Apoptosis-Associated Protein Domains: History
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There are proteins or families of proteins that regulate the caspase activation pathways, namely, the extrinsic or intrinsic pathways, and they are identified depending on their amino acid sequence or homologue. The interactions facilitated by these protein families are driven through protein domains that are linked with the regulation of apoptosis, such as death domains (DDs), caspase recruitment domains (CARDs), death effector domains (DEDs), BCL-2 family proteins and of IAP-family proteins.

  • apoptosis
  • signaling
  • therapy
  • proteins
  • cell death

1. Death Domain Proteins

DDs consist of a condensed bundle of six alpha helices that interact among themselves and form an oligomer. The differences in surface residue decide the specificity for partner selection in the death domain [71]. Several TNF family members of the cytokine receptor have their death domains in the cytosolic face. The TNF receptor family, including TNFR1, DR3 and DR6, works by binding to adapter protein, namely, TRADD, which contains its homologous death domain. The DD of TRADD is able to bind with other proteins containing DD, such as Fadd, an adapter protein. The FADD protein has a DD through which it associates with the TNF family and, hence, links TNF family receptors to caspases [72]. Hence, this protein plays a role of mediator. Additionally, a similar example is seen in case of RAIDD, which is also known as CRADD. This is an adapter protein containing DD along with CARDs. It aids in linking the death receptor family with procaspases by binding with the CARD domain of pro-caspase-2 [73]. Moreover, TRAIL-R1 (DR4) and TRAIL-R2 (DR5) are involved in the induction of apoptosis by binding to the TRAIL ligand. TRAIL ligand can also bind with DcR1, DcR2 and osteoprotegerin (OPG), which are also TNF family proteins, but they are considered as decoy receptors as they do not carry a death signal [74]. Fas, is also a member of TNF receptor family and is considered a potential stimulator of apoptosis. It is crucial in the homeostasis of the immune system, removing autoreactive lymphocytes and reducing immune response once foreign antigens are removed [75,76]. There are some other proteins that have DD and are involved in apoptosis, such as DAP kinase, but their mechanism is not well understood. However, it has been reported that they activate caspase-8, which disturbs the cytoskeleton [54]. Due to the importance of DD proteins, any imbalance or defect in the regulation and functioning of these proteins leads to human diseases. For instance, upregulation in the expression of FasR or FasL on lymphocytes has been known to be associated with HIV infection. Moreover, mutation in death domain of the Fas gene can cause lymphoproliferative syndrome, an autoimmune syndrome and malignancies [77].

2. Death Effector Domain Proteins

The death effector domain (DED) shares similarity with DD proteins in terms of structure. It exists in initiator caspases, namely, caspase-8 and caspase-10. There are two tandem DED motifs present in the prodomain region of initiator caspases. Due to the presence of DED in caspase-8 and caspase-10, they are able to make an interaction with DED of FADD, and thereby allow their association with the death receptor complexes [78]. The DED family proteins regulate apoptosis either by increasing caspase activation or preventing caspase activation by TNF family death receptors. One of the DED-containing proteins is FLIP, which is also known as FLAME, CLARP, CASH, CASPER, I-FLICE, MRIT or Usurpin [79]. It has been documented to be involved in suppressing apoptosis in cancer. The amino acid sequence of FLIP shares similarity with pro caspase-8 and -10 and, hence, competes with these caspases for binding to FADD, thereby silencing death receptor signaling. The increasing level of FLIP in tumor cells results in the development of resistance against apoptosis induction by Fas-expressing CTLs [80]. This FLIP-associated Fas resistance makes the tumor cell tolerate FasL expression, utilizing this death ligand as a weapon to nearby normal cells, and to stimulate apoptosis of immune cells. However, there are strategies for down-regulating the expression of FLIP employing antisense technology and drugs that restore sensitivity of tumor cell lines toward apoptosis towards FasL [81].

3. CARD-Family Proteins

There are numerous pro-caspases, namely, caspase-1, -2, -4, -5 and -9, which have CARDs in their N-terminal prodomains. The CARD has six helices present in DED and DD. CARD-family proteins are crucial in caspase activation via homotypic interactions throughout animal evolution. An example of caspase activation can be seen in CED-4 in C. elegans and APAF-1 in humans [82]. Both the proteins carry a CARD domain along with a nucleotide-binding oligomerization domain, called the NB-ARC (Nucleotide-binding domain homologous to APAF-1, CED-4 and plant R gene products) [83]. The CARD of APAF-1 associates with the CARD of pro-caspase-9, and upon oligomerization, they activate caspases. APAF-1 contains several WD-40 regulatory domains, which makes it dependent on cytochrome c. It has been seen that the oligomerization of APAF-1 is dependent upon dATP and cytochrome c, and once it is oligomerized, it binds and activates caspase-9 [82]. Cancer cells have shown innumerable mechanisms for preventing caspase activation via APAF-1, such as the inhibition of APAF-1 gene by methylation, increased expression of heat shock proteins that inhibit their functioning, increased expression of the tumor-up-regulated CARD-containing antagonist of caspase nine (TUCAN), which contains CARDs and is an antagonist of caspase-9, phosphorylating caspase-9 causing inhibition of their activity, and the association of CARDs with IAP-family proteins [73].

4. Inhibitor of Apoptosis Proteins

The inhibitor of apoptosis proteins or IAPs constitutes a family of suppressors of apoptosis that are conserved throughout the evolution. The main function of these proteins is the inhibition of caspases endogenously [84]. All IAPs have a common baculovirus inhibitor of the apoptosis protein repeat (BIR) domain, which is crucial for inhibiting apoptosis. However, only the presence of the BIR domain does not indicate anti-apoptotic activity as this domain is involved in regulating cell cycle with no impact on cell death [84]. Apart from the BIR domain, the IAP family of proteins also consists of other domains, such as RING zinc-fingers, CARDs, Ubiquitin-conjugating enzyme (E2s) domains and putative nucleotide-binding domains [85]. The RINGs and Apollon (BIR domain protein) are associated with ubiquitination machinery. Several IAPs, such as the X-linked inhibitor of the apoptosis protein (XIAP), cIAP1 and cIAP2 bind directly to the initiator caspase-9 and even to the executioner caspase-3 and caspase-7, thereby inhibiting their function. Different domains are required for inhibiting caspases; for instance, in the case of XIAP, a second BIR domain is required to inhibit caspase-3 and -7, while a third BIR domain is crucial for suppressing caspase-9 [86]. The IAP family members, namely, Livin and survivin, have one BIR domain and suppress caspase-9, but not caspase-3 and -7. The IAPs are highly selective towards specific caspases; therefore, overexpression of IAPs can inhibit some of the apoptotic pathways but not all [87,88]. However, baculovirus p35 protein represents a broad-spectrum activity against most of the caspases, but no cellular homologue of this protein has yet been found. Further, IAPs target apoptosis mediated by intrinsic or extrinsic pathways, because their target, i.e., effector caspases are associated with these two pathways. It has been documented that overexpression of IAP family members is associated with cancers [89]. For instance, survivin, Livin, XIAP and cIAP1 overexpression are shown in melanomas and tumor cells [87,88]. However, antisense-mediated targeting of XIAP or cIAP1 can stimulate apoptosis in tumor cell lines. There are endogenous antagonists of IAPs, such as SMAC (DIABLO) and HTRA2 (OMI), which function in promoting apoptosis. These antagonists compete with caspases to bind to IAPs, thereby not allowing caspases from binding to IAP and, hence, favoring apoptosis [90].

5. BCL-2 Family Proteins

The BCL-2-family proteins are associated with the mitochondrial dependent pathway of apoptosis. Some proteins of this family, such as BCL-2, BCL-XL and BAK have a patch of hydrophobic amino acids at the C-terminal end through which these are linked with the outer mitochondrial membrane [91]. However, this hydrophobic patch is absent in BID, BIM and BAD, but they are associated with mitochondria via specific stimuli [92]. The BCL-2-family proteins are found to be conserved in the evolution of metazoan, and their homologues are present in vertebrates as well as invertebrates. Innumerable animal virus genomes, such as herpes simplex virus, Epstein-Barr virus (EBV) and Kaposi sarcoma herpes virus contain BCL-2 homologs [93]. There are 26 members of the BCL-2 family that are known currently. Their genes code for anti-apoptotic and pro-apoptotic proteins are, namely, BCL-2, BFL-1 (A1), BCL-XL, MCL-1, BCL-w, BCL-B and BAX, BOK (MTD), BAK, BAD, BIM, BID, BIK, BCL-XS, NIP3, HRK, PUMA, APR (NOXA), BCL-Gs, p193, NIX (BNIP) and BCL-RAMBO (MIL) [91,94,95,96]. The BCL-2 family members with their location and functions are summarized in Table 2. Few of the BCL-2 family genes generate more than two proteins via alternative splicing, which shows contrasting effects on apoptotic regulation, such as BCL-XL versus BCL-XS [97].
Table 2. The BCL-2 family proteins with their location and functions *.
BCL-2 Protein Location Roles Refs
BAX Cytosol Liberation of apoptogenic factors and induction of caspases [98]
BAK Integral mitochondrial membrane protein Conformational changes in BAK take place to form larger complexes in apoptosis and create pores in the mitochondrial membrane to liberate apoptogenic factors to promote apoptosis [99]
BID Cytosol and membrane Directly activate BAX [100]
BCL-2 Mitochondria, nucleus, endoplasmic reticulum Prevents apoptosis by maintaining integrity of mitochondrial membrane integrity [101,102]
BCL-XL Mitochondrial transmembrane Prevents release of cytochrome c via mitochondrial pore, thereby inhibiting activation of caspases by cytochrome c [102]
MCL-1 Nucleus, mitochondria Associated with BAK1, BCL-2-associated death promoter, NOXA, BCL2L11 and PCNA [103,104]
BCL-w/BCL2L2 Mitochondrion Under cytotoxic conditions downregulate apoptosis [105]
A1/BFL-1 Mitochondria, nucleus unknown [106,107]
BIM/BCL2L11 Mitochondria Interacts with BCL-2 or BCL-XL and prevents their anti-apoptotic actions [108]
PUMA Mitochondria unknown; regulated by p53 transcriptionally [109,110]
BAD Mitochondria Generate a complex with BCL-2 and BCL-XL, inhibits them, thereby promoting BAX/BAK-mediated apoptosis [111]
BIK/BLK Endoplasmic reticulum unknown [112]
NOXA/PMAIP1 Mitochondria unknown [47,113]
BMF Mitochondria unknown [114]
* Abbreviations used are: A1/BFL-1, BCL-2-related protein A1; BAD, BCL-2-associated agonist of cell death; BAK, BCL-2 antagonist killer; BAX, BCL-2-associated protein X; BCL-2, B-cell lymphoma 2; BCL-w/BCL2L2, BCL-2-like 2; BCL-XL, B-cell lymphoma-extra-large; BID, BH3-interacting domain death agonist; BIK/BLK, BCL-2-interacting killer; BIM/BCL2L11, BCL-2-interacting protein BIM; BMF, BCL-2-modifying factor; MCL-1, myeloid cell leukemia 1; NOXA/ PMAIP1, phorbol-12-myristate-13acetate-induced protein 1; PUMA, p53-upregulated modulator of apoptosis.
Altogether, this section highlights the importance of DDs, CARDs, DEDs, BCL-2 family proteins and of IAP-family proteins in the regulation of apoptosis, and their possible link with lymphoproliferative syndrome, autoimmune syndrome and other malignancies. The next section focuses on the significance of apoptosis in carcinogenesis.

This entry is adapted from the peer-reviewed paper 10.3390/sci4020015

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