Enolase, also known as phosphopyruvate hydratase, is a metalloenzyme that catalyzes the dehydration of 2-phospho-D-glycerate (PGA) to phosphoenolpyruvate in the second half of the glycolitic pathway. In the reverse reaction (anabolic pathway), which occurs during gluconeogenesis, the same enzyme catalyzes the hydration of PEP to PGA.
The enzyme can be found from archeabacteria to mammals and its sequence is highly conserved. In mammals, three different genes, ENO1, ENO2 and ENO3 encodes for three isoforms of the enzyme: alpha-enolase (ENOA), gamma-enolase (ENOG) and beta-enolase (ENOB) with high sequence identity. The expression of these isoforms is tissue-specific: alpha-enolase is present in almost all adult tissues, beta-enolase is expressed in muscle tissues and gamma-enolase is found in neuron and neuroendocrine tissues.
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CHEMICAL STRUCTURE AND IMAGES
The monomer of ENOA consists of a smaller N-terminal domain (residues 1-133) and a larger C-terminal domain (residues 141-431). In eukarya the enzymatically enolase exists in a dimeric form in which two subunits face each other in a antiparallel manner; some eubacterial enolases, by contrast, are octameric.
A significant number of proteins, glycolytic enzymes and other cytosolic proteins that lack an N-terminal signal peptide reach the surface of eukaryotic cells. In mammals cells, some export routes of unconventional protein secretion were postulated: membrane blebbing, membrane flip-flop, endosomal recycling or a plasma membrane transporter. In yeast, using proteome chips, enolase and other glycolitic proteins were identified as phospholipid-binding proteins, supporting their interaction with membranes. In yeast, enolase amino acids 1 to 169 are sufficient to direct the protein to the cell surface and it doesn’t seem to be a integral protein, but a membrane associated one. How ENOA is displayed on the cell surface remains unknown. One possibility is that phosphoinositides could recruit enolase to plasma membrane and then it could be translocated.
ENOA structure details
ENOA aminoacids Percentage
SYNTHESIS AND TURNOVER
mRNA and protein synthesis
- ENOG lacks Ser 419 that is phosphorylated in ENOA
- The alpha/alpha homodimer is expressed in embryo and in most adult tissues.
- The alpha/beta heterodimer and the beta/beta homodimer are found in striated muscle
- The alpha/gamma heterodimer and the gamma/gamma homodimer in neurons.
Multifunctional enzyme that, as well as its role in glycolysis, plays a part in various processes such as growth control, hypoxia tolerance and allergic responses. May also function in the intravascular and pericellular fibrinolytic system due to its ability to serve as a receptor and activator of plasminogen on the cell surface of several cell-types such as leukocytes and neurons. Stimulates immunoglobulin production. Ref.3 Ref.18 Ref.20 Ref.21 Ref.22
MBP1 binds to the myc promoter and acts as a transcriptional repressor. May be a tumor suppressor.
Growing evidence makes clear that ENOA is a multifunctional protein. In hypoxic condition, ENOA act as a stress protein that is up-regulated via activation of the transcription factor hypoxia-inducible factor-1 (HIF-1) and may provide protection to the cells by increasing anaerobic metabolism. Several other functions have been reported for ENOA. In S. cervesiae is a heat shock protein (HSP48) thought to be involved in both thermal tolerance and growth control in this organism. ENOA has been identified as the eye lens crystallin τ in reptiles and birds; furthermore it has been described as a centrosome component in HeLa cells.
ENOA is considered a moonlighting protein, indeed it is not only a cytoplasmatic, but also a surface protein. It is present on the plasma membrane of many prokaryotic and eukaryotic cells where it acts as a strong plasminogen receptor. ENOA has been identified on the surface of pathogens like Gram-positive bacteria and Candida albicans, parasites like Plasmodium falciparum, yeast cells, hematopoietic cells like neutrophils, monocytes, T cells and B cells , neuron cells, endothelial cells and breast tumor cells, lung and pancreatic cancer cells.
ENOA is also a secreted protein; in fact it was detected in exosomes and culture medium of melanoma and non-small cell lung carcinoma (NSCLC) cells.
The monomeric form of ENOA is catalytically inefficient, but it could be more suitable and have a freely accessible subunit-interacting surface with other proteins. Anyway it has been recently shown that, in MCF-7 breast cancer cells, the surface protein maintain the catalytic activity, this suggests that its location in the plasma membrane doesn’t change the active centre of the enzyme. As ENOA undergoes several post-translational modifications, it is likely that some of these may stabilize the monomeric form of the protein and enable it to interact with other proteins to reach the cell surface. In Streptococcus species, the membrane translocation of the enzyme involves the hydrophobic domain of ENOA and is allowed through post-translational modifications such as acylation or phosphorylation.
ENOA present on cell surface acts as a strong plasminogen-binding receptor. Plasminogen binds to many cell types with high capacity (105-107 binding sites/cell), no single molecule can account for total plasminogen binding to cells. Both gangliosides and proteins provide cellular plasminogen binding sites. However, proteins such as ENOA, with carboxyl terminal lysine are predominantly responsible for the ability of cells to enhance plasminogen activation.
The binding of plasminogen to cell surface has profibrinolytic conseguences: enhancement of plasminogen activation, protection of plasmin from its inhibitor alpha-2-antiplasmin and enhancement of proteolytic activity of cell-bound plasmin. Proteolysis mediated by cell-associated plasmin contributes to physiological processes, such as tissue remodelling and embryogenesis, and to pathophysiological processes such as cell invasion, metastasis and inflammatory response. Noteworthy in a variety of neoplastic conditions a positive correlation exists between elevated levels of plasminogen activation and malignancy. Higher expression levels of urokinase plasminogen activator and/or plasminogen activator inhibitor-1 (PAI-1) in tumor tissues correlate with tumor aggressiveness and poor prognosis; this has been observed in breast, ovarian, esophageal, gastric, colorectal, lung and liver cancers. By cooperating with uPA, ENOA may concentrate plasminogen in tumor microenvironment to facilitate the generation of proteolytically active plasmin and may help tumor cell spreading. Indeed ENOA takes part, with uPAR (urokinase plasminogen activator receptor), integrins and some cytoskeletal proteins, to a multiprotein complex, called metastasome, responsible for adhesion, migration and proliferation in ovarian cancer cells. In pancreatic ductal adenocarcinoma (PDAC) several studies have revealed a de-regulated expression of many proteins involved in the plasminogen pro-fibrinolitic cascade (annexin A2, PAI- 2, uPA, uPAR, MMP-1 and MMP-10) and their expression has also been correlated with patients survival. Moreover it has been published that PDAC cells tPA is able to activate a mitogenic signal mediated by ERK-1/2 through the epidermal growth factor receptor (EGFR) and annexin A2 (ANXA2). Probably this proteins form a complex that includes also ENOA, indeed it has been shown that ENOA pulls down together with ANXA2, cytokeratin 8 and tPA in graft membrane fractions of PDAC cells.
ENOA auto-antibodies have been recognized as common markers of systemic autoimmune and inflammatory disorders. Anti-ENOA auto-antibodies have been initially reported in sera from patients that reacted with centrosomes in systemic rheumatic diseases. Then, anti-ENOA auto-antibodies have been shown to be a minor target antigen of anti-neutrophil cytoplasm antibodies (ANCA) in systemic vasculitides, ulcerative colitis and Chron’s disease, and primary sclerosing cholangitis. Since them, anti-ENOA auto-antibodies have also been found in sera of a large variety of autoimmune and inflammatory disorders such as systemic lupus erythematosus (SLE), mixed cryoglobulnemia (MC), systemic sclerosis (SSc), rheumatoid arthritis (RA), Behcet’s disease, multiple sclerosis,Hashimoto's encephalopathy (HE), endometriosis and cancer associated retinopathy (CAR). In healthy controls, auto-antibodies against ENOA have been found in 0 to 6%.
Moreover, reactivities between purified and recombinant ENOA differ, suggesting the predominant recognition of post-translationally or post-traductionally modified forms of ENOA, such as citrullination. Epitope-mapping was performed in CAR and in endometriosis and differences in epitopes recognition has been shown between patients and healthy controls. Anti-ENOA auto-antibodies might be produced after a contact with bacteria or yeast and cross react with human ENOA. In autoimmune and inflammatory diseases, anti-ENOA could induce endothelial injury through the generation of immune complexes and activation of the complement cascade, inhibit the binding of plasminogen to ENOA with perturbations of the intravascular and pericellular fibrinolytic system, and induce cell death through an apoptotic process.
Moreover, the overexpression of ENOA is associated with tumor development and this could be linked to a process known as aerobic glycolysis or Warburg effect. Warburg observed that cancer cells consume more glucose than normal cells and the way cancer cells use to generate ATP is through conversion of pyruvate to lactic acid even under normal oxygen supply. Not much about the mechanism of Warburg effect has been known until recently that upregulation of glycolytic enzymes by Hypoxia-inducible factor (HIF) has been identified. When the size of solid tumor is greater than 1 mm, cells face hypoxic stress due to slow-growing of blood vessel. One way of cell’s response to hypoxia is through the HIF-regulated gene expression to modulate several biological processes such as angiogenesis, proliferation, migration, apoptosis and metabolism. However, hypoxia is not the only condition directly responsible for HIF activation and subsequent changes in glucose metabolism, infact tumour suppressor and oncogene activity can even influence HIF activity. The ENOA promoter shows an Hypoxia Responsive Element and it has been shown to be upregulated at mRNA and protein level in different tumors; among them: breast cancer, hepatocellular carcinoma, colon carcinoma , lung cancer and PDAC.
Results from a bioinformatic study using gene chips and EST databases support a correlation between ENOA expression and tumorigenicity since overexpression of ENOA has been found in 18 out of 24 types of cancer. Furthermore, the clinical correlation of ENOA expression to tumor status has just been recently reported. It has been shown that ENOA expression in NSCLC cells is inversely correlated with patients prognosis; in hepatitis C virus-related hepatocellular carcinoma it has also been shown that expression of ENOA correlated positively with tumor size and venous invasion. Moreover increased ENOA mRNA expression has been preferentially detected in estrogen receptor-positive breast tumors; this overexpression correlates with a poor prognosis and confers tamoxifen treatment resistance.
ENOA can be classified as a tumor-associated antigen if we consider its overexpression together with the development of autoantibody against it in cancer patients. Each of the aspect previously mentioned can be a possible explanation for the ability of ENOA to induce an humoral response. Indeed ENOA is overexpressed in malignant cancer cells and aberrantly localize on the cell surface, it is found in exosomes in a p53-dependent manner after DNA damage and it is also a protein with many described post-translational modifications. Moreover, anti-ENOA auto-antibodies have been reported in sera from PDAC, lung cancer , leukaemia and melanoma patients.
α-Enolase: a promising therapeutic and diagnostic tumor target. 2011
Taken as a whole, these findings illustrate the multifunctional properties of ENOA in tumorigenesis, and its key implications in cancer proliferation, invasion and immune response. In cancer cells, ENOA is overexpressed and localizes on their surface, where it acts as a key protein in tumor metastasis, promoting cellular metabolism in anaerobic conditions and driving tumor invasion through plasminogen activation and extracellular matrix degradation. It also displays a characteristic pattern of PTMs, namely acetylation, methylation and phosphorylation, that regulate protein functions and immunogenicity. In several kinds of tumor, patients develop an integrated response of CD4+, CD8+ T cells and B cells against ENOA, together with anti-ENOA autoantibodies in their sera. Clinical correlations propose ENOA as a novel target for cancer immunotherapy. In pancreatic cancer, for example, the pancreas-specific Ser 419 phosphorylated ENOA is upregulated and induces the production of autoantibodies with diagnostic and prognostic value
Production of autoantibodies to phosphorylated ENOA in pancreatic cancer.
ENOA is overexpressed in tumor cells compared with normal tissues and it is present on the surface of different cell types where it acts as a plasminogen receptor.
ENOA is phosphorylated on Ser 419 only in pancreatic tissues, the overexpression of this post-translationally modified ENOA in tumor condition induces the production of autoantibodies with clinical relevance in pancreatic cancer patients.