A B23-interacting sequence as a tool to visualize protein interactions in a cellular context

Abstract Delineating the protein network associated with long non-coding RNAs (lncRNAs) is fundamental to understanding the functional mechanisms of lncRNAs. Current methods to identify lncRNA-binding proteins either rely on crosslinking-mediated complex co-precipitation or require extensive molecular engineering, leading to drawbacks such as loss of cellular context and low capture efficiency. Here we describe a CRISPR-Assisted RNA-Protein Interaction Detection method (CARPID), which leverages CRISPR/CasRx-based RNA targeting and proximity labeling, to rapidly capture binding proteins of specific lncRNAs in their native cellular context followed by LC-MS/MS identification. Applied to a variety of lncRNAs of different lengths and subcellular localizations, CARPID is proven to be a reliable and robust tool to discover the binding proteins of lncRNAs inside living cells.

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mARVCF cellular localisation and binding to cadherins is influenced by the cellular context but not by alternative splicing

Journal of Cell Science ◽

10.1242/jcs.114.21.3873 ◽

2001 ◽

Vol 114 (21) ◽

pp. 3873-3884 ◽

Cited By ~ 1

Author(s):

Zoe Waibler ◽

Annette Schäfer ◽

Anna Starzinski-Powitz

Keyword(s):

Alternative Splicing ◽

Protein Interactions ◽

Coiled Coil ◽

Distinct Pattern ◽

Subcellular Localisation ◽

Protein Protein Interactions ◽

Protein Protein Interaction ◽

C Terminus ◽

Coiled Coil Region ◽

Cellular Context

ARVCF, a member of the catenin family, is thought to contribute to the morphoregulatory function of the cadherin-catenin complex. Recently, we reported the isolation and characterisation of murine ARVCF (mARVCF), particularly its interaction with M-cadherin. Here, we describe the identification of novel mARVCF isoforms that arise by alternative splicing. At the N-terminus, alternative splicing results in the inclusion or omission of a coiled-coil region probably important for protein-protein interactions. At the C-terminus, four isoforms also differ by domains potentially important for selective protein-protein interaction. The eight putative mARVCF isoforms were expressed as EGFP-fusion proteins in six different cell lines that exhibit a distinct pattern of cadherins. Apparently, binding of the mARVCF isoforms to M-, N-, or E-cadherin is generally unaffected by their altered N- and C-termini, as revealed by the MOM recruitment assay. However, mARVCF isoforms reproducibly exhibit differential localisation in distinct cellular environments. For example, mARVCF isoforms are unable to colocalise with N-cadherin in EJ28 carcinoma cells but do so in HeLa cells. Our results suggest that the subcellular localisation of mARVCF may be determined not only by the presence or absence of an appropriate interaction partner, in this case cadherins, but also by the cellular context.

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Elucidating Carbohydrate-Protein Interactions Using Nanoparticle-Based Approaches

Frontiers in Chemistry ◽

10.3389/fchem.2021.669969 ◽

2021 ◽

Vol 9 ◽

Author(s):

Dongyoon Kim ◽

Nowras Rahhal ◽

Christoph Rademacher

Keyword(s):

Protein Interactions ◽

Early Recognition ◽

Particle Surface ◽

Biological Response ◽

Cellular Interactions ◽

Molecular Fingerprints ◽

Cellular Immunology ◽

Promising Tool ◽

Cellular Context ◽

Molecular Patterns

Carbohydrates are present on every living cell and coordinate important processes such as self/non-self discrimination. They are amongst the first molecular determinants to be encountered when cellular interactions are initiated. In particular, they resemble essential molecular fingerprints such as pathogen-, danger-, and self-associated molecular patterns guiding key decision-making in cellular immunology. Therefore, a deeper understanding of how cellular receptors of the immune system recognize incoming particles, based on their carbohydrate signature and how this information is translated into a biological response, will enable us to surgically manipulate them and holds promise for novel therapies. One approach to elucidate these early recognition events of carbohydrate interactions at cellular surfaces is the use of nanoparticles coated with defined carbohydrate structures. These particles are captured by carbohydrate receptors and initiate a cellular cytokine response. In the case of endocytic receptors, the capturing enables the engulfment of exogenous particles. Thereafter, the particles are sorted and degraded during their passage in the endolysosomal pathway. Overall, these processes are dependent on the nature of the endocytic carbohydrate receptors and consequently reflect upon the carbohydrate patterns on the exogenous particle surface. This interplay is still an under-studied subject. In this review, we summarize the application of nanoparticles as a promising tool to monitor complex carbohydrate-protein interactions in a cellular context and their application in areas of biomedicine.

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CBRPP: a new RNA-centric method to study RNA-protein interactions

10.1101/2020.04.09.033290 ◽

2020 ◽

Cited By ~ 2

Author(s):

Yunfei Li ◽

Shengde Liu ◽

Lili Cao ◽

Yujie Luo ◽

Hongqiang Du ◽

...

Keyword(s):

Gene Expression ◽

Protein Interactions ◽

High Specificity ◽

Cellular Mechanism ◽

Cross Linking ◽

Cell Dysfunction ◽

Cellular Context ◽

Enzyme Labels ◽

Target Rna

AbstractRNA-protein interactions play essential roles in tuning gene expression at RNA level and modulating the function of proteins. Abnormal RNA-protein interactions lead to cell dysfunction and human diseases. Therefore, mapping networks of RNA-protein interactions is crucial for understanding cellular mechanism and pathogenesis of diseases. Different practical protein-centric methods for studying RNA-protein interactions has been reported, but few RNA-centric methods exist. Here, we developed CRISPR-based RNA proximity proteomics (CBRPP), a new RNA-centric method to identify proteins associated with the target RNA in native cellular context without cross-linking or RNA manipulation in vitro. CBRPP is based on a fusion of dCas13 and proximity-based labeling (PBL) enzyme. dCas13 can deliver PBL enzyme to the target RNA with high specificity, while PBL enzyme labels the surrounding proteins of the target RNA, which are then identified by mass spectrometry.

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Towards an Ideal In Cell Hybridization-Based Strategy to Discover Protein Interactomes of Selected RNA Molecules

International Journal of Molecular Sciences ◽

10.3390/ijms23020942 ◽

2022 ◽

Vol 23 (2) ◽

pp. 942

Author(s):

Michele Spiniello ◽

Mark Scalf ◽

Amelia Casamassimi ◽

Ciro Abbondanza ◽

Lloyd M. Smith

Keyword(s):

Protein Interactions ◽

Rna Binding ◽

Rna Binding Proteins ◽

Cell Hybridization ◽

Rna Molecules ◽

Cellular Context ◽

Protein Binders ◽

Non Coding Rnas ◽

Technical Features ◽

Experimental Strategies

RNA-binding proteins are crucial to the function of coding and non-coding RNAs. The disruption of RNA–protein interactions is involved in many different pathological states. Several computational and experimental strategies have been developed to identify protein binders of selected RNA molecules. Amongst these, ‘in cell’ hybridization methods represent the gold standard in the field because they are designed to reveal the proteins bound to specific RNAs in a cellular context. Here, we compare the technical features of different ‘in cell’ hybridization approaches with a focus on their advantages, limitations, and current and potential future applications.

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Prediction of kinase-specific phosphorylation sites through an integrative model of protein context and sequence

10.1101/043679 ◽

2016 ◽

Author(s):

Ralph Patrick ◽

Coralie Horin ◽

Bostjan Kobe ◽

Kim-Anh Lê Cao ◽

Mikael Bodén

Keyword(s):

Protein Interactions ◽

Binding Sites ◽

Protein Function ◽

Computational Prediction ◽

Alternative Methods ◽

Integrative Model ◽

Model Organisms ◽

Phosphorylation Sites ◽

Nuclear Localisation ◽

Cellular Context

AbstractThe identification of kinase substrates and the specific phosphorylation sites they regulate is an important factor in understanding protein function regulation and signalling pathways. Computational prediction of kinase targets – assigning kinases to putative substrates, and selecting from protein sequence the sites that kinases can phosphorylate – requires the consideration of both the cellular context that kinases operate in, as well as their binding affinity. This consideration enables investigation of how phosphorylation influences a range of biological processes.We report here a novel probabilistic model for the classification of kinase-specific phosphorylation sites from sequence across three model organisms: human, mouse and yeast. The model incorporates position-specific amino acid frequencies, and counts of co-occurring amino acids from kinase binding sites in a kinase‐ and family-specific manner. We show how this model can be seamlessly integrated with protein interactions and cell-cycle abundance profiles. When evaluating the prediction accuracy of our method, PhosphoPICK, on an independent hold-out set of kinase-specific phosphorylation sites, we found it achieved an average specificity of 97% while correctly predicting 32% of true positives. We also compared PhosphoPICK’s ability, through cross-validation, to predict kinase-specific phosphorylation sites with alternative methods, and found that at high levels of specificity PhosphoPICK outperforms alternative methods for most comparisons made.We investigated the relationship between experimentally confirmed phosphorylation sites and predicted nuclear localisation signals by predicting the most likely kinases to be regulating the phosphorylated residues immediately upstream or downstream from the localisation signal. We show that kinases PKA, Akt1 and AurB have an over-representation of predicted binding sites at particular positions downstream from predicted nuclear localisation signals, demonstrating an important role for these kinases in regulating the nuclear import of proteins.PhosphoPICK is freely available online as a web-service at http://bioinf.scmb.uq.edu.au/phosphopick.

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A reference map of the human protein interactome

10.1101/605451 ◽

2019 ◽

Cited By ~ 10

Author(s):

Katja Luck ◽

Dae-Kyum Kim ◽

Luke Lambourne ◽

Kerstin Spirohn ◽

Bridget E. Begg ◽

...

Keyword(s):

Protein Interactions ◽

Protein Function ◽

Molecular Mechanisms ◽

Human Genetics ◽

Genomic Variation ◽

Small Scale ◽

Human Interactome ◽

Tissue Specific ◽

Cellular Context ◽

Reference Map

AbstractGlobal insights into cellular organization and function require comprehensive understanding of interactome networks. Similar to how a reference genome sequence revolutionized human genetics, a reference map of the human interactome network is critical to fully understand genotype-phenotype relationships. Here we present the first human “all-by-all” binary reference interactome map, or “HuRI”. With ~53,000 high-quality protein-protein interactions (PPIs), HuRI is approximately four times larger than the information curated from small-scale studies available in the literature. Integrating HuRI with genome, transcriptome and proteome data enables the study of cellular function within essentially any physiological or pathological cellular context. We demonstrate the use of HuRI in identifying specific subcellular roles of PPIs and protein function modulation via splicing during brain development. Inferred tissue-specific networks reveal general principles for the formation of cellular context-specific functions and elucidate potential molecular mechanisms underlying tissue-specific phenotypes of Mendelian diseases. HuRI thus represents an unprecedented, systematic reference linking genomic variation to phenotypic outcomes.

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Transient binding of an activator BH3 domain to the Bak BH3-binding groove initiates Bak oligomerization

The Journal of Cell Biology ◽

10.1083/jcb.201102027 ◽

2011 ◽

Vol 194 (1) ◽

pp. 39-48 ◽

Cited By ~ 96

Author(s):

Haiming Dai ◽

Alyson Smith ◽

X. Wei Meng ◽

Paula A. Schneider ◽

Yuan-Ping Pang ◽

...

Keyword(s):

Cytochrome C ◽

Protein Interactions ◽

Point Mutations ◽

Protein Protein Interactions ◽

Cytochrome C Release ◽

Interaction Point ◽

Bh3 Domain ◽

Cellular Context ◽

Binding Groove

The mechanism by which the proapoptotic Bcl-2 family members Bax and Bak release cytochrome c from mitochondria is incompletely understood. In this paper, we show that activator BH3-only proteins bind tightly but transiently to the Bak hydrophobic BH3-binding groove to induce Bak oligomerization, liposome permeabilization, mitochondrial cytochrome c release, and cell death. Analysis by surface plasmon resonance indicated that the initial binding of BH3-only proteins to Bak occurred with similar kinetics with or without detergent or mitochondrial lipids, but these reagents increase the strength of the Bak–BH3-only protein interaction. Point mutations in Bak and reciprocal mutations in the BH3-only proteins not only confirmed the identity of the interacting residues at the Bak–BH3-only protein interface but also demonstrated specificity of complex formation in vitro and in a cellular context. These observations indicate that transient protein–protein interactions involving the Bak BH3-binding groove initiate Bak oligomerization and activation.

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Characterization of microtubules by dark-field STEM and replication

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s042482010008506x ◽

1991 ◽

Vol 49 ◽

pp. 152-153

Author(s):

S.B. Andrews ◽

R.D. Leapman ◽

P.E. Gallant ◽

T.S. Reese

Keyword(s):

Protein Interactions ◽

Low Dose ◽

Unit Length ◽

Dark Field ◽

Carbon Films ◽

Microtubule Associated Proteins ◽

Molecular Weights ◽

Freeze Dried ◽

Protein Motors ◽

Associated Proteins

As part of a study on protein interactions involved in microtubule (MT)-based transport, we used the VG HB501 field-emission STEM to obtain low-dose dark-field mass maps of isolated, taxol-stabilized MTs and correlated these micrographs with detailed stereo images from replicas of the same MTs. This approach promises to be useful for determining how protein motors interact with MTs. MTs prepared from bovine and squid brain tubulin were purified and free from microtubule-associated proteins (MAPs). These MTs (0.1-1 mg/ml tubulin) were adsorbed to 3-nm evaporated carbon films supported over Formvar nets on 600-m copper grids. Following adsorption, the grids were washed twice in buffer and then in either distilled water or in isotonic or hypotonic ammonium acetate, blotted, and plunge-frozen in ethane/propane cryogen (ca. -185 C). After cryotransfer into the STEM, specimens were freeze-dried and recooled to ca.-160 C for low-dose (<3000 e/nm2) dark-field mapping. The molecular weights per unit length of MT were determined relative to tobacco mosaic virus standards from elastic scattering intensities. Parallel grids were freeze-dried and rotary shadowed with Pt/C at 14°.

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Spot protein-protein interactions... fast

PSI Structural Genomics Knowledgebase ◽

10.1038/th_psisgkb.2010.08 ◽

2010 ◽

Author(s):

Maria Hodges

Keyword(s):

Protein Interactions ◽

Protein Protein Interactions

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