scholarly journals A rapid and accurate approach for Prediction of interactomes from co-elution data (PrInCE)

2017 ◽  
Author(s):  
R. Greg Stacey ◽  
Michael A. Skinnider ◽  
Nichollas E. Scott ◽  
Leonard J. Foster
Keyword(s):  
Protein Interactions ◽  
Isotope Labeling ◽  
Protein Complexes ◽  
Affinity Purification ◽  
Ease Of Use ◽  
Label Free ◽  
Protein Protein Interactions ◽  
Bioinformatics Pipeline ◽  
Vast Number ◽  
Free Data

AbstractBackgroundAn organism’s protein interactome, or complete network of protein-protein interactions, defines the protein complexes that drive cellular processes. Techniques for studying protein complexes have traditionally applied targeted strategies such as yeast two-hybrid or affinity purification-mass spectrometry to assess protein interactions. However, given the vast number of protein complexes, more scalable methods are necessary to accelerate interaction discovery and to construct whole interactomes. We recently developed a complementary technique based on the use of protein correlation profiling (PCP) and stable isotope labeling in amino acids in cell culture (SILAC) to assess chromatographic co-elution as evidence of interacting proteins. Importantly, PCP-SILAC is also capable of measuring protein interactions simultaneously under multiple biological conditions, allowing the detection of treatment-specific changes to an interactome. Given the uniqueness and high dimensionality of co-elution data, new tools are needed to compare protein elution profiles, control false discovery rates, and construct an accurate interactome.ResultsHere we describe a freely available bioinformatics pipeline, PrInCE, for the analysis of co-elution data. PrInCE is a modular, open-source library that is computationally inexpensive, able to use label and label-free data, and capable of detecting tens of thousands of protein-protein interactions. Using a machine learning approach, PrInCE offers greatly reduced run time, better performance, prediction of protein complexes, and greater ease of use over previous bioinformatics tools for co-elution data. PrInCE is implemented in Matlab (version R2015b). Source code and standalone executable programs for Windows and Mac OSX are available at https://github.com/fosterlab/PrInCE, where usage instructions can be found. An example dataset and output are also provided for testing purposes.ConclusionsPrInCE is the first fast and easy-to-use data analysis pipeline that predicts interactomes and protein complexes from co-elution data. PrInCE allows researchers without bioinformatics proficiency to analyze high-throughput co-elution datasets.

scholarly journals PIPINO: A Software Package to Facilitate the Identification of Protein-Protein Interactions from Affinity Purification Mass Spectrometry Data

2016 ◽  
Vol 2016 ◽  
pp. 1-13
Author(s):  
Stefan Kalkhof ◽  
Stefan Schildbach ◽  
Conny Blumert ◽  
Friedemann Horn ◽  
Martin von Bergen ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Protein Interactions ◽  
Isotope Labeling ◽  
Affinity Purification ◽  
Interaction Network ◽  
Mass Spectrometry Data ◽  
Protein Protein Interactions ◽  
Protein Protein Interaction ◽  
Binding Behavior ◽  
Bona Fide

The functionality of most proteins is regulated by protein-protein interactions. Hence, the comprehensive characterization of the interactome is the next milestone on the path to understand the biochemistry of the cell. A powerful method to detect protein-protein interactions is a combination of coimmunoprecipitation or affinity purification with quantitative mass spectrometry. Nevertheless, both methods tend to precipitate a high number of background proteins due to nonspecific interactions. To address this challenge the software Protein-Protein-Interaction-Optimizer (PIPINO) was developed to perform an automated data analysis, to facilitate the selection of bona fide binding partners, and to compare the dynamic of interaction networks. In this study we investigated the STAT1 interaction network and its activation dependent dynamics. Stable isotope labeling by amino acids in cell culture (SILAC) was applied to analyze the STAT1 interactome after streptavidin pull-down of biotagged STAT1 from human embryonic kidney 293T cells with and without activation. Starting from more than 2,000 captured proteins 30 potential STAT1 interaction partners were extracted. Interestingly, more than 50% of these were already reported or predicted to bind STAT1. Furthermore, 16 proteins were found to affect the binding behavior depending on STAT1 phosphorylation such as STAT3 or the importin subunits alpha 1 and alpha 6.

scholarly journals Characterization of major chromatin assembly proteins in tetrahymena thermophile using proeomic and molecular evolutionary analyses

2021 ◽  
Author(s):  
Syed N Shah
Keyword(s):  
Protein Interactions ◽  
Protein Complexes ◽  
Affinity Purification ◽  
Regulation Of Gene Expression ◽  
Chromatin Assembly ◽  
Histone Chaperones ◽  
Protein Protein Interactions ◽  
Selective Forces ◽  
Assembly Proteins

Histones H3/H4 are deposited onto DNA in a replication-dependent or independent fashion by the CAF1 and HIRA protein complexes. Despite the identification of these protein complexes, mechanistic details remain unclear. Recently, we showed that in T. thermophila histone chaperones Nrp1, Asf1 and the Impβ6 importin function together to transport newly synthesized H3/H4 from the cytoplasm to the nucleus. To characterize chromatin assembly proteins in T.thermophila, I used affinity purification combined with mass spectrometry to identify protein-protein interactions of Nrp1, Cac2 subunit of CAF1, HIRA and histone modifying Hat1-complex in T. thermophila. I found that the three-subunit T.thermophila CAF1 complex interacts with Casein Kinase 2 (CKII), possibly accounting for previously reported human CAF1phosphorylation. I also found that Hat2 subunit of HAT1 complex is also shared by CAF1 complex as its Cac3 subunit. This suggests that Hat2/Cac3 might exist in two separate pools of protein complexes. Remarkably, proteomic analysis of Hat2/Cac3 in turn revealed that it forms several complexes with other proteins including SIN3, RXT3, LIN9 and TESMIN, all of which have known roles in the regulation of gene expression. Finally, I asked how selective forces might have impacted on the function of proteins involved in H3/H4 transport. Focusing on NASP which possesses several TPR motifs, I showed that its protein-protein interactions are conserved in T. thermophila. Using molecular evolutionary methods I show that different TPRs in NASP evolve at different rates possibly accounting for the functional diversity observed among different family members.

Label-free quantitative proteomic analysis of the YAP/TAZ interactome

2014 ◽  
Vol 306 (9) ◽  
pp. C805-C818 ◽  
Author(s):  
Priyanka Kohli ◽  
Malte P. Bartram ◽  
Sandra Habbig ◽  
Caroline Pahmeyer ◽  
Tobias Lamkemeyer ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Protein Interactions ◽  
Affinity Purification ◽  
Single Copy ◽  
Single Step ◽  
Mass Spectrometry Analysis ◽  
Single Shot ◽  
Label Free ◽  
Protein Protein Interactions ◽  
Spectrometry Analysis

The function of an individual protein is typically defined by protein-protein interactions orchestrating the formation of large complexes critical for a wide variety of biological processes. Over the last decade the analysis of purified protein complexes by mass spectrometry became a key technique to identify protein-protein interactions. We present a fast and straightforward approach for analyses of interacting proteins combining a Flp-in single-copy cellular integration system and single-step affinity purification with single-shot mass spectrometry analysis. We applied this protocol to the analysis of the YAP and TAZ interactome. YAP and TAZ are the downstream effectors of the mammalian Hippo tumor suppressor pathway. Our study provides comprehensive interactomes for both YAP and TAZ and does not only confirm the majority of previously described interactors but, strikingly, revealed uncharacterized interaction partners that affect YAP/TAZ TEAD-dependent transcription. Among these newly identified candidates are Rassf8, thymopoetin, and the transcription factors CCAAT/enhancer-binding protein (C/EBP)β/δ and core-binding factor subunit β (Cbfb). In addition, our data allowed insights into complex stoichiometry and uncovered discrepancies between the YAP and TAZ interactomes. Taken together, the stringent approach presented here could help to significantly sharpen the understanding of protein-protein networks.

scholarly journals Probing conformational hotspots for the recognition and intervention of protein complexes by lysine reactivity profiling

2021 ◽  
Author(s):  
Zheyi Liu ◽  
Wenxiang Zhang ◽  
Binwen Sun ◽  
Yaolu Ma ◽  
Min He ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Small Molecule ◽  
Protein Interactions ◽  
Isotope Labeling ◽  
Protein Complexes ◽  
Protein Protein Interactions ◽  
Active Compounds

A mass spectrometry-based two-step isotope labeling-lysine reactivity profiling strategy is developed to probe the molecular details of protein–protein interactions and evaluate the conformational interventions by small-molecule active compounds.

scholarly journals Investigation of protein–protein interactions by isotope‒edited Fourier transformed infrared spectroscopy

2004 ◽  
Vol 18 (3) ◽  
pp. 397-406 ◽  
Author(s):  
Tiansheng Li
Keyword(s):  
Ftir Spectroscopy ◽  
Conformational Changes ◽  
Protein Interactions ◽  
Isotope Labeling ◽  
Structural Information ◽  
Protein Complexes ◽  
Secondary Structures ◽  
Receptor Complex ◽  
Protein Protein Interactions

Recent advance in FTIR spectroscopy has shown the usefulness of13C uniform isotope labeling in proteins to study protein–protein interactions.13C uniform isotope labeling can significantly resolve the spectral overlap in the amide I/I′ region in the spectra of protein–protein complexes, and therefore allows more accurate determination of secondary structures of individual protein component in the complex than does the conventional FTIR spectroscopy. Only a limited number of biophysical techniques can be used effectively to obtain structural information of large protein–protein complex in solution. Though X‒ray crystallography and NMR have been used to provide structural information of proteins at atomic resolution, they are limited either by the ability of protein to crystallize or the large molecular weight of protein. Vibrational spectroscopy, including FTIR and Raman spectroscopies, has been extensively employed to investigate secondary structures and conformational dynamics of protein–protein complexes. However, significant spectral overlap in the amide I/Iʹ region in the spectra of protein–protein complexes often hinders the utilization of vibrational spectroscopy in the study of protein–protein complex. In this review, we shall discuss our recent work involving the application of isotope labeled FTIR to the investigation of protein–protein complexes such as cytokine–receptor complexes. One of the examples involves G‒CSF/receptor complex. To determine unambiguously the conformations of G‒CSF and the receptor in the complex, we have prepared uniformly13C/15N isotope labeled G‒CSF to resolve its amide Iʹ band from that of its receptor in the IR spectrum of the complex. Conformational changes and structural stability of individual protein subunit in G‒CSF/receptor complex have then been investigated by using FTIR spectroscopy (Li et al.,Biochemistry29 (1997), 8849–8859). Another example involves BDNF/trkB complex in which13C/15N uniformly labeled BDNF is complexed with its receptor trkB (Li et al.,Biopolymers67(1) (2002), 10–19). Interactions of13C/15N uniformly labeled brain‒derived neurotrophic factor (BDNF) with the extracellular domain of its receptor, trkB, have been investigated by employing FTIR spectroscopy. Conformational changes and structural stability and dynamics of BDNF/trkB complex have been determined unambiguously by FTIR spectroscopy, since amide I/Iʹ bands of13C/15N labeled BDNF are resolved from those of the receptor. Together, those studies have shown that isotope edited FTIR spectroscopy can be successfully applied to the determination of protein secondary structures of protein complexes containing either the same or different types of secondary structures. It was observed that13C/15N uniform labeling also affects significantly the frequency of amide IIʹ band, which may permit the determination of hydrogen–deuterium exchange in individual subunit of protein–protein complexes.

scholarly journals Characterization of major chromatin assembly proteins in tetrahymena thermophile using proeomic and molecular evolutionary analyses

2021 ◽  
Author(s):  
Syed N Shah
Keyword(s):  
Protein Interactions ◽  
Protein Complexes ◽  
Affinity Purification ◽  
Regulation Of Gene Expression ◽  
Chromatin Assembly ◽  
Histone Chaperones ◽  
Protein Protein Interactions ◽  
Selective Forces ◽  
Assembly Proteins

Histones H3/H4 are deposited onto DNA in a replication-dependent or independent fashion by the CAF1 and HIRA protein complexes. Despite the identification of these protein complexes, mechanistic details remain unclear. Recently, we showed that in T. thermophila histone chaperones Nrp1, Asf1 and the Impβ6 importin function together to transport newly synthesized H3/H4 from the cytoplasm to the nucleus. To characterize chromatin assembly proteins in T.thermophila, I used affinity purification combined with mass spectrometry to identify protein-protein interactions of Nrp1, Cac2 subunit of CAF1, HIRA and histone modifying Hat1-complex in T. thermophila. I found that the three-subunit T.thermophila CAF1 complex interacts with Casein Kinase 2 (CKII), possibly accounting for previously reported human CAF1phosphorylation. I also found that Hat2 subunit of HAT1 complex is also shared by CAF1 complex as its Cac3 subunit. This suggests that Hat2/Cac3 might exist in two separate pools of protein complexes. Remarkably, proteomic analysis of Hat2/Cac3 in turn revealed that it forms several complexes with other proteins including SIN3, RXT3, LIN9 and TESMIN, all of which have known roles in the regulation of gene expression. Finally, I asked how selective forces might have impacted on the function of proteins involved in H3/H4 transport. Focusing on NASP which possesses several TPR motifs, I showed that its protein-protein interactions are conserved in T. thermophila. Using molecular evolutionary methods I show that different TPRs in NASP evolve at different rates possibly accounting for the functional diversity observed among different family members.

Protein–protein interactions

2010 ◽  
Vol 38 (4) ◽  
pp. 875-878 ◽  
Author(s):  
Mike P. Williamson ◽  
Michael J. Sutcliffe
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Present Article ◽  
High Throughput ◽  
Protein Interactions ◽  
Metabolic Pathway ◽  
Protein Complexes ◽  
Affinity Purification ◽  
Good Evidence ◽  
Protein Protein Interactions ◽  
Two Hybrid

In the present article, we describe the two standard high-throughput methods for identification of protein complexes: two-hybrid screens and TAP (tandem affinity purification) tagging. These methods have been used to characterize the interactome of Saccharomyces cerevisiae, showing that the majority of proteins are part of complexes, and that complexes typically consist of a core to which are bound ‘party’ and ‘dater’ proteins. Complexes typically are merely the sum of their parts. A particularly interesting type of complex is the metabolon, containing enzymes within the same metabolic pathway. There is reasonably good evidence that metabolons exist, but they have not been detected using high-thoughput assays, possibly because of their fragility.

scholarly journals Recent advances in proximity-based labeling methods for interactome mapping

F1000Research ◽  
2019 ◽  
Vol 8 ◽  
pp. 135 ◽  
Author(s):  
Laura Trinkle-Mulcahy
Keyword(s):  
Protein Interactions ◽  
Large Scale ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Protein Complexes ◽  
Affinity Purification ◽  
Small Scale ◽  
Protein Protein Interactions ◽  
Large Scale Network ◽  
Complementary Approach

Proximity-based labeling has emerged as a powerful complementary approach to classic affinity purification of multiprotein complexes in the mapping of protein–protein interactions. Ongoing optimization of enzyme tags and delivery methods has improved both temporal and spatial resolution, and the technique has been successfully employed in numerous small-scale (single complex mapping) and large-scale (network mapping) initiatives. When paired with quantitative proteomic approaches, the ability of these assays to provide snapshots of stable and transient interactions over time greatly facilitates the mapping of dynamic interactomes. Furthermore, recent innovations have extended biotin-based proximity labeling techniques such as BioID and APEX beyond classic protein-centric assays (tag a protein to label neighboring proteins) to include RNA-centric (tag an RNA species to label RNA-binding proteins) and DNA-centric (tag a gene locus to label associated protein complexes) assays.

scholarly journals Lights, Camera, Interaction: Studying Protein–Protein Interactions of the ER Protein Translocase in Living Cells

2021 ◽  
Vol 22 (19) ◽  
pp. 10358
Author(s):  
Mark Sicking ◽  
Martin Jung ◽  
Sven Lang
Keyword(s):  
Membrane Proteins ◽  
Protein Interactions ◽  
Mammalian Cells ◽  
Protein Complexes ◽  
Living Cells ◽  
Ease Of Use ◽  
Protein Protein Interactions ◽  
Regulatory Subunits ◽  
Complex Interactions ◽  
Er Membrane

Various landmark studies have revealed structures and functions of the Sec61/SecY complex in all domains of live demonstrating the conserved nature of this ancestral protein translocase. While the bacterial homolog of the Sec61 complex resides in the plasma membrane, the eukaryotic counterpart manages the transfer of precursor proteins into or across the membrane of the endoplasmic reticulum (ER). Sec61 complexes are accompanied by a set of dynamically recruited auxiliary proteins assisting the transport of certain precursor polypeptides. TRAP and Sec62/Sec63 are two auxiliary protein complexes in mammalian cells that have been characterized by structural and biochemical methods. Using these ER membrane protein complexes for our proof-of-concept study, we aimed to detect interactions of membrane proteins in living mammalian cells under physiological conditions. Bimolecular luminescence complementation and competition was used to demonstrate multiple protein–protein interactions of different topological layouts. In addition to the interaction of the soluble catalytic and regulatory subunits of the cytosolic protein kinase A, we detected interactions of ER membrane proteins that either belong to the same multimeric protein complex (intra-complex interactions: Sec61α–Sec61β, TRAPα–TRAPβ) or protein complexes in juxtaposition (inter-complex interactions: Sec61α–TRAPα, Sec61α–Sec63, and Sec61β–Sec63). In the process, we established further control elements like synthetic peptide complementation for expression profiling of fusion constructs and protease-mediated reporter degradation demonstrating the cytosolic localization of a reporter complementation. Ease of use and flexibility of the approach presented here will spur further research regarding the dynamics of protein–protein interactions in response to changing cellular conditions in living cells.

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