Erika Sandmeier

Neurotoxicity of prion peptide 106-126 not confirmed.

Prion‐related diseases are accompanied by neurodegeneration, astroglial proliferation and formation of proteinase K‐resistant aggregates of the scrapie isoform of the prion protein (PrPSc). The synthetic PrP fragment 106‐126 was reported to be neurotoxic towards cultured rat hippocampal neurons (Forloni, G., Angeretti, N., Chiesa, R., Monzani, E., Salmona, M., Bugiani, O. and Tagliavini, F. (1993) Nature 362, 543–546) and mouse cortical cells (Brown, D.R., Herms, J. and Kretzschmar, H.A. (1994) Neuroreport 5, 2057–2060). However, we found the viability of these and other neuronal cell types not to be impaired in the presence of PrP106‐126 under widely varied sets of conditions. Aged preparations of the peptide as well as synthetic deamidated and isomerized derivatives that correspond to the aging products of the peptide proved also to lack neurotoxicity. Apparently, PrP106‐126 cannot serve as a model for the interaction of PrP with neuronal cells.

Active-site Arg → Lys Substitutions Alter Reaction and Substrate Specificity of Aspartate Aminotransferase

Arg386 and Arg292of aspartate aminotransferase bind the α and the distal carboxylate group, respectively, of dicarboxylic substrates. Their substitution with lysine residues markedly decreased aminotransferase activity. Thek cat values with l-aspartate and 2-oxoglutarate as substrates under steady-state conditions at 25 °C were 0.5, 2.0, and 0.03 s−1 for the R292K, R386K, and R292K/R386K mutations, respectively, k cat of the wild-type enzyme being 220 s−1. Longer dicarboxylic substrates did not compensate for the shorter side chain of the lysine residues. Consistent with the different roles of Arg292 and Arg386 in substrate binding, the effects of their substitution on the activity toward long chain monocarboxylic (norleucine/2-oxocaproic acid) and aromatic substrates diverged. Whereas the R292K mutation did not impair the aminotransferase activity toward these substrates, the effect of the R386K substitution was similar to that on the activity toward dicarboxylic substrates. All three mutant enzymes catalyzed as side reactions the β-decarboxylation of l-aspartate and the racemization of amino acids at faster rates than the wild-type enzyme. The changes in reaction specificity were most pronounced in aspartate aminotransferase R292K, which decarboxylated l-aspartate tol-alanine 15 times faster (k cat = 0.002 s−1) than the wild-type enzyme. The rates of racemization of l-aspartate, l-glutamate, andl-alanine were 3, 5, and 2 times, respectively, faster than with the wild-type enzyme. Thus, Arg → Lys substitutions in the active site of aspartate aminotransferase decrease aminotransferase activity but increase other pyridoxal 5′-phosphate-dependent catalytic activities. Apparently, the reaction specificity of pyridoxal 5′-phosphate-dependent enzymes is not only achieved by accelerating the specific reaction but also by preventing potential side reactions of the coenzyme substrate adduct.

Active Immunization of Mice with an Aβ-Hsp70 Vaccine

Heat-shock proteins are highly immunogenic. Complexed with an antigen, they act as adjuvants, inducing a humoral and cellular immune response against both the antigen and the chaperone. In this study, we produced an Hsp70-supported vaccine to induce the generation of antibodies against amyloid-β (Aβ) peptides, the major constituent of β-amyloid plaques in Alzheimer’s disease. The vaccine consisted of synthetic human Aβ42 covalently cross-linked with DnaK, an Hsp70 homolog of Escherichia coli. Active immunization of mice with this vaccine resulted in the generation of antibodies against Aβ, that were detectable in sera after the first booster immunization. Antibody titers varied markedly with the genetic background of the mice. Prophylactic short-term immunization of transgenic mice (APP tg2576) before the onset of plaques, however, did not prevent amyloid plaque deposition. There were no differences in the plaque load and in the level of Triton X-100-soluble Aβ peptides in the brains of immunized and control-treated transgenic mice. Unexpectedly, the level of formic-acid soluble Aβ peptides tended to be higher in immunized mice. The reason for the increase may be an enhanced deposition of Aβ in the small cerebral blood vessels. These data emphasize the need for anti-Aβ antibodies that remove Aβ peptides from the central nervous system without negative side effects.