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Intracellular accumulation and resistance to degradation of the Alzheimer amyloid A4/beta protein
M F Knauer1, B Soreghan, D Burdick
1Department of Molecular Biology and Biochemistry, University of California, Irvine 92717.
Abstract:
The A4 or beta protein is a peptide that constitutes the major protein component of senile plaques in Alzheimer disease. The A4/beta protein is derived from a larger, transmembrane amyloid precursor protein (APP). The putative abnormal processing events leading to amyloid accumulation are largely unknown. Here we report that a 42-residue synthetic peptide, beta 1-42, corresponding to one of the longer forms of the A4/beta protein, accumulates in cultured human skin fibroblasts and is stable for at least 3 days. The peptide appears to accumulate intracellularly, since it does not accumulate under conditions that prevent endocytosis and accumulation is correlated with the acquisition of resistance to removal by trypsin digestion. This intracellular accumulation is also correlated with the ability of the peptide to aggregate as determined by SDS/polyacrylamide gel electrophoresis. At low concentrations of the beta 1-42 peptide, which favor the nonaggregated state, no accumulation is observed. Shorter peptide analogs (28 or 39 residues) that are truncated at the C terminus, which lack the ability to aggregate in SDS gels, fail to accumulate. The accumulated intracellular beta 1-42 peptide is in an aggregated state and is contained in a dense organellar compartment that overlaps the distribution of late endosomes or secondary lysosomes. Immunofluorescence of the internalized peptide in permeabilized cells reveals that it is contained in granular deposits, consistent with localization in late endosomes or secondary lysosomes. Sequence analysis indicates that some of the internalized peptide is subject to N-terminal trimming. These results suggest that the aggregated A4/beta protein may be resistant to degradation and suggest that the A4/beta protein may arise, at least in part, by endosomal or lysosomal processing of APP. Our results also suggest that relatively nonspecific proteolysis may be sufficient to generate the A4/beta protein if this part of APP is selectively resistant to proteolysis.
Insights
The beta 1-42 peptide, a component of Alzheimer's disease plaques, accumulates intracellularly in fibroblasts. Aggregated beta 1-42 is resistant to degradation, suggesting endosomal processing of amyloid precursor protein (APP).
Area of Science:
- Neuroscience
- Cell Biology
- Biochemistry
Background:
- Alzheimer disease is characterized by senile plaques composed of the A4/beta protein.
- The A4/beta protein originates from the amyloid precursor protein (APP).
- The precise mechanisms of abnormal APP processing leading to amyloid accumulation remain unclear.
Purpose of the Study:
- To investigate the cellular processing and accumulation of the A4/beta protein.
- To determine the aggregation properties and cellular localization of synthetic beta 1-42 peptide.
- To explore the potential role of endosomal/lysosomal pathways in A4/beta protein generation.
Main Methods:
- Utilized cultured human skin fibroblasts to study synthetic beta 1-42 peptide.
- Assessed peptide accumulation under conditions affecting endocytosis and proteolysis (trypsin digestion).
- Analyzed peptide aggregation using SDS/polyacrylamide gel electrophoresis and immunofluorescence microscopy.
Main Results:
- Synthetic beta 1-42 peptide accumulated intracellularly in fibroblasts and was stable for at least 3 days.
- Accumulation correlated with resistance to trypsin digestion and peptide aggregation.
- Aggregated beta 1-42 localized to late endosomes or secondary lysosomes, with some N-terminal trimming observed.
Conclusions:
- Aggregated A4/beta protein appears resistant to degradation.
- Endosomal or lysosomal processing of APP may contribute to A4/beta protein formation.
- Non-specific proteolysis could generate A4/beta protein if specific APP regions are resistant to degradation.