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.

scholarly journals Interfacial Peptides as Affinity Modulating Agents of Protein-Protein Interactions

Biomolecules ◽  
2022 ◽  
Vol 12 (1) ◽  
pp. 106
Author(s):  
Pavel V. Ershov ◽  
Yuri V. Mezentsev ◽  
Alexis S. Ivanov
Keyword(s):  
Protein Interactions ◽  
Targeted Delivery ◽  
Protein Complexes ◽  
Protein Protein Interactions ◽  
Good Efficiency ◽  
Cellular Targets ◽  
Proteolytic Stability ◽  
Clinically Significant

The identification of disease-related protein-protein interactions (PPIs) creates objective conditions for their pharmacological modulation. The contact area (interfaces) of the vast majority of PPIs has some features, such as geometrical and biochemical complementarities, “hot spots”, as well as an extremely low mutation rate that give us key knowledge to influence these PPIs. Exogenous regulation of PPIs is aimed at both inhibiting the assembly and/or destabilization of protein complexes. Often, the design of such modulators is associated with some specific problems in targeted delivery, cell penetration and proteolytic stability, as well as selective binding to cellular targets. Recent progress in interfacial peptide design has been achieved in solving all these difficulties and has provided a good efficiency in preclinical models (in vitro and in vivo). The most promising peptide-containing therapeutic formulations are under investigation in clinical trials. In this review, we update the current state-of-the-art in the field of interfacial peptides as potent modulators of a number of disease-related PPIs. Over the past years, the scientific interest has been focused on following clinically significant heterodimeric PPIs MDM2/p53, PD-1/PD-L1, HIF/HIF, NRF2/KEAP1, RbAp48/MTA1, HSP90/CDC37, BIRC5/CRM1, BIRC5/XIAP, YAP/TAZ–TEAD, TWEAK/FN14, Bcl-2/Bax, YY1/AKT, CD40/CD40L and MINT2/APP.

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.

scholarly journals Evolutionary diversification of protein–protein interactions by interface add-ons

2017 ◽  
Vol 114 (40) ◽  
pp. E8333-E8342 ◽  
Author(s):  
Maximilian G. Plach ◽  
Florian Semmelmann ◽  
Florian Busch ◽  
Markus Busch ◽  
Leonhard Heizinger ◽  
...  
Keyword(s):  
Protein Interaction ◽  
Protein Interactions ◽  
Protein Complexes ◽  
Evolutionary Strategy ◽  
Structural Level ◽  
Protein Protein Interactions ◽  
Protein Protein Interaction ◽  
Evolutionary Diversification

Cells contain a multitude of protein complexes whose subunits interact with high specificity. However, the number of different protein folds and interface geometries found in nature is limited. This raises the question of how protein–protein interaction specificity is achieved on the structural level and how the formation of nonphysiological complexes is avoided. Here, we describe structural elements called interface add-ons that fulfill this function and elucidate their role for the diversification of protein–protein interactions during evolution. We identified interface add-ons in 10% of a representative set of bacterial, heteromeric protein complexes. The importance of interface add-ons for protein–protein interaction specificity is demonstrated by an exemplary experimental characterization of over 30 cognate and hybrid glutamine amidotransferase complexes in combination with comprehensive genetic profiling and protein design. Moreover, growth experiments showed that the lack of interface add-ons can lead to physiologically harmful cross-talk between essential biosynthetic pathways. In sum, our complementary in silico, in vitro, and in vivo analysis argues that interface add-ons are a practical and widespread evolutionary strategy to prevent the formation of nonphysiological complexes by specializing protein–protein interactions.

scholarly journals Flotillin mediated membrane fluidity controls peptidoglycan synthesis and MreB movement

2019 ◽  
Author(s):  
Aleksandra Zielińska ◽  
Abigail Savietto ◽  
Anabela de Sousa Borges ◽  
Denis Martinez ◽  
Melanie Berbon ◽  
...  
Keyword(s):  
Membrane Fluidity ◽  
Protein Interactions ◽  
Protein Complexes ◽  
Direct Control ◽  
Protein Protein Interactions ◽  
Cellular Compartment ◽  
Peptidoglycan Synthesis ◽  
Synthetic Proteins ◽  
Formation Of Complexes

AbstractThe bacterial plasma membrane is an important cellular compartment. In recent years it has become obvious that protein complexes and lipids are not uniformly distributed within membranes. Current hypotheses suggest that flotillin proteins are required for the formation of complexes of membrane proteins including cell-wall synthetic proteins. We show here that bacterial flotillins are important factors for membrane fluidity homeostasis. Loss of flotillins leads to a decrease in membrane fluidity that in turn leads to alterations in MreB dynamics and, as a consequence, in peptidoglycan synthesis. These alterations are reverted when membrane fluidity is restored by a chemical fluidizer. In vitro, the addition of a flotillin increases membrane fluidity of liposomes. Our data support a model in which flotillins are required for direct control of membrane fluidity rather than for the formation of protein complexes via direct protein-protein interactions.

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 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 Interactions of invadopodia scaffold protein TKS4 with intersectin family proteins

2019 ◽  
Vol 25 ◽  
pp. 126-130
Author(s):  
S. V. Kropyvko ◽  
T. A. Gryaznova ◽  
A. V. Rynditch
Keyword(s):  
Protein Interactions ◽  
Protein Complexes ◽  
Cancer Cell Line ◽  
Adaptor Proteins ◽  
Metastatic Potential ◽  
Adapter Proteins ◽  
Protein Protein Interactions ◽  
Membrane Structures ◽  
Active Forms Of Oxygen

Aim. TKS4 is one of the key proteins of invadopodies, specialized membrane structures that provide invasiveness and metastatic potential of malignant cells. This protein is a substrate for the tyrosine kinase SRC, which is involved in the formation of the membrane bends, affects cellular production of active forms of oxygen, interacts with membrane metallоproteinases causing degradation of the extracellular matrix, but its role in invasive structures has not yet been fully understood. Further study of molecular components of invadopodies and their specific interactions provides better understanding of mechanisms of cellular invasion. Methods. Protein-protein interactions were identified by in vitro precipitation of protein complexes using GST-fused proteins and co-immunoprecipitation. Results. Adapter proteins ITSN1 and ITSN2 are new partners of TKS4. Interactions between intersectins and TKS4 are mediated by the SH3A, SH3C and SH3E domains of ITSN1 or ITSN2. TKS4 phosphorylation on tyrosine residues does not affect interactions between TKS4 and intersectins. Conclusions. In this study, we have showed that TKS4 interacts with endocytic adaptor proteins of the intersectin family, ITSN1 and ITSN2. We have also demonstrated that TKS4 and ITSN1 can form a complex in invasive MDA-MB-231 breast cancer cell line. Keywords: TKS4, ITSN1, ITSN2, protein-protein interactions.

scholarly journals A cell-free approach to accelerate the study of protein–protein interactions in vitro

2013 ◽  
Vol 3 (5) ◽  
pp. 20130018 ◽  
Author(s):  
E. Sierecki ◽  
N. Giles ◽  
M. Polinkovsky ◽  
M. Moustaqil ◽  
K. Alexandrov ◽  
...  
Keyword(s):  
Drug Discovery ◽  
Single Molecule ◽  
Protein Interactions ◽  
Protein Complexes ◽  
Protein Interaction Networks ◽  
The Novel ◽  
Protein Protein Interactions ◽  
Single Molecule Fluorescence Spectroscopy ◽  
A Cell

Protein–protein interactions are highly desirable targets in drug discovery, yet only a fraction of drugs act as binding inhibitors. Here, we review the different technologies used to find and validate protein–protein interactions. We then discuss how the novel combination of cell-free protein expression, AlphaScreen and single-molecule fluorescence spectroscopy can be used to rapidly map protein interaction networks, determine the architecture of protein complexes, and find new targets for drug discovery.

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