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Background: The differential diagnostic role of plasma developmental endothelial locus-1 (Del-1) was proposed in our previous study. Therefore the current study aimed to confirm the diagnostic role and explore the prognostic role of exosomal Del-1 in a prospective cohort of female patients with breast cancer., Patients and Methods: To determine the optimal sampling time for the postoperative Del-1 measurements, blood was serially collected on days 1, 3, 5, and 7 after surgery in 22 patients (cohort 1). Thereafter, 111 female patients with breast cancer were prospectively enrolled (cohort 2) to compare exosomal Del-1 levels before and after surgery., Results: Among the subsequent prospective cohort, 107 patients (96.4%) showed a high exosomal Del-1 level (optical density [OD] value > 0.5) at the time of diagnosis. Of these patients, 101 (94.6%) in this high-level group showed normalized Del-1 levels postoperatively, representing a significant difference (mean OD value, 1.232 vs. 0.196; P < .00001). High postoperative Del-1 level was significantly associated with a worse disease-free survival adjusted to the clinicopathological characteristics (hazard ratio, 24.0; P = .0011)., Conclusion: This study confirmed the normalization of exosomal Del-1 after surgery, indicating exosomal Del-1 as a potent diagnostic biomarker for breast cancer. In addition, because a high Del-1 level after surgery was associated with early relapse, this suggests exosomal Del-1 as a potential prognostic marker by identifying the existence of residual cancer., Competing Interests: Disclosure This study was supported by the National Research Foundation. The authors have stated that they have no conflicts of interest., (Copyright © 2021. Published by Elsevier Inc.)

The aim of this study was to identify potential proteomic biomarkers for chronic active antibody-mediated rejection (CAMR) in kidney transplant recipients (KTRs). Among 385 KTRs enrolled in a cross-sectional multicenter study, 26 KTRs with biopsy-proven CAMR, 57 KTRs with long-term graft survival (LGS), and 10 rejection-free matched KTRs were included. A proteomic approach was employed to measure urinary extracellular vesicle (EV) changes in the KTRs. The urinary EVs were trypsin-digested using a gel-assisted protocol and quantified by label-free liquid chromatography with tandem mass spectrometry, using a data-dependent acquisition (DDA) mode. Western blot analysis was performed to confirm the protein levels for each candidate biomarker. Analysis of the isolated EV proteins revealed 93 and 97 proteins in the CAMR and LGS patients, respectively. Proteins that were identical in both groups were excluded and only high-significance proteins with a fold change of at least 1.5 were selected as candidate biomarkers. Six proteins (APOA1, TTR, PIGR, HPX, AZGP1, and CP) that were distinguishable between CAMR and LGS were selected. The proteins were confirmed by immunoblot analyses using independently acquired urinary EV samples. AZGP1 in particular was found to be a CAMR-specific proteomic biomarker that was distinguishable from the rejection-free control group with matching kidney function, duration of transplantation, and age. We identified and validated six proteomic biomarkers for CAMR and clarified one CAMR-specific proteomic biomarker in KTRs. Further clinical trials are needed before these rejection-specific biomarkers can be applied for the early prediction, diagnosis, and monitoring of the clinical response of KTRs to the treatment of CAMR., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Aims: Peroxiredoxin5 (Prdx5), a thioredoxin peroxidase, is an antioxidant enzyme that is widely studied for its antioxidant properties and protective roles in neurological and cardiovascular disorders. This study is aimed at investigating the functional significance of Prdx5 in mitochondria and at analyzing its roles in ciliogenesis during the process of vertebrate development., Results: We found that several Prdx genes were strongly expressed in multiciliated cells in developing Xenopus embryos, and their peroxidatic functions were crucial for normal cilia development. Depletion of Prdx5 increased levels of cellular reactive oxygen species (ROS), consequently leading to mitochondrial dysfunction and abnormal cilia formation. Proteomic and transcriptomic approaches revealed that excessive ROS accumulation on Prdx5 depletion subsequently reduced the expression level of pyruvate kinase (PK), a key metabolic enzyme in energy production. We further confirmed that the promotor activity of PK was significantly reduced on Prdx5 depletion and that the reduction in PK expression and its promoter activity led to ciliary defects observed in Prdx5-depleted cells., Innovation: Our data revealed the novel relationship between ROS and Prdx5 and the consequent effects of this interaction on vertebrate ciliogenesis. The normal process of ciliogenesis is interrupted by the Prdx5 depletion, resulting in excessive ROS levels and suggesting cilia as vulnerable targets of ROS., Conclusion: Prdx5 plays protective roles in mitochondria and is critical for normal cilia development by regulating the levels of ROS. The loss of Prdx5 is associated with excessive production of ROS, resulting in mitochondrial dysfunction and aberrant ciliogenesis.

Inhibitors of the secretion of cancer exosomes, which promote cancer progression and metastasis, may not only accelerate exosome biology research but also offer therapeutic benefits for cancer patients. Here we identify sulfisoxazole (SFX) as an inhibitor of small extracellular vesicles (sEV) secretion from breast cancer cells through interference with endothelin receptor A (ETA). SFX, an FDA-approved oral antibiotic, showed significant anti-tumor and anti-metastatic effects in mouse models of breast cancer xenografts, the reduced expression of proteins involved in biogenesis and secretion of sEV, and triggered co-localization of multivesicular endosomes with lysosomes for degradation. We demonstrate the important role of ETA, as target of SFX, by gain- and loss-of-function studies of the ETA protein, through a direct binding assay, and pharmacological and genetic approaches. These findings may provide a foundation for sEV-targeted cancer therapies and the mechanistic studies on sEV biology.

Background: Small extracellular vesicles (small-EVs) are membranous vesicles that contain unique information regarding the condition of cells and contribute to the recruitment and reprogramming of components associated with the tumor environment. Therefore, many researchers have suggested that small-EV proteins are potential biomarkers for diseases such as cancer. Colon cancer (CC) is one of the most common causes of cancer-related deaths worldwide. Biomarkers such as carcinoembryonic antigen (CEA) show low sensitivity (~ 40%), and thus the demand for novel biomarkers for CC diagnosis is increasing., Methods: In this study, we identified biomarkers for diagnosing CC through proteomic analysis of small-EVs from CC cell lines. These small-EVs were characterized by western blot analysis, nanoparticle tracking analysis, and transmission electron microscopy and analyzed using mass spectrometry., Results: Five selected proteins were found to be upregulated in CC by western blot analysis. Among the candidate proteins, tetraspanin 1 (TSPAN1) was found to be upregulated in plasma EVs from CC patients compared to those from healthy controls (HCs) with 75.7% sensitivity., Conclusions: These results suggest that TSPAN1 is a potent non-invasive biomarker for CC detection. Our experimental strategy provides useful insights into the identification of cancer-specific non-invasive biomarkers.

Exosomes are small extracellular membrane vesicles released from endosomes of various cells and could be found in most body fluids. The main functions of exosomes have been recognized as important mediators of intercellular communication and as potential biomarkers of various disease states. This study investigated whether exogenous exosomes from mice with acetaminophen (APAP)-induced liver injury can damage the recipient hepatic cells or promote hepatotoxicity in mice. We observed that exogenous exosomes derived from APAP-exposed mice were internalized into the primary mouse hepatocytes or HepG2 hepatoma cells and significantly decreased the viability of these recipient cells. They also elevated mRNA transcripts and proteins associated with the cell death signaling pathways in primary hepatocytes or HepG2 cells via exosomes-to-cell communications. In addition, confocal microscopy of ex vivo liver section showed that exogenously added exosomes were accumulated in recipient hepatocytes. Furthermore, plasma reactive oxygen species and hepatic TNF-α/IL-1β production were elevated in APAP-exosomes recipient mice compared to control-exosomes recipient mice. The levels of apoptosis-related proteins such as phospho-JNK/JNK, Bax, and cleaved caspase-3 were increased in mouse liver received APAP-exosomes. These results demonstrate that exogenous exosomes from APAP-exposed mice with acute liver injury are functional and stimulate cell death or toxicity of the recipient hepatocytes and mice.

Drug- and alcohol-induced liver injury are a leading cause of liver failure and transplantation. Emerging evidence suggests that extracellular vesicles (EVs) are a source of biomarkers because they contain unique proteins reflecting the identity and tissue-specific origin of the EV proteins. This study aimed to determine whether potentially hepatotoxic agents, such as acetaminophen (APAP) and binge alcohol, can increase the amounts of circulating EVs and evaluate liver-specific EV proteins as potential biomarkers for liver injury. The circulating EVs, isolated from plasma of APAP-exposed, ethanol-fed mice, or alcoholic hepatitis patients versus normal control counterparts, were characterized by proteomics and biochemical methods. Liver specific EV proteins were analyzed by immunoblots and ELISA. The amounts of total and liver-specific proteins in circulating EVs from APAP-treated mice significantly increased in a dose- and time-dependent manner. Proteomic analysis of EVs from APAP-exposed mice revealed that the amounts of liver-specific and/or hepatotoxic proteins were increased compared to those of controls. Additionally, the increased protein amounts in EVs following APAP exposure returned to basal levels when mice were treated with N-acetylcysteine or glutathione. Similar results of increased amounts and liver-specific proteins in circulating EVs were also observed in mice exposed to hepatotoxic doses of thioacetamide or d-galactosamine but not by non-hepatotoxic penicillin or myotoxic bupivacaine. Additionally, binge ethanol exposure significantly elevated liver-specific proteins in circulating EVs from mice and alcoholics with alcoholic hepatitis, compared to control counterparts. These results indicate that circulating EVs in drug- and alcohol-mediated hepatic injury contain liver-specific proteins that could serve as specific biomarkers for hepatotoxicity.

Ubiquitination, a post-translational modification, involves the covalent attachment of ubiquitin to the target protein. The ubiquitin-proteasome pathway and the endosome-lysosome pathway control the degradation of the majority of eukaryotic proteins. Our previous study illustrated that δ-catenin ubiquitination occurs in a glycogen synthase kinase-3 (GSK-3) phosphorylation-dependent manner. However, the molecular mechanism of δ-catenin ubiquitination is still unknown. Here, we show that the lysine residues required for ubiquitination are located mainly in the C-terminal portion of δ-catenin. In addition, we provide evidence that β-TrCP-1 interacts with δ-catenin and functions as an E3 ligase, mediating δ-catenin ubiquitin-proteasome degradation. Furthermore, we prove that both the ubiquitin-proteasome pathway and the lysosome degradation pathway are involved in δ-catenin degradation. Our novel findings on the mechanism of δ-catenin ubiquitination will add a new perspective to δ-catenin degradation and the effects of δ-catenin on E-cadherin involved in epithelial cell-cell adhesion, which is implicated in prostate cancer progression., (Copyright © 2016. Published by Elsevier B.V.)

Extracellular vesicles (EVs) secreted from cancer cells have potential for generating cancer biomarker signatures. Fibronectin (FN) was selected as a biomarker candidate, due to the presence in surface on EVs secreted from human breast cancer cell lines. A subsequent study used two types of enzyme-linked immunosorbent assays (ELISA) to determine the presence of these proteins in plasma samples from disease-free individuals (n=70), patients with BC (n=240), BC patients after surgical resection (n=40), patients with benign breast tumor (n=55), and patients with non-cancerous diseases (thyroiditis, gastritis, hepatitis B, and rheumatoid arthritis; n=80). FN levels were significantly elevated (p< .0001) at all stages of BC, and returned to normal after tumor removal. The diagnostic accuracy for FN detection in extracellular vesicles (ELISA method 1) (area under the curve, 0.81; 95% CI, 0.76 to 0.86; sensitivity of 65.1% and specificity of 83.2%) were also better than those for FN detection in the plasma (ELISA method 2) (area under the curve, 0.77; 95% CI, 0.72 to 0.83; sensitivity of 69.2% and specificity of 73.3%) in BC. The diagnostic accuracy of plasma FN was similar in both the early-stage BC and all BC patients, as well as in the two sets. This liquid biopsy to detect FN on circulating EVs could be a promising method to detect early breast cancer., Competing Interests: The authors have declared that no competing interests exist.

Purpose: Currently, there are no molecular biomarkers for the early detection of breast cancer. This study focused on identifying surface proteins found on circulating extracellular vesicles (EVs) for detecting early-stage breast cancer., Experimental Design: Circulating EVs, isolated from the plasma of 10 patients with breast cancer (stages I and II) and 5 healthy controls, were analyzed using LC-MS/MS. Developmental endothelial locus-1 protein (Del-1) was selected as a candidate biomarker. Two different ELISAs were used to measure Del-1 in plasma samples from healthy controls (n= 81), patients with breast cancer (n= 269), breast cancer patients after surgical resection (n= 50), patients with benign breast tumors (n= 64), and patients with noncancerous diseases (n= 98) in two cohorts., Results: Plasma Del-1 levels were significantly higher (P< 0.0001) in patients with breast cancer than in all controls and returned to almost normal after tumor removal. The diagnostic accuracy of Del-1 was AUC, 0.961 [95% confidence interval (CI), 0.924-0.983], sensitivity of 94.70%, and specificity of 86.36% in test cohort and 0.968 (0.933-0.988), 92.31%, and 86.62% in validation cohort for early-stage breast cancer by one type of ELISA. Furthermore, Del-1 maintained diagnostic accuracy for patients with early-stage breast cancer using the other type of ELISA [0.946 (0.905-0.972), 90.90%, and 77.14% in the test cohort; 0.943 (0.900-0.971), 89.23%, and 80.99% in the validation cohort]., Conclusions: Del-1 on circulating EVs is a promising marker to improve identification of patients with early-stage breast cancer and distinguish breast cancer from benign breast tumors and noncancerous diseases., (©2015 American Association for Cancer Research.)

Cancer cell-derived extracellular vesicles have been linked to the pathogenesis of various cancers; however, the role of extracellular vesicles in tumorigenesis remains unclear. To identify extracellular vesicle proteins involved in cancer metastasis, quantitative proteomic analyses were performed on extracellular vesicles derived from two representative breast cancer cell lines: the less invasive MCF-7 and the invasive MDA-MB-231. Proteomic analysis allowed for the identification of 270 proteins in the extracellular vesicles. Here we report a new function of EDIL3 on extracellular vesicles, which are sufficient for enhancement of cell invasion and for acceleration of lung metastasis in vivo. This invasion is most likely mediated via the integrin-FAK signaling cascade in breast cancer cells. However, these effects are suppressed when EDIL3 is inactivated, providing evidence for a critical role of EDIL3 in development of cancer. Consistently, in human patients with metastatic breast cancer, the levels of EDIL3 on circulating extracellular vesicles are significantly elevated. This information is a remarkable breakthrough in understanding of the molecular mechanism underlying metastasis of breast cancer as well as in the research for cancer biomarkers using circulating extracellular vesicles. Furthermore, targeting EDIL3 on extracellular vesicles may lead to a new therapeutic option for treatment of breast cancer., (Copyright © 2015 Elsevier B.V. All rights reserved.)

O-linked β-N-acetylglucosaminylation (O-GlcNAcylation) regulates many cellular processes including the cell cycle, cell signaling, and protein trafficking. Dysregulation of O-GlcNAcylation may be involved in the development of insulin resistance and type 2 diabetes. Therefore, it is necessary to identify cellular proteins that are induced by elevated O-GlcNAcylation. Here, using adenosine 5'-triphosphate affinity chromatography, we employed a proteomic approach in order to identify differentially expressed proteins in response to treatment with the O-GlcNAcase inhibitor, O-(2-acetamido-2-deoxy-d-glucopyranosylidene)amino-N-phenylcarbamate (PUGNAc), in mouse C2C12 myotube cells. Among 205 selected genes, we identified 68 nucleotide-binding proteins, 14 proteins that have adenosinetriphosphatase activity, and 10 proteins with ligase activity. Upregulation of proteins, including ubiquitin-activating enzyme E1, proteasome subunit 20S, cullin-associated NEDD8-dissociated protein 1, ezrin, and downregulation of the protein nucleoside diphosphate kinase B, were confirmed by western blot analysis. In particular, we found that the protein ubiquitination level in C2C12 cells was increased by PUGNAc treatment. This is the first report of quantitative proteomic profiles of myotube cells after treatment with PUGNAc, and our results demonstrate the potential to enhance understanding of the relationship between insulin resistance, O-GlcNAc, and PUGNAc in the future., (Copyright © 2015 John Wiley & Sons, Ltd.)

Extracellular vesicles (EVs) such as exosomes are secretory vesicles that act as autocrine, paracrine, or endocrine messengers; mediate intercellular cross-talk; and carry a cargo of various proteins. Because EVs can be transported to recipient cells via circulation, many researchers have been studying EVs from immune cells or cancer cells. Adipocytes are also considered endocrine cells and secrete adipokines such as adiponectin, regulating a variety of intracellular signaling pathways. Expansion of adipose tissue in obesity alters adipokine secretion, thereby increasing the risk of metabolic diseases. Characterization of adipocyte-derived exosomes is necessary to explain the communication between adipocytes and other cell types. In the present study, to identify proteins associated with adipocyte-derived exosomes, we isolated exosomes from adipose tissue of obese diabetic and obese nondiabetic rats. We identified proteins by analyzing exosomes from obese rats with type 2 diabetes and their matched control littermates using nano-liquid chromatography with tandem mass spectrometry coupled with label-free relative quantification. We identified 509 proteins from adipocytes including 81 known adipokines; ~78% of all the identified proteins were categorized as exosome-associated proteins. Among the protein profiles, we uncovered 128 upregulated and 72 downregulated proteins, which are differentially expressed in OLETF adipocyte-derived exosomes. This study seems to demonstrate for the first time hundreds of proteins in exosomes released by adipocytes in obese rats and rats with type 2 diabetes. Thus, protein profiles of exosomes from adipocytes possibly indicate the transmission of signals as part of cell-cell communication and should further our understanding of obesity- and diabetes-related diseases.

Matrix metalloproteinases (MMPs) are zinc-dependent endopeptidases that regulate cell-matrix composition and are also involved in processing various bioactive molecules such as cell-surface receptors, chemokines, and cytokines. Our group recently reported that MMP-3, -8, and -9 are upregulated during microglial activation and play a role as proinflammatory mediators (Lee et al., 2010, 2014). In particular, we demonstrated that MMP-8 has tumor necrosis factor alpha (TNF-α)-converting enzyme (TACE) activity by cleaving the prodomain of TNF-α and that inhibition of MMP-8 inhibits TACE activity. The present study was undertaken to compare the effect of MMP-8 inhibitor (M8I) with those of inhibitors of other MMPs, such as MMP-3 (NNGH) or MMP-9 (M9I), in their regulation of TNF-α activity. We found that the MMP inhibitors suppressed TNF-α secretion from lipopolysaccharide (LPS)-stimulated BV2 microglial cells in an order of efficacy: M8I>NNGH>M9I. In addition, MMP inhibitors suppressed the activity of recombinant TACE protein in the same efficacy order as that of TNF-α inhibition (M8I>NNGH>M9I), proving a direct correlation between TACE activity and TNF-α secretion. A subsequent pro-TNF-α cleavage assay revealed that both MMP-3 and MMP-9 cleave a prodomain of TNF-α, suggesting that MMP-3 and MMP-9 also have TACE activity. However, the number and position of cleavage sites varied between MMP-3, -8, and -9. Collectively, the concurrent inhibition of MMP and TACE by NNGH, M8I, or M9I may contribute to their strong anti-inflammatory and neuroprotective effects.

Glutathione S-transferase π-1 (GSTP-1) is a member of the glutathione S-transferase enzyme superfamily, which catalyzes the conjugation of electrophiles to glutathione during the process of detoxification. In this study, the epigenetic alterations of GSTP-1 expression in human colorectal cancers and the underlying mechanisms were investigated. In 10 colon cancer patients, proteomic analysis revealed that expression of GSTP-1 protein was higher in tumor tissues than in paired adjacent normal tissues. Likewise, in 7 of 10 colon cancer patients, GSTP-1 protein expression was more than 1.5-fold higher in tumor tissues than in adjacent normal tissues, as determined by western blotting. Immunohistochemical data confirmed that GSTP-1 protein was expressed at higher levels in colon cancer tissues compared to normal mucosa. GSTP-1 enzyme activity was closely correlated with GSTP-1 protein expression in colon cancer patients. Consistent with this, GSTP-1 mRNA, protein and activity levels were higher in the colorectal cancer cell lines Caco-2, HCT-116, HT-29, SNU-407 and SNU-1033 compared to the normal colon cell line FHC. Methylation-specific PCR results indicated that the high levels of GSTP-1 in human colorectal cancer cell lines were likely due to the lower degree of promoter methylation in colon cancer cell lines compared to the normal colon cell line, consistent with findings in colon cancer patients. Moreover, the levels of specific activator-protein complexes and histone marks were higher in human colorectal cancer cells compared to the normal human colon cell line, whereas the repressor protein complexes exhibited the opposite pattern. Furthermore, chromatin immunoprecipitation assays demonstrated that expression levels of the transcription factors AP-1 and SP-1 were correlated with the upregulation of GSTP-1 expression in colorectal cancer cells. Finally, knockdown of GSTP-1 promoted the sensitivity of SNU-407 cells to the anticancer agent 5-fluorouracil. These data indicate that GSTP-1 may serve as a clinically useful biomarker of colon cancer and a target for anti-colon cancer drugs.

Matrix metalloproteinases (MMPs) play important roles in normal brain development and synaptic plasticity, although aberrant expression of MMPs leads to brain damage, including blood-brain barrier disruption, inflammation, demyelination, and neuronal cell death. In this article, we report that MMP-8 is upregulated in LPS-stimulated BV2 microglial cells and primary cultured microglia, and treatment of MMP-8 inhibitor (M8I) or MMP-8 short hairpin RNA suppresses proinflammatory molecules, particularly TNF-α secretion. Subsequent experiments showed that MMP-8 exhibits TNF-α-converting enzyme (TACE) activity by cleaving the prodomain of TNF-α (A(74)/Q(75), A(76)/V(77) residues) and, furthermore, that M8I inhibits TACE activity more efficiently than TAPI-0, a general TACE inhibitor. Biochemical analysis of the underlying anti-inflammatory mechanisms of M8I revealed that it inhibits MAPK phosphorylation, NF-κB/AP-1 activity, and reactive oxygen species production. Further support for the proinflammatory role of microglial MMP-8 was obtained from an in vivo animal model of neuroinflammatory disorder. MMP-8 is upregulated in septic conditions, particularly in microglia. Administration of M8I or MMP-8 short hairpin RNA significantly inhibits microglial activation and expression/secretion of TNF-α in brain tissue, serum, and cerebrospinal fluid of LPS-induced septic mice. These results demonstrate that MMP-8 critically mediates microglial activation by modulating TNF-α activity, which may explain neuroinflammation in septic mouse brain., (Copyright © 2014 by The American Association of Immunologists, Inc.)

Azathioprine, an immunosuppressant, has gained a prominent position in the clinic for prevention of graft rejection in organ transplants, as well as dermatological autoimmune diseases. However, according to a number of research reports, hepatotoxicity, as one of the side effects, is a major obstacle in azathioprine therapy. In this study, an integrated toxicoproteomic and toxicotranscriptomic analysis was performed using rat primary hepatocytes, in order to gain insight into the in-depth pathway map related to azathioprine-induced hepatotoxicity. For proteomic and transcriptomic analysis, rat primary hepatocytes were exposed to azathioprine at IC20 concentration for 24 h. In particular, 2D LC-MS/MS and informatics-assisted label-free strategy for proteomic analysis were applied in order to increase the number of identified proteins and to improve the confidence of the quantitation results. Among 119 differentially identified protein species, 69 were upregulated and 50 were downregulated in the azathioprine-treated group. At the mRNA level, results of transcriptomic analysis showed increased transcription of 340 genes and decreased transcription of 63 genes in the azathioprine-treated group. Based on the analysis of transcriptomic and proteomic results using the DAVID program, drug metabolism/oxidative stress enzymes, xenobiotic metabolism by cytochrome P450, fatty acid metabolism, primary bile acid biosynthesis, contraction, inflammation metabolism, and mitogen-activated protein kinase (MAPK) kinase (ERK/JNK/p38 kinase) pathways were affected in azathioprine-treated hepatotoxicity. The effects on genes and proteins related to several important pathways were confirmed by real-time PCR and immunoblot analysis, respectively. This study is the first to report on relevant pathways related to azathioprine-induced hepatotoxicity through performance of integrated transcriptomic and proteomic analyses., (© 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

O-GlcNAc (2-acetamino-2-deoxy-β-D-glucopyranose), an important modification for cellular processes, is catalyzed by O-GlcNAc transferase and O-GlcNAcase. O-(2-acetamido-2-deoxy-D-glucopyranosylidene) amino-N-phenylcarbamate (PUGNAc) is a nonselective inhibitor of O-GlcNAcase, which increases the level of protein O-GlcNAcylation and is known to induce insulin-resistance in adipose cells due to uncharacterized targets of this inhibitor. In this study, using ATP affinity chromatography, we applied a targeted proteomic approach for identification of proteins induced by treatment with PUGNAc. For optimization of proteomic methods using ATP affinity chromatography, comparison of two cell lines (3T3-L1 adipocytes and C2C12 myotubes) and two different digestion steps was performed using four different structures of immobilized ATP-bound resins. Using this approach, based on DNA sequence homologies, we found that the identified proteins covered almost half of ATP-binding protein families classified by PROSITE. The optimized ATP affinity chromatography approach was applied for identification of proteins that were differentially expressed in 3T3-L1 adipocytes following treatment with PUGNAc. For label-free quantitation, a gel-assisted method was used for digestion of the eluted proteins, and analysis was performed using two different MS modes, data-independent (671 proteins identified) and data-dependent (533 proteins identified) analyses. Among identified proteins, 261 proteins belong to nucleotide-binding proteins and we focused on some nucleotide-binding proteins, ubiquitin-activation enzyme 1 (E1), Hsp70, vasolin-containing protein (Vcp), and Hsp90, involved in ubiquitin-proteasome degradation and insulin signaling pathways. In addition, we found that treatment with PUGNAc resulted in increased ubiquitination of proteins in a time-dependent manner, and a decrease in both the amount of Akt and the level of phosphorylation of Akt, a key component in insulin signaling, through downregulation of Hsp90. In this study, based on a targeted proteomic approach using ATP affinity chromatography, we found four proteins related to ubiquitination and insulin signaling pathways that were induced by treatment with PUGNAc. This result would provide insight into understanding functions of PUGNAc in 3T3-L1 cells., (© 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Mercury is a well-recognized health hazard and a deleterious environmental contaminant. Exposure to mercury can cause neurotoxic manifestations, nephrotoxicity, and immune function alterations; however, the mechanisms and related proteins responsible for these effects are still unclear. Our goal is to understand the relationship between the toxicity of mercury and the proteins affected by this toxic heavy metal and to define biomarkers for mercury intoxication. Two different forms of mercury, organic methylmercury or inorganic mercury sulfide, were orally administered to the mice for 4 weeks. To reduce complexity of the serum proteome, we enriched glycoproteins from mice serum with lectin concanavalin A resin, and the tryptic peptides of the purified glycoproteins were subjected to nanoultra performance liquid chromatography-Quadrupole time-of-flight for identification and label-free quantification. In this study, we characterized approximately 209 proteins from mice serum, and, among them, 21 proteins were differentially expressed in organic methylmercury-treated mice serum compared with the control group. Two proteins, serum amyloid P component (SAP) and inter α-trypsin inhibitor heavy chain 4 (ITI-H4), were upregulated in organic methylmercury-treated mice and confirmed with different doses of both types of mercury by Western blot analysis. Results of immunohistochemistry also confirmed the validity of SAP and ITI-H4 as biomarker candidates for organic methylmercury exposure. Findings of this study may assist in understanding the relationship between toxicity of mercury and upregulated proteins in mouse serum. Furthermore, the proteins identified here might be used as biomarker candidates in mercury intoxication.

Androgenetic alopecia (AGA) is the most common and progressive disorder of hair loss with psychological effects. However, the exact mechanisms of baldness have not yet been elucidated. Until now, there has been no report using current proteomic approaches to examine balding and non-balding mesenchyme-derived DPCs simultaneously. To achieve the goal of identifying differentially expressed proteins in balding DPCs compared to non-balding DPCs, we present a strategy combining gel-assisted digestion and automatic on-line two-dimensional reversed phase-reversed phase (2D RP-RP) LC-MS/MS with informatics-assisted label-free protein quantitation. This strategy efficiently quantified the proteins of balding and non-balding DPCs from patients, and 128 up-regulated and 12 down-regulated proteins among 690 distinct proteins were identified in balding DPCs compared to non-balding DPCs. Up-regulated proteins belonging to these pathways in the balding DPCs, including argininosuccinate synthase 1 (ASS1), phosphoribosylaminoimidazole carboxylase (PAICS; ADE2), cytoskeleton-associated protein 4 (CKAP4), gelsolin (GSN), Ras GTPase-activating-like protein (IQGAP1), and S-phase kinase-associated protein 1 (SKP1), were confirmed by Western blot analysis. Gelsolin (GSN), Ras GTPase-activating-like protein (IQGAP1), plectin-1 (PLEC1), and nonerythroid α-spectrin (SPTAN1) were confirmed by immunofluorescence analysis. This proteomic approach to DPCs should help in understanding the pathogenesis of AGA., Biological Significance: The results demonstrated the first deep proteomic approach to mesenchyme-derived dermal papilla cells (DPCs), which are a useful model system to investigate the mechanisms of baldness, from human hair and also identified differentially expressed proteins in balding DPCs compared to non-balding DPCs. This study should help in understanding the pathogenesis of androgenetic alopecia (AGA) and furthermore some proteins differentially expressed could be studied as therapeutic targets for AGA., (Crown Copyright © 2013. Published by Elsevier B.V. All rights reserved.)

Hepatocytes are used widely as a cell model for investigation of xenobiotic metabolism and the toxic mechanism of drugs. Simvastatin is the first statin drug used extensively in clinical practice for control of elevated cholesterol or hypercholesterolemia. However, it has also been reported to cause adverse effects in liver due to cellular damage. In this study, for proteomic and transcriptomic analysis, rat primary hepatocytes were exposed to simvastatin at IC20 concentration for 24 h. Among a total of 607 differentially expressed proteins, 61 upregulated and 29 downregulated proteins have been identified in the simvastatin-treated group. At the mRNA level, results of transcriptomic analysis revealed 206 upregulated and 41 downregulated genes in the simvastatin-treated group. Based on results of transcriptomic and proteomic analysis, NRF2-mediated oxidative stress response, xenobiotics by metabolism of cytochrome P450, fatty acid metabolism, bile metabolism, and urea cycle and inflammation metabolism pathways were focused using IPA software. Genes (FASN, UGT2B, ALDH1A1, CYP1A2, GSTA2, HAP90, IL-6, IL-1, FABP4, and ABC11) and proteins (FASN, CYP2D1, UG2TB, ALDH1A1, GSTA2, HSP90, FABP4, and ABCB11) related to several important pathways were confirmed by real-time PCR andWestern blot analysis, respectively. This study will provide new insight into the potential toxic pathways induced by simvastatin., (© 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

This study presents a proteomic method that differentiates between matched normal and breast tumor tissues from ductal carcinoma in situ (DCIS) and invasive carcinoma from Korean women, to identify biomarker candidates and to understand pathogenesis of breast cancer in protein level. Proteins from tissues obtained by biopsy were extracted by RIPA buffer, digested by the gel-assisted method, and analyzed by nano-UPLC-MS/MS. From proteomic analysis based on label-free quantitation strategy, a non-redundant list of 298 proteins was identified from the normal and tumor tissues, and 244 proteins were quantified using IDEAL-Q software. Hierarchical clustering analysis showed two patterns classified as two groups, invasive carcinoma and DCIS, suggesting a difference between two carcinoma at the protein expression level as expected. Differentially expressed proteins in tumor tissues compared to the corresponding normal tissues were related to three biological pathways: antigen-processing and presentation, glycolysis/gluconeogenesis, and complement and coagulation cascades. Among them, the up-regulation of calreticulin (CRT) and protein disulfide isomerase A3 (PDIA3) was confirmed by Western blot analysis. In conclusion, this study showed the possibility of identifying biomarker candidates for breast cancer using tissues and might help to understand the pathophysiology of this cancer at the protein level.

Chemotherapy-induced apoptosis of tumor cells enhances the antigen presentation and sensitizes tumor cells to T cell-mediated cytotoxicity. Here we harnessed the apoptosis of tumor cells as a homing signal for the delivery of T cells to tumor. Jurkat T cells were anchored with ApoPep-1, an apoptosis-targeted peptide ligand, using the biocompatible anchor for membrane (BAM), an oleyl acid derivative. The ApoPep-1-BAM conjugate was efficiently anchored to cell membrane, while little anchoring was obtained with ApoPep-1 alone. The retention period of the ApoPep-1-BAM conjugate on cell membrane was approximately 80 and 40 min in the absence and presence of serum, respectively. ApoPep-1 was resistant to degradation in serum until 2h. The apoptosis-targeted T cells that were anchored with the ApoPep-1-BAM preferentially bound to apoptotic tumor cells over living cells. When intravenously injected into tumor-bearing mice, the number of apoptosis-targeted T cells and in vivo fluorescence signals by the homing of the cells to doxorubicin-treated tumor were higher than those of untargeted T cells. Accumulation of apoptosis-targeted T cells at other organs such as liver was not detected. These results suggest that the chemotherapy-induced apoptosis and subsequent enhancement of T cell delivery to tumor by the membrane anchoring of the apoptosis-targeted peptide could be a novel strategy for cancer immunotherapy., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Cisplatin is used widely for treatment of a variety of cancer diseases. Recently, however, the use of cisplatin is restricted because of its adverse effects such as hepatotoxicity. There is no study with current proteomics technology to evaluate cisplatin-induced hepatotoxicity, even if some studies have reported on the hepatotoxicity. In this study, proteomic as well as genomic analyses have been used for identification of proteins and genes that respond to cisplatin treatment in rat primary hepatocytes. To investigate the hepatotoxic effects of cisplatin, rat primary hepatocytes were treated with an IC(20) concentration for 24 h. From proteomic analysis based on label-free quantitation strategy, cisplatin induced 76 up-regulated and 19 down-regulated proteins among 325 distinct proteins. In the mRNA level, genomic analysis revealed 72 up-regulated and 385 down-regulated genes in the cisplatin-treated group. Based on these two analyses, 19 pathways were commonly altered, whereas seven pathways were identified only by proteomic analysis, and 19 pathways were identified only by genomic analysis. Overall, this study explained the mechanism of cisplatin-induced hepatotoxicity with two points of view: well known pathways including drug metabolism, fatty acid metabolism, and glycolysis/TCA cycle and little known pathways including urea cycle and inflammation metabolism, for hepatotoxicity of other toxic agents. Up-regulated proteins detected by proteomic analysis in the cisplatin-treated group: FBP1 (fructose 1,6-bisphosphatase 1), FASN (fatty acid synthase), CAT (catalase), PRDX1 (peroxiredoxin-1), HSPD1 (60-kDa heat shock protein), MDH2 (malate dehydrogenase 2), and ARG1 (arginase 1), and also down-regulated proteins in the cisplatin-treated group: TPM1 (tropomyosin 1), TPM3 (tropomyosin 3), and CTSB (cathepsin B), were confirmed by Western blot analysis. In addition, up-regulated mRNAs detected by microarray analysis in the cisplatin-treated group: GSTA2, GSTT2, YC2, TXNRD1, CYP2E1, CYP2C13, CYP2D1, ALDH17, ARG1, ARG2, and IL-6, and also down-regulated mRNAs: CYP2C12, CYP26B1, TPM1, and TPM3, were confirmed by RT-PCR analysis. In case of PRDX1, FASN, and ARG1, they were further confirmed by immunofluorescence analysis. Through the integrated proteomic and genomic approaches, the present study provides the first pathway map related to cisplatin-induced hepatotoxicity, which may provide new insight into the mechanism of hepatotoxicity.

A number of highly abundant proteins in urine have been identified through proteomics approaches, and some have been considered as disease-biomarker candidates. These molecules might be clinically useful in diagnosis of various diseases. However, none has proven to be specifically indicative of perturbations of cellular processes in cells associated with urogenital diseases. Exosomes could be released into urine which flows through the kidney, ureter, bladder and urethra, with a process of filtration and reabsorption. Urinary exosomes have been recently suggested as alternative materials that offer new opportunities to identify useful biomarkers, because these exosomes secreted from epithelial cells lining the urinary track might reflect the cellular processes associated with the pathogenesis of diseases in their donor cells. Proteomic analysis of such urinary exosomes assists the search of urinary biomarkers reflecting pathogenesis of various diseases and also helps understanding the function of urinary exosomes in urinary systems. Thus, it has been recently suggested that urinary exosomes are one of the most valuable targets for biomarker development and to understand pathophysiology of relevant diseases., (Copyright © 2011 Wiley Periodicals, Inc.)

To identify biomarker candidates associated with early IgA nephropathy (IgAN) and thin basement membrane nephropathy (TBMN), the most common causes presenting isolated hematuria in childhood, a proteomic approach of urinary exosomes from early IgAN and TBMN patients was introduced. The proteomic results from the patients were compared with a normal group to understand the pathophysiological processes associated with these diseases at the protein level. The urinary exosomes, which reflect pathophysiological processes, collected from three groups of young adults (early IgAN, TBMN, and normal) were trypsin-digested using a gel-assisted protocol, and quantified by label-free LC-MS/MS, using an MS(E) mode. A total of 1877 urinary exosome proteins, including cytoplasmic, membrane, and vesicle trafficking proteins, were identified. Among the differentially expressed proteins, four proteins (aminopeptidase N, vasorin precursor, α-1-antitrypsin, and ceruloplasmin) were selected as biomarker candidates to differentiate early IgAN from TBMN. We confirmed the protein levels of the four biomarker candidates by semi-quantitative immunoblot analysis in urinary exosomes independently prepared from other patients, including older adult groups. Further clinical studies are needed to investigate the diagnostic and prognostic value of these urinary markers for early IgAN and TBMN. Taken together, this study showed the possibility of identifying biomarker candidates for human urinary diseases using urinary exosomes and might help to understand the pathophysiology of early IgAN and TBMN at the protein level., (Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Rosiglitazone, one of the thiazolidinedione (TZD), is an oral antidiabetic drug that activates a gamma isoform of peroxisome proliferator-activated receptor (PPARγ). To identify target proteins induced by rosiglitazone in adipocytes, we first performed simultaneous in-depth proteomic profiling of cytosolic proteins and secreted proteins (secretome) from 3T3-L1 adipocytes using a label-free quantification method with nano-UPLC MS/MS. In total, we identified 646 proteins from 3T3-L1 adipocytes, of which 172 and 162 proteins were upregulated and downregulated >1.5-fold, respectively, in rosiglitazone-treated cells, as compared to controls. Some differentially expressed proteins in particular, including fatty acid translocase (FAT)/CD36, fatty acid binding protein, lipoprotein lipase, acetyl CoA acyltransferase, carnitine O-palmitoyltransferase 2, sterol carrier protein, adiponectin, and phosphoenolpyruvate carboxykinase could explain the current action mechanism of TZDs. Furthermore, this study is the first to report on two potential target proteins of rosiglitazone, such as adenomatosis polyposis coli 2 (APC2), and eukaryotic translation initiation factor 5A-1 (eIF5A) related to apoptosis and cell division. Our data clearly suggest that in-depth proteomic approaches using cytosolic and secreted proteins are important and necessary for identification of drug targets at the protein level.

The mutation or overexpression of alpha-synuclein protein plays a pivotal role in the pathogenesis of Parkinson's disease. In our preliminary experiments, we found that alpha-synuclein induced the expression of matrix metalloproteinases (MMPs) (MMP-1, -3, -8, and -9) in rat primary cultured microglia. Thus, the current study was undertaken to determine the roles of MMPs in alpha-synuclein-induced microglial activation. The inhibition of MMP-3, -8, or -9 significantly reduced NO and reactive oxygen species levels and suppressed the expression of TNF-alpha and IL-1beta. Notably, MMP-8 inhibitor suppressed TNF-alpha production more efficaciously than MMP-3 or MMP-9 inhibitors. Inhibition of MMP-3 or -9 also suppressed the activities of MAPK, NF-kappaB, and AP-1. Previously, protease-activated receptor-1 (PAR-1) has been associated with the actions of MMPs, and thus, we further investigated the role of PAR-1 in alpha-synuclein-induced inflammatory reactions. A PAR-1-specific inhibitor and a PAR-1 antagonist significantly suppressed cytokine levels, and NO and reactive oxygen species production in alpha-synuclein-treated microglia. Subsequent PAR-1 cleavage assay revealed that MMP-3, -8, and -9, but not alpha-synuclein, cleaved the synthetic peptide containing conventional PAR-1 cleavage sites. These results suggest that MMPs secreted by alpha-synuclein-stimulated microglia activate PAR-1 and amplify microglial inflammatory signals in an autocrine or paracrine manner. Furthermore, our findings suggest that modulation of the activities of MMPs and/or PAR-1 may provide a new therapeutic strategy for Parkinson's disease.

Many different types of urine proteome studies have been done, but urine glycoprotein studies are insufficient. Therefore, we studied the glycoproteins from rat urine, which could be used to identify biomarkers in an animal model. First, urinary proteins were prepared by using the dialysis and lyophilizing methods from rat urine. Glycoproteins enriched with lectin affinity purification, concanavalin A, jacalin and wheat germ agglutinin from the urinary proteins were separated by means of reverse-phase fast protein LC (FPLC) or 1-D PAGE. Each FPLC fraction and 1-D PAGE gel band were trypsin-digested and analyzed by means of nanoLC-MS/MS. LC-MS/MS analyses were carried out by using linear ion trap MS. A total of 318 rat urinary glycoproteins were identified from the FPLC fractions and gel bands; approximately 90% of identified proteins were confirmed as glycoproteins in Swiss-Prot. Many glycoproteins, known as biomarkers, including C-reactive protein, uromodulin, amyloid beta A4 protein, alpha-1-inhibitor 3, vitamin D-binding protein, kallikrein 3 and fetuin-A were identified in this study. By studying urinary glycoproteins collected from rat, these results may help to assist in identifying urinary biomarkers regarding various types of disease models.