhang ◽  
Jochen Spiegel ◽  
Sergio Martínez Cuesta ◽  
Santosh Adhikari ◽  
Shankar Balasubramanian
Keyword(s):  
Protein Interactions ◽  
Protein Profiling ◽  
Live Cells ◽  
Interacting Proteins ◽  
Chemical Profiling ◽  
G Quadruplex ◽  
Functional Classes ◽  
Chemical Strategy

AbstractDNA–protein interactions regulate critical biological processes. Identifying proteins that bind to specific, functional genomic loci is essential to understand the underlying regulatory mechanisms on a molecular level. Here we describe a co-binding-mediated protein profiling (CMPP) strategy to investigate the interactome of DNA G-quadruplexes (G4s) in native chromatin. CMPP involves cell-permeable, functionalized G4-ligand probes that bind endogenous G4s and subsequently crosslink to co-binding G4-interacting proteins in situ. We first showed the robustness of CMPP by proximity labelling of a G4 binding protein in vitro. Employing this approach in live cells, we then identified hundreds of putative G4-interacting proteins from various functional classes. Next, we confirmed a high G4-binding affinity and selectivity for several newly discovered G4 interactors in vitro, and we validated direct G4 interactions for a functionally important candidate in cellular chromatin using an independent approach. Our studies provide a chemical strategy to map protein interactions of specific nucleic acid features in living cells.

An organic molecular compound for in-situ identification of mitochondrial G-quadruplexes in live cells

2022 ◽  
Author(s):  
Xiaomeng Guo ◽  
Hongbo Chen ◽  
Yan Liu ◽  
Dawei Yang ◽  
Qian Li ◽  
...  
Keyword(s):  
Mitochondrial Gene ◽  
Critical Role ◽  
Diagnosis And Treatment ◽  
Live Cells ◽  
Molecular Compound ◽  
Gene Replication ◽  
Promising Target ◽  
G Quadruplex

Emerging studies have shown that mitochondrial G-quadruplex plays a critical role in regulating mitochondrial gene replication and transcription, which makes it a promising target for the diagnosis and treatment of...

scholarly journals Assay Systems for Profiling Deubiquitinating Activity

2020 ◽  
Vol 21 (16) ◽  
pp. 5638
Author(s):  
Jinhong Cho ◽  
Jinyoung Park ◽  
Eunice EunKyeong Kim ◽  
Eun Joo Song
Keyword(s):  
Protein Degradation ◽  
Protein Interactions ◽  
Live Cells ◽  
Deubiquitinating Enzyme ◽  
Protein Protein Interactions ◽  
Deubiquitinating Enzymes ◽  
Cell Lysates ◽  
Cellular Processes

Deubiquitinating enzymes regulate various cellular processes, particularly protein degradation, localization, and protein–protein interactions. The dysregulation of deubiquitinating enzyme (DUB) activity has been linked to several diseases; however, the function of many DUBs has not been identified. Therefore, the development of methods to assess DUB activity is important to identify novel DUBs, characterize DUB selectivity, and profile dynamic DUB substrates. Here, we review various methods of evaluating DUB activity using cell lysates or purified DUBs, as well as the types of probes used in these methods. In addition, we introduce some techniques that can deliver DUB probes into the cells and cell-permeable activity-based probes to directly visualize and quantify DUB activity in live cells. This review could contribute to the development of DUB inhibitors by providing important information on the characteristics and applications of various probes used to evaluate and detect DUB activity in vitro and in vivo.

Functional Display of Bioactive Peptides on the vGFP Scaffold.

2021 ◽  
Author(s):  
Sharon Min Qi Chee ◽  
Jantana Wongsantichon ◽  
Sze Yi Lau ◽  
Barindra Sana ◽  
Yuri Frosi ◽  
...  
Keyword(s):  
Protein Interactions ◽  
Bioactive Peptides ◽  
Negative Regulator ◽  
Live Cells ◽  
Structural Studies ◽  
Protein Protein Interactions ◽  
Target Interaction ◽  
High Affinity ◽  
Novel Scaffold

Abstract Grafting bioactive peptides into recipient protein scaffolds can often increase their activities by conferring enhanced stability and cellular longevity. Here, we describe use of vGFP as a novel scaffold to display peptides. vGFP comprises GFP fused to a bound high affinity Enhancer nanobody that potentiates its fluorescence. We show that peptides inserted into the linker region between GFP and the Enhancer are correctly displayed for on-target interaction, both in vitro and in live cells by pull-down, measurement of target inhibition and imaging analyses. This is further confirmed by structural studies highlighting the optimal display of a vGFP-displayed peptide bound to Mdm2, the key negative regulator of p53 that is often overexpressed in cancer. We also demonstrate a potential biosensing application of the vGFP scaffold by showing target-dependent modulation of intrinsic fluorescence. vGFP is relatively thermostable, well-expressed and inherently fluorescent. These properties make it a useful scaffold to add to the existing tool box for displaying peptides that can disrupt clinically relevant protein-protein interactions.

Autoactivation of matriptase in vitro: requirement for biomembrane and LDL receptor domain

2007 ◽  
Vol 293 (1) ◽  
pp. C95-C105 ◽  
Author(s):  
Ming-Shyue Lee ◽  
I-Chu Tseng ◽  
Youhong Wang ◽  
Ken-ichi Kiyomiya ◽  
Michael D. Johnson ◽  
...  
Keyword(s):  
Protein Interactions ◽  
Ldl Receptor ◽  
Subcellular Fractionation ◽  
Live Cells ◽  
Protein Protein Interactions ◽  
Hepatocyte Growth Factor Activator ◽  
Domain 3 ◽  
Ph Range ◽  
Receptor Domain

In live cells, autoactivation of matriptase, a membrane-bound serine protease, can be induced by lysophospholipids, androgens, and the polyanionic compound suramin. These structurally distinct chemicals induce different signaling pathways and cellular events that somehow, in a cell type-specific manner, lead to activation of matriptase immediately followed by inhibition of matriptase by hepatocyte growth factor activator inhibitor 1 (HAI-1). In the current study, we established an analogous matriptase autoactivation system in an in vitro cell-free setting and showed that a burst of matriptase activation and HAI-1-mediated inhibition spontaneously occurred in the insoluble fractions of cell homogenates and that this in vitro activation could be attenuated by a soluble suppressive factor(s) in cytosolic fractions. Immunofluorescence staining and subcellular fractionation studies revealed that matriptase activation occurred in the perinuclear regions. Solubilization of matriptase from cell homogenates by Triton X-100 or sonication of cell homogenates completely inhibited the effect, suggesting that matriptase activation requires proper lipid bilayer microenvironments, potentially allowing appropriate interactions of matriptase zymogens with HAI-1 and other components. Matriptase activation occurred in a narrow pH range (from pH 5.2 to 7.2), with a sharp increase in activation at the transition from pH 5.2 to 5.4, and could be completely suppressed by moderately increased ionic strength. Protease inhibitors only modestly affected activation, whereas 30 nM (5 μg/ml) of anti-matriptase LDL receptor domain 3 monoclonal antibodies completely blocked activation. These atypical biochemical features are consistent with a mechanism for autoactivation of matriptase that requires protein-protein interactions but not active proteases.

scholarly journals Harnessing molecular motors for nanoscale pulldown in live cells

2017 ◽  
Vol 28 (3) ◽  
pp. 463-475 ◽  
Author(s):  
Jonathan E. Bird ◽  
Melanie Barzik ◽  
Meghan C. Drummond ◽  
Daniel C. Sutton ◽  
Spencer M. Goodman ◽  
...  
Keyword(s):  
Protein Interactions ◽  
Molecular Motors ◽  
Protein Complexes ◽  
Software Tool ◽  
Epifluorescence Microscopy ◽  
Live Cells ◽  
Data Sets ◽  
Domain Mapping ◽  
Myosin Motors

Protein–protein interactions (PPIs) regulate assembly of macromolecular complexes, yet remain challenging to study within the native cytoplasm where they normally exert their biological effect. Here we miniaturize the concept of affinity pulldown, a gold-standard in vitro PPI interrogation technique, to perform nanoscale pulldowns (NanoSPDs) within living cells. NanoSPD hijacks the normal process of intracellular trafficking by myosin motors to forcibly pull fluorescently tagged protein complexes along filopodial actin filaments. Using dual-color total internal reflection fluorescence microscopy, we demonstrate complex formation by showing that bait and prey molecules are simultaneously trafficked and actively concentrated into a nanoscopic volume at the tips of filopodia. The resulting molecular traffic jams at filopodial tips amplify fluorescence intensities and allow PPIs to be interrogated using standard epifluorescence microscopy. A rigorous quantification framework and software tool are provided to statistically evaluate NanoSPD data sets. We demonstrate the capabilities of NanoSPD for a range of nuclear and cytoplasmic PPIs implicated in human deafness, in addition to dissecting these interactions using domain mapping and mutagenesis experiments. The NanoSPD methodology is extensible for use with other fluorescent molecules, in addition to proteins, and the platform can be easily scaled for high-throughput applications.

scholarly journals Mapping the mammalian ribosome quality control complex interactome using proximity labeling approaches

2018 ◽  
Vol 29 (10) ◽  
pp. 1258-1269 ◽  
Author(s):  
Nathan Zuzow ◽  
Arit Ghosh ◽  
Marilyn Leonard ◽  
Jeffrey Liao ◽  
Bing Yang ◽  
...  
Keyword(s):  
Quality Control ◽  
Protein Interactions ◽  
Mammalian Cells ◽  
Interacting Proteins ◽  
S6 Kinase ◽  
Ribosomal Protein S6 ◽  
Independent Functions ◽  
Control Complex

Previous genetic and biochemical studies from Saccharomyces cerevisiae have identified a critical ribosome-associated quality control complex (RQC) that facilitates resolution of stalled ribosomal complexes. While components of the mammalian RQC have been examined in vitro, a systematic characterization of RQC protein interactions in mammalian cells has yet to be described. Here we utilize both proximity-labeling proteomic approaches, BioID and APEX, and traditional affinity-based strategies to both identify interacting proteins of mammalian RQC members and putative substrates for the RQC resident E3 ligase, Ltn1. Surprisingly, validation studies revealed that a subset of substrates are ubiquitylated by Ltn1 in a regulatory manner that does not result in subsequent substrate degradation. We demonstrate that Ltn1 catalyzes the regulatory ubiquitylation of ribosomal protein S6 kinase 1 and 2 (RPS6KA1, RPS6KA3). Further, loss of Ltn1 function results in hyperactivation of RSK1/2 signaling without impacting RSK1/2 protein turnover. These results suggest that Ltn1-mediated RSK1/2 ubiquitylation is inhibitory and establishes a new role for Ltn1 in regulating mitogen-activated kinase signaling via regulatory RSK1/2 ubiquitylation. Taken together, our results suggest that mammalian RQC interactions are difficult to observe and may be more transient than the homologous complex in S. cerevisiae and that Ltn1 has RQC-independent functions.

scholarly journals Functional display of bioactive peptides on the vGFP scaffold

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Sharon Min Qi Chee ◽  
Jantana Wongsantichon ◽  
Lau Sze Yi ◽  
Barindra Sana ◽  
Yuri Frosi ◽  
...  
Keyword(s):  
Protein Interactions ◽  
Bioactive Peptides ◽  
Negative Regulator ◽  
Live Cells ◽  
Structural Studies ◽  
Protein Protein Interactions ◽  
Target Interaction ◽  
High Affinity ◽  
Novel Scaffold

AbstractGrafting bioactive peptides into recipient protein scaffolds can often increase their activities by conferring enhanced stability and cellular longevity. Here, we describe use of vGFP as a novel scaffold to display peptides. vGFP comprises GFP fused to a bound high affinity Enhancer nanobody that potentiates its fluorescence. We show that peptides inserted into the linker region between GFP and the Enhancer are correctly displayed for on-target interaction, both in vitro and in live cells by pull-down, measurement of target inhibition and imaging analyses. This is further confirmed by structural studies highlighting the optimal display of a vGFP-displayed peptide bound to Mdm2, the key negative regulator of p53 that is often overexpressed in cancer. We also demonstrate a potential biosensing application of the vGFP scaffold by showing target-dependent modulation of intrinsic fluorescence. vGFP is relatively thermostable, well-expressed and inherently fluorescent. These properties make it a useful scaffold to add to the existing tool box for displaying peptides that can disrupt clinically relevant protein–protein interactions.

scholarly journals Nuclear membrane-tethered FRAP method for measuring protein complex off-rates in live cells

2021 ◽  
Author(s):  
Lindsey R. Pack ◽  
Leighton H. Daigh ◽  
Mingyu Chung ◽  
Tobias Meyer
Keyword(s):  
Protein Interactions ◽  
Nuclear Membrane ◽  
Protein Complex ◽  
Protein Complexes ◽  
Live Cells ◽  
Cdk Inhibitors ◽  
Cyclin Dependent Kinase ◽  
Protein Protein Interactions ◽  
The Stability

Abstract Understanding the stability or binding affinity of protein complex members is important for understanding their regulation and roles in cells. While there are many biochemical methods to measure protein-protein interactions in vitro, these methods often rely on the ability to robustly purify components individually. Moreover, few methods have been developed to study protein complexes within live cells. Binding parameters for cyclin-dependent kinase (CDK) complexes have been challenging to measure due to difficulty expressing and purifying CDKs separately from activating cyclins. Here, we develop a method to measure off-rates of protein complex components in live-cells. Our method relies on the stable tethering of CDK to the inner nuclear membrane (Figure 1), and the utilization of FRAP to measure the off-rate of soluble, fluorescently-tagged CDK binding proteins. We use this method to study dimeric CDK complexes, measuring the off-rates of cyclins or INK4 CDK inhibitor p16 from CDKs, and trimeric CDK complexes, measuring the off-rate of cyclins and CIP/KIP CDK inhibitors p21 and p27 when bound together.

Specific receptor for hydrazine: mapping the in situ release of hydrazine in live cells and in an in vitro enzymatic assay

2016 ◽  
Vol 52 (36) ◽  
pp. 6166-6169 ◽  
Author(s):  
Firoj Ali ◽  
Anila H. A. ◽  
Nandaraj Taye ◽  
Devraj G. Mogare ◽  
Samit Chattopadhyay ◽  
...  
Keyword(s):  
Hepg2 Cells ◽  
Enzymatic Assay ◽  
Live Cells ◽  
Specific Receptor ◽  
Specific Detection ◽  
Physiological Conditions

New chemodosimetric reagent for the specific detection of hydrazine in physiological conditions as well as for the mapping of its in situ generation in live Hct116 and HepG2 cells by enzymatic transformations.

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