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.

Probing the 14-3-3 Isoform-Specificity Profile of Interactions Stabilized by Fusicoccin A

2020 ◽  
Author(s):  
James Frederich ◽  
Ananya Sengupta ◽  
Josue Liriano ◽  
Ewa A. Bienkiewicz ◽  
Brian G. Miller
Keyword(s):  
Protein Interactions ◽  
Neurological Diseases ◽  
Adapter Proteins ◽  
Protein Protein Interactions ◽  
Specific Expression ◽  
Individual Client ◽  
Isoform Specificity ◽  
Fusicoccin A

Fusicoccin A (FC) is a fungal phytotoxin that stabilizes protein–protein interactions (PPIs) between 14-3-3 adapter proteins and their phosphoprotein interaction partners. In recent years, FC has emerged as an important chemical probe of human 14-3-3 PPIs implicated in cancer and neurological diseases. These previous studies have established the structural requirements for FC-induced stabilization of 14-3-3·client phosphoprotein complexes; however, the effect of different 14-3-3 isoforms on FC activity has not been systematically explored. This is a relevant question for the continued development of FC variants because there are seven distinct isoforms of 14-3-3 in humans. Despite their remarkable sequence and structural similarities, a growing body of experimental evidence supports both tissue-specific expression of 14-3-3 isoforms and isoform-specific functions <i>in vivo</i>. Herein, we report the isoform-specificity profile of FC <i>in vitro</i>using recombinant human 14-3-3 isoforms and a focused library of fluorescein-labeled hexaphosphopeptides mimicking the C-terminal 14-3-3 recognition domains of client phosphoproteins targeted by FC in cell culture. Our results reveal modest isoform preferences for individual client phospholigands and demonstrate that FC differentially stabilizes PPIs involving 14-3-3s. Together, these data provide strong motivation for the development of non-natural FC variants with enhanced selectivity for individual 14-3-3 isoforms.

scholarly journals Probing the 14-3-3 Isoform-Specificity Profile of Interactions Stabilized by Fusicoccin A

2020 ◽  
Author(s):  
James Frederich ◽  
Ananya Sengupta ◽  
Josue Liriano ◽  
Ewa A. Bienkiewicz ◽  
Brian G. Miller
Keyword(s):  
Protein Interactions ◽  
Neurological Diseases ◽  
Adapter Proteins ◽  
Protein Protein Interactions ◽  
Specific Expression ◽  
Individual Client ◽  
Isoform Specificity ◽  
Fusicoccin A

Fusicoccin A (FC) is a fungal phytotoxin that stabilizes protein–protein interactions (PPIs) between 14-3-3 adapter proteins and their phosphoprotein interaction partners. In recent years, FC has emerged as an important chemical probe of human 14-3-3 PPIs implicated in cancer and neurological diseases. These previous studies have established the structural requirements for FC-induced stabilization of 14-3-3·client phosphoprotein complexes; however, the effect of different 14-3-3 isoforms on FC activity has not been systematically explored. This is a relevant question for the continued development of FC variants because there are seven distinct isoforms of 14-3-3 in humans. Despite their remarkable sequence and structural similarities, a growing body of experimental evidence supports both tissue-specific expression of 14-3-3 isoforms and isoform-specific functions <i>in vivo</i>. Herein, we report the isoform-specificity profile of FC <i>in vitro</i>using recombinant human 14-3-3 isoforms and a focused library of fluorescein-labeled hexaphosphopeptides mimicking the C-terminal 14-3-3 recognition domains of client phosphoproteins targeted by FC in cell culture. Our results reveal modest isoform preferences for individual client phospholigands and demonstrate that FC differentially stabilizes PPIs involving 14-3-3s. Together, these data provide strong motivation for the development of non-natural FC variants with enhanced selectivity for individual 14-3-3 isoforms.

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 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.

Probing the 14-3-3 Isoform-Specificity Profile of Interactions Stabilized by Fusicoccin A

2020 ◽  
Author(s):  
James Frederich ◽  
Ananya Sengupta ◽  
Josue Liriano ◽  
Ewa A. Bienkiewicz ◽  
Brian G. Miller
Keyword(s):  
Protein Interactions ◽  
Neurological Diseases ◽  
Adapter Proteins ◽  
Protein Protein Interactions ◽  
Specific Expression ◽  
Individual Client ◽  
Isoform Specificity ◽  
Fusicoccin A

Fusicoccin A (FC) is a fungal phytotoxin that stabilizes protein–protein interactions (PPIs) between 14-3-3 adapter proteins and their phosphoprotein interaction partners. In recent years, FC has emerged as an important chemical probe of human 14-3-3 PPIs implicated in cancer and neurological diseases. These previous studies have established the structural requirements for FC-induced stabilization of 14-3-3·client phosphoprotein complexes; however, the effect of different 14-3-3 isoforms on FC activity has not been systematically explored. This is a relevant question for the continued development of FC variants because there are seven distinct isoforms of 14-3-3 in humans. Despite their remarkable sequence and structural similarities, a growing body of experimental evidence supports both tissue-specific expression of 14-3-3 isoforms and isoform-specific functions <i>in vivo</i>. Herein, we report the isoform-specificity profile of FC <i>in vitro</i>using recombinant human 14-3-3 isoforms and a focused library of fluorescein-labeled hexaphosphopeptides mimicking the C-terminal 14-3-3 recognition domains of client phosphoproteins targeted by FC in cell culture. Our results reveal modest isoform preferences for individual client phospholigands and demonstrate that FC differentially stabilizes PPIs involving 14-3-3s. Together, these data provide strong motivation for the development of non-natural FC variants with enhanced selectivity for individual 14-3-3 isoforms.

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 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.

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

eLife ◽  
2020 ◽  
Vol 9 ◽  
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

The 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.

scholarly journals Synthesis of Novel Suramin Analogs With Anti-Proliferative Activity via FGF1 and FGFRD2 Blockade

2022 ◽  
Vol 9 ◽  
Author(s):  
Nuzhat Parveen ◽  
Yan-Liang Lin ◽  
Ruey-Hwang Chou ◽  
Chung-Ming Sun ◽  
Chin Yu
Keyword(s):  
Cell Proliferation ◽  
Protein Interactions ◽  
Binding Sites ◽  
Proliferative Activity ◽  
Three Dimensional ◽  
Cancer Cell Line ◽  
Protein Protein Interactions ◽  
Docking Simulations ◽  
Three Dimensional Models

A promising approach in cancer therapy is the inhibition of cell proliferation using small molecules. In this study, we report the synthesis of suramin derivatives and their applications. We used NMR spectroscopy and docking simulations to confirm binding sites and three-dimensional models of the ligand-protein complex. The WST-1 assay was used to assess cell viability and cell proliferation in vitro to evaluate the inhibition of protein–protein interactions and to investigate the anti-proliferative activities in a breast cancer cell line. All the suramin derivatives showed anti-proliferative activity by blocking FGF1 binding to its receptor FGFRD2. The dissociation constant was measured by fluorescence spectroscopy. The suramin compound derivatives synthesized herein show potential as novel therapeutic agents for their anti-proliferative activity via the inhibition of protein–protein interactions. The cytotoxicity of these suramin derivatives was lower than that of the parent suramin compound, which may be considered a significant advancement in this field. Thus, these novel suramin derivatives may be considered superior anti-metastasis molecules than those of suramin.

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