Μελέτη της έκφρασης και της ενεργότητας της L-Dopa αποκαρβοξυλάσης σε ανθρώπινα κύτταρα

Abstract

L-3.4-dihydroxyphenyl-alanine decarboxylase (L-Dopa decarboxylase, orDDC, EC 4.1.1.26) is the enzyme which catalyzes the decarboxylation of L-Dopa todopamine. Dopamine is considered to be one of the major mediators of the neuralimmunecommunication since it has been found to be a regulating factor of theproliferation and differentiation of different leukocyte subtypes. Leukocytes interactwith dopamine when, upon activation, cross the blood-brain barrier, but also inperipheral tissues as the white blood cells are capable of endogenous synthesis,secretion and uptake of catecholamines. The regulatory effect of dopamine occurs viadopamine receptors which are identified in the plasma membrane of white blood cells.Although several experimental data suggest the communication between the nervousand immune systems, the biosynthetic pathway of catecholamines in white bloodcells, has not yet been fully elucidated. Additionally, the experimental data relating tothe regulatory effect of dopamine appear contradictory. The need to furtherinvestigate the role of dopamine on immune system becomes more relevant whensome of the most important neurodegenerative diseases such as Parkinson's diseaseand schizophrenia are well-correlated with severe abnormalities of immune functions.Therefore, fully clarification of the biochemical pathways dopamine is involved inmay help in the understanding of the pathogenesis and treatment of manyneurodegenerative diseases. Based on the above and on recent studies of ourlaboratory which revealed for the first time the expression of the full-length DDC andthe alternative transcript lacking exon 3 in human peripheral leukocytes, the purposeof the present Ph.D. thesis was to further investigate the expression, the subcellulardistribution and the enzymatic activity regulation of DDC in immune cells of humanorigin.The results of this dissertation are summarized as follows: Study of the expression, topology and enzymatic activity of DDC inhuman peripheral white blood cells. In order to study the subcellular distribution of DDC in human peripheralwhite blood cells, samples of homogenized white blood cells were treated withthe non-ionic detergent Triton X-114. The enzyme recovered both in thedetergent enriched and highly hydrophobic phases, indicating the association ofthe molecule with the membrane fraction and the existence of multiple DDCsubpopulations with variable degrees of hydrophobicity. Western blot analysisresulted in the detection of the alternative DDC isoform, alt-DDC. The enzymewas found to be active towards the decarboxylation of L-Dopa to DA (mU / mg =3,67 ± 0,31). Although the obtained activity values were found to be considerablylower than the activity values observed in human embryonic kidney K293 cells,which were used as positive controls, the data strongly suggest the endogenousdopamine production in these types of cells. Study of the expression, topology and enzymatic activity of DDC inU937cell line.To further investigate the expression, topology, and enzymatic activity ofDDC in immune cells, U937 cell line was used as a model of macrophagefunction. DDC was detected for the first time in the cell line U937 byimmonoblotting experiments. The nature of DDC mRNA expressed in U937 cellline was investigated by reverse transcription-polymerase chain reactionexperiments and further Western bot analysis which lead to the detection of theneural-type DDC transcript lacking exon 3 and the protein isoform alt-DDC. Thefull-length transcript and the non-neural transcript were not detected. Thesubcellular distribution of DDC in U937 cell line was investigated using the nonionicreagent Triton X-114. Treatment of samples with the detergent resulted inthe recovery of DDC in the hydrophilic, hydrophobic, and the strong hydrophobicfraction, indicating the association of the enzyme with the membranes of U937cell line. The membrane-associated DDC subpopulation was found to be releasedfrom the membrane fraction into the soluble fraction in a pH dependent mannerwith an optimum release at pH 6.0–6.5. Determination of DDC enzymaticactivity in total homogenate, the soluble and membrane fraction of U937 cellssuggests that DDC is enzymatically active towards the decarboxylation of LDopato dopamine in all the subcellular fractions. The enzymatic activity levels of DDC in the membrane fraction were determined by 70% higher than theenzymatic activity values in total homogenate and the soluble fraction of the cellline. The solubilized molecule was found to be enzymatically active towards thedecarboxylation of L-Dopa with a peak of activity between pH 6.0–6.5.Incubation of the membrane fraction in the presence of the soluble fractionresulted in a decrease of DDC activity suggesting the existence of a regulatorymolecule of DDC activity in the soluble fraction of U937 cell line. Exogenousadministration of the DDC inhibitor, carbidopa, to U937 cell culture for 24 hourswas found to be cytotoxic in a dose-dependent manner, suggesting that DDC maybe involved in immune cell survival and cell death mechanisms. Similarly,incubation of U937 cells in the presence of increasing dopamine concentrationsfor 24 hours lead to cell death. These results support the hypothesis thatmacrophages are able to uptake extracellular dopamine, which regulatesproliferation and cell death in immune cells. Isolation and biochemical characterization of a DDC enzymaticactivity inhibitor from U937 cell line.The presence of the inhibitor of the enzymatic activity of DDC in U937 cellline was originally detected after the incubation of homogenized U937 cellsamples in the presence homogenized HEK-293 cell samples which are known tocontain high DDC activity levels. Fractionation experiments of U937 cellhomogenate demonstrated the exclusive localization of the endogenous inhibitorof the enzymatic activity of DDC in the soluble fraction, suggesting thesubcellular topology of the inhibitor. Further studies through temperature-inducedphase separation in the presence of the non-ionic detergent Triton X-114 led tothe recovery of the inhibitory activity in the aqueous phase of Triton X-114,confirming the hydrophilic nature of the inhibitory molecule. Treatment of thesoluble fraction of macrophages (SFM) with serine proteases such as, trypsin,chymotrypsin and proteinase K, resulted in the total loss of inhibitory activity,suggesting the protein nature of the inhibitor. Treatment of the SFM with RNAseA verified the protein nature of the regulatory molecule. The inhibitory activity ofthe molecule was found to be regulated by pre-incubation time, pH, temperatureand protein concentration. Using genetically modified cell lines (CHO) to model stable DDC and alt-DDC expressing cells, it was indicated that the regulatorymolecule inhibits the enzymatic activity of the full length DDC as well as itstruncated form, alt-DDC. Purification was achieved by means of ammoniumsulphate precipitation, phenyl sepharose hydrophobic chromatography andsubsequent extraction from a 2D non denaturing polyacrylamide gel. Thispurification scheme resulted in a 4.761-fold purification of the DDC activityinhibitory molecule. The molecular mass of the inhibitor was estimated at 67kDa, following analysis of the samples on SDS-PAGE. The inhibitory activity ofthe purified molecule was found to be affected by various parameters, such as,protein concentration, incubation time, pH and temperature. Kinetic analysisexperiments on DDC activity inhibition by the purified inhibitor showed that themode of inhibition was competitive with a Ki value of 10.59 ιΜ. Massspectrometry results suggested that the inhibitor corresponds to serum albumin.Highly purified human serum albumin was tested for its inhibitory effect onhuman DCC activity (HEK-297 cells homogenate). Moreover, human serumalbumin was found to inhibit the enzymatic activity of DDC derived from all thesubcellular fractions of U937 cell line. Incubation of CHO cells transfected eitherwith DDC or alt-DDC in the presence of the inhibitor indicated that human serumalbumin inhibits the enzymatic activity of both full-length DDC, and alt-DDC.The purification and biochemical characterization of human serum albouminas an inhibitor of the enzymatic activity of L-Dopa decarboxylase demonstrates aspecial interest, because it could be included in novel experimental approaches,which will have as a final target the development of novel pharmacological andtherapeutic means for the combat of neurodegenerative and neoplasmic diseases.