Apoptotic Protease-Activating Factor-1

Created by Michael Hong

      APAF-1, or Apoptotic Protease-Activating Factor-1, is a protein that helps relay the apoptosis death signal in humans. Under normal cellular conditions, APAF-1 is located in the cytosol and exists in its auto-inhibited, or closed, conformation. Activation of apoptosis causes the mitochondria to release Cytochrome C into the cytosol where it binds APAF-1 and causes it to change into its open, or active, conformation. This conformational change exposes oligomerization activating sites and allows for the formation of the heptamer apoptosome. This wheel-like structure recruits and activates procaspase-9, which in turn activates downstream executioner caspases that continue the apoptosis process.

      APAF-1 obviously plays an important role in programmed cell death and helps maintain a variety of homeostatic functions. Considering APAF-1’s intrinsic ability to activate or inhibit apoptosis, studying APAF-1 could lead to serious advances in modern medicine. First, further investigation of APAF-1 could lead to new advances in the understanding and treatment of cancer. Cancerous cells have the uncommon ability to evade apoptosis and they gain this ability from mutations to proto-oncogenes that normally maintain and control apoptosis. Studying APAF-1 could provide a greater understanding as to how apoptosis can be induced or even restored in cancerous cells to target tumors. Further research of APAF-1 could even lead to a greater understanding about how cancer originates in otherwise normal cells. APAF-1 can also be studied to develop our understanding of neurodegenerative disorders like Alzheimer’s disease and Parkinson’s disease. Both of these life-altering conditions result from the degeneration of different components of the central nervous system. Thus, apoptosis is active when it should be dormant. Perhaps further research of APAF-1 could help correct these diseases or even reveal the underlying causes.

       Regulation of apoptosis is a necessary homeostatic function for all multicellular organisms and the general mechanism to activate caspases is conserved across species7. Accordingly, each apoptotic protein, including APAF-1, must have evolved from an ancestral protein and must share some similarities with evolutionarily related species. CED-4, or corpse engulfment protein-4, is a homolog of APAF-1 found in the nematode C. elegans. CED-4 is only 549 residues long but shares forty-five percent of its amino acid sequence with APAF-1 in the first 500 residues of the polypeptide7. CED-4 normally exists as a self-inactivated homodimer in the cytosol and is activated by apoptosis initiating stimuli to produce an apoptosome. In contrast to APAF-1, which forms a heptamer apoptosome, CED-4 polymerizes to form an octamer composed of four of the initial CED-4 homodimers. The CED-4 apoptosome recruits and activates CED-3, a proteolytic caspase that is homologous to procaspase-9 in humans.Both CED-4 and APAF-1 contain a caspase recruitment domain (CARD) at the N terminus of the polypeptide that helps form the caspase activating apoptosome. The evolutionary retention of the  sequence is logical since the activation of caspases is the ultimate goal of apoptosis. Both proteins also share common nucleotide binding regions and other helical domains7. Finally, both proteins depend on the binding of ATP to the nucleotide binding site for the activation of the protein.

       The overall function of APAF-1 is largely determined by the structure of its three major domains. The first 94 amino acid residues of the primary sequence form the N-terminal CARD, or the caspase recruitment domain. The CARD is composed of six alpha helices that are tightly packed with one another. The next 495 residues form the NOD, or the nucleotide-binding and oligomerization domain, which is composed of four sub-domains. When reading the primary sequence from the N-terminus to the C-terminus, the four sub-domains are the nucleotide-binding domain (NBD), the helical domain 1 (HD1), the winged-helix domain (WHD) and the helical domain 2 (HD2). The primary sequence of these amino acids predominantly folds into a series of alpha helices linked together by beta turns. A nucleotide binding site is located in the NBD and normally binds ADP. As will be discussed, ATP replaces ADP during the conformational change. Lastly, the final 680 residues of the primary amino acid sequence form the WD40 domain, which is almost exclusively composed of beta sheets and beta turns. The regulatory WD40 domain contains twelve WD repeats (tryptophan-aspartic acid) and two sub-domains called WD1 and WD2. The WD40 domain contains all five disulfide bridges in APAF-1; C633 binds C663, C675 binds C705, C704 binds C749, C761 binds C804, and C803 binds C846.

       Implicit to its name, the CARD recruits and activates caspase molecules to continue the downstream apoptosis pathway. The six, anti-parallel alpha helices of the CARD are arranged like a six pack of cans. Accordingly, there are an abundance of hydrophobic residues located at the contact areas of these alpha helices that allow them to pack together via hydrophobic interactions. The two alpha helices in the center of the “six-pack” structure exhibit the most hydrophobic regions and thus the most hydrophobic interactions4. Under normal conditions, it is important that the CARD remains buried in the interior of the APAF-1 protein as to prevent the activation of apoptosis. The CARD is predominantly surrounded and stabilized by the winged helical sub-domain (WHD) of the NOD. These domains interact with one another via “thirteen inter-domain hydrogen bonds and some van der Waals contact.”4 There are several key amino acids in the CARD and WHD that contribute to this interaction and promote the burial of the CARD in the interior of APAF-1. For example, Glutamic acid-78 (E78) of the CARD hydrogen bonds to arginine-428 (R428) of the WHD and R52 of the CARD bonds to D413 of the WHD.  

       The WD40 domain is also important to APAF-1 because it is responsible for the auto-inhibitory mechanism that prevents the onset of apoptosis. The WD40 domain acts like a lock and prevents the movement of other domains in the closed conformation of APAF-1. As I will discuss, movement of the NOD sub-domains exposes the oligomerization activating regions and ultimately allows for the formation of the heptameric apoptosome. Upon activation of apoptosis, Cytochrome C binds to the WD repeated regions of the WD40 domain and moves the WD40 domain to allow for the free rotation of the NBD and HD1 sub-domains. As the NBD rotates, the nucleotide binding site is exposed. The nucleotide binding site is formed at the junction of three NOD sub-domains; the NBD, the HD1 and the WHD. The NBD contains one aspartic acid residue (D244) and the WHD contains two, D392 and D439. The negatively charged oxygen atoms of the carboxyl groups on each aspartic acid residue form a triangle with one another that creates some electrostatic repulsion. This repulsion is stabilized by the positively charged side chain of a nearby arginine residue (R265) that protrudes from the NBD into the nucleotide binding site. ADP fits into this nucleotide binding site but does not form any covalent bonds to any of these amino acid residues. Rather, ADP has multiple oxygen atoms that hydrogen bond to different residues, including V127, G157, G159, K160, S161, and S325. All of these residues are located in the NOD.  

   In the absence of apoptosis, ADP occupies the nucleotide binding site and the surrounding domains act to shield ADP from the periphery. However, when APAF-1 begins to change into its open conformation, ADP is exposed and is replaced by ATP. The additional negatively charged phosphate group of ATP interfere with the natural stability of the nucleotide binding site and competes with the aspartic acid residues for the positively charged side chain of the arginine residue. This destabilization results in more electrostatic repulsion and causes the NOD sub-domains to repel one another. As the sub-domains move apart, they continue to open the conformation and “expose the contact areas for oligomerization”.1

       Rotation of the NOD sub-domains also exposes the CARD that was initially buried within the NOD. Once the CARD and the oligomerization activating regions are both exposed, APAF-1 molecules oligomerize with one another until they form a heptamer called the caspase-activating apoptosome1. As mentioned, caspase recruitment domains are composed of six amphipathic alpha helices. However, the majority of these residues are hydrophobic, and when the CARD is exposed during the conformational change, the hydrophobic regions will aggregate with hydrophobic CARDs of other APAF-1 proteins to prevent a decrease in the entropy of the solvent. There are also hydrophobic patches on the exterior surfaces of the CARD helices that are normally buried by the winged-helical domain (WHD). Some hydrophobic residues that contribute to the formation of the CARD octamer are I30, F34, I37, V69, F71, L76, L83, A84, A85, L86, I91, and P92.

       The CARD also contains acidic patches on the exterior regions of the alpha helices that help the CARD recruit procaspase-9. Amino acid residues D27, E39, E40 and E41 create the acidic region that interacts with a basic patch of arginine residues on procaspase-9. The seven, oligomerized CARD domains occupy the center of the wheel-like apoptosome and bind procaspase-9 to continue the downstream activation of executioner caspases.