Biomedical subjects

Mirjam Koker

Publications and source records attributed to Mirjam Koker.

5 recordsLinked to original sources

PubMed2006

GGA1 acts as a spatial switch altering amyloid precursor protein trafficking and processing.

The beta-amyloid (Abeta) precursor protein (APP) is cleaved sequentially by beta-site of APP-cleaving enzyme (BACE) and gamma-secretase to release the Abeta peptides that accumulate in plaques in Alzheimer's disease (AD). GGA1, a member of the Golgi-localized gamma-ear-containing ARF-binding (GGA) protein family, interacts with BACE and influences its subcellular distribution. We now report that overexpression of GGA1 in cells increased the APP C-terminal fragment resulting from beta-cleavage but surprisingly reduced Abeta. GGA1 confined APP to the Golgi, in which fluorescence resonance energy transfer analyses suggest that the proteins come into close proximity. GGA1 blunted only APP but not notch intracellular domain release. These results suggest that GGA1 prevented APP beta-cleavage products from becoming substrates for gamma-secretase. Direct binding of GGA1 to BACE was not required for these effects, but the integrity of the GAT (GGA1 and TOM) domain of GGA1 was. GGA1 may act as a specific spatial switch influencing APP trafficking and processing, so that APP-GGA1 interactions may have pathophysiological relevance in AD.

ADP-Ribosylation Factors↗

PubMed2006

Interaction of the cytosolic domains of sorLA/LR11 with the amyloid precursor protein (APP) and beta-secretase beta-site APP-cleaving enzyme.

sorLA is a recently identified neuronal receptor for amyloid precursor protein (APP) that is known to interact with APP and affect its intracellular transport and processing. Decreased levels of sorLA in the brain of Alzheimer's disease (AD) patients and elevated levels of amyloid-beta peptide (Abeta) in sorLA-deficient mice point to the importance of the receptor in this neurodegenerative disorder. We analyzed APP cleavage in an APP-shedding assay and found that both sorLA and, surprisingly, a sorLA tail construct inhibited APP cleavage in a beta-site APP-cleaving enzyme (BACE)-dependent manner. In line with this finding, sorLA and the sorLA tail significantly reduced secreted Abeta levels when BACE was overexpressed, suggesting that sorLA influences beta-cleavage. To understand the effect of sorLA on APP cleavage by BACE, we analyzed whether sorLA interacts with APP and/or BACE. Because both full-length sorLA and sorLA C-terminal tail constructs were functionally relevant for APP processing, we analyzed sorLA-APP for a potential cytoplasmatic interaction domain. sorLA and C99 coimmunoprecipitated, pointing toward the existence of a new cytoplasmatic interaction site between sorLA and APP. Moreover, sorLA and BACE also coimmunoprecipitate. Thus, sorLA interacts both with BACE and APP and might therefore directly affect BACE-APP complex formation. To test whether sorLA impacts BACE-APP interactions, we used a fluorescence resonance energy transfer assay to evaluate BACE-APP interactions in cells. We discovered that sorLA significantly reduced BACE-APP interactions in Golgi. We postulate that sorLA acts as a trafficking receptor that prevents BACE-APP interactions and hence BACE cleavage of APP.

Amyloid Precursor Protein Secretases↗

PubMed2005

Comparative analysis of the human and chicken prion protein copper binding regions at pH 6.5.

Recent experimental evidence supports the hypothesis that prion proteins (PrPs) are involved in the Cu(II) metabolism. Moreover, the copper binding region has been implicated in transmissible spongiform encephalopathies, which are caused by the infectious isoform of prion proteins (PrP(Sc)). In contrast to mammalian PrP, avian prion proteins have a considerably different N-terminal copper binding region and, most interestingly, are not able to undergo the conversion process into an infectious isoform. Therefore, we applied x-ray absorption spectroscopy to analyze in detail the Cu(II) geometry of selected synthetic human PrP Cu(II) octapeptide complexes in comparison with the corresponding chicken PrP hexapeptide complexes at pH 6.5, which mimics the conditions in the endocytic compartments of neuronal cells. Our results revealed that structure and coordination of the human PrP copper binding sites are highly conserved in the pH 6.5-7.4 range, indicating that the reported pH dependence of copper binding to PrP becomes significant at lower pH values. Furthermore, the different chicken PrP hexarepeat motifs display homologous Cu(II) coordination at sub-stoichiometric copper concentrations. Regarding the fully cation-saturated prion proteins, however, a reduced copper coordination capability is supposed for the chicken prion protein based on the observation that chicken PrP is not able to form an intra-repeat Cu(II) binding site. These results provide new insights into the prion protein structure-function relationship and the conversion process of PrP.

Animals↗

PubMed2004

Synthetic human prion protein octapeptide repeat binds to the proteinase K active site.

Proteinase K is widely used in tests for the presence of infectious prion protein causing fatal spongiform encephalopathies. To investigate possible interactions between the enzyme and the functionally important N-terminal prion domain, we crystallized mercury-inhibited proteinase K in the presence of the synthetic peptides GGGWGQPH and HGGGW. The octapeptide sequence is identical to that of a single octapeptide repeat (OPR) from the physiologically important OPR region. Here, we present the first direct evidence for the complex formation between a proteolytic enzyme and a segment of human prion molecule. The X-ray structures of the complexes at 1.4 and 1.8A resolution, respectively, revealed that in both cases the segment GGG is strongly bound as a real substrate at the substrate recognition site of the proteinase forming an antiparallel beta-strand between the two parallel strands of Asn99-Tyr104 and Ser132-Gly136. The complex is stabilized through an extended H-bonding network.

Binding Sites↗

PubMed2004

Oligomerization of the proteolytic products is an intrinsic property of prion proteins.

In the present study we show that the oligomerization of the proteolytic products is an intrinsic property of prion proteins. No such oligomerization was observed for the proteolytic products of other proteins after identical treatment. The rate of enzymatic hydrolysis of recombinant human (rhPrP) (23-231) and golden hamster (rmaPrP) (23-231) prion proteins as well as that of rmaPrP (90-231), corresponding to the infectious fragment of the scrapie form, drastically increases in the presence of chemical chaperones like dimethyl sulphoxide and glycerol as well as in 20% ethanol. A bacterial proteinase, termed "prionase," has a superior efficiency towards prion proteins in comparison to proteinase K and subtilisin DY. The early steps in the proteolysis by the latter enzymes have been identified. The results have potential impact on the treatment of scrapie-infected materials.

Animals↗