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George Pyrowolakis,,
and
Stefan Jentsch*
Department of Molecular Cell Biology, Max Planck Institute of Biochemistry, Martinsried, Germany
Received 19 May 2004/ Returned for modification 26 June 2004/ Accepted 4 August 2004
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ABSTRACT |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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INTRODUCTION |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-amino groups) of substrate proteins, thereby forming branched isopeptide-linked protein complexes. Substrates can be modified either by a single ubiquitin moiety (monoubiquitylation) or by a multiubiquitin chain, in which several ubiquitin moieties are linked together by isopeptide bonds (multiubiquitylation). Proteins modified by multiubiquitin chains are usually targeted to the 26 proteasome for degradation (14). In contrast, monoubiquitylation and modification by noncanonical (lysine 63-linked) multiubiquitin chains mediate nonproteolytic functions, including protein sorting, gene silencing, signal transduction, and DNA repair (19). Ubiquitylation is ATP dependent and requires the sequential activities of usually three enzymes, termed ubiquitin-activating enzyme (E1), ubiquitin-conjugating enzyme (E2), and ubiquitin ligase (E3) (14). The E1 enzyme hydrolyses ATP and forms a thioester-linked complex between its active-site cysteine residue and the C-terminal glycine residue of ubiquitin. Ubiquitin is then transferred onto a cysteine residue of one of several E2 enzymes. These proteins are usually small proteins within the 14- to 35-kDa size range and are characterized by a so-called ubiquitin-conjugating (UBC) domain, which carries the active-site cysteine residue. Subsequently, an E3 enzyme binds both the E2 and the substrate and mediates the transfer of ubiquitin to a lysine residue of the substrate. The E3 enzymes also exist as a large protein family, and these enzymes in particular contribute to the substrate specificity of the system (14). Some substrates additionally require E4 enzymes for multiubiquitylation (16).
Previously, we cloned an unusual member of the UBC protein family from mouse by a homology-based strategy with sequences for yeast UBC domains as probes (12). This enzyme, called BRUCE (for baculovirus inhibitor of apoptosis repeat [BIR] repeat-containing ubiquitin-conjugating enzyme), is a giant 528-kDa protein which bears a UBC domain close to its C terminus. In addition, BRUCE carries a single BIR domain close to its amino (N)-terminal end. The BIR domain, a zinc-binding fold, is a hallmark of inhibitor of apoptosis proteins (IAPs), which bind and thereby inhibit caspases through this domain (9, 25, 27). IAPs belong to a larger group of so-called BIR domain-containing proteins, which also includes the mammalian protein survivin and the BIR1 proteins from the yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe (27). In contrast to the IAPs, these proteins do not seem to block apoptosis but play a role in cytokinesis (27). BRUCE is highly conserved, and homologs have been identified in humans and Drosophila (1, 3, 28). The protein is absent in Caenorhabditis elegans, however. BRUCE is a peripheral membrane protein and localizes specifically to the trans-Golgi network (TGN) and vesicles (12). In neuronal cells, BRUCE is also found at vesicular structures in axons and dendrites (12).
BRUCE was recently shown to possess antiapoptotic activity (2, 3, 20). Similar to other IAPs, BRUCE binds and thereby inhibits caspases via its BIR domain (2). BRUCE inhibits most strongly the effector caspase 3 and binds to its processed, active form but not to the proform of the caspase. In HeLa and 293T cells, overexpression of BRUCE blocks apoptosis induced by various stimuli, including UV light and TRAIL (2). Moreover, antisense oligonucleotides against BRUCE sensitize cells against apoptosis induced by DNA-damaging agents (3), and downregulation by RNA interference or ubiquitin- or proteasome-dependent degradation of BRUCE promotes apoptosis (20). BRUCE also has activity to monoubiquitylate the proapoptotic, mitochondrion-derived protein Smac, which depends on the integrity of both the BIR and the UBC domains (2). Notably, this reaction is catalyzed by E1 and BRUCE alone and does not require additional E2 or E3 enzymes. Thus, BRUCE combines in a single large polypeptide E2 and E3 activities and functions as a chimeric E2/E3 ubiquitin ligase (2).
In this work we investigated the function of BRUCE and the consequences of its loss in vivo by generation and characterization of BRUCE knockout mice. We show that these mice exhibit perinatal lethality and growth deficiencies, which are linked to a defect in proper placental development. Surprisingly, no evidence for an elevated apoptosis rate was detectable in embryonic and extraembryonic tissues and knockout fibroblasts, which may be due to possible overlapping IAP activities. This suggests that the observed defects of BRUCE-deficient mice may not be exclusively linked to BRUCE's IAP activity but possibly may be linked to its ubiquitin ligase activity at endomembranes.
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MATERIALS AND METHODS |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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phage library with cDNA fragments according to standard procedures. Three partially overlapping clones spanning exon 2 to 6 (5, 7, and 9) were subcloned into pBluescript SK(+) and analyzed by restriction mapping, Southern blotting, sequencing, and PCR. A 5-kb XbaI-EcoRI fragment from clone 7 spanning parts of intron 3 and exon 4 was subcloned into pBluescript SK(+) and elongated by a 1-kb PCR fragment generated with primer jp71 (GCT GGA GAA GTA TTG CTT TGC) and mutagenic primer ko2XbaI*.la.asense (GGT CTA GAG GTA AGA CAA TCT GTC C) to introduce an XbaI site and a stop codon into exon 4. This construct was further elongated by a 2.2-kb PCR fragment generated with primers ko2XbaI*.la.sense (ACC TCT AGA CCA GTG CAC GTC CAG) and ko2SalItd.la.asense (GAC GTC GAC GAC AGC CTT ATG AGA CTA CCT GG) to introduce the XbaI site and the stop codon into exon 4 and a SalI site into intron 4. These three regions together represent the 5' arm of homology. A neoR-tk cassette containing the neomycin resistance gene under control of the herpes simplex virus promoter, the 1.1-kb 3' homology arm derived from exon 6 and intronic sequences by PCR with primers ko2XbaItd.sa.asense (CCG TCT AGA CTG TAC TGT AAA TAC TTC CAT GC) and ko2XbaItd.sa.sense (CCG TCT AGA TTA TGA TGT TAG TCC TAT GTC CTA C), and the negative selectable marker HSV-TK (herpes simplex virus thymidine kinase) was then placed into the SalI site of the pBluescript SK(+) construct. Thus, in the resulting targeting vector, exon 4 contains an introduced stop codon, whereas exon 5 is replaced with a neoR cassette.
Gene targeting in ES cells.
Fifty micrograms of linearized (NotI) plasmid DNA was electroporated into 107 R1 embryonic stem (ES) cells suspended in phosphate-buffered saline (PBS). The ES cells were then cultured on dishes treated with gelatin. Following double selection with G418 (300 µg/ml) (Sigma) and ganciclovir (2 µM) (Sigma), 64 ES cell clones were picked, expanded on mitomycin-treated embryonic fibroblasts, and analyzed by Southern blotting of genomic PvuII-digested DNA. An external probe comprising a 0.4-kb BamHI fragment of a PCR-amplified 7 sequence (using primers jp101 [GAG CCT GTG ATT TAT AGT GTA C] and jp136 [CTT CAG CTG GAT AGT GAG TTC C]) was used to identify the 2.9-kb fragment of the wild-type (WT) allele and the 2.3-kb fragment of the mutated allele. In addition, a genomic PCR was performed to screen for homologous recombination events. One primer was chosen to be outside the targeting vector, and the other was chosen to be within the neoR cassette (outer.33, TCT TCT AAA TTA AGG CTT CAT AGT AGA TCT TGG; jp82, GCC TCT CCA CCC AAG CGG CCG GAG AAC CTG CGT GC). For higher sensitivity, a second round of PCR was performed to amplify a 1.5-kb genomic fragment (inner.22, GTT GTA CAC TAT AAA CAG G; jp83, GCA ATC CAT CTT GTT CAA TGG C).
Generation of BRUCE-deficient mice. Two different ES cell clones with the targeted BRUCE allele were injected into 3.5-day-old C57BL/6 blastocysts and then reimplanted into 2.5-day pseudopregnant NMRI females. The spermatides of an infertile male chimera (germ line transmission was observed in the coat color of the F1 offspring) were used for in vitro fertilization of C57BL/6 oocytes, and fertilized oocytes were reimplanted into pseudopregnant B6D2F2 females. Male chimeras were then backcrossed to C57BL/6 females, and heterozygous male and female mice were interbred to produce homozygous mutant offspring. The genotypes of the mice were assessed by Southern blotting and by PCR analysis with genomic DNA from tail biopsies of 21-day-old pups with primers wt-0 (GTG TCT CCA CCT AAC CTA TGC) and wt-3.as (GGT GAT AAA ATC CAG CTT GAG C) to detect a 556-bp band for the WT allele and with additional primers neo4 (GGC TAT TCG GCT ATG ACT GGG C) and neo5 (GGG TAG CCA ACG CTA TGT CCT G) to amplify a 624-bp band for the mutated allele.
Weight study and histological examination. After cervical dislocation of pregnant mice, embryos and placentas at different developmental stages (embryonic day 9.5 [E9.5] to E19.5) were dissected out of uterus and decidua and cleared of membranes and umbilical cords in PBS. Yolk sac was used for genotyping. After excess buffer was blotted with a paper towel, embryos and placentas were weighed. For histological examination, dissected tissue was fixed in Bouin's or 10% neutral buffered formalin overnight and then dehydrated and embedded in paraffin. Serial sections (4 to 8 µm thick) were stained with hematoxylin and eosin by using routine procedures.
Protein analysis and Western blotting. Immunoblot analysis of BRUCE protein was performed either on E10 whole-embryo lysates obtained by solubilization of the embryo in sodium dodecyl sulfate (SDS) sample buffer or on extraembryonic tissue lysates obtained by homogenization of the tissue in PBS (500 µl per 100 mg of tissue, containing complete [Roche] protease inhibitor cocktail) followed by solubilization in SDS sample buffer. For fibroblast extracts, cells were harvested and counted, and equal numbers of cells were solubilized in SDS sample buffer. Proteins were resolved by SDS-polyacrylamide gel electrophoresis (in a 4 to 20% polyacrylamide gradient) and electroblotted onto polyvinylidene difluoride membrane (Immobilon P; Millipore). Blots were probed with a monoclonal antibody against the N terminus of BRUCE (Transduction Laboratories) and a rabbit polyclonal (affinity-purified) antibody recognizing the C terminus of BRUCE (12). Equal loading of the sample was assessed by probing blots with a goat antiserum against actin (I-19; Santa Cruz). Bands were visualized with peroxidase-conjugated secondary antibodies and the ECL detection kit (Amersham). Other antibodies used for immunoblotting were anti-XIAP (Transduction Laboratories), anti-cIAP1 (PharMingen), anti-cIAP2 (R&D Systems), antisurvivin (A-19; Santa Cruz), and anti-caspase 3 (K-19; Santa Cruz).
BrdU labeling, immunohistochemistry, and TUNEL assay. Pregnant mice from heterozygous matings were injected intraperitoneally at E11.5, E13.5, E15.5, and E17.5 with 5-bromo-2-deoxyuridine (BrdU) (Sigma) at a dose of 50 µg per g of body weight. Pregnant mice were sacrificed 4 h after injection, and the uteri were removed. Embryos and corresponding placentas were shock frozen on dry ice and cut on a cryostat in 10- to 15-µm-thick sections. Detection of the incorporated BrdU was performed with a BrdU in situ detection kit (BD PharMingen) according to the manufacturer's instructions, followed by microscopic examination. For immunohistochemistry, the placenta was dissected on E9.5 to E17.5, fixed in 10% neutral buffered formalin, paraffin embedded, and serially sectioned (8 µm thick). Immunohistochemistry on dewaxed and rehydrated sections was performed with the Vectastain Universal Quick kit in combination with the M.O.M. Basic kit (Vector Laboratories) according to the manufacturer's protocol. Affinity-purified antilaminin antibody (1:25) (Sigma) was used as the primary antibody. Immune complexes were visualized with diaminobenzidine tetrahydrochloride precipitates, and the sections were subsequently counterstained with nuclear fast red. For analysis of programmed cell death, terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling (TUNEL) assays were performed with the ApopTag in situ apoptosis detection kit (Oncor) according to the manufacturer's instructions. To determine apoptotic cell death, in addition to TUNEL staining, caspase 3 activity was detected by using an antibody that specifically binds to the cleaved and thereby activated form of caspase 3 (anti-active caspase 3 pAb; Promega). Immunohistochemistry was performed after permeabilization of the cells with 0.1% Triton X-100 by using the Vectastain Universal Quick kit (Vector Laboratories).
In situ hybridization.
Hybridization of paraffin-embedded sections was performed essentially as described previously (6) 33P-labeled antisense and sense mRNA probes were transcribed in vitro by using T7, T3, or SP6 RNA polymerase (Roche) from plasmids containing cDNA sequences of mPl-1, Tpbp (4311), Flt-1, Mash 2, Gcm1, eHand, Flk-1, and BRUCE. For hybridization, sections were dewaxed and pretreated as described previously (6). Briefly, slides were pretreated with proteinase K (20 µg/ml) for 7 min, postfixed in 4% paraformaldehyde in PBS, and acetylated in acetic acid anhydride diluted 1:400 in 0.1 M triethanolamine (pH 8.0). After washing, slides were dehydrated in ethanol and air dried. Hybridizations were performed in defined hybridization buffer (InnoGenex) supplemented with 
3 x 107 cpm of sense or antisense riboprobe per ml at 57.5°C overnight. Slides were washed by standard protocols, treated with RNase A (20 µg/ml) for 30 min at 37°C, washed again in SSC buffer (2x, 1x, and 0.5x; 1x SSC is 0.15 M NaCl plus 0.015 M sodium citrate), and at least at 65°C in 0.1x SSC containing 0.1 mM dithiothreitol. Slides were finally rinsed, dehydrated, air dried, coated with autoradiographic emulsion (LM-1; Amersham), developed after 2 to 6 weeks, and counterstained with cresyl violet.
Cell culture of primary embryonic fibroblasts.
Primary mouse embryonic fibroblasts (MEFs) (at least four independent lines per genotype) were isolated from day 13.5 embryos. Part of the embryo was used for genotyping. The remaining embryonic tissue was minced by using a pair of scissors, syringe, and needle (19 gauge) and immersed in culture medium (Dulbecco's modified Eagle medium supplemented with 10% fetal calf serum, 2 mM glutamine, 50 µg of penicillin per ml, and 50 µg of streptomycin per ml). Cells were then incubated at 37°C with 95% humidity and 7.5% CO2. A single-cell suspension of MEFs (excluding the embryonic cell clumps) was plated onto a new dish before reaching confluency, and these cells were regarded as passage 1 cells. In the growth curve experiments, passage 2 cells (four lines per genotype) were plated at 1 x 105, 3 x 105, and 9 x 105 cells per 6-cm-diameter culture dish and counted on successive days. In parallel the same MEF lines were immortalized as described previously (26). The number of cells in S phase in asynchronous cultures of fibroblasts was determined by using the BrdU in situ detection kit (BD PharMingen) according to the manufacturer's instructions, followed by microscopic examination of a randomly chosen area. For analysis of programmed cell death in asynchronous cultures of fibroblasts, TUNEL assays were performed with the ApopTag in situ apoptosis detection kit (Oncor) according to the manufacturer's instructions. Apoptosis was induced in primary MEFs by cultivating the cells for 0 to 36 h in growth medium containing different concentrations of various inducers of apoptosis (actinomycin D [Sigma], 0.01 to 2.5 µg/ml; anisomycin [Sigma], 0.05 to 2.5 µM; brefeldin A [Sigma], 0.1 to 20 µg/ml; calcimycin [Calbiochem], 0.05 to 5.0 µM; camptothecin [Calbiochem], 0.1 to 20 µg/ml, UV radiation, 10 to 500 J/m2; etoposide [Sigma], 0.5 to 100 µM; tunicamycin [Sigma], 0.1 to 10 µg/ml; H2O2 [Merck], 10 to 500 µM; tumor necrosis factor alpha [TNF-
] [R&D Systems], 1.0 ng/ml to 1.0 µg/ml [alone or in combination with 1.0 µg of cycloheximide]; cycloheximide [Sigma], 1.0 to 50 µg/ml; and hamster anti-mouse Fas [PharMingen], 1.0 to 100 ng/ml). Measurements of cell death were performed at different time points in a colorimetric assay with crystal violet (0.5% [wt/vol] in 40% methanol in PBS). Cell death was expressed as the optical density at 595 nm.
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RESULTS |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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phage-based libraries. The genomic locus of BRUCE spans 175 kb on chromosome 17 and includes a 14,535-bp open reading frame (12). In order to design a knockout construct, we characterized the genomic organization of BRUCE's 5' region. To this end, we cloned genomic sequences corresponding to the 5' region from a library containing liver DNA of the mouse line 129/SvJ and characterized two nonconsecutive genomic regions in more detail. The first 20-kb region (R1) encompassed exon 1, whereas the second region of 30 kb (R2) contained exon 2 to exon 6 (Fig. 1A). We noticed that the first half of BRUCE's BIR domain is encoded by exon 5. Exon 6 corresponds to the metal-binding fold of the BIR domain; however it does not encode it completely. Region R1, and to a lesser extent also R2, contained multiple copies of B1 and B2 elements of the SINE family and were intensely rich in GC content. Based on this information, we chose a knockout strategy that took away a region encompassing exon 4 to exon 6, thereby eliminating essential portions of the BIR domain. In addition, a stop codon was introduced in the open reading frame of exon 4, which is expected to result in a complete null allele. The targeting construct (Fig. 1A) contained a neomycin resistance gene (neoR) flanked by sequences corresponding to segments containing exon 4 and exon 6, followed by a thymidine kinase cassette (TK).
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BRUCE deficiency results in retarded growth and perinatal lethality. Phenotypically normal heterozygotes were intercrossed to produce BRUCE-deficient mice. We observed Mendelian inheritance up to the time of birth (Table 1). However, during or shortly after birth BRUCE null mice died, and no surviving knockout offspring could be obtained, demonstrating that the absence of BRUCE causes perinatal death. Tissue samples of knockout (BRUCE–/–) embryos verified that no detectable BRUCE protein was expressed (Fig. 1B), thus confirming that the targeting strategy led to a complete BRUCE knockout.
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In situ hybridization analysis with a radiolabled antisense probe revealed a relatively high expression level of BRUCE in the different layers of the WT placenta, and also in the visceral yolk sac, throughout the different developmental ages (Fig. 4). Within the labyrinth and the chorionic plate, BRUCE mRNA is detectable in endothelial cells as well, but predominantly in labyrinthine and chorionic trophoblast cells. Increased levels of BRUCE mRNA were found in the spongiotrophoblast layer, whereas in trophoblast giant cells BRUCE expression was reduced. Notably, BRUCE expression is particularly abundant in those tissues that exhibit defects in the targeted mutants.
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The development of the placental vascular network was further investigated immunohistochemically by using an antibody to laminin, which stains the basal lamina of fetal capillaries in the labyrinth layer. In mature WT placenta the fetal capillaries were elongated, of similar size, and evenly distributed throughout the labyrinth from the chorionic plate to the spongiotrophoblast layer. In contrast, the fetal blood vessels in mutant placenta showed less branching. They were highly variable in thickness and formed a less dense vasculature than in WT placenta (Fig. 6B). In addition, the elimination of the nuclei from red blood cells seemed to be delayed in BRUCE mutant placenta. In fact, in WT placenta, all red blood cells are nucleated before E11.5. However, between E12 and E15 these cells enucleate, and after E16 all red blood cells in the embryonic circulation are nonnucleated (15). In BRUCE mutants about 50% of cells in fetal blood vessels of E15.5 placenta were still nucleated, whereas in the WT almost all red blood cells had already lost their nuclei. Even at stage E17.5, single nucleated red blood cells were visible in the mutant labyrinth (Fig. 5C).
Apoptosis and proliferation. Recently, it was shown that BRUCE inhibits apoptosis, that it possesses IAP activity, and that overexpression of the protein blocks apoptosis (2, 3, 20). We therefore considered the possibility that the observed abnormalities of BRUCE-deficient mice are caused by an increase of apoptosis events. To address this question, the rate of apoptosis in WT and BRUCE mutant placenta, specifically within the spongiotrophoblast and the labyrinth layer, was analyzed. Yet, by using a TUNEL assay and immunohistochemistry to monitor activated caspase 3 levels, no significant differences between BRUCE-deficient and control placentas were found (Fig. 7A and B). Apoptosis could be observed at equally low rates in WT and mutant maternal decidual tissue, yolk sac, chorionic plate, spongiotrophoblasts, and labyrinth layer. Other IAP family members, which might overlap with BRUCE function, are expressed at equal levels in WT and knockout placenta (Fig. 7C). In contrast, we detected significant differences in cell proliferation between WT and BRUCE-deficient cells as monitored by BrdU incorporation. Analysis of E11.5 to E17.5 placenta indicated a changed BrdU index of BRUCE-deficient trophoblasts in comparison to WT littermate controls (Fig. 8A and B). At early stages, the number of proliferating cells was significantly reduced within the mutant labyrinth and almost no proliferating cells were visible within the spongiotrophoblast layer, whereas in the corresponding WT layers high proliferation rates could be observed. The proliferating activity in the labyrinth of knockout placenta was reduced to 60% at E11.5 and to 55% at developmental stage E13.5. At later stages (E15.5 and E17.5), when cells in the mature WT placenta already ceased proliferating, BrdU-positive cells were still detectable in the mutant labyrinth (Fig. 8B). This effect seems to be predominantly restricted to trophoblast cells, since the numbers of proliferating endothelial cells within the chorionic plates of WT and knockout placenta were similar (Fig. 8C). However, intensive trophoblast proliferation and branching are a prerequisite for vascularization of the labyrinth (22), and thus the number of proliferating endothelial and trophoblast cells within the labyrinth layer seems to be reduced to the same extent. From these findings we conclude that the abnormalities observed in BRUCE mutant placenta are perhaps caused by an altered proliferation and/or differentiation pattern of trophoblast cells and are not due to enhanced apoptosis in this tissue.
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70% of the normal culture density of WT cells (Fig. 9A).
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(Fig. 9C) or Fas, brefeldin A, or camptothecin (not shown). Next, we asked whether other IAP family members are able to compensate for the lack of BRUCE, as, for example, reported for the knockout of XIAP (11). To address this question, we investigated the levels of XIAP, c-IAP2, and survivin in WT or BRUCE-deficient MEFs that were either untreated or treated with TNF-. No increase of IAP levels could be detected in mutant cells compared to the WT control (Fig. 9D). Moreover, caspase 3 levels also were similar in WT and knockout fibroblasts after induction of apoptosis. |
DISCUSSION |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The basis for the observed placenta defect of BRUCE–/– mice is currently unclear. BRUCE combines in a single protein two distinct activities (2, 12). It functions as an antiapoptotic IAP due to the presence of an N-terminal BIR domain, and it possesses ubiquitylation activity, which depends on the C-terminal UBC domain. Due to the design of our targeting vector, we generated a complete BRUCE null mutant, and therefore both activities of BRUCE are absent in the mutant. Downregulation of BRUCE in HeLa cells by RNAi treatment promotes apoptosis (20), and loss-of-function mutants of dBruce, the Drosophila homolog, also are prone to apoptosis in certain settings (1, 28). However, we observed no evidence for a higher apoptosis rate in BRUCE-deficient embryonic and extraembryonic tissues. Our data rather suggest that the observed placenta deficiencies are perhaps linked to differential proliferation or differentiation defects of diploid trophoblast cells, which may affect the normal branching of the embryonic labyrinthine vascular system.
BRUCE–/– animals display a slight delay in erythrocyte maturation, characterized by increased numbers of nucleated red blood cells, predominantly in the peripheral circulation. In addition, knockout embryos are visually paler than the littermate controls. But, as shown for Rb or SOCS3-deficient mice (7, 21) erythropoeisis defects might be secondary to placental defects. Oxygen and/or nutrient deprivation, as well as a possibly altered placental secretion of erythropoietic differentiation factors, could increase the number of immature red blood cells.
Growth defects could also be observed in cultures of BRUCE-deficient primary embryonic fibroblasts. However, BRUCE–/– MEFs also are not detectably prone to apoptotic stimuli compared to MEFs from WT mice. A possible explanation for the lack of discernible apoptosis phenotypes in BRUCE-deficient cells is that other IAPs may compensate for a BRUCE deficiency in certain cells. Indeed, previous work has shown that IAPs have overlapping functions. For example, mice deficient in XIAP, the strongest IAP known, do not exhibit higher apoptosis rates, due to an apparent overlap with cIAP1 and cIAP2 (11). In case of BRUCE deficiency, we observed no upregulation of other IAPs such as XIAP or cIAP2 in extraembryonic tissue as well as in isolated fibroblasts. However, this might not be surprising given the fact that BRUCE is about 10-fold less potent than XIAP in blocking apoptosis (2). Alternatively, as shown for Drosophila dBruce (1), murine BRUCE might act as a local inhibitor of specific activated caspases that also function in nonapoptotic pathways.
Another possibility is that the observed defects are caused by the absence of BRUCE's ubiquitylation activity. Currently the only known substrate for BRUCE-dependent ubiquitylation is Smac. However, analogous to other ubiquitin ligases, BRUCE may have additional substrates. Notably, Smac is only monoubiquitylated by BRUCE. Since monoubiquitylation does not usually promote proteasomal degradation, BRUCE's activity could play a nonproteolytical role. BRUCE localizes specifically to the TGN and vesicular structures. Therefore, it will be interesting to see whether BRUCE's IAP and ubiquitylation activity is specifically important for TGN function, for example, TGN maintenance or protein sorting.
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ACKNOWLEDGMENTS |
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We especially thank K.-A. Nave and M. Klugmann for their generous help in introducing us to mouse knockout techniques. We thank F. Zimmermann for excellent transgenic and in vitro fertilization service; J. Putzke, B. Kunkel, N. Borchers, and M. Moser for help with histology techniques; C. Hitz (Flt-1 and Flk-1), D. I. Linzer (Pl-1), J. Rossant (4311), A. Joyner (Mash2), and M. Wegner (Gcm1) for kindly providing cDNA hybridization probes; and T. Bartke, C. Pohl, K.-A. Nave, W. Wurst, T. Floss, and C. Hitz for discussions.
The work was supported by grants from the Deutsche Forschungsgemeinschaft and Fonds der Chemischen Industrie (to S.J.).
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FOOTNOTES |
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K.L. and G.P. contributed equally to this work.
Present address: Biozentrum, University of Basel, Department of Cell Biology, Basel, Switzerland.
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