Abstract: FR-PO231

Apolipoprotein L-1 and the Wound Healing Pathway: A Fight for Survival

Session Information

Category: Cell Biology

  • 202 Apoptosis, Proliferation, Autophagy, Cell Senescence, Cell Transformation

Authors

  • Giovinazzo, Joseph A., City University of New york, New York, New York, United States
  • Thomson, Russell P., City University of New york, New York, New York, United States
  • Almanzar, Anibelky, City University of New york, New York, New York, United States
  • Singh, Simranjit, NYU Medical Center, New York, New York, United States
  • Kang, Ji Won, City University of New york, New York, New York, United States
  • Raper, Jayne, City University of New york, New York, New York, United States
Background

APOL1 is an innate immunity protein that forms pores in trypanosomes. Variants of APOL1 have been linked to kidney disease, yet the mechanism responsible remains controversial. We tested the hypothesis that APOL1 toxicity is cell intrinsic and dependent upon secretion via the Golgi. Along the secretory pathway, APOL1 is acidified and then neutralized upon delivery to the plasma membrane, wherein the cation selective pore initiates wound repair via the influx of Ca2+.

Methods

HEK293 cells were transfected with APOL1 and its variants, including deletion of the signal peptide. 24-48h later cell toxicity and viability were measured. Treatment with ammonium chloride was performed 2h prior to transfection. Recombinant APOL1 was purified from E. coli and reconstituted in planar lipid bilayers to measure ion channel conductivity and selectivity. HEK293 cells that stably express APOL1 were generated using the FlpIn recombinase system. The cells were transfected with the fluorescent calcium reporter gCAMP6f. Expression of APOL1 was induced at several timepoints and then read in a fluorescent plate reader. Activity of β-hexosaminidase (β-hex) was assayed in the supernatant of transfected cells at various timepoints after APOL1 expression.

Results

Deletion of the signal peptide led to a significant reduction of toxicity across all variants. Pre-treatment of cells with ammonium chloride reduced the toxicity of APOL1 by 50%. In planar lipid bilayers, rAPOL1 of all three major variants allowed for the passage of Ca2+. In stably transfected cells, induction of APOL1 expression lead to an increase in cytoplasmic Ca2+. In a cell, this would cause lysosomes to fuse with the plasma membrane to initiate removal of the wound and repair the membrane. Indeed, release of the lysosomal enzyme β-hexosaminidase, a marker of wound-healing, was detected prior to cell death.

Conclusion

These data support a model of APOL1 mediated cell death that requires acidic activation along the secretory pathway prior to forming pores at the plasma membrane. Increases in Ca2+ flux and the release of lysosomal enzymes prior to cell death indicate activation of the wound healing pathway by APOL1 pore-formation. Maintaining the balance between secretion and excessive pore-formation of APOL1 and the ability to remove and repair the wounds are key to cell survival.

Funding

  • Other NIH Support