Parkin interacts with Ambra1 to induce mitophagy

Mutations in the gene encoding Parkin are a major cause of recessive Parkinson's disease. Recent work has shown that Parkin translocates from the cytosol to depolarized mitochondria and induces their autophagic removal (mitophagy). However, the molecular mechanisms underlying Parkin-mediated mitophagy are poorly understood. Here, we investigated whether Parkin interacts with autophagy-regulating proteins. We purified Parkin and associated proteins from HEK293 cells using tandem affinity purification and identified the Parkin interactors using mass spectrometry. We identified the autophagy-promoting protein Ambra1 (activating molecule in Beclin1-regulated autophagy) as a Parkin interactor. Ambra1 activates autophagy in the CNS by stimulating the activity of the class III phosphatidylinositol 3-kinase (PI3K) complex that is essential for the formation of new phagophores. We found Ambra1, like Parkin, to be widely expressed in adult mouse brain, including midbrain dopaminergic neurons. Endogenous Parkin and Ambra1 coimmunoprecipitated from HEK293 cells, SH-SY5Y cells, and adult mouse brain. We found no evidence for ubiquitination of Ambra1 by Parkin. The interaction of endogenous Parkin and Ambra1 strongly increased during prolonged mitochondrial depolarization. Ambra1 was not required for Parkin translocation to depolarized mitochondria but was critically important for subsequent mitochondrial clearance. In particular, Ambra1 was recruited to perinuclear clusters of depolarized mitochondria and activated class III PI3K in their immediate vicinity. These data identify interaction of Parkin with Ambra1 as a key mechanism for induction of the final clearance step of Parkin-mediated mitophagy

HUWE1 E3 ligase promotes PINK1/PARKINindependent mitophagy by regulating AMBRA1 activation via IKKa

The selective removal of undesired or damaged mitochondria by autophagy, known as mitophagy, is crucial for cellular homoeostasis, and prevents tumour diffusion, neurodegeneration and ageing. The pro-autophagic molecule AMBRA1 (autophagy/beclin-1 regulator-1) has been defined as a novel regulator of mitophagy in both PINK1/PARKIN-dependent and -independent systems. Here, we identified the E3 ubiquitin ligase HUWE1 as a key inducing factor in AMBRA1-mediated mitophagy, a process that takes place independently of the main mitophagy receptors. Furthermore, we show that mitophagy function of AMBRA1 is post-translationally controlled, upon HUWE1 activity, by a positive phosphorylation on its serine 1014. This modification is mediated by the IKKα kinase and induces structural changes in AMBRA1, thus promoting its interaction with LC3/GABARAP (mATG8) proteins and its mitophagic activity. Altogether, these results demonstrate that AMBRA1 regulates mitophagy through a novel pathway, in which HUWE1 and IKKα are key factors, shedding new lights on the regulation of mitochondrial quality control and homoeostasis in mammalian cells

AMBRA1 is able to induce mitophagy via LC3 binding, regardless of PARKIN and p62/SQSTM1

Damaged mitochondria are eliminated by mitophagy, a selective form of autophagy whose dysfunction associates with neurodegenerative diseases. PINK1, PARKIN and p62/SQTMS1 have been shown to regulate mitophagy, leaving hitherto ill-defined the contribution by key players in 'general' autophagy. In basal conditions, a pool of AMBRA1 - an upstream autophagy regulator and a PARKIN interactor - is present at the mitochondria, where its pro-autophagic activity is inhibited by Bcl-2. Here we show that, upon mitophagy induction, AMBRA1 binds the autophagosome adapter LC3 through a LIR (LC3 interacting region) motif, this interaction being crucial for regulating both canonical PARKIN-dependent and -independent mitochondrial clearance. Moreover, forcing AMBRA1 localization to the outer mitochondrial membrane unleashes a massive PARKIN- and p62-independent but LC3-dependent mitophagy. These results highlight a novel role for AMBRA1 as a powerful mitophagy regulator, through both canonical or noncanonical pathways

Evidence for the involvement of lipid rafts localized at the ER-mitochondria associated membranes in autophagosome formation

Mitochondria-associated membranes (MAMs) are subdomains of the endoplasmic reticulum (ER) that interact with mitochondria. This membrane scrambling between ER and mitochondria appears to play a critical role in the earliest steps of autophagy. Recently, lipid microdomains, i.e. lipid rafts, have been identified as further actors of the autophagic process. In the present work, a series of biochemical and molecular analyses has been carried out in human fibroblasts with the specific aim of characterizing lipid rafts in MAMs and to decipher their possible implication in the autophagosome formation. In fact, the presence of lipid microdomains in MAMs has been detected and, in these structures, a molecular interaction of the ganglioside GD3, a paradigmatic “brick” of lipid rafts, with core-initiator proteins of autophagy, such as AMBRA1 and WIPI1, was revealed. This association seems thus to take place in the early phases of autophagic process in which MAMs have been hypothesized to play a key role. The functional activity of GD3 was suggested by the experiments carried out by knocking down ST8SIA1 gene expression, i.e., the synthase that leads to the ganglioside formation. This experimental condition results in fact in the impairment of the ER-mitochondria crosstalk and the subsequent hindering of autophagosome nucleation. We thus hypothesize that MAM raft-like microdomains could be pivotal in the initial organelle scrambling activity that finally leads to the formation of autophagosome. Introduction The interaction of the endoplasmic reticulum (ER) with mito- chondria occurs via certain subdomains of the ER, named mitochondria-associated membranes (MAMs), which allow membrane “scrambling” between these organelles and contrib- utes to the complex series of ER functions.1-3 Indeed, several regions of close apposition between the ER and mitochondria were detected by studies carried out several years ago.4,5 How- ever, since these studies provided only ultrastructural observa- tions, these reports remained neglected for a long time. In particular, while morphological evidence of the physical juxta- position between ER and mitochondria was described since 1959,6 it was experimentally proven only 30 y later. In fact, ana- lyzing ER fractions copurified with mitochondria in velocity sedimentation assays, mainly from rat liver cells, it was observed that mitochondria can tightly be associated with ele- ments of the ER and that the communication and intermixing between ER and mitochondria can be mediated by MAMs.7-12 These works also showed that these cosedimenting fractions were enriched in enzymes responsible for the synthesis of lipids. These findings suggested that MAMs could act as sites

Characterization of Ambra1 heterozygous mice as genetic mouse model of female-specific autism

Autism is known as a heritable neurodevelopmental disorder, diagnosed prior to the age of three years in humans based on three major domains: (1) impairment in social interaction (2) communication deficits (3) restricted interests and repetitive behaviors. Since it is a very heterogeneous disorder with various causes and different combinations of phenotypes, it is also called autism spectrum disorder (ASD). Monogenic heritable forms of ASD enable us to develop genetic mouse models of autism in order to obtain mechanistic insight in this disorder. Ambra1 is a positive regulator of Beclin1, a major player in the formation of autophagosomes during the process of autophagy. While Ambra1 null mutation leads to embryonic lethality, we could show that Ambra1 heterozygous mice (Ambra1+/-) display autism-like behavior only in females. Purpose of this thesis was therefore to characterize this mouse line further. It turned out that communication deficits, measured by ultrasound vocalization, start in the neonatal stage of females, while physical or neurological development is normal in Ambra1+/-. Female Ambra1 mutants had a stronger reduction in Ambra1 expression than male mutants, which gives first hints of the female-specific autism-like behavior in this mouse line. Mild enlargement of whole brain and hippocampus was detected in both Ambra1+/- males and females, with no change of ventricle size. Since β-galactosidase, used as reporter expressed under the Ambra1 promoter, was found only in neuronal cells, I focused on understanding the neural mechanism of its phenotype. Short-term and long-term synaptic plasticity in the hippocampus was normal for males and females of both genotypes. However, the power of gamma oscillations (γ-power), indicative of change in the balance of excitation and inhibition, was age-dependently altered in Ambra1+/- females only. However, this difference was not detected in male. Moreover, increased susceptibility to seizures, a known comorbid condition of ASD was restricted to females, suggesting an association between autism-like behavior, gamma oscillation and seizure propensity in female Ambra1+/- mice. Next, I approached the neuronal substrate of these three phenotypes by morphological analysis of hippocampal pyramidal neurons, such as dendritic arborization and synapse number. A genotype-associated difference of dendritic arborization was detected in neither males nor females. The quantification of spines or synapses and cellular electrophysiology are still on-going. First signals point to an imbalance between excitation and inhibition as a cause of the female autism-like behavior in Ambra1+/- mice

Ambra1 is an essential regulator of autophagy and apoptosis in SW620 cells: Pro-survival role of Ambra1

Recent research has revealed a role for Ambra1, an autophagy-related gene-related (ATG) protein, in the autophagic pro-survival response, and Ambra1 has been shown to regulate Beclin1 and Beclin1-dependent autophagy in embryonic stem cells. However, whether Ambra1 plays an important role in the autophagy pathway in colorectal cancer cells is unknown. In this study, we hypothesized that Ambra1 is an important regulator of autophagy and apoptosis in CRC cell lines. To test this hypothesis, we confirmed autophagic activity in serum-starved SW620 CRC cells by assessing endogenous microtubule-associated protein 1 light chain 3 (LC3) localization, the presence of autophagosomes (transmission electron microscopy) and LC3 protein levels (Western blotting). Ambra1 expression was detected by Western blot in SW620 cells treated with staurosporine or etoposide. Calpain and caspase inhibitors were employed to verify whether calpains and caspases were responsible for Ambra1 cleavage. To examine the role of Ambra1 in apoptosis, Ambra1 knockdown cells were treated with staurosporine and etoposide. Cell apoptosis and viability were measured by annexin-V and PI staining and MTT assays. We determined that serum deprivation-induced autophagy was associated with Ambra1 upregulation in colorectal cancer cell lines. Ambra1 expression decreased during staurosporine- or etoposide-induced apoptosis. Calpains and caspases may be responsible for Ambra1 degradation. When Ambra1 expression was reduced by siRNA, SW620 cells were more sensitive to staurosporine- or etoposide-induced apoptosis. In addition, starvation-induced autophagy decreased. Finally, Co-immunoprecipitation of Ambra1 and Beclin1 demonstrated that Ambra1 and Beclin1 interact in serum-starved or rapamycin-treated SW620 cells, suggesting that Ambra1 regulates autophagy in CRC cells by interacting with Beclin1. In conclusion, Ambra1 is a crucial regulator of autophagy and apoptosis in CRC cells that maintains the balance between autophagy and apoptosis

The autophagy protein Ambra1 regulates gene expression by supporting novel transcriptional complexes

Ambra1 is considered an autophagy and trafficking protein with roles in neurogenesis and cancer cell invasion. Here, we report that Ambra1 also localizes to the nucleus of cancer cells, where it has a novel nuclear scaffolding function that controls gene expression. Using biochemical fractionation and proteomics, we found that Ambra1 binds to multiple classes of proteins in the nucleus, including nuclear pore proteins, adaptor proteins such as FAK and Akap8, chromatin-modifying proteins, and transcriptional regulators like Brg1 and Atf2. We identified biologically important genes, such as Angpt1, Tgfb2, Tgfb3, Itga8, and Itgb7, whose transcription is regulated by Ambra1-scaffolded complexes, likely by altering histone modifications and Atf2 activity. Therefore, in addition to its recognized roles in autophagy and trafficking, Ambra1 scaffolds protein complexes at chromatin, regulating transcriptional signaling in the nucleus. This novel function for Ambra1, and the specific genes impacted, may help to explain the wider role of Ambra1 in cancer cell biology

Characterization of Ambra1 heterozygous mice as genetic mouse model of female-specific autism

Autism is known as a heritable neurodevelopmental disorder, diagnosed prior to the age of three years in humans based on three major domains: (1) impairment in social interaction (2) communication deficits (3) restricted interests and repetitive behaviors. Since it is a very heterogeneous disorder with various causes and different combinations of phenotypes, it is also called autism spectrum disorder (ASD). Monogenic heritable forms of ASD enable us to develop genetic mouse models of autism in order to obtain mechanistic insight in this disorder. Ambra1 is a positive regulator of Beclin1, a major player in the formation of autophagosomes during the process of autophagy. While Ambra1 null mutation leads to embryonic lethality, we could show that Ambra1 heterozygous mice (Ambra1+/-) display autism-like behavior only in females. Purpose of this thesis was therefore to characterize this mouse line further. It turned out that communication deficits, measured by ultrasound vocalization, start in the neonatal stage of females, while physical or neurological development is normal in Ambra1+/-. Female Ambra1 mutants had a stronger reduction in Ambra1 expression than male mutants, which gives first hints of the female-specific autism-like behavior in this mouse line. Mild enlargement of whole brain and hippocampus was detected in both Ambra1+/- males and females, with no change of ventricle size. Since β-galactosidase, used as reporter expressed under the Ambra1 promoter, was found only in neuronal cells, I focused on understanding the neural mechanism of its phenotype. Short-term and long-term synaptic plasticity in the hippocampus was normal for males and females of both genotypes. However, the power of gamma oscillations (γ-power), indicative of change in the balance of excitation and inhibition, was age-dependently altered in Ambra1+/- females only. However, this difference was not detected in male. Moreover, increased susceptibility to seizures, a known comorbid condition of ASD was restricted to females, suggesting an association between autism-like behavior, gamma oscillation and seizure propensity in female Ambra1+/- mice. Next, I approached the neuronal substrate of these three phenotypes by morphological analysis of hippocampal pyramidal neurons, such as dendritic arborization and synapse number. A genotype-associated difference of dendritic arborization was detected in neither males nor females. The quantification of spines or synapses and cellular electrophysiology are still on-going. First signals point to an imbalance between excitation and inhibition as a cause of the female autism-like behavior in Ambra1+/- mice

HUWE1 controls MCL1 stability to unleash AMBRA1-induced mitophagy

Receptor-mediated mitophagy is a crucial process involved in mitochondria quality control. AMBRA1 is a mitophagy receptor for the selective removal of damaged mitochondria in mammalian cells. A critical unresolved issue is how AMBRA1-mediated mitophagy is controlled in response to cellular stress. Here, we investigated the role of BCL2-family proteins on AMBRA1-dependent mitophagy and showed that MCL1 delays AMBRA1-dependent mitophagy. Indeed, MCL1 overexpression is sufficient to inhibit recruitment to mitochondria of the E3 Ubiquitin ligase HUWE1, a crucial dynamic partner of AMBRA1, upon AMBRA1-mediated mitophagy induction. In addition, we found that during mitophagy induced by AMBRA1, MCL1 levels decreased but were sustained by inhibition of the GSK-3β kinase, which delayed AMBRA1-mediated mitophagy. Also, we showed that MCL1 was phosphorylated by GSK-3β at a conserved GSK-3 phosphorylation site (S159) during AMBRA1-mediated mitophagy and that this event was accompanied by HUWE1-dependent MCL1 degradation. Altogether, our results demonstrate that MCL1 stability is regulated by the kinase GSK-3β and the E3 ubiquitin ligase HUWE1 in regulating AMBRA1-mediated mitophagy. Our work thus defines MCL1 as an upstream stress-sensitive protein, functional in AMBRA1-mediated mitophagy

PIKES Analysis Reveals Response to Degraders and Key Regulatory Mechanisms of the CRL4 Network

Co-opting Cullin4 RING ubiquitin ligases (CRL4s) to inducibly degrade pathogenic proteins is emerging as a promising therapeutic strategy. Despite intense efforts to rationally design degrader molecules that co-opt CRL4s, much about the organization and regulation of these ligases remains elusive. Here, we establish protein interaction kinetics and estimation of stoichiometries (PIKES) analysis, a systematic proteomic profiling platform that integrates cellular engineering, affinity purification, chemical stabilization, and quantitative mass spectrometry to investigate the dynamics of interchangeable multiprotein complexes. Using PIKES, we show that ligase assemblies of Cullin4 with individual substrate receptors differ in abundance by up to 200-fold and that Cand1/2 act as substrate receptor exchange factors. Furthermore, degrader molecules can induce the assembly of their cognate CRL4, and higher expression of the associated substrate receptor enhances degrader potency. Beyond the CRL4 network, we show how PIKES can reveal systems level biochemistry for cellular protein networks important to drug development