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Regulation of Cell Growth Laboratory, Center for Cancer Research, National Cancer Institute at Frederick, National Institutes of Health, Frederick, Maryland, USA; and
* Department of Cell Biology, Institute of Basic Medical Sciences, Beijing 100850, China
1Correspondence: NCI-Frederick, 1050 Boyles St., Building 560, Room 22-45, Frederick, MD 21702, USA. E-mail: yangyili{at}ncifcrf.gov
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ABSTRACT |
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 TOP ABSTRACT THE UBIQUITINATION SYSTEMUBIQUITINATION IS ASSOCIATED...THE RING-CONTAINING IAPS -...CONCLUSIONSREFERENCES |
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Key Words: ubiquitination • caspase • IAP • ring finger protein • ubiquitin protein ligase (E3)
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THE UBIQUITINATION SYSTEM |
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TOPABSTRACTTHE UBIQUITINATION SYSTEMUBIQUITINATION IS ASSOCIATED...THE RING-CONTAINING IAPS -...CONCLUSIONSREFERENCES |
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-amino group of a lysine residue in the substrate. The same set of enzymes also catalyzes the formation of the isopeptide bond between G76 and the lysine residue (usually the K48) of previously conjugated ubiquitin, leading to formation of polyubiquitin chain (1)
. Other factors may participate in elongation of the polyubiquitin chains under certain circumstances. For example, yeast protein Ufd2 is required for efficient polyubiquitination of substrate catalyzed by Ufd4 (E3) and Ubc4 (E2) (2)
. Proteins attached with polyubiquitin chains usually are degraded by the proteasome, a large multri-subunit complex consisting of 20S catalytic core and two 19S subunits that recognize ubiquitinated proteins and remove attached ubiquitins (3)
. The catalytic core has many different types of proteolytic activities to digest substrate proteins. Its activities can be modified by incorporation of different catalytic subunits and by association with alternative accessory factors to perform specific functions such as antigen processing. An interesting issue is how the polyubiquitinated proteins encounter and recognize proteasomes. Recent studies have indicated that proteins like Cdc48/VCP may act as chaperones to facilitate the degradation of ubiquitinated proteins at proteasomes (4)
. Tagging proteins with ubiquitins is involved in many other cellular events such as activation of kinases and transcription factors, DNA repair, endocytosis, and intracellular trafficking of proteins (5)
. Therefore, like phosphorylation, ubiquitination is a mechanism of signal transduction in the cells. It is not a surprise that cells have a large number of deubiquitination enzymes (DUBs) to facilitate the degradation of ubiquitinated proteins and regulate the functions of various proteins (Fig. 1
) (6
, 7)
.
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The E1 enzyme initiates the ubiquitination process by binding with ubiquitin in the presence of ATP and forming a thiol ester bond between Cys residue in its active site and G76 of ubiquitin. There is only a single E1 gene in most organisms. Studies in cells harboring temperature-sensitive mutation of E1 gave the first definitive evidence that ubiquitination is essential for the cell cycle (1)
. E2 accepts activated ubiquitin from E1 with its own Cys residue. However, there are multiple E2s in eukaryotic cells due to presence of isoforms and the emergence of new genes during evolution. There are at least 11 E2s in Saccharomyces cerevisiae and >25 E2s in mammalian cells. All E2s share a characteristic UBC domain consisting of 
150 amino acid residues that contains the active site and interacts with ubiquitin, E1, and E3. In vitro and in vivo experiments demonstrated that although E2s are interchangeable under certain circumstances, different E2s are often required for the ubiquitination of different substrates. For example, Ubc3 is essential for degradation of cell cycle-related proteins such as G1 cyclins whereas Ubc7 is mainly involved in ER-associated protein degradation (8)
.
E3 is the enzyme that recognizes target proteins and transfers activated ubiquitin to them. Two types of E3s have been identified so far. The HECT domain E3 was initially discovered when studying the oncogenic human papillomaviruses (HPV) (9)
. E6 protein encoded by HPV genome bound to a cellular protein named E6-AP (E6-associated protein) that functioned as E3 to catalyze the ubiquitination and degradation of tumor suppressor p53 in the host cells. The carboxyl terminal of E6-AP is required for its E3 activity and contains the Cys that participates in the transfer of ubiquitin to target proteins. Subsequent studies found that other E3s had a region of 350 amino acid residues homologous to the carboxyl-terminal of E6-AP, and the name HECT (homologous to E6-AP carboxyl terminus) domain E3 was coined (10)
. It has been estimated there are probably >30 HECT domain E3s in mammalian cells. The amino terminal of many HECT domain E3s have substrate binding region such as the WW domain, characterized by a pair of tryptophan (W) residues and conserved prolines, which recognizes phosphorylated proline-rich regions in the target proteins (11)
. Besides regulating p53, E6-AP ubiquitinates some other proteins, and its mutation leads to the severe neurological disease Angelman syndrome (12)
. Another extensively studied HECT domain E3 Nedd4 regulates the level of epithelial Na+ channel (ENaC) through ubiquitination. Mutation of Nedd4 leads to Liddle’s syndrome (hypertension and hypokalemic alkalosis) due to increased expression and activity of ENaC on the cell surface (13)
.
Ring finger consists of eight conserved Cys and His residues forming a cross-brace structure to chelate two Zinc ions. Numerous studies during the last 3 years demonstrated that some ring finger-containing proteins are E3s that catalyze autoubiquitination and/or the ubiquitination of their substrates. For example, oncogenic protein Mdm2 promotes the ubiquitination and degradation of p53, a major mechanism regulating intracellular level of p53 (14
, 15)
. The carboxyl-terminal ring finger of Mdm2 is required for ubiquitination of p53 in the presence of E1 and E2, whereas the amino-terminal of Mdm2 is the major p53 binding site. Ring finger-containing protein c-Cbl ubiquitinates some growth factor receptors, leading to their down-regulation (16)
. Mutational analysis and crystal structure revealed that ring finger is responsible for binding with E2 (17)
and therefore brings E2 and substrate together. Although recombinant ring finger fusion proteins can promote their autoubiquitination in vitro, it appears that ring finger does not directly participate in the transfer of ubiquitin from E2 to substrates, and it is not known yet whether the ring finger activates E2s. Small ring finger proteins Rbx1 and Apc11 are essential components of multri-subunit E3s SCF (Skp1/Cullin/F-box protein), APC (anaphase-promoting complex), and VHL-CBC (von Hippel-Lindau/Cul2/elongin B/elongin C) (16)
. The cullin protein in these E3s forms a scaffold that associates with the ring finger protein at one end of the molecule and the substrate-recognizing protein at the other (18)
. The ring finger-containing proteins bind with E2 and function similarly to the ring fingers in the single polypeptide E3s, such as Mdm2 and c-Cbl. These complex E3s can bind many different substrate-recognizing subunits (such as different F-box proteins in SCF E3s) and therefore ubiquitinate a variety of target proteins. There are an estimated 200 ring finger proteins in mammalian cells, the majority of which have not been characterized. Based on the structure of ring finger and its interaction with E2s, it is conceivable that we could learn to predict whether a particular ring finger confers E3 activity. Two alternatives of the ring finger domain, the U-box and PHD domains, can also bring E3 activity to proteins (19
20
21)
. It is likely that both U-box and PHD domains use mechanisms similar to that of ring finger to facilitate ubiquitination, as the structure of the PHD domain is similar to that of the ring finger; the U-box shares significant sequence homology with ring finger and, like ring finger, can directly bind to E2 (22
23
24)
.
Two types of ubiquitin-related proteins have been identified in cells. The ubiquitin-like molecules (UBLs) are small proteins structurally similar to ubiquitin; like ubiquitin, they can be conjugated to various target proteins. Modification with UBLs does not target proteins for degradation; rather, it affects their functions in many different ways. For example, sumoylation, modifying proteins with the small ubiquitin-related modifier (SUMO), affects protein and protein interaction, translocation, activities of transcription factors, and may save the target proteins from ubiquitination and degradation (25)
. Sumoylation uses enzymes similar to those participating in ubiquitination to activate and ligate SUMO to target proteins, suggesting these two processes are evolutionally closely related. There are growing lists of proteins that contain ubiquitin domain (termed UDPs) and not known to be conjugated to other proteins. It remains to be determined whether the ubiquitin domain plays a unique role in the function of various UDPs (26
, 27)
.
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UBIQUITINATION IS ASSOCIATED WITH APOPTOSIS |
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TOPABSTRACTTHE UBIQUITINATION SYSTEMUBIQUITINATION IS ASSOCIATED...THE RING-CONTAINING IAPS -...CONCLUSIONSREFERENCES |
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. In these dying cells, there were increased ubiquitinating enzymes activities as well as increased proteasome components and their proteolytic activities. Despite many negative results, a correlation between apoptosis and increased ubiquitin or ubiquitinated proteins was further shown in several other model systems, such as muscles from dystrophin-deficient mice, thyroxin- or serum deprivation-induced cell death, and apoptosis triggered by DNA-damaging agents (29)
. Antisense oligonucleotides of ubiquitin can inhibit protein ubiquitination and apoptosis induced by 
-radiation in lymphocytes (30)
, indicating protein ubiquitination is essential for lymphocyte apoptosis. Consistent with these results, proteasome inhibitors effectively blocked DNA-damaging agents and glucocorticoid-induced thymocyte death (31)
and nerve growth factor withdrawal-induced sympathetic neuron death (32)
, suggesting that proteasome degradation is required for mitochondria-dependent apoptosis. Subsequent studies indicated that glucocorticoids increased proteasomal proteolytic activity at the premitochondria step of thymocyte apoptosis (33)
. It is not clear what the target molecules of the increased ubiquitination and proteasome activity are or how the ubiquitination and proteasome system interacts with the apoptosis machinery.
The tremendous progress in understanding the ubiquitination process over the last couple of years made it possible to identify ubiquitination substrates and study the role of ubiquitination in regulating the levels and functions of many apoptosis-related proteins. The tumor suppressor p53 plays a critical role in maintaining the integrity of genome and preventing tumor development. When cells are subjected to DNA-damaging agents, oncogene activation, hypoxia, and lack of growth factors, the intracellular level of p53 increases rapidly to induce growth arrest and/or apoptosis (34)
. It has been demonstrated that the level of p53 is mainly regulated at the post-transcriptional stage through ubiquitination-dependent degradation (14
, 15)
. The ring-containing protein Mdm2 is the E3 that ubiquitinates p53 and keeps it at a low level in unstimulated cells. Ubiquitination of p53 by Mdm2 also facilitates the export of p53 from nucleus to cytoplasm, where it could not activate transcription of target genes (35)
. Upon stimulation of cells with chemotherapeutic agents or radiation, phosphorylation of p53 prevents its interaction with Mdm2 and leads to inhibition of ubiquitination and degradation. Under certain circumstances, such as oncogene activation, the expression of an Mdm2 binding protein, ARF, is increased, which directly inhibits Mdm2’s E3 activity and therefore increases intracellular p53 level (36)
. Deubiquitination enzyme herpes virus-associated ubiquitin-specific protease (HAUSP) can specifically remove ubiquitin from p53 and stabilize it, even in the presence of Mdm2 (7)
. Overexpression of HAUSP suppressed colony formation in a p53-dependent manner, presumably through p53-induced growth arrest and/or apoptosis. It is not known yet whether or how HAUSP is regulated by p53-inducing stimuli such as DNA damage and oncogene activation. On the other hand, increased p53 up-regulates Mdm2 gene transcription, forming a negative feedback to prevent the unwanted high level of p53 in the cells. The importance of regulating p53 level by Mdm2 was illustrated by the observation that the early embryonic lethality of Mdm2-deficient mice was completely rescued by the simultaneous deletion of p53 (35)
. Surprisingly, deletion of Mdm2 homologue MdmX also led to embryonic death, which can be rescued by loss of p53 (37
38
39)
. Although MdmX has a ring finger and can bind to p53, it does not possess E3 activity toward p53. Recent studies indicated that MdmX formed a heterodimer with Mdm2 that led to its stabilization (40)
. This is likely because Mdm2 promotes its own ubiquitination and degradation in the absence of MdmX. However, overexpressed MdmX might compete with Mdm2 in binding with p53 and therefore inhibit Mdm2-mediated p53 ubiquitination and degradation, as earlier studies reported. Like mutation of p53 itself, abnormalities in these regulating mechanisms such as enhanced p53 degradation due to expression of E6 after infection of oncogenic HPV, loss of ARF and amplification of Mdm2 or MdmX gene have been demonstrated to play critical roles in the development of certain tumors (41
, 42)
. It is conceivable that dysfunction of p53 pathway may contribute to the development of most, if not all, human cancers.
Bcl-2 family proteins are critical regulators of apoptosis (43)
. The anti-apoptotic members of the family such as Bcl-2, Bcl-xL, and Bcl-w all contain the conserved Bcl-2 homology (BH) regions BH1, BH2, and BH3, which form a hydrophobic groove that binds to BH3 domain of other family members (44)
. The Bcl-2 family has two types of proapoptotic members: the Bax and Bak subfamily and the BH3-only proapoptotic members such as Bid, Bad, Bim/Bod, and PUMA (45)
. Studies with gene-targeted cells indicated that the presence of Bax or Bak is required for many forms of apoptosis (46
, 47)
, and each type of cell needs at least one of the anti-apoptotic Bcl-2 family member s to survive (43
, 48)
. The BH3-only proteins are activated in response to various apoptotic stimuli and are able to induce apoptosis through interacting directly with Bax and Bak (48)
or binding to the hydrophobic groove of anti-apoptotic members such as Bcl-2 or Bcl-xL and removing the inhibition of these anti-apoptotic molecules on Bax and Bak. Besides being controlled through transcription, phosphorylation, and proteolytic cleavage, it is becoming evident that Bcl-2 family members are regulated by ubiquitination and proteasome degradation systems. Bcl-2 was ubiquitinated and degraded upon stimulation of human umbilical vein endothelial cells with TNF-
(49)
. Expression of Bcl-2 that had all four lysine residues mutated conferred significantly more resistance to apoptosis than that of wild-type Bcl-2, suggesting down-regulation of Bcl-2 is required for TNF- to induce apoptosis in the cells. It appeared that the increased ubiquitination resulted from dephosphorylation of Bcl-2 induced by TNF- (50)
. MAP kinase (ERK1/2) can phosphorylate Bcl-2 in vitro and in transfected cells, and its inhibition by a specific inhibitor or TNF- correlated with Bcl-2 dephosphorylation and degradation. Mutated Bcl-2 with threonine/serine of all three potential MAP kinase sites changed into aspartic acid residues (phospho-mimetic mutation) was resistant to TNF--induced degradation, whereas changes of PKC and cAMP-dependent protein kinase sites did not affect the stability of Bcl-2. MAP kinase-specific phosphatase-3 is likely to be the enzyme responsible for removal of phosphorus from Bcl-2. Proapoptotic Bc-2 family members Bax and Bik are degraded by the ubiquitination and proteasome systems (51
, 52)
. Their accumulation in the cells might be at least partially responsible for the apoptosis induced by proteasome inhibitors. Decreased intracellular level of Bax was accompanied by increased Bax-degrading activity in aggressive human prostate adenocarcinomas (51)
, indicating degradation of proapoptotic Bax might be the survival mechanism of some tumor cells. Bid is a BH3 region-only Bcl-2 family member located primarily in the cytoplasm. After activation of caspase 8, the carboxyl terminal of Bid is cleavaged to generate the truncated Bid (tBid), which then translocates to mitochondria and leads to the release of apoptosis-promoting molecules. Like Bax and Bik, tBid is degraded through ubiquitination and the proteasome system (53)
. Expression of the mutated tBid that could not be ubiquitinated enhanced cytochrome c release and cell death. These studies demonstrated that the ubiquitination and proteasome systems could change the ratio of Bcl-2 family members and therefore alter the susceptibility of cells to various apoptotic stimuli. It is hoped that we will identify in the not-so-distant future the enzymes responsible for ubiquitination of Bcl-2 family members, which could provide valuable targets for modulating apoptosis of normal and tumor cells.
Many molecules of the extrinsic apoptosis pathway are closely related to the ubiquitination and proteasome system. For example, the intracellular domains of Fas and type I TNF receptor bind with SUMO-1 and Ubc9 (54
, 55)
, suggesting that they are targets of SUMO modification or may function as a scaffold to promote sumoylation. The death receptor-associated protein Daxx can also bind with SUMO-1 and Ubc9 (56)
and was conjugated with SUMO-1 (57)
. Since the SUMO-1 and Ubc9 binding site is overlapped with the Fas binding region of Daxx, it is conceivable that SUMO association or sumoylation may affect signal transduction of death receptor. Since we now know that sumoylation modulates many proteins and cellular processes, the mechanisms and physiological significance of the earlier transfection experiments indicating that overexpression of SUMO-1 altered the susceptibility to apoptosis remain to be determined. Another Fas-associated protein, FAF1, contains an ubiquitin-like domain required for the apoptosis induced by its overexpression (27)
. cFLIP contains two death effector domains and acts as a dominant negative inhibitor of death receptor-mediated activation of caspase 8 (58)
. Lack of this protein made cells become highly sensitive to FasL or TNF (59)
. The resistance of certain melanoma cells to the death receptor ligand TRAIL has been attributed to the expression of cFLIP (60)
. It was shown recently that agents that bind to the peroxisome proliferation-activated receptor sensitize tumor, but not untransformed, cells to TRAIL (61)
. The increased sensitivity was closely correlated with their ability to reduce the level of c-FLIP and could be overcome by enforced expression of c-FLIP. Further studies indicated that the down-regulation of c-FLIP was due to its ubiquitination and subsequent proteasomal degradation. However, it has not been addressed yet how these compounds selectively induced the ubiquitination and degradation of c-FLIP and which E3 is responsible for its ubiquitination. Since other chemotherapeutic agents synergize with TRAIL in killing tumor cells (62)
, it would be interesting to explore whether they also cause ubiquitination and degradation of c-FLIP.
Ubiquitination is a critical process in regulating many signal transduction pathways, transcription factors, and adaptor molecules that have profound effects on the growth and death of cells. For example, suppressors of cytokine signaling (SOCS) are a family of intracellular proteins that contain a center SH2 domain and characteristic carboxyl-terminal SOCS box (63)
. They are often induced by cytokines and in turn suppress the Jak-Stat pathways that mediate the signal of cytokines, forming a negative feedback to limit cytokines’ biological effects. Some of the SOCS family members directly bind to Jaks and inhibit their kinase activities; others occupy the cytokine receptors and block the recruitment of Stats. The SOCS domain can bind to elongins B and C, which are components of complex E3 VHL-CBC (64
, 65)
. It has been shown that SOCS1 can act as an adaptor to facilitate ubiquitination and degradation of proteins bound to its SH2 domain, such as oncogenic fusion protein Tel-JAK2 (66
, 67)
and guanine nucleotide exchange factor Vav (68)
. It is now believed that SOCS-induced ubiquitination and degradation is an important mechanism to down-regulate cytokine’s effects. Ubiquitination also affects signal transduction in a nondegradating manner. For example, TRAF6 is an important signaling mediator for Toll-like and IL-1 receptors as well as the TNF receptor family. The ring finger-dependent E3 activity of TRAF6, which catalyzes the formation of K-63 polyubiquitin chain, is required for activating TAK1, which in turn activates I
B kinase (IKK) and kinase for JNK (69
, 70)
. Activated IKK phosphorylates IB and leads to its ubiquitination and degradation, which enable the activation of NFB, a critical apoptosis-regulating transcription factor (71)
.
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THE RING-CONTAINING IAPS – E3S AS APOPTOSIS INHIBITORS AND BEYOND |
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TOPABSTRACTTHE UBIQUITINATION SYSTEMUBIQUITINATION IS ASSOCIATED...THE RING-CONTAINING IAPS -...CONCLUSIONSREFERENCES |
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70 amino acid residues (72)
. Since the founding member was discovered in baculovirus almost a decade ago, cellular IAPs have been identified in species from yeast to human (73)
. Among the eight mammalian IAPs, cIAP1, cIAP2, XIAP, and NAIP have three BIR domains, whereas survivin, BRUCE, ILP-2, and ML-IAP only have one (73
74
75)
. Based mainly on transfection experiments, many IAPs have been shown to be bona fide apoptosis inhibitors. XIAP appeared to be the most potent one and blocked cell death through inhibiting caspases 3, 7, and 9. Whereas the BIR3 of XIAP directly bound to the small subunit of caspase 9, it was the linker region between BIR1 and BIR2, which contacted with caspase 3 or 7, and prevented their active sites from binding with substrates (76)
. Perhaps to avoid the inhibition of extrinsic and intrinsic death pathways at the same time, testis expresses ILP-2 (that contains one BIR3-like BIR), which inhibits caspase 9 but not caspase 3, and therefore prevents only mitochondria-dependent apoptosis (74)
. Besides acting as endogenous caspases inhibitors, IAP family proteins such as XIAP, cIAP1, and cIAP2 participate in receptor-mediated signal transduction (77)
. A subgroup of IAPs, including survivin in mammalian cells and BIR-1 and BIR-2 in Caenorhabditis elegans, appears to function not mainly as caspases inhibitors, but to regulate mitotic spindle formation and cytokinesis (78
79
80)
. This unique feature has led to the suggestion that these proteins should simply be called BIR domain-containing proteins (81)
.
The majority of the caspase-inhibiting IAPs possess a carboxyl-terminal ring finger motif. In an effort to understand why proteasome inhibitors could effectively block thymocyte apoptosis, we found that XIAP, cIAP1, and cIAP2 were degraded in a proteasome-dependent manner when the thymocytes were stimulated with apoptotic stimuli such as glucocorticoids or etoposide (82)
. It was found that these IAPs have ring finger-dependent E3 activities that catalyze autoubiquitination in vitro and in cells. Overexpression cIAP1 caused its autoubiquitination and degradation. This may explain why transfection of cIAP1 or cIAP2 failed to protect cells from Bax, TNF-, or Sindbis virus-induced apoptosis whereas expression of ring domain-deleted cIAP1 or cIAP2 did (83)
. In contrast to wild-type XIAP, mutated XIAP without E3 activity was not down-regulated by apoptotic stimuli in T cell hybridomas and provided better protection against apoptosis (82)
, indicating that autoubiquitination and degradation of XIAP are required for these cells to undergo apoptosis. The involvement of IAP-associated E3 activity in inhibiting apoptosis is further supported by the finding that cIAP2 can promote mono-ubiquitination of caspase 3 and 7 (84)
and that XIAP catalyzes ubiquitination and degradation of caspase 3 (85)
. However, mice deficient in XIAP developed normally and cells from XIAP-/- mice are not more sensitive to apoptotic stimuli (86)
, suggesting there might be compensation mechanisms during development.
The importance of eliminating IAPs during apoptosis is clearly illustrated by studies using Drosophila, which has two ring finger-containing IAPs: DIAP1 and DIAP2. Pioneer researches found that induction of apoptosis in Drosophila requires Reaper, Hid, and Grim; their overexpression resulted in excess cell death that could be suppressed by expression of DIAP1 (87
, 88)
. Further studies demonstrated that DAP1 is essential for the survival of Drosophila embryonic cells. Reaper, Him, and Grim all have the so-called RHG motif at their amino termini, which could interact with DIAP1. They induce apoptosis through direct interaction with DIAP1 and suppressing its caspase-inhibiting activity (89
, 90)
. Recently, a series of studies using different in vivo and in vitro systems demonstrated that Reaper, Hid, and Grim not only bound with DIAP1, but also down-regulated DIAP1 (91)
. Intriguingly, in some systems, Reaper and Grim but not Hid induced the ubiquitination and degradation of DIAP1 (92
93
94)
; in other systems, it was Hid but not Reaper and Grim that caused DIAP1 ubiquitination and degradation in a ring-dependent manner (95)
. In either situation, DIAP1 degradation is required for apoptosis to occur. Reaper and Grim also caused general suppression of protein translation, which contributed to the reduction of short-lived DIAP1 (94
, 95)
. Genetic modifier screen found that certain mutations of DIAP ring finger enhanced Reaper-induced cell death but inhibited Hid-induced apoptosis (96)
, suggesting that additional factors are involved in their interactions with DIAP1. At least two E2s—Morgue and UBCD1—can bind DIAP1 and promote its autoubiquitination; the reduction of Morgue or UBCD1 suppresses Reaper and Grim-induced DIAP1 down-regulation and apoptosis (92
, 93
, 97)
. Morgue has an F-box, suggesting it might be a substrate-recognizing subunit of a SCF E3. If so, it would be interesting to know whether this putative E3 participates in the ubiquitination and degradation of DIAP1. Finally, like mammalian IAPs, DIAP1 binds with Drosophila caspase Dronc and induces its ubiquitination in a ring-dependent manner (96)
. This interaction is likely important as DIAP1 binding protein Jafrac2 competes with Dronc for the binding of DIAP1 and promotes cell death (98)
.
SMAC (second mitochondria-derived activator of caspase, also known as Diablo) is synthesized as a 239 amino acid precursor protein (99
, 100)
. The first 55 amino acids of the molecule are required for its import into mitochondria, where the sequence is cleaved. Upon apoptotic stimuli such as DNA damage and serum deprivation, matured SMAC is released from the mitochondria and removes the inhibition of XIAP on caspase 9 (101
102
103)
. Apparently this is because the amino terminal of SMAC has a motif similar to that of the small subunit of caspase 9, which is responsible for binding of caspase 9 to the groove on the surface of XIAP BIR3 domain (104)
. This four amino acid motif is similar to the RHG motif of Drosophila proteins Reaper, Hid, and Grim, raising the possibility that SMAC may be able to promote the ubiquitination and degradation of XIAP. Though this remains to be proved or disproved, it has been shown that XIAP is able to promote the ubiquitination and degradation of SMAC (105)
. Although XIAP-deficient mice developed normally and cells from these mice are not more sensitive to apoptotic stimuli (86)
, three different laboratories have demonstrated recently that the SMAC and XIAP interaction does play an important role in apoptosis. It was found that TRAIL (TNF-related apoptosis-inducing ligand) could not induce apoptosis effectively in Bax-deficient cells, apparently due to formation of p20/p12 caspase 3 but not the fully active p17/p12 dimer. This incompletely processed p20/p12 caspase 3 was associated with XIAP due to the lack of release of SMAC from mitochondria in the absence of Bax (106)
. When SMAC was expressed in the cytoplasm of these Bax -/- cells, TRAIL could induce the generation of p17/p12 caspase 3 as well as apoptosis, indicating that interaction of SMAC with XIAP is required for full activation of caspase and a cell death program. Consistent with these results, Fas engagement could not induce full processing of caspase 3 in Jurkat cells that overexpress Bcl-2 or Bcl-xL (107)
, which prevented the release of SMAC from mitochondria. In this report, the ability of SMAC to remove the inhibition of XIAP on the full activation of caspase 3 was directly demonstrated through reconstitution of all the components in vitro. Similar results were observed in hepatocytes from Bid-deficient mice (108)
, since Bid-mediated activation of the mitochondria death pathway is required for anti-Fas antibody to induce apoptosis in hepatocytes. Serine protease Omi/HtrA2 also contains the four amino acid motif that interacts with XIAP and blocks its ability to inhibit caspase (109
110
111
112)
. Like SMAC, Omi/HtrA2 is released from mitochondria upon apoptotic stimuli. Enforced expression of Omi/HtrA2-sensitized cells for apoptosis, and decreased Omi/HtrA2 made cells more resistant to death stimuli. The physiological role of Omi/HtrA2 and its relationship with SMAC remain to be determined.
The E3 activity of IAPs may also regulate apoptosis indirectly. The type I TNF receptor (TNF-RI) has an intracellular death domain and is able to activate caspase 8 to induce apoptosis. Through adaptor molecules TRADD and TRAF2, it can activate Jun kinase and induce NFB, which are anti-apoptotic under many circumstances. The importance of TRAF2 in protecting cells from apoptosis is clearly illustrated by the observation that cells from TRAF2-deficient mice are significantly more sensitive to TNF--induced apoptosis (113)
. Type II TNF receptor (TNF-RII) does not have the death domain and associates with the complex of TRAF1/TRAF2/cIAP1/cIAP2 (114)
. Paradoxically, although engagement of TNF-RII leads to activation of NFB, it enhances TNF-RI induced apoptosis in both normal and transformed cells (115
, 116)
. It has been shown that in the presence of TNF-RII and TNF-RI, TNF- caused ubiquitination and proteasomal degradation of TRAF2, which was accompanied by quicker reduction of Jun kinase activity than that in cells expressing only TNF-RI (117)
. Further studies demonstrated that cIAP1 ubiquitinated TRAF2 in vitro and in vivo in a ring finger-dependent manner. Overexpression of cIAP1 that does not possess E3 activity suppressed TNF--induced down-regulation of TRAF2 and cell death, indicating that cIAP1-mediated ubiquitination and degradation of TRAF2 are responsible for the enhanced apoptosis. Therefore, cIAP1 acts as a proapoptotic molecule rather than an inhibitor of apoptosis under this scenario. It has been shown that cIAP1 can be cleaved by caspases to generate a carboxyl-terminal fragment that includes the ring domain. In transfection experiments, this fragment suppressed the anti-apoptotic activity of the cIAP1 BIR domain (83)
. Although the physiological significance of this observation remains to be established, it shows again that cIAP1 can have proapoptotic effect during apoptotic cell death, presumably due to its E3 activity.
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CONCLUSIONS |
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TOPABSTRACTTHE UBIQUITINATION SYSTEMUBIQUITINATION IS ASSOCIATED...THE RING-CONTAINING IAPS -...CONCLUSIONSREFERENCES |
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). These findings not only advanced our understanding of apoptosis regulation, but provided a unique opportunity for therapeutic intervention. For example, targeting the ring finger of Mdm2 could increase the intracellular level of p53, which in turn induces apoptosis in transformed cells. Due to the specificity of the ubiquitination reaction, especially the interaction between E3 and substrate and the critical role of apoptosis in various pathological conditions, it is conceivable that targeting the ubiquitination process could become an effective therapeutics in the not-so-distant future.
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ACKNOWLEDGMENTS |
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Received for publication October 14, 2002. Accepted for publication January 9, 2003.
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TOPABSTRACTTHE UBIQUITINATION SYSTEMUBIQUITINATION IS ASSOCIATED...THE RING-CONTAINING IAPS -...CONCLUSIONSREFERENCES |
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B activity. Annu. Rev. Immunol. 18,621-663[CrossRef][Medline]
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 S. Sekiguchi, J. Kwon, E. Yoshida, H. Hamasaki, S. Ichinose, M. Hideshima, M. Kuraoka, A. Takahashi, Y. Ishii, S. Kyuwa, et al. Localization of Ubiquitin C-Terminal Hydrolase L1 in Mouse Ova and Its Function in the Plasma Membrane to Block Polyspermy Am. J. Pathol., November 1, 2006; 169(5): 1722 - 1729. [Abstract] [Full Text] [PDF]
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 D. Versari, J. Herrmann, M. Gossl, D. Mannheim, K. Sattler, F. B. Meyer, L. O. Lerman, and A. Lerman Dysregulation of the Ubiquitin-Proteasome System in Human Carotid Atherosclerosis Arterioscler. Thromb. Vasc. Biol., September 1, 2006; 26(9): 2132 - 2139. [Abstract] [Full Text] [PDF]
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 M. Noguchi, K. Kabayama, S. Uemura, B.-w. Kang, M. Saito, Y. Igarashi, and J.-i. Inokuchi Endogenously produced ganglioside GM3 endows etoposide and doxorubicin resistance by up-regulating Bcl-2 expression in 3LL Lewis lung carcinoma cells Glycobiology, July 1, 2006; 16(7): 641 - 650. [Abstract] [Full Text] [PDF]
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 S. R. Powell The ubiquitin-proteasome system in cardiac physiology and pathology Am J Physiol Heart Circ Physiol, July 1, 2006; 291(1): H1 - H19. [Abstract] [Full Text] [PDF]
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 E. Ruiz, A. Gordillo-Moscoso, E. Padilla, S. Redondo, E. Rodriguez, F. Reguillo, A. M. Briones, C. van Breemen, E. Okon, and T. Tejerina Human vascular smooth muscle cells from diabetic patients are resistant to induced apoptosis due to high bcl-2 expression. Diabetes, May 1, 2006; 55(5): 1243 - 1251. [Abstract] [Full Text] [PDF]
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 F. He, B. J. Fenner, A. K. Godwin, and J. Kwang White Spot Syndrome Virus Open Reading Frame 222 Encodes a Viral E3 Ligase and Mediates Degradation of a Host Tumor Suppressor via Ubiquitination. J. Virol., April 1, 2006; 80(8): 3884 - 3892. [Abstract] [Full Text] [PDF]
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 S. Yamamoto, Y. Tomita, T. Uruno, Y. Hoshida, Y. Qiu, N. Iizuka, I. Nakamichi, A. Miyauchi, and K. Aozasa Increased Expression of Valosin-Containing Protein (p97) Is Correlated With Disease Recurrence in Follicular Thyroid Cancer Ann. Surg. Oncol., November 1, 2005; 12(11): 925 - 934. [Abstract] [Full Text] [PDF]
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 C. Gulmann, V. Espina, E. Petricoin III, D. L. Longo, M. Santi, T. Knutsen, M. Raffeld, E. S. Jaffe, L. A. Liotta, and A. L. Feldman Proteomic Analysis of Apoptotic Pathways Reveals Prognostic Factors in Follicular Lymphoma Clin. Cancer Res., August 15, 2005; 11(16): 5847 - 5855. [Abstract] [Full Text] [PDF]
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T. Minami, M. Adachi, R. Kawamura, Y. Zhang, Y. Shinomura, and K. Imai Sulindac Enhances the Proteasome Inhibitor Bortezomib-Mediated Oxidative Stress and Anticancer Activity Clin. Cancer Res., July 15, 2005; 11(14): 5248 - 5256. [Abstract] [Full Text] [PDF]
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R. Lotan, A. Rotem, H. Gonen, J. P. M. Finberg, S. Kemeny, H. Steller, A. Ciechanover, and S. Larisch Regulation of the Proapoptotic ARTS Protein by Ubiquitin-mediated Degradation J. Biol. Chem., July 8, 2005; 280(27): 25802 - 25810. [Abstract] [Full Text] [PDF]
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 J. Kwon, K. Mochida, Y.-L. Wang, S. Sekiguchi, T. Sankai, S. Aoki, A. Ogura, Y. Yoshikawa, and K. Wada Ubiquitin C-Terminal Hydrolase L-1 Is Essential for the Early Apoptotic Wave of Germinal Cells and for Sperm Quality Control During Spermatogenesis Biol Reprod, July 1, 2005; 73(1): 29 - 35. [Abstract] [Full Text] [PDF]
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 E. J. Song, S.-H. Yim, E. Kim, N.-S. Kim, and K.-J. Lee Human Fas-Associated Factor 1, Interacting with Ubiquitinated Proteins and Valosin-Containing Protein, Is Involved in the Ubiquitin-Proteasome Pathway Mol. Cell. Biol., March 15, 2005; 25(6): 2511 - 2524. [Abstract] [Full Text] [PDF]
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A. Gaikwad, A. Poblenz, V. Haridas, C. Zhang, M. Duvic, and J. Gutterman Triterpenoid Electrophiles (Avicins) Suppress Heat Shock Protein-70 and X-Linked Inhibitor of Apoptosis Proteins in Malignant Cells by Activation of Ubiquitin Machinery: Implications for Proapoptotic Activity Clin. Cancer Res., March 1, 2005; 11(5): 1953 - 1962. [Abstract] [Full Text] [PDF]
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 N. Machuy, B. Thiede, K. Rajalingam, C. Dimmler, O. Thieck, T. F. Meyer, and T. Rudel A Global Approach Combining Proteome Analysis and Phenotypic Screening with RNA Interference Yields Novel Apoptosis Regulators Mol. Cell. Proteomics, January 1, 2005; 4(1): 44 - 55. [Abstract] [Full Text] [PDF]
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C. Gorbea, G. M. Goellner, K. Teter, R. K. Holmes, and M. Rechsteiner Characterization of Mammalian Ecm29, a 26 S Proteasome-associated Protein That Localizes to the Nucleus and Membrane Vesicles J. Biol. Chem., December 24, 2004; 279(52): 54849 - 54861. [Abstract] [Full Text] [PDF]
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 I. V. Davydov, D. Woods, Y. J. Safiran, P. Oberoi, H. O. Fearnhead, S. Fang, J. P. Jensen, A. M. Weissman, J. H. Kenten, and K. H. Vousden Assay for Ubiquitin Ligase Activity: High-Throughput Screen for Inhibitors of HDM2 J Biomol Screen, December 1, 2004; 9(8): 695 - 703. [Abstract] [PDF]
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W. Martinet, M. De Bie, D. M. Schrijvers, G. R.Y. De Meyer, A. G. Herman, and M. M. Kockx 7-Ketocholesterol Induces Protein Ubiquitination, Myelin Figure Formation, and Light Chain 3 Processing in Vascular Smooth Muscle Cells Arterioscler. Thromb. Vasc. Biol., December 1, 2004; 24(12): 2296 - 2301. [Abstract] [Full Text] [PDF]
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E Tian, K. G. T. Hagen, L. Shum, H. C. Hang, Y. Imbert, W. W. Young Jr, C. R. Bertozzi, and L. A. Tabak An Inhibitor of O-Glycosylation Induces Apoptosis in NIH3T3 Cells and Developing Mouse Embryonic Mandibular Tissues J. Biol. Chem., November 26, 2004; 279(48): 50382 - 50390. [Abstract] [Full Text] [PDF]
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S. Yamamoto, Y. Tomita, Y. Hoshida, N. Iizuka, M. Monden, S. Yamamoto, K. Iuchi, and K. Aozasa Expression Level of Valosin-Containing Protein (p97) Is Correlated With Progression and Prognosis of Non-Small-Cell Lung Carcinoma Ann. Surg. Oncol., July 1, 2004; 11(7): 697 - 704. [Abstract] [Full Text] [PDF]
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M. Adachi, Y. Zhang, X. Zhao, T. Minami, R. Kawamura, Y. Hinoda, and K. Imai Synergistic Effect of Histone Deacetylase Inhibitors FK228 and m-Carboxycinnamic Acid Bis-Hydroxamide with Proteasome Inhibitors PSI and PS-341 against Gastrointestinal Adenocarcinoma Cells Clin. Cancer Res., June 1, 2004; 10(11): 3853 - 3862. [Abstract] [Full Text] [PDF]
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 R. Sasson, E. Rimon, A. Dantes, T. Cohen, V. Shinder, A. Land-Bracha, and A. Amsterdam Gonadotrophin-induced gene regulation in human granulosa cells obtained from IVF patients. Modulation of steroidogenic genes, cytoskeletal genes and genes coding for apoptotic signalling and protein kinases Mol. Hum. Reprod., May 1, 2004; 10(5): 299 - 311. [Abstract] [Full Text] [PDF]
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Y. Tsujimoto, Y. Tomita, Y. Hoshida, T. Kono, T. Oka, S. Yamamoto, N. Nonomura, A. Okuyama, and K. Aozasa Elevated Expression of Valosin-Containing Protein (p97) Is Associated with Poor Prognosis of Prostate Cancer Clin. Cancer Res., May 1, 2004; 10(9): 3007 - 3012. [Abstract] [Full Text] [PDF]
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Q.-H. Yang and C. Du Smac/DIABLO Selectively Reduces the Levels of c-IAP1 and c-IAP2 but Not That of XIAP and Livin in HeLa Cells J. Biol. Chem., April 23, 2004; 279(17): 16963 - 16970. [Abstract] [Full Text] [PDF]
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 D. Perrelet, F. E. Perrin, P. Liston, R. G. Korneluk, A. MacKenzie, M. Ferrer-Alcon, and A. C. Kato Motoneuron Resistance to Apoptotic Cell Death In Vivo Correlates with the Ratio between X-Linked Inhibitor of Apoptosis Proteins (XIAPs) and Its Inhibitor, XIAP-Associated Factor 1 J. Neurosci., April 14, 2004; 24(15): 3777 - 3785. [Abstract] [Full Text] [PDF]
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T. Iwakuma and G. Lozano MDM2, An Introduction Mol. Cancer Res., December 1, 2003; 1(14): 993 - 1000. [Abstract] [Full Text] [PDF]
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