Glycation and glycoxidation protein products are formed upon binding of sugars to NH 2 groups of lysine and arginine residues and have been shown to accumulate during aging and in pathologies such as Alzheimer's disease and diabetes. Because the proteasome is the major intracellular proteolytic system involved in the removal of altered proteins, the effect of intracellular glycation on proteasome function has been analyzed in human dermal fibroblasts subjected to treatment with glyoxal that promotes the formation of N⑀-carboxymethyl-lysine adducts on proteins. The three proteasome peptidase activities were decreased in glyoxal-treated cells as compared with control cells, and glyoxal was also found to inhibit these peptidase activities in vitro. In addition, the activity of glucose-6-phosphate dehydrogenase, a crucial enzyme for the regulation of the intracellular redox status, was dramatically reduced in glyoxal-treated cells. Further analysis was performed to determine whether glycated proteins are substrates for proteasome degradation. In contrast to the oxidized glucose-6-phosphate dehydrogenase, both N⑀-carboxymethyl-lysine-and fluorescent-glycated enzymes were resistant to degradation by the 20 S proteasome in vitro, and this resistance was correlated with an increased conformational stability of the glycated proteins. These results provide one explanation for why glycated proteins build up both as a function of disease and aging. Finally, N⑀-carboxymethyl-lysine-modified proteins were found to be ubiquitinated in glyoxal-treated cells suggesting a potential mechanism by which these modified proteins may be marked for degradation.

Responses of three bioluminescent Ca(2+) sensors were studied in vitro and in neurons from brain slices. These sensors consisted of tandem fusions of green fluorescent protein (GFP) with the photoproteins aequorin, obelin, or a mutant aequorin with high Ca(2+) sensitivity. Kinetics of GFP-obelin responses to a saturating Ca(2+) concentration were faster than those of GFP-aequorin at all Mg(2+) concentrations tested, whereas GFP-mutant aequorin responses were the slowest. GFP-photoproteins were efficiently expressed in pyramidal neurons following overnight incubation of acute neocortical slices with recombinant Sindbis viruses. Expression of GFP-photoproteins did not result in conspicuous modification of morphological or electrophysiological properties of layer V pyramidal cells. The three sensors allowed the detection of Ca(2+) transients associated with action potential discharge in single layer V pyramidal neurons. In these neurons, depolarizing steps of increasing amplitude elicited action potential discharge of increasing frequency. Bioluminescent responses of the three sensors were similar in several respects: detection thresholds, an exponential increase with stimulus intensity, photoprotein consumptions, and kinetic properties. These responses, which were markedly slower than kinetics measured in vitro, increased linearly during the action potential discharge and decayed exponentially at the end of the discharge. Onset slopes increased with stimulus intensity, whereas decay kinetics remained constant. Dendritic light emission contributed to whole-field responses, but the spatial resolution of bioluminescence imaging was limited to the soma and proximal apical dendrite. Nonetheless, the high signal-to-background ratio of GFP-photoproteins allowed the detection of Ca(2+) transients associated with 5 action potentials in single neurons upon whole-field bioluminescence recordings.

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To investigate the role of some tertiary interactions, the disulfide bonds, in the early stages of refolding of hen lysozyme, we report the kinetics of reoxidation of denatured and reduced lysozyme under the same refolding conditions as those previously used to investigate the kinetics of regain of its circular dichroism~CD!, fluorescence, and activity. At different stages of the refolding, the oxidation of the protein was blocked by alkylation of the free cysteines with iodoacetamide and the various oxidation states present in the samples were identified by electrospray-mass spectrometry. Thus, it was possible to monitor the appearance and0or disappearance of the species with 0 to 4 disulfide bonds. Using a simulation program, these kinetics were compared with those of regain of far-UV CD, fluorescence, and enzymatic activity and were discussed in terms of a refined model for the refolding of reduced hen egg white lysozyme.