Abstract
Autophagy is an intracellular catabolic system. It delivers cellular components to lysosomes for degradation and supplies nutrients that promote cell survival under stress conditions. Although much is known regarding starvation-induced autophagy, the regulation of autophagy by cellular energy level is less clear. BRUCE is an ubiquitin conjugase and ligase with multi-functionality. It has been reported that depletion of BRUCE inhibits starvation-induced autophagy by blockage of the fusion step. Herein we report a new function for BRUCE in the dual regulation of autophagy and cellular energy. Depletion of BRUCE alone (without starvation) in human osteosarcoma U2OS cells elevated autophagic activity as indicted by the increased LC3B-II protein and its autophagic puncta as well as further increase of both by chloroquine treatment. Such elevation results from enhanced induction of autophagy since the numbers of both autophagosomes and autolysosomes were increased, and recruitment of ATG16L onto the initiating membrane structure phagophores was increased. This concept is further supported by elevated lysosomal enzyme activities. In contrast to starvation-induced autophagy, BRUCE depletion did not block fusion of autophagosomes with lysosomes as indicated by increased lysosomal cleavage of the GFP-LC3 fusion protein. Mechanistically, BRUCE depletion lowered the cellular energy level as indicated by both a higher ratio of AMP/ATP and the subsequent activation of the cellular energy sensor AMPK (pThr-172). The lower energy status co-occurred with AMPK-specific phosphorylation and activation of the autophagy initiating kinase ULK1 (pSer-555). Interestingly, the higher autophagic activity by BRUCE depletion is coupled with enhanced cisplatin resistance in human ovarian cancer PEO4 cells. Taken together, BRUCE depletion promotes induction of autophagy by lowering cellular energy and activating the AMPK-ULK1-autophagy axis, which could contribute to ovarian cancer chemo-resistance. This study establishes a BRUCE-AMPK-ULK1 axis in the regulation of energy metabolism and autophagy, as well as provides insights into cancer chemo-resistance.
Highlights
The BIR repeat containing ubiquitin-conjugating enzyme (BRUCE) is a high molecular mass protein (528 kDa) with multiple cellular functions
We observed a significant increase in the number and intensity of LC3 puncta in BRUCE depleted cells compared to control (-DOX) (Fig 1A and 1B)
These data indicate that BRUCE depletion promotes the increase of autophagy activity rather than blockage of the fusion step when cells are cultured in complete medium
Summary
The BIR repeat containing ubiquitin-conjugating enzyme (BRUCE) is a high molecular mass protein (528 kDa) with multiple cellular functions. BRUCE suppresses apoptosis by ubiquitinating and promoting proteasomal degradation of pro-apoptotic caspase-9 and Smac/Diablo (IAP antagonist) [11,12,13] [6, 9]. The Bruce KO mice are embryonic lethal and die on E16.5–17.5 Prior to their death, the viable homozygous KO embryos and the extraembryonic tissues of placenta and yolk sac exhibit increased levels of apoptosis and reduced amount of cell proliferation [3, 4, 9, 14]. The anti-apoptotic function of BRUCE is evolutionarily conserved in mammals and Drosophila, as its Drosophila homolog dBruce suppresses cell death induced by Reaper and Grim, which are the functional homologues of Smac/DIABLO [15, 16]
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