Sequence-dependent internalization of aggregating peptides.
Recently, a number of aggregation disease polypeptides have been shown to spread from cell to cell, thereby displaying prionoid behavior. Studying aggregate internalization, however, is often hampered by the complex kinetics of the aggregation process, resulting in the concomitant uptake of aggregates of different sizes by competing mechanisms, which makes it difficult to isolate pathway-specific responses to aggregates. We designed synthetic aggregating peptides bearing different aggregation propensities with the aim of producing modes of uptake that are sufficiently distinct to differentially analyze the cellular response to internalization. We found that small acidic aggregates (≤500 nm in diameter) were taken up by nonspecific endocytosis as part of the fluid phase and traveled through the endosomal compartment to lysosomes. By contrast, bigger basic aggregates (>1 μm) were taken up through a mechanism dependent on cytoskeletal reorganization and membrane remodeling with the morphological hallmarks of phagocytosis. Importantly, the properties of these aggregates determined not only the mechanism of internalization but also the involvement of the proteostatic machinery (the assembly of interconnected networks that control the biogenesis, folding, trafficking, and degradation of proteins) in the process; whereas the internalization of small acidic aggregates is HSF1-independent, the uptake of larger basic aggregates was HSF1-dependent, requiring Hsp70. Our results show that the biophysical properties of aggregates determine both their mechanism of internalization and proteostatic response. It remains to be seen whether these differences in cellular response contribute to the particular role of specific aggregated proteins in disease.
From the Switch Laboratory, VIB, Leuven, Belgium, the Switch Laboratory, Department of Cellular and Molecular Medicine, KU Leuven, B-3000 Leuven, Belgium.
José R
JR
Couceiro
From the Switch Laboratory, VIB, Leuven, Belgium, the Switch Laboratory, Department of Cellular and Molecular Medicine, KU Leuven, B-3000 Leuven, Belgium.
Rodrigo
R
Gallardo
From the Switch Laboratory, VIB, Leuven, Belgium, the Switch Laboratory, Department of Cellular and Molecular Medicine, KU Leuven, B-3000 Leuven, Belgium.
Frederik
F
De Smet
From the Switch Laboratory, VIB, Leuven, Belgium, the Switch Laboratory, Department of Cellular and Molecular Medicine, KU Leuven, B-3000 Leuven, Belgium.
Greet
G
De Baets
the Electron Microscopy Facility (EMoNe), KU Leuven Centre for Human Genetics, B-3000 Leuven, Belgium, the VIB BIO Imaging Core, VIB, B-3000 Leuven, Belgium.
Pieter
P
Baatsen
the Laboratory for Membrane Trafficking, KU Leuven and VIB-Centre for the Biology of Disease, B-3000 Leuven, Belgium.
Wim
W
Annaert
the VIB Inflammation Research Center, 9052 Ghent, Belgium, and the Department of Biomedical Molecular Biology, Ghent University, 9052 Ghent, Belgium.
Kenny
K
Roose
the VIB Inflammation Research Center, 9052 Ghent, Belgium, and the Department of Biomedical Molecular Biology, Ghent University, 9052 Ghent, Belgium.
Xavier
X
Saelens
From the Switch Laboratory, VIB, Leuven, Belgium, the Switch Laboratory, Department of Cellular and Molecular Medicine, KU Leuven, B-3000 Leuven, Belgium.
Joost
J
Schymkowitz
From the Switch Laboratory, VIB, Leuven, Belgium, the Switch Laboratory, Department of Cellular and Molecular Medicine, KU Leuven, B-3000 Leuven, Belgium, frederic.rousseau@switch.vib-kuleuven.be.
Frederic
F
Rousseau
D008841:Actin Cytoskeleton / Q000737:chemistry / Q000187:drug effects / Q000378:metabolism; D000584:Amiloride / Q000031:analogs & derivatives / Q000494:pharmacology; D000595:Amino Acid Sequence; D015638:Cytochalasin D / Q000494:pharmacology; D004268:DNA-Binding Proteins / Q000378:metabolism; D004705:Endocytosis / Q000187:drug effects / Q000502:physiology*; D011992:Endosomes / Q000187:drug effects / Q000378:metabolism*; D057809:HEK293 Cells; D018840:HSP70 Heat-Shock Proteins / Q000378:metabolism; D000076249:Heat Shock Transcription Factors; D006801:Humans; D006835:Hydrazones / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008148:Lovastatin / Q000494:pharmacology; D008247:Lysosomes / Q000187:drug effects / Q000378:metabolism*; D008969:Molecular Sequence Data; D010455:Peptides / Q000138:chemical synthesis / Q000737:chemistry / Q000378:metabolism*; D066329:Protein Aggregates; D011485:Protein Binding; D017510:Protein Folding; D021381:Protein Transport / Q000187:drug effects / Q000502:physiology; D059748:Proteolysis; D013329:Structure-Activity Relationship; D014157:Transcription Factors / Q000378:metabolism
D004268:DNA-Binding Proteins; C000619706:HSF1 protein, human; D018840:HSP70 Heat-Shock Proteins; D000076249:Heat Shock Transcription Factors; D006835:Hydrazones; C511794:N'-(3,4-dihydroxybenzylidene)-3-hydroxy-2-naphthahydrazide; D010455:Peptides; D066329:Protein Aggregates; D014157:Transcription Factors; D015638:Cytochalasin D; D000584:Amiloride; D008148:Lovastatin; C039614:ethylisopropylamiloride
Aggresome; Amyloid; Internalization; Molecular Chaperone; Peptide; Peptide Transport; Prionoid; Protein Aggregation