No abstract

Lysine acetylation is a widespread posttranslational modification affecting many biological pathways. Recent studies indicate that acetylated lysine residues mainly exhibit low acetylation occupancy, but challenges in sample preparation and analysis make it difficult to confidently assign these numbers, limiting understanding of their biological significance. Here, we tested three common sample preparation methods to determine their suitability for assessing acetylation stoichiometry in three human cell lines, identifying the acetylation occupancy in more than 1,300 proteins from each cell line. The stoichiometric analysis in combination with quantitative proteomics also enabled us to explore their functional roles. We found that higher abundance of the deacetylase sirtuin 1 (SIRT1) correlated with lower acetylation occupancy and lower levels of ribosomal proteins, including those involved in ribosome biogenesis and rRNA processing. Treatment with the SIRT1 inhibitor EX-527 confirmed SIRT1's role in the regulation of pre-rRNA synthesis and processing. Specifically, proteins involved in pre-rRNA transcription, including subunits of the polymerase I and SL1 complexes and the RNA polymerase I-specific transcription initiation factor RRN3, were up-regulated after SIRT1 inhibition. Moreover, many protein effectors and regulators of pre-rRNA processing needed for rRNA maturation were also up-regulated after EX-527 treatment with the outcome that pre-rRNA and 28S rRNA levels also increased. More generally, we found that SIRT1 inhibition down-regulates metabolic pathways, including glycolysis and pyruvate metabolism. Together, these results provide the largest data set thus far of lysine acetylation stoichiometry (available via ProteomeXchange with identifier PXD005903) and set the stage for further biological investigations of this central posttranslational modification.

The molecular processes and proteomic markers leading to tumor progression (TP) in cervical cancer (CC) are either unknown or only partially understood. TP affects metabolic and regulatory mechanisms that can be identified as proteomic changes. To identify which proteins are differentially expressed and to understand the mechanisms of cancer progression, we analyzed the dynamics of the tumor proteome in CC cell lines. This analysis revealed two proteins that are up-regulated during TP, GSTM3 and GSTP1. These proteins are involved in cell maintenance, cell survival and the cellular stress response via the NF-κB and MAP kinase pathways during TP. Furthermore, GSTM3 and GSTP1 knockdown showed that evasion of apoptosis was affected, and tumor proliferation was significantly reduced. Our data indicate the critical role of GST proteins in the regulation and progression of cervical cancer cells. Hence, we suggest GSTM3 and GSTP1 as novel biomarkers and potential therapeutic targets for treating cervical cancer.SignificanceCC is particularly hazardous in the advanced stages, and there are few therapeutic strategies specifically targeting these stages. We performed analyses on CC tumor proteome dynamics and identified GSTM3 and GSTP1 as novel potential therapeutic targets. Knockdown of these proteins showed that they are involved in cell survival, cell proliferation and cellular evasion of apoptosis.

Background/Aim: To date, several proteomics studies in cervical cancer (CC) have focused mainly on squamous cervical cancer (SCC). Our study aimed to discover and clarify differences in SCC and CAD that may provide valuable information for the identification of proteins involved in tumor progression, in CC as a whole, or specific for SCC or CAD. Materials and Methods: Total protein extracts from 15 individual samples corresponding to 5 different CC tissue types were compared with a non-cancerous control group using bidimensional liquid chromatography-mass spectrometry (2D LC-MS/MS), isobaric tags for relative and absolute quantitation (ITRAQ), principal component analysis (PCA) and gene set enrichment analysis (GSEA). Results: A total of 622 statistically significant different proteins were detected. Exocytosis-related proteins were the most over-represented, accounting for 25% of the identified and quantified proteins. Based on the experimental results, reticulocalbin 3 (RCN3) and Ras-related protein Rab-14 (RAB14) were chosen for further downstream in vitro and vivo analyses. RCN3 was overexpressed in all CC tissues compared to the control and RAB14 was overexpressed in squamous cervical cancer (SCC) compared to invasive cervical adenocarcinoma (CAD). In the tumor xenograft experiment, RAB14 protein expression was positively correlated with increased tumor size. In addition, RCN3-expressing HeLa cells induced a discrete size increment compared to control, at day 47 after inoculation. Conclusion: RAB14 and RCN3 are suggested as potential biomarkers and therapeutic targets in the treatment of CC.Cervical cancer (CC) is the fourth most frequently diagnosed cancer in women, with an estimated 265,000 deaths worldwide (1). A large majority (around 80%) of the global burden occurs in the less developed countries (2). Careful retrospective investigations have strongly implicated highrisk human papillomavirus (HPV) with cervical carcinogenesis (3). Although CC diagnosis and treatment programs have decreased the number of cases and mortality rates, the prognosis and complete response to treatment remain poor in CC variants (4,5). Approximately 80% of the CC cases worldwide are of the squamous cervical cancer (SCC) type, with the remainder being invasive cervical adenocarcinoma (CAD) (6). The incidence of CAD has increased, especially in younger women (7). During the last two decades, the relationship between CAD and SCC and all CCs has doubled even in developed countries. This epidemiological evidence suggests that current screening practices are inadequate to identify a significant proportion of CAD precursor lesions (8).In a population-based study in Korean women, it was shown that the prevalence of HPV in CAD tissue is approximately 90% (9). The infection of HPV 16 and/or HPV 18 accounted for 78% of HPV-positive CADs, and multiple HPV types were found in the remaining 13% of cases (9). Risk factors for developing CC that have been described in SCC include the use of oral contraceptives for more than five years, smokin...