Global Profiling of Lysine Acetylation and Lactylation in Kupffer Cells.

Among the various cell types that constitute the liver, Kupffer cells (KCs) are responsible for the elimination of gut-derived foreign products. Protein lysine acetylation (Kac) and lactylation (Kla) are dynamic and reversible post-translational modifications, and various global acylome studies have been conducted for liver and liver-derived cells. However, no such studies have been conducted on KCs. In this study, we identified 2198 Kac sites in 925 acetylated proteins and 289 Kla sites in 181 lactylated proteins in immortalized mouse KCs using global acylome technology. The subcellular distributions of proteins with Kac and Kla site modifications differed. Similarly, the specific sequence motifs surrounding acetylated or lactylated lysine residues also showed differences. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses were performed to better understand the differentially expressed proteins in the studies by Kac and Kla. In the newly identified Kla, we found K82 lactylation in the high-mobility group box-1 protein in the neutrophil extracellular trap formation category using KEGG enrichment analyses. Here, we report the first proteomic survey of Kac and Kla in KCs.

Anti-Metastatic Effect of Pyruvate Dehydrogenase Kinase 4 Inhibition in Bladder Cancer via the ERK, SRC, and JNK Pathways.

Bladder cancer is a common global cancer with a high percentage of metastases and high mortality rate. Thus, it is necessary to identify new biomarkers that can be helpful in diagnosis. Pyruvate dehydrogenase kinase 4 (PDK4) belongs to the PDK family and plays an important role in glucose utilization in living organisms. In the present study, we evaluated the role of PDK4 in bladder cancer and its related protein changes. First, we observed elevated PDK4 expression in high-grade bladder cancers. To screen for changes in PDK4-related proteins in bladder cancer, we performed a comparative proteomic analysis using PDK4 knockdown cells. In bladder cancer cell lines, PDK4 silencing resulted in a lower rate of cell migration and invasion. In addition, a PDK4 knockdown xenograft model showed reduced bladder cancer growth in nude mice. Based on our results, PDK4 plays a critical role in the metastasis and growth of bladder cancer cells through changes in ERK, SRC, and JNK.

Characterization of the RAS/RAF/ERK Signal Cascade as a Novel Regulating Factor in Alpha-Amanitin-Induced Cytotoxicity in Huh-7 Cells.

The well-known hepatotoxicity mechanism resulting from alpha-amanitin (α-AMA) exposure arises from RNA polymerase II (RNAP II) inhibition. RNAP Ⅱ inhibition occurs through the dysregulation of mRNA synthesis. However, the signaling pathways in hepatocytes that arise from α-AMA have not yet been fully elucidated. Here, we identified that the RAS/RAF/ERK signaling pathway was activated through quantitative phosphoproteomic and molecular biological analyses in Huh-7 cells. Bioinformatics analysis showed that α-AMA exposure increased protein phosphorylation in a time-dependent α-AMA exposure. In addition, phosphorylation increased not only the components of the ERK signaling pathway but also U2AF65 and SPF45, known splicing factors. Therefore, we propose a novel mechanism of α-AMA as follows. The RAS/RAF/ERK signaling pathway involved in aberrant splicing events is activated by α-AMA exposure followed by aberrant splicing events leading to cell death in Huh-7 cells.

Comparative Secretome Profiling and Mutant Protein Identification in Metastatic Prostate Cancer Cells by Quantitative Mass Spectrometry-based Proteomics.

BACKGROUND: Secreted proteins play an important role in promoting cancer (PCa) cell migration and invasion. Proteogenomics helps elucidate the mechanism of diseases, discover therapeutic targets, and generate biomarkers for diagnosis through protein variations. MATERIALS AND METHODS: We carried out mass a spectrometry-based proteomic analysis of the conditioned media (CM) from two human prostate cancer cell lines, belonging to different metastatic sites, to identify potential metastatic and/or aggressive factors. RESULTS: We identified a total of 598 proteins, among which 561 were quantified based on proteomic analysis. Among the quantified proteins, 128 were up-regulated and 83 were down-regulated in DU145/PC3 cells. Six mutant peptides were identified in the CM of prostate cancer cell lines using proteogenomics approach. CONCLUSION: This is the first proteogenomics study in PCa aiming at exploring a new type of metastatic factor, which are mutant peptides, predicting a novel biomarker of metastatic PCa for diagnosis, prognosis and drug targeting.

Quantitative Proteomic Analysis of Changes Related to Age and Calorie Restriction in Rat Liver Tissue.

Calorie restriction (CR) is the most frequently studied mechanism for increasing longevity, protecting against stress, and delaying age-associated diseases. Most studies have initiated CR in young animals to determine the protective effects against aging. Although aging phenomena are well-documented, the molecular mechanisms of aging and CR remain unclear. In this study, we observe changes in hepatic proteins upon age-related and diet-restricted changes in the rat liver using quantitative proteomics. Quantitative proteomes are measured using tandem mass tag labeling followed by LC-MS/MS. We compare protein levels in livers from young (6 months old) and old (25 months old) rats with 40% calorie-restricted (YCR and OCR, respectively) or ad libitum diets. In total, 44 279 peptides and 3134 proteins are identified and 260 differentially expressed proteins are found. Functional enrichment analysis show that these proteins are mainly involved in glucose and fatty acid metabolism-related processes, consistent with the theory that energy metabolism regulation is dependent on age-related and calorie-restricted changes in liver tissue. In addition, proteins mediating inflammation and gluconeogenesis are increased in OCR livers, but not YCR livers. These results show that CR in old rats might not have antiaging benefits because liver inflammation is increased.

Proteomics approach to identify novel metastatic bone markers from the secretome of PC-3 prostate cancer cells.

Prostate cancer is the leading type of cancer diagnosed, and the most frequent cause of worldwide male cancer-related deaths annually. The limitations of current prostate cancer screening tests demand the identification of novel biomarkers for the early diagnosis of prostate cancer bone metastasis. In the present study, we performed a proteomic analysis of secreted proteins from the prostate cancer bone metastasis cell line, PC-3, and the normal prostate cell line, RWPE-1. We thus quantified 917 proteins, of which 68 were found to be secreted at higher levels by PC-3 than by RWPE-1 cells via LC-MS/MS. To characterize the highly secreted proteins in the PC-3 cell line and thereby identify biomarker proteins, we divided the quantifiable proteins into four quantitative categories (Q1-Q4). The KEGG lysine degradation and osteoclast differentiation pathways were demonstrated to be enriched in the highly secreted Q4 protein group. Transforming growth factor (TGF) beta family proteins related to osteoclast differentiation were identified as key regulators of PC-3 cell proliferation. Immunoblotting was used to confirm the observed high level of pentraxin, follistatin, TGF-beta family members, and serpin B3 secretion by PC-3 cells. From the collective results of the present study, we suggest that serpin B3 is a promising novel biomarker candidate for the diagnosis of prostate cancer bone metastasis.

Comprehensive Analysis of in Vivo Phosphoproteome of Mouse Liver Microsomes.

Protein phosphorylation at serine, threonine, and tyrosine residues are some of the most widespread reversible post-translational modifications. Microsomes are vesicle-like bodies, not ordinarily present within living cells, which form from pieces of the endoplasmic reticulum (ER), plasma membrane, mitochondria, or Golgi apparatus of broken eukaryotic cells. Here we investigated the total phosphoproteome of mouse liver microsomes (MLMs) using TiO2 enrichment of phosphopeptides coupled to on-line 2D-LC-MS/MS. In total, 699 phosphorylation sites in 527 proteins were identified in MLMs. When compared with the current phosphoSitePlus database, 155 novel phosphoproteins were identified in MLM. The distributions of phosphosites were 89.4, 8.0, and 2.6% for phosphoserine, phosphotheronine, and phosphotyrosine, respectively. By Motif-X analysis, eight Ser motifs and one Thr motif were found, and five acidic, two basophilic-, and two proline-directed motifs were assigned. The potential functions of phosphoproteins in MLM were assigned by Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis. In GO annotation, phosphorylated microsomal proteins were involved in mRNA processing, mRNA metabolic processes, and RNA splicing. In the KEGG pathway analysis, phosphorylated microsomal proteins were highly enriched in ribosome protein processing in ER and ribosomes and in RNA transport. Furthermore, we determined that 52 and 23 phosphoproteins were potential substrates of cAMP-dependent protein kinase A and casein kinase II, respectively, many of which are 40S/60S ribosomal proteins. Overall, our results provide an overview of features of protein phosphorylation in MLMs that should be a valuable resource for the future understanding of protein synthesis or translation involving phosphorylation.