A structural model of 20S immunoproteasomes: effect of LMP2 codon 60 polymorphism on expression, activity, intracellular localisation and insight into the regulatory mechanisms

The immunoproteasome subunit low molecular weight protein 2 (LMP2) codon 60 polymorphism has been associated with autoimmune diseases. It has also been demonstrated to influence susceptibility to TNF-alpha-induced apoptosis in blood cells and proteasome activity in aged human brain. In the present study, an in silico model of immunoproteasome was used to examine the effect of the R60H polymorphism in the LMP2 subunit. The investigation of immunoproteasome expression, activity and intracellular localisation in an in vitro cellular model, namely lymphoblastoid cell lines, showed no major variations in functionality and amount, while a significant difference in antibody affinity was apparent. These data were integrated with previous results obtained in different tissues and combined with a structural model of the LMP2 polymorphism. Accordingly, we identified three prospective mechanisms that could explain the biological data for the polymorphism, such as modulation of the binding affinity of a putative non-catalytic modifier site on the external surface of the immunoproteasome core, or the modification of any channel between alpha and beta rings.     

Rapid caspase-3 activation during apoptosis revealed using fluorescence-resonance energy transfer

Caspase-3 is a crucial component of the apoptotic machinery in many cell types. Here, we report the timescale of caspase-3 activation in single living cells undergoing apoptosis. This was achieved by measuring the extent of fluorescence resonance energy transfer within a recombinant substrate containing cyan fluorescent protein (CFP) linked by a short peptide possessing the caspase-3 cleavage sequence, DEVD, to yellow fluorescent protein (YFP; i.e. CFP–DEVD–YFP). We demonstrate that, once initiated, the activation of caspase-3 is a very rapid process, taking 5 min or less to reach completion. Furthermore, this process occurs almost simultaneously with a depolarization of the mitochondrial membrane potential. These events occur just prior to the characteristic morphological changes associated with apoptosis. Our results clearly demonstrate that, once initiated, the commitment of cells to apoptosis is a remarkably rapid event when visualized at the single cell level.     

In vitro mucosal digestion of synthetic gliadin-derived peptides in celiac disease

Two celiac-active synthetic peptides derived from the A-gliadin structure corresponding to residues 8–19 (LQPQNPSQQQPQ) and to 11–19 were digestedin vitro with small intestinal mucosa from children with celiac disease in remission and from normal children. The products of digestion were separated into two fractions on the basis of M_r<400 and M_r>400 by gel permeation chromatography and subjected to amino acid analysis. After digestion of the dodecapeptide with celiac mucosa, 71±14% (molar) of the total digestion products remained in the M_r>400 fraction. Glutamine, proline, serine, and asparagine were the major amino acids present. Glutamine, proline, and leucine were the major amino acids in the M_r<400 fraction. The M_r>400 fraction from the celiac mucosal digestion of the nonapeptide was of similar composition to the corresponding fraction from the dodecapeptide and represented 78±15% of the total products. Digestion of the two peptides with normal mucosa gave lower amounts of products in the M_r>400 fraction, but they were of similar composition to the corresponding fractions from the celiac mucosal digestion. Peptides such as NPSQQQP and QNPSQQQ may be present in the M_r>400 fractions since glutamine and proline are present in the approximate ratio of 21, respectively. The results indicate a defect in the mucosal digestion of peptides which are active in an animal model of celiac disease.     

Studies on the photooxidation of tryptophan

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1. From the study of the photooxidation of tryptophan in three different aqueous systems, it has been concluded that the primary reaction is one which leads to the formation of formylkynurenine. Secondary reactions result in the formation of a variety of simpler amino acids together with a complex mixture of aromatic and polymeric materials.     

Interaction of amphipathic peptides with an immobilised model membrane

Summary The interaction of melittin and a truncated analogue of melittin with an immobilised phosphatidylcholine monolayer has been studied using dynamic elution techniques. The melittin analogue (21Q analogue) had five amino acids omitted from the C-terminal region of melittin. The influence of temperature and methanol concentration on the binding affinity of the two peptides was determined and compared to the binding behaviour of two control moleculesN-acetyltryptophanamide and diphenylalanine. Both peptides exhibited non-linear dependence of affinity on % methanol at different temperatures, whileN-acetyltryptophanamide and diphenylalanine exhibited linear behaviour. In addition, both melittin and the 21Q analogue exhibited significant band broadening under a range of experimental conditions, which was not evident forN-acetyltryptophanamide and diphenylalanine. As melittin is known to adopt a significant degree of α-helical conformation in the presence of lipids, the results suggest that melittin and the 21Q analogue adopt different conformations and orientations upon binding to the immobilised phosphatidylcholine surface. Overall, the results of this study demonstrate that the immobilised lipid monolayer provides a powerful system to rapidly assess the affinity of peptides for different lipid surfaces.     

Sulfate Conjugation of Dopamine in Rat Brain: Regional Distribution of Activity and Evidence for Neuronal Localization

Brain tissue contains at least two forms of phenolsulfotransferase that are involved in the sulfate conjugation of biogenic amines and their metabolites. Two apparent Km values were obtained for p-nitrophenol at pH 7.4 (0.6 microM and 0.3 mM) but only one enzyme had the capacity to conjugate dopamine (Km = 130 microM). Dopamine sulfotransferase activity was found to vary 17-fold in different brain regions, with the highest levels in diencephalon, hippocampus, and striatum. To determine the cellular localization of the enzymes, phenolsulfotransferase activity was measured in striatum following selective destruction of striatal neurons by stereotaxic injection of 2 micrograms kainic acid. Fourteen days after injection the catecholamine sulfotransferase activity in the lesioned striatum was reduced to approximately 40-50% of that in the control contralateral striatum. There was a statistically significant correlation between the ratio of lesioned to control activity for the sulfotransferase and the neuronal marker enzymes glutamate decarboxylase and neuron-specific enolase. p-Nitrophenol sulfotransferase activity was also decreased in the lesioned striatum. These results suggest that PST activity is present within the kainic acid-sensitive neurons of the striatum. The regional variation in activity, together with the results of the kainic acid studies, suggest that sulfate conjugation of biogenic amines and their metabolites in brain may take place within specific types of neurons.     

Distinct Cellular Localization of Membrane-Bound and Soluble Forms of Catechol-O-Methyltransferase in Brain

Abstract: The cellular localization of the two forms of catechol- O -methyltransferase (COMT) was investigated by measuring their activities in rat striatum following unilateral stereotaxic injection of kainic acid, which causes degeneration of striatal neurons followed by proliferation of astroglial cells. Membrane-bound COMT activity was decreased in the lesioned striatum, while soluble COMT activity was increased. There was a statistically significant correlation between the ratio of lesioned to control activity for membrane-bound COMT and the neuronal marker enzyme glutamate decarboxylase. Similarly the increase in soluble COMT activity paralleled that of the astroglial marker enzyme, glutamine synthetase. These results indicate that the K _m membrane-bound catechol- O -methyltransferase may be localized predominantly in neurons, whereas the high-K_m soluble enzyme is found in glial cells.     

Enhanced degradation of oxidized glutamine synthetase in vitro and after microinjection into hepatoma cells

Mixed-function oxidation of Escherichia coli glutamine synthetase has previously been suggested to mark the enzyme for intracellular degradation, and in vitro studies have demonstrated that oxidation renders the enzyme susceptible to proteolytic attack. In this study, the susceptibility of glutamine synthetase to degradation by purified proteases has been compared with the rate of degradation after microinjection into hepatoma cells. Upon exposure to an ascorbate mixed-function oxidation system the enzyme rapidly loses most of its activity, but further oxidation is required to cause susceptibility to extensive proteolytic attack either by a high-molecular-weight liver cysteine proteinase or by trypsin. The rate of degradation of biosynthetically 14C-labeled native and oxidized glutamine synthetase preparations after injection into hepatoma cells parallels their susceptibility to proteolysis in vitro. Native enzyme preparations and enzyme oxidatively inactivated, but not susceptible to extensive degradation by purified proteases, had similar intracellular half-lives; however, oxidized enzyme preparations that were susceptible to proteolytic breakdown in vitro were degraded almost ten times faster than the native enzyme within the growing hepatoma cells. These results suggest that the same features of the oxidized enzyme that render it susceptible to proteolysis in vitro are also recognized by the intracellular degradation system. In addition, they show that loss of enzyme activity does not necessarily imply decreased metabolic stability.