Proteomic and metabonomic analysis uncovering Enterovirus A71 reprogramming host cell metabolic pathway.

Enterovirus A71 (EV71) infection can cause hand, foot, and mouth disease (HFMD) and severe neurological complications in children. However, the biological processes regulated by EV71 remain poorly understood. Herein, proteomics and metabonomics studies were conducted to uncover the mechanism of EV71 infection in rhabdomyosarcoma (RD) cells and identify potential drug targets. Differential expressed proteins from enriched membrane were analyzed by isobaric tags for relative and absolute quantitation (iTRAQ)-based proteomics technology. Twenty-six differential proteins with 1.5-fold (p < 0.05) change were detected, including 14 upregulated proteins and 12 downregulated proteins. The upregulated proteins are mainly involved in metabolic process, especially in the glycolysis pathway. Alpha-enolase (ENO1) protein was found to increase with temporal dependence following EV71 infection. The targeted metabolomics analysis revealed that glucose absorption and glycolysis metabolites were increased after EV71 infection. The glycolysis pathway was inhibited by knocking down ENO1 or the use of a glycolysis inhibitor (dichloroacetic acid [DCA]); and we found that EV71 infection was inhibited by depleting ENO1 or using DCA. Our study indicates that EV71 may reprogram glucose metabolism by activating glycolysis, and EV71 infection can be inhibited by interrupting the glycolysis pathway. ENO1 may be a potential target against EV71, and DCA could act as an inhibitor of EV71.

ENO1 Promotes OSCC Migration and Invasion by Orchestrating IL-6 Secretion from Macrophages via a Positive Feedback Loop.

Oral squamous cell carcinoma (OSCC) has a five-year survival rate of less than 50% due to its susceptibility to invasion and metastasis. Crosstalk between tumor cells and macrophages has been proven to play a critical role in tumor cell migration and invasion. However, the specific mechanisms by which tumor cells interact with macrophages have not been fully elucidated. This study sought to investigate the regulatory mechanism of tumor cell-derived alpha-enolase (ENO1) in the interaction between tumor cells and macrophages during OSCC progression. Small interfering RNA (siRNA) transfection and recombinant human ENO1 (rhENO1) stimulation were used to interfere with the interaction between tumor cells and macrophages. Our results showed that ENO1 was expressed higher in CAL27 cells than in HaCaT cells and regulated lactic acid release in CAL27 cells. Conditioned medium of macrophages (Macro-CM) significantly up-regulated the ENO1 mRNA expression and protein secretion in CAL27 cells. ENO1 promoted the migration and invasion of tumor cells by facilitating the epithelial-mesenchymal transition (EMT) through macrophages. ENO1 orchestrated the IL-6 secretion of macrophages via tumor cell-derived lactic acid and the paracrine ENO1/Toll-like receptor (TLR4) signaling pathway. In turn, IL-6 promoted the migration and invasion of tumor cells. Collectively, ENO1 promotes tumor cell migration and invasion by orchestrating IL-6 secretion of macrophages via a dual mechanism, thus forming a positive feedback loop to promote OSCC progression. ENO1 might be a promising therapeutic target which is expected to control OSCC progression.

The effects of maternal anti-alpha-enolase antibody expression on the brain development in offspring.

Anti-alpha-enolase autoantibodies have not only been found to play an important role in autoimmune diseases but also cause neurological damage in adults. In this study, a pregnant mouse model with high serum alpha-enolase (ENO1)-specific antibody (ENO1Ab) was established by immunization with ENO1 protein to explore the effects of maternal circulatory ENO1Ab on the brain development in offspring. The pups showed impaired learning and memory abilities with obviously thinner tight junctions in the brain tissue. IgG deposits colocalized with both ENO1 protein and complement 3 (C3), and the membrane attack complex was obviously detectable in the brain tissues of pups from dams with high serum ENO1Ab expression. Our findings suggest that highly expressed ENO1Ab in the maternal circulation can pass through the blood-placenta-barrier and the compromised blood-brain barrier into the brain tissues of offspring and may cause neurological development impairment mainly through complement-dependent cytotoxicity.

Identification and functional analysis of senescence-associated secretory phenotype of premature senescent hepatocytes induced by hexavalent chromium.

Hexavalent chromium [Cr(VI)] is a common heavy metal pollutant that can cause a number of human disease, including inflammation and cancer. Senescent cells can secrete a variety of molecules known as senescence-associated secretory phenotype (SASP). Our previous studies have confirmed that Cr(VI) can induce premature senescence in L02 hepatocytes, but the composition and the function of the related SASP are still unknown. In order to understand the components of SASP secreted by senescent L02 hepatocytes under the action of Cr(VI), we applied LC-MS/MS-based label-free protein quantification. We found that three SASP components including Coactosin-like protein 1 (COTL1), Alpha-enolase (ENO1), and Peroxiredoxin 2 (PRDX2) were up-regulated, which were confirmed by western blotting and qRT-PCR. Evidence suggested that SASP may promote the development of tumor through chronic inflammatory response, therefore we identified and analyzed the potential biological functions and signaling pathways of these three SASP components using GO and KEGG methods. The interaction between SASP components was analyzed by STRING, and verified by Co-IP. We also found that ENO1 and PRDX2, which have direct interaction, can inhibit the growth and proliferation of wildtype hepatocytes and premature senescent hepatocytes, but can promote the proliferation and behavioral changes of liver tumor cells. The present study provides valuable clues for elucidation of the carcinogenic mechanism of Cr(VI), especially for further prevention and targeted treatment of Cr(VI)-related cancer.

Alpha-enolase in viral target cells suppresses the human immunodeficiency virus type 1 integration.

BACKGROUND: A protein exhibiting more than one biochemical function is termed a moonlighting protein. Glycolytic enzymes are typical moonlighting proteins, and these enzymes control the infection of various viruses. Previously, we reported that glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and alpha-enolase (ENO1) are incorporated into human immunodeficiency virus type 1 (HIV-1) particles from viral producer cells and suppress viral reverse transcription independently each other. However, it remains unclear whether these proteins expressed in viral target cells affect the early phase of HIV-1 replication. RESULTS: Here we show that the GAPDH expression level in viral target cells does not affect the early phase of HIV-1 replication, but ENO1 has a capacity to suppress viral integration in viral target cells. In contrast to GAPDH, suppression of ENO1 expression by RNA interference in the target cells increased viral infectivity, but had no effect on the expression levels of the HIV-1 receptors CD4, CCR5 and CXCR4 and on the level of HIV-1 entry. Quantitative analysis of HIV-1 reverse transcription products showed that the number of copies of the late products (R/gag) and two-long-terminal-repeat circular forms of viral cDNAs did not change but that of the integrated (Alu-gag) form increased. In contrast, overexpression of ENO1 in viral target cells decreased viral infectivity owing to the low viral integration efficiency. Results of subcellular fractionation experiments suggest that the HIV integration at the nucleus was negatively regulated by ENO1 localized in the nucleus. In addition, the overexpression of ENO1 in both viral producer cells and target cells most markedly suppressed the viral replication. CONCLUSIONS: These results indicate that ENO1 in the viral target cells prevents HIV-1 integration. Importantly, ENO1, but not GAPDH, has the bifunctional inhibitory activity against HIV-1 replication. The results provide and new insights into the function of ENO1 as a moonlighting protein in HIV-1 infection.

Non-paraneoplastic autoimmune retinopathy that developed in fellow eye 10 years after onset in first eye: a case report.

BACKGROUND: Evidence-based criteria for the treatment of autoimmune retinopathy (AIR) have not been established. The pathology and clinical features of each antibody causing AIR, and its long-term course are still undetermined. We report our findings in a case of non-paraneoplastic AIR (npAIR) that developed in the fellow eye 10 years after the onset in the first eye. CASE PRESENTATION: Our patient had photophobia in both eyes and a rapidly progressing visual field defect in his right eye at the initial examination. He was diagnosed with non-paraneoplastic AIR based on the clinical findings and immunoblot analyses for anti-retinal antibodies, and he was treated with steroids. Ten years later, a visual field defect developed in the fellow eye, and a diagnosis of npAIR was made. Immunoblot analyses were positive for anti-α-enolase antibodies. He was treated with steroids, immunosuppressants, and plasma exchange. However, the response to the treatment was poor and both eyes eventually became blind. CONCLUSIONS: As best we know, this is the first case report of npAIR that developed in the fellow eye over 10 years after the development in the first eye. Long-term follow-up and a search for tumor lesions are necessary in cases of npAIR. Further understanding of the long-term course of AIR can contribute to an understanding of the pathology and treatment of npAIR.

Generation and Characterization of Single Chain Variable Fragment against Alpha-Enolase of Candida albicans.

Candida albicans (C. albicans) is an opportunistic human pathogen responsible for approximately a half of clinical candidemia. The emerging Candida spp. with resistance to azoles is a major challenge in clinic, suggesting an urgent demand for new drugs and therapeutic strategies. Alpha-enolase (Eno1) is a multifunctional protein and represents an important marker for invasive candidiasis. Thus, C. albicans Eno1 (CaEno1) is believed to be an important target for the development of therapeutic agents and antibody drugs. Recombinant CaEno1 (rCaEno1) was first used to immunize chickens. Subsequently, we used phage display technology to construct two single chain variable fragment (scFv) antibody libraries. A novel biopanning procedure was carried out to screen anti-rCaEno1 scFv antibodies, whose specificities were further characterized. The polyclonal IgY antibodies showed binding to rCaEno1 and native CaEno1. A dominant scFv (CaS1) and its properties were further characterized. CaS1 attenuated the growth of C. albicans and inhibited the binding of CaEno1 to plasminogen. Animal studies showed that CaS1 prolonged the survival rate of mice and zebrafish with candidiasis. The fungal burden in kidney and spleen, as well as level of inflammatory cytokines were significantly reduced in CaS1-treated mice. These results suggest CaS1 has potential of being immunotherapeutic drug against C. albicans infections.

Nell-1-ΔE, a novel transcript of Nell-1, inhibits cell migration by interacting with enolase-1.

NELL-1 is a secreted protein that was originally found to be upregulated in pathologically fusing and fused sutures in non-syndromic unilateral coronal synostosis patients. Apart from the ability of NELL-1 to promote osteogenesis in long and craniofacial bones, NELL-1 reportedly inhibits the formation of several benign and malignant tumors. We previously identified a novel transcript of Nell-1 that lacked a calcium-binding epidermal growth factor (EGF)-like domain compared with full-length Nell-1; this new transcript was named Nell-1-ΔE. Three obvious structural differences between these two isoforms were revealed by homology modeling. Furthermore, the recombinant Nell-1-ΔE protein, but not the full-length Nell-1 protein, inhibited cell migration in vitro. However, full-length Nell-1 and Nell-1-ΔE proteins were present in similar subcellular locations and displayed similar expression patterns in both the intracellular and extracellular spaces. The results from the co-immunoprecipitation and liquid chromatography/tandem mass spectrometry analyses using two cell lines demonstrated that Nell-1-ΔE but not full-length Nell-1 interacted with enolase-1 in the extracellular spaces of both cell lines. The results of wound healing assays using ENO-1-overexpressing cells treated with full-length Nell-1/Nell-1-ΔE suggested that Nell-1-ΔE inhibited cell migration by interacting with ENO-1. Our study indicated that the novel transcript Nell-1-ΔE, but not full-length Nell-1, might be a candidate tumor suppressor factor for basic research and clinical practice.

Alpha-enolase regulates hepatitis B virus replication through suppression of the interferon signalling pathway.

Persistent chronic infection with hepatitis B virus (HBV) is a major risk factor for the development of HBV-related diseases. The molecular mechanisms that underlie HBV infection and associated carcinogenesis are not fully understood. The aim of this study was to explore the role of ENO1 in HBV replication processes. Here, we examined ENO1 expression levels in HBV-infected and non-HBV-infected liver tissues and cells by Western blot analysis, real-time PCR and immunohistochemistry. In addition, HBsAg and HBeAg in the media of transfected HepG2.2.15 cells were detected using an electrochemical luminescence analyser within 48 hours after ENO1-specific siRNA transfection. The expression levels of HBV DNA, type I interferon and 5 downstream IFN-stimulated genes in HepG2.2.15 cells were examined using real-time PCR. We found ENO1 expression was upregulated in the HBV-infected liver tissues and cells. Silencing of ENO1 resulted in a significant reduction in HBV replication, and this siRNA-mediated reaction also caused the upregulation of expression of type I interferon and downstream IFN-stimulated genes. Therefore, we come to the conclusion ENO1 is involved in HBV replication. It is therefore likely that HBV replication is enhanced following suppression of the IFN signalling pathway. However, the mechanisms that underpin ENO1-mediated modulation of the IFN signalling pathway remain to be elucidated.

The immunoregulatory role of alpha enolase in dendritic cell function during Chlamydia infection.

BACKGROUND: We have previously reported that interleukin-10 (IL-10) deficient dendritic cells (DCs) are potent antigen presenting cells that induced elevated protective immunity against Chlamydia. To further investigate the molecular and biochemical mechanism underlying the superior immunostimulatory property of IL-10 deficient DCs we performed proteomic analysis on protein profiles from Chlamydia-pulsed wild-type (WT) and IL-10-/- DCs to identify differentially expressed proteins with immunomodulatory properties. RESULTS: The results showed that alpha enolase (ENO1), a metabolic enzyme involved in the last step of glycolysis was significantly upregulated in Chlamydia-pulsed IL-10-/- DCs compared to WT DCs. We further studied the immunoregulatory role of ENO1 in DC function by generating ENO1 knockdown DCs, using lentiviral siRNA technology. We analyzed the effect of the ENO1 knockdown on DC functions after pulsing with Chlamydia. Pyruvate assay, transmission electron microscopy, flow cytometry, confocal microscopy, cytokine, T-cell activation and adoptive transfer assays were also used to study DC function. The results showed that ENO1 knockdown DCs had impaired maturation and activation, with significant decrease in intracellular pyruvate concentration as compared with the Chlamydia-pulsed WT DCs. Adoptive transfer of Chlamydia-pulsed ENO1 knockdown DCs were poorly immunogenic in vitro and in vivo, especially the ability to induce protective immunity against genital chlamydia infection. The marked remodeling of the mitochondrial morphology of Chlamydia-pulsed ENO1 knockdown DCs compared to the Chlamydia-pulsed WT DCs was associated with the dysregulation of translocase of the outer membrane (TOM) 20 and adenine nucleotide translocator (ANT) 1/2/3/4 that regulate mitochondrial permeability. The results suggest that an enhanced glycolysis is required for efficient antigen processing and presentation by DCs to induce a robust immune response. CONCLUSIONS: The upregulation of ENO1 contributes to the superior immunostimulatory function of IL-10 deficient DCs. Our studies indicated that ENO1 deficiency causes the reduced production of pyruvate, which then contributes to a dysfunction in mitochondrial homeostasis that may affect DC survival, maturation and antigen presenting properties. Modulation of ENO1 thus provides a potentially effective strategy to boost DC function and promote immunity against infectious and non-infectious diseases.

Antibodies against citrullinated alpha enolase peptides in primary Sjogren's syndrome.

Citrullinated alpha enolase (CEP-1) has been designated as a major antigenic target of antibodies against citrullinated proteins (ACPA) in patients with rheumatoid arthritis (RA). Our aim is to determine the prevalence of anti-CEP-1 in a cohort of ACPA positive (ACPA+) primary Sjogren's syndrome (pSS) patients. Anti-CEP1 titers were determined by ELISA in sera from 15 ACPA+ and 45 ACPA- age/sex matched pSS; 12 ACPA+ RA patients and 30 healthy controls (HC). Increased anti-CEP-1 antibody titers were detected in nine out of the 15 (60%) ACPA+ pSS patients and 5 out of 12 (41.7%) ACPA+ RA patients; no reactivities were detected in ACPA- pSS patients and HC. Anti-CEP-1 antibodies in the setting of pSS were associated with higher urine pH levels at baseline. CEP-1 is a major antigenic target of ACPA in patients with pSS characterizing a subgroup with distinct laboratory features.

Virion-incorporated alpha-enolase suppresses the early stage of HIV-1 reverse transcription.

Human immunodeficiency virus type-1 (HIV-1) particles contain not only viral-encoded but also host-encoded proteins. Interestingly, several studies showed that host proteins play a critical role in viral infectivity, replication and/or immunoreactivity in the next target cells. Here, we show that alpha-enolase (ENO1) is incorporated into HIV-1 virions and the virion-incorporated ENO1 prevents the early stage of HIV-1 reverse transcription. We found that viral particles contain two isoforms of ENO1 with different isoelectric points by two-dimensional electrophoresis. Suppression of ENO1 expression by RNA interference in the HIV-1 producer cells decreased ENO1 incorporation into virions without altering the packaging of viral structural proteins and viral production but increased viral infectivity. Although the low-level-ENO1-packaging virus maintained comparable levels of reverse transcriptase activity, viral genomic RNA and tRNALys3 packaging to the control virus, its levels of early cDNA products of reverse transcription were higher than those of the control virus. In contrast, the high-level-ENO1-packaging virus, which was produced from ENO1-overexpressing cells, showed decreased infectivity and the levels of early cDNA products. Taken together, these findings reveal a novel function of ENO1 as a negative regulation factor targeting HIV-1 reverse transcription.

Over-Expression of Alpha-Enolase as a Prognostic Biomarker in Patients with Pancreatic Cancer.

Background: Alpha-enolase is an important glycolytic enzyme, and its aberrant expression has been associated with multiple tumor progression. However, few studies investigated the expression of alpha-enolase and its clinical significance in pancreatic cancer (PC). Objectives: To evaluate alpha-enolase level in PC tissues by immunohistochemical (IHC) analysis, and investigate the association of alpha-enolase expression with clinicopathologic features. Methods: The alpha-enolase levels in pancreatic cancer tissues were analyzed by using the Oncomine database. The expression of alpha-enolase, Ki67 and p53 in pancreatic cancer and adjacent normal tissues were evaluated by IHC using the corresponding primary antibodies on the commercial tissue arrays. We also examined their association with clinicopathologic parameters, and explored their prognostic value in PC. Results: We identified an elevation of alpha-enolase mRNA level in pancreatic cancer independent datasets from Oncomine. IHC analysis showed that alpha-enolase protein levels were elevated in 47% (n=100) PC tissue samples, but there was weak or no staining in the normal tissues. Statistical analysis revealed that high levels of alpha-enolase were significantly associated with Stage and Lymph node metastasis. Correlation analysis indicated that over-expression of alpha-enolase was positively associated with Ki67 expression and inversely correlated with p53 expression. Furthermore, membranous expression of alpha-enolase was also observed in 29.8% (14/47) total alpha-enolase positive samples, and was significantly associated with Lymph node metastasis. Kaplan-Meier survival analysis demonstrated that high total alpha-enolase expression was significantly associated with unfavorable survival, while membranous alpha-enolase expression was significantly associated with better survival of PC patients. Multivariate Cox analysis demonstrated that total alpha-enolase expression was an independent prognostic factor for PC patients. Conclusions: Our results suggested that alpha-enolase level was significantly elevated in pancreatic cancer tissues, which was closely associated with PC progression. It might be a candidate target for targeted pancreatic cancer treatments.

Analysis of A549 cell proteome alteration in response to recombinant influenza A virus nucleoprotein and its interaction with cellular proteins, a preliminary study.

Influenza A virus undergoes frequent changes of antigenicity and contributes to seasonal epidemics or unpredictable pandemics. Nucleoprotein, encoded by gene segment 5, is an internal protein of the virus and is conserved among strains of different host origins. In the current study, we analyzed the differentially expressed proteins in A549 cells transiently transfected with the recombinant nucleoprotein of influenza A virus by 2D gel electrophoresis. The resolved protein spots on gel were identified by MALDI-TOF/Mass spectrometry analysis. The majority of the host proteins detected to be differentially abundant in recombinant nucleoprotein-expressing cells as compared to vector-transfected cells are the proteins of metabolic pathways, glycolytic enzymes, molecular chaperones and cytoskeletal proteins. We further demonstrated the interaction of virus nucleoprotein with some of the identified host cellular proteins. In vitro binding assay carried out using the purified recombinant nucleoprotein (pET29a+NP-His) and A549 cell lysate confirmed the interaction between nucleoprotein and host proteins, such as alpha enolase 1, pyruvate kinase and ß-actin. The preliminary data of our study provides the information on virus nucleoprotein interaction with proteins involved in glycolysis. However, studies are ongoing to understand the significance of these interactions in modulating the host factors during virus replication.

Effect of prolonged exposure to sublethal concentrations of DDT and DDE on protein expression in human pancreatic beta cells.

Pollution of the environment represents one of less explored potential reasons for the worldwide epidemic of type 2 diabetes. One of the most prevalent organochlorine pollutants remains the pesticide DDT and its degradation product DDE. Despite some epidemiologic correlations between levels of DDT and DDE in human organism and the prevalence of diabetes, there is almost no information about the exact targets of these compounds inside pancreatic beta cells. To detect functional areas of pancreatic beta cells that could be affected by exposure to DDT and DDE, we analyzed changes in protein expression in the NES2Y human pancreatic beta cell line exposed to three sublethal concentrations (0.1 µM, 1 µM, 10 µM) of DDT and DDE for 1 month. Protein separation and identification was achieved using high-resolution 2D-electrophoresis, computer analysis and mass spectrometry. With these techniques, four proteins were found downregulated after exposure to 10 µM DDT: three cytoskeletal proteins (cytokeratin 8, cytokeratin 18 and actin) and one protein involved in glycolysis (alpha-enolase). Two proteins were downregulated after exposure to 10 µM DDE: cytokeratin 18 and heterogenous nuclear ribonucleoprotein H1 (HNRH1). These changes correlate with previously described effects of other stress conditions (e.g. exposure to palmitate, hyperglycemia, imidazoline derivative, and cytokines) on protein expression in pancreatic beta cells. We conclude that cytoskeletal proteins and their processing, glucose metabolism, and mRNA processing may represent targets affected by exposure to conditions hostile to pancreatic beta cells, including exposure to DDT and DDE.

Decreased expression of alpha-enolase inhibits the proliferation of hypoxia-induced rheumatoid arthritis fibroblasts-like synoviocytes.

OBJECTIVES: To investigate the effect of decreased alpha-enolase (ENO1) expression on rheumatoid arthritis fibroblasts-like synoviocytes (RA-FLSs) proliferation in response to hypoxia, and elucidate the possible mechanisms involved. METHODS: RA-FLSs and osteoarthritis fibroblasts-like synoviocytes (OA-FLSs) were cultured in tri-gas incubators with different oxygen concentrations (3% O2, 7% O2, and 21% O2). 3% O2 (hypoxia) and 7% O2 conditions simulated intra-articular oxygen concentrations as observed in RA and healthy individual, respectively. 21% O2 represented oxygen condition for normal cell culture. ENO1-knockdown FLSs were established using ENO1-siRNA. The expression level of ENO1 was detected using reverse transcription polymerase chain reaction or RT-PCR and Western blot. Proliferation and apoptosis of RA-FLSs and OA-FLSs were assessed using 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2 H-tetrazolium or MTS assay and flow cytometry, respectively. Western blot analysis was used to detect key proteins involved in apoptosis. RESULTS: ENO1 gene expression was remarkably upregulated, as well as its translation into protein, in RA-FLSs and OA-FLSs that were cultured in 3% O2 concentration. RA-FLSs and OA-FLSs that were cultured under hypoxic conditions hyperproliferated compared with similar cells under normaxic conditions. Neither 7% O2 nor 21% O2 condition had any significant effect on ENO1 expression. ENO1-siRNA-transfected FLSs, but not control-siRNA FLSs, showed markedly decreased proliferation. Additionally, ENO1 expression was found to promote significantly higher expression levels of the anti-apoptotic proteins Bcl-2, surviving, and cyclinB1, but inhibited the expression of cleaved caspase3. CONCLUSION: ENO1 may be crucial in the regulation of the proliferation and survival of synovial fibroblasts.

Hep88 mAb-initiated paraptosis-like PCD pathway in hepatocellular carcinoma cell line through the binding of mortalin (HSPA9) and alpha-enolase.

BACKGROUND: Hepatocellular carcinoma (HCC) is the most prevalent hepatic cancer worldwide. Currently, a targeted therapy via monoclonal antibodies (mAbs) specific to tumor-associated antigen is undergoing continual development in HCC treatment. METHODS: In this regard, after establishing and consequently exploring Hep88 mAb's tumoricidal effect on hepatocellular carcinoma cell line (HepG2 cell line), the Hep88 mAb's specific antigens from both membrane and cytoplasmic fractions of HepG2 cell line were identified by 2-D gel electrophoresis and western blot analysis. After in-gel digestion and subsequent analysis by liquid chromatography-mass spectrometry (LC-MS), mortalin (HSPA9) and alpha-enolase were identified. The recombinant proteins specific to Hep88 mAb were cloned and expressed in E. coli BL21(DE3). Moreover, alteration of HepG2 and Chang liver cell line after being induced by Hep88 mAb for 1-3 days was investigated using a transmission electron microscope. RESULTS: The result demonstrated that Hep88 mAb can bind to the recombinant mortalin (HSPA9) and alpha-enolase. In addition, the gradual appearing of mitochondria vacuolization and endoplasmic reticulum dilatation were observed. Those characteristics might be explained by the paraptosis-like program cell death (PCD), which is induced by the binding of Hep88 mAb to mortalin (HSPA9). Mortalin depletion resulting from the formation of Hep88 mAb-mortalin (HSPA9) complex might initiate transcription-independence of p53-mediated apoptosis. Additionally, Hep88mAb-alpha-enolase complex might initiate HepG2 cells energy exhaustion by glycolysis pathway obstruction. CONCLUSION: These fascinating results imply that Hep88 mAb might be a promising tool for the development of an effective treatment of HCC in the next decade.

Serum Th1, Th2 and Th17 cytokine profiles and alpha-enolase levels in recurrent aphthous stomatitis.

BACKGROUND: All aspects of aetiopathogenesis of recurrent aphthous stomatitis (RAS) have not been elucidated. RAS and Behçet's disease (BD) have clinical and immunological characteristics in common. Although T17 cytokines and alpha-enolase have been shown to play effective roles in BD and many other autoinflammatory diseases recently, their roles in RAS have not been studied extensively. In the present study, we investigated levels of several Th1, Th2 and Th17 pathways related cytokines and alpha-enolase to elucidate pathogenesis of RAS and to obtain data about possible treatment alternatives for the condition. METHODS: Serum interleukin-1, interleukin-13, interleukin-17, interleukin-18, interferon gamma and alpha-enolase levels in 24 patients with RAS, 30 patients with BD and 20 healthy controls were measured. RESULTS: Serum interleukin-1, interleukin-13, interleukin-17, interleukin-18, interferon gamma and alpha-enolase levels were higher in patients with RAS and patients with BD than in healthy controls (P < 0.005). CONCLUSION: Like Th1 and Th2 cells, Th17 cells were found to be effective in pathogenesis of RAS. In addition, alpha-enolase, the levels of which were high, may play an important role in etio-pathogenesis of RAS. Further studies to be designed in the light of these findings are required to shed light on pathogenesis and treatment of the condition.

High Expression of ENO1 and Low Levels of Circulating Anti-ENO1 Autoantibodies in Patients with Myelodysplastic Neoplasms and Acute Myeloid Leukaemia.

In solid tumours, high expression of the glycolytic enzyme, α-enolase (ENO1), predicts for poor patient overall survival (OS), and circulating autoantibodies to ENO1 correlate positively with diagnosis and negatively with advanced disease. Although ENO1 is one of the most highly expressed genes in acute myeloid leukaemia (AML), its potential role as a biomarker in AML or its precursor, myelodysplastic neoplasms (MDS), has not been investigated. A meta-analysis of nine AML online datasets (n = 1419 patients) revealed that high ENO1 expression predicts for poor OS (HR = 1.22, 95% CI: 1.10-1.34, p < 0.001). Additionally, when compared to AML in remission (n = 5), ENO1 protein detected by immunohistochemistry was significantly higher at diagnosis in bone marrow from both AML (n = 5, p < 0.01) and MDS patients (n = 12, p < 0.05), and did not correlate with percentage of blasts (r = 0.28, p = 0.21). AML patients (n = 34) had lower circulating levels of ENO1 autoantibodies detected by ELISA compared to 26 MDS and 18 controls (p = 0.003). However, there was no difference in OS between AML patients with high vs. low levels of anti-ENO1 autoantibodies (p = 0.77). BM immunostaining for ENO1 and patient monitoring of anti-ENO1 autoantibody levels may be useful biomarkers for MDS and AML.

Tumour-specific phosphorylation of serine 419 drives alpha-enolase (ENO1) nuclear export in triple negative breast cancer progression.

BACKGROUND: The glycolytic enzyme alpha-enolase is a known biomarker of many cancers and involved in tumorigenic functions unrelated to its key role in glycolysis. Here, we show that expression of alpha-enolase correlates with subcellular localisation and tumorigenic status in the MCF10 triple negative breast cancer isogenic tumour progression model, where non-tumour cells show diffuse nucleocytoplasmic localisation of alpha-enolase, whereas tumorigenic cells show a predominantly cytoplasmic localisation. Alpha-enolase nucleocytoplasmic localisation may be regulated by tumour cell-specific phosphorylation at S419, previously reported in pancreatic cancer. RESULTS: Here we show ENO1 phosphorylation can also be observed in triple negative breast cancer patient samples and MCF10 tumour progression cell models. Furthermore, prevention of alpha-enolase-S419 phosphorylation by point mutation or a casein kinase-1 specific inhibitor D4476, induced tumour-specific nuclear accumulation of alpha-enolase, implicating S419 phosphorylation and casein kinase-1 in regulating subcellular localisation in tumour cell-specific fashion. Strikingly, alpha-enolase nuclear accumulation was induced in tumour cells by treatment with the specific exportin-1-mediated nuclear export inhibitor Leptomycin B. This suggests that S419 phosphorylation in tumour cells regulates alpha-enolase subcellular localisation by inducing its exportin-1-mediated nuclear export. Finally, as a first step to analyse the functional consequences of increased cytoplasmic alpha-enolase in tumour cells, we determined the alpha-enolase interactome in the absence/presence of D4476 treatment, with results suggesting clear differences with respect to interaction with cytoskeleton regulating proteins. CONCLUSIONS: The results suggest for the first time that tumour-specific S419 phosphorylation may contribute integrally to alpha-enolase cytoplasmic localisation, to facilitate alpha-enolase's role in modulating cytoskeletal organisation in triple negative breast cancer. This new information may be used for development of triple negative breast cancer specific therapeutics that target alpha-enolase.