scholarly journals Zebrafish xenografts to isolate unique human breast cancer metastatic cell populations

2021 ◽  
Author(s):  
Jerry Xiao ◽  
Joseph R. McGill ◽  
Apsra Nasir ◽  
Alexander Lekan ◽  
Bailey Johnson ◽  
...  
Keyword(s):  
Protein Interactions ◽  
Cancer Metastasis ◽  
Circulatory System ◽  
Cell Populations ◽  
Protein Protein Interactions ◽  
Zebrafish Model ◽  
Metastatic Cell ◽  
Metastatic Cells

Cancer metastasis is a critical culprit frequently blamed for treatment failure, drug resistance, poor prognosis, and high mortality rate among all human cancers. Laboratory efforts to isolate metastatic cell populations have typically been confined to mouse models, which are time-consuming and expensive. Here, we present a model system based on xenografting zebrafish embryos to select for cells that are predisposed to progress through the early stages of metastasis. This model requires only 3-5 days to achieve distinct intravasation to the zebrafish circulatory system. The metastatic cells are easily tracked in real-time as they migrate, as well as isolated and propagated in vitro. Once expanded, molecular characterization of the serially derived invasive cell populations from the tails of the zebrafish accurately predicts genes, signaling pathways, protein-protein interactions, and differential splicing products that are important for an invasive phenotype. This zebrafish model therefore offers a high-throughput and robust method for identifying gene targets critical for cancer metastasis.

scholarly journals Perturbation of BRMS1 interactome reveals pathways that impact cell migration

2021 ◽  
Author(s):  
Rosalyn Zimmermann ◽  
Mihaela E. Sardiu ◽  
Christa A. Manton ◽  
Md. Sayem Miah ◽  
Charles A.S. Banks ◽  
...  
Keyword(s):  
Cell Migration ◽  
Protein Interactions ◽  
Cancer Metastasis ◽  
Breast Cancer Metastasis ◽  
Metastasis Suppressor ◽  
Protein Protein Interactions ◽  
Multiple Cancer ◽  
C Terminus ◽  
Metastasis Suppression

AbstractBreast Cancer Metastasis Suppressor 1 (BRMS1) expression has been associated with longer patient survival in multiple cancer types. Understanding BRMS1 at the protein level will provide insights into both mechanism of action and enhance potential therapeutic development. We previously mapped the C-terminus of BRMS1 as critical for metastasis suppression and hypothesized that critical protein interactions in this region will explain function. These studies indicate that phosphorylation status at S237 regulates BRMS1 interactions related to a variety of biological processes, phenotypes [cell cycle (e.g., CDKN2A), DNA repair (e.g., BRCA1)], and metastasis [(e.g., TCF2 and POLE2)]. Presence of the C-terminal site appears to be critical for BRMS1 directed metastasis suppression, as demonstrated by in vitro migration assays. These assays demonstrated that presence of S237 directly decreased MDA-MB-231 migration. This study furthers our understanding of BRMS1’s molecular role, as it demonstrates that BRMS1 C-terminus is involved in direct protein-protein interactions. Several of the interacting proteins are associated with cancer and metastasis, which may result in metastasis suppression as suggested by in vitro findings.Abstract FigureGraphical AbstractUtilizing BRMS1 mutants to mimic-phosphorylation, this study demonstrates that S237-phosphorylation disrupts BRMS1 protein-protein interactions. The disruption includes both known Sin3/HDAC interactors as well as additionally previously unidentified Sin3-indepedent binding partners (indicated by increased opacity). It is revealed that BRMS1-phosphorylation status also more greatly inhibits cell migration (indicated by +) compared to the unphosphorylated state, suggesting that phosphorylation plays a role in BRMS1 metastatsis suppresion function, potentially though altered protein interactions.

scholarly journals Label-free methods for optical in vitro characterization of protein–protein interactions

2021 ◽  
Author(s):  
Fabian Soltermann ◽  
Weston B. Struwe ◽  
Philipp Kukura
Keyword(s):  
Protein Interactions ◽  
Label Free ◽  
Protein Protein Interactions ◽  
Cellular Processes ◽  
P Protein ◽  
In Vitro Characterization ◽  
And Function

Protein–protein interactions are involved in the regulation and function of the majority of cellular processes.

scholarly journals Structural and functional characterization of Rpn12 identifies residues required for Rpn10 proteasome incorporation

2012 ◽  
Vol 448 (1) ◽  
pp. 55-65 ◽  
Author(s):  
Jonas Boehringer ◽  
Christiane Riedinger ◽  
Konstantinos Paraskevopoulos ◽  
Eachan O. D. Johnson ◽  
Edward D. Lowe ◽  
...  
Keyword(s):  
Protein Interactions ◽  
Functional Characterization ◽  
Ubiquitin Proteasome System ◽  
26S Proteasome ◽  
Structural Motif ◽  
Protein Protein Interactions ◽  
Ubiquitin Proteasome

The ubiquitin–proteasome system targets selected proteins for degradation by the 26S proteasome. Rpn12 is an essential component of the 19S regulatory particle and plays a role in recruiting the extrinsic ubiquitin receptor Rpn10. In the present paper we report the crystal structure of Rpn12, a proteasomal PCI-domain-containing protein. The structure helps to define a core structural motif for the PCI domain and identifies potential sites through which Rpn12 might form protein–protein interactions. We demonstrate that mutating residues at one of these sites impairs Rpn12 binding to Rpn10 in vitro and reduces Rpn10 incorporation into proteasomes in vivo.

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.

scholarly journals Perturbation of BRMS1 interactome reveals pathways that impact metastasis

PLoS ONE ◽  
2021 ◽  
Vol 16 (11) ◽  
pp. e0259128
Author(s):  
Rosalyn C. Zimmermann ◽  
Mihaela E. Sardiu ◽  
Christa A. Manton ◽  
Md. Sayem Miah ◽  
Charles A. S. Banks ◽  
...  
Keyword(s):  
Protein Interactions ◽  
Cancer Metastasis ◽  
Breast Cancer Metastasis ◽  
Metastasis Suppressor ◽  
Protein Protein Interactions ◽  
Multiple Cancer ◽  
C Terminus ◽  
Therapeutic Development

Breast Cancer Metastasis Suppressor 1 (BRMS1) expression is associated with longer patient survival in multiple cancer types. Understanding BRMS1 functionality will provide insights into both mechanism of action and will enhance potential therapeutic development. In this study, we confirmed that the C-terminus of BRMS1 is critical for metastasis suppression and hypothesized that critical protein interactions in this region would explain its function. Phosphorylation status at S237 regulates BRMS1 protein interactions related to a variety of biological processes, phenotypes [cell cycle (e.g., CDKN2A), DNA repair (e.g., BRCA1)], and metastasis [(e.g., TCF2 and POLE2)]. Presence of S237 also directly decreased MDA-MB-231 breast carcinoma migration in vitro and metastases in vivo. The results add significantly to our understanding of how BRMS1 interactions with Sin3/HDAC complexes regulate metastasis and expand insights into BRMS1’s molecular role, as they demonstrate BRMS1 C-terminus involvement in distinct protein-protein interactions.

scholarly journals Rational discovery of antimetastatic agents targeting the intrinsically disordered region of MBD2

2019 ◽  
Vol 5 (11) ◽  
pp. eaav9810 ◽  
Author(s):  
Min Young Kim ◽  
Insung Na ◽  
Ji Sook Kim ◽  
Seung Han Son ◽  
Sungwoo Choi ◽  
...  
Keyword(s):  
Protein Interactions ◽  
Drug Targets ◽  
Cancer Metastasis ◽  
Epithelial Mesenchymal Transition ◽  
Tumor Model ◽  
Intrinsically Disordered Protein ◽  
Protein Protein Interactions ◽  
Mesenchymal Transition ◽  
Intrinsically Disordered

Although intrinsically disordered protein regions (IDPRs) are commonly engaged in promiscuous protein-protein interactions (PPIs), using them as drug targets is challenging due to their extreme structural flexibility. We report a rational discovery of inhibitors targeting an IDPR of MBD2 that undergoes disorder-to-order transition upon PPI and is critical for the regulation of the Mi-2/NuRD chromatin remodeling complex (CRC). Computational biology was essential for identifying target site, searching for promising leads, and assessing their binding feasibility and off-target probability. Molecular action of selected leads inhibiting the targeted PPI of MBD2 was validated in vitro and in cell, followed by confirming their inhibitory effects on the epithelial-mesenchymal transition of various cancer cells. Identified lead compounds appeared to potently inhibit cancer metastasis in a murine xenograft tumor model. These results constitute a pioneering example of rationally discovered IDPR-targeting agents and suggest Mi-2/NuRD CRC and/or MBD2 as a promising target for treating cancer metastasis.

scholarly journals Characterization of A-kinase-anchoring disruptors using a solution-based assay

2006 ◽  
Vol 400 (3) ◽  
pp. 493-499 ◽  
Author(s):  
Anne J. Stokka ◽  
Frank Gesellchen ◽  
Cathrine R. Carlson ◽  
John D. Scott ◽  
Friedrich W. Herberg ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Protein Interactions ◽  
Protein Protein Interactions ◽  
Regulatory Subunits ◽  
Anchoring Proteins ◽  
Ic50 Values ◽  
Order Of Magnitude ◽  
Dependent Protein

Subcellular localization of PKA (cAMP-dependent protein kinase or protein kinase A) is determined by protein–protein interactions between its R (regulatory) subunits and AKAPs (A-kinase-anchoring proteins). In the present paper, we report the development of the Amplified Luminescent Proximity Homogeneous Assay (AlphaScreen™) as a means to characterize AKAP-based peptide competitors of PKA anchoring. In this assay, the prototypic anchoring disruptor Ht31 efficiently competed in RIIα isoform binding with RII-specific and dual-specificity AKAPs (IC50 values of 1.4±0.2 nM and 6±1 nM respectively). In contrast, RIα isoform binding to a dual-specific AKAP was less efficiently competed (IC50 of 156±10 nM). Characterization of two RI-selective anchoring disruptors, RIAD (RI-anchoring disruptor) and PV-38 revealed that RIAD (IC50 of 13±1 nM) was 20-fold more potent than PV-38 (IC50 of 304±17 nM) and did not compete in the RIIα–AKAP interaction. We also observed that the kinetics of RII displacement from pre-formed PKA–AKAP complexes and competition of RII–AKAP complex formation by Ht31 differed by an order of magnitude when the component parts were mixed in vitro. No such difference in potency was seen for RIα–AKAP complexes. Thus the AlphaScreen assay may prove to be a valuable tool for detailed characterization of a variety of PKA–AKAP complexes.

scholarly journals The Carboxy-terminal Cysteine of the Tetraspanin L6 Antigen Is Required for Its Interaction with SITAC, a Novel PDZ Protein

2000 ◽  
Vol 11 (12) ◽  
pp. 4217-4225 ◽  
Author(s):  
Maria Borrell-Pagès ◽  
Juan Fernández-Larrea ◽  
Aldo Borroto ◽  
Federico Rojo ◽  
José Baselga ◽  
...  
Keyword(s):  
Protein Interactions ◽  
Pdz Domains ◽  
Protein Protein Interactions ◽  
Subcellular Compartment ◽  
Natural Ligand ◽  
Pdz Protein ◽  
Carboxy Terminal ◽  
High Degree

PDZ domains are protein modules that mediate protein-protein interactions. Here, we present the identification and characterization of a protein similar to the recently identified PDZ-containing protein TACIP18, which we have named SITAC (similar to TACIP18). SITAC is preferentially expressed in cells of the digestive tract, associated with intracellular membranes. Despite the high degree of sequence identity between the PDZ domains of TACIP18 and those of SITAC, none of the known ligands of the former shows interaction with the latter, as judged by two-hybrid analysis. SITAC interacts with peptides containing bulky hydrophobic amino acids two positions upstream of the C-terminal residue. Surprisingly, SITAC also shows interaction with peptides ending in C, a previously unacknowledged ability of PDZ domains. The sequence -Y-X-C-COOH, bound in vitro by SITAC, is present in the member of the tetraspanin superfamily, the L6 antigen. Coimmunoprecipitation experiments show that SITAC interacts with L6A, but not with an L6A C-terminal mutant, confirming the capacity of SITAC to interact with proteins ending in C. Confocal analysis shows that the interaction between L6A and SITAC is necessary for the precise colocalization of both molecules in the same subcellular compartment. In summary, the characterization of the protein SITAC has unveiled novel sequences recognized by PDZ domains, and it suggests that L6A is a natural ligand of this PDZ protein.

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 Identification and Biochemical Characterization of High Mobility Group Protein 20A as a Novel Ca2+/S100A6 Target

Biomolecules ◽  
2021 ◽  
Vol 11 (4) ◽  
pp. 510
Author(s):  
Maho Yamamoto ◽  
Rina Kondo ◽  
Haruka Hozumi ◽  
Seita Doi ◽  
Miwako Denda ◽  
...  
Keyword(s):  
Protein Interactions ◽  
Biochemical Characterization ◽  
S100 Protein ◽  
High Mobility ◽  
Dependent Manner ◽  
Protein Signaling ◽  
Protein Protein Interactions ◽  
High Mobility Group Protein ◽  
Pulldown Assay

During screening of protein-protein interactions, using human protein arrays carrying 19,676 recombinant glutathione s-transferase (GST)-fused human proteins, we identified the high-mobility protein group 20A (HMG20A) as a novel S100A6 binding partner. We confirmed the Ca2+-dependent interaction of HMG20A with S100A6 by the protein array method, biotinylated S100A6 overlay, and GST-pulldown assay in vitro and in transfected COS-7 cells. Co-immunoprecipitation of S100A6 with HMG20A from HeLa cells in a Ca2+-dependent manner revealed the physiological relevance of the S100A6/HMG20A interaction. In addition, HMG20A has the ability to interact with S100A1, S100A2, and S100B in a Ca2+-dependent manner, but not with S100A4, A11, A12, and calmodulin. S100A6 binding experiments using various HMG20A mutants revealed that Ca2+/S100A6 interacts with the C-terminal region (residues 311–342) of HMG20A with stoichiometric binding (HMG20A:S100A6 dimer = 1:1). This was confirmed by the fact that a GST-HMG20A mutant lacking the S100A6 binding region (residues 311–347, HMG20A-ΔC) failed to interact with endogenous S100A6 in transfected COS-7 cells, unlike wild-type HMG20A. Taken together, these results identify, for the first time, HMG20A as a target of Ca2+/S100 proteins, and may suggest a novel linkage between Ca2+/S100 protein signaling and HMG20A function, including in the regulation of neural differentiation.

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