Direct molecular fishing in molecular partners investigation in protein–protein and protein–peptide interactions

Ivanov, A. S.; Ершов, П. В.; Molnar, A. A.; Mezentsev, Yu. V.; Kaluzhskiy, Leonid; Yablokov, E. O.; Florinskaya, A. V.; Gnedenko, O. V.; Медведев, А. Е.; Kozin, Sergey A.; Mitkevich, Vladimir A.; Makarov, Alexander А.; Gilep, Andrei A.; Luschik, A.Ya.; Гайдукевич, И. В.; Usanov, Sergey A.

doi:10.1134/s1068162016010052

Cited by 15 publications

(14 citation statements)

References 43 publications

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“…However, it should be noted that proteins identified by direct molecular fishing can only be considered as potential protein partners of TBXAS1, since not only real partner proteins, but also simultaneously “fished” extraneous proteins composing micelles or supramolecular complexes may be isolated from the lysate due to the features of this techniques [ 17 ]. Among our “fished” proteins ( table ), these are SERPINA1, SERPINA3, APOH, FGA, and FN1, which are involved in the blood clotting cascade [ 29 , 30 ], as well as serum proteins (HP, SAA1, CP), which can have a high nonspecific adsorption level.…”

Section: Resultsmentioning

confidence: 99%

“…Previously, we developed an integrated approach to the discovery of novel protein partners interacting with a target protein which is based on the use of direct molecular fishing on the affinity sorbent with the immobilized target protein (or peptide) as a ligand, mass spectrometric identification of the isolated proteins, and validation of the potential PPIs by surface plasmon resonance (SPR) [ 15 - 17 ].…”

Section: Introductionmentioning

confidence: 99%

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Direct Molecular Fishing of New Protein Partners for Human Thromboxane Synthase

Svirid¹,

Ershov²,

Yablokov³

et al. 2017

Acta Naturae

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Thromboxane synthase (TBXAS1) catalyzes the isomerization reaction of prostaglandin H 2 producing thromboxane A 2 , the autocrine and paracrine factor in many cell types. A high activity and metastability by these arachidonic acid derivatives suggests the existence of supramolecular structures that are involved in the regulation of the biosynthesis and directed translocation of thromboxane to the receptor. The objective of this study was to identify TBXAS1 protein partners from human liver tissue lysate using a complex approach based on the direct molecular fishing technique, LC-MS/MS protein identification, and protein-protein interaction validation by surface plasmon resonance (SPR). As a result, 12 potential TBXAS1 protein partners were identified, including the components regulating cytoskeleton organization (BBIP1 and ANKMY1), components of the coagulation cascade of human blood (SERPINA1, SERPINA3, APOH, FGA, and FN1), and the enzyme involved in the metabolism of xenobiotics and endogenous bioregulators (CYP2E1). SPR validation on the Biacore 3000 biosensor confirmed the effectiveness of the interaction between CYP2E1 (the enzyme that converts prostaglandin H 2 to 12-HHT/thromboxane A 2 proantagonist) and TBXAS1 (K d = (4.3 ± 0.4) × 10 -7 M). Importantly, the TBXAS1•CYP2E1 complex formation increases fivefold in the presence of isatin (indole-2,3-dione, a low-molecular nonpeptide endogenous bioregulator, a product of CYP2E1). These results suggest that the interaction between these hemoproteins is important in the regulation of the biosynthesis of eicosanoids. KEYWORDS Thromboxane synthase (CYP5A1, TBXAS1), cytochrome P450, surface plasmon resonance, direct molecular fishing, protein partners, isatin. ABBREVIATIONS Protein-protein interaction (PPI), surface plasmon resonance (SPR), association rate constant (k on ), dissociation rate constant (k off ), equilibrium dissociation constant (K d ).

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Section: Resultsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Direct Molecular Fishing of New Protein Partners for Human Thromboxane Synthase

Svirid¹,

Ershov²,

Yablokov³

et al. 2017

Acta Naturae

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“…In subsequent studies we actively used the SPR technology not only at the final stage of research to validate paired interactions between the target protein and its isolated/identified protein partners, but also at other stages of direct molecular fishing [ 13 ]. These stages are marked with red numbers in Figure 1 .…”

Section: Application Of the Spr Technology At Various Stages Of DImentioning

confidence: 99%

“…The practice of direct molecular fishing of proteins from the whole tissue lysate (liver tissue lysate as an example) has shown that from dozens to hundreds of proteins can be fished using a target bait protein [ 12 , 13 ]. In addition, the protein composition of the “fished material” for each bait protein is quite specific—lists of identified proteins coincide only by 2–5%.…”

Section: Spr-based Analytical Fishingmentioning

confidence: 99%

SPR Biosensors in Direct Molecular Fishing: Implications for Protein Interactomics

Florinskaya

Ершов

Mezentsev

et al. 2018

Sensors

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We have developed an original experimental approach based on the use of surface plasmon resonance (SPR) biosensors, applicable for investigation of potential partners involved in protein–protein interactions (PPI) as well as protein–peptide or protein–small molecule interactions. It is based on combining a SPR biosensor, size exclusion chromatography (SEC), mass spectrometric identification of proteins (LC-MS/MS) and direct molecular fishing employing principles of affinity chromatography for isolation of potential partner proteins from the total lysate of biological samples using immobilized target proteins (or small non-peptide compounds) as ligands. Applicability of this approach has been demonstrated within the frame of the Human Proteome Project (HPP) and PPI regulation by a small non-peptide biologically active compound, isatin.

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“…In this study, using a surface plasmon resonance (SPR) based biosensor we have investigated the specificity and molecular mechanism responsible for the affinity-enhancing effect of isatin on the interaction of FECH and ADR. Since FECH can exist in monomeric and dimeric forms [ 10 , 11 ], we have initially investigated which complexes (dimeric or trimeric) FECH formed during interaction with ADR, and other tested proteins (cytochrome b 5 type B (CYB5B) and SMAD family member 4 (SMAD4)) [ 12 , 13 , 14 ]. Experiments have shown that CYB5B and SMAD4 can interact with both the monomeric and dimeric forms of FECH, with the formation of heterodimeric and heterotrimeric complexes, respectively.…”

Section: Introductionmentioning

confidence: 99%

Mechanism of the Affinity-Enhancing Effect of Isatin on Human Ferrochelatase and Adrenodoxin Reductase Complex Formation: Implication for Protein Interactome Regulation

Ершов

Veselovsky

Mezentsev

et al. 2020

IJMS

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Isatin (indole-2, 3-dione) is a non-peptide endogenous bioregulator exhibiting a wide spectrum of biological activity, realized in the cell via interactions with numerous isatin-binding proteins, their complexes, and (sub) interactomes. There is increasing evidence that isatin may be involved in the regulation of complex formations by modulating the affinity of the interacting protein partners. Recently, using Surface Plasmon Resonance (SPR) analysis, we have found that isatin in a concentration dependent manner increased interaction between two human mitochondrial proteins, ferrochelatase (FECH), and adrenodoxine reductase (ADR). In this study, we have investigated the affinity-enhancing effect of isatin on the FECH/ADR interaction. The SPR analysis has shown that FECH forms not only homodimers, but also FECH/ADR heterodimers. The affinity-enhancing effect of isatin on the FECH/ADR interaction was highly specific and was not reproduced by structural analogues of isatin. Bioinformatic analysis performed using three dimensional (3D) models of the interacting proteins and in silico molecular docking revealed the most probable mechanism involving FECH/isatin/ADR ternary complex formation. In this complex, isatin is targeted to the interface of interacting FECH and ADR monomers, forming hydrogen bonds with both FECH and ADR. This is a new regulatory mechanism by which isatin can modulate protein–protein interactions (PPI).

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Direct molecular fishing in molecular partners investigation in protein–protein and protein–peptide interactions

Cited by 15 publications

References 43 publications

Direct Molecular Fishing of New Protein Partners for Human Thromboxane Synthase

Direct Molecular Fishing of New Protein Partners for Human Thromboxane Synthase

SPR Biosensors in Direct Molecular Fishing: Implications for Protein Interactomics

Mechanism of the Affinity-Enhancing Effect of Isatin on Human Ferrochelatase and Adrenodoxin Reductase Complex Formation: Implication for Protein Interactome Regulation

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