共查询到20条相似文献,搜索用时 31 毫秒
1.
Freyberg MA Kaiser D Graf R Vischer P Friedl P 《Biochemical and biophysical research communications》2000,271(3):584-588
Recently, it was reported that the offset of hemodynamic forces induces an unusual pattern of apoptosis in vascular endothelium (1). Although the apoptotic trigger covers all cells and is maintained for a longer time period, only few cells become apoptotic. So, in contrast to common apoptosis inducers, the lack of hemodynamic forces initiates only a low basal level of apoptosis, however steadily increases with time, this way preventing the complete vessel destruction upon an only transient offset of blood flow. The molecular means by which the mechanical stimulus and apoptosis are smoothly coupled have now been identified as an autocrine loop of thrombospondin-1 (TSP-1) and the alpha(v)beta(3) integrin/integrin-associated protein (IAP) complex as its receptor. Vascular EC (EC) secrete TSP-1 only in postconfluent static monolayers and not under flow. This also holds true for the IAP whereas the alpha(v)beta(3) integrin is present under static conditions, as well as under flow, assigning the IAP an essential and new switch function in the receptor complex. 相似文献
2.
A common mechanism for the mechanosensitive regulation of apoptosis in different cell types and for different mechanical stimuli 总被引:1,自引:0,他引:1
Graf R Apenberg S Freyberg M Friedl P 《Apoptosis : an international journal on programmed cell death》2003,8(5):531-538
In all kinds of tissue cells are influenced by mechanical forces. In vivo fibroblasts are exposed to mechanical tension and endothelial cells are subjected directly to hemodynamic flow. It has been shown that disturbance of the mechanical stimulus leads to apoptosis by induction of an autocrine loop with thrombospondin-1 as ligand and an integrin/integrin associated protein (CD47) complex as receptor. In the present study the nature of the mechanical stimulus has been exchanged for these two cell types. If fibroblasts are subjected to laminar flow apoptosis decreases about 20-fold whereas turbulence leads to an significant increase compared with the static conditions. If endothelial cells grown on thin silicone membranes are exposed to permanent and pulsatile uniaxial strain, the cells are completely devoid of apoptosis. The thrombospondin-1 secretion as well as the expression of CD47 occurs exclusively under mechanical relaxation respectively turbulence. So different types of cells seem to share a common sense deciding whether a mechanical stimulus induces or suppresses apoptosis and use a common molecular machinery for the regulation of the process. 相似文献
3.
Farhan Basit Robin Humphreys Simone Fulda 《The Journal of biological chemistry》2012,287(46):38767-38777
Searching for new strategies to trigger apoptosis in rhabdomyosarcoma (RMS), we investigated the effect of two novel classes of apoptosis-targeting agents, i.e. monoclonal antibodies against TNF-related apoptosis-inducing ligand (TRAIL) receptor 1 (mapatumumab) and TRAIL receptor 2 (lexatumumab) and small-molecule inhibitors of inhibitor of apoptosis (IAP) proteins. Here, we report that IAP inhibitors synergized with lexatumumab, but not with mapatumumab, to reduce cell viability and to induce apoptosis in several RMS cell lines in a highly synergistic manner (combination index <0.1). Cotreatment-induced apoptosis was accompanied by enhanced activation of caspase-8, -9, and -3; loss of mitochondrial membrane potential; and caspase-dependent apoptosis. In addition, IAP inhibitor and lexatumumab cooperated to stimulate the assembly of a cytosolic complex containing RIP1, FADD, and caspase-8. Importantly, knockdown of RIP1 by RNA interference prevented the formation of the RIP1·FADD·caspase-8 complex and inhibited subsequent activation of caspase-8, -9, and -3; loss of mitochondrial membrane potential; and apoptosis upon treatment with IAP inhibitor and lexatumumab. In addition, RIP1 silencing rescued clonogenic survival of cells treated with the combination of lexatumumab and IAP inhibitor, thus underscoring the critical role of RIP1 in cotreatment-induced apoptosis. By comparison, the TNFα-blocking antibody Enbrel had no effect on IAP inhibitor/lexatumumab-induced apoptosis, indicating that an autocrine TNFα loop is dispensable. By demonstrating that IAP inhibitors and lexatumumab synergistically trigger apoptosis in a RIP1-dependent but TNFα-independent manner in RMS cells, our findings substantially advance our understanding of IAP inhibitor-mediated regulation of TRAIL-induced cell death. 相似文献
4.
Harpel JG Schultz-Cherry S Murphy-Ullrich JE Rifkin DB 《Biochemical and biophysical research communications》2001,284(1):11-14
We have found that the enhanced activation of latent TGF-beta by human breast carcinoma cell lines either treated with tamoxifen or deprived of estrogen is dependent upon thrombospondin-1 (TSP-1) since activation was blocked by anti-TSP-1 antibodies or by a TSP antagonist peptide. However, TGF-beta formation upon tamoxifen exposure to estrogen withdrawal is associated with decreased levels of soluble TSP-1. A concomitant increase in the expression of the TSP-1 receptors alphavbeta3 and integrin-associated protein (IAP) occurs under these conditions, and antibodies to TSP-1 or to these receptors inhibit increased TGF-beta formation. Therefore, increased cell surface associated TSP-1 enhances latent TGF-beta activation. 相似文献
5.
The migration of vascular smooth muscle cells from the media to intima and their subsequent proliferation are critical causes of arterial wall thickening. In atherosclerotic lesions increases in the thickness of the vascular wall and the impairment of oxygen diffusion capacity result in the development of hypoxic lesions. We investigated the effect of hypoxia on the migration of human coronary artery smooth muscle cells (CASMCs) via HIF-1alpha-dependent expression of thrombospondin-1 (TSP-1). When the cells were cultured under hypoxic conditions, mRNA and protein levels of TSP-1, and mRNA levels of integrin beta(3) were increased with the increase in HIF-1alpha protein. DNA synthesis and migration of the cells were stimulated under the conditions, and a neutralizing anti-TSP-1 antibody apparently suppressed the migration, but not DNA synthesis. The migration was also inhibited by RGD peptide that binds to integrin beta(3). Furthermore, the migration was completely suppressed in HIF-1alpha-knockdown cells exposed to hypoxia, while it was significantly enhanced in HIF-1alpha-overexpressing cells. These results suggest that the hypoxia induces the migration of CASMCs, and that the migration is elicited by TSP-1 of which induction is fully dependent on the stabilization of HIF-1alpha, in autocrine regulation. Thus we suggest that HIF-1alpha plays an important role in the pathogenesis of atherosclerosis. 相似文献
6.
Vince JE Wong WW Khan N Feltham R Chau D Ahmed AU Benetatos CA Chunduru SK Condon SM McKinlay M Brink R Leverkus M Tergaonkar V Schneider P Callus BA Koentgen F Vaux DL Silke J 《Cell》2007,131(4):682-693
XIAP prevents apoptosis by binding to and inhibiting caspases, and this inhibition can be relieved by IAP antagonists, such as Smac/DIABLO. IAP antagonist compounds (IACs) have therefore been designed to inhibit XIAP to kill tumor cells. Because XIAP inhibits postmitochondrial caspases, caspase 8 inhibitors should not block killing by IACs. Instead, we show that apoptosis caused by an IAC is blocked by the caspase 8 inhibitor crmA and that IAP antagonists activate NF-kappaB signaling via inhibtion of cIAP1. In sensitive tumor lines, IAP antagonist induced NF-kappaB-stimulated production of TNFalpha that killed cells in an autocrine fashion. Inhibition of NF-kappaB reduced TNFalpha production, and blocking NF-kappaB activation or TNFalpha allowed tumor cells to survive IAC-induced apoptosis. Cells treated with an IAC, or those in which cIAP1 was deleted, became sensitive to apoptosis induced by exogenous TNFalpha, suggesting novel uses of these compounds in treating cancer. 相似文献
7.
Vascular endothelial cells express a functional fas-receptor due to lack of hemodynamic forces 总被引:1,自引:0,他引:1
Freyberg MA Kaiser D Graf R Friedl P 《Apoptosis : an international journal on programmed cell death》2001,6(5):339-343
The fas system is present in atherosclerotic lesions. However, its role in the initiation and progression is still unclear. Here we show that in endothelial cells (EC) the expression of the fas receptor is regulated by flow conditions. The EC of the vascular system are regularly exposed to a range of hemodynamic forces with great impact on cellular structures and functions. Recently it was reported that in endothelial cells the lack of hemodynamic forces as well as irregular flow conditions trigger apoptosis by induction of a mechanosensitive autocrine loop of thrombospondin-1 and the V3 integrin/integrin-associated protein complex. Here we show that EC cultivated under regular laminar flow conditions are devoid of the fas-receptor whereas cultivation under static conditions as well as under turbulence leads to its expression. Stimulation of the fas-receptor by its ligand increases the amount of apoptotic cells by twofold; the increase can be prevented by blocking the fas-receptor. The availability of the expressed fas receptor for stimulation by its ligand hints at a role as a tool for progression of atherosclerosis. 相似文献
8.
Vince JE Chau D Callus B Wong WW Hawkins CJ Schneider P McKinlay M Benetatos CA Condon SM Chunduru SK Yeoh G Brink R Vaux DL Silke J 《The Journal of cell biology》2008,182(1):171-184
Synthetic inhibitor of apoptosis (IAP) antagonists induce degradation of IAP proteins such as cellular IAP1 (cIAP1), activate nuclear factor kappaB (NF-kappaB) signaling, and sensitize cells to tumor necrosis factor alpha (TNFalpha). The physiological relevance of these discoveries to cIAP1 function remains undetermined. We show that upon ligand binding, the TNF superfamily receptor FN14 recruits a cIAP1-Tnf receptor-associated factor 2 (TRAF2) complex. Unlike IAP antagonists that cause rapid proteasomal degradation of cIAP1, signaling by FN14 promotes the lysosomal degradation of cIAP1-TRAF2 in a cIAP1-dependent manner. TNF-like weak inducer of apoptosis (TWEAK)/FN14 signaling nevertheless promotes the same noncanonical NF-kappaB signaling elicited by IAP antagonists and, in sensitive cells, the same autocrine TNFalpha-induced death occurs. TWEAK-induced loss of the cIAP1-TRAF2 complex sensitizes immortalized and minimally passaged tumor cells to TNFalpha-induced death, whereas primary cells remain resistant. Conversely, cIAP1-TRAF2 complex overexpression limits FN14 signaling and protects tumor cells from TWEAK-induced TNFalpha sensitization. Lysosomal degradation of cIAP1-TRAF2 by TWEAK/FN14 therefore critically alters the balance of life/death signals emanating from TNF-R1 in immortalized cells. 相似文献
9.
The cell biology of thrombospondin-1. 总被引:41,自引:0,他引:41
Thrombospondin-1 (TSP-1) is a matricellular protein that regulates cellular phenotype during tissue genesis and repair. It acts as a molecular facilitator by bringing together cytokines, growth factors, matrix components, membrane receptors and extracellular proteases. TSP-1 binds to a wide variety of integrin and non-integrin cell surface receptors. The binding sites for these receptors on TSP-1 are dispersed throughout the molecule, with most domains binding multiple receptors. In some cases, TSP-1 binds to multiple receptors concurrently, and recent data indicate that there is cross-talk between the receptor systems. Thus, TSP-1 may function to direct the clustering of receptors to specialized domains for adhesion and signal transduction. 相似文献
10.
Ishibashi Y Yoshimura K Nishikawa A Claus S Laudanna C Relman DA 《Cellular microbiology》2002,4(12):825-833
Bordetella pertussis, the causative agent of whooping cough, adheres to human monocytes by means of filamentous haemagglutinin (FHA), a bacterial surface protein that is recognized by complement receptor type 3 (CR3, alphaMbeta2 integrin). Previous work has shown that an FHA Arg-Gly-Asp (RGD, residues 1097-1099) site interacts with a complex composed of leucocyte response integrin (LRI, alphavbeta3 integrin) and integrin-associated protein (IAP, CD47) on human monocytes, resulting in enhancement of CR3-mediated bacterial binding. However, the pathway that mediates alphavbeta3-alphaMbeta2 integrin signalling remains to be characterized. Here we describe the involvement of phosphatidylinositol 3-kinase (PI3-K) in this pathway. Wortmannin and LY294002, inhibitors of PI3-K, reduced alphavbeta3/IAP-upregulated, CR3-associated bacterial binding to human monocytes. B. pertussis infection of human monocytes resulted in a marked recruitment of cellular PI3-K to the sites of B. pertussis contact. In contrast, cells infected with an isogenic strain carrying a G1098A mutation at the FHA RGD site did not show any recruitment of PI3-K. We found that ligation of FHA by alphavbeta3/IAP induced RGD-dependent tyrosine phosphorylation of a 60 kDa protein, which associated with IAP and PI3-K in human monocytes. These results suggest that PI3-K and a tyrosine phosphorylated 60 kDa protein may be involved in this biologically important integrin signalling pathway. 相似文献
11.
Rege TA Stewart J Dranka B Benveniste EN Silverstein RL Gladson CL 《Journal of cellular physiology》2009,218(1):94-103
Thrombospondin-1 (TSP-1) treatment of dermal microvascular endothelial cells (MvEC) has been shown to upregulate Fas ligand (FasL) and to induce apoptosis by a mechanism that requires caspase-8 activity. We have examined the potential anti-angiogenic effects of TSP-1 on primary human brain MvEC. The addition of TSP-1 to primary human brain MvEC cultured as monolayers on type 1 collagen, induced cell death and apoptosis (evidenced by caspase-3 cleavage) in a dose- (5-30 nM) and time-dependent (maximal at 17 h) manner. TSP-1 treatment for 17 h induced caspase-3 cleavage that required caspase-8 activity and the tumor necrosis factor receptor 1 (TNF-R1). We did not find a requirement for Fas, or the tumor necrosis-related apoptosis-inducing ligand receptors (TRAIL-R) 1 and 2. We confirmed the findings using caspase inhibitors, blocking antibodies and small interfering RNA (siRNA). Further analysis indicated that the TSP-1 induction of caspase-3 cleavage of primary human brain MvEC adherent to collagen required the synthesis of new message and protein, and that TSP-1 induced the expression of TNFalpha mRNA and protein. Consistent with these findings, when the primary human brain MvEC were propagated on collagen gels mAb anti-TNF-R1 reversed the inhibitory effect, in part, of TSP-1 on tube formation and branching. These data identify a novel mechanism whereby TSP-1 can inhibit angiogenesis-through induction of apoptosis in a process mediated by TNF-R1. 相似文献
12.
We study effects of oscillatory convective flow in extracellular space on the velocity of chemical signal propagation having a form of a front wave above a cellular layer. We found that the time-averaged propagation velocity under oscillatory flow for a particular Péclet number amplitude is slower than the velocity under steady laminar flow regime for the same value of the Péclet number, but significantly faster than under no-flow conditions. We derive asymptotic values of the propagation velocity and asymptotic characteristics of the corresponding concentration fronts in high- and low-frequency regimes and show that the reason for the observed velocity increase under the oscillatory flow stems from a nonlinear dependence of the propagation velocity on the Péclet number, particularly from the convex character of the dependence. Our findings suggest that the specific responses of cellular cultures to different flow conditions in the extracellular space (for example, expression of atherosclerosis protective genes under steady laminar flow but not under oscillatory flow) is a consequence of a nonlinear coupling between the extracellular transport and complex intracellular reaction cascades forming a positive feedback loop of the autocrine signaling. This mechanism can operate independently of, or in conjunction with, a direct stress-sensing due to mechanotransduction. 相似文献
13.
Analysis of the tumor-associated antigen TSP-180. Identity with alpha 6-beta 4 in the integrin superfamily 总被引:26,自引:0,他引:26
S J Kennel L J Foote R Falcioni A Sonnenberg C D Stringer C Crouse M E Hemler 《The Journal of biological chemistry》1989,264(26):15515-15521
The tumor-associated antigen complex, TSP-180, was previously defined in carcinoma cell lines and found to be expressed in higher amounts in tumor than in normal tissue. Here, the mouse TSP-180 complex is shown to consist of three related proteins (bands 1, 2, and 3) associated with a distinct protein (band 5) that is probably derived from a precursor protein (band 4). All of these proteins are cell surface glycoproteins, and the largest protein (band 1) can be readily labeled with 32PO4. The mouse TSP-180 complex described here strongly resembles the recently described human integrin alpha 6-beta 4 complex. This homology was confirmed using two distinct rat anti-alpha 6 monoclonal antibodies, each of which recognized both human alpha 6-beta 4 and mouse TSP-180 complexes. Furthermore, the TSP-180 band 5 protein (mouse alpha 6) had an N-terminal sequence identical to that of human alpha 6. Finally, two different monoclonal antibodies are described, 346-11A and 439-9B, which directly recognize the multiple forms of mouse and human beta 4 proteins, respectively. 相似文献
14.
We have investigated the effects of expression of the viral proteins CrmA, P35 and IAP, and the three mammalian IAP homologues (MIHA, MIHB and MIHC), on the regulation of apoptosis induced by either the overexpression of caspases (ICE, CPP32 and Nedd2), by serum-deprivation, or by gamma-irradiation in NIH3T3 fibroblasts. As previously shown, CrmA strongly inhibited ICE-induced apoptosis but was ineffective against Nedd2- or CPP32-mediated apoptosis. P35, IAP and MIHA protected cells from apoptosis induced by the three caspases to varying extents but MIHB and MIHC were largely ineffective. NIH3T3 cells expressing P35 and MIHA, but not IAP, CrmA, MIHB and MIHC, showed enhanced cell survival under serum-deprived conditions. In addition, P35, CrmA and MIHA could provide substantial protection against death induced by gamma-irradiation. These results suggest the presence of multiple apoptotic pathways with differential sensitivity to various naturally occurring apoptosis inhibitors. 相似文献
15.
Expression of the 50-kDa integrin-associated protein on myeloid cells and erythrocytes. 总被引:10,自引:0,他引:10
C Rosales H D Gresham E J Brown 《Journal of immunology (Baltimore, Md. : 1950)》1992,149(8):2759-2764
Integrin-associated protein (IAP) is a 50-kDa intrinsic membrane protein that is involved in signal transduction during neutrophil activation by a variety of Arg-Gly-Asp-containing ligands. However, IAP does not itself directly bind these ligands, which are instead recognized by the leukocyte response integrin (LRI). In fact, IAP is more widely expressed than the LRI and is even on erythrocytes, which express no known integrins. This suggests that IAP may have additional functions besides signal transduction in association with the LRI. In this work we have quantitated IAP expression on several myeloid cells and cell lines, as well as erythrocytes, using a mAb which recognizes this protein. These data show that there are about 10,000 IgG-binding sites on each erythrocyte and 20 times that number on the myeloid cell lines U937 and HL60. Normal PMN and monocytes express about 25,000 IgG-binding sites. There are twice as many Fab'-binding sites on each cell, suggesting that each IgG binds via both Ag-combining sites. Binding data using several mAb to IAP suggest that IAP undergoes a temperature-dependent conformational change. Unlike some integrin receptors, IAP is not stored in a regulated secretory compartment in polymorphonuclear leukocytes (PMN). In addition, there is no evidence for internalization or shedding of IAP upon PMN activation. These data show that IAP is expressed at significant levels on myeloid cells and erythrocytes and that its expression is unaffected by the state of PMN activation. 相似文献
16.
Thrombospondin-1 (TSP-1) is known to be subject to three unusual carbohydrate modifications: C-mannosylation, O-fucosylation, and O-glucosylation. We now describe a fourth: O-β-N-acetylglucosaminylation. Previously, O-β-N-acetylglucosamine (O-β-GlcNAc) was found on a threonine in the loop between the fifth and sixth cysteines of the 20(th) epidermal growth factor (EGF)-like module of Drosophila Notch. A BLAST search based on the Drosophila Notch loop sequence identified a number of human EGF-like modules that contain a similar sequence, including EGF-like module 1 of TSP-1 and its homolog, TSP-2. TSP-1, which has a potentially modifiable serine in the loop, reacted in immuno-blots with the CTD110.6 anti-O-GlcNAc antibody. Antibody reactivity was diminished by treatment of TSP-1 with β-N-acetylhexosaminidase. TSP-2, which lacks a potentially modifiable serine/threonine in the loop, did not react with CTD110.6. Analysis of tandem modules of TSP-1 localized reactivity of CTD110.6 to EGF-like module 1. Top-down mass spectrometric analysis of EGF-like module 1 demonstrated the expected modifications with glucose (+162 Da) and xylose (+132 Da) separately from modification with N-acetyl hexosamine (+203 Da). Mass spectrometric sequence analysis localized the +203-Da modification to Ser580 in the sequence (575)CPPGYSGNGIQC(586). These results demonstrate that O-β-N-acetylglucosaminylation can occur on secreted extracellular matrix proteins as well as on cell surface proteins. 相似文献
17.
We study effects of oscillatory convective flow in extracellular space on the velocity of chemical signal propagation having a form of a front wave above a cellular layer. We found that the time-averaged propagation velocity under oscillatory flow for a particular Péclet number amplitude is slower than the velocity under steady laminar flow regime for the same value of the Péclet number, but significantly faster than under no-flow conditions. We derive asymptotic values of the propagation velocity and asymptotic characteristics of the corresponding concentration fronts in high- and low-frequency regimes and show that the reason for the observed velocity increase under the oscillatory flow stems from a nonlinear dependence of the propagation velocity on the Péclet number, particularly from the convex character of the dependence. Our findings suggest that the specific responses of cellular cultures to different flow conditions in the extracellular space (for example, expression of atherosclerosis protective genes under steady laminar flow but not under oscillatory flow) is a consequence of a nonlinear coupling between the extracellular transport and complex intracellular reaction cascades forming a positive feedback loop of the autocrine signaling. This mechanism can operate independently of, or in conjunction with, a direct stress-sensing due to mechanotransduction. 相似文献
18.
Greenaway J Lawler J Moorehead R Bornstein P Lamarre J Petrik J 《Journal of cellular physiology》2007,210(3):807-818
VEGF is a potent pro-angiogenic factor whose effects are opposed by a host of anti-angiogenic proteins, including thrombospondin-1 (TSP-1). We have previously shown that VEGF has important extravascular roles in the ovary and that VEGF and TSP-1 are inversely expressed throughout the ovarian cycle. To date, however, a causal interaction between TSP-1 and VEGF has not been identified. Here, we show that TSP-1 has a direct inhibitory effect on VEGF by binding the growth factor and internalizing it via LRP-1. Mice lacking TSP-1 are subfertile and exhibited ovarian hypervascularization and altered ovarian morphology. Treatment of ovarian cells with TSP-1 decreased VEGF levels and rendered the cells more susceptible to TNFalpha-induced apoptosis. Knockdown of TSP-1, through RNA interference, resulted in overexpression of VEGF and reduced cytokine-induced apoptosis. In conclusion, we demonstrate a direct inhibitory effect of TSP-1 on VEGF in the ovary. TSP-1's regulation of VEGF appears to be an important mediator of ovarian angiogenesis and follicle development. 相似文献
19.
Taba Y Miyagi M Miwa Y Inoue H Takahashi-Yanaga F Morimoto S Sasaguri T 《American journal of physiology. Heart and circulatory physiology》2003,285(1):H38-H46
Laminar shear stress strongly inhibits vascular endothelial cell apoptosis by unknown mechanisms. We reported that shear stress stimulates endothelial cells to produce 15-deoxy-Delta12,14-prostaglandin J2 (15d-PGJ2) by elevating the expression level of lipocalin-type prostaglandin D synthase. To investigate the role of 15d-PGJ2 produced in the vascular wall, we examined the effect of 15d-PGJ2 on endothelial cell apoptosis. We induced apoptosis in human umbilical vein endothelial cells (HUVECs) by growth factor deprivation. 15d-PGJ2 strongly inhibited DNA ladder formation, nuclear fragmentation, and caspase-3-like activity in HUVECs. To elucidate the mechanism by which 15d-PGJ2 inhibits endothelial cell apoptosis, we examined expression of the inhibitor of apoptosis proteins (IAP) cellular-IAP1 (c-IAP1), c-IAP2, x-linked IAP, and survivin in HUVECs. In parallel with the inhibition of apoptosis, 15d-PGJ2 elevated the expression level of c-IAP1 protein in a dose- and time-dependent manner without changing the mRNA level. Laminar shear stress also induced c-IAP1 expression. Chase experiments with the use of cycloheximide revealed that 15d-PGJ2 and shear stress both inhibited the proteolytic degradation of c-IAP1 protein. These results suggested that 15d-PGJ2 inhibits endothelial cell apoptosis through, at least in part, c-IAP1 protein stabilization. This mechanism might be involved in the antiapoptotic effect of laminar shear stress. 相似文献
20.
Blankenship JW Varfolomeev E Goncharov T Fedorova AV Kirkpatrick DS Izrael-Tomasevic A Phu L Arnott D Aghajan M Zobel K Bazan JF Fairbrother WJ Deshayes K Vucic D 《The Biochemical journal》2009,417(1):149-160
A family of anti-apoptotic regulators known as IAP (inhibitor of apoptosis) proteins interact with multiple cellular partners and inhibit apoptosis induced by a variety of stimuli. c-IAP (cellular IAP) 1 and 2 are recruited to TNFR1 (tumour necrosis factor receptor 1)-associated signalling complexes, where they mediate receptor-induced NF-kappaB (nuclear factor kappaB) activation. Additionally, through their E3 ubiquitin ligase activities, c-IAP1 and c-IAP2 promote proteasomal degradation of NIK (NF-kappaB-inducing kinase) and regulate the non-canonical NF-kappaB pathway. In the present paper, we describe a novel ubiquitin-binding domain of IAPs. The UBA (ubiquitin-associated) domain of IAPs is located between the BIR (baculovirus IAP repeat) domains and the CARD (caspase activation and recruitment domain) or the RING (really interesting new gene) domain of c-IAP1 and c-IAP2 or XIAP (X-linked IAP) respectively. The c-IAP1 UBA domain binds mono-ubiquitin and Lys(48)- and Lys(63)-linked polyubiquitin chains with low-micromolar affinities as determined by surface plasmon resonance or isothermal titration calorimetry. NMR analysis of the c-IAP1 UBA domain-ubiquitin interaction reveals that this UBA domain binds the classical hydrophobic patch surrounding Ile(44) of ubiquitin. Mutations of critical amino acid residues in the highly conserved MGF (Met-Gly-Phe) binding loop of the UBA domain completely abrogate ubiquitin binding. These mutations in the UBA domain do not overtly affect the ubiquitin ligase activity of c-IAP1 or the participation of c-IAP1 and c-IAP2 in the TNFR1 signalling complex. Treatment of cells with IAP antagonists leads to proteasomal degradation of c-IAP1 and c-IAP2. Deletion or mutation of the UBA domain decreases this degradation, probably by diminishing the interaction of the c-IAPs with the proteasome. These results suggest that ubiquitin binding may be an important mechanism for rapid turnover of auto-ubiquitinated c-IAP1 and c-IAP2. 相似文献