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1.
Cell death in higher organisms is negatively regulated by Inhibitor of Apoptosis Proteins (IAPs), which contain a ubiquitin ligase motif, but how ubiquitin-mediated protein degradation is regulated during apoptosis is poorly understood. Here, we report that Drosophila melanogaster IAP1 (DIAP1) auto-ubiquitination and degradation is actively regulated by Reaper (Rpr) and UBCD1. We show that Rpr, but not Hid (head involution defective), promotes significant DIAP1 degradation. Rpr-mediated DIAP1 degradation requires an intact DIAP1 RING domain. Among the mutations affecting ubiquitination, we found ubcD1, which suppresses rpr-induced apoptosis. UBCD1 and Rpr specifically bind to DIAP1 and stimulate DIAP1 auto-ubiquitination in vitro. Our results identify a novel function of Rpr in stimulating DIAP1 auto-ubiquitination through UBCD1, thereby promoting its degradation. 相似文献
2.
Upon activation of the Fas apoptotic signaling pathway, Bid, a "BH3 domain-only" pro-apoptotic molecule, is cleaved by caspase-8 into a 6.5-kDa N-terminal and a 15-kDa BH3 domain-containing C-terminal fragment, referred to as t(n)-Bid and t(c)-Bid, respectively. t(c)-Bid is a more potent inducer of apoptosis than full-length Bid, suggesting that the N-terminal region of Bid has an inhibitory effect on its pro-apoptotic activity. Here, we report the identification of a novel BH3-like motif (amino acid residues 35-43) in t(n)-Bid. Although Bid does not homodimerize, t(n)-Bid is able to associate avidly with t(c)-Bid. Site-directed mutagenesis revealed that both the novel BH3-like and BH3 domains are necessary for direct binding between t(n)-Bid and t(c)-Bid. While full-length Bid does not associate with t(n)-Bid, substitution of Leu(35), a critical residue in mediating t(n)-Bid/t(c)-Bid interaction, with Ala in full-length Bid is sufficient to establish Bid/t(n)-Bid interaction. Interestingly, the L35A Bid mutant is as effective as t(c)-Bid in inducing apoptosis and binding Bcl-X(L). We propose that the intramolecular interaction involving the BH3-like and BH3 domains serves to regulate the pro-apoptotic potential of Bid. 相似文献
3.
《Current biology : CB》2023,33(16):3522-3528.e7
4.
Tienan Wang Bo Yan Liang Lou Xi Lin Tao Yu Shuting Wu Qing Lu Wei Liu Zhibin Huang Mingjie Zhang Wenqing Zhang Zilong Wen 《遗传学报》2019,46(6):291-299
Microglia are tissue-resident macrophages residing in the central nervous system(CNS) and play critical roles in removing cellular debris and infectious agents as well as regulating neurogenesis and neuronal activities. Yet, the molecular basis underlying the establishment of microglia pool and the maintenance of their homeostasis in the CNS remain largely undefined. Here we report the identification and characterization of a mutant zebrafish, which harbors a point mutation in the nucleotide-binding oligomerization domain(NOD) like receptor gene nlrc3-like, resulting in the loss of microglia in a temperature sensitive manner. Temperature shift assay reveals that the late onset of nlrc3-like deficiency leads to excessive microglia cell death. Further analysis shows that the excessive microglia death in nlrc3-like deficient mutants is attributed, at least in part, to aberrant activation of canonical inflammasome pathway. Our study indicates that proper regulation of inflammasome cascade is critical for the maintenance of microglia homeostasis. 相似文献
5.
Arimura S Fujimoto M Doniwa Y Kadoya N Nakazono M Sakamoto W Tsutsumi N 《The Plant cell》2008,20(6):1555-1566
Mitochondrial fission is achieved partially by the activity of self-assembling dynamin-related proteins (DRPs) in diverse organisms. Mitochondrial fission in Arabidopsis thaliana is mediated by DRP3A and DRP3B, but the other genes and molecular mechanisms involved have yet to be elucidated. To identify these genes, we screened and analyzed Arabidopsis mutants with longer and fewer mitochondria than those of the wild type. ELM1 was found to be responsible for the phenotype of elongated mitochondria. This phenotype was also observed in drp3a plants. EST and genomic sequences similar to ELM1 were found in seed plants but not in other eukaryotes. ELM1:green fluorescent protein (GFP) was found to surround mitochondria, and ELM1 interacts with both DPR3A and DRP3B. In the elm1 mutant, DRP3A:GFP was observed in the cytosol, whereas in wild-type Arabidopsis, DRP3A:GFP localized to the ends and constricted sites of mitochondria. These results collectively suggest that mitochondrial fission in Arabidopsis is mediated by the plant-specific factor ELM1, which is required for the relocalization of DRP3A (and possibly also DRP3B) from the cytosol to mitochondrial fission sites. 相似文献
6.
Dullard promotes degradation and dephosphorylation of BMP receptors and is required for neural induction 总被引:1,自引:0,他引:1
Bone morphogenetic proteins (BMPs) regulate multiple biological processes, including cellular proliferation, adhesion, differentiation, and early development. In Xenopus development, inhibition of the BMP pathway is essential for neural induction. Here, we report that dullard, a gene involved in neural development, functions as a negative regulator of BMP signaling. We show that Dullard promotes the ubiquitin-mediated proteosomal degradation of BMP receptors (BMPRs). Dullard preferentially complexes with the BMP type II receptor (BMPRII) and partially colocalizes with the caveolin-1-positive compartment, suggesting that Dullard promotes BMPR degradation via the lipid raft-caveolar pathway. Dullard also associates with BMP type I receptors and represses the BMP-dependent phosphorylation of the BMP type I receptor. The phosphatase activity of Dullard is essential for the degradation of BMP receptors and neural induction in Xenopus. Together, these observations suggest that Dullard is an essential inhibitor of BMP receptor activation during Xenopus neuralization. 相似文献
7.
Yeast contains at least 24 snRNAs, many of which are dispensable for viability. We recently demonstrated that a small subset of these RNAs has a functional binding site for the Sm antigen, a hallmark of metazoan snRNAs involved in mRNA processing. Here we show that one of these snRNAs, snR7, is required for growth. To determine the biochemical basis of lethality in cells lacking snR7, we engineered the conditional synthesis of snR7 by fusing the snRNA coding sequences to the yeast GAL1 control region. Cells depleted for the SNR7 gene product by growth on glucose for five generations show marked accumulation of unspliced mRNA precursors from the four intron-containing genes tested. In some cases, intron-exon 2 lariats also accumulate. We have identified a 70 nucleotide domain within snR7 with limited sequence-specific but striking structural homology to the mammalian snRNA U5. We conclude that mRNA splicing in yeast requires the function of a U5-like snRNA. 相似文献
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The Par-3 NTD adopts a PB1-like structure required for Par-3 oligomerization and membrane localization 总被引:1,自引:0,他引:1
The evolutionarily conserved Par-3/Par-6/aPKC complex is essential for the establishment and maintenance of polarity of a wide range of cells. Both Par-3 and Par-6 are PDZ domain containing scaffold proteins capable of binding to polarity regulatory proteins. In addition to three PDZ domains, Par-3 also contains a conserved N-terminal oligomerization domain (NTD) that is essential for proper subapical membrane localization and consequently the functions of Par-3. The molecular basis of NTD-mediated Par-3 membrane localization is poorly understood. Here, we describe the structure of a monomeric form of the Par-3 NTD. Unexpectedly, the domain adopts a PB1-like fold with both type-I and type-II structural features. The Par-3 NTD oligomerizes into helical filaments via front-to-back interactions. We further demonstrate that the NTD-mediated membrane localization of Par-3 in MDCK cells is solely attributed to its oligomerization capacity. The data presented in this study suggest that the Par-3 NTD is likely to facilitate the assembly of higher-order Par-3/Par-6/aPKC complex with increased avidities in targeting the complex to the subapical membrane domain and in binding to other polarity-regulating proteins. 相似文献
10.
Membrane-bound domain of HMG CoA reductase is required for sterol-enhanced degradation of the enzyme 总被引:32,自引:0,他引:32
3-Hydroxy-3-methylglutaryl coenzyme A reductase (HMG CoA reductase) is a single polypeptide chain with two contiguous domains: a soluble domain (548 amino acids) that catalyzes the rate-controlling step in cholesterol synthesis and a membrane-bound domain (339 amino acids) that anchors the protein to the endoplasmic reticulum (ER). HMG CoA reductase is degraded at least 10-fold more rapidly than other ER proteins; degradation is accelerated in the presence of cholesterol. To understand this controlled degradation, we transfected reductase-deficient Chinese hamster ovary (CHO) cells with a plasmid expression vector containing a reductase cDNA that lacks the segment encoding the membrane domain. The plasmid produced a truncated reductase (37 kd smaller than normal) that was enzymatically active with normal kinetics; most of the truncated enzyme was found in the cytosol. The truncated enzyme was degraded one-fifth as fast as the holoenzyme; degradation was no longer accelerated by sterols. We conclude that the membrane-bound domain of reductase plays a crucial role in the rapid and regulated degradation of this ER protein. 相似文献
11.
Xue Xia Sarah Katzenell Erin F. Reinhart Katherine M. Bauer Maria Pellegrini 《Autophagy》2018,14(9):1620-1628
Mitochondria are targeted for degradation by mitophagy, a selective form of autophagy. In Saccharomyces cerevisiae, mitophagy is dependent on the autophagy receptor, Atg32, an outer mitochondrial membrane protein. Once activated, Atg32 recruits the autophagy machinery to mitochondria, facilitating mitochondrial capture in phagophores, the precursors to autophagosomes. However, the mechanism of Atg32 activation remains poorly understood. To investigate this crucial step in mitophagy regulation, we examined the structure of Atg32. We have identified a structured domain in Atg32 that is essential for the initiation of mitophagy, as it is required for the proteolysis of the C-terminal domain of Atg32 and the subsequent recruitment of Atg11. The solution structure of this domain was determined by NMR spectroscopy, revealing that Atg32 contains a previously undescribed pseudo-receiver (PsR) domain. Our data suggests that the PsR domain of Atg32 regulates Atg32 activation and the initiation of mitophagy.
Abbreviations:AIM: Atg8-interacting motif; GFP: green fluorescent protein; LIR: LC3-interacting region; NMR: nuclear magnetic resonance; NOESY: nuclear Overhauser effect spectroscopy; PDB: protein data bank; PsR: pseudo-receiver; RMSD: root-mean-square deviation 相似文献
12.
Tobias Schenk Laura Trimborn Song Chen Christian Schenkel Ute Hoecker 《Plant physiology》2021,187(1):276
Arabidopsis (Arabidopsis thaliana) CONSTITUTIVELY PHOTOMORPHOGENIC1 (COP1) and members of the SUPPRESSOR OF PHYTOCHROMEA-105 (SPA) protein family form an E3 ubiquitin ligase that suppresses light signaling in darkness by polyubiquitinating positive regulators of the light response. COP1/SPA is inactivated by light to allow photomorphogenesis to proceed. Mechanisms of inactivation include light-induced degradation of SPA1 and, in particular, SPA2, corresponding to a particularly efficient inactivation of COP1/SPA2 by light. Here, we show that SPA3 and SPA4 proteins are stable in the light, indicating that light-induced destabilization is specific to SPA1 and SPA2, possibly related to the predominant function of SPA1 and SPA2 in dark-grown etiolating seedlings. SPA2 degradation involves cullin and the COP10-DEETIOLATED-DAMAGED-DNA BINDING PROTEIN (DDB1) CDD complex, besides COP1. Consistent with this finding, light-induced SPA2 degradation required the DDB1-interacting Trp-Asp (WD)-repeat domain of SPA2. Deletion of the N-terminus of SPA2 containing the kinase domain led to strong stabilization of SPA2 in darkness and fully abolished light-induced degradation of SPA2. This prevented seedling de-etiolation even in very strong far-red and blue light and reduced de-etiolation in red light, indicating destabilization of SPA2 through its N-terminal domain is essential for light response. SPA2 is exclusively destabilized by phytochrome A in far-red and blue light. However, deletion of the N-terminal domain of SPA2 did not abolish SPA2-phytochrome A interaction in yeast nor in vivo. Our domain mapping suggests there are two SPA2-phytochrome A interacting domains, the N-terminal domain and the WD-repeat domain. Conferring a light-induced SPA2-phyA interaction only via the WD-repeat domain may thus not lead to COP1/SPA2 inactivation. Light inactivates the COP1/SPA2 repressor of photomorphogenesis through cullin- and CDD-mediated degradation of SPA2, whereas the family members SPA3 and SPA4 are stable in the light. 相似文献
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Although it is well established that epidermal growth factor receptors (EGFRs) are asymmetrically expressed at the basolateral plasma membrane in polarized epithelial cells, how this process is regulated is not known. The purpose of this study was to address the mechanism of directed EGFR basolateral sorting using the Madin-Darby canine kidney (MDCK) cell model. The first set of experiments established sorting patterns for endogenous canine EGFRs. The polarity of the canine EGFR was not quantitatively affected by differences in electrical resistance exhibited by the MDCK I and MDCK II cell strains. In both cases, greater than 90% of total surface EGFRs was localized to the basolateral surface. Canine EGFRs sort directly to the basolateral membrane from the trans-Golgi network with a halftime of approximately 45 min and have an approximate t1/2 of 12.5 h once reaching the basolateral surface. Human holoreceptors expressed in stably transfected MDCK cells also localize to the basolateral membrane with similar efficiency. To identify EGFR sequences necessary for basolateral sorting, MDCK cells were transfected with cDNAs coding for cytoplasmically truncated human receptor proteins. Human EGFRs truncated at Arg-651 were localized predominantly at the apical surface of filter-grown cells, whereas receptors truncated at Leu-723 were predominantly basolateral. These results suggest that the cytoplasmic juxtamembrane domain contains a positive basolateral sorting determinant. Moreover, the EGFR ectodomain or transmembrane domain may possess a cryptic sequence that specifically interacts with the apical sorting machinery once the dominant basolateral sorting signal is removed. Further elucidation of the precise loacation of these signals will enhance our basic understanding of regulated plasma membrane sorting, as well as the functional consequences of inappropriate EGFR expression associated with certain pathophysiologic and malignant states. © 1995 Wiley-Liss, Inc. 相似文献
15.
Grim encodes a protein required for programmed cell death in Drosophila, whose proapoptotic activity is conserved in mammalian cells. Two proapoptotic domains are relevant for Grim killing function; the N-terminal region, which induces apoptosis by disrupting inhibitor of apoptosis protein (IAP) blockage of caspase activity, and the internal GH3 domain, which triggers a mitochondrial pathway. We explored the role of these two domains in heterologous killing of mammalian cells by Grim. The GH3 domain is essential for Grim proapoptotic activity in mouse cells, whereas the N-terminal domain is dispensable. The GH3 domain is required and sufficient for Grim targeting to mitochondria and for cytochrome c release in a caspase- and N-terminal-independent, IAP-insensitive manner. These Grim GH3 activities do not require Bax or Bak function, revealing GH3 activity as the first proapoptotic stimulus able to trigger the mitochondrial death pathway in mammalian cells in the absence of multidomain proapoptotic Bcl-2 proteins. 相似文献
16.
M. R. K. Alley 《Molecular microbiology》2001,40(6):1335-1343
We have fused GFP to the C-terminus of McpA to study chemoreceptor polar localization in Caulobacter crescentus. The full-length McpA-GFP fusion is polarly localized and methylated. The methylation is dependent on the chemoreceptor methyltransferase (cheR) and chemoreceptor methylesterase (cheB) genes present in the mcpA operon. C-terminal and internal deletions of McpA were constructed and fused to the N-terminus of GFP to identify the domains required for polar localization. When the R1 methylation domain was deleted, the McpA-GFP fusion was still polarly localized, suggesting that this domain is dispensable for polar localization. However, when the highly conserved domain (HCD), which is involved in interacting with CheW, was deleted either by an internal deletion or C-terminal deletion, the resulting McpA-GFP fusions were completely delocalized. When the mcpA operon, which contains the cheW and cheA homologues, was deleted, the full-length McpA-GFP fusion was delocalized. Although additional chemotaxis genes are required for the polar localization of McpA-GFP, the presence of the single polar flagellum is not required. However, in filamentous cells, which are frequently found in C. crescentus fliF mutants, the McpA-GFP fusion was observed at mid-cell positions. 相似文献
17.
The ARF tumor suppressor, encoded by the CDKN2A gene, has a well-defined role regulating TP53 stability; this activity maps to exon 1β of CDKN2A. In contrast, little is known about the function(s) of exon 2 of ARF, which contains the majority of mutations in human cancer. In addition to controlling TP53 stability, ARF also has a role in the induction of autophagy. However, whether the principal molecule involved is full-length ARF, or a small molecular weight variant called smARF, has been controversial. Additionally, whether tumor-derived mutations in exon 2 of CDKN2A affect ARF’s autophagy function is unknown. Finally, whereas it is known that silencing or inhibiting TP53 induces autophagy, the contribution of ARF to this induction is unknown. In this report we used multiple autophagy assays to map a region located in the highly conserved 5′ end of exon 2 of CDKN2A that is necessary for autophagy induction by both human and murine ARF. We showed that mutations in exon 2 of CDKN2A that affect the coding potential of ARF, but not p16INK4a, all impair the ability of ARF to induce autophagy. We showed that whereas full-length ARF can induce autophagy, our combined data suggest that smARF instead induces mitophagy (selective autophagy of mitochondria), thus potentially resolving some confusion regarding the role of these variants. Finally, we showed that silencing Tp53 induces autophagy in an ARF-dependent manner. Our data indicated that a conserved domain in ARF mediates autophagy, and for the first time they implicate autophagy in ARF’s tumor suppressor function. 相似文献
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Abstract The Dickkopf (Dkk) family is composed of four main members (Dkk1–4), which typically regulate Wnt/β-catenin signaling. An exception is Dkk3, which does not affect Wnt/β-catenin signaling and whose function is poorly characterized. Here, we describe the Xenopus dkk3 homolog and characterize its expression and function during embryogenesis. Dkk3 is maternally expressed and zygotically in the cement gland, head mesenchyme, and heart. We show that depletion of Dkk3 in Xenopus embryos by Morpholino antisense oligonucleotides induces axial defects as a result of Spemann organizer and mesoderm inhibition. Dkk3 depletion leads to down-regulation of Activin/Nodal signaling by reducing levels of Smad4 protein. Dkk3 overexpression can rescue phenotypic effects resulting from overexpression of the Smad4 ubiquitin ligase Ectodermin. Furthermore, depletion of Dkk3 up-regulates FGF signaling, while Dkk3 overexpression reduces it. These results indicate that Dkk3 modulates FGF and Activin/Nodal signaling to regulate mesoderm induction during early Xenopus development. 相似文献
20.
A bipartite nuclear localization signal is required for p53 nuclear import regulated by a carboxyl-terminal domain. 总被引:8,自引:0,他引:8
Abnormal p53 cellular localization has been considered to be one of the mechanisms that could inactivate p53 function. To understand the regulation of p53 cellular trafficking, we have previously identified two p53 domains involved in its localization. A basic domain, Lys(305)-Arg(306), is required for p53 nuclear import, and a carboxyl-terminal domain, namely the cytoplasmic sequestration domain (CSD) from residues 326-355, could block the nuclear import of Lys(305) or Arg(306) mutated p53. To characterize further the function of these two domains, we demonstrate in this report that the previously described major nuclear localization signal works together with Lys(305)-Arg(306) to form a bipartite and functional nuclear localization sequence (NLS) for p53 nuclear import. The CSD could block the binding of p53 to the NLS receptor, importin alpha, and reduce the efficiency of p53 nuclear import in MCF-7, H1299, and Saos-2 cells. The blocking effect of the CSD is not due to the enhancement of nuclear export or oligomerization of the p53. These results indicate that the CSD can regulate p53 nuclear import by controlling access of the NLS to importin alpha binding. 相似文献