The first step in glycolysis is the phosphorylation of glucose, catalyzed by hexokinase (HK). There are four isoforms of HKs. While every kind of cell has HK1, HK2 is expressed at high levels in only a limited number of adult tissues, including adipose, skeletal, lung and cardiac muscle tissue. HK2 is considered the inducible HK form, because it can be up-regulated approximately 100-fold in Th17 cells. Therefore, HK2 is a key rate-limiting factor in their development. An inhibitor of all HKs, 2-deoxy-D-glucose (2-DG), is reported to ameliorate inflammation in experimental autoimmune encephalomyelitis model mice by enhancing Tregs and suppressing Th17 lymphocyte differentiation. A specific inhibitor of HK2, instead, is 3-bromopyruvate which has been investigated on experimental models of rheumatoid arthrotis (RA). Glycolysis inhibition could become a feasible therapeutic strategy for RA, since it has already reported that blocking glycolysis with dichloroacetate (DCA), a pyruvate dehydrogenase kinase inhibitor, ameliorates experimental autoimmune arthritis in a mouse model.
Indeed, lymphocytes infiltrated in the synovial tissue of RA patients express HK2. In contrast, lymphocytes infiltrated in the synovium in OA patients do not express this enzyme. Inhibiting glycolysis with 2-DG was previously shown to decreases Th17 cell differentiation. We CD4+ T cells are cultured under Th17 conditions, BrPA dose-dependently inhibited the differentiation of Th17 cells, while enhancing the differentiation of Treg cells. IL-17 is the signature cytokine of the Th17 cell population, and is implicated in the pathogenesis of numerous autoimmune diseases. BrPA has another additional effect: it suppresses dendritic cell activation in the similar way to that of 2-DG. Tolerogenic dendritic cells (tol-DCs) have important role in tolerance and along with Th17 lymphocytes, they play a co-operative role in the arthritis. In addition, this sugar suppresses the DC activation induced by Toll like receptor 4 (TLR4) signaling. Considering that both Th17 cells and DC activation are metabolically depend on glycolysis, researchers think that BrPA is a rational therapeutic effect on inflammatory arthritis by metabolically suppressing the activation loop between Th17 and DC.
The fact that glycolysis enzyme regulate immune functioning and autoimmunity as well is fascinating and is corroborated by another phenomenon: protein moonlightning. This encompasses the concept that one protein may perform more than one funtion, especially in a cellular or biological context differing from the firstly acknowledged one. For example, aconitase is a key enzyme of the tricarboxylic acid cycle inside mitochondria, to produce energy from glucose. An additional function in this compartment is the regulation of iron homeostasis. Within these organelles, the LON protease (needed to regulate maturation of mitochondrial proteins) may work also as a molecular chaperone. Delving into trascriptional variances, the mitogen kinase ERK2 and the regulatory subunits of the cyclic AMP-dependent kinase (PKA) may work also as transcriptional repressors. On the other hand, the tumor suppressor Rb (retinoblastoma) and its major effectors E2Fs take role into the wide network of the DNA repair machinery. At least half of glycolytic enzymes are moonlightning proteins as well. The first studied in this regard might have been the dehydrogenase GAPDH.
This small enzyme catalyzes the conversion of glyceraldehyde 3-phosphate to glycerate 1,3-bisphosphate in glycolysis in the cytoplasm. However, other activities have been reported on this protein, that seem orchestrated by reactive oxidant species (ROS). These include direct binding to mRNAs, interference with the onset of a programmed cell death metabolic program, DNA replication and repair and telomere maintainance. On the cell surface, GAPDH works also as a transferrin receptor in macrophages. Phosphogluco-isomerase was serendipitously discovered in 1991 outside cells and characterized as an autocrine motility factor (AMF), working on a cellular receptor called gp78. Later it was identified as the actual component of the glycolytic chain, and deeper investigations reported how this protein influences tumor migration, cell proliferation, angiogenesis and resistance to chemotherapy-induced cell death. Its receptor, infact, is a member of the family of receptors (7TM) that bind many neurotransmitters, peptides and metabolites.
Aldolase is positioned midway in the glycolytic pathway and catalyzes the reversible cleavage of fructose-1,6-bisphosphate (FBP) to dihydroxyacetone-3-phosphate and glyceraldehyde-3-phosphate. Its muscle isoform (ALDOA), which is the most abundant aldolase isoform in almost all cancers, can organize actin filaments and regulate Wnt and p53 signaling. It has been also discovered that ALDOA is involved in progression of the S/G1 phase of the cell cycle. Finally, this protein affects activities of the energy sensor AMPK, which act as a cancer suppressor Enolase is another glycolysis moonlighting proteins in many eukaryote species, as well as prokaryotes. Inside the cell, it catalyzes the conversion of 2-phosphoglycerate to phosphoenolpyruvate. When displayed on the cell surface, it binds to host proteins. When this enzyme is temporarily in an inhibited status, it may translocate in cell nucleus where it works as a repressor of gene expression. When it is coded in its shorter form, enolase matches for the so called Myc-binding protein 1 (MBP-1), which is able to affect the activity of the famous metab-oncogene c-Myc.
Finally the pyruvate kinase component of the glycolytic chain is another enzymatic wonder. PKM2 is reported to interact with the C‐terminal residues of Oct‐4 and enhances its transactivation. This transcription factor is important in maintaining pluripotency in embryonic stem cells. Once in the cell nucleus, PKM2 has been shown to interact with its transcriptional activator HIF1α. This interaction increases transcriptional activity of HIF1α, which results in higher expression of genes involved in hypoxia adaptation. However, there more to that since very recently scientists from the University of Sao Paulo discovered the role of PKM2 in the development and maintenance of the exacerbated inflammation typical of autoimmune diseases. In the specific, beside glycolysis it acts in parallel with the differentiation Th17 helper T cells. When T-cell-specific PKM2 was excluded in vitro, Th17 differentiation was impaired and disease symptoms were attenuated, reducing Th17-mediated inflammation and demyelination. In tests on mice modified to not express the enzyme, disease development was reduced by more than 50%.
There is a logic into the moonlight phenomenon, despite the fact that it might seem fuzzy. Evolution has a way to find the most effective yet economical manner to orchstrate all biophenomena of living organisms. Since glucose metabolism is being discovered as a central ruler of immune development and maturation, the “system” must ave envised a protocol to take advantage of existing proteins, bestowing onto them additional rules along the evolutionary path. What better choices than to exploit the same glycolytic enzyme for the process of glucose-driven immune maturation or switch into autoimmunity? This way the cells has a limited number of genes to be regulated in a better manner, than a larger size and more complicated network into it. This concept might be or become the initial explanation why most part of our DNA is not actually conding genes, what it has been scientifically nicknamed “junk DNA”.
In our fuzzy logic.
- Edited by Dr. Gianfrancesco Cormaci, PhD, specialist in Clinical Biochemistry.
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