Abstract
Previously we reported that adipocyte SNAP23 (synaptosome-associated protein of 23 kDa) deficiency blocks the activation of macroautophagy, leading to an increased abundance of BAX, a pro-death Bcl-2 family member, and activation and adipocyte cell death both in vitro and in vivo Here, we found that knockdown of SNAP23 inhibited the association of the autophagosome regulators ATG16L1 and ATG9 compartments by nutrient depletion and reduced the formation of ATG16L1 membrane puncta. ATG16L1 knockdown inhibited autophagy flux and increased BAX protein levels by suppressing BAX degradation. The elevation in BAX protein had no effect on BAX activation or cell death in the nutrient-replete state. However, following nutrient depletion, BAX was activated with a concomitant induction of cell death. Co-immunoprecipitation analyses demonstrated that SNAP23 and ATG16L1 proteins form a stable complex independent of nutrient condition, whereas in the nutrient-depleted state, BAX binds to SNAP23 to form a ternary BAX-SNAP23-ATG16L1 protein complex. Taken together, these data support a model in which SNAP23 plays a crucial function as a scaffold for ATG16L1 necessary for the suppression of BAX activation and induction of the intrinsic cell death program.
Highlights
Nonical autophagy pathway required for the lysosomal degradation of the pro-apoptotic protein BAX [1]
We reported that SNAP23 deficiency results in an ATG9-dependent, but ATG7-independent, induced intrinsic cell adipocyte cell death caused by the inhibition of a nonca
We previously reported that adipocyte-specific SNAP23 knockout mice within in the first several months of age develop generalized lipodystrophy caused by an elevation of the BAX protein, BAX activation, and the BAX-dependent adipocyte cell death program that occurs during periods of nutrient deprivation in vivo and in vitro [1]
Summary
SNARE proteins comprise a large family of proteins (;60 members in mammalian cells) that primarily function in the fusion of membrane compartments with each other [2,3,4,5,6,7]. To characterize the potential interaction and morphological changes associated with SNAP23 deficiency, we transfected NIH3T3 fibroblast SNAP23 knockdown (SNAP23 shRNA) and control (NM shRNA) cells with CFP-ATG16L1 and RFPATG9 (under nutrient-depleted conditions to active autophagy (Fig. 1A). Consistent with several previous studies, ATG16L1 deficiency suppressed autophagy as determined by the inhibition of LC3-II flux and inhibition of p62 degradation (Fig. 3, G–J) Because BAX was substantially activated only in the ATG16L1 knockdown cells under nutrient-depleted conditions, there was a concomitant increase in cell death (Fig. 5, A, panel 4, and B). The induction of cell death by nutrient depletion of ATG16L1-deficient cells was confirmed by propidium iodide labeling in independent clones of NM shRNA and ATG16L1 shRNA (Fig. S4). All data represent the means 6 standard deviation. *, P 0.05 by unpaired two-tailed Student’s t test
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