Oxidation of protein-bound methionine in Photofrin-photodynamic therapy-treated human tumor cells explored by methionine-containing peptide enrichment and quantitative proteomics approach

Abstract

In Photofrin-mediated photodynamic therapy (PDT), cell fate can be modulated by the subcellular location of Photofrin. PDT triggers oxidative damage to target cells, including the methionine (Met) oxidation of proteins. Here, we developed a new Met-containing peptide enrichment protocol combined with SILAC-based quantitative proteomics, and used this approach to explore the global Met oxidation changes of proteins in PDT-treated epidermoid carcinoma A431 cells preloaded with Photofrin at the plasma membrane, ER/Golgi, or ubiquitously. We identified 431 Met-peptides corresponding to 302 proteins that underwent severe oxidation upon PDT and observed overrepresentation of proteins related to the cell surface, plasma membrane, ER, Golgi, and endosome under all three conditions. The most frequently oxidized Met-peptide sequence was “QAMXXMM-E/G/M-S/G-A/G/F-XG”. We also identified several hundred potential Photofrin-binding proteins using affinity purification coupled with LC-MS/MS, and confirmed the bindings of EGFR and cathepsin D with Photofrin. The enzyme activities of both proteins were significantly reduced by Photofrin-PDT. Our results shed light on the global and site-specific changes in Met-peptide oxidation among cells undergoing Photofrin-PDT-mediated oxidative stress originating from distinct subcellular sites, and suggest numerous potential Photofrin-binding proteins. These findings provide new insight into the molecular targets through which Photofrin-PDT has diverse effects on target cells.

TheRhodobacter sphaeroidesmethionine sulfoxide reductase MsrP can reduceR- andS-diastereomers of methionine sulfoxide from a broad-spectrum of protein substrates

“…Interestingly, we observed from our IceLogo analysis that Met sensitive to oxidation are generally presented in a polar amino acid environment and can be found in cluster (Figure 4). These properties might be common to all Met in proteins as similar results were found in human cells (Ghesquière et al, 2011; Hsieh et al, 2017) and plants (Jacques et al, 2015). Moreover, oxidized Met efficiently reduced by the RsMsrP were also found in cluster in polar environment and our analysis shows that the presence of Thr and Pro in N-terminal side of a MetO strongly decrease RsMsrP efficiency (Table 1, Figure 5 and S6).…”

Rhodobacter sphaeroides methionine sulfoxide reductase P reduces R- and S-diastereomers of methionine sulfoxide from a broad-spectrum of protein substrates

“…were found in human cells [41,42] and plants [43]. Moreover, oxidized Met efficiently reduced by the RsMsrP were also found clustered in polar environments and our analysis shows that the presence of Thr and Pro in N-terminal side of a MetO strongly decrease RsMsrP efficiency (Table 1, Figure 5 and Supplementary Figure S6).…”

Susceptibility of Protein Methionine Oxidation in Response to Hydrogen Peroxide Treatment–Ex Vivo Versus In Vitro: A Computational Insight

“…In previous studies using proteomic data derived from H 2 O 2 -stressed Jurkat cells, it was noticed that acidic amino acids closely surrounded the oxidized methionine (MetO) whereas tyrosine, tryptophan and histidine were underrepresented in the neighborhood of MetO [18,31]. The observation that acidic residues are frequently found in the vicinity of MetO sites has also been reported in the human carcinoma A431 cell line where methionines were photooxidized within living cells pre-treated with Photofrin [21]. Interestingly, when we analyzed the data provided by a proteome-wide study of in vivo methionine oxidation in Arabidopsis plants subjected to high light irradiation [20], we again observed that both glutamate and aspartate were significantly overrepresented in the neighborhood of MetO (Figure S1).…”

“…On the other hand, there is abundant evidence that the dysregulation of this methionine oxidation-reduction cycle could be the basis of multiple diseases [17]. Not surprisingly, the interest in characterizing methionine oxidation has been revived and a number of proteome-wide studies of methionine oxidation has been reported in the past few years [18][19][20][21][22][23][24]. In an additional effort to identify, classify and document sulfoxidized proteins/sites in different organisms and under different experimental conditions, we have recently built a database, MetOSite, that provides easy access to information on experimentally confirmed sulfoxidized methionine sites [25].…”