Direct Molecular Fishing of Protein Partners for Proteins Encoded by Genes of Human Chromosome 18 in HepG2 Cell Lysate

Ершов, П. В.; Mezentsev, Yu. V.; Yablokov, E. O.; Kaluzhskiy, Leonid; Florinskaya, A. V.; Gnedenko, O. V.; Згода, В. Г.; Вахрушев, И. В.; Раева, О. С.; Yarygin, K. N.; Гилеп, А. А.; Usanov, Sergey A.; Медведев, А. Е.; Иванов, А. С.

doi:10.1134/s1068162019010059

Cited by 5 publications

(8 citation statements)

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“…It was found that two enzymes with 20% amino acid sequence identity had both common and tissue-specific protein partners [ 122 , 123 , 124 ]. In general, the identification of PPIs of a target protein by affinity chromatography and mass spectrometry showed that the number of its potential protein partners can reach several dozen [ 122 , 125 , 126 , 127 ], and the complete experimental validation of all combinations of binary PPIs is an overly complex methodological problem.…”

Section: Discussionmentioning

confidence: 99%

Enzymes in the Cholesterol Synthesis Pathway: Interactomics in the Cancer Context

et al. 2021

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A global protein interactome ensures the maintenance of regulatory, signaling and structural processes in cells, but at the same time, aberrations in the repertoire of protein–protein interactions usually cause a disease onset. Many metabolic enzymes catalyze multistage transformation of cholesterol precursors in the cholesterol biosynthesis pathway. Cancer-associated deregulation of these enzymes through various molecular mechanisms results in pathological cholesterol accumulation (its precursors) which can be disease risk factors. This work is aimed at systematization and bioinformatic analysis of the available interactomics data on seventeen enzymes in the cholesterol pathway, encoded by HMGCR, MVK, PMVK, MVD, FDPS, FDFT1, SQLE, LSS, DHCR24, CYP51A1, TM7SF2, MSMO1, NSDHL, HSD17B7, EBP, SC5D, DHCR7 genes. The spectrum of 165 unique and 21 common protein partners that physically interact with target enzymes was selected from several interatomic resources. Among them there were 47 modifying proteins from different protein kinases/phosphatases and ubiquitin-protein ligases/deubiquitinases families. A literature search, enrichment and gene co-expression analysis showed that about a quarter of the identified protein partners was associated with cancer hallmarks and over-represented in cancer pathways. Our results allow to update the current fundamental view on protein–protein interactions and regulatory aspects of the cholesterol synthesis enzymes and annotate of their sub-interactomes in term of possible involvement in cancers that will contribute to prioritization of protein targets for future drug development.

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Section: Discussionmentioning

confidence: 99%

Enzymes in the Cholesterol Synthesis Pathway: Interactomics in the Cancer Context

et al. 2021

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“…The PTGIS protein was covalently immobilized on the activated CNBr-sepharose 4B via amino groups of the protein. A similar protocol has already been used to immobilize recombinant thromboxane synthase [16] and some recombinant proteins encoded by genes on Chromosome 18 [12,14,25] on this sorbent. The specificity of direct molecular fishing was analyzed by SPR.…”

Section: Resultsmentioning

confidence: 99%

“…We have previously demonstrated the applicability of different approaches to direct molecular fishing [12,13,14] for isolation and identification of previously unknown and real protein partners for the bait proteins from tissue lysates. These approaches include a combination of paramagnetic nanoparticle technology, affinity chromatography, LC/MS-MS and surface plasmon resonance (SPR) optical biosensors [12,15,16,17].…”

Section: Introductionmentioning

confidence: 99%

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

Ершов

Mezentsev

Kopylov

et al. 2019

Biology

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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.

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“…The subinteractome of each target protein is the entire combination of other proteins forming direct stable and dynamic binary and higher‐order molecular complexes with it (Ershov et al, 2018; Ivanov et al, 2016). Proteins, which have been isolated from tissue and/or cell lysates, using a target protein as an affinity bait, represent all potential spectrum of protein partners.…”

Section: Binary Ppis Involving Cyp5a1 and Cyp8a1mentioning

confidence: 99%

A new insight into subinteractomes of functional antagonists: Thromboxane (CYP5A1) and prostacyclin (CYP8A1) synthases

Ершов

Yablokov

Zgoda

et al. 2021

Cell Biology International

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The current article aims to summarize all possible spectrum of protein–protein interactions for thromboxane A synthase (CYP5A1) and prostacyclin synthase (CYP8A1). These enzymes metabolize the same substrate (prostaglandin H2) and can participate in cardiovascular, inflammatory, immune processes, and apoptosis modulation, as well as significantly influence the risk of cancers. Binary protein–protein and multiprotein complexes are of great importance in enzyme‐regulating and signal‐transduction pathways. However, protein partners of CYP5A1 and CYP8A1 are not yet fully identified, although both synthases are considered as prospective drug targets. At least 36 novel protein partners of CYP5A1 and CYP8A1 were revealed from different tissue types using an approach based on affinity isolation and mass spectrometry. Enrichment analysis showed that these proteins have different molecular functions: folding (refolding), unfolded protein and chaperon binding, protein transport (export/import), posttranslational modification, protein domain‐specific binding, antioxidant activity, and glutathione homeostasis. A significant part of them, belonging to molecular chaperones, were common partners for CYP5A1 and CYP8A1, while other proteins were unique with the tissue‐dependent distribution. New aspects of CYP5A1 and CYP8A1 interactomics and hetero‐complex formation with different protein partners, including cytochrome P450s are discussed.

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Direct Molecular Fishing of Protein Partners for Proteins Encoded by Genes of Human Chromosome 18 in HepG2 Cell Lysate

Cited by 5 publications

References 50 publications

Enzymes in the Cholesterol Synthesis Pathway: Interactomics in the Cancer Context

Enzymes in the Cholesterol Synthesis Pathway: Interactomics in the Cancer Context

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

A new insight into subinteractomes of functional antagonists: Thromboxane (CYP5A1) and prostacyclin (CYP8A1) synthases

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