Renal Risk Variants of Apolipoprotein L-1 Form Channels at the Plasma Membrane that Lead to a Cytotoxic Influx of Calcium

<p>Apolipoprotein L-1 (APOL1) is a secreted protein that provides protection against several protozoan parasites due to its channel forming properties. Recently evolved variants, G1 and G2, increase kidney disease risk when present in two copies. In mammalian cells, overexpression of G1 and G2, but not wild-type G0, leads to swelling and eventual lysis. However, the mechanism of cell death remains elusive with multiple pathways being invoked, such as autophagic cell death mediated by a BH3 domain in APOL1, which we evaluated in this study. We hypothesized that the common trigger for these pathways is the APOL1 cation channel, which is pH-gated and requires acidification followed by neutralization to function, conditions met along the secretory pathway. Testing ion selectivity in planar lipid bilayers revealed that all APOL1 variants conduct Ca2+. Furthermore, we discovered that cellular toxicity of G1 and G2 was Ca2+-dependent and led to a cytosolic Ca2+ influx, without ER Ca2+ release, quantified via high-throughput livecell microscopy. This influx preceded swelling and required prior plasma membrane (PM) localization, demonstrated by expression of tagged APOL1 in the Retention Using Selective Hooks system. G0 was also cytotoxic if acidified and neutralized after PM localization, suggesting regulation along the secretory pathway that G1 and G2 evade. Furthermore, we report that PM wound repair mediated by ESCRT-III promotes cell survival against APOL1 cytotoxicity, and that the putative BH3 domain is dispensable for APOL1-mediated cell death. These results indicate that APOL1 channel activity drives cytotoxicity via Ca2+ influx, and as a potent signaling molecule, Ca2+ may activate previously reported cell death pathways.</p>

Name thesis:degree_name

Doctor of Philosophy

Level thesis:degree_level

Doctoral

Discipline thesis:degree_discipline

Biology

Grantor

The Graduate School and University Center of The City University of New York

Year dc:date.available

2018

Author dc:creator

• Giovinazzo, Joseph A

Advisor dc:contributor.advisor

• Jayne Raper

Committee members dc:contributor.committeemember

• Mitchell Goldfarb
• Paul Feinstein
• Vincent Racaniello
• David Friedman

• Biochemistry
• Cell Biology
• Molecular Biology
• APOL1
• calcium
• kidney disease
• cell death

Repository record dc:identifier

https://academicworks.cuny.edu/gc_etds/2954

OAI identifier oai:identifier

oai:academicworks.cuny.edu:gc_etds-3970