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Cervical cancer is the third most prevalent cancer in females (2018) with an estimation of 569,847 incidences and 311,365 deaths worldwide despite the rapid advancement of current technology in treating cervical cancer. Radiotherapy and chemotherapy pose side effects and subsequently hinder treatment efficacy. Therefore, taken together with the previous reports of the plants’ ability in treating cancers, Streblus asper is suggested to be a potential candidate for cervical cancer. This study was conducted to investigate the anti-cervical cancer potential of Streblus asper through the identification of key proteins and their expression that are regulated in the treatment using mice xenograft model. By employing the use of Liquid Chromatography Mass Spectrometry (LCMS), several proteins associated with cancer growth mechanisms were successfully identified. Four-hundred and fifty-two proteins common to both groups were identified, and 122 proteins were found able to be quantified. Among those proteins, 52 proteins were expressed more than 2-fold changes and 12 proteins were selected based on its established relationship with cancers, including annexin A2, 14-3-3 protein, transgelin-2, galectin-1, keratin, heat shock protein 10 and 70, glucose regulated protein (78kDa), gelsolin, alpha enolase, cofilin-1, vimentin, and calreticulin. All these proteins were downregulated upon treatment of cervical cancer tumour by Streblus asper. Pathway enrichment analysis revealed 40 related pathways which include among others, metabolism of protein, post-translational protein modification, cellular responses to external stimuli and stress, cell cycle, and apoptosis. These analyses may improve our molecular insight of the mechanisms involved in the treatment of cervical cancer tumour by Streblus asper extract.

The major proteases that constitute the fibrinolysis system are tightly regulated. Protease inhibitors target plasmin, the protease responsible for fibrin degradation, and the proteases that convert plasminogen into plasmin, including tissue-type plasminogen activator (tPA) and urokinase-type plasminogen activator (uPA). A second mechanism by which fibrinolysis is regulated involves exosite interactions, which localize plasminogen and its activators to fibrin, extracellular matrix (ECM) proteins, and cell surfaces. Once plasmin is generated in association with cell surfaces, it may cleave transmembrane proteins, activate growth factors, release growth factors from ECM proteins, remodel ECM, activate metalloproteases, and trigger cell-signaling by cleaving receptors in the Proteaseactivated Receptor (PAR) family. These processes are all implicated in cancer. It is thus not surprising that a family of structurally diverse but functionally similar cell-surface proteins, called Plasminogen Receptors (PlgRs), which increase the catalytic efficiency of plasminogen activation, have received attention for their possible function in cancer and as targets for anticancer drug development. In this review, we consider four previously described PlgRs, including: α-enolase, annexin-A2, Plg-RKT, and cytokeratin-8, in human cancer. To compare the PlgRs, we mined transcriptome profiling data from The Cancer Genome Atlas (TCGA) and searched for correlations between PlgR expression and patient survival. In glioma, the expression of specific PlgRs correlates with tumor grade. In a number of malignancies, including glioblastoma and liver cancer, increased expression of α-enolase or annexin-A2 is associated with an unfavorable prognosis. Whether these correlations reflect the function of PlgRs as receptors for plasminogen or other activities is discussed.

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.

Alpha-enolase (Eno) is an ubiquitary glycolytic enzyme playing multiple functions that go well beyond its principal metabolic role of energy supplier during glycolysis. Eno is localized in the cytoplasm, but also expressed on the cell membrane, where it binds plasminogen allowing its activation. Its shorter form, in the nucleus, acts as transcription factor. In inflammatory conditions, Eno undergoes post-translational modifications, such as citrullination, oxidation and phosphorylation. Eno is also an autoantigen in different disorders. In fact, autoantibodies to Eno have been detected in rheumatoid arthritis, lupus nephritis, primary glomerulonephritis, cancer, infections and other disorders, and in many cases they represent specific markers to be utilized in clinical practice. Anti-Eno antibodies in the different clinical conditions are not equal: they differ in isotype and often recognize different epitopes on the enzyme. IgG1 and IgG3 are prevalent in Rheumatoid Arthritis, IgG2 in Lupus nephritis and IgG4 in primary autoimmune glomerulopathy. This review analyzes the characteristics of anti-Eno autoantibodies in autoimmune disorders and cancer, describing their fine specificity and isotype restriction. The post-translational modifications that are target of autoantibodies are also discussed, as they represent the basis for elucidating the molecular mechanisms responsible for epitope generation. Despite an impressive amount of experimental work on anti-Eno antibodies, it is still necessary to validate the use of anti-Eno antibodies as biomarkers of selected diseases and extend the knowledge on the mechanisms of anti-Eno autoantibody production. Strategies that downmodulate the immune response to Eno may represent in the future novel approaches in the treatment of autoimmune disorders.

Renal α-enolase has variable expression in inflammatory and neoplastic diseases. Therefore, in order to define the distribution of α-enolase in renal tissues of cats, an immunohistochemistry assay was validated and described here. Tissues from 29 cats with IRIS Stage 2-4 CKD, 8 control cats2 years of age, and 4 control cats10 years of age were assessed. Interstitial nephritis was the predominant histopathological finding in the CKD group. The control cats2 years of age had moderate α-enolase immunoreactivity in tubular epithelium but staining was absent to mild in glomeruli. In contrast, α-enolase was moderate to high in tubular epithelium and glomeruli in control cats10 years of age. In cats with CKD, α-enolase was decreased in tubules that were degenerative or atrophic, similar to normal tubules in control groups, and moderate to high in glomeruli. When compared between the study groups, the results suggest that alpha-enolase decreases in damaged tubules and increases in the glomeruli of older cats prior to the development of detectable CKD. Further studies will be required to determine whether these findings relate to the pathogenesis or could be used in the diagnosis of feline CKD.

Differentiating stem cells must coordinate their metabolism and fate trajectories. Here, we report that the cat-alytic activity of the glycolytic enzyme Enolase 1 (ENO1) is directly regulated by RNAs leading to metabolic rewiring in mouse embryonic stem cells (mESCs). We identify RNA ligands that specifically inhibit ENO1???s enzymatic activity in vitro and diminish glycolysis in cultured human cells and mESCs. Pharmacological in-hibition or RNAi-mediated depletion of the protein deacetylase SIRT2 increases ENO1???s acetylation and en-hances its RNA binding. Similarly, induction of mESC differentiation leads to increased ENO1 acetylation, enhanced RNA binding, and inhibition of glycolysis. Stem cells expressing mutant forms of ENO1 that escape or hyper-activate this regulation display impaired germ layer differentiation. Our findings uncover acetylation-driven riboregulation of ENO1 as a physiological mechanism of glycolytic control and of the regulation of stem cell differentiation. Riboregulation may represent a more widespread principle of biological control.

We used small-angle X-ray scattering to study the self-association of enolase from Trichomonas vaginalis as a function of the protein concentration and cosolute type. We observed coexisting monomer...

Background: Serum tumor markers for the early detection of hepatocellular carcinoma is limited. This study analyzed the distribution and differences of ENO1 in the serum of healthy people, patients with chronic hepatitis B, cirrhosis and hepatocellular carcinoma. Furthermore, the distribution and correlation of ENO1 in the clinical data of patients with hepatocellular carcinoma were explored and its clinical diagnostic effect as well as combined diagnostic effect with AFP were compared. In addition, the effect of ENO1 on the survival of patients with hepatocellular carcinoma were also evaluated. The study will provide data basis for the application of ENO1 in the diagnosis of hepatocellular carcinoma, and provide new ideas for the study of molecular markers and therapeutic targets for hepatocellular carcinoma.Methods: In accordance with the inclusion and exclusion criteria, Patients diagnosed at the Infectious Diseases Department of the General Hospital of Ningxia Medical University and the healthy check-up population at the medical check-up centre from May 2012 to March 2017 were screened for inclusion in the study. T The final study subjects were 181, 28 patients with chronic hepatitis B (CHB), 31 patients with liver cirrhosis (LC), 104 patients with liver cancer (HCC) and 18 healthy people (NC). Clinical data and sera were collected from all study subjects, tissue specimens were collected from some patients. The levels of serum ENO1 and AFP were measured by ELISA. QRT-PCR and Western Blot t were used to detect the relative expression of ENO1 mRNA and protein in liver cancer tissues and paracancerous tissues. Statistical analysis was performed by t-test, bivariate Spearman correlation test, curve regression analysis, ROC curve analysis and survival curve analysis.Results: The relative expression of ENO1 mRNA and protein was significantly upregulated in hepatocellular carcinoma tissues relative to paraneoplastic tissues (p0.05), there were statistically significant differences (pConclusion: Serum ENO1 levels were significantly correlated with the occurrence, progression and metastasis of hepatocellular carcinoma, staging and postoperative survival of patients. ENO1 may be a candidate diagnostic marker for early diagnosis and evaluation of hepatocellular carcinoma progression, and can be used in combination with AFP as an auxiliary diagnostic function.

PURPOSE: To predict potential inhibitors of alpha-enolase to reduce plasminogen binding of Streptococcus pneumoniae (S. pneumoniae) that may lead as an orally active drug. S. pneumoniae remains dominant in causing invasive diseases. Fibrinolytic pathway is a critical factor of S. pneumoniae to invade and progression of disease in the host body. Besides the low mass on the cell surface, alpha-enolase possesses significant plasminogen binding among all exposed proteins. METHODS: In-silico based drug designing approach was implemented for evaluating potential inhibitors against alpha-enolase based on their binding affinities, energy score and pharmacokinetics. Lipinski’s rule of five (LRo5) and Egan’s (Brain Or IntestinaL EstimateD) BOILED-Egg methods were executed to predict the best ligand for biological systems. RESULTS: Molecular docking analysis revealed, Sodium (1,5-dihydroxy-2-oxopyrrolidin-3-yl)-hydroxy-dioxidophosphanium (SF-2312) as a promising inhibitor that fabricates finest attractive charges and conventional hydrogen bonds with S. pneumoniae alpha-enolase. Moreover, the pharmacokinetics of SF-2312 predict it as a therapeutic inhibitor for clinical trials. Like SF-2312, phosphono-acetohydroxamate (PhAH) also constructed adequate interactions at the active site of alpha-enolase, but it predicted less favourable than SF-2312 based on binding affinity. CONCLUSION: Briefly, SF-2312 and PhAH ligands could inhibit the role of alpha-enolase to restrain plasminogen binding, invasion and progression of S. pneumoniae. As per our investigation and analysis, SF-2312 is the most potent naturally existing inhibitor of S. pneumoniae alpha-enolase in current time. GRAPHICAL ABSTRACT: [Image: see text]

Background Tumor-associated antigens (TAAs) can be targeted in cancer therapy. We previously identified a monoclonal antibody (mAb) 12C7, which presented anti-tumor activity in lung cancer stem cells (LCSCs). Here, we aimed to identify the target antigen for 12C7 and confirm its role in LCSCs. Methods Immunofluorescence was used for antigen localization. After targeted antigen purification by electrophoresis and immunoblot, the antigen was identified by LC-MALDI-TOF/TOF mass spectrometry, immunofluorescence, and immunoprecipitation. The overexpression or silence of ENO1 was induced by lentiviral transduction. Self-renewal, growth, and invasion of LCSCs were evaluated by sphere formation, colony formation, and invasion assay, respectively. High-throughput transcriptome sequencing (RNA-seq) and bioinformatics analysis were performed to analyze downstream targets and pathways of targeted antigen. Results Targeted antigen showed a surface antigen expression pattern, and the 43–55 kDa protein band was identified as α-enolase (ENO1). Self-renewal, growth, and invasion abilities of LCSCs were remarkably inhibited by ENO1 downregulation, while enhanced by ENO1 upregulation. RNA-seq and bioinformatics analysis eventually screened 4 self-renewal-related and 6 invasion-related differentially expressed genes. GSEA analysis and qRT-PCR verified that ENO1 regulated self-renewal, invasion-related genes, and pathways. KEGG pathway analysis and immunoblot demonstrated that ENO1 inactivated AMPK pathway and activated mTOR pathway in LCSCs. Conclusions ENO1 is identified as a targeted antigen of mAb 12C7 and plays a pivotal role in facilitating self-renewal, growth, and invasion of LCSCs. These findings provide a potent therapeutic target for the stem cell therapy for lung cancer and have potential to improve the anti-tumor activity of 12C7.

Influenza A virus, a respiratory pathogen manipulates various host cellular processes to establish a successful infection in a host. We had reported earlier the interaction of influenza A virus nucleoprotein with host glycolytic enzymes; alpha enolase and pyruvate kinase in A549 cells. Matrix protein (M1), another multifunctional protein encoded by genome segment 7 forms the inner layer of the virion and interacts with the ribonucleoprotein complex. Nucleoprotein and matrix protein, major structural components of the virion together contribute to the stability of the capsid. Thus, we have investigated the interaction of viral matrix protein with host glycolytic enzymes; alpha enolase and pyruvate kinase. Results had demonstrated differential expression of these two glycolytic enzymes in response to matrix protein and their interaction with matrix protein by in vitro binding, co-immunoprecipitation and co-localization studies. Our results confirmed that viral matrix protein interacts with host glycolytic enzymes in association with viral nucleoprotein.

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.

Objective This study sought to explore the role of alpha-Enolase 1 (ENO1) in retinoblastoma (RB) which remains uncertain. Methods The expression of ENO1 in RB cell lines was examined by RT-qPCR and western blot. The biological function of ENO1 on cell proliferation in RB was determined in vitro. The predicted target of ENO1 was validated by dual-luciferase reporter assay and rescue experiment. The validation of clinical tissue samples was performed by RT-qPCR. Results Elevated ENO1 could promote the proliferation of RB cells. The dual luciferase reporter assay confirmed that ENO1 is the target for miR-22-3p. In rescue experiment, the result also indicated that miR-22-3p inhibits the proliferation of RB cells by negatively regulating the expression of ENO1. These differences were statistically significant (P Conclusion ENO1 functions as an oncogene in RB and inhibiting ENO1 by miR-22-3p suppresses the proliferation of RB cell lines. miR-22-3p/ENO1 pathway may serve as a novel target in RB.

Background Astrocytes contribute to the crosstalk that generates chronic neuro-inflammation in neurological diseases; however, compared with microglia, astrocytes respond to a more limited continuum of innate immune system stimulants. Recent studies suggest that the fibrinolysis system may regulate inflammation. The goal of this study was to test whether fibrinolysis system components activate astrocytes and if so, elucidate the responsible biochemical pathway. Methods Primary cultures of astrocytes and microglia were prepared from neonatal mouse brains. The ability of purified fibrinolysis system proteins to elicit a pro-inflammatory response was determined by measuring expression of the mRNAs encoding tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), and chemokine (C-C motif) ligand 2 (CCL2). IκBα phosphorylation also was measured. Plasminogen activation in association with cells was detected by chromogenic substrate hydrolysis. The activity of specific receptors was tested using neutralizing antibodies and reagents. Results Astrocytes expressed pro-inflammatory cytokines when treated with plasminogen but not when treated with agonists for Toll-like Receptor-4 (TLR4), TLR2, or TLR9. Microglia also expressed pro-inflammatory cytokines in response to plasminogen; however, in these cells, the response was observed only when tissue-type plasminogen activator (tPA) was added to activate plasminogen. In astrocytes, endogenously produced urokinase-type plasminogen activator (uPA) converted plasminogen into plasmin in the absence of tPA. Plasminogen activation was dependent on the plasminogen receptor, α-enolase, and the uPA receptor, uPAR. Although uPAR is capable of directly activating cell-signaling, the receptor responsible for cytokine expression and IκBα phosphorylation response to plasmin was Protease-activated Receptor-1 (PAR-1). The pathway, by which plasminogen induced astrocyte activation, was blocked by inhibiting any one of the three receptors implicated in this pathway with reagents such as εACA, α-enolase-specific antibody, uPAR-specific antibody, the uPA amino terminal fragment, or a pharmacologic PAR-1 inhibitor. Conclusions Plasminogen may activate astrocytes for pro-inflammatory cytokine expression through the concerted action of at least three distinct fibrinolysis protease receptors. The pathway is dependent on uPA to activate plasminogen, which is expressed endogenously by astrocytes in culture but also may be provided by other cells in the astrocytic cell microenvironment in the CNS.

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.

BACKGROUND Members of the Bacteroides fragilis group are the most important components of the normal human gut microbiome, but are also major opportunistic pathogens that are responsible for significant mortality, especially in the case of bacteraemia and other severe infections, such as intra-abdominal abscesses. Up to now, several virulence factors have been described that might explain the involvement of B. fragilis in these infections. The secretion of extracellular membrane vesicles (EMVs) has been proposed to play a role in pathogenesis and symbiosis in gram-negative bacteria, by releasing soluble proteins and other molecules. In B. fragilis, these vesicles are known to have haemagglutination and sialidosis activities, and also contain a capsular polysaccharide (PSA), although their involvement in virulence is still not clear. OBJECTIVE The aim of this study was to identify proteins in the EMV of the 638R B. fragilis strain by mass spectrometry, and also to assess for the presence of Bfp60, a surface plasminogen (Plg) activator, previously shown in B. fragilis to be responsible for the conversion of inactive Plg to active plasmin, which can also bind to laminin-1. METHODS B. fragilis was cultured in a minimum defined media and EMVs were obtained by differential centrifugation, ultracentrifugation, and filtration. The purified EMVs were observed by both transmission electron microscopy (TEM) and immunoelectron microscopy (IM). To identify EMV constituent proteins, EMVs were separated by 1D SDS-PAGE and proteomic analysis of proteins sized 35 kDa to approximately 65 kDa was performed using mass spectrometry (MALDI-TOF MS). FINDINGS TEM micrographs proved the presence of spherical vesicles and IM confirmed the presence of Bfp60 protein on their surface. Mass spectrometry identified 23 proteins with high confidence. One of the proteins from the B. fragilis EMVs was identified as an enolase P46 with a possible lyase activity. MAIN CONCLUSIONS Although the Bfp60 protein was not detected by proteomics, α-enolase P46 was found to be present in the EMVs of B. fragilis. The P46 protein has been previously described to be present in the outer membrane of B. fragilis as an iron-regulated protein.

Computational approach is used to identify the binding region of alpha-enolase over cell-surface protein of neutrophil. The product of alpha-enolase gene binds the one of the cell surface protein of neutrophil known as myoblast. After the binding on myoblast, neutrophil structure gets change and mobilized chromatin fibers came out to eliminate pathogen though NETosis. Thus, over study revealed that alpha-enolase of Streptococcus Pneumoniae is one of the major factor in inducing NETs during innate immune response.

We herein analyzed the relationships among the immunohistochemical expression of alpha-enolase (ENO1) and clinicopathological factors in order to define the significance of ENO1 in lung adenocarcinomas (ADCs). ENO1 expression was detected in most of the ADCs examined (95.8%), but not in bronchial and alveolar epithelia. ENO1 expression was typically observed in the cytoplasm among most ADCs (95.8%), but was also detected in the nucleus (56.3%). The levels were significantly higher in terminal respiratory unit (TRU) cytological subtype ADCs. Neither cytoplasmic nor nuclear expression was associated with any other clinicopathological factors including post-operative survival and growth activity. These results suggest that ENO1 is a crucial factor promoting neoplastic transformation exclusively in TRU subtype ADCs. We also investigated the potential utility of the immunohistochemical expression of ENO1 to differentiate TRU-type ADC cells from the reactive hyperplasia of pneumocytes and bronchiolar epithelial cells because difficulties are associated with discriminating these lesions in small biopsy specimens. The sensitivity and specificity of ENO1 (cytoplasmic/nuclear) were 87.5%/37.5% and 88.9%/100%, respectively, which are superior to those of p53 (18.8% and 100%). ENO1 has potential as a biomarker to assist in the histopathological detection of TRU subtype ADC cells.

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.

// Li Cui 1, 2, 3 , Naseim Elzakra 1, 2 , Shuaimei Xu 4 , Gary Guishan Xiao 5 , Yan Yang 1, 6 , Shen Hu 1, 2 1 UCLA School of Dentistry, Los Angeles, CA 90095, USA 2 UCLA Jonsson Comprehensive Cancer Center, Los Angeles, CA 900953, USA 3 Department of Dentistry, Maoming People’s Hospital, Maoming 525000, China 4 Guangdong Provincial Stomatological Hospital, Guangzhou 510000, China 5 School of Pharmaceutical Science and Technology, Dalian University of Technology, Dalian, 116024 China 6 Department of Stomatology, Zhongnan Hospital, Wuhan University, Wuhan 430071, China Correspondence to: Shen Hu, email: shenhu@ucla.edu Yan Yang, email: yanyangwhu@yahoo.com Keywords: Rho GDP-dissociation inhibitor 2, alpha-enolase, cofilin-1, primary Sjogren’s syndrome, mucosal-associated lymphoid tissue lymphoma Received: December 15, 2016 Accepted: January 24, 2017 Published: February 22, 2017 ABSTRACT Primary Sjogren’s syndrome (pSS) is a chronic autoimmune disease which might progress to mucosal-associated lymphoid tissue lymphoma (pSS/MALT). Diagnosis of pSS requires an invasive tissue biopsy and a delay in diagnosis of pSS has been frequently reported. In this study, four proteins including cofilin-1, alpha-enolase, annexin A2 and Rho GDP-dissociation inhibitor 2 (RGI2) were found to be over-expressed in pSS and pSS/MALT by 2D gel electrophoresis/mass spectrometry, and the finding was verified by the microarray analysis and western blotting results. We then developed enzyme-linked immunosorbent assays for autoantibodies including anti-cofilin-1, anti-alpha-enolase and anti-RGI2 with good quantitative ability. The expression levels of salivary anti-cofilin-1, anti-alpha-enolase and anti-RGI2 were found to be the highest in pSS/MALT patients and lowest in healthy controls. The combination of these three antiantibodies yielded an “area under the curve” (AUC) value of 0.94 with an 86% sensitivity and 93% specificity in distinguishing patients with pSS from healthy controls, an AUC value of 0.99 with a 95% sensitivity and 94% specificity in distinguishing patients with pSS/MALT from healthy controls and an AUC value of 0.86 with a 75% sensitivity and 94% specificity in distinguishing pSS/MALT patients from pSS patients. Collectively, we have successfully identified a panel of potential autoantigens that are progressively up-regulated during the development of pSS and its progression to MALT lymphoma. The autoantibody biomarkers may be used to help diagnose pSS and predict its progression to MALT lymphoma.

Alpha-enolase (ENO1) is a metabolic enzyme involved in the synthesis of pyruvate. It also acts as a plasminogen receptor and mediates the activation of plasmin and extracellular matrix degradation. In tumor cells, ENO1 is up-regulated and supports the Warburg effect; it is expressed at the cell surface, where it promotes cancer invasion, and is subjected to a specific array of post-translational modifications, namely acetylation, methylation and phosphorylation. ENO1 overexpression and post-translational modifications could be of diagnostic and prognostic value in many cancer types. Information on the biochemical, proteomics and immunological characterization of ENO1, and particularly its ability to trigger a strong specific humoral and cellular immune response, make this ubiquitous protein an interesting tumor target; DNA vaccination with ENO1 in preclinical models efficiently delays the development of very aggressive tumors such as pancreatic cancer. This review aims to analyze the main stages by which the tumor associated antigen (TAA) ENO1 has become a promising target that opens potential avenues for cancer immunotherapy.

OBJECTIVES The aim of this study was to explore the auxiliary diagnostic value of neutrophil gelatinase-associated lipocalin (NGAL) and anti-citrullinated alpha-enolase peptide 1 (CEP-1) in lower respiratory tract infections (LRTIs). METHODS Blood samples were collected from 99 in-patients with LRTIs [62 community-acquired pneumonia (CAP), 14 acute exacerbated chronic obstructive pulmonary diseases (AECOPD), 23 other diseases] and 50 healthy subjects. NGAL, CEP-1 and IL-6 were measured and compared. IL-6 was tested by electrochemiluminescence assay kit on Roche E601 immunology analyzer, CEP-1 was assessed with enzyme-linked immunosorbent assay kit, and NGAL was detected by latex immunoturbidimetric assay kit on Beckman Coulter AU2700. RESULTS Compared with healthy controls, NGAL and IL-6 levels were significantly increased in the patients with LRTIs, the area under the curves (AUC) was 0.97 and 0.88 respectively (P

Background:Anti-citrullinated alpha enolase antibodies have been investigated in rheumatoid arthritis and associated with bone erosion and interstitial lung disease but little is known about their prevalence and role in connective tissue diseases (CTDs).Objectives:The aim of this study was to investigate the prevalence and relevance of anti-CEP1 antibodies in CTDs.Methods:Serum samples from five independent patient cohorts were assessed: 1) established (est) primary Sjogren’s syndrome (pSS) N=78, 2) est-systemic lupus erythematosus (SLE) N=52, 3) est-systemic sclerosis (SSc) N=71, 4) pSS at disease onset N=30, 5) SLE at disease onset N=46 (cohorts 4 and 5 had at least 3 years of follow-up). Samples from ninety sex and age matched healthy donors (HD) and 200 patients with est-RA (disease controls) were also tested. Anti-CEP1 IgG antibodies were measured with a commercially available ELISA kit (Euroimmun, Luebeck, Germany).Results:Anti-CEP1 titer was significantly higher in est-pSS, est-SLE and est-SSc compared to HD, significantly lower in est-pSS and est-SSc compared to est-RA and comparable in est-SLE versus est-RA. We divided patients in every CTD group based on whether their anti-CEP1 titer was below or above the 25th, 50th and 75th percentile. In est-SLE anti-CEP1 values over the 25th percentile were associated with articular involvement (odds ratio, OR (95% confidence interval, CI)=11.5; 1.9-70.6, p=0.008). In est-pSS, no relationship between anti-CEP1>25th percentile and articular involvement was found but rather an association with rheumatoid factor positivity (OR (95% CI)=4.8, 1.6-14.1, p=0.004) and salivary gland swelling (OR (95% CI)=6.2, 1.3-29.1, p=0.021). In est-SSc no difference could be detected across the 3 groups. Anti-CEP-1 titers in pSS and SLE at onset did not differ from each other, were comparable also to those of HD and significantly lower than those of est-pSS, est-SLE and est-RA patients (all pConclusion:Anti-CEP-1 antibodies can be detected in CTDs at different title during the disease course and may increase overtime, at least in pSS. Although anti-CEP1 antibodies are associated with specific clinical manifestation in est-CTDs, such as articular involvement in est-SLE, they seem to lack a predictive value for future manifestations when measured at disease onset.References:[1]Alunno A, Bistoni O, Pratesi F et al Rheumatology (Oxford) 2018.[2]Manca ML, Alunno A, D’Amato C et al. Joint Bone Spine 2018.Disclosure of Interests:None declared

Celiac disease is a life-long intestinal autoimmune disease, characterized by the gluten intolerance and chronic enteric inflammation. Traditionally presented by intestinal manifestations, however, a shift toward extra intestinal presentation is taking place. One of the affected organs is the nervous systems presented by neuropsychiatric manifestations, hence the mechanism and pathways are not clear. The presence of neuronal and alpha-enolases and their corresponding antibodies were noticed in the mucosa and serum of celiac disease patients, as well as in other various autoimmune diseases with psycho-neurological manifestations. The aims of the present review are to screen the literature on different isoforms of enolase, mainly alpha enolase, and their specific antibodies and to suggest their potential pathophysiological mechanisms relaying the enolases to intestinal or extraintestinal celiac disease manifestations. The shared aspects between the enolases and celiac disease and the cross-talks between alpha-enolase and tissue transglutaminase suggest new potential pathophysiological mechanisms that might drive celiac disease evolvement., Highlights • Celiac disease is an autoimmune disease affecting mainly the small intestine and also the nervous systems. • Neuronal and alpha-enolases and their corresponding antibodies are present in celiac patient's mucosa and serum. • The shared aspects between the enolases and celiac disease might open new therapeutic aspects for celiac disease.

Alpha-enolase (ENO1), also known as 2-phospho-D-glycerate hydrolase, is a metalloenzyme that catalyzes the conversion of 2-phosphoglyceric acid to phosphoenolpyruvic acid in the glycolytic pathway. It is a multifunctional glycolytic enzyme involved in cellular stress, bacterial and fungal infections, autoantigen activities, the occurrence and metastasis of cancer, parasitic infections, and the growth, development and reproduction of organisms. This article mainly reviews the basic characteristics and biological functions of ENO1.

Energy metabolism in cancer cells is often increased to meet their higher proliferative rate and biosynthesis demands. Suppressing cancer cell metabolism using agents like metformin has become an attractive strategy for treating cancer patients. We showed that a novel ginsenoside derivative, Rh2E2, is as effective as aspirin in preventing the development of AOM/DSS-induced colorectal cancer and suppresses tumor growth and metastasis in a LLC-1 xenograft. A sub-chronic and acute toxicity LD50 test of Rh2E2 showed no harmful reactions at the maximum oral dosage of 5000 mg/kg body weight in mice. Proteomic profiling revealed that Rh2E2 specifically inhibited ATP production in cancer cells via down-regulation of metabolic enzymes involving glycolysis, fatty acid β-oxidation and the tricarboxylic acid cycle, leading to specific cytotoxicity and S-phase cell cycle arrest in cancer cells. Those findings suggest that Rh2E2 possesses a novel and safe anti-metabolic agent for cancer patients by specific reduction of energy-based metabolism in cancer cells.

Alpha-enolase (ENO1) is a ubiquitous protein. Patients with autoimmune thyroiditis-associated encephalopathy have high serum ENO1Ab titers. We aimed to explore whether ENO1Ab was the pathogenic antibody in the thyroid and brain. The serum ENO1Ab titers were significantly increased in the mice immunized with Thyroglobulin (Tg). And in the mice immunized with ENO1, serum levels of both TgAb and thyroid-stimulating hormone (TSH) were significantly increased. Obvious CD16+ cell infiltration, IgG deposit and cleaved caspase-3 were observed in the thyroid of ENO1-immunized mice. Spatial learning and memory abilities and synaptic functions were impaired in ENO1-immunized mice. Furthermore, the expression levels of Iba-1, GFAP, interlukin-6, CDK5, and phosphorylated tau were increased, and endothelial tight junction proteins were decreased in the brain of ENO1-immunized mice. These results suggest that ENO1Ab can cause thyrocyte damage via ADCC effect and impair cerebral function by disrupting the blood-brain barrier.

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&ndash, 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.

Female and male mice of the BTBR T+Itpr3tf/J (BTBR) strain have behaviors that resemble autism spectrum disorder. In comparison to C57BL/6 (B6) mice, BTBR mice have elevated humoral immunity, in that they have naturally high serum IgG levels and generate high levels of IgG antibodies, including autoantibodies to brain antigens. This study focused on the specificities of autoantibodies and the immune cells and their transcription factors that might be responsible for the autoantibodies. BTBR IgG autoantibodies bind to neurons better than microglia and with highest titer to nuclear antigens. Two of the antigens identified were alpha-enolase (ENO1) and dihydrolipoyllysine-residue succinyltransferase component of 2-oxoglutarate dehydrogenase complex, mitochondrial (DLST). Surprisingly based on IgG levels, the blood and spleens of BTBR mice have more CD4+ and CD8+ T cells, but fewer B cells than B6 mice. The high levels of autoantibodies in BTBR relates to their splenic T follicular helper (Tfh) cell levels, which likely are responsible for the higher number of plasma cells in BTBR mice than B6 mice. BTBR mice have increased gene expression of interleukin-21 receptor (Il-21r) and Paired Box 5 (Pax5), which are known to aid B cell differentiation to plasma cells, and an increased Lysine Demethylase 6B (Kdm6b)/DNA Methyltransferase 1 (Dnmt1) ratio, which increases gene expression. Identification of gene expression and immune activities of BTBR mice may aid understanding of mechanisms associated with autism since neuroimmune network interactions have been posited and induction of autoantibodies may drive the neuroinflammation associated with autism., Highlights • BTBR mice have more autoantibodies, e.g., anti-α-enolase and anti-DLST than B6 mice. • T follicular helper cells are increased in BTBR mice compared to B6 mice. • BTBR mice have more plasma cells with ​Pax5 ​and ​IL-21r ​than B6 mice. • A high ​Kdm6b ​to ​Dmnt1 ​ratio influences the BTBR’s increase in plasma cells.

Severe acute pancreatitis (SAP) associated acute lung injury (ALI) accounts for about 70% mortality of SAP patients. However, there are no precise biomarkers for the disease currently. Herein, we evaluated the potential of gamma-enolase (ENO2), against its universal isoform alpha-enolase (ENO1), as a marker of SAP–ALI in a rat model. Firstly, 16 male Sprague–Dawley rats were randomly divided into two groups, Sham (n = 8) and SAP–ALI (n = 8), for pancreatitis induction. Ultra-structure examination by electron microscopy and HE staining were used for lung injury assessment. Lung tissue expressions of alpha-enolase and gamma-enolase were evaluated by qRT-PCR and immunohistochemistry. In a prospective validation experiment, 28 rats were used: sham (n = 8), SAP–ALI at 3 h (3 h, n = 10), and SAP–ALI at 24 h (24 h, n = 10). Lung tissue damage, tissue expression and circulating alpha-enolase and gamma-enolase levels were evaluated. Elevated serum levels of α-amylase and TNF-α were observed in SAP rats but not in sham-operated rats. Histological examination of pancreatic and lung tissues indicated marked damage in SAP rats. While alpha-enolase was universally expressed, gamma-enolase was expressed only in damaged lung tissues. Gamma-enolase was detected in lung tissues, BALF, and serum as early as 3 h post-surgery when physical pathological damage was not apparent. Unlike alpha-enolase, secreted and/or circulating gamma-enolase level progressively increased, especially in serum, as lung damage progressed. Thus, gamma-enolase may signal and correlate lung tissue damage well before obvious physical pathological tissue damage and might be a candidate diagnostic and/or prognostic marker.

Objective Identification of molecular signatures having key roles in hypobaric hypoxia by analysing the salivary proteome. Saliva holds a promising future in the search for new clinical biomarkers that are easily accessible, less complex, accurate, and cost effective as well as being non-invasive. Methodology We employed qualitative proteomics approach to develop discriminatory biomarker signatures from human saliva exposed to hypobaric hypoxia. Salivary proteins were analyzed and compared between age-matched healthy subjects exposed to high altitude (∼13700 ft) for seven days (HAD7) with control subjects at sea level (Normoxia) by using 2-Dimensional gel electrophoresis/Mass Spectrometry approach. Results Several proteins with significant differential expression were found. The up-regulated proteins were apoptosis inducing factor-2, cystatin S, cystatin SN and carbonic anhydrase 6. The down regulated proteins were polymeric immunoglobulin receptor, alpha-enolase and prolactin-inducible protein. Further confirmation of the altered proteins such as alpha enolase, carbonic anhydrase 6, prolactin-inducible protein, apoptosis inducing factor 2, cystatin S and cystatin SN were performed using immunoblotting. The expression patterns of the selected proteins observed by immunoblot were in concurrence with 2-Dimesional gel electrophoresis results, therefore affirming the authenticity of the proteomic investigation. Conclusion This study provides the proof of concept of salivary biomarkers for the non-invasive detection of hypobaric hypoxia induced effects. It is highly feasible to turn these biomarkers into an applicable clinical test after large scale validation.

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.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.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.ENO1 may be crucial in the regulation of the proliferation and survival of synovial fibroblasts.

Background Anti-cyclic citrullinated peptide (anti-CCP) auto-antibodies (auto-Abs) represent the current gold standard for the diagnosis of rheumatoid arthritis (RA). However, growing evidence suggests that a variety of other citrullinated or not citrullinated self-proteins may act as autoantigens and lead to the production of auto-Abs. The identification of the diagnostic and/or prognostic value of such novel auto-Abs is under intense investigation. We recently demonstrated that RA patients display a higher prevalence of auto-Abs against the interferon-inducible protein 16 (anti-IFI16) but these auto-Abs do not have a good diagnostic value (1). Recent data showed that auto-Abs against citrullinated alpha-enolase (anti-CEP1) are associated with erosive RA (2). Objectives The purpose of this study was to investigate the possible prognostic value of anti-CEP-1 and anti-IFI16 as well as the clinical implication of their association with anti-CCP in a cohort or RA patients. Methods Two hundred and fifty two RA patients were enrolled and serum samples were obtained. Auto-Abs were assessed as follows: anti-CCP EDIA 2nd generation ELISA kit (Eurodiagnostica); anti-CEP-1 IgG ELISA kit (Euroimmun). In a subgroup of 113 patients also anti-IFI16 auto-Abs were assessed with an in-house ELISA kit (1). Clinical and serological records of patients were collected and statistical analysis was performed with SPSS 21.0 software. Results One hundred and twenty patients (44%) displayed anti-CEP-1 auto-Abs and of these 97 patients (87%) also displayed anti-CCP. Logistic regression analysis revealed an association between both auto-Abs and RA-associated pulmonary disease (odds ratio-OR=2.9; 95% CI=1.06–7.9; p=0.04). We also confirmed that anti-CEP-1 are associated with erosive RA but of interest to a greater extent compared to anti-CCP (anti-CEP-1: OR=4.12; p=0.04; anti-CCP: OR=2.1; p=0.03). The analysis that included anti-IFI16 auto-Abs revealed that a small proportion of patients display all the three auto-Abs (9%) but the triple positivity was significantly associated with male gender (OR=3.5; p=0.02), the presence of rheumatoid nodules (OR=5,3; p=0.015) and pulmonary involvement (OR=2.6; p=0.007). Anti-IFI16 auto-Abs were associated to male gender independently of the presence of the other two auto-Abs. Conclusions Our study demonstrated that anti-CEP-1 auto-Abs may participate to the development of RA-associated pulmonary manifestation together with anti-CCP and that the assessment of multiple auto-Abs in daily practice may help clinician to stratify RA patients at identify those at higher risk to develop extra-articular manifestations. References Alunno A et al. Arthritis Care Res (Hoboken) 2016;68(4):440–5. Montes A et al. Arthritis Rheum 2012;64(10):3102–3110. Disclosure of Interest None declared

Summary Glycolytic enzymes are attractive anticancer targets. They also carry out numerous, nonglycolytic "moonlighting" functions in cells. In this study, we investigated the anticancer activity of the triazine small molecule, GAPDS, that targets the glycolytic enzyme glyceraldehyde 3-phosphate dehydrogenase (GAPDH). GAPDS showed greater toxicity against cancer cells compared to a known GAPDH enzyme inhibitor. GAPDS also selectively inhibited cell migration and invasion. Our analysis showed that GAPDS treatment reduced GAPDH levels in the cytoplasm, which would modulate the secondary, moonlighting functions of this enzyme. We then used GAPDS as a probe to demonstrate that a moonlighting function of GAPDH is tubulin regulation, which may explain its anti-invasive properties. We also observed that GAPDS has potent anticancer activity in vivo. Our study indicates that strategies to target the secondary functions of anticancer candidates may yield potent therapeutics and useful chemical probes.

Background: Cisplatin and other anticancer drugs are important in the treatment of head and neck squamous cell carcinoma; however, some tumours develop drug resistance. If chemoresistance could be determined before treatment, unnecessary drug administration would be avoided. Here, we investigated chemoresistance factors by comprehensive analyses at the protein level. Methods: Four human carcinoma cell lines were used: cisplatin-sensitive UM-SCC-23, UM-SCC-23-CDDPR with acquired cisplatin resistance, naturally cisplatin-resistant UM-SCC-81B, and UM-SCC-23/WR with acquired 5-fluorouracil resistance. Extracted proteins were labelled with iTRAQ and analysed by tandem mass spectrometry to identify resistance. Protein expression was confirmed by western blotting and functional analysis was carried out using siRNA. Results: Thirteen multiple-drug resistance proteins were identified, as well as seven proteins with specific resistance to cisplatin, including α-enolase. Differential expression of these proteins in cisplatin-resistant and -sensitive cell lines was confirmed by western blotting. Functional analysis for α-enolase by siRNA showed that cisplatin sensitivity significantly was increased in UM-SCC-81B and slightly in UM-SCC-23-CDDPR but not in UM-SCC-23/WR cells. Conclusions: We identified proteins thought to mediate anticancer drug resistance using recent proteome technology and identified α-enolase as a true cisplatin chemoresistance factor. Such proteins could be used as biomarkers for anticancer agent resistance and as targets of cancer therapy.

Celik, Ahmet/0000-0001-6779-0674 WOS: 000342753600007 PubMed: 24801453 BackgroundAll aspects of aetiopathogenesis of recurrent aphthous stomatitis (RAS) have not been elucidated. RAS and Behcet'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. MethodsSerum 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. ResultsSerum 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

Introduction. Bone marrow plasmacytoid dendritic cells (pDCs) in patients with multiple myeloma (MM) both promote tumor growth, survival, and drug resistance, as well as induce decreased T/NK effector cell function and immune suppression. Delineation of the mechanism(s) mediating pDCs-MM-T-NK cells interactions may therefore identify novel therapeutic targets to enhance anti-MM immunity. Using gene expression profiling, we show that pDC-MM interactions trigger significant upregulation of the immunosuppressive metabolic enzyme alpha-Enolase (ENO1) in both pDCs and MM cells. ENO-1 functions as a glycolytic enzyme and plasminogen receptor which is overexpressed on the surface of tumor cells and MM pDCs. Here, we utilized our coculture models of patient autologous pDC-T-NK-MM cells to examine whether targeting ENO1, either alone or in combination can enhance anti-MM immunity in the BM milieu. Methods Gene expression profiles of MM cells cultured in the presence vs absence of pDCs were compared, and a heat map was generated (>1.5-fold change was considered significant, CI > 95%). MM cells were co-cultured with pDCs for 24h, followed by multicolor flow analyses to determine the pDC-induced change in ENO1 expression. Cytotoxic T lymphocyte (CTL) and NK cell activity assays: MM patient BM CD8+ T or NK-cells were cocultured with autologous pDCs (pDC1:T/NK10 ratio) in the presence or absence of ENO1 inhibitor/ENO1i ENOblock (0.2 µM) or anti-ENO1 Ab for 5 days; MM pre-stained cells were added for 24h (10T/NK:1MM), followed by quantification of viable MM cells by FACS. Anti-PD-L1 Ab (5 ug/ml) or HDAC 6 selective inhibitor ACY-241 (0.2 uM) were utilized for combination studies with ENO1i. CD107a expression was quantified in a degranulation assay. Results GEP analysis showed that pDCs induce upregulation of ENO1 transcript in MM (1.8-fold vs MM alone; n = 3; CI > 95%). Protein expression analysis showed that ENO1 is expressed in both pDC and MM cells; and importantly, that pDC-MM coculture further increases ENO1 expression in MM cells (5-6-fold; n=3; p = 0.003). The ENO+ MM cell population is also increased after pDC-MM cell coculture (3-4-fold vs MM; mean ± SD; n = 3; p = 0.005). Blockade of ENO1 with ENO inhibitor (ENOi) activates pDCs, as evidenced by increase in pDCs maturation/activation markers (CD80/CD83/CD86). Importantly, ENOi restores the ability of pDCs to trigger T cell activation and proliferation (n = 3; p = 0.018). Above all, ENOi increases pDC-induced MM-specific CD8+ CTL activity (p = 0.006), as well as NK cell-mediated cytolytic activity against autologous tumor cells (p = 0.005). Moreover, pDC-mediated MM-specific CD8+ CTL activity was effective even against allogeneic HLA-A2+ U266 MM cells (p = 0.008). Consistent with CTL and NK cell activation, ENO1i increases expression of surface CD107a on CD8+ T and NK cells (p = 0.006 and p= 0.0112, respectively; n = 7). The combination of ENO1i and anti-PD-L1 Ab induces more robust allogeneic and autologous MM-specific CD8+ CTL activity than ENO1i alone (% MM lysis: ENO1i: 34%; ENO1i + anti-PD-L1 Ab: 54%; n = 7; p = 0.0013). Finally, the combination of ENOi and ACY-241 also enhances anti-MM immune responses (% MM lysis: ENO1i: 35%; ENO1i + ACY-241: 54%; n = 8; p = 0.0044). Conclusions Our preclinical data provide the basis for novel immune-based therapeutic approaches targeting immunosuppressive metabolic alpha-enolase enzyme ENO1 to restore anti-MM immunity and improve patient outcome. Disclosures Chauhan: Stemline Therapeutics: Consultancy; C4 Therapeutics.: Equity Ownership. Anderson:Sanofi-Aventis: Other: Advisory Board; Bristol-Myers Squibb: Other: Scientific Founder; Oncopep: Other: Scientific Founder; Amgen: Consultancy, Speakers Bureau; Janssen: Consultancy, Speakers Bureau; Takeda: Consultancy, Speakers Bureau; Celgene: Consultancy, Speakers Bureau.

In order to extend the knowledge of rabies pathogenesis, a two-dimensional electrophoresis/mass spectrometry based postmortem comparative proteomics analysis was carried out on human brain samples. Alteration in expression profile of several proteins was detected. Proteins related to cytoskeleton, metabolism, proteasome and immune regulatory systems showed the most changes in expression levels. Among these groups, the cytoskeleton related proteins (dynein light chain, β-centractin, tubulin alpha-1C chain and destrin) and metabolism associated proteins (fatty acid-binding protein, macrophage migration inhibitory factor, glutamine synthetase and alpha enolase) were the main altered proteins. These alterations may be considered as an evidence of disturbances in neuronal key processes including axonal transport, synaptic activity, signaling and metabolic pathways in rabies virus infected human brain.

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Infertility affects approximately 10% of couples at reproductive age. Semen constituents may be potential immunogenic structures for women. The aim of our work is to detect and identify sperm proteins interacting with serum IgG antibodies from women with fertility disorders. The biochemical characterization of sperm antigens was performed using one and two dimensional gel electrophoresis, both of which were followed by immunoblotting. The IgG-binding proteins of interest were identified using mass spectrometry. From the serum pool of 30 infertile women, we detected sperm antigens within a relative molecular mass range between 30–80 kDa with an isoelectric point of 4–7. No antigens were detected using the serum pool from 10 fertile women (control group). Heat shock proteins (HSP 70) were identified as major sperm antigens associated with female immune infertility. Additionally, we report for the first time that alpha-enolase is a significant sperm antigen from the serum pool of infertile women. We suggest that the IgG-binding proteins identified in our study are related to immune infertility in the case of certain women with abnormally high levels of IgG antibodies linked to sperm proteins. Our results might be useful in the diagnoses of female immune infertility and may provide potential targets for further therapeutic treatment.

Enolase is a component of the glycolysis pathway and a "moonlighting" protein, with important roles in diverse cellular processes that are not related to its function in glycolysis. However, small molecule tools to probe enolase function have been restricted to crystallography or enzymology. In this study, we report the discovery of the small molecule "ENOblock", which is the first, nonsubstrate analogue that directly binds to enolase and inhibits its activity. ENOblock was isolated by small molecule screening in a cancer cell assay to detect cytotoxic agents that function in hypoxic conditions, which has previously been shown to induce drug resistance. Further analysis revealed that ENOblock can inhibit cancer cell metastasis in vivo. Moreover, an unexpected role for enolase in glucose homeostasis was revealed by in vivo analysis. Thus, ENOblock is the first reported enolase inhibitor that is suitable for biological assays. This new chemical tool may also be suitable for further study as a cancer and diabetes drug candidate.

Brucella abortus, the causative agent of bovine brucellosis, invades and replicates within cells inside a membrane-bound compartment known as the Brucella containing vacuole (BCV). After trafficking along the endocytic and secretory pathways, BCVs mature into endoplasmic reticulum-derived compartments permissive for bacterial replication. Brucella Type IV Secretion System (VirB) is a major virulence factor essential for the biogenesis of the replicative organelle. Upon infection, Brucella uses the VirB system to translocate effector proteins from the BCV into the host cell cytoplasm. Although the functions of many translocated proteins remain unknown, some of them have been demonstrated to modulate host cell signaling pathways to favor intracellular survival and replication. BPE123 (BAB2_0123) is a B. abortus VirB-translocated effector protein recently identified by our group whose function is yet unknown. In an attempt to identify host cell proteins interacting with BPE123, a pull-down assay was performed and human alpha-enolase (ENO-1) was identified by LC/MS-MS as a potential interaction partner of BPE123. These results were confirmed by immunoprecipitation assays. In bone-marrow derived macrophages infected with B. abortus, ENO-1 associates to BCVs in a BPE123-dependent manner, indicating that interaction with translocated BPE123 is also occurring during the intracellular phase of the bacterium. Furthermore, ENO-1 depletion by siRNA impaired B. abortus intracellular replication in HeLa cells, confirming a role for α-enolase during the infection process. Indeed, ENO-1 activity levels were enhanced upon B. abortus infection of THP-1 macrophagic cells, and this activation is highly dependent on BPE123. Taken together, these results suggest that interaction between BPE123 and host cell ENO-1 contributes to the intracellular lifestyle of B. abortus.

Salmonella enteric serovar Typhi Ty2 is a human specific pathogen and an etiological agent for typhoid fever. Most of Salmonella serotypes produce glycogen which has a comparatively minor role in virulence and colonization, but has a more significant role in survival. Enzymes present in glycolytic pathway of bacteria help bacteria to survive by activating other factors inside host. Numerous pathogenic bacteria species intervene with the plasminogen system, and this plasminogen-enolase association may play a critical role in the virulence of S. Typhi by causing direct damage to the host cell extracellular matrix, possibly by enzymic degradation of extracellular matrix proteins or other protein constituents. In this study, molecular modelling of enolase of Salmonella has been accomplished in silico by comparative modelling; we have then analyzed Human alpha enolase which is a homodimer and serves on epithelial cells with our model. Both Structures were docked by D-tartronate semialdehyde phosphate (TSP) and 3-aminoenolpyruvate phosphate (AEP) enolase inhibitors. Our study shows that salmonella enolase and human enolase have different active sites in their structure. This will help in development of new ligands, more suitable for inhibiting bacterial survival inside host as vaccines for typhoid fever are not fully protective. The study also confirmed that enolase Salmonella and Human Plasminogen suggested direct physical interaction between both of them as the activation loop of plasminogen residues showed conformational changes similar to the tissue type plasminogen activator. Various computational biology tools were used for our present study such as Modeller, Molegro Virtual Docker, Grommacs.

Objective To assess the prevalence of anti-alpha-enolase antibody in systemic autoimmune diseases in Chinese patients and its role in endothelial cell apoptosis. Methods The reactivity of anti-alpha-enolase antibody in a variety of autoimmune disorders in Chinese patients was evaluated by dot blot assay. Endothelial cell apoptosis was investigated by in vitro incubation of endothelial cells with IgG purified from anti-alpha-enolase antibody-positive sera, with or without pre-incubation with recombinant alpha-enolase. Results Anti-alpha-enolase antibody was prevalent in different systemic autoimmune diseases with relatively high reactivity in Chinese patients. In vitro incubation of endothelial cells with IgG containing anti-alpha-enolase antibody induced apoptosis in a time- and dose-dependent manner. Apoptosis was partly inhibited by pre-incubation of the endothelial cells with recombinant alpha-enolase. Conclusions Our data suggest that alpha-enolase is a common auto-antigen recognized by anti-endothelial cell antibodies in connective tissue disease. Interaction between alpha-enolase and its autoantibody plays a role in endothelial cell apoptosis. Changes other than cell killing may contribute to the pathogenesis of endothelial damage and microvascular lesions.

α-enolase (ENOA) is a metabolic enzyme involved in the synthesis of pyruvate. It also acts as a plasminogen receptor and thus mediates activation of plasmin and extracellular matrix degradation. In tumor cells, EΝΟΑ is upregulated and supports anaerobic proliferation (Warburg effect), it is expressed at the cell surface, where it promotes cancer invasion, and is subjected to a specific array of post-translational modifications, namely acetylation, methylation and phosphorylation. Both ENOA overexpression and its post-translational modifications could be of diagnostic and prognostic value in cancer. This review will discuss recent information on the biochemical, proteomics and immunological characterization of ENOA, particularly its ability to trigger a specific humoral and cellular immune response. In our opinion, this information can pave the way for effective new therapeutic and diagnostic strategies to counteract the growth of the most aggressive human disease.

Antibodies against citrullinated proteins (ACPAs) are highly specific for RA. Since the discovery of these antibodies, several of studies that focused on the presence and identity of citrullinated proteins in the joints of RA patients have been carried out. The best-known antigens that bind ACPAs are citrullinated filaggrin, Type II collagen (CII), alpha-enolase, fibrinogen and vimentin. This review compares citrullinated filaggrin, CII, alpha-enolase and fibrinogen with vimentin in their contribution to ACPA triggering, and gives an overview of the literature in which the role of citrullinated and non-citrullinated vimentin in the onset of ACPA production and the pathogenesis of RA is discussed.

Trichomonas vaginalis is a parasitic protozoan causing the most common human sexually transmitted disease (STD), trichomoniasis. The exact mechanism of its pathogenesis is still obscure. Alpha-enolase plays a pivotal role in the host-pathogen interaction, and as a surface receptor of several protists mediating plasminogen binding. In view of identifying plasminogen binding sites of T. vaginalis alpha-enolase, homology modeling and docking studies were conducted to obtain modeled structures of the T. vaginalis alpha-enolase-plasminogen complex. Modeling templates were searched by using BLAST, followed by multiple sequence alignment. The atomic coordinates of Escherichia coli enolase was retrieved from Protein Data Bank. Molecular structures of T. vaginalis alpha-enolase were modeled by using restraint-based modeling, followed by energy minimization using MODELLER program. The quality and stereochemistry of the models were evaluated by program PROCHECK. After addition of Mg2+, the selected model further refined by energy minimization employing NAMD program. The VMD program was used to superimpose structure of T. vaginalis alpha-enolase model with crystal structures of enolases from E. coli and S. pneumoniae. T. vaginalis alpha-enolase model was docked to human plasminogen for protein-protein interaction using Hex 5.1. Mark Gerstein’s calc-surface program was used to calculate the solvent accessibility at the interface of T. vaginalis alpha-enolase and human plasminogen before and after docking. The finding provides new insights for interaction at the protein-protein interface. Our theoretical prediction is consistent with preexisting biochemical data. The predicted interaction complex can be of great assistance in understanding structural insights, probably being necessary to an interaction between pathogen and host-component. The ability of T. vaginalis alpha-enolase to bind plasminogen may be indicative of being a key player in invasion of this pathogen to host. Conclusively, this work theoretically establishes the T. vaginalis alpha-enolase as a novel surface-linked virulence factor.