Who Put the “A” in Atg12: Autophagy or Apoptosis?

Abstract

Much deliberation surrounds how the two homeostatic pathways, autophagy and apoptosis, converge; in the December 9 issue of Molecular Cell, Rubinstein et al. (2011) identify a proapoptotic role for the autophagic protein Atg12, based on a BH3-like domain, which enables binding and inhibition of antiapoptotic Bcl-2 family proteins.

Autophagy plays an important role in intracellular homeostasis by regulating destruction and reconstruction of important organelles and components. Apoptosis plays an important role in organismal homeostasis by controlling elimination of cells, maintaining cell number in the face of ongoing cellular proliferation. Increasingly, biologists are identifying ways in which these two different and important cellular functions are connected.

The mitochondrial pathway of apoptosis is regulated by a family of proteins termed the Bcl-2 family. They are divided into proapoptotic members and antiapoptotic members (Adams and Cory, 2007). The Bcl-2 homology 3 (BH3) domain is required for the proapoptotic function of the BH3-only proteins, so named as they possess only the BH3 of the four Bcl-2 homology regions. The BH3-only proteins exert their prodeath function either by inhibition of the antiapoptotic Bcl-2 family members (including Bcl-2 and Mcl-1) or by directly activating multidomain proapoptotic effector proteins like Bax and Bak (Letai et al., 2002). Activation of Bax and Bak results in their oligomerization, which in turn causes mitochondrial outer membrane permeabilization (MOMP). For most purposes, MOMP may be considered the point of commitment to cell death via the mitochondrial apoptotic pathway.

In times of stress or starvation, autophagy can be exploited to facilitate cell survival, enabling utilization of building blocks for alternative important functions (Kroemer and Levine, 2008). A ubiquitin-like process is involved in the conjugation of Atg12 to Atg5. The Atg12-Atg5 complex then binds with Atg16 to enable autophagosome formation (see Figure 1). In this issue of Molecular Cell, Rubinstein and colleagues demonstrate that the autophagy-related gene 12 (Atg12) can bind to antiapoptotic Bcl-2 family members to promote apoptosis.

Figure 1. Proposed Model of the Autophagic and Apoptotic Role of Atg12.

Autophagic stimuli lead to the formation of autophagsomes. On the left side of the diagram is the list of interactions required to form the autophagosome. PE is phosphatidylethanolamine. On the right side is the proposed mechanism by which Atg12 affects apoptosis by interacting with Bcl-2 and Mcl-1. Once caspases are activated, this can lead to the cleavage of Beclin-1, which can then block autophagy and enhance apoptosis.

The authors performed an RNAi screen using genes with known autophagic function to identify genes that could regulate both autophagy and mitochondrial apoptosis. The screen identified Atg12 as a proapoptotic molecule. Knockdown of Atg12 by siRNA protected against mitochondrial apoptosis demonstrated in response to a wide variety of apoptotic insults, including the pan-kinase inhibitor staurosporine, the topisomerase 2 inhibitor etoposide, paclitaxel, TNF-α, and tunicamycin.

The proapoptotic activity of Atg12 was dependent on a domain described as “BH3-like,” due mainly to its possession of a LXXXXD amino acid sequence motif present in all BH3 domains thus far characterized. Of course, this is not a very stringent standard, as thousands of proteins in the proteome possess this same sequence, and it is unlikely that most of them are truly used to interact with Bcl-2 family proteins. A true BH3 domain has a conserved sequence of LXXXXD along with an α-helical structure that enables binding into the hydrophobic cleft of the antiapoptotic family members (Sattler et al., 1997). The BH3-like domain of Atg12 has the conserved LXXXXD sequence; however, there is a proline two residues after the aspartic acid that prevents the α-helical structure found in true BH3 domains.

Whether or not “BH3” belongs in its name, this region does appear to be critical for interactions with antiapoptotic proteins. Amino acid substitutions within the BH3-like region interfered with interaction with Bcl-2 and Mcl-1. Moreover, the BH3-mimetic Bcl-2 antagonist ABT-737 competitively inhibits the interaction of Atg12 with Bcl-2, but not Mcl-1. Importantly, mutation of the BH3-like region that affected Atg12 binding to Bcl-2 did not have an effect on autophagy, demonstrating that two distinct parts of the protein may play roles in the two distinct homeostatic pathways.

A recent publication has implicated Atg12 in control of cell death (Radoshevich et al., 2010), but in a manner apparently distinct from what is reported in Rubinstein et al. (2011). Radoshevich et al. showed that conjugation of Atg12 to Atg3 enhanced mitochondrial apoptosis and that preventing the Atg3-Atg12 complex protected against mitochondrial apoptosis potentially by increasing the levels of the antiapoptotic protein Bcl-XL (Radoshevich et al., 2010). In contrast, the proapoptotic effect of Atg12 identified by Rubinstein et al. did not require Atg12 to be in a complex. In fact, the uncomplexed form alone seemed to have proapoptotic function.

As with any novel interaction, there are many types of follow-up experiments that will be important to validate its significance. At the biochemical level, structural data will be key to examining the testable hypotheses provoked by the authors’ computer modeling of the interaction. At the cellular level, it will be important to test in many different contexts whether Atg12 acts as a significant modulator of apoptotic signaling to the mitochondrion.

There is a broader question of why eukaryotic biology has selected for control of apoptosis and autophagy by some of the same proteins. Bcl-2 can inhibit both apoptosis (by binding proapoptotic Bcl-2 family proteins) (Yang et al., 1995) and autophagy (by binding Beclin-1) (Pattingre et al., 2005). BH3-only proteins can promote both apoptosis and autophagy by inhibiting those same binding interactions (Maiuri et al., 2007). It may be that Atg12, which promotes initiation of autophagy, also promotes apoptosis. On the other hand, proapoptotic caspases, which are responsible for most phenotypes associated with apoptosis downstream of MOMP, inhibit autophagy by cleavage of Beclin-1, which also produces a prodeath protein that can enhance apoptosis (Wirawan et al., 2010).

One conjecture to unite these findings is that in the face of stresses that provoke a sublethal dose of death signaling, the cell attempts to mitigate long-term damage by seeking what catabolic benefits may be found in autophagy. However, once the stress is overwhelming and the cell has committed to cell death after MOMP, autophagy is futile, and it is beneficial to confirm and support apoptosis. Perhaps autophagy might even interfere with subsequent catabolism of high-energy macromolecules by whatever cell phagocytoses the apoptotic cell in the organismal setting and is thus selected against after MOMP.

Rubinstein et al. remind us that although we biologists find it convenient to subdivide the cellular workings with labels such as autophagy and apoptosis, nature has no obligation to obey such boundaries. In fact, some of the most interesting biology may take place where those boundaries are crossed.

This is a commentary on article Rubinstein AD, Eisenstein M, Ber Y, Bialik S, Kimchi A. The autophagy protein Atg12 associates with antiapoptotic Bcl-2 family members to promote mitochondrial apoptosis.. Mol Cell. 2011;44(5):698-709.