The cholesterol transfer protein GRAMD1A regulates autophagosome biogenesis

Abstract

The cholesterol transfer protein GRAMD1A regulates autophagosome biogenesis Nature Chemical Biology, 15 (7): 710-720 Editorial SummaryThe cholesterol transfer protein GRAMD1A was identified as the target of the autophagy inhibitors autogramin-1 and 2. GRAMD1A is required for autophagosome biogenesis, and autogramins represent tool compounds for studying this process. AbstractAutophagy mediates the degradation of damaged proteins, organelles and pathogens and plays a key role in health and disease. The identification of new mechanisms involved in autophagy regulation is of major interest. In particular little is known about the roles of lipids and lipid binding proteins in the early steps of autophagosome biogenesis. Through target agnostic, high-content, image-based identification of indicative phenotypic changes induced by small molecules, we have identified autogramins as a novel autophagy inhibitor class. Autogramins selectively target the recently discovered cholesterol transfer protein GRAM domain containing protein 1A (GRAMD1A), which had not been implicated in autophagy before, and directly compete with cholesterol binding to the GRAMD1A StART domain. GRAMD1A accumulates at sites of autophagosome initiation, affects cholesterol distribution in response to starvation and is required for autophagosome biogenesis. These findings identify a novel biological function of GRAMD1A and a new role for cholesterol in autophagy.

A DNA-Based FLIM Reporter for Simultaneous Quantification of Lysosomal pH and Ca2+ during Autophagy Regulation

“…Meanwhile, autophagy level was confirmed by the amount of light chain 3 (LC3), a membrane microtubule-associated protein 1. LC3 is the credible biomarker of autophagy, which can lose peptide fragments to produce LC3-I, and the produced LC3-I can bind with phosphatidyl ethanolamine (PE) to form LC3-II during autophagy ( Cui et al., 2018 ; Laraia et al., 2019 ; Li et al., 2018 ). As shown in Figures 4 J and 4K, the level of LC3-II/β-actin gradually went up from 0.92 ± 0.05 to 1.17 ± 0.06 according to the quantification of western blot as lysosomal pH increased from 4.00 to 4.90.…”

Phenotyping Reveals Targets of a Pseudo‐Natural‐Product Autophagy Inhibitor

“…We compared the cell painting profile of azaquindole‐1 against other recently identified autophagy inhibitors (see Figures S10 and S11). Our analysis revealed that azaquindole‐1 ( 10 w‐j ) was biosimilar to oxautin‐1, autoquin, and autophinib.…”

Phenotyping Reveals Targets of a Pseudo‐Natural‐Product Autophagy Inhibitor

“…We compared the cell painting profile of azaquindole-1a gainst other recently identified autophagy inhibitors [11,29,38,39,45,46] (see Figures S10 and S11). Our analysis revealed that azaquindole-1 (10 w-j)w as biosimilar to oxautin-1, autoquin, and autophinib.N otably,a utophinib is ak nown VPS34 inhibitor, and the cell painting analysis further validates this earlier finding.T he ability of the cell painting assay data to suggest and subsequently identify molecular targets is therefore apparent.…”