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Secretory ribonucleases are internalized by a dynamin-independent endocytic pathway
Marcia C Haigis1, Ronald T Raines
1Department of Biochemistry, University of Wisconsin-Madison, Madison, Wisconsin 53706, USA.
Abstract:
Cytosolic internalization is a requirement for the toxicity of secretory ribonucleases. Here, we investigate the mechanism of internalization of Onconase (ONC), a toxic protein, and ribonuclease A (RNase A), a nontoxic homolog. Microscopy studies indicate that both ribonucleases readily bind to the cell surface and are internalized via acidic vesicles. Blocking dynamin-dependent endocytosis prevents transferrin internalization but does not hinder RNase A internalization. ONC and G88R RNase A, which is a toxic variant, demonstrate enhanced cytotoxicity in the absence of clathrin- and dynamin-mediated endocytosis. The cytosolic entry of ribonucleases does not require an acidic environment or transport to the ER and probably occurs from endosomes. Thus, common proteins - secretory ribonucleases - enter the cytosol by a pathway that is distinct from that of other known toxins.
Insights
Secretory ribonucleases, like Onconase (ONC), enter cells through acidic vesicles. Their toxic mechanism involves cytosolic entry via a pathway distinct from other toxins.
Area of Science:
- Cell biology
- Protein biochemistry
- Toxicology
Background:
- Cytosolic internalization is crucial for the toxicity of secretory ribonucleases.
- Onconase (ONC) is a toxic secretory ribonuclease, while ribonuclease A (RNase A) is a nontoxic homolog.
Purpose of the Study:
- To investigate the internalization mechanism of Onconase (ONC) and ribonuclease A (RNase A).
- To elucidate the pathway of cytosolic entry for secretory ribonucleases.
Main Methods:
- Microscopy studies to observe ribonuclease binding and internalization.
- Endocytosis pathway analysis using dynamin and clathrin inhibitors.
- Cytotoxicity assays under different endocytosis conditions.
Main Results:
- Both ONC and RNase A bind to the cell surface and are internalized via acidic vesicles.
- Dynamin-dependent endocytosis is not required for RNase A internalization.
- Toxic ONC and a variant G88R RNase A show enhanced cytotoxicity independently of clathrin and dynamin pathways.
Conclusions:
- Secretory ribonucleases enter the cytosol through a pathway distinct from other known toxins.
- Cytosolic entry likely occurs from endosomes and does not require an acidic environment or ER transport.