Overview

Autophagy is an essential process that consists of selective degradation of cellular components. There are at least three different types of autophagy described and possibly more. These autophagy types include macroautophagy (hereafter referred to as autophagy), microautophagy and chaperone-mediated autophagy. The initial step of autophagy is the surrounding and sequestering of cytoplasmic organelles and proteins within an isolation membrane (phagophore). Potential sources for the membrane to generate the phagophore include the Golgi complex, endosomes, the endoplasmic reticulum (ER), mitochondria and the plasma membrane.The nascent membranes are fused at their edges to form double-membrane vesicles, called autophagosomes. Autophagosomes undergo a stepwise maturation process, including fusion with acidified endosomal and/or lysosomal vesicles, eventually leading to the delivery of cytoplasmic contents to lysosomal components, where they fuse, then degrade and are recycled . The process of mammalian autophagy is divided into six principal steps: initiation , nucleation , elongation , closure, maturation  and degradation or extrusion.(1)

it has been well demonstrated that autophagy depends on Atg5/Atg7, is associated with microtubule-associated protein light chain 3 (LC3) truncation and lipidation, and may originate directly from the ER membrane and other membrane organelles. Furthermore, recent study has identified a Atg5/Atg7-independent pathway of autophagy.This pathway of autophagy was not associated with LC3 processing but appeared to involve autophagosome formation from late endosomes and the trans-Golgi.Atg7-independent autophagy had been implicated in mitochondrial clearance from reticulocytes. The exact molecular basis of Atg5/Atg7-independent autophagy remains to be elucidated. Interestingly, Beclin 1 is required for Atg5/Atg7-dependent and -independent autophagy. However, the presence of Beclin 1-independent autophagy is evaluated in various experimental setting, further complicates discernment of the role of these individual pathways. (2)

The mammalian autophagy gene Beclin 1, an ortholog of the Atg6/ vacuolar protein sorting (Vps)-30 protein in yeast, was cloned through a yeast two-hybrid screen in 1998 by Beth Levine's group. Beclin 1 is important for localization of autophagic proteins to a pre-autophagosomal structure (PAS), depending on interaction with the class III type phosphoinositide 3-kinase (PI3KC3)/Vps34. Together they form the Beclin 1-Vps34-Vps15 core complex (2)

General concepts

Beclin 1 is a novel Bcl-2-homology (BH)-3 domain only protein. The embryonic phenotype of Beclin 1 null mice is even more severe than that of other autophagy gene-deficient mice, which die in early embryonic development (E7.5 or earlier) with defects in proamniotic canal closure. Beclin 1 is expressed in many human and murine tissues, and is localized primarily within cytoplasmic structures, including the ER, mitochondria and the perinuclear membrane. In human colon cancer tissue observed via immunohistochemistry, Beclin 1 is distributed within the plasma-membrane, the cytoplasm and the nucleus. Beclin 1 contains three identified structural domains: a BH3 domain (amino acids 114–123) at the N-terminus, a central coiled-coil domain (CCD, amino acids 144–269) and an evolutionarily conserved domain (ECD, amino acids 244–337) . The ECD is essential for Beclin 1's ability to mediate autophagy and to inhibit tumorigenesis. Beclin 1 also contains a short leucine-rich amino acid sequence that is responsible for its efficient nuclear export signal (NES) . Mutations of the Beclin 1 NES interfere with its abilities to promote nutrient deprivation-induced autophagy and suppress tumorigenesis. Anti-apoptotic Bcl-2 family members interact with the BH3 domain of Beclin 1. The activating molecule in Beclin 1-regulated autophagy (Ambra1)/UV radiation resistance-associated gene (UVRAG)/Atg14L interact with the CCD domain, and PI3KC3/Vps34 interacts with the ECD and CCD domains. (3,4,5)

Interactions

p65, one of the canonical NF-κB pathway components, directly binds the Beclin 1 promoter and upregulates its mRNA and protein levels, leading to autophagy in T cells. However, the relationship between NF-κB and regulation of autophagy seems puzzling and complex. NF-κB has emerged as a negative regulator of autophagy, as induced by tumor necrosis factor, reactive oxygen species (ROS) and starvation in some cell lines. On the other hand, other studies suggest NF-κB and its counter-regulator IKK have an activating role in autophagy.Constitutively, active IKK subunits stimulate autophagy. Remarkably, NF-κB p65 positively modulates Beclin 1 transcription and autophagy. Further study, likely evaluating regulatable systems in various cellular targets in vivo, will be required to sort out the complexity. (6,7)

Recent work suggests that E2F1 binds Atg1/ULK1, LC3, DRAM1, BNIP3, ULK2, Atg4, Atg7, GABARAPL2, Atg9A, Atg10, Atg12, p73, Apaf1 and caspase promoters, increasing their expression and regulating autophagy and apoptosis.A ChIP-on-chip study suggested that Beclin 1 could be an E2F target. Indeed, E2Fs can directly transactivate the Beclin 1 promoter .When E2F-1 or -2 or -3 is depleted, Beclin 1 mRNA and protein levels are significantly reduced in the U2OS cell line.(6)

Autophagy: induction-inhibition

Although more and more Beclin 1-binding proteins and complexes are being identified in mammals, each of the individual complexes seems to recycle various elements derived from other cellular processes. Beclin 1 (Atg6/Vps30), PI3KC3/Vps34 and Vps15  have been predicted to regulate autophagy in a similar manner to yeast .(7)

The phosphoinositol-3-kinase (PI3K) family is divided into three different classes in mammals – class I, class II and class III. Yeast cells have only a solitary class III PI3K and Vps34. Vps34 forms at least two distinct PI3K complexes in yeast : complex I consisting of Vps34, Vps15, Atg6/Vps30 and Atg14, and complex II containing Vps34, Vps15, Atg6/Vps30 and Vps38 (Itakura et al.). Complexes I and II function in autophagy and the Vps pathway, respectively. Complex II is more abundant than complex I, suggesting that Atg6/Vps30-Vps34-Vps15 is primarily involved in the Vps pathways under normal conditions and that yeast autophagy is strictly regulated at a basal level in yeast. Interestingly, recent studies show that Vps34 is phosphorylated on Thr159 by cyclin-dependent kinase 1 (Cdk1), which negatively regulates its interaction with Beclin 1 during mitosis in mammalian cells . Vps34 also appears to have a role in other membrane-trafficking processes distinct from autophagy. It would be interesting to know if Vps34 phosphorylation affects these processes, and if cell cycle proteins are required for autophagy. (8,9)

 

Mutations of either the BH3-only domain within Beclin 1, or the BH3 receptor domain within Bcl-2 or Bcl-XL, disrupted the Beclin 1–Bcl-2 complex, resulting in the stimulation of autophagy. The relationship between Beclin 1 and Bcl-2/Bcl-XL is complicated. Beclin 1 cannot neutralize the anti-apoptotic function of Bcl-2, which is exerted at the mitochondrial membrane. In contrast, Bcl-2 or Bcl-XL reduces the pro-autophagic activity of Beclin 1 (Pattingre et al.; Maiuri et al.). Interestingly, ER-localized Bcl-2, but not mitochondrial-localized Bcl-2, inhibits autophagy,which is consistent with the older notion that ER-associated class III PI3K activity may be crucial in the nucleation of autophagosome formation. Beclin 1 can colocalize with Bcl-XL within mitochondria via its BH3 domain,suggesting a differential role of Bcl-XL in Beclin 1 complex when compared with Bcl-2. (9)

 

Footnotes

1. Zhang J, Randall MS, Loyd MR, Dorsey FC, Kundu M, Cleveland JL, et al. Mitochondrial clearance is regulated by Atg7-dependent and -independent mechanisms during reticulocyte maturation. Blood. 2009;114:157–164. [PMC free article] [PubMed]
2. Scarlatti F, Maffei R, Beau I, Codogno P, Ghidoni R. Role of non-canonical Beclin 1-independent autophagy in cell death induced by resveratrol in human breast cancer cells. Cell Death Differ. 2008;15:1318–1329. [PubMed]
3. Yu L, McPhee CK, Zheng L, Mardones GA, Rong Y, Peng J, et al. Termination of autophagy and reformation of lysosomes regulated by mTOR. Nature. 2010;465:942–946. [PMC free article] [PubMed]
4. Liang XH, Kleeman LK, Jiang HH, Gordon G, Goldman JE, Berry G, et al. Protection against fatal Sindbis virus encephalitis by beclin, a novel Bcl-2-interacting protein. J Virol. 1998;72:8586–8596. [PMC free article] [PubMed]
5. He C, Levine B. The Beclin 1 interactome. Curr Opin Cell Biol. 2010;22:140–149. [PMC free article] [PubMed]
6. Oberstein A, Jeffrey PD, Shi Y. Crystal structure of the Bcl-XL-Beclin 1 peptide complex: Beclin 1 is a novel BH3-only protein. J Biol Chem. 2007;282:13123–13132. [PubMed]
7. Yue Z, Jin S, Yang C, Levine AJ, Heintz N. Beclin 1, an autophagy gene essential for early embryonic development, is a haploinsufficient tumor suppressor. Proc Natl Acad Sci USA. 2003;100:15077–15082. [PMC free article] [PubMed]
8. Li BX, Li CY, Peng RQ, Wu XJ, Wang HY, Wan DS, et al. The expression of beclin 1 is associated with favorable prognosis in stage IIIB colon cancers. Autophagy. 2009;5:303–306. [PubMed]
9. Liang XH, Yu J, Brown K, Levine B. Beclin 1 contains a leucine-rich nuclear export signal that is required for its autophagy and tumor suppressor function. Cancer Res. 2001;61:3443–3449. [PubMed]