Flow-cytometric assessment of cellular poly(ADP-ribosyl)ation capacity in peripheral blood lymphocytes

BACKGROUND: Poly(ADP-ribosyl)ation is a posttranslational modification of nuclear proteins catalysed by poly(ADP-ribose) polymerases (PARPs), using NAD(+ )as a substrate. Activation of PARP-1 is in immediate response to DNA damage generated by endogenous and exogenous damaging agents. It has been implicated in several crucial cellular processes including DNA repair and maintenance of genomic stability, which are both intimately linked with the ageing process. The measurement of cellular poly(ADP-ribosyl)ation capacity, defined as the amount of poly(ADP-ribose) produced under maximal stimulation, is therefore relevant for research on ageing, as well as for a variety of other scientific questions. RESULTS: This paper reports a new, robust protocol for the measurement of cellular poly(ADP-ribosyl)ation capacity in PBMC or Jurkat T-cells using flow cytometry, based on a previously established immuno-dot-blot assay. In order to validate the new assay, we determined the dose-response curve of 3-aminobenzamide, a well-known competitive PARP inhibitor, and we derived an IC(50 )that is very close to the published value. When testing a set of PBMC samples taken from fifteen healthy young human donors, we could confirm the presence of a substantial interindividual variation, as previously observed using a radiometric assay. CONCLUSION: The methodology described in this paper should be generally useful for the determination of cellular poly(ADP-ribosyl)ation capacity in a wide variety of settings, especially for the comparison of large sets of samples, such as population studies. In contrast to previously published radiometric or immuno-dot-blot assays, the new FACS-based method allows (i) selective analysis of mononuclear cells by gating and (ii) detection of a possible heterogeneity in poly(ADP-ribosyl)ation capacity between cells of the same type

Funktionale Analyse von Mutanten in der Transmembranregion-1 und in der Octarepeat-Region des Prion-Proteins

The cellular prion protein (PrPPcP) is a glycosyl-phosphatidyl-inositol (GPI)-anchored 35 kDa glycoprotein located on the outer surface of the plasma membrane and plays an essential role in the pathogenesis of several inherited and transmissible neurodegenerative diseases such as Creutzfeldt-Jakob disease (CJD) and Gerstmann-Sträussler-Scheinker syndrome (GSSS). Despite being the subject of many recent studies, the physiological function of PrPPcP remains largely unresolved. Several candidate functions have been discussed, including binding and internalisation of copper or other metals, superoxide dismutase-like activity, signal transduction and regulation of cellular antioxidant activities. The transmembrane (TM1) region of PrPPcP (codons 110-135) should play a key role in PrPPcP function because of its high conservation throughout evolution. Moreover it contains an array of hydrophobic amino acids, and peptides derived from this region are neurotoxic.The aim of the present study was to try to elucidate a possible role of PrPPcP in the regulation mitochondrial membrane potential (), in the regulation of the basal level of endogenous reactive oxygen species (ROS) and in antioxidative defence. For this purpose transiently transfected mouse neuroblastoma cells overexpressing PrP, either as wild-type (wt) protein or as a deletion mutant lacking codons 114-121 (henceforth called Δ8TM1-PrP), were analysed. In addition a deletion mutant in the octarepeat region (octa-PrP) was studied. The results showed that wt-PrP and 8TM1-PrP have no impact on . Likewise, overexpression of PrP (wt or mutant) had no impact on endogenous ROS levels. However, under conditions of oxidative stress induced by H2O2 treatment of the cells, ROS levels were lower in cells transfected with wt-PrP or Δ8TM1-PrP expression plasmids.Increased phosphorylation of ERK2 (p42) and decreased phosphorylation of JNK1/JNK2/3 seemed to be linked to this protective effect of PrPc.Two other systems derived from transgenic Δ8TM1-PrP mouse brain (whole-brain or cerebellar granular neurones [CGN]) were also studied, but no biological impact of the transgene 8TM1-PrP was observable, most likely due to the low expression level.In conclusion, the protective effect of PrPPcP against oxidative stress implicates its octarepeat region but not its TM1 domain; it does not manifest as lowered basal ROS level; and it seems to involve the MAPK pathway, especially p42 and JNK1/JNK2/3

The octarepeat region of prion protein, but not the TM1 domain, is important for the antioxidant effect of prion protein

The cellular prion protein (PrPc) plays a crucial role in the pathogenesis of prion diseases, but its physiological function is far from understood. Several candidate functions have been proposed including binding and internalization of metal ions, a superoxide dismutase-like activity, regulation of cellular antioxidant activities, and signal transduction. The transmembrane (TM1) region of PrPc (residues 110 135) is particularly interesting because of its very high evolutionary conservation. We investigated a possible role of TM1 in the antioxidant defense, by assessing the impact of overexpressing wt-PrP or deletion mutants in N2A mouse neuroblastoma cells on intracellular reactive oxygen species (ROS) levels. Under conditions of oxidative stress, intracellular ROS levels were significantly lowered in cells overexpressing either wild-type PrPc (wt-PrP) or a deletion mutant affecting TM1 (Δ8TM1-PrP), but, as expected, not in cultures overexpressing a deletion mutant lacking the octapeptide region (Δocta-PrP). Overexpression of wt-PrP, Δ8TM1-PrP, or Δocta-PrP did not affect basal ROS levels. Interestingly, the mitochondrial membrane potential was significantly lowered in Δocta-PrP-transfected cultures in the absence of oxidative stress.We conclude that the protective effect of PrPc against oxidative stress involves the octarepeat region but not the TM1 domain nor the highaffinity copper binding site described for human residues His96/His111

Flow-cytometric assessment of cellular poly(ADP-ribosyl)ation capacity in peripheral blood lymphocytes

Abstract Background Poly(ADP-ribosyl)ation is a posttranslational modification of nuclear proteins catalysed by poly(ADP-ribose) polymerases (PARPs), using NAD+ as a substrate. Activation of PARP-1 is in immediate response to DNA damage generated by endogenous and exogenous damaging agents. It has been implicated in several crucial cellular processes including DNA repair and maintenance of genomic stability, which are both intimately linked with the ageing process. The measurement of cellular poly(ADP-ribosyl)ation capacity, defined as the amount of poly(ADP-ribose) produced under maximal stimulation, is therefore relevant for research on ageing, as well as for a variety of other scientific questions. Results This paper reports a new, robust protocol for the measurement of cellular poly(ADP-ribosyl)ation capacity in PBMC or Jurkat T-cells using flow cytometry, based on a previously established immuno-dot-blot assay. In order to validate the new assay, we determined the dose-response curve of 3-aminobenzamide, a well-known competitive PARP inhibitor, and we derived an IC50 that is very close to the published value. When testing a set of PBMC samples taken from fifteen healthy young human donors, we could confirm the presence of a substantial interindividual variation, as previously observed using a radiometric assay. Conclusion The methodology described in this paper should be generally useful for the determination of cellular poly(ADP-ribosyl)ation capacity in a wide variety of settings, especially for the comparison of large sets of samples, such as population studies. In contrast to previously published radiometric or immuno-dot-blot assays, the new FACS-based method allows (i) selective analysis of mononuclear cells by gating and (ii) detection of a possible heterogeneity in poly(ADP-ribosyl)ation capacity between cells of the same type.</p