Dataset Information

Comprehensive Profiling of Proteome and Ubiquitome Changes in Human Lens Epithelial Cell Line after Ultraviolet-B Irradiation.

ABSTRACT: Ultraviolet-B (UVB) is a recognized risk factor for age-related cataract (ARC) and can cause various changes, including ubiquitination, in lens epithelial cells (LECs). However, the relationship between ubiquitination and ARC is unclear. Herein, we used UVB-irradiated human lens epithelial cell line (SRA01/04) representing the cell model of ARC to investigate the profile changes in the proteome and ubiquitome. A total of 552 differentially expressed proteins (DEPs) and 871 differentially ubiquitinated proteins (DUPs) were identified, and 9 ubiquitination motifs were found. Bioinformatics analysis revealed diverse pathways and biological processes of differential proteins and several DNA damage repair proteins that were potentially mediated via ubiquitin-proteasome pathway. We validated the decreased protein expression of DNA-directed RNA polymerase II subunit RPB2 (POLR2B) in both human cataract capsule tissues and UVB-treated SRA01/04 cells and found that treatment with proteasome inhibitor (MG-132) could reverse the protein level of POLR2B in UVB-irradiated SRA01/04 cells. Our data provide novel information regarding protein expressions and ubiquitination modifications in UVB-induced oxidative damage model. This study might offer a cell-level reference to further investigate the pathogenesis of ARC.

SUBMITTER: Chen X

PROVIDER: S-EPMC7758888 | biostudies-literature | 2020 Dec

REPOSITORIES: biostudies-literature

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Comprehensive Profiling of Proteome and Ubiquitome Changes in Human Lens Epithelial Cell Line after Ultraviolet-B Irradiation.

Chen Xiaojuan X Li Pengfei P Zhang Guowei G Kang Lihua L Qin Bai B Mao Xinmu X Qin Miaomiao M Cao Yu Y Wang Ying Y Guan Huaijin H

ACS omega 20201207 50

Ultraviolet-B (UVB) is a recognized risk factor for age-related cataract (ARC) and can cause various changes, including ubiquitination, in lens epithelial cells (LECs). However, the relationship between ubiquitination and ARC is unclear. Herein, we used UVB-irradiated human lens epithelial cell line (SRA01/04) representing the cell model of ARC to investigate the profile changes in the proteome and ubiquitome. A total of 552 differentially expressed proteins (DEPs) and 871 differentially ubiquit ...[more]

PMID: 33376855

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Project description:PURPOSE: Epidemiological and experimental studies have revealed that exposure to ultraviolet B (UVB) light can induce cataractogenesis. The objective of this study was to determine gene expression changes in human lens epithelial cells in response to UVB exposure and identify factors that can be involved in UVB-induced cataractogenesis. METHODS: SV40 T-antigen-transformed human lens epithelial cells (SRA01/04) were irradiated at various UVB-energy levels (10-80 mJ/cm²) and checked for viability. An irradiation condition of 30 mJ/cm² was adopted for transcriptome analysis. Total RNAs isolated from UVB-exposed and unexposed cells at 12 h and 24 h after UVB exposure were examined for global gene expression changes using Affymetrix Human Gene 1.0 ST array. mRNA levels of specific genes were examined by RT-PCR and real-time PCR, and protein levels in the conditioned media were assayed by ELISA. To examine mRNA expression in human lens, primary cultured human lens epithelial (HLE) cells were prepared from surgically removed lens epithelium, and used for UVB-irradiation and expression analysis. Effects of certain gene products on SRA01/04 cell metabolism were examined using commercially available recombinant proteins. RESULTS: Expression of most the genes analyzed was essentially unchanged (between 0.5 and 2.0 fold) in UVB-irradiated cells compared to non-irradiated cells at both 12 and 24 h after UVB exposure. Sixty one and 44 genes were upregulated more than twofold by UVB exposure at 12 h and 24 h, respectively. Emphasis was placed on genes encoding extracellular proteins, especially growth factors and cytokines. A total of 18 secreted protein genes were upregulated more than twofold at either or both time points. Amphiregulin (AREG) and growth differentiation factor 15 (GDF15) were chosen because of their higher upregulation and novelty, and their upregulation was confirmed in SRA01/04 cells using RT-PCR and real-time PCR analysis. AREG and GDF15 protein levels in conditioned media significantly increased at all UVB-energy points at 24 h, while they were scarcely detectable at 12 h. AREG and GDF15 mRNA levels were also significantly upregulated in UVB-irradiated primary cultured HLE cells compared with the corresponding control culture. AREG significantly stimulated ³H-thymidine and ³H-leucine uptake in SRA01/04 cells as did a positive control epidermal growth factor (EGF). Recombinant GDF15 did not stimulate ³H-thymidine incorporation at any concentration tested, but significantly stimulated ³H-leucine uptake. RT-PCR analysis demonstrated that primary cultured HLE and SRA01/04 cells expressed not only epidermal growth factor receptor (EGFR) mRNA but also transforming growth factor ? receptors (TGFBR1 and TGFBR2) mRNAs. CONCLUSIONS: These results indicate that AREG and GDF15 produced in response to UVB exposure can affect the growth and protein synthesis of lens epithelial cells, suggesting that they have autocrine and paracrine roles related to pathological changes of lens tissue during long-term UVB exposure.

Dataset Information

Comprehensive Profiling of Proteome and Ubiquitome Changes in Human Lens Epithelial Cell Line after Ultraviolet-B Irradiation.

Publications

Comprehensive Profiling of Proteome and Ubiquitome Changes in Human Lens Epithelial Cell Line after Ultraviolet-B Irradiation.

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