ABSTRACT
O-linked ß-N-acetylglucosaminylation (O-GlcNAcylation) is a reversible post-translational modification on serine and threonine residues of cytosolic, nuclear and mitochondrial proteins. O-GlcNAcylation level is regulated by OGT (O-GlcNAc transferase), which adds GlcNAc on proteins, and OGA (O-GlcNAcase), which removes it. Abnormal level of protein O-GlcNAcylation has been observed in numerous cancer cell types, including cervical cancer cells. In the present study, we have evaluated the effect of increasing protein O-GlcNAcylation on cervical cancer-derived CaSki cells. We observed that pharmacological enhancement of protein O-GlcNAcylation by Thiamet G (an inhibitor of OGA) and glucosamine (which provides UDP-GlcNAc substrate to OGT) increases CaSki cells proliferation, migration and survival. Moreover, we showed that increased O-GlcNAcylation promotes IGF-1 receptor (IGF1R) autophosphorylation, possibly through inhibition of protein tyrosine-phosphatase 1B activity. This was associated with increased IGF-1-induced phosphatidyl-Inositol 3-phosphate production at the plasma membrane and increased Akt activation in CaSki cells. Finally, we showed that protein O-GlcNAcylation and Akt phosphorylation levels were higher in human cervical cancer samples compared to healthy cervix tissues, and a highly positive correlation was observed between O-GlcNAcylation level and Akt phosphorylation in theses tissues. Together, our results indicate that increased O-GlcNAcylation, by activating IGF1R/ Phosphatidyl inositol 3-Kinase (PI-3K)/Akt signaling, may participate in cervical cancer cell growth and proliferation.
Subject(s)
Acetylglucosamine , Uterine Cervical Neoplasms , Acetylglucosamine/metabolism , Cervix Uteri/metabolism , Female , Humans , Inositol/metabolism , N-Acetylglucosaminyltransferases/genetics , Protein Processing, Post-Translational , Proto-Oncogene Proteins c-akt/metabolism , Receptor, IGF Type 1/metabolism , Uterine Cervical Neoplasms/metabolismABSTRACT
In this work, we studied a recombinant mu-class glutathione transferase of 25.5 kDa from Taenia solium metacestode (rTs25GST1-1) that follows MichaelisMenten kinetics with 1-chloro-2,4-dinitrobenzene (CDNB). The kinetic parameters obtained for rTs25GST1-1 with CDNB and GSH were V(max) =12.04 µmol/min/mg and K(m)=1.38 mM, and V(max) =10.20 µmol/min/mg and K(m)=0.90, respectively. The optimal activity was found at pH 8 in the 37-40 °C temperature range. Circular dichroism studies for rTs25GST1-1 at different pH showed that it maintains a typical α-helix structure between pH 6.5-7.5, but loses it between pH 8 and 8.5. Thermal CD assays showed rTs25GST1-1 barely changed its secondary structure. Unfolding/refolding assays showed that rTs25GST1-1 retained its structure up to 40 °C without loss of its activity. Additionally, exposure of rTs25GST1-1 to cumene hydroperoxide did not produce significant changes in its structure and only affected 50% of its activity.
Subject(s)
Glutathione Transferase/chemistry , Glutathione Transferase/metabolism , Taenia solium/enzymology , Animals , Circular Dichroism , Dinitrochlorobenzene/metabolism , Enzyme Stability , Glutathione Transferase/genetics , Glutathione Transferase/isolation & purification , Hydrogen-Ion Concentration , Kinetics , Molecular Weight , Protein Conformation , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/isolation & purification , Recombinant Proteins/metabolism , TemperatureABSTRACT
Glutathione transferases (GSTs) are essential enzymes in many organisms due their diverse functions and, in helminths they are the main detoxification system. For Taenia solium, two cytosolic GSTs with molecular masses of 25.5 and 26.5 kDa (Ts26GST) have been found. Ts26GST was cloned to be studied in its recombinant form (recTs26GST). Although the primary structure is related to the mu class, the kinetic parameters for CDNB (V(max)=51.5 micromol min(-1)mg(-1); K(m)=1.06 mM; k(cat)= 22.2s(-1)) are related with some alpha GSTs. The substrate and inhibitor class markers reaffirmed these bimodal characteristics. Inhibition studies with anthelminthics indicate that recTs26GST is sensitive to mebendazole, displaying a non competitive inhibition pattern suggesting that at least two molecules are binding to recTs26GST. On the other hand, the kinetic curves for CDNB and GSH showed a positive cooperativity that was corroborated using fluorometric assays. Those assays indicate that CDNB binding is highly influenced by GSH, probably by modulation of the Ts26GST conformational ensamble.
Subject(s)
Glutathione Transferase/chemistry , Recombinant Proteins/chemistry , Taenia solium/enzymology , Animals , Enzyme Activation , Kinetics , Taenia solium/geneticsABSTRACT
The glutathione transferase (GST) system of parasites represents the main detoxification mechanism of hydrophobic and electrophilic compounds. Parasites lack the CYP450 activity, hence part of its function has been taken over by other enzymes including GSTs. Cytosolic GSTs (cGSTs) are found in this system and constitute a versatile and numerous group that in parasites display many peculiarities in contrast to mammalian cGSTs. This review summarizes aspects of the biochemistry of parasite cGSTs such as substrate specificities, inhibitor sensitivities, classification, kinetics and catalysis, as well as some aspects of their protective role.
