Membrane curvature response in autophagy

Membrane trafficking is key for signal transduction, cargo transportation, and in the case of autophagy, delivering cytoplasmic substrates to the lysosome for degradation. Autophagy requires the formation of a unique double membrane vesicle, the autophagosome. However, the mechanism by which the autophagosome forms is unknown. Our recent study focused on the role of Barkor/Atg14(L) in targeting the autophagy-specific class III phosphatidylinositol-3-kinase (PtdIns3KC3) complex to the early autophagosome has implicated this complex in autophagosome formation. This study found that the BATS domain of Barkor targets the PtdIns3KC3 complex to early autophagic structures and senses highly curved membranes enriched in phosphatidylinositol-3-phosphate (PtdIns(3)P). Consequently, this study uncovered an exciting new role for the PtdIns3KC3 complex as a potential inducer of autophagosome formation.     

Membrane curvature response in autophagy

Membrane trafficking is key for signal transduction, cargo transportation, and in the case of autophagy, delivering cytoplasmic substrates to the lysosome for degradation. Autophagy requires the formation of a unique double membrane vesicle, the autophagosome. However, the mechanism by which the autophagosome forms is unknown. Our recent study focused on the role of Barkor/Atg14(L) in targeting the autophagy-specific class III phosphatidylinositol-3-kinase (PtdIns3KC3) complex to the early autophagosome has implicated this complex in autophagosome formation. This study found that the BATS domain of Barkor targets the PtdIns3KC3 complex to early autophagic structures and senses highly curved membranes enriched in phosphatidylinositol-3-phosphate (PtdIns(3)P). Consequently, this study uncovered an exciting new role for the PtdIns3KC3 complex as a potential inducer of autophagosome formation.     

Bif-1 interacts with Beclin 1 through UVRAG and regulates autophagy and tumorigenesis

Autophagy is an evolutionarily conserved 'self-eating' process. Although the genes essential for autophagy (named Atg) have been identified in yeast, the molecular mechanism of how Atg proteins control autophagosome formation in mammalian cells remains to be elucidated. Here, we demonstrate that Bif-1 (also known as Endophilin B1) interacts with Beclin 1 through ultraviolet irradiation resistance-associated gene (UVRAG) and functions as a positive mediator of the class III PI(3) kinase (PI(3)KC3). In response to nutrient deprivation, Bif-1 localizes to autophagosomes where it colocalizes with Atg5, as well as microtubule-associated protein light chain 3 (LC3). Furthermore, loss of Bif-1 suppresses autophagosome formation. Although the SH3 domain of Bif-1 is sufficient for binding to UVRAG, both the BAR and SH3 domains are required for Bif-1 to activate PI(3)KC3 and induce autophagosome formation. We also observed that Bif-1 ablation prolongs cell survival under starvation conditions. Moreover, knockout of Bif-1 significantly enhances the development of spontaneous tumours in mice. These findings suggest that Bif-1 joins the UVRAG-Beclin 1 complex as a potential activator of autophagy and tumour suppressor.     

Autophagic and tumour suppressor activity of a novel Beclin1-binding protein UVRAG

Autophagy, the degradation of cytoplasmic components, is an evolutionarily conserved homeostatic process involved in environmental adaptation, lifespan determination and tumour development. The tumor suppressor Beclin1 is part of the PI(3) kinase class III (PI(3)KC3) lipid-kinase complex that induces autophagy. The autophagic activity of the Beclin1-PI(3)KC3 complex, however, is suppressed by Bcl-2. Here, we report the identification of a novel coiled-coil UV irradiation resistance-associated gene (UVRAG) as a positive regulator of the Beclin1-PI(3)KC3 complex. UVRAG, a tumour suppressor candidate that is monoallelically mutated at high frequency in human colon cancers, associates with the Beclin1-Bcl-2-PI(3)KC3 multiprotein complex, where UVRAG and Beclin1 interdependently induce autophagy. UVRAG-mediated activation of the Beclin1-PI(3)KC3 complex promotes autophagy and also suppresses the proliferation and tumorigenicity of human colon cancer cells. These results identify UVRAG as an essential component of the Beclin1-PI(3)KC3 lipid kinase complex that is an important signalling checkpoint for autophagy and tumour-cell growth.     

The RUN domain of rubicon is important for hVps34 binding, lipid kinase inhibition, and autophagy suppression

The class III phosphatidylinositol 3-kinase (PI3KC3) plays a central role in autophagy. Rubicon, a RUN domain-containing protein, is newly identified as a PI3KC3 subunit through its association with Beclin 1. Rubicon serves as a negative regulator of PI3KC3 and autophagosome maturation. The molecular mechanism underlying the PI3KC3 and autophagy inhibition by Rubicon is largely unknown. Here, we demonstrate that Rubicon interacts with the PI3KC3 catalytic subunit hVps34 via its RUN domain. The RUN domain contributes to the efficient inhibition of PI3KC3 lipid kinase activity by Rubicon. Furthermore, a Rubicon RUN domain deletion mutant fails to complement the autophagy deficiency in Rubicon-depleted cells. Hence, these results reveal a critical role of the Rubicon RUN domain in PI3KC3 and autophagy regulation.     

Beclin 1 bridges autophagy, apoptosis and differentiation

Beclin 1 is a critical component in the class III PI3 kinase complex (PI3KC3) that induces the formation of autophagosomes in mammalian systems. Autophagic triggers upregulate Beclin 1, which in turn binds to PI3KC3 or Bcl-X(L) to form complexes of Beclin 1-PI3KC3 or Beclin 1-Bcl-X(L) that are physically and functionally independent from each other. Contrary to the previous observations that Beclin 1 binding to Bcl-2 family members is apoptotic and antiautophagic, we found that autophagic trigger-induced Beclin 1-binding to Bcl-X(L) is antiapoptotic and has no effect on autophagy, suggesting a convertible role of the Beclin 1-Bcl-X(L) complex in response to autophagy stimuli. Both autophagy and differentiation cascades require upregulation of Beclin 1. While the basal Beclin 1 level does not cause autophagy or differentiation, depletion of Beclin 1 cripples both autophagy and differentiation capabilities, but activates apoptosis. These results demonstrate that Beclin 1 is essential for autophagy, differentiation and antiapoptosis, and may play an important role in coordinating inputs for cellular decisions to signaling machinery that mediates different cellular cascades.     

Beclin 1 bridges autophagy,apoptosis and differentiation

Beclin 1 is a critical component in the class III PI3 kinase complex (PI3KC3) that induces the formation of autophagosomes in mammalian systems. Autophagic triggers upregulate Beclin 1, which in turn binds to PI3KC3 or Bcl-X_L to form complexes of Beclin 1-PI3KC3 or Beclin 1-Bcl-X_L that are physically and functionally independent from each other. Contrary to the previous observations that Beclin 1 binding to Bcl-2 family members is apoptotic and antiautophagic, we found that autophagic trigger-induced Beclin 1-binding to Bcl-X_L is antiapoptotic and has no effect on autophagy, suggesting a convertible role of the Beclin 1-Bcl-X_L complex in response to autophagy stimuli. Both autophagy and differentiation cascades require upregulation of Beclin 1. While the basal Beclin 1 level does not cause autophagy or differentiation, depletion of Beclin 1 cripples both autophagy and differentiation capabilities, but activates apoptosis. These results demonstrate that Beclin 1 is essential for autophagy, differentiation and antiapoptosis, and may play an important role in coordinating inputs for cellular decisions to signaling machinery that mediates different cellular cascades.

Addendum to: Wang J, Lian H, Zhao Y, Kauss MA, Spindel S. Vitamin D3 induces autophagy of human myeloid leukemia cells. J Biol Chem 2008; doi:10.1074/jbc.     

Traf6 and A20 differentially regulate TLR4-Induced autophagy by affecting the ubiquitination of Beclin 1

Toll-like receptor 4 (TLR4) signaling triggers autophagy, which has been linked to both adaptive and innate immunity. Engagement of TLR4 recruits to the receptor complex Beclin 1, a key component of a class III phosphatidylinositol 3-kinase complex (PI3KC3) that initiates autophagosome formation. Recently, we found that tumor necrosis factor receptor (TNFR)-associated factor 6 (TRAF6)-mediates Lys⁶³ (K63)-linked ubiquitination of Beclin 1 is crucial for TLR4-triggered autophagy in macrophages. We identified two TRAF6-binding motifs in Beclin 1 that facilitate the binding of TRAF6 and the ubiquitination of Beclin 1. A lysine located in the Bcl-2 homology 3 (BH3) domain of Beclin 1 serves as a major site for K63-linked ubiquitination. Opposing TRAF6, the deubiquitinating enzyme A20 reduces the extent of K63-linked ubiquitination of Beclin 1 and limits the induction of autophagy in response to TLR4 signaling. Furthermore, treatment of macrophages with either interferon- or interleukin-1 triggers the K63-linked ubiquitination of Beclin 1 and the formation of autophagosomes. These results indicate that the status of K63-linked ubiquitination of Beclin 1 plays a key role in regulating autophagy during inflammatory responses.     

An insight into the mechanistic role of Beclin 1 and its inhibition by prosurvival Bcl-2 family proteins

A multiprotein complex composed of Beclin 1, PI(3)KC3 and UVRAG promotes autophagosome formation, while this activity is suppressed by a cohort of antiapoptotic Bcl-2 family members. Recently, we showed that a viral Bcl-2 of murine γ-herpesvirus 68, known as M11, binds to Beclin 1 with markedly high affinity in comparison with cellular Bcl-2 or Bcl-X_L that interacts with Beclin 1 weakly.1 Furthermore, the binding affinity directly correlated with the potency of inhibition of autophagosome formation in cells. Herein, we present additional data showing that Beclin 1 forms a large homo-oligomer, and this oligomerization is partly disrupted by the binding of M11. Oligomerized Beclin 1 is proposed to serve as a platform enabling a concerted action of many molecules of the associating proteins, including Bif-1 that could be directly involved in autophagosome biogenesis on membranes owing to its BAR domain.

Addendum to: Ku B, Woo J-S, Liang C, Lee K-H, Hong H-S, Xiaofei E, Kim K-S, Jung JU, Oh B-H. Structural and biochemical bases for the inhibition of autophagy and apoptosis by viral BCL-2 of murine γ-herpesvirus 68. PLoS Pathog 2008; 4:e25.     

HMGB1: a novel Beclin 1-binding protein active in autophagy

The autophagosome delivers damaged cytoplasmic constituents and proteins to the lysosome or to the extracellular space. Beclin 1, an essential: autophagic protein, is a BH3-only protein that binds Bcl-2 anti-apoptotic family members and has a critical role in the initiation of autophagy. How the Beclin 1 complex specifically promotes autophagy remains largely unknown. We have found that high mobility group box 1 (HMGB1), a chromatin-associated nuclear protein and extracellular damage associated molecular pattern molecule (DAMP), is a novel Beclin 1-binding protein important in sustaining autophagy. HMGB1 shares considerable sequence homology with Beclin 1 in yeast, mice and human, representing an evolutionarily conserved regulatory step in early autophagosome formation. Endogenous HMGB1 competes with Bcl-2 for interaction with Beclin 1, and orients Beclin 1 to autophagosomes. Moreover, the intramolecular disulfide bridge (C23/45) of HMGB1 is required for binding to Beclin 1 and sustaining autophagy. Taken together, these findings indicate that endogenous HMGB1 functions as an autophagy effector by regulation of autophagosome formation.     

An insight into the mechanistic role of Beclin 1 and its inhibition by prosurvival Bcl-2 family proteins

A multiprotein complex composed of Beclin 1, PI(3)KC3 and UVRAG promotes autophagosome formation, while this activity is suppressed by a cohort of antiapoptotic Bcl-2 family members. Recently, we showed that a viral Bcl-2 of murine gamma-herpesvirus 68, known as M11, binds to Beclin 1 with markedly high affinity in comparison with cellular Bcl-2 or Bcl-X(L) that interacts with Beclin 1 weakly.(1) Furthermore, the binding affinity directly correlated with the potency of inhibition of autophagosome formation in cells. Herein, we present additional data showing that Beclin 1 forms a large homo-oligomer, and this oligomerization is partly disrupted by the binding of M11. Oligomerized Beclin 1 is proposed to serve as a platform enabling a concerted action of many molecules of the associating proteins, including Bif-1 that could be directly involved in autophagosome biogenesis on membranes owing to its BAR domain.     

HMGB1: A novel Beclin 1-binding protein active in autophagy

The autophagosome delivers damaged cytoplasmic constituents and proteins to the lysosome or to the extracellular space. Beclin 1, an essential

autophagic protein, is a BH3-only protein that binds Bcl-2 anti-apoptotic family members and has a critical role in the initiation of autophagy. How the Beclin 1 complex specifically promotes autophagy remains largely unknown. We have found that high mobility group box 1 (HMGB1), a chromatin-associated nuclear protein and extracellular damage associated molecular pattern molecule (DAMP), is a novel Beclin 1-binding protein important in sustaining autophagy. HMGB1 shares considerable sequence homology with Beclin 1 in yeast, mice and human, representing an evolutionarily conserved regulatory step in early autophagosome formation. Endogenous HMGB1 competes with Bcl-2 for interaction with Beclin 1, and orients Beclin 1 to autophagosomes. Moreover, the intramolecular disulfide bridge (C23/45) of HMGB1 is required for binding to Beclin 1 and sustaining autophagy. Taken together, these findings indicate that endogenous HMGB1 functions as an autophagy effector by regulation of autophagosome formation.     

The E1B19K oncoprotein complexes with Beclin 1 to regulate autophagy in adenovirus-infected cells

The mechanisms underlying adenovirus-mediated autophagy are currently unknown. Recently, members of the Bcl-2 protein family have been associated with autophagy. It was also reported that the Bcl-2 homology-3 (BH3) domain encompassed by both Beclin 1 and Bcl-2-like proteins is essential for their pro-autophagy or anti-autophagy functions. Here, we report for the first time that E1B19K, the adenovirus BH3 domain protein, interacts with Beclin 1 to initiate autophagy. Using immunoprecipitation assays we showed that expression of E1B19K in the host cell disrupted the physical interactions between Beclin 1 and Bcl-2 proteins. The displacement of Bcl-2 was coincident with the recruitment of PI3KC3 to the Beclin 1/E1B19K complexes. As a result of the changes in the components of the Beclin 1 interactome, there was activation of PI3KC3, as showed by the identification of PI3K-mediated lipid phosphorylation, and subsequent formation of autophagosomes. Importantly, the BH3 functional domain of E1B19K protein was required for the heterodimerization with Beclin 1. We also showed that transfer of E1B19K was sufficient to trigger autophagy in cancer cells. Consistent with these data, mutant adenoviruses encompassing a deletion of the E1B19K gene produced a marked deficiency in the capability of the virus to induce autophagy as showed by examining the lipidation and cleavage of LC3-I as well as the subcellular localization of LC3-II, the decrease in the levels of p62, and the formation of autophagosomes. Our work offers new information on the mechanisms of action of the adenoviral E1B19K protein as partner of Beclin 1 and positive regulator of autophagy.     

Beclin1: a role in membrane dynamics and beyond

Beclin1(Atg6) is a well-known key regulator of autophagy. Although Beclin1 is enzymatically inert, it governs the autophagic process by regulating PtdIns3KC3-dependent generation of phosphatidylinositol3-phosphate (PtdIns(3)P) and the subsequent recruitment of additional Atg proteins that orchestrate autophagosome formation. Furthermore, Beclin1 is implicated in numerous biological processes, including adaptation to stress, development, endocytosis, cytokinesis, immunity, tumorigenesis, ageing and cell death. Whether all of these processes involve only the autophagy-inducing function of Beclin1 is now being seriously questioned, because Beclin1 appears to exercise several non-autophagy functions. Therefore, we should broaden our view of Beclin1 as a specialized molecule in autophagy to that of a multifunctional protein. The central role of Beclin1 in multiple signaling events obviously requires tight regulation at multiple levels. Its function is kept in check by diverse mechanisms, such as epigenetic silencing, microRNA regulation, post-translational modifications, and protein-protein interactions. Interestingly, multiple diseases are associated with deficiency or malfunction of Beclin1, which makes it a potentially valuable target for various therapies, including anti-cancer treatment. In this review, we focus on Beclin1 as a multifunctional protein, discuss the variety of mechanisms by which it is controlled, and give an overview of Beclin1-associated pathologies.     

Regulation of the autophagic PI3KC3 complex by laforin/malin E3-ubiquitin ligase,two proteins involved in Lafora disease

Lafora progressive myoclonus epilepsy is a fatal rare neurodegenerative disorder characterized by the accumulation of insoluble abnormal glycogen deposits in the brain and peripheral tissues. Mutations in at least two genes are responsible for the disease: EPM2A, encoding the glucan phosphatase laforin, and EPM2B, encoding the RING-type E3-ubiquitin ligase malin. Both laforin and malin form a functional complex in which laforin recruits the substrates to be ubiquitinated by malin. We and others have described that, in cellular and animal models of this disease, there is an autophagy impairment which leads to the accumulation of dysfunctional mitochondria. In addition, we established that the autophagic defect occurred at the initial steps of autophagosome formation. In this work, we present evidence that in cellular models of the disease there is a decrease in the amount of phosphatidylinositol-3P. This is probably due to defective regulation of the autophagic PI3KC3 complex, in the absence of a functional laforin/malin complex. In fact, we demonstrate that the laforin/malin complex interacts physically and co-localizes intracellularly with core components of the PI3KC3 complex (Beclin1, Vps34 and Vps15), and that this interaction is specific and results in the polyubiquitination of these proteins. In addition, the laforin/malin complex is also able to polyubiquitinate ATG14L and UVRAG. Finally, we show that overexpression of the laforin/malin complex increases PI3KC3 activity. All these results suggest a new role of the laforin/malin complex in the activation of autophagy via regulation of the PI3KC3 complex and explain the defect in autophagy described in Lafora disease.     

Unleashing the Ambra1-Beclin 1 complex from dynein chains: Ulk1 sets Ambra1 free to induce autophagy

The Beclin 1-VPS34 complex plays a crucial role in the induction of the autophagic process by generating PtdIns(3)P-rich membranes, which act as platforms for ATG protein recruitment and autophagosome nucleation. Several cofactors, such as Ambra1, ATG14 and UVRAG, are necessary for Beclin 1 complex activity. However, the mechanism by which Beclin 1 complex activity is: stimulated by autophagic stimuli has not yet been fully elucidated. Recently, we reported that autophagosome formation in mammalian cells is primed by Ambra1 release from the dynein motor complex. We found that Ambra1 specifically binds the dynein motor complex under normal conditions through a direct interaction with DLC1. When autophagy is induced, Ambra1-DLC1 are released from the dynein complex in an ULK1-dependent manner, and relocalize to the endoplasmic reticulum, thus enabling autophagosome nucleation. In addition, we found that both DLC1 downregulation and Ambra1 mutations in its DLC1-binding sites strongly enhance autophagosome formation. Ambra1 is therefore not only a cofactor of Beclin 1 in favoring its kinase-associated activity, but also a crucial upstream regulator of autophagy initiation.     

Dapper1 promotes autophagy by enhancing the Beclin1-Vps34-Atg14L complex formation

Autophagy is an intracellular degradation process to clear up aggregated proteins or aged and damaged organelles. The Beclin1-Vps34-Atg14L complex is essential for autophagosome formation. However, how the complex formation is regulated is unclear. Here, we show that Dapper1 (Dpr1) acts as a critical regulator of the Beclin1-Vps34-Atg14L complex to promote autophagy. Dpr1 ablation in the central nervous system results in motor coordination defect and accumulation of p62 and ubiquitinated proteins. Dpr1 increases autophagosome formation as indicated by elevated puncta formation of LC3, Atg14L and DFCP1 (Double FYVE-containing protein 1). Conversely, loss of Dpr1 impairs LC3 lipidation and causes p62/SQSTM1 accumulation. Dpr1 directly interacts with Beclin1 and Atg14L and enhances the Beclin1-Vps34 interaction and Vps34 activity. Together, our findings suggest that Dpr1 enhances the Atg14L-Beclin1-Vps34 complex formation to drive autophagy.     

UVRAG: a new player in autophagy and tumor cell growth

Autophagy has a well-documented role in the maintenance of homeostasis and the response to stressful environments and it is often deregulated in various human diseases including cancer. The regulation of the Beclin 1-PI3KC3 complex lipid kinase activity is a critical element in the autophagy signaling pathway. Previous studies(1) have demonstrated that Beclin 1-PI3KC3-mediated autophagy is negatively regulated by a proto-oncogene Bcl-2. We have recently identified a novel coiled-coil UVRAG tumor suppressor candidate, which positively engages in Beclin 1-dependent autophagy. UVRAG interacts with Beclin 1, leading to activation of autophagy and thereof inhibition of tumorigenesis. This finding adds a new player to the emerging picture of the autophagy network, under-scoring the importance of the coordinated activity between Bcl-2 and UVRAG in the regulation of Beclin 1-PI3KC3-mediated autophagy and tumor cell control.