Chaperone-like protein p32 regulates ULK1 stability and autophagy

Abstract

Mitophagy mediates clearance of dysfunctional mitochondria, and represents one type of mitochondrial quality control, which is essential for optimal mitochondrial bioenergetics. p32, a chaperone-like protein, is crucial for maintaining mitochondrial membrane potential and oxidative phosphorylation. However, the relationship between p32 and mitochondrial homeostasis has not been addressed. Here, we identified p32 as a key regulator of ULK1 stability by forming complex with ULK1. p32 depletion potentiated K48-linked but impaired K63-linked polyubiquitination of ULK1, leading to proteasome-mediated degradation of ULK1. As a result, silencing p32 profoundly impaired starvation-induced autophagic flux and the clearance of damaged mitochondria caused by mitochondrial uncoupler. Importantly, restoring ULK1 expression in p32-depleted cells rescued autophagy and mitophagy defects. Our findings highlight a cytoprotective role of p32 under starvation conditions by regulating ULK1 stability, and uncover a crucial role of the p32-ULK1-autophagy axis in coordinating stress response, cell survival and mitochondrial homeostasis. Mitophagy is a selective form of autophagy by which mitochondria are degraded in autolysosomes. p32 is a critical regulator of mitochondrial bioenergetics. 1 It primarily localizes to the mitochondrial matrix, but has also been reported to be present in other subcellular locations. 2-5 Many human tumors exhibit higher p32 expression levels than their nonmalignant counterpart tissues. 6-9 Depleting p32 in human cancer cells strongly shifts their metabolism from oxidative phosphorylation to glycolysis. 1 Consistently, p32 knockout causes mid-gestation lethality of knockout embryos and defects in oxidative phosphorylation. Mouse embryonic fibroblasts (MEFs) generated from p32 knockout embryos exhibited impaired ATP production and reduced mitochondrial membrane potential, which is in agreement with the observation that p32 silencing leads to increased mitochondrial fragmentation. 10,11 Notably, p32 was found to form protein complex with a variety of molecules 7,12,13 and has been suggested that it may act as a multifunctional chaperone protein. [12][13][14] ULK1 has a crucial role in mitophagy induction. 15 Despite the pivotal role of ULK1 in mitochondrial clearance, little is known as how ULK1 itself is regulated. ULK1 is a relatively stable protein and is subject to proteasome-mediated degradation. Post-translational modifications including K63-linked ubiquitylation 16,17 and phosphorylation [18][19][20] have been reported to modulate the rates of ULK1 turnover and kinase activity in different cellular contexts. Hsp90 and Cdc37 have been shown to regulate ULK1 stability and activity by forming complex with ULK1, which subsequently influences Atg13-mediated mitophagy. 21 Here, we found p32 regulates ULK1 stability by forming protein complex with ULK1. The interaction between ULK1 and p32 is crucial for maintaining the steady-state levels and activity of ULK1. We further show that p32 ablation results in a ...

Suppressed translation and ULK1 degradation as potential mechanisms of autophagy limitation under prolonged starvation

“…Based on several previous reports, [14][15][16] the ULK1 protein could be degraded either as a part of the autophagosomal membrane or in proteasomes. Therefore, we tested the effect of lysosomal and proteasomal degradation inhibitors bafilomycin A 1 and MG132 on stabilization of the ULK1 protein.…”

“…13 Some other proteins, including HSP90 and CDC37 and the recently identified chaperone-like protein C1QBP/p32, also regulate ULK1 stability and autophagy. 14,15 It has also been reported that binding of Atg8 to Atg1/ULK1 in an Atg8-intearcting motif/LC3-interacting region delivers Atg1/ULK1 within an autophagosome for lysosomal degradation in yeast and mammals, 16 suggesting a negative feedback loop in autophagy regulation.…”

Reactivation of latently infected HIV-1 viral reservoirs and correction of aberrant alternative splicing in the LMNA gene via AMPK activation: Common mechanism of action linking HIV-1 latency and Hutchinson–Gilford progeria syndrome

“…Further evidence that AMPK activation may indeed lead to an upregulation of p32 activity is demonstrated by the direct phosphorylation of p32 by ATM (Ataxia-telangiectasia mutated, an upstream activator of AMPK) and p32's association with TFAM, a mitochondrial transcription factor necessary for mitochondrial DNA transcription and biogenesis whose expression is increased by AMPK activation [51,165]. Particularly compelling is the recent finding that p32 is essential in the regulation of autophagy by forming a complex with and stabilizing the activity of ULK1, a serine-threonine kinase that is necessary for autophagic induction [166]. Indeed, p32 silencing significantly impaired both starvation-and rapamycin-induced autophagic flux [166].…”

“…Particularly compelling is the recent finding that p32 is essential in the regulation of autophagy by forming a complex with and stabilizing the activity of ULK1, a serine-threonine kinase that is necessary for autophagic induction [166]. Indeed, p32 silencing significantly impaired both starvation-and rapamycin-induced autophagic flux [166]. Because AMPK has also been shown to regulate autophagy by phosphorylating and activating ULK1 and both rapamycin (mTOR inhibitor) and sulforaphane (isothiocyanate found in cruciferous vegetables) have each been shown to enhance autophagic clearance of progerin, a novel AMPK-p32 axis is further implicated, potentially decreasing progerin production via alternative splicing modulation while enhancing progerin clearance through autophagic induction [32,167,168].…”

“…Because FTIs have been shown to reduce nuclear blebbing and restore nuclear architecture in HGPS by blocking farnesylation of progerin and the combined use of a statin and an aminobisphosphonate corrects mitochondrial dysfunction and improves aging-like phenotypes in a mouse model of human premature aging, inhibition of farnesylation and geranylgeranylation may indeed be translated by the cell as a signal of cellular stress, leading to compensatory activation of AMPK, upregulation of mitochondrial biogenesis and ATP synthesis, possible correction of aberrant alternative splicing in the LMNA gene, and induction of T cell activation-induced latent HIV-1 reactivation [25,31,166]. Indeed, a recently completed clinical trial in HGPS patients involving the use of the FTI lonafarnib, an FTI that was shown in preliminary data to also weakly reactivate latent HIV-1, lead to an increase in weight gain, bone structure, improvements in arterial stiffness, or sensorineural hearing, lending further evidence to a novel pathophsyiolgical connection between HGPS and HIV-1 latency [179,180].…”