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Peng Shang, Mallika Valapala, Stacey L Hose, Jun Wan, Jiang Qian, Yuri V Sergeev, J. Samuel Zigler, Jr., Debasish Sinha; βA3/A1-Crystallin/SLC36A4/V-ATPase complex in the RPE is a novel therapeutic target for AMD. Invest. Ophthalmol. Vis. Sci. 2017;58(8):3384.
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We have previously demonstrated that βA3/A1-crystallin in the RPE is localized to the lysosomal lumen, where it regulates endo-lysosomal acidification by binding to ATP6V0A1/V0-ATPase. It is now clear that amino acids and V-ATPase are essential for mechanistic target of rapamycin, complex 1 (mTORC1) signaling. This study investigated whether βA3/A1-crystallin interacts with the amino acid transporter SLC36A4 (solute-linked carrier 36 family, member 4) and thereby regulates the amino acid pool in the lysosomes of RPE.
Co-immunoprecipitation was performed to investigate the binding between βA3/A1-crystallin and SLC36A4. An L-amino acid quantification kit was used to measure intracellular L-amino-acid concentration in RPE cells from both Cryba1fl/fl (control) and Cryba1 (gene encoding βA3/A1-crystallin) knockout (KO) mice. Autophagy was induced by 24h fasting of animals or by withholding serum and L-glutamine from cell cultures to investigate the mTORC1 pathway.
We showed that βA3/A1-crystallin binds to SLC36A4 in RPE cells from Cryba1fl/fl mice, but not in cells from Cryba1 KO mice. SLC36A4 expression both at the mRNA and protein level was down-regulated in Cryba1fl/fl mice after 24h fasting. However, in Cryba1 KO mice, SLC36A4 expression was up-regulated after 24h fasting. Cryba1 KO mice appear to have a lower basal level of SLC36A4 expression compared to control mice. Our data also indicate that after 24h fasting the concentration of L-amino acids in RPE cells of Cryba1 KO mice was elevated, whereas this increase was not seen in RPE cells of Cryba1fl/fl mice. After autophagy induction, the level of phosphorylated mTORC1(Ser2448) and phosphorylated p70s6k (T421/S424) decreased in RPE cells of Cryba1fl/fl mice, but stayed at consistently higher levels in RPE cells of Cryba1 KO mice, suggesting mTORC1 activation and autophagy inhibition in Cryba1 KO RPE cells. Moreover, the protein levels of Ragulator and Rag GTPases were generally higher in Cryba1 KO mice, especially after autophagy induction.
We observed that βA3/A1-crystallin binds to SLC36A4, providing a direct link between amino acid availability and mTORC1 signaling. Therefore, targeting the βA3/A1-crystallin/SLC36A4/V-ATPase complex in the RPE may be an effective means of preventing or delaying the progression of age-related macular degeneration.
This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.
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