Mass spectrometry-based quantification of the cellular response to ultraviolet radiation in HeLa cells

<div><p>Ultraviolet (UV) irradiation is a common form of DNA damage that can cause pyrimidine dimers between DNA, which can cause gene mutations, even double-strand breaks and threaten genome stability. If DNA repair systems default their roles at this stage, the organism can be damaged and result in disease, especially cancer. To better understand the cellular response to this form of damage, we applied highly sensitive mass spectrometry to perform comparative proteomics of phosphorylation in HeLa cells. A total of 4367 phosphorylation sites in 2100 proteins were identified, many of which had not been reported previously. Comprehensive bioinformatics analysis revealed that these proteins were involved in many important biological processes, including signaling, localization and cell cycle regulation. The nuclear pore complex, which is very important for RNA transport, was changed significantly at phosphorylation level, indicating its important role in response to UV-induced cellular stress. Protein–protein interaction network analysis and DNA repair pathways crosstalk were also examined in this study. Proteins involved in base excision repair, nucleotide repair and mismatch repair changed their phosphorylation pattern in response to UV treatment, indicating the complexity of cellular events and the coordination of these pathways. These systematic analyses provided new clues of protein phosphorylation in response to specific DNA damage, which is very important for further investigation. And give macroscopic view on an overall phosphorylation situation under UV radiation.</p></div

Network of phosphoproteins derived from data and the expanding view of phosphorylation level changes for parts of representative proteins.

<p>The different colors represent different ratios from -2 to +2. The highlight part are mismatch repair related protein—MSH6 network picture, DNA replication related protein—POLA1 and POLE network and nuclear pore complex protein—Nup153, Nup50, Nup188 and Nup214 network part.</p

Influenced phosphoproteins inrelated to DNA repair pathway under UV treatment.

<p>Diamond represents mismatch repair proteins, ellipse represents base excision repair proteins and rectangle stands for nucleotide excision repair proteins. The different colors represent different ratios of phosphorylation level from -2 to +2.</p

Quantitative overview of phosphorylated peptides and proteins in <i>HeLa</i> cells following UV treatment.

<p>(A) Number of phosphorylated proteins and proteins’sites quantified in HeLa cells in response to UV treatment. (B) Distribution of identified phosphorylated peptides at Serine, Threonine and Tyrosine sites. (C) Distribution of phosphorylated and non-phosphorylated amino acids in secondary structure. (D) Comparison of phosphorylated peptides identified in this study (I) and Phospho.ELM database (II) (<a href="http://phospho.elm.eu.org/" target="_blank">http://phospho.elm.eu.org/</a>).Validation of phosphorylation sites in RPA1 (S38) and RFC3 (T76) by western blot. Plasmids with 3xFLAG-S38A or 3xFLAG-T76A mutation were transfected to HeLa cells and precipitated by M2 beads. The phosphorylation level between wildtype and mutant proteins were evaluated by western blotting using specific antibody.</p

Phosphorylation-specific motifs using the Motif-X algorithm.

<p>(A) Pro-directed motif centered on Thr and Ser with a strong preference for additional Pro residues C-terminal to the phosphate. (B) Double-phosphorylation motifs found in our study.</p

Mn(II) and Fe(II) modulate the binding activity of DR2539 <i>in vivo</i>.

<p>(A) Effects of divalent metals (50 µM) on expression of pRAZH in <i>D. radiodurans</i>. Data shown are the means ± standard deviations of three independent experiments. (B) Effects of Mn(II) (squares) and Fe(II) (circles) on the expression of pRAZH in wild-type samples expressing DR2539. (C) Effects of Mn(II) (squares) and Fe(II) (circles) on the expression of pRAZH in the <i>dr2539</i> null mutant. (D) Rea-time PCR analysis of the <i>dr1709</i> gene expression using <i>dr0089</i> as internal control gene. Longitudinal axes indicate the change fold of <i>dr1709</i> mRNA relative to controls. Control cells were cultured in medium without Mn(II). *, <i>P</i><0.05 relative to control. The data are the means ± standard deviations of three independent experiments.</p

Sequence alignment of the metal binding sites of DR2539 with other DxtR/MntR family members.

<p>ScaR (<i>Streptococcus gordonii</i>), SloR (<i>Streptococcus suis</i>), LCAS (<i>Lactobacillus casei</i>), SirR (<i>Corynebacterium glutamicum</i>), TroR (<i>Treponema pallidum</i>), TroR (<i>Treponema denticola</i>), IdeR (<i>Mycobacterium tuberculosis</i>), DtxR (<i>Corynebacterium diptheriae</i>), DR2539 (<i>Deinococcus radiodurans</i>), MntR (<i>Staphylococcus aureus</i>), MntR (<i>Escherichia coli</i>), and MntR (<i>Bacillus subtilis</i>). The sequences were aligned using the CLUSTAL W software. Residues shaded with black represent metal-binding sites that have been studied while residues shaded with grey represent predicted metal binding sites.</p

DR0865 binds to the promoter of MntABC in an ion-dependent manner.

<p>(A) and (B) DR0865 binding to p2523 and p2284 as the concentration of DR0865 increased. (C) Wild-type R1, <i>dr2539</i> null mutant (<b>Δ</b><i>dr2539</i>), and <i>dr0865</i> null mutant (<b>Δ</b><i>dr0865</i>) were cultured on TGY plates overlaid with filter discs saturated with 1 M solution MnCl₂. (D) The zone of inhibition was measured from edge of disc after three days. *, <i>P</i><0.05. Data represent the means±deviations of three independent experiments.</p

His98 plays an important role in DNA binding activity of DR2539.

<p>(A) 10 µl cell dilution was dripped on the TGY plate to which 6 mM of Mn(II) had been added. The cells were cultured for 3 days. (B) H98Y mutant and wild-type DR2539 proteins were incubated with p1709b at different concentrations of Mn(II). (C) Quantification of the fluorescence intensity of binding bands was performed using ImageJ. *, <i>P</i><0.05.</p

DR2539 binds to the MntH promoter DNA fragment in a Mn(II)- and Fe(II)-dependent manner.

<p>(A) Schematic of <i>dr1709</i> promoter (p1709a and p1709b) DNA sequence region. The inverted repeat region is shown by the inverted arrows. (B) DR2539 binding to p1709b with increasing quantities of DR2539 and 25 µM Mn(II). (C) and (D) EMSA analysis was performed using DR2539 and p1709b with increasing concentration of Mn(II) or Fe(II). RBS, ribosome binding site; Start, transcription start codon. p1709a and p1709b sequence regions are underlined by straight lines and dashed lines, respectively.</p