Abstract: TH-PO363
Darunavir Localizes to Cytoplasmic Stress Granules in Human Proximal Tubular Cells
Session Information
- Pharmacology (PharmacoKinetics, -Dynamics, -Genomics)
November 07, 2019 | Location: Exhibit Hall, Walter E. Washington Convention Center
Abstract Time: 10:00 AM - 12:00 PM
Category: Pharmacology (PharmacoKinetics, -Dynamics, -Genomics)
- 1800 Pharmacology (PharmacoKinetics, -Dynamics, -Genomics)
Authors
- Rosales, Alan, Albert Einstein College of Medicine, Bronx, New York, United States
- Tandoh, Buadi K., Albert Einstein College of Medicine, Bronx, New York, United States
- Gao, Xiaobo, Albert Einstein College of Medicine, Bronx, New York, United States
- Karttunen, Heidi, Albert Einstein College of Medicine, Bronx, New York, United States
- Ross, Michael J., Albert Einstein College of Medicine/ Montefiore Medical Center , Bronx, New York, United States
Background
HIV protease inhibitors (PI) have off-target effects on many cellular pathways data from our laboratory demonstrated that darunavir (DRV), the most commonly used PI, protects renal epithelial cells from HIV-induced injury and inflammatory responses via mechanisms independent of HIV protease. Since the mechanism by which DRV protects kidney cells from injury is poorly understood, we performed studies to identify cellular protein targets of DRV in human renal epithelial cells.
Methods
We used the Direct Magnetic IP/Co-IP Kit to covalently link DRV to NHS-activated magnetic beads. DRV bound– and unbound– beads were incubated with HPT1b whole cell lysate from immortalized human proximal tubular cells (HPT1b). Bound proteins were eluted and analyzed by mass spectrometry. DRV was also covalently linked to NHS-AlexaFluor 488, and cellular localization of DRV-AF488 and stress granule protein G3BP1 were analyzed by fluorescence microscopy.
Results
52 proteins were identified in all 3 samples from DRV-bound beads at 100-fold or greater abundance than control samples. 23 of the 52 proteins are RNA-binding proteins, most of which are components of cytoplasmic stress granules (SG), including canonical SG proteins G3BP1, G3BP2, and Caprin1. DRV-G3BP1 interaction was confirmed by western blotting of protein eluted from DRV- and control-conjugated beads.
To determine if DRV colocalizes with G3BP1 at SG, SG formation was induced in HPT1b cells by incubation with NaAsO2. Fluorescence microscopy localized G3BP1 and DRV-AF488 in the cytoplasm and in cytoplasmic punctae, consistent with SG localization. HIV transduction of HPT1b cells increased G3BP1 punctate staining, demonstrating that HIV induced SG formation. We examined phosphorylation of G3BP1, which promotes SG disassembly. A reduction in p-G3BP1 (Ser149) was observed in HIV transduced HPT1b cells treated with DRV, suggesting that DRV prevents SG disassembly.
Conclusion
These data demonstrate that DRV localizes preferentially to SG in renal epithelial cells. Since SG are intracellular domains that regulate response to stress, DRV may attenuate renal epithelial injury via novel effects upon SG dynamics. Additional studies are needed to determine the role of SG proteins in mediating the protective effects of DRV against kidney injury.
Funding
- NIDDK Support