Comparative study of the human keratinocytes proteome of the HaCaT line: identification of proteins encoded by genes of 18 chromosomes under the influence of detergents

Kisrieva, Yu. S.; Samenkova, N. F.; Larina, O B; Zgoda, Victor G.; Karuzina, I I; Русанов, А. Л.; Лузгина, Н. Г.; Петушкова, Н. А.

doi:10.18097/pbmc20206606469

Cited by 3 publications

(5 citation statements)

References 18 publications

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“…S1, S2). Таким образом, количество белков, идентифицированных в кератиноцитах НаСаТ до и после обработки SDS с применением пробоподготовки на основе солюбилизации с помощью ионного детергента и 1DE-гель концентрирования, заметно превышает количество идентификаций, полученных при экстракции белков с помощью осмотического шока (990 и 1223 белка соответственно до и после воздействия SDS) [16].…”

Section: сравнительный анализ протеома Hacat до и после воздействия Sdsunclassified

“…THOC1 сверхэкспрессируется при различных раковых заболеваниях [39] и может принимать участие в процессах неопластической трансформации. В нашем раннем исследовании протеома кератиноцитов НаСаТ с использованием пробоподготовки на основе осмотического шока и трипсинолиза в растворе белок THOC1 не был зарегистрирован [16]. В настоящей работе THOC1 определен с низким значением NSAF (0.0019), таким образом, наш метод позволил зарегистрировать низкопредставленные белки, кодируемые генами хромосомы 18 человека.…”

Section: Aнализ обогащения Gounclassified

“…В отличие от нашего более раннего исследования протеома HaCaT, использование в данной работе метода подготовки образцов, основанного на солюбилизации белков в присутствии 0.2% SDS, и процедуре 1DE-гель концентрирования с последующим LC-MS/MS анализом, позволило дополнительно идентифицировать 18 белков, кодируемых генами хромосомы 18: SEH1, TIM21, SPB4, RB27B, AFG32, THOC1, MAOM, MBD2, UBP14, SPB12, IR3IP, DSC3, F210A, RAB31, DSG1, PTGR3, SC11C, DSC1. В образцах клеток, подвергнутых осмотическому шоку (замораживанию-оттаиванию) и расщеплению трипсином в растворе, были идентифицированы такие белки, как ROCK1, RHG28, DSC2, MTCL1, а также TBB6, MARE2, CTIF [16]. В настоящей работе в контрольных клетках идентифицированы белки NDUV2, LMAN1 и KSDR , которые не были обнаружены в предыдущем исследовании.…”

Section: Aнализ обогащения Gounclassified

“…Белки, кодируемые генами хромосомы 18, представляют интерес, поскольку, согласно базе данных UniProtKB [15], они связаны с целым рядом заболеваний, в том числе со злокачественными новообразованиями. Список белков, кодируемых генами хромосомы 18, полученный с помощью метода 1DE-гель концентрирования, сравнивали с ранее полученными нами результатами идентификации белков после суспендирования клеток HaCaT в воде с тройным замораживанием-оттаиванием и расщеплением трипсином в растворе [16]. Дифференциально экспрессируемые в ответ на воздействие SDS белки были рассмотрены в контексте специфичности кожного канцерогенеза.…”

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Comparative Analysis of the Proteomic Profile of HaCaT Keratinocytes Using a 1DE Concentrating Gel

Kisrieva

Samenkova

Shkrigunov

et al. 2023

BMCRM

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Using tandem mass spectrometry with electrospray ionization, a comparative analysis of HaCaT keratinocyte proteins was carried out before and after exposure of cells to sodium dodecyl sulfate (25 mg/ml) for 48 hours; proteins encoded by human chromosome 18 genes were chosen as the comparison proteins. A total of 2418 proteins were detected in the HaCaT immortalized human keratinocytes, 70% of these proteins were identified by two or more unique peptides. Panoramic mass spectrometry analysis identified 38 proteins encoded by chromosome 18 genes, 27 proteins were common to control HaCaT cells and HaCaT cells exposed to SDS. Using the Metascape database (https://metascape.org), an enrichment analysis of GO terms of the Biological Process category of chromosome 18 gene encoded proteins of HaCaT keratinocytes was performed before and after the SDS exposure. The SDS exposure resulted in a slight enrichment of the GO term "response to stimulus" (GO:0050896) and the related GO term "negative regulation of biological process" (GO:0048519). We found decreased expression levels of membrane proteins encoded by chromosome 18 genes related to cell-cell adhesion (GO:0098609), such as DSC1, DSC3, and DSG1. A decrease in the expression level of desmosomal cadherins is characteristic of malignant neoplasms developing from epithelial tissue cells of various internal organs, mucous membranes, and skin. The method of preparation of HaCaT keratinocyte samples used in this work increased the sensitivity of proteomic analysis of cell culture and made it possible to identify twice as many proteins in one gel strip as compared to the number of proteins (1284) in HaCaT samples subjected to osmotic shock and cleavage by trypsin in solution.

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Section: сравнительный анализ протеома Hacat до и после воздействия Sdsunclassified

Section: Aнализ обогащения Gounclassified

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Comparative Analysis of the Proteomic Profile of HaCaT Keratinocytes Using a 1DE Concentrating Gel

Kisrieva

Samenkova

Shkrigunov

et al. 2023

BMCRM

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“…SDS is believed to be a frequently used detergent for cell lysis (release of soluble proteins) and membrane protein solubilization in proteomic analysis of various human tissues and cells 15 , 16 . SDS is a strong lysis agent that works with most cell types 17 .…”

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Comparative proteoinformatics revealed the essentials of SDS impact on HaCaT keratinocytes

Shkrigunov

Kisrieva

Samenkova

et al. 2022

Sci Rep

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There is no direct evidence supporting that SDS is a carcinogen, so to investigate this fact, we used HaCaT keratinocytes as a model of human epidermal cells. To reveal the candidate proteins and/or pathways characterizing the SDS impact on HaCaT, we proposed comparative proteoinformatics pipeline. For protein extraction, the performance of two sample preparation protocols was assessed: 0.2% SDS-based solubilization combined with the 1DE-gel concentration (Protocol 1) and osmotic shock (Protocol 2). As a result, in SDS-exposed HaCaT cells, Protocol 1 revealed 54 differentially expressed proteins (DEPs) involved in the disease of cellular proliferation (DOID:14566), whereas Protocol 2 found 45 DEPs of the same disease ID. The ‘skin cancer’ term was a single significant COSMIC term for Protocol 1 DEPs, including those involved in double-strand break repair pathway (BIR, GO:0000727). Considerable upregulation of BIR-associated proteins MCM3, MCM6, and MCM7 was detected. The eightfold increase in MCM6 level was verified by reverse transcription qPCR. Thus, Protocol 1 demonstrated high effectiveness in terms of the total number and sensitivity of MS identifications in HaCaT cell line proteomic analysis. The utility of Protocol 1 was confirmed by the revealed upregulation of cancer-associated MCM6 in HaCaT keratinocytes induced by non-toxic concentration of SDS. Data are available via ProteomeXchange with identifier PXD035202.

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Identification of protein components of the transformation system in the cell line of immortalized human keratinocytes HaCaT exposed to surfactants

Shkrigunov,

Vavilov,

Samenkova

et al. 2024

BIOMED KHIM

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Using the method of shotgun mass spectrometry, we have evaluated changes in the proteomic profile of HaCat cells in response to the treatment with sodium dodecyl sulfate (anionic surfactant) and Triton-X100 (non-ionic surfactant) in two concentrations (12.5 µg/mL and 25.0 µg/mL). The study revealed induction of orphan CYP2S1 (biotransformation phase I) in response to Triton-X100. We have identified proteins of II (glutathione-S-transferases, GSTs) and III (solute carrier proteins, SLCs) biotransformation phases, as well as antioxidant proteins (peroxiredoxins, PRDXs; catalase, CAT; thioredoxin, TXN). Thus, proteins of all three xenobiotic detoxification phases were detected. The presented results suggest a new prospect of using HaCaT keratinocytes as a model of human epidermis for studying the metabolism of drugs/toxicants in human skin in vitro.

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Comparative study of the human keratinocytes proteome of the HaCaT line: identification of proteins encoded by genes of 18 chromosomes under the influence of detergents

Cited by 3 publications

References 18 publications

Comparative Analysis of the Proteomic Profile of HaCaT Keratinocytes Using a 1DE Concentrating Gel

Comparative Analysis of the Proteomic Profile of HaCaT Keratinocytes Using a 1DE Concentrating Gel

Comparative proteoinformatics revealed the essentials of SDS impact on HaCaT keratinocytes

Identification of protein components of the transformation system in the cell line of immortalized human keratinocytes HaCaT exposed to surfactants

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