Biomedical subjects

Andrea Schlicksupp

Publications and source records attributed to Andrea Schlicksupp.

5 recordsLinked to original sources

PubMed2004

Human BACE forms dimers and colocalizes with APP.

Beta-site APP-cleaving enzyme (BACE) is a membrane-bound aspartyl protease with no strict primary preference for cleavage. The molecular mechanisms that link the gamma-secretase multicomponent amyloid precursor protein (APP) processing complex to biochemical properties of BACE generating the N terminus of the amyloid beta-peptide have not, as yet, been identified. We found that in human brain tissue, BACE occurred as a dimer. The overall stability of the BACE homodimer was based on intermolecular interactions that were not affected by high salt, nonionic detergents or reducing conditions. BACE homodimers could only partially be separated even under strong denaturing conditions and revealed dramatic differences in the surface charge distribution compared with the monomer. In contrast, the soluble ectodomain of truncated BACE revealed a seemingly lower avidity to the prototypic aspartate protease inhibitor pepstatin and exclusively occurred in the monomeric form. Immunocytochemical studies colocalized APP and BACE in the plasma membrane of cells expressing endogenous levels of BACE and overexpressing APP. In cells that were cotransfected with APP and a putative active site D289A mutant of BACE, colocalization persisted. Remaining enzyme activity was found to be attributable to the mutant protease. Accordingly, inactivation of the carboxyl-terminal active site motif of BACE without an impairment of overall enzyme activity suggests that the enzyme may act as a dimer. Thus, homodimerization of BACE may help the enzyme to acquire specific mechanisms to associate with its substrates to exert catalytic activity.

Amyloid Precursor Protein Secretases↗

PubMed2003

Kinetic analysis of the interaction of the copper chaperone Atox1 with the metal binding sites of the Menkes protein.

Excess copper is effluxed from mammalian cells by the Menkes or Wilson P-type ATPases (MNK and WND, respectively). MNK and WND have six metal binding sites (MBSs) containing a CXXC motif within their N-terminal cytoplasmic region. Evidence suggests that copper is delivered to the ATPases by Atox1, one of three cytoplasmic copper chaperones. Attempts to monitor a direct Atox1-MNK interaction and to determine kinetic parameters have not been successful. Here we investigated interactions of Atox1 with wild-type and mutated pairs of the MBSs of MNK using two different methods: yeast two-hybrid analysis and real-time surface plasmon resonance (SPR). A copper-dependent interaction of Atox1 with the MBSs of MNK was observed by both approaches. Cys to Ser mutations of conserved CXXC motifs affected the binding of Atox1 underlining the essentiality of Cys residues for the copper-induced interaction. Although the yeast two-hybrid assay failed to show an interaction of Atox1 with MBS5/6, SPR analysis clearly demonstrated a copper-dependent binding with all six MBSs highlighting the power and sensitivity of SPR as compared with other, more indirect methods like the yeast two-hybrid system. Binding constants for copper-dependent chaperone-MBS interactions were determined to be 10-5-10-6 m for all the MBSs representing relatively low affinity binding events. The interaction of Atox1 with pairs of the MBSs was non-cooperative. Therefore, a functional difference of the MBSs in the MNK N terminus cannot be attributed to cooperativity effects or varying affinities of the copper chaperone Atox1 with the MBSs.

Adenosine Triphosphatases↗

PubMed2002

Identification of a beta-secretase activity, which truncates amyloid beta-peptide after its presenilin-dependent generation.

The beta-amyloid precursor protein (beta APP) is proteolytically processed by two secretase activities to produce the pathogenic amyloid beta-peptide (A beta). N-terminal cleavage is mediated by beta-secretase (BACE) whereas C-terminal intramembraneous cleavage is exerted by the presenilin (PS) gamma-secretase complex. The A beta-generating gamma-secretase cleavage principally occurs after amino acid 40 or 42 and results in secretion of A beta-(1-40) or A beta-(1-42). Upon overexpression of BACE in cultured cells we unexpectedly noticed a reduction of secreted A beta-(1-40/42). However, mass spectrometry revealed a truncated A beta species, which terminates at amino acid 34 (A beta-(1-34)) suggesting an alternative gamma-secretase cut. Indeed, expression of a loss-of-function variant of PS1 inhibited not only the production of A beta-(1-40) and A beta-(1-42) but also that of A beta-(1-34). However, expression levels of BACE correlate with the amount of A beta-(1-34), and A beta-(1-34) is produced at the expense of A beta-(1-40) and A beta-(1-42). Since this suggested that BACE is involved in a C-terminal truncation of A beta, we incubated purified BACE with A beta-(1-40) in vitro. Under these conditions A beta-(1-34) was generated. Moreover, when conditioned media containing Abeta-(1-40) and A beta-(1-42) were incubated with cells expressing a loss-of-function PS1 variant together with BACE, A beta-(1-34) was efficiently produced in vivo. These data demonstrate that an apparently gamma-secretase-dependent A beta derivative is produced after the generation of the non-truncated A beta via an additional and unexpected activity of BACE.

Alzheimer Disease↗

PubMed2002

Evidence for a copper-binding superfamily of the amyloid precursor protein.

The amyloid precursor protein (APP) copper-binding domain (CuBD) has been shown to reduce Cu(II) to Cu(I) and to mediate copper-induced oxidation in vitro. However, little is known about copper binding to the homologous domains of APP and APP family paralogs and orthologs (including amyloid precursor-like proteins from Drosophila melanogaster, Xenopus laevis, and Caenorhabditis elegans) and their effects on Cu-induced oxidation and Cu(I) formation. Here, we show that APP homologues with and without conserved histidine residues at positions 147, 149, and 151 all bind Cu(II). Oxidized peptides were the kinetically favored products of the redox reaction of CuBDs promoting the reduction of Cu(II) to Cu(I). These results reveal a molecular phylogeny-based divergence that has taken place between the ancestral Drosophila APPL and C. elegans APL-1 and the recently evolved APP lineage of CuBDs. Whereas higher species CuBDs have a decreased affinity for Cu(II) and high Cu(II) reducing activities, ancestral CuBDs form very tight binding sites for Cu(II) ions and have low Cu(II) reducing activities. Thus, the APP lineage displays a gain in activity toward promoting Cu(II) reduction and Cu(I) release. The findings suggests that the Cu(II)-binding equilibrium at the phylogenetic stage of Drosophila APPL and C. elegans APL-1 is shifted from the exchangeable Cu(II) pool to the tightly bound, nonexchangeable pool and that ancestral CuBDs may exert antioxidation activities in vivo. The more recently evolved homologues of human APP appear to take advantage of unique redox properties for yet unknown biological functions.

Amino Acid Sequence↗

PubMed2002

Possible mechanisms of APP-mediated oxidative stress in Alzheimer's disease.

Oxidative stress was presented to play an important role in the pathogenesis of Alzheimer's disease (AD), especially in the early evolution of AD amyloidogenesis and not only as a consequence thereof. The effect of oxidative stress catalysed by transition metals appears to have a critical relevance in AD. Metal-ion homeostasis is severely dysregulated in AD and it was found that experimentally induced disturbances in the homeostasis of Zn(II) and Cu(II) affect the amyloid precursor protein (APP) metabolism. APP itself binds Zn(II) and Cu(II) at nanomolar concentrations and an altered APP metabolism or expression level is believed to result in neurotoxic processes.

Alzheimer Disease↗