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A modular toolkit to inhibit proline-rich motif-mediated protein-protein interactions.

ABSTRACT: Small-molecule competitors of protein-protein interactions are urgently needed for functional analysis of large-scale genomics and proteomics data. Particularly abundant, yet so far undruggable, targets include domains specialized in recognizing proline-rich segments, including Src-homology 3 (SH3), WW, GYF, and Drosophila enabled (Ena)/vasodilator-stimulated phosphoprotein (VASP) homology 1 (EVH1) domains. Here, we present a modular strategy to obtain an extendable toolkit of chemical fragments (ProMs) designed to replace pairs of conserved prolines in recognition motifs. As proof-of-principle, we developed a small, selective, peptidomimetic inhibitor of Ena/VASP EVH1 domain interactions. Highly invasive MDA MB 231 breast-cancer cells treated with this ligand showed displacement of VASP from focal adhesions, as well as from the front of lamellipodia, and strongly reduced cell invasion. General applicability of our strategy is illustrated by the design of an ErbB4-derived ligand containing two ProM-1 fragments, targeting the yes-associated protein 1 (YAP1)-WW domain with a fivefold higher affinity.

SUBMITTER: Opitz R

PROVIDER: S-EPMC4413326 | biostudies-literature | 2015 Apr

REPOSITORIES: biostudies-literature

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A modular toolkit to inhibit proline-rich motif-mediated protein-protein interactions.

Opitz Robert R Müller Matthias M Reuter Cédric C Barone Matthias M Soicke Arne A Roske Yvette Y Piotukh Kirill K Huy Peter P Beerbaum Monika M Wiesner Burkhard B Beyermann Michael M Schmieder Peter P Freund Christian C Volkmer Rudolf R Oschkinat Hartmut H Schmalz Hans-Günther HG Kühne Ronald R

Proceedings of the National Academy of Sciences of the United States of America 20150406 16

Small-molecule competitors of protein-protein interactions are urgently needed for functional analysis of large-scale genomics and proteomics data. Particularly abundant, yet so far undruggable, targets include domains specialized in recognizing proline-rich segments, including Src-homology 3 (SH3), WW, GYF, and Drosophila enabled (Ena)/vasodilator-stimulated phosphoprotein (VASP) homology 1 (EVH1) domains. Here, we present a modular strategy to obtain an extendable toolkit of chemical fragments ...[more]

PMID: 25848013

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Project description:BackgroundThe bimolecular fluorescence complementation (BiFC) assay has emerged as one of the most popular methods for analysing protein-protein interactions (PPIs) in plant biology. This includes its increasing use as a tool for dissecting the molecular mechanisms of chloroplast function. However, the construction of chloroplast fusion proteins for BiFC can be difficult, and the availability and selection of appropriate controls is not trivial. Furthermore, the challenges of performing BiFC in restricted cellular compartments has not been specifically addressed.ResultsHere we describe the development of a flexible modular cloning-based toolkit for BiFC (MoBiFC) and proximity labelling in the chloroplast and other cellular compartments using synthetic biology principles. We used pairs of chloroplast proteins previously shown to interact (HSP21/HSP21 and HSP21/PTAC5) and a negative control (HSP21/ΔPTAC5) to develop standardised Goldengate-compatible modules for the assembly of protein fusions with fluorescent protein (FP) fragments for BiFC expressed from a single multigenic T-DNA. Using synthetic biology principles and transient expression in Nicotiana benthamiana, we iteratively improved the approach by testing different FP fragments, promoters, reference FPs for ratiometric quantification, and cell types. A generic negative control (mCHERRY) was also tested, and modules for the identification of proximal proteins by Turbo-ID labelling were developed and validated.ConclusionsMoBiFC facilitates the cloning process for organelle-targeted proteins, allows robust ratiometric quantification, and makes available model positive and negative controls. Development of MoBiFC underlines how Goldengate cloning approaches accelerate the development and enrichment of new toolsets, and highlights several potential pitfalls in designing BiFC experiments including the choice of FP split, negative controls, cell type, and reference FP. We discuss how MoBiFC could be further improved and extended to other compartments of the plant cell and to high throughput cloning approaches.

Project description:Background Proline-rich coiled-coil 2C (PRRC2C) is located in the chromosome region lq where hepatocellular carcinoma (HCC) frequently undergoes genomic fragment amplification, but its role in HCC is unknown. In this study, we aimed to explore the correlation of PRRC2C with HCC diagnosis and progression, as well as its influence on the biological behavior of HCC cells. Methods The Cancer Genome Atlas (TCGA) RNA-sequencing datasets of 371 cases of primary liver cancer and 50 normal liver tissue specimens were obtained to analyze correlation between PRRC2C expression and HCC staging, grades, and overall survival. After confirming expression of PRRC2C in HCC cells, PRRC2C silencing was performed. Celigo cell counting, cell clone formation, MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay and Flow cytometry were used to detect the cell proliferation and apoptosis; wound healing and Transwell assays were used to detect the invasion abilities of cells. Xenograft transplantation in nude mice was performed to investigate the impact of PRRC2C knockdown on tumorigenic capabilities. In addition, the expression levels of EMT (epithelial-mesenchymal transition)-related genes, including E-cadherin, N-cadherin, Twistl, Snail, Slug, and Smad2/3/4, were detected. Results Analysis of TCGA data sets revealed that patients with high PRRC2C expression had significantly shorter overall survival. PRRC2C was abundantly expressed in four human hepatocarcinoma cell lines. After knockdown PRRC2C, the proliferation of HCC cells were suppressed and the numbers of apoptotic cells increased. Migration and invasion ability of HCC cells were inhibited by PRRC2C knockdown. Meanwhile, PRRC2C silencing inhibited the tumor formation (indicated by reduced tumor volume and weight compared to the control group) in BALB/c (Bagg Albino Laboratory-bred strain) nude mice. The expressions of EMT-related genes N-cadherin and Vimentin were significantly lower in the PRRC2C knockdown group than in the control group. Conclusions PRRC2C promotes the proliferation and metastasis of liver cancer cells and inhibited apoptosis, potentially through upregulation of EMT related N-cadherin and Vimentin.

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A modular toolkit to inhibit proline-rich motif-mediated protein-protein interactions.

Publications

A modular toolkit to inhibit proline-rich motif-mediated protein-protein interactions.

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