scholarly journals Affinity Isolation and Mass Spectrometry Identification of Prostacyclin Synthase (PTGIS) Subinteractome

Biology ◽  
2019 ◽  
Vol 8 (2) ◽  
pp. 49 ◽  
Author(s):  
Pavel V. Ershov ◽  
Yuri V. Mezentsev ◽  
Arthur T. Kopylov ◽  
Evgeniy O. Yablokov ◽  
Andrey V. Svirid ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Protein Interactions ◽  
Functional Coupling ◽  
Protein Protein Interactions ◽  
Affinity Isolation ◽  
Protein Partners ◽  
Mass Spectrometry Identification ◽  
Potent Vasodilator ◽  
Rat Tissue ◽  
Prostacyclin Synthase

Prostacyclin synthase (PTGIS; EC 5.3.99.4) catalyzes isomerization of prostaglandin H2 to prostacyclin, a potent vasodilator and inhibitor of platelet aggregation. At present, limited data exist on functional coupling and possible ways of regulating PTGIS due to insufficient information about protein–protein interactions in which this crucial enzyme is involved. The aim of this study is to isolate protein partners for PTGIS from rat tissue lysates. Using CNBr-activated Sepharose 4B with covalently immobilized PTGIS as an affinity sorbent, we confidently identified 58 unique proteins by mass spectrometry (LC-MS/MS). The participation of these proteins in lysate complex formation was characterized by SEC lysate profiling. Several potential members of the PTGIS subinteractome have been validated by surface plasmon resonance (SPR) analysis. SPR revealed that PTGIS interacted with full-length cytochrome P450 2J2 and glutathione S-transferase (GST). In addition, PTGIS was shown to bind synthetic peptides corresponding to sequences of for GSTA1, GSTM1, aldo-keto reductase (AKR1A1), glutaredoxin 3 (GLRX3) and histidine triad nucleotide binding protein 2 (HINT2). Prostacyclin synthase could potentially be involved in functional interactions with identified novel protein partners participating in iron and heme metabolism, oxidative stress, xenobiotic and drugs metabolism, glutathione and prostaglandin metabolism. The possible biological role of the recognized interaction is discussed in the context of PTGIS functioning.

Structure, Function, Involvement in Diseases and Targeting of 14-3-3 Proteins: An Update

2018 ◽  
Vol 25 (1) ◽  
pp. 5-21 ◽  
Author(s):  
Ylenia Cau ◽  
Daniela Valensin ◽  
Mattia Mori ◽  
Sara Draghi ◽  
Maurizio Botta
Keyword(s):  
Protein Interactions ◽  
Molecular Mechanisms ◽  
Physiological Role ◽  
Specific Protein ◽  
Protein Protein Interactions ◽  
Cellular Processes ◽  
Protein Partners ◽  
High Sequence Homology ◽  
Small Molecule Modulators

14-3-3 is a class of proteins able to interact with a multitude of targets by establishing protein-protein interactions (PPIs). They are usually found in all eukaryotes with a conserved secondary structure and high sequence homology among species. 14-3-3 proteins are involved in many physiological and pathological cellular processes either by triggering or interfering with the activity of specific protein partners. In the last years, the scientific community has collected many evidences on the role played by seven human 14-3-3 isoforms in cancer or neurodegenerative diseases. Indeed, these proteins regulate the molecular mechanisms associated to these diseases by interacting with (i) oncogenic and (ii) pro-apoptotic proteins and (iii) with proteins involved in Parkinson and Alzheimer diseases. The discovery of small molecule modulators of 14-3-3 PPIs could facilitate complete understanding of the physiological role of these proteins, and might offer valuable therapeutic approaches for these critical pathological states.

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 Mass spectrometry-based cross-linking study shows that the Psb28 protein binds to cytochrome b559 in Photosystem II

2017 ◽  
Vol 114 (9) ◽  
pp. 2224-2229 ◽  
Author(s):  
Daniel A. Weisz ◽  
Haijun Liu ◽  
Hao Zhang ◽  
Sundarapandian Thangapandian ◽  
Emad Tajkhorshid ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Photosystem Ii ◽  
Protein Interactions ◽  
Protein Complexes ◽  
Protein Docking ◽  
Cross Linking ◽  
Protein Protein Interactions ◽  
Cytochrome B559 ◽  
Reaction Center Complex ◽  
Assembly Intermediate

Photosystem II (PSII), a large pigment protein complex, undergoes rapid turnover under natural conditions. During assembly of PSII, oxidative damage to vulnerable assembly intermediate complexes must be prevented. Psb28, the only cytoplasmic extrinsic protein in PSII, protects the RC47 assembly intermediate of PSII and assists its efficient conversion into functional PSII. Its role is particularly important under stress conditions when PSII damage occurs frequently. Psb28 is not found, however, in any PSII crystal structure, and its structural location has remained unknown. In this study, we used chemical cross-linking combined with mass spectrometry to capture the transient interaction of Psb28 with PSII. We detected three cross-links between Psb28 and the α- and β-subunits of cytochrome b559, an essential component of the PSII reaction-center complex. These distance restraints enable us to position Psb28 on the cytosolic surface of PSII directly above cytochrome b559, in close proximity to the QB site. Protein–protein docking results also support Psb28 binding in this region. Determination of the Psb28 binding site and other biochemical evidence allow us to propose a mechanism by which Psb28 exerts its protective effect on the RC47 intermediate. This study also shows that isotope-encoded cross-linking with the “mass tags” selection criteria allows confident identification of more cross-linked peptides in PSII than has been previously reported. This approach thus holds promise to identify other transient protein–protein interactions in membrane protein complexes.

scholarly journals Exploring the Interactome of Cytochrome P450 2E1 in Human Liver Microsomes with Chemical Cross-Linking Mass Spectrometry

2021 ◽  
Author(s):  
Dmitri R. Davydov ◽  
Bikash Dangi ◽  
Guihua Yue ◽  
Bhagwat Prasad ◽  
Viktor G. Zgoda
Keyword(s):  
Mass Spectrometry ◽  
Cytochrome P450 ◽  
Protein Interactions ◽  
Human Liver ◽  
Liver Microsomes ◽  
Human Liver Microsomes ◽  
Cross Linking ◽  
Cytochrome P450 2E1 ◽  
Protein Protein Interactions ◽  
Chemical Cross Linking

This study aimed on exploration of the system-wide effects of the alcohol-induced increase in the content of cytochrome P450 2E1 (CYP2E1) in the human liver on drug metabolism. Using membrane incorporation of purified CYP2E1 modified with photoreactive crosslinkers benzophenone-4-maleimide (BPM) and 4-(N-succinimidylcarboxy)benzophenone (BPS), we explored the array of its protein-protein interactions (proteome) in human liver microsomes (HLM) with chemical cross-linking mass spectrometry (CXMS). Exposure of bait-incorporated HLM samples to light was followed by isolation of the His-tagged bait protein and its cross-linked aggregates on Ni-NTA agarose. Analyzing the individual bands of SDS-PAGE slabs of thereby isolated protein with the toolset of untargeted proteomics, we detected the cross-linked dimeric and trimeric complexes of CYP2E1 with other drug-metabolizing enzymes. Among the most extensively cross-linked partners of CYP2E1 are cytochromes P450 2A6, 3A4, 2C9, and 4A11. We also detected the conjugates of CYP2E1 with UDP-glucuronosyltransferases (UGTs) 1A6, 1A9, 2B4, 2B15, and 2B17. These results demonstrate the exploratory power of the proposed CXMS strategy and corroborate the concept of tight functional integration in the human drug-metabolizing ensemble through protein-protein interactions of the constituting enzymes. Of particular interest is the observation of efficient cross-linking of CYP2E1 with CYP4A11. This enzyme plays a central role in the synthesis of vasoactive eicosanoids and its interactions with alcohol-inducible CYP2E1 may shed light on the mechanisms of alcohol-induced hypertension.

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