Identification of differentially expressed proteins by treatment with PUGNAc in 3T3‐L1 adipocytes through analysis of ATP‐binding proteome

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.     

Proteomics and PUGNAcity will overcome questioning of insulin resistance induction by nonselective inhibition of O‐GlcNAcase

PTMs are the ultimate elements that perfect the existence and the activity of proteins. Owing to PTM, not less than 500 millions biological activities arise from approximately 20 000 protein‐coding genes in human. Hundreds of PTM were characterized in living beings among which is a large variety of glycosylations. Many compounds have been developed to tentatively block each kind of glycosylation so as to study their biological functions but due to their complexity, many off‐target effects were reported. Insulin resistance exemplifies this problem. Several independent groups described that inhibiting the removal of O‐GlcNAc moieties using O‐(2‐acetamido‐2‐deoxy‐d‐glucopyranosylidene)amino‐N‐phenylcarbamate (PUGNAc), a nonselective inhibitor of the nuclear and cytoplasmic O‐GlcNAcase, induced insulin resistance both in vivo and ex vivo. The development of potent and highly selective O‐GlcNAcase inhibitors called into question that elevated O‐GlcNAcylation levels are responsible for insulin resistance; these compounds not recapitulating the insulin‐desensitizing effect of PUGNAc. To tackle this intriguing problem, a South Korean group recently combined ATP‐affinity chromatography and gel‐assisted digestion to identify proteins, differentially expressed upon treatment of 3T3‐L1 adipocytes with PUGNAc, involved in protein turnover and insulin signaling.     

O‐GlcNAcylation of protein kinase A catalytic subunits enhances its activity: a mechanism linked to learning and memory deficits in Alzheimer's disease

Alzheimer's disease (AD) is characterized clinically by memory loss and cognitive decline. Protein kinase A (PKA)‐CREB signaling plays a critical role in learning and memory. It is known that glucose uptake and O‐GlcNAcylation are reduced in AD brain. In this study, we found that PKA catalytic subunits (PKAcs) were posttranslationally modified by O‐linked N‐acetylglucosamine (O‐GlcNAc). O‐GlcNAcylation regulated the subcellular location of PKAcα and PKAcβ and enhanced their kinase activity. Upregulation of O‐GlcNAcylation in metabolically active rat brain slices by O‐(2‐acetamido‐2‐deoxy‐d ‐glucopyranosylidenamino) N‐phenylcarbamate (PUGNAc), an inhibitor of N‐acetylglucosaminidase, increased the phosphorylation of tau at the PKA site, Ser214, but not at the non‐PKA site, Thr205. In contrast, in rat and mouse brains, downregulation of O‐GlcNAcylation caused decreases in the phosphorylation of CREB at Ser133 and of tau at Ser214, but not at Thr205. Reduction in O‐GlcNAcylation through intracerebroventricular injection of 6‐diazo‐5‐oxo‐l ‐norleucine (DON), the inhibitor of glutamine fructose‐6‐phosphate amidotransferase, suppressed PKA‐CREB signaling and impaired learning and memory in mice. These results indicate that in addition to cAMP and phosphorylation, O‐GlcNAcylation is a novel mechanism that regulates PKA‐CREB signaling. Downregulation of O‐GlcNAcylation suppresses PKA‐CREB signaling and consequently causes learning and memory deficits in AD.     

Proteomic analysis and abrogated expression of O‐GlcNAcylated proteins associated with primary breast cancer

O‐GlcNAcylation is a dynamic PTM of nuclear and cytoplasmic proteins, regulated by O‐GlcNAc transferase (OGT) and O‐GlcNAcase, which catalyze the addition and removal of O‐GlcNAc, respectively. This modification is associated with glucose metabolism, which plays important roles in many diseases including cancer. Although emerging evidence reveals that some tumor‐associated proteins are O‐GlcNAc modified, the total O‐GlcNAcylation in cancer is still largely unexplored. Here, we demonstrate that O‐GlcNAcylation was increased in primary breast malignant tumors, not in benign tumors and that this augmentation was associated with increased expression of OGT level. Using 2D O‐GlcNAc immnoblotting and LC‐MS/MS analysis, we successfully identified 29 proteins, with seven being uniquely O‐GlcNAcylated or associated with O‐GlcNAcylation in cancer. Of these identified proteins, some were related to the Warburg effect, including metabolic enzymes, proteins involved in stress responses and biosynthesis. In addition, proteins associated with RNA metabolism, gene expression, and cytoskeleton were highly O‐GlcNAcylated or associated with O‐GlcNAcylation. Moreover, OGT knockdown showed that decreasing O‐GlcNAcylation was related to inhibition of the anchorage‐independent growth in vitro. These data indicate that aberrant protein O‐GlcNAcylation is associated with breast cancer. Abnormal modification of these O‐GlcNAc‐modified proteins might be one of the vital malignant characteristics of cancer.     

γ‐Secretase Inhibition Induces Muscle Hypertrophy in a Notch‐Independent Mechanism

A wide variety of cellular processes and signaling events are regulated by the proteolytic enzyme γ‐secretase. Notch‐1 is one of the substrates of γ‐secretase and its role in the regulation of muscle differentiation has been well described. Importantly, besides Notch‐1, a number of proteins have been identified to undergo proteolysis by γ‐secretase. To date, the specific role of γ‐secretase during embryonic skeletal muscle differentiation has not been studied. Therefore, we address this question through the analysis of in vitro grown chick myogenic cells during the formation of multinucleated myotubes. The γ‐secretase inhibitor DAPT (N‐N[‐(3,5‐Difluorophenacetyl‐l ‐alanyl)]‐S‐328 phenylglycine‐t‐butyl‐ester) induces muscle hypertrophy. Knockdown of Notch‐1 using siRNA specific to chick shows no significant effect in myotube size, suggesting that γ‐secretase‐dependent effects on muscle hypertrophy in chick myogenic cells are Notch‐1‐independent. We also investigate the effects of γ‐secretase inhibition in the whole proteomic profile of chick myogenic cells. We identified 276 differentially expressed proteins from Label‐free proteomic approach. Data overview of interaction network obtained from STRING show that after γ‐secretase inhibition cells exhibited imbalance in protein metabolism, cytoskeleton/adhesion, and Sonic Hedgehog signaling. The collection of these results provides new insights into the role of γ‐secretase in skeletal muscle hypertrophy.     

Evaluation of serum‐free differentiation conditions for C2C12 myoblast cells assessed as to active tension generation capability

We have compared several serum‐free media for the differentiation of C2C12 myoblasts and assessed the extent of differentiation in several ways including as to active tension generation capability. C2C12 cells were allowed to differentiate in Dulbecco's modified Eagle's medium (DMEM) containing Ham's F‐12 (F‐12), AIM‐V (AIM), 0.2% Ultroser‐G in DMEM (Ult‐G), and 0.1% Sericin in DMEM (Sericin), compared with in DMEM supplemented with 2% horse serum (HS) or 2% calf serum (CS). C2C12 differentiation was assessed as the extent of myotube formation, glucose metabolism, protein expression, sarcomere formation, and active tension generation. All serum‐free media examined were capable of inducing myotube formation and the expression of muscle‐specific proteins. All serum‐free media except for F‐12 gave the sarcomere structure. Active tension generation was observed for cells that differentiated in AIM and Ult‐G, but the active tension generated by C2C12 cells that differentiated in Ult‐G was only ～25% in the case of myotubes that formed in HS. The addition of Ult‐G to the AIM resulted in improvement of the active tension generation capability, the active tension generated being ～3.4× compared to that in HS. The approach for assessing muscle cell differentiation presented in this study will be suitable for other studies that involve the differentiation of muscle cells. Biotechnol. Bioeng. 2010;107: 894–901. © 2010 Wiley Periodicals, Inc.     

Phosphorylation coexists with O‐GlcNAcylation in a plant virus protein and influences viral infection

Phosphorylation and O‐GlcNAcylation are two widespread post‐translational modifications (PTMs), often affecting the same eukaryotic target protein. Plum pox virus (PPV) is a member of the genus Potyvirus which infects a wide range of plant species. O‐GlcNAcylation of the capsid protein (CP) of PPV has been studied extensively, and some evidence of CP phosphorylation has also been reported. Here, we use proteomics analyses to demonstrate that PPV CP is phosphorylated in vivo at the N‐terminus and the beginning of the core region. In contrast with the ‘yin–yang’ mechanism that applies to some mammalian proteins, PPV CP phosphorylation affects residues different from those that are O‐GlcNAcylated (serines Ser‐25, Ser‐81, Ser‐101 and Ser‐118). Our findings show that PPV CP can be concurrently phosphorylated and O‐GlcNAcylated at nearby residues. However, an analysis using a differential proteomics strategy based on iTRAQ (isobaric tags for relative and absolute quantitation) showed a significant enhancement of phosphorylation at Ser‐25 in virions recovered from O‐GlcNAcylation‐deficient plants, suggesting that crosstalk between O‐GlcNAcylation and phosphorylation in PPV CP takes place. Although the preclusion of phosphorylation at the four identified phosphotarget sites only had a limited impact on viral infection, the mimicking of phosphorylation prevents PPV infection in Prunus persica and weakens infection in Nicotiana benthamiana and other herbaceous hosts, prompting the emergence of potentially compensatory second mutations. We postulate that the joint action of phosphorylation and O‐GlcNAcylation in the N‐proximal segment of CP allows a fine‐tuning of protein stability, providing the amount of CP required in each step of viral infection.     

Identification of secreted proteins regulated by cAMP in glioblastoma cells using glycopeptide capture and label‐free quantification

Exposure of glioblastoma U87MG cells to a cAMP analog leads to a decrease in proliferation, invasion, and angiogenic potential. Here, we apply a label‐free MS‐based approach to identify formerly N‐linked glycopeptides that change in abundance upon cAMP treatment. Over 150 unique glycopeptides in three biological repetitions were quantified, leading to the identification of 14 upregulated proteins and 21 downregulated proteins due to cAMP treatment. Of these, eight have been validated, either through comparison with microarray data or by Western blot. We estimate our ability to identify differentially expressed peptides at greater than 85% in a single biological repetition, while the analysis of multiple biological repetitions lowers the false positive rate to ～2%. Many of the proteins identified in this study are involved in cell signaling and some, such as Tenascin C, Cathepsin L, Neuroblastoma suppressor of tumorigenicity, and AXL/UFO tyrosine–protein kinase receptor, have been previously shown to be involved in glioblastoma progression. We also identify several semitryptic peptides that increase in abundance upon cAMP treatment, suggesting that cAMP regulates protease activity in these cells. Overall, these results demonstrate the benefits of using a highly specific enrichment method for quantitative proteomic experiments.     

Proteome profiling reveals potential toxicity and detoxification pathways following exposure of BEAS-2B cells to engineered nanoparticle titanium dioxide

Oxidative stress is known to play important roles in engineered nanomaterial‐induced cellular toxicity. However, the proteins and signaling pathways associated with the engineered nanomaterial‐mediated oxidative stress and toxicity are largely unknown. To identify these toxicity pathways and networks that are associated with exposure to engineered nanomaterials, an integrated proteomic study was conducted using human bronchial epithelial cells, BEAS‐2B and nanoscale titanium dioxide. Utilizing 2‐DE and MS, we identified 46 proteins that were altered at protein expression levels. The protein changes detected by 2‐DE/MS were verified by functional protein assays. These identified proteins include some key proteins involved in cellular stress response, metabolism, adhesion, cytoskeletal dynamics, cell growth, cell death, and cell signaling. The differentially expressed proteins were mapped using Ingenuity Pathway Analyses^? canonical pathways and Ingenuity Pathway Analyses tox lists to create protein‐interacting networks and proteomic pathways. Twenty protein canonical pathways and tox lists were generated, and these pathways were compared to signaling pathways generated from genomic analyses of BEAS‐2B cells treated with titanium dioxide. There was a significant overlap in the specific pathways and lists generated from the proteomic and the genomic data. In addition, we also analyzed the phosphorylation profiles of protein kinases in titanium dioxide‐treated BEAS‐2B cells for a better understanding of upstream signaling pathways in response to the titanium dioxide treatment and the induced oxidative stress. In summary, the present study provides the first protein‐interacting network maps and novel insights into the biological responses and potential toxicity and detoxification pathways of titanium dioxide.     

Insight into a strategy for attenuating AmpC‐mediated β‐lactam resistance: Structural basis for selective inhibition of the glycoside hydrolase NagZ

NagZ is an exo‐N‐acetyl‐β‐glucosaminidase, found within Gram‐negative bacteria, that acts in the peptidoglycan recycling pathway to cleave N‐acetylglucosamine residues off peptidoglycan fragments. This activity is required for resistance to cephalosporins mediated by inducible AmpC β‐lactamase. NagZ uses a catalytic mechanism involving a covalent glycosyl enzyme intermediate, unlike that of the human exo‐N‐acetyl‐β‐glucosaminidases: O‐GlcNAcase and the β‐hexosaminidase isoenzymes. These latter enzymes, which remove GlcNAc from glycoconjugates, use a neighboring‐group catalytic mechanism that proceeds through an oxazoline intermediate. Exploiting these mechanistic differences we previously developed 2‐N‐acyl derivatives of O‐(2‐acetamido‐2‐deoxy‐D ‐glucopyranosylidene)amino‐N‐phenylcarbamate (PUGNAc), which selectively inhibits NagZ over the functionally related human enzymes and attenuate antibiotic resistance in Gram‐negatives that harbor inducible AmpC. To understand the structural basis for the selectivity of these inhibitors for NagZ, we have determined its crystallographic structure in complex with N‐valeryl‐PUGNAc, the most selective known inhibitor of NagZ over both the human β‐hexosaminidases and O‐GlcNAcase. The selectivity stems from the five‐carbon acyl chain of N‐valeryl‐PUGNAc, which we found ordered within the enzyme active site. In contrast, a structure determination of a human O‐GlcNAcase homologue bound to a related inhibitor N‐butyryl‐PUGNAc, which bears a four‐carbon chain and is selective for both NagZ and O‐GlcNAcase over the human β‐hexosamnidases, reveals that this inhibitor induces several conformational changes in the active site of this O‐GlcNAcase homologue. A comparison of these complexes, and with the human β‐hexosaminidases, reveals how selectivity for NagZ can be engineered by altering the 2‐N‐acyl substituent of PUGNAc to develop inhibitors that repress AmpC mediated β‐lactam resistance.     

Proteomic identification of proteins translocated to membrane microdomains upon treatment of fibroblasts with the glycosphingolipid,C8‐β‐D‐lactosylceramide

Plasma membrane (PM) microdomains, including caveolae and other cholesterol‐enriched subcompartments, are involved in the regulation of many cellular processes, including endocytosis, attachment and signaling. We recently reported that brief incubation of human skin fibroblasts with the synthetic glycosphingolipid, D‐erythro‐octanoyl‐lactosylceramide (C8‐D ‐e‐LacCer), stimulates endocytosis via caveolae and induces the appearance of micron‐size microdomains on the PM. To further understand the effects of C8‐D ‐e‐LacCer treatment on PM microdomains, we used a detergent‐free method to isolate microdomain‐enriched membranes from fibroblasts treated ±C8‐D ‐e‐LacCer, and performed 2‐DE and mass spectrophotometry to identify proteins that were altered in their distribution in microdomains. Several proteins were identified in the microdomain‐enriched fractions, including lipid transfer proteins and proteins related to the functions of small GTPases. One protein, Rho‐associated protein kinase 2 (ROCK2), was verified by Western blotting to occur in microdomain fractions and to increase in these fractions after D ‐e‐LacCer treatment. Immunofluorescence revealed that ROCK2 exhibited an increased localization at or near the PM in C8‐D ‐e‐LacCer‐treated cells. In contrast, ROCK2 distribution in microdomains was decreased by treatment of cells with C8‐L ‐threo‐lactosylceramide, a glycosphingolipid with non‐natural stereochemistry. This study identifies new microdomain‐associated proteins and provides evidence that microdomains play a role in the regulation of the Rho/ROCK signaling pathway.     

Disruption of O‐GlcNAc homeostasis during mammalian oocyte meiotic maturation impacts fertilization

Meiotic maturation and fertilization are metabolically demanding processes, and thus the mammalian oocyte is highly susceptible to changes in nutrient availability. O‐GlcNAcylation—the addition of a single sugar residue (O‐linked β‐N‐acetylglucosamine) on proteins—is a posttranslational modification that acts as a cellular nutrient sensor and likely modulates the function of oocyte proteins. O‐GlcNAcylation is mediated by O‐GlcNAc transferase (OGT), which adds O‐GlcNAc onto proteins, and O‐GlcNAcase (OGA), which removes it. Here we investigated O‐GlcNAcylation dynamics in bovine and human oocytes during meiosis and determined the developmental sequelae of its perturbation. OGA, OGT, and multiple O‐GlcNAcylated proteins were expressed in bovine cumulus oocyte complexes (COCs), and they were localized throughout the gamete but were also enriched at specific subcellular sites. O‐GlcNAcylated proteins were concentrated at the nuclear envelope at prophase I, OGA at the cortex throughout meiosis, and OGT at the meiotic spindles. These expression patterns were evolutionarily conserved in human oocytes. To examine O‐GlcNAc function, we disrupted O‐GlcNAc cycling during meiotic maturation in bovine COCs using Thiamet‐G (TMG), a highly selective OGA inhibitor. Although TMG resulted in a dramatic increase in O‐GlcNAcylated substrates in both cumulus cells and the oocyte, there was no effect on cumulus expansion or meiotic progression. However, zygote development was significantly compromised following in vitro fertilization of COCs matured in TMG due to the effects on sperm penetration, sperm head decondensation, and pronuclear formation. Thus, proper O‐GlcNAc homeostasis during meiotic maturation is important for fertilization and pronuclear stage development.     

Trafficking of Acetyl‐C16‐Ceramide‐NBD with Long‐Term Stability and No Cytotoxicity into the Golgi Complex

The Golgi complex plays a prominent role in the modification and sorting of lipids and proteins, and is a highly dynamic organelle that is dispersed and rearranged before and after mitosis. Several reagents including 4‐nitrobenzo‐2‐oxa‐1,3‐diazole‐labeled C6‐ceramide (NBD‐C6‐ceramide, a ceramide having an NBD‐bound C6‐N‐acyl chain) and Golgi‐specific proteins that emit fluorescence are used as Golgi markers. In the present study, we synthesized a new ceramide analog, acetyl‐C16‐ceramide‐NBD (a ceramide having an acetylated C‐1 hydroxyl group, C16‐N‐acyl chain, and NBD‐bound C15‐sphingosine), and showed that it preferentially accumulated in the Golgi complex without cytotoxicity for over 24 h. Pathways for cellular uptake and interorganelle trafficking of acetyl‐C16‐ceramide‐NBD were investigated. Acetyl‐C16‐ceramide‐NBD was transported to the Golgi complex via ceramide transport proteins. In contrast to NBD‐C6‐ceramide, acetyl‐C16‐ceramide‐NBD was resistant to ceramide metabolic enzymes such as sphingomyelin synthase and glucosylceramide synthase. Because of its weaker cytotoxicity and resistance to ceramide metabolic enzymes, the localization of the Golgi complex could be observed in acetyl‐C16‐ceramide‐NBD‐labeled cells before and after mitosis.      

Conformational flexibility of the glycosidase NagZ allows it to bind structurally diverse inhibitors to suppress β‐lactam antibiotic resistance

NagZ is an N‐acetyl‐β‐d ‐glucosaminidase that participates in the peptidoglycan (PG) recycling pathway of Gram‐negative bacteria by removing N‐acetyl‐glucosamine (GlcNAc) from PG fragments that have been excised from the cell wall during growth. The 1,6‐anhydromuramoyl‐peptide products generated by NagZ activate β‐lactam resistance in many Gram‐negative bacteria by inducing the expression of AmpC β‐lactamase. Blocking NagZ activity can thereby suppress β‐lactam antibiotic resistance in these bacteria. The NagZ active site is dynamic and it accommodates distortion of the glycan substrate during catalysis using a mobile catalytic loop that carries a histidine residue which serves as the active site general acid/base catalyst. Here, we show that flexibility of this catalytic loop also accommodates structural differences in small molecule inhibitors of NagZ, which could be exploited to improve inhibitor specificity. X‐ray structures of NagZ bound to the potent yet non‐selective N‐acetyl‐β‐glucosaminidase inhibitor PUGNAc (O‐(2‐acetamido‐2‐deoxy‐d ‐glucopyranosylidene) amino‐N‐phenylcarbamate), and two NagZ‐selective inhibitors – EtBuPUG, a PUGNAc derivative bearing a 2‐N‐ethylbutyryl group, and MM‐156, a 3‐N‐butyryl trihydroxyazepane, revealed that the phenylcarbamate moiety of PUGNAc and EtBuPUG completely displaces the catalytic loop from the NagZ active site to yield a catalytically incompetent form of the enzyme. In contrast, the catalytic loop was found positioned in the catalytically active conformation within the NagZ active site when bound to MM‐156, which lacks the phenylcarbamate extension. Displacement of the catalytic loop by PUGNAc and its N‐acyl derivative EtBuPUG alters the active site conformation of NagZ, which presents an additional strategy to improve the potency and specificity of NagZ inhibitors.     

OGT Controls the Expression and the Glycosylation of E‐cadherin,and Affects Glycosphingolipid Structures in Human Colon Cell Lines

Colorectal cancer (CRC) affects both women and men living in societies with a high sedentary lifestyle. Amongst the phenotypic changes exhibited by tumor cells, a wide range of glycosylation has been reported for colon cancer‐derived cell lines and CRC tissues. These aberrant modifications affect different aspects of glycosylation, including an increase in core fucosylation and GlcNAc branching on N‐glycans, alteration of O‐glycans, upregulated sialylation, and O‐GlcNAcylation. Although O‐GlcNAcylation and complex glycosylations differ in many aspects, sparse evidences report on the interference of O‐GlcNAcylation with complex glycosylation. Nevertheless, this relationship is still a matter of debate. Combining different approaches on three human colon cell lines (HT29, HCT116 and CCD841CoN), it is herein reported that silencing O‐GlcNAc transferase (OGT, the sole enzyme driving O‐GlcNAcylation), only slightly affects overall N‐ and O‐glycosylation patterns. Interestingly, silencing of OGT in HT29 cells upregulates E‐cadherin (a major actor of epithelial‐to‐mesenchymal transition) and changes its glycosylation. On the other hand, OGT silencing perturbs biosynthesis of glycosphingolipids resulting in a decrease in gangliosides and an increase in globosides. Together, these results provide novel insights regarding the selective regulation of complex glycosylations by O‐GlcNAcylation in colon cancer cells.     

Proteomic analysis of differentially expressed proteins in human cholangiocarcinoma cells treated with Clonorchis sinensis excretory–secretory products

Severe Clonorchis sinensis infection is a significant risk factor for malignant changes in bile ducts and surrounding liver tissues occurring as a result of direct contact with C. sinensis worms and their excretory–secretory products (ESP). However, the intrinsic molecular mechanisms involved in these processes remain obscure. To determine the effects of C. sinensis infection on protein expression in host bile duct epithelium, we examined proteomic profile changes in the human cholangiocarcinoma cell line (HuCCT1) treated with ESP at 24 h. Using a combination of 2‐DE, quantitative image and MALDI‐TOF MS analysis, we identified 83 proteins that were translationally modulated in response to ESP, among which 49 were up‐regulated and 34 down‐regulated. These proteins were classified under various biological categories, including metabolism, cell structure and architecture, proteolysis, protein modification, transport, signal transduction, and reactive oxygen species (ROS) detoxification. In particular, ESP induced the expression of redox‐regulating proteins, including peroxiredoxins (Prdx 2, 3, and 6) and thioredoxin 1 (Trx 1), possibly via intracellular ROS generation. Application of the proteomic approach to identify ESP response proteins should be a prerequisite before further investigation to clarify the molecular pathways and mechanisms involved in C. sinensis infection of host cells. J. Cell. Biochem. 108: 1376–1388, 2009. © 2009 Wiley‐Liss, Inc.