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Proteolytic processing of the APP gene family members APLP-1 and APLP-2
PROTEOLYTIC PROCESSING OF THE APP GENE FAMILY MEMBERS APLP-I AND APLP-2 SirnoneEggert, Friedrich B M Reinhard, Urziv of Heidelberg, Heidelberg Gmnany; Krz~~sztoffaligu, Tirraerztliche Hochschuk Hannover, Hannover Germany; Joachim Stumm, St&m Scheuermann, Andreas Weidemann, Univ of Heidelberg, Heidelberg GfY~tlU~l~ The P-amyloid precursor protein (APP) involved in Alzheimer’s disease is a member of a gene family consisting of APP and two closely related molecules termed APP-like proteins, APLP-1 and APLP-2. Little is known on the metabolism of these APP-related proteins although further characterization of the proteolytic processing will allow to evaluate therapeutical strategies that are aimed at inhibiting APP secretases. Epitope-tagged APLP-1 and APLP-2 were expressed in COS-7 and SH-SYSY cells followed by analysis of the C-terminal fragments. As deduced from their size by comparison with m vim translated deletion mutants and in analogy to APP, most of the fragments observed for APLP-I and APLP-2 were classified as cleavage products by one or several a-secretases. For APLP-2, additional bands were detected with a higher molecular weight that may represent cleavage product? by b-secretases. Additionally. APLP-I and APLP-2 are cleaved close to the C-termmal end of their predicted transmembrane domains in a similar position as described for Notch and APP (termed APP E-cleavage site). By employing Brefeldin A and performing pulse-chase experiments, the cellular compartments in which the proteins are proteolytically converted. were identified within the late secretory pathway. Incubation of cells with known APP y-secretase inhibitors including MDL28170 resulted in an accumulati?~ of APLP-1 and APLP-2 C-terminal fragments suggesting a conversion by y-aecretase-like proteases. The same accumulation was observed when a dominant negative PSI Asp/Ala mutant was co-expressed with APLP-2. Thus. after APP and Notch, APLP-2 represents the third protein of which the proteolytic processing is dependent on the expression of functionally active presenilin-1. The% rrwlts demonstrate that the proteolytic processing of APP, APLP-1 and APLP-2 hharea many similarities as expected for members of the same gene family.
(1206)WITHDRAWN (12071NEUROTOXIC TERMINALLY
AND FIBRILLOGENIC PROPERTIES OF AMINOMODIFIED AMYLOID P-PEPTIDES.
Proteolytlc proceaung of amyloid precursor protem (APP) generates amyloid p (A(rl) peptldes in soluble form. Amino-terminally truncated and pyroglutamate modified AP peptider (~3.42/40 and p I l-42) are abundantly prewnt in the brain parenchyma of Allheimer’s dwease (AD) patients and are the ma,jor Ai3 species found in AD brains being present in early deposits and diffuse plaques. These species are increased in patients carrying presenilins 1 (PSI) mutations and in Down syndrome (DS) subjects in an age (and severity)-related manner, suggesting that these &forms are pathogenically important. Little IS known about fibrillogenic properties, catabolic procasing and toxicity of N-truncated AP peptides. Electron Microscopy analysis of synthetic A@ peptides shows a very different fiber morphology between ~3-40 and I-40 or l-42 AP peptides, but similar kinetic. The hypothesis that the peculiar amino-terminal modification could make these peptides resistant to protease action other than pyroglutamate specific ones has been recently raised. Western Blotting and MALDITOF analysis shows that the AP ~3.40 peptide is more resistant to degradation by primary cultures of rat astrocyte\ than AP l-40 or A@ l-42. Furthermore, the truncated peptide shows a significantly higher toxiaty than AP I-40 or l-42 AP peptides on hippocampal neuronal cultures and neuronally differentiated PC12 cells. The ~3-40 Ap peptide ir toac also on astrocyte cultures. Confocal microscopy imaging shows that aggregated AP peptides are present either on the membrane surface of treated neurons and astrocytes and also as internalized aggregates (mainly by astrocytes). These data suggest that the formation of amino-terminally modified AP peptides might influence the persistence of AP, favoring the aggregation process and the consequent amyloidotic deposit formation and neurotoxicity.
FUNCTIONAL ANALYSIS OF THE MACROMOLECULAR COM112081 PLEXES INVOLVING THE APP BINDING ADAPTOR FE65
The short, 47 amino acid-long C-terminal tail of the amyloid precursor protein (APP) is capable of interactions with at least three different cytosolic adaptors containing
PTB domains: the Fe65, X1 1 and m-dab proteins. Since the binding of the three proteins is dependent on the YENPTY sequence on APP, it is expected that their interactiona are not simultaneous. Indeed, it has been shown that Fe65 and Xl 1 proteins have opposite effects on the proteolytic processing of APP. A possible explanation may reside in the recruitment, by either Fe65 or Xl 1, of APP in different macromolecular complexes, depending on the interaction of different sets of proteins with the other protein-protein interaction domains of the two adaptors. In fact, Xl 1 contains a pair of PDZ domains, and an N-terminal region that is also engaged in protein-protein interactions. On the other hand, Fe65 contains one WW domaIn, which interacts with Mena, a protein involved in the remodeling of the actm cytoskeleton, and a second PTB module (PrBl), which is N-terminal to tix domain interacting with APP (pTB2). The Fe65 protein, through the PTBI domain can interact, both in the nucleus and in the cytosol, with the CPZ/LSF/LBPl transcription factor and, at the membrane level, with LRP, the LDL receptor-Related Protein. The trafficking of Fe65 between cytosol and nucleus is potentially regulated by a shon region of the protein, which is also a substrate of different protein kinase acttvities. It could be relevant, for the understanding of Alzheimer’s disease pathogenic mechanwns, to analyze the effects of this trafficking on the regulation of the proteolytic activities leading to amyloid peptide generation and on the potential signal tmnsduction mechaniw\ that may involve APP.
(1206)WITHDRAWN (12071NEUROTOXIC TERMINALLY
AND FIBRILLOGENIC PROPERTIES OF AMINOMODIFIED AMYLOID P-PEPTIDES.
Proteolytlc proceaung of amyloid precursor protem (APP) generates amyloid p (A(rl) peptldes in soluble form. Amino-terminally truncated and pyroglutamate modified AP peptider (~3.42/40 and p I l-42) are abundantly prewnt in the brain parenchyma of Allheimer’s dwease (AD) patients and are the ma,jor Ai3 species found in AD brains being present in early deposits and diffuse plaques. These species are increased in patients carrying presenilins 1 (PSI) mutations and in Down syndrome (DS) subjects in an age (and severity)-related manner, suggesting that these &forms are pathogenically important. Little IS known about fibrillogenic properties, catabolic procasing and toxicity of N-truncated AP peptides. Electron Microscopy analysis of synthetic A@ peptides shows a very different fiber morphology between ~3-40 and I-40 or l-42 AP peptides, but similar kinetic. The hypothesis that the peculiar amino-terminal modification could make these peptides resistant to protease action other than pyroglutamate specific ones has been recently raised. Western Blotting and MALDITOF analysis shows that the AP ~3.40 peptide is more resistant to degradation by primary cultures of rat astrocyte\ than AP l-40 or A@ l-42. Furthermore, the truncated peptide shows a significantly higher toxiaty than AP I-40 or l-42 AP peptides on hippocampal neuronal cultures and neuronally differentiated PC12 cells. The ~3-40 Ap peptide ir toac also on astrocyte cultures. Confocal microscopy imaging shows that aggregated AP peptides are present either on the membrane surface of treated neurons and astrocytes and also as internalized aggregates (mainly by astrocytes). These data suggest that the formation of amino-terminally modified AP peptides might influence the persistence of AP, favoring the aggregation process and the consequent amyloidotic deposit formation and neurotoxicity.
FUNCTIONAL ANALYSIS OF THE MACROMOLECULAR COM112081 PLEXES INVOLVING THE APP BINDING ADAPTOR FE65
The short, 47 amino acid-long C-terminal tail of the amyloid precursor protein (APP) is capable of interactions with at least three different cytosolic adaptors containing
PTB domains: the Fe65, X1 1 and m-dab proteins. Since the binding of the three proteins is dependent on the YENPTY sequence on APP, it is expected that their interactiona are not simultaneous. Indeed, it has been shown that Fe65 and Xl 1 proteins have opposite effects on the proteolytic processing of APP. A possible explanation may reside in the recruitment, by either Fe65 or Xl 1, of APP in different macromolecular complexes, depending on the interaction of different sets of proteins with the other protein-protein interaction domains of the two adaptors. In fact, Xl 1 contains a pair of PDZ domains, and an N-terminal region that is also engaged in protein-protein interactions. On the other hand, Fe65 contains one WW domaIn, which interacts with Mena, a protein involved in the remodeling of the actm cytoskeleton, and a second PTB module (PrBl), which is N-terminal to tix domain interacting with APP (pTB2). The Fe65 protein, through the PTBI domain can interact, both in the nucleus and in the cytosol, with the CPZ/LSF/LBPl transcription factor and, at the membrane level, with LRP, the LDL receptor-Related Protein. The trafficking of Fe65 between cytosol and nucleus is potentially regulated by a shon region of the protein, which is also a substrate of different protein kinase acttvities. It could be relevant, for the understanding of Alzheimer’s disease pathogenic mechanwns, to analyze the effects of this trafficking on the regulation of the proteolytic activities leading to amyloid peptide generation and on the potential signal tmnsduction mechaniw\ that may involve APP.