scholarly journals Cooperative stabilisation of 14-3-3σ protein–protein interactions via covalent protein modification

2021 ◽  
Vol 12 (39) ◽  
pp. 12985-12992
Author(s):  
Marta Falcicchio ◽  
Jake A. Ward ◽  
Sara Y. Chothia ◽  
Jaswir Basran ◽  
Alisha Mohindra ◽  
...  
Keyword(s):  
Cancer Cells ◽  
Protein Interactions ◽  
Protein Modification ◽  
Protein Protein Interactions ◽  
Partner Protein ◽  
Fusicoccin A

The aminothiol WR-1065 covalently modifies 14-3-3σ to stabilse its interactions with p53 and ERα. It enhances the effect of fusicoccin A via a cooperative mechanism that leads to 14-3-3 partner-protein specific activty against cancer cells.

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 Cytosolic localization and in vitro assembly of human de novo thymidylate synthesis complex

2020 ◽  
Author(s):  
Sharon Spizzichino ◽  
Dalila Boi ◽  
Giovanna Boumis ◽  
Roberta Lucchi ◽  
Francesca R. Liberati ◽  
...  
Keyword(s):  
Cancer Cells ◽  
Protein Interactions ◽  
De Novo ◽  
Proximity Ligation Assay ◽  
Protein Protein Interactions ◽  
Aggregation State ◽  
Entire Complex ◽  
The Individual ◽  
Thymidylate Synthesis

ABSTRACTDe novo thymidylate synthesis is a crucial pathway for normal and cancer cells. Deoxythymidine monophosphate (dTMP) is synthesized by the combined action of three enzymes: thymidylate synthase (TYMS), serine hydroxymethyltransferase (SHMT) and dihydrofolate reductase (DHFR), targets of widely used chemotherapeutics such as antifolates and 5-fluorouracil. These proteins translocate to the nucleus after SUMOylation and are suggested to assemble in this compartment into the thymidylate synthesis complex (dTMP-SC). We report the intracellular dynamics of the complex in lung cancer cells by in situ proximity ligation assay, showing that it is also detected in the cytoplasm. We have successfully assembled the dTMP synthesis complex in vitro, employing tetrameric SHMT1 and a bifunctional chimeric enzyme comprising human TYMS and DHFR. We show that the SHMT1 tetrameric state is required for efficient complex assembly, indicating that this aggregation state is evolutionary selected in eukaryotes to optimize protein-protein interactions. Lastly, our results on the activity of the complete thymidylate cycle in vitro, provide a useful tool to develop drugs targeting the entire complex instead of the individual components.

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 The Crystal Structure of Klebsiella pneumoniae FeoA Reveals a Site For Protein-Protein Interactions

2019 ◽  
Author(s):  
Richard O. Linkous ◽  
Alexandrea E. Sestok ◽  
Aaron T. Smith
Keyword(s):  
Crystal Structure ◽  
Klebsiella Pneumoniae ◽  
Protein Interactions ◽  
Pathogenic Bacteria ◽  
Protein Protein Interactions ◽  
Anaerobic Conditions ◽  
Small Peptide ◽  
Host Environment ◽  
Partner Protein ◽  
A Site

ABSTRACTIn order to establish infection, pathogenic bacteria must obtain essential nutrients such as iron. Under acidic and/or anaerobic conditions, most bacteria utilize the Feo system in order to acquire ferrous iron (Fe2+) from their host environment. The mechanism of this process, including its regulation, remains poorly understood. In this work, we have determined the crystal structure of FeoA from the nosocomial agent Klebsiella pneumoniae (KpFeoA). Our structure reveals an SH3-like domain that mediates interactions between neighboring polypeptides via intercalations into a Leu zipper motif. Using docking of a small peptide corresponding to a postulated FeoB partner binding site, we demonstrate the KpFeoA can assume both ‘open’ and ‘closed’ conformations, controlled by peptide binding. We propose a model in which a ‘C-shaped’ clamp along the FeoA surface mediates interactions with its partner protein, FeoB. These findings are the first to demonstrate atomic-level details of FeoA-based protein-protein interactions, which could be exploited for future antibiotic developments.

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 A High-Throughput Solid-Phase Microplate Protein-Binding Assay to Investigate Interactions between Myofilament Proteins

2011 ◽  
Vol 2011 ◽  
pp. 1-8 ◽  
Author(s):  
Brandon J. Biesiadecki ◽  
J.-P. Jin
Keyword(s):  
Protein Binding ◽  
Protein Interactions ◽  
Protein Modification ◽  
Binding Assay ◽  
Solid Phase ◽  
Regulatory Protein ◽  
Protein Isoforms ◽  
Protein Protein Interactions ◽  
Protein Binding Assay ◽  
Phase Protein

To understand the structure-function relationship of muscle-regulatory-protein isoforms, mutations, and posttranslational modifications, it is necessary to probe functional effects at the level of the protein-protein interaction. Traditional methodologies assessing such protein-protein interactions are laborious and require significant amounts of purified protein, while many current methodologies require costly and specialized equipment or modification of the proteins, which may affect their interaction. To address these issues, we developed a novel method of microplate-based solid-phase protein-binding assay over the recent years. This method assesses specific protein-protein interactions at physiological conditions, utilizes relatively small amounts of protein, is free of protein modification, and does not require specialized instrumentation. Here we present detailed methodology for the solid-phase protein-binding assay with examples that we have successfully applied to quantify interactions of myofilament-regulatory proteins. We further provide considerations for optimization of the assay conditions and its broader application in studies of other protein-protein interactions.

scholarly journals BIRC3 and BIRC5: multi‐faceted inhibitors in cancer

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Raffaele Frazzi
Keyword(s):  
Cancer Cells ◽  
Protein Interactions ◽  
Point Mutations ◽  
Therapeutic Targets ◽  
Progression Free Survival ◽  
Lymphocytic Leukemia ◽  
Low Grade ◽  
Main Body ◽  
Protein Protein Interactions ◽  
Smac Mimetics

Abstract Background The evasion from apoptosis is a common strategy adopted by most tumors, and inhibitors of apoptosis proteins (IAPs) are among the most studied molecular and therapeutic targets. BIRC3 (cellular IAP2) and BIRC5 (survivin) are two of the eight members of the human IAPs family. This family is characterized by the presence of the baculoviral IAP repeat (BIR) domains, involved in protein-protein interactions. In addition to the BIR domains, IAPs also contain other important domains like the C-terminal ubiquitin-conjugating (UBC) domain, the caspase recruitment (CARD) domain and the C-terminal Ring zinc-finger (RING) domain. Main body BIRC3 and BIRC5 have been characterized in some solid and hematological tumors and are therapeutic targets for the family of drugs called “Smac mimetics”. Many evidences point to the pro-survival and antiapoptotic role of BIRC3 in cancer cells, however, not all the data are consistent and the resulting picture is heterogeneous. For instance, BIRC3 genetic inactivation due to deletions or point mutations is consistently associated to shorter progression free survival and poor prognosis in chronic lymphocytic leukemia patients. BIRC3 inactivation has also been associated to chemoimmunotherapy resistance. On the contrary, the progression from low grade gliomas to high grade gliomas is accompanied by BIRC3 expression increase, which bears relevant prognostic consequences. Due to the relationship between BIRC3, MAP3K14 and the non-canonical NF-kB pathway, BIRC3 inactivation bears consequences also on the tumor cells relying on NF-kB pathway to survive. BIRC5, on the contrary, is commonly considered an anti-apoptotic molecule, promoting cell division and tumor progression and it is widely regarded as potential therapeutic target. Conclusions The present manuscript collects and reviews the most recent literature concerning the role played by BIRC3 and BIRC5 in cancer cells, providing useful information for the choice of the best therapeutic targets.

scholarly journals Designed nanomolar small-molecule inhibitors of Ena/VASP EVH1 interaction impair invasion and extravasation of breast cancer cells

2020 ◽  
Vol 117 (47) ◽  
pp. 29684-29690
Author(s):  
Matthias Barone ◽  
Matthias Müller ◽  
Slim Chiha ◽  
Jiang Ren ◽  
Dominik Albat ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Cancer Cells ◽  
Protein Interactions ◽  
Breast Cancer Cells ◽  
Cancer Metastasis ◽  
Cancer Therapies ◽  
Protein Protein Interactions ◽  
Zebrafish Model ◽  
Therapeutic Concept ◽  
Protein Protein Interaction

Battling metastasis through inhibition of cell motility is considered a promising approach to support cancer therapies. In this context, Ena/VASP-depending signaling pathways, in particular interactions with their EVH1 domains, are promising targets for pharmaceutical intervention. However, protein–protein interactions involving proline-rich segments are notoriously difficult to address by small molecules. Hence, structure-based design efforts in combination with the chemical synthesis of additional molecular entities are required. Building on a previously developed nonpeptidic micromolar inhibitor, we determined 22 crystal structures of ENAH EVH1 in complex with inhibitors and rationally extended our library of conformationally defined proline-derived modules (ProMs) to succeed in developing a nanomolar inhibitor (Kd=120 nM,MW=734Da). In contrast to the previous inhibitor, the optimized compounds reduced extravasation of invasive breast cancer cells in a zebrafish model. This study represents an example of successful, structure-guided development of low molecular weight inhibitors specifically and selectively addressing a proline-rich sequence-recognizing domain that is characterized by a shallow epitope lacking defined binding pockets. The evolved high-affinity inhibitor may now serve as a tool in validating the basic therapeutic concept, i.e., the suppression of cancer metastasis by inhibiting a crucial protein–protein interaction involved in actin filament processing and cell migration.

How Cancer Cells Resist Chemotherapy: Design and Development of Drugs Targeting Protein-Protein Interactions

2019 ◽  
Vol 19 (6) ◽  
pp. 394-412 ◽  
Author(s):  
Vadim V. Tarasov ◽  
Vladimir N. Chubarev ◽  
Ghulam Md Ashraf ◽  
Samira A. Dostdar ◽  
Alexander V. Sokolov ◽  
...  
Keyword(s):  
Drug Resistance ◽  
Clinical Outcome ◽  
Cancer Cells ◽  
Protein Interactions ◽  
Resistance Mechanisms ◽  
Protein Protein Interactions ◽  
Treatment Regimens ◽  
Pubmed Search ◽  
Bona Fide ◽  
Improve Clinical Outcome

Background:Resistance toward chemotherapeutics is one of the main obstacles on the way to effective cancer treatment. Personalization of chemotherapy could improve clinical outcome. However, despite preclinical significance, most of the potential markers have failed to reach clinical practice partially due to the inability of numerous studies to estimate the marker’s impact on resistance properly.Objective:The analysis of drug resistance mechanisms to chemotherapy in cancer cells, and the proposal of study design to identify bona fide markers.Methods:A review of relevant papers in the field. A PubMed search with relevant keywords was used to gather the data. An example of a search request: drug resistance AND cancer AND paclitaxel.Results:We have described a number of drug resistance mechanisms to various chemotherapeutics, as well as markers to underlie the phenomenon. We also proposed a model of a rational-designed study, which could be useful in determining the most promising potential biomarkers.Conclusion:Taking into account the most reasonable biomarkers should dramatically improve clinical outcome by choosing the suitable treatment regimens. However, determining the leading biomarkers, as well as validating of the model, is a work for further investigations.

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