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Discovery of Missing Methylation Sites on Endogenous Peptides of Human Cell Lines.

ABSTRACT: Methylation of proteins has considerable impacts on physiological processes including signal transduction, DNA damage repair, transcriptional regulation, gene activation, and inhibition of gene expression. However, the traditional proteomics-based approach suffers from limited identification rates of these critical methylation sites on endogenous peptides. In this work, a peptidomics-based workflow was established to discover and characterize the global methylome of endogenous peptides in human cells. The reliability of our strategy was validated by methyl-SILAC labeling, resulting in 83% true-positive identifications in the HeLa cell line. We applied this approach to seven human cell lines, and 700 methylated forms on 646 putative methylation sites were identified in total, with over 61% of the methylation sites being newly identified. This study provides a complementary strategy for a traditional proteomics-based approach that enables identification of missing methylation sites and creates a first methylome draft of endogenous peptides of human cell lines, offering a valuable resource for in-depth studies of biological functions of methylated endogenous peptides.

SUBMITTER: Yan X

PROVIDER: S-EPMC7316123 | biostudies-literature | 2019 Dec

REPOSITORIES: biostudies-literature

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Discovery of Missing Methylation Sites on Endogenous Peptides of Human Cell Lines.

Yan Xin X Li Lingjun L Jia Chenxi C

Journal of the American Society for Mass Spectrometry 20190819 12

Methylation of proteins has considerable impacts on physiological processes including signal transduction, DNA damage repair, transcriptional regulation, gene activation, and inhibition of gene expression. However, the traditional proteomics-based approach suffers from limited identification rates of these critical methylation sites on endogenous peptides. In this work, a peptidomics-based workflow was established to discover and characterize the global methylome of endogenous peptides in human ...[more]

PMID: 31429054

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Project description:Human endogenous retroviruses (HERVs) are transcribed in many cancers including prostate cancer. Human endogenous retrovirus K (HERV-K) of the HML2 subtype is the most recently integrated and most intact retrovirus in the human genome, with many of the viral genomes encoding full- or partial-length viral proteins. To assess transcripts of HERV-K in prostate cancer cell lines and identify the specific HERV-K elements in the human genome that are transcribed, reverse transcriptase-PCR (RT-PCR) and cDNA sequencing were undertaken. Strand-specific RT-PCR, plasmid subcloning, and cDNA sequencing detected the presence of HERV-K(HML2) coding strand transcripts within four prostate cell lines (LNCaP, DU145, PC3, and VCaP). RT-PCR across splice junctions revealed splicing variants for env gene mRNA in three cell lines, two involving previously undescribed alternative splice sites. To determine the HERV-K loci from which the transcripts arose, RepeatMasker was used to compile a list of over 200 HERV-K internal genome segment fragments and over 1,000 HERV-K solo long terminal repeat (LTR) fragments in the human genome. Surprisingly, the sequences identified from internal positions of the viral genome were mostly smaller segments, while the LTRs were relatively intact. Possible reasons for this are discussed. The transcripts in the cell lines tested, arose from several HERV-K loci, with some proviruses being detected in multiple cell lines and others in only one of the four used. In some instances, transcripts from viral antisense strands was also detected. In addition, transcripts from both strands of solo LTRs were detected. These data show that transcripts from HERV-K loci commonly occur in prostate cancer cell lines and that transcription of either strand can occur. They also emphasize the importance of single nucleotide level analysis to identify the specific, individual HERV-K loci that are transcribed, and indicate that HERV-K expression in prostate cancer warrants further study.

Project description:BackgroundHypoxia is an important factor that contributes to tumour aggressiveness and correlates with poor prognosis and resistance to conventional therapy. Therefore, identifying hypoxic environments within tumours is extremely useful for understanding cancer biology and developing novel therapeutic strategies. Several studies have suggested that carbonic anhydrase 9 (CA9) is a reliable biomarker of hypoxia and a potential therapeutic target, while pimonidazole has been identified as an exogenous hypoxia marker. However, other studies have suggested that CA9 expression is not directly induced by hypoxia and it is not expressed in all types of tumours. Thus, in this study, we focused on endoplasmic reticulum disulphide oxidase 1α (ERO1α), a protein that localises in the endoplasmic reticulum and is involved in the formation of disulphide bonds in proteins, to determine whether it could serve as a potential tumour-hypoxia biomarker.MethodsUsing quantitative real-time polymerase chain reaction, we analysed the mRNA expression of ERO1α and CA9 in different normal and cancer cell lines. We also determined the protein expression levels of ERO1α and CA9 in these cell lines by western blotting. We then investigated the hypoxia-inducible ERO1α and CA9 expression and localisation in HCT116 and HeLa cells, which express low (CA9-low) and high (CA9-high) levels of CA9, respectively. A comparative analysis was performed using pimonidazole, an exogenous hypoxic marker, as a positive control. The expression and localisation of ERO1α and CA9 in tumour spheres during hypoxia were analysed by a tumour sphere formation assay. Finally, we used a mouse model to investigate the localisation of ERO1α and CA9 in tumour xenografts using several cell lines.ResultsWe found that ERO1α expression increased under chronic hypoxia. Our results show that ERO1α was hypoxia-induced in all the tested cancer cell lines. Furthermore, in the comparative analysis using CA9 and pimonidazole, ERO1α had a similar localisation to pimonidazole in both CA9-low and CA9-high cell lines.ConclusionERO1α can serve as a novel endogenous chronic hypoxia marker that is more reliable than CA9 and can be used as a diagnostic biomarker and therapeutic target for cancer.

Dataset Information

Discovery of Missing Methylation Sites on Endogenous Peptides of Human Cell Lines.

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Discovery of Missing Methylation Sites on Endogenous Peptides of Human Cell Lines.

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