Autogramin-2 is a new class ofautophagy inhibitor with IC50 value of 0.27 ± 0.09 µM (measured by amino acid starvation induced autophagy) and 0.14 ± 0.08 µM (measured by Rapamycin induced autophagy) through targeting GRAMD1A.
Autophagy is a catabolic process aimed at recycling cellular components and damaged organelles in response to diverse conditions of stress, such as nutrient deprivation, viral infection and genotoxic stress. Autophagy mediates the degradation of damaged proteins, organelles and pathogens, and plays a key role in health and disease[1]. Autogramin-2 potently inhibits autophagy induced by either starvation (IC50=0.27 μM) or mTORC1 inhibition (Rapamycin; IC50=0.14 μM).The PH-GRAM construct alone did not transfer cholesterol. The StART domains of GRAMD1B and GRAMD1C also transferred cholesterol, but this could not be inhibited by autogramin-2 or was only inhibited to a minor extent. Autogramin-2 did not compete with binding of 22-NBD-cholesterol to GRAMD1B or GRAMD1C, further confirming its selectivity for GRAMD1A.Autogramins selectively target the recently discovered cholesterol transfer protein GRAM domain-containing protein 1A (GRAMD1A, which had not previously been implicated in autophagy), and directly compete with cholesterol binding to the GRAMD1A StART domain. The positive control 25-hydroxycholesterol was able to compete with binding of 22-NBD-cholesterol to all three GRAMD1 proteins, while no oxysterols hydroxylated in the A- or B-ring were able to compete.GRAMD1A accumulates at sites of autophagosome initiation, affects cholesterol distribution in response to starvation and is required for autophagosome biogenesis. in the presence of 25-hydroxycholesterol or autogramin-2, hydrogen–deuterium exchange was reduced for amino acids 397–405, 426–440, 464–473 and 507–529[2].
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