共查询到20条相似文献,搜索用时 31 毫秒
1.
To detect the posttranslational N-myristoylation of caspase substrates, the susceptibility of the newly exposed N-terminus of known caspase substrates to protein N-myristoylation was evaluated by in vivo metabolic labeling with [(3)H]myristic acid in transfected cells using a fusion protein in which the query sequence was fused to a model protein. As a result, it was found that the N-terminal nine residues of the newly exposed N-terminus of the caspase-cleavage product of cytoskeletal actin efficiently direct the protein N-myristoylation. Metabolic labeling of COS-1 cells transiently transfected with cDNA coding for full-length truncated actin (tActin) revealed the efficient incorporation of [(3)H]myristic acid into this molecule. When COS-1 cells transiently transfected with cDNA coding for full-length actin were treated with staurosporine, an apoptosis-inducing agent, an N-myristoylated tActin was generated. Immunofluorescence staining coupled with MitoTracker or fluorescence tagged-phalloidin staining revealed that exogenously expressed tActin colocalized with mitochondria without affecting cellular and actin morphology. Taken together, these results demonstrate that the C-terminal 15 kDa fragment of cytoskeletal actin is posttranslationally N-myristoylated upon caspase-mediated cleavage during apoptosis and targeted to mitochondria. 相似文献
2.
Sakurai N Moriya K Suzuki T Sofuku K Mochiki H Nishimura O Utsumi T 《Analytical biochemistry》2007,362(2):236-244
To establish a simple and sensitive method to detect protein N-myristoylation, the usefulness of a newly developed cell-free protein synthesis system derived from insect cells for detecting protein N-myristoylation by in vitro metabolic labeling was examined. The results showed that in vitro translation of cDNA coding for N-myristoylated protein in the presence of [(3)H]myristic acid followed by SDS-PAGE and fluorography is a useful method for rapid detection of protein N-myristoylation. Differential labeling of N-myristoylated model proteins with [(3)H]leucine, [(3)H]myristic acid, and [(35)S]methionine revealed that the removal of the initiating Met during the N-myristoylation reaction could be detected using this system. Analysis of the N-myristoylation of a series of model proteins with mutated N-myristoylation motifs revealed that the amino acid sequence requirements for the N-myristoylation reaction in this system are quite similar to those observed in the rabbit reticulocyte lysate system. N-myristoylation of tBid (a posttranslationally N-myristoylated cytotoxic protein that could not be expressed in transfected cells) was successfully detected in this assay system. Thus, metabolic labeling in an insect cell-free protein synthesis system is an effective strategy to detect co- and posttranslational protein N-myristoylation irrespective of the cytotoxicity of the protein. 相似文献
3.
Suzuki T Ito M Ezure T Shikata M Ando E Utsumi T Tsunasawa S Nishimura O 《Proteomics》2006,6(16):4486-4495
To evaluate the ability of an insect cell-free protein synthesis system to generate proper N-terminal cotranslational protein modifications such as removal of the initiating Met, N-acetylation, and N-myristoylation, several mutants were constructed using truncated human gelsolin (tGelsolin) as a model protein. Tryptic digests of these mutants were analyzed by MALDI-TOF MS and MALDI-quadrupole-IT-TOF MS. The wild-type tGelsolin, which is an N-myristoylated protein, was found to be N-myristoylated when myristoyl-CoA was added to the in vitro translation reaction mixture. N-myristoylation did not occur on the Gly-2 to Ala mutant, in which the N-myristoylation motif was disrupted, whereas this mutant was found to be N-acetylated after removal of the initiating Met. Analyses of Gly-2 to His and Leu-3 to Asp mutants revealed that the amino acids at positions 2 and 3 strongly affect the susceptibility of the nascent peptide chain to removal of the initiating Met and to N-acetylation, respectively. These results suggest that N-terminal modifications occurring in the insect cell-free protein synthesis system are quite similar to those observed in the mammalian protein synthesis system. Thus, a combination of the cell-free protein synthesis system with MS is an effective strategy to analyze protein modifications. 相似文献
4.
In eukaryotic cellular proteins, protein N-myristoylation has been recognized as a protein modification that occurs mainly on cytoplasmic or nucleoplasmic proteins. In this study, to search for a eukaryotic N-myristoylated transmembrane protein, the susceptibility of the N-terminus of several G-protein-coupled receptors (GPCRs) to protein N-myristoylation was evaluated by in vitro and in vivo metabolic labeling. It was found that the N-terminal 10 residues of B96Bom, a Bombyx mori GPCR, efficiently directed the protein N-myristoylation. Analysis of a tumor necrosis factor (TNF) fusion protein with the N-terminal 90 residues of B96Bom at its N-terminus revealed that (a) transmembrane domain 1 of B96Bom functioned as a type I signal anchor sequence, (b) the N-myristoylated N-terminal domain (58 residues) was translocated across the membrane, and (c) two N-glycosylation motifs located in this domain were efficiently N-glycosylated. In addition, when Ala4 in the N-myristoylation motif of B96Bom90-TNF, Met-Gly-Gln-Ala-Ala-Thr(1-6), was replaced with Asn to generate a new N-glycosylation motif, Asn-Ala-Thr(4-6), efficient N-glycosylation was observed on this newly introduced N-glycosylation site in the expressed protein. These results indicate that the N-myristoylated N-terminus of B96Bom is translocated across the membrane and exposed to the extracellular surface. To our knowledge, this is the first report showing that a eukaryotic transmembrane protein can be N-myristoylated and that the N-myristoylated N-terminus of the protein can be translocated across the membrane. 相似文献
5.
To establish a non-radioactive, cell-free detection system for protein N-myristoylation, metabolic labeling in a cell-free protein synthesis system using bioorthogonal myristic acid analogues was performed. After Cu(I)-catalyzed azide–alkyne cycloaddition (CuAAC) with a biotin tag, the tagged proteins were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) and blotted on a polyvinylidene fluoride (PVDF) membrane, and then protein N-myristoylation was detected by enhanced chemiluminescence (ECL) using horseradish peroxidase (HRP)-conjugated streptavidin. The results showed that metabolic labeling in an insect cell-free protein synthesis system using an azide analogue of myristic acid followed by CuAAC with alkynyl biotin was the most effective strategy for cell-free detection of protein N-myristoylation. To determine whether the newly developed detection method can be applied for the detection of novel N-myristoylated proteins from complementary DNA (cDNA) resources, four candidate cDNA clones were selected from a human cDNA resource and their susceptibility to protein N-myristoylation was evaluated using the newly developed strategy. As a result, the products of three cDNA clones were found to be novel N-myristoylated protein, and myristoylation-dependent specific intracellular localization was observed for two novel N-myristoylated proteins. Thus, the metabolic labeling in an insect cell-free protein synthesis system using bioorthogonal azide analogue of myristic acid was an effective strategy to identify novel N-myristoylated proteins from cDNA resources. 相似文献
6.
Background
N-myristoylation is a crucial covalent modification of numerous eukaryotic and viral proteins that is catalyzed by N-myristoyltransferase (NMT). Prokaryotes are lacking endogeneous NMT activity. Recombinant production of N-myristoylated proteins in E. coli cells can be achieved by coexpression of heterologous NMT with the target protein. In the past, dual plasmid systems were used for this purpose.Methodology/Principal Findings
Here we describe a single vector system for efficient coexpression of substrate and enzyme suitable for production of co- or posttranslationally modified proteins. The approach was validated using the HIV-1 Nef protein as an example. A simple and efficient protocol for production of highly pure and completely N-myristoylated Nef is presented. The yield is about 20 mg myristoylated Nef per liter growth medium.Conclusions/Significance
The single vector strategy allows diverse modifications of target proteins recombinantly coexpressed in E. coli with heterologous enzymes. The method is generally applicable and provides large amounts of quantitatively processed target protein that are sufficient for comprehensive biophysical and structural studies. 相似文献7.
Koko Moriya Mayumi KimotoKanako Matsuzaki Aya KiwadoEmi Takamitsu Toshihiko Utsumi 《Analytical biochemistry》2016
To establish a strategy to identify dually fatty acylated proteins from cDNA resources, seven N-myristoylated proteins with cysteine (Cys) residues within the 10 N-terminal residues were selected as potential candidates among 27 N-myristoylated proteins identified from a model human cDNA resource. Seven proteins C-terminally tagged with FLAG tag or EGFP were generated and their susceptibility to protein N-myristoylation and S-palmitoylation were evaluated by metabolic labeling with [3H]myristic acid or [3H]palmitic acid either in an insect cell-free protein synthesis system or in transfected mammalian cells. As a result, EEPD1, one of five proteins (RFTN1, EEPD1, GNAI1, PDE2A, RNF11) found to be dually acylated, was shown to be a novel dually fatty acylated protein. Metabolic labeling experiments using G2A and C7S mutants of EEPD1-EGFP revealed that the palmitoylation site of EEPD1 is Cys at position 7. Analysis of the intracellular localization of EEPD1 C-terminally tagged with FLAG tag or EGFP and its G2A and C7S mutants revealed that the dual acylation directs EEPD1 to localize to the plasma membrane. Thus, dually fatty acylated proteins can be identified from cDNA resources by cell-free and cellular metabolic labeling of N-myristoylated proteins with Cys residue(s) close to the N-myristoylated N-terminus. 相似文献
8.
9.
Erwan Beauchamp Xavier Tekpli Gaëlle Marteil Dominique Lagadic-Gossmann Philippe Legrand Vincent Rioux 《Biochimie》2009,91(11-12):1411-1419
This study was designed to analyze the effect of myristic acid on ceramide synthesis and its related lipoapoptosis pathway. It was previously observed that myristic acid binds dihydroceramide Δ4-desaturase 1 (DES1) through N-myristoylation and activates this enzyme involved in the final de novo ceramide biosynthesis step. In the present study, we show first by immunofluorescence microscopy and subcellular fractionation that DES1 myristoylation targets part of the recombinant protein to the mitochondria in COS-7 cells. In addition, native dihydroceramide Δ4-desaturase activity was found in both the endoplasmic reticulum and mitochondria in rat hepatocytes. Dihydroceramide conversion to ceramide was increased in COS-7 cells expressing DES1 and incubated with myristic acid. The expression of the wild-type myristoylable DES1-Gly alone, but not the expression of the unmyristoylable mutant DES1-Ala, induced apoptosis of COS-7 cells. Finally, myristic acid alone also increased the production of cellular ceramide and had an apoptotic effect. This effect was potentiated on caspase activity when the myristoylable form of DES1 was expressed. Therefore, these results suggest that the myristoylation of DES1 can target the enzyme to the mitochondria leading to an increase in ceramide levels which in turn contributes to partially explain the apoptosis effect of myristic acid in COS-7 cells. 相似文献
10.
Boccellino M Giuberti G Quagliuolo L Marra M D'Alessandro AM Fujita H Giovane A Abbruzzese A Caraglia M 《Journal of cellular physiology》2004,201(1):71-83
We have previously reported that interferon-alpha (IFNalpha) induces apoptosis and EGF can antagonize this effect in human epidermoid cancer KB cells. Since apoptosis occurs together with cytoskeleton reorganization we have evaluated if IFNalpha and EGF could modulate cell remodeling in our experimental conditions. We have found that 48 h 1,000 IU/ml IFNalpha induced structural reorganization of stress fibers and membrane delocalization and partial capping of the actin severing protein gelsolin. The transfection of KB cells with both a wild type (WT) or a C-terminal truncated form of gelsolin caused overexpression of the protein and an increase of both the spontaneous and IFNalpha-induced apoptosis and cell cytoskeletal modifications. In fact, after 48 h of treatment IFNalpha induced 45% of apoptotic cell death in parental cells while an approximately 80% of cell population was apoptotic in transfected cells. These effects occurred together with an increase of the expression and consequent degradation of gelsolin. Again the addition of EGF to IFNalpha-treated transfected cells caused a recovery of the apoptosis. Notably, IFNalpha and EGF did not modify the expression of other molecules associated to cytoskeleton such as focal adhesion kinase and vinculin. In the same experimental conditions IFNalpha induced also gelsolin cleavage that occurred together with caspase-3 activation and release of cytochrome c. All these effects were antagonized by the exposure of IFNalpha-treated KB to 10 nM EGF for the last 12 h. Moreover, the specific inhibition of caspase-3 with 20 microM DEVD completely abrogated apoptosis and gelsolin cleavage induced by IFNalpha. In conclusion, our data are the first demonstration that IFNalpha can induce morphological cell changes that are peculiar of apoptosis onset through the caspase-3-mediated cleavage of gelsolin. Furthermore, we have demonstrated that EGF is able to antagonize these effects through the inhibition of caspase-3 activation. 相似文献
11.
Morita D Igarashi T Horiike M Mori N Sugita M 《Journal of immunology (Baltimore, Md. : 1950)》2011,187(2):608-612
The use of the host cellular machinery is essential for pathogenic viruses to replicate in host cells. HIV and SIV borrow the host-derived N-myristoyl-transferase and its substrate, myristoyl-CoA, for coupling a saturated C(14) fatty acid (myristic acid) to the N-terminal glycine residue of the Nef protein. This biochemical reaction, referred to as N-myristoylation, assists its targeting to the plasma membrane, thereby supporting the immunosuppressive activity proposed for the Nef protein. In this study, we show that the host immunity is equipped with CTLs capable of sensing N-myristoylation of the Nef protein. A rhesus macaque CD8(+) T cell line was established that specifically recognized N-myristoylated, but not unmodified, peptides of the Nef protein. Furthermore, the population size of N-myristoylated Nef peptide-specific T cells was found to increase significantly in the circulation of SIV-infected monkeys. Thus, these results identify N-myristoylated viral peptides as a novel class of CTL target Ag. 相似文献
12.
Earlier immunolocalization experiments showed that the extreme cationic C-terminus of the rat intestinal mucin Muc2 (RMC) was present at the base of intestinal goblet cells in the vicinity of ER and golgi compartments, but was not found with the rest of the mucin in apical storage granules. This prompted us to investigate the possibility that an early proteolytic cleavage reaction occurs post-translationally. A plasmid pRMC, encoding the C-terminal 534 amino acids of the mucin, was expressed in COS-7 cells and was shown to undergo cleavage at an R-T-R-R sequence located within the C-terminal 14 amino acids. Cleavage did not occur with the construct RMCfH, a furin site-mutated (A-T-A-A) counterpart of pRMCH (poly His6 tagged RMC). Addition of a furin inhibitor to COS-7 cell incubations also prevented cleavage of RMC and RMCH products. 35S pulse-chase kinetic experiments revealed that a truncated mutant lacking the C-terminal 14 amino acids (pRMCDeltaCT) forms faulty (doublet) dimers in the ER. These were not secreted as efficiently as the normal dimer of wild-type (pRMC) constructs. Thus the cationic C-terminus of rMuc2 apppears to facilitate the correct formation of normal Muc2 domain dimers. 相似文献
13.
The amyloid precursor protein (APP) undergoes "alternative" proteolysis mediated by caspases. Three major caspase recognition sites have been identified in the APP, i.e. one at the C terminus (Asp720) and two at the N terminus (Asp197 and Asp219). Caspase cleavage at Asp720 has been suggested as leading to increased production of Abeta. Thus, we set out to determine which putative caspase sites in APP, if any, are cleaved in Chinese hamster ovary cell lines concurrently with the increased Abeta production that occurs during apoptosis. We found that cleavage at Asp720 occurred concurrently with caspase 3 activation and the increased production of total secreted Abeta and Abeta1-42 in association with staurosporine- and etoposide-induced apoptosis. To investigate the contribution of caspase cleavage of APP to Abeta generation, we expressed an APP mutant truncated at Asp720 that mimics APP caspase cleavage at the C-terminal site. This did not increase Abeta generation but, in contrast, dramatically decreased Abeta production in Chinese hamster ovary cells. Furthermore, the ablation of caspase-dependent cleavage at Asp720, Asp197, and Asp219 (by site-directed mutagenesis) did not prevent enhanced Abeta production following etoposide-induced apoptosis. These findings indicate that the enhanced Abeta generation associated with apoptosis does not require cleavage of APP at its C-terminal (Asp720) and/or N-terminal caspase sites. 相似文献
14.
15.
Chlamydomonas alpha-tubulin is posttranslationally modified by acetylation on the epsilon-amino group of a lysine 总被引:25,自引:0,他引:25
Previous work has shown that the principal alpha-tubulin within Chlamydomonas reinhardtii flagellar axonemes differs from the major alpha-tubulin in the cell body. These two variants of alpha-tubulin are related to one another since posttranslational modification of the cell body form converts it to the axonemal form. When flagella are induced to assemble in the absence of de novo protein synthesis, tritiated acetate can be used to posttranslationally label alpha-tubulin in vivo, and under these conditions, no other flagellar polypeptides exhibit detectable labeling [L'Hernault, S. W., & Rosenbaum, J. L. (1983) J. Cell Biol. 97, 258-263]. In the present report, this labeling method has been used to provide material for chemical analysis of the tritiated moiety that is posttranslationally added to flagellar alpha-tubulin. This radioactivity was volatile after acid hydrolysis, suggesting that the posttranslational modification is the addition of neither an amino acid nor carbohydrate. Treatment of posttranslationally 3H-labeled alpha-tubulin with hydrazine yields radioactive acetylhydrazine, indicating that the moiety involved in posttranslational modification is an acetyl group. Analysis of complete proteolytic digests by thin-layer chromatography has revealed that this acetyl group is located on the epsilon-amino group of a flagellar alpha-tubulin lysine residue. 相似文献
16.
Phospholipase D plays an anti-apoptotic role but little is known about dynamics of phospholipase D turnover during apoptosis. We have recently identified phospholipase D1 as a new substrate of caspases which generates the N-terminal and C-terminal fragment of phospholipase D1. In the present study, we tried to investigate whether association of the caspase cleavage fragments may be involved in regulation of apoptosis. Ectopically expressed C-terminal fragment, but not N-terminal fragment of phospholipase D1, is exclusively imported into the nucleus via a nuclear localization sequence; however, endogenous C-terminal fragment of phospholipase D1 from etoposide-induced apoptotic cells and Alzheimer's disease brain tissues with active caspase-3, was localized in the cytosolic fraction as well as the nuclear fraction. Intermolecular association between the two fragments of phospholipase D1 through hydrophobic residues within the catalytic motif inhibited nuclear localization of C-terminal fragment of phospholipase D1, and two catalytic motif and nuclear localization sequence regulated nuclocytoplasmic shuttling of phospholipase D1. Moreover, hydrophobic residues involved in the intermolecular association are also required for both its enzymatic activity and anti-apoptotic function. Taken together, we demonstrate that interdomain association and dissociation of phospholipase D1 might provide new insights into modulation of apoptosis. 相似文献
17.
Marissen WE Guo Y Thomas AA Matts RL Lloyd RE 《The Journal of biological chemistry》2000,275(13):9314-9323
Induction of apoptosis in a variety of cell types leads to inhibition of protein synthesis. Recently, the cleavage of eukaryotic translation initiation factor 4G (eIF4G) by caspase 3 was described as a possible event contributing to translation inhibition. Here, we report the cleavage of another initiation factor in apoptotic cells, eIF2alpha, that could contribute to regulation of translation during apoptosis. This cleavage event could be completely inhibited by pretreatment of HeLa cells with Z-VAD-fmk. In vitro analysis using purified eIF2 and purified caspases showed cleavage of eIF2alpha by caspase 3, 6, 8, and 10 but not 9. Caspase 3 most efficiently cleaved eIF2alpha and this could be inhibited by addition of Ac-DEVD-CHO in vitro. Comparison of cleavage of phosphorylated versus nonphosphorylated eIF2alpha revealed a modest preference of the caspases for the nonphosphorylated form. When eIF2. 2B complex was used as substrate, only caspase 3 was capable of eIF2alpha cleavage, which was not affected by phosphorylation of the alpha subunit. The eIF2.GDP binary complex was cleaved much less efficiently by caspase 3. Sequence analysis of the cleavage fragment suggested that the cleavage site is located in the C-terminal portion of the protein. Analysis showed that after caspase cleavage, exchange of GDP bound to eIF2 was very rapid and no longer dependent upon eIF2B. Furthermore, in vitro translation experiments indicated that cleavage of eIF2alpha results in functional alteration of the eIF2 complex, which no longer stimulated upstream AUG selection on a mRNA containing a viral internal ribosome entry site and was no longer capable of stimulating overall translation. In conclusion, we describe here the cleavage of a translation initiation factor, eIF2alpha that could contribute to inhibition or alteration of protein synthesis during the late stages of apoptosis. 相似文献
18.
MAGI-1 is a membrane-associated guanylate kinase (MAGUK) protein present at adherent and tight junctions, where it acts as a structural and signaling scaffold. During apoptosis, MAGI-1 is cleaved by caspases at Asp761 into N- and C-terminal cleavage products, allowing further dismantling of the cell junctions, one of the key features of apoptosis. Here, we investigated the cellular distribution and possible proapototic role of MAGI-1 caspase cleavage products. Full-length MAGI-1 exhibited submembrane localization, while the N-terminal caspase cleavage product of MAGI-1 is translocated to the cytosol and the C-terminal caspase cleavage product accumulates in the nucleus. When overexpressed in MDCK cells, both N- and C-terminal MAGI-1 caspase cleavage products exhibited minor proapoptotic activity, although their role in apoptosis is probably more passive. 相似文献
19.
20.
Overexpression of BCL2 blocks TNF-related apoptosis-inducing ligand (TRAIL)-induced apoptosis in human lung cancer cells 总被引:9,自引:0,他引:9
Sun SY Yue P Zhou JY Wang Y Choi Kim HR Lotan R Wu GS 《Biochemical and biophysical research communications》2001,280(3):788-797
The tumor necrosis factor (TNF) related apoptosis-inducing ligand (TRAIL or Apo2L) and its receptors are members of the tumor necrosis factor superfamily. TRAIL triggers apoptosis by binding to its two proapoptotic receptors DR4 and DR5, a process which is negatively regulated by binding of TRAIL to its two decoy receptors TRID and TRUNDD. Here, we show that TRAIL effectively induces apoptosis in H460 human non-small-cell lung carcinoma cells via cleavage of caspases 8, 9, 7, 3, and BID, release of cytochrome c from the mitochondria, and cleavage of poly (ADP-ribose) polymerase (PARP). However, overexpression of Bcl2 blocked TRAIL-induced apoptosis in H460 cells, which correlated with the Bcl2 protein levels. Importantly, the release of cytochrome c and cleavage of caspase 7 triggered by TRAIL were considerably blocked in Bcl2 overexpressing cells as compared to vector control cells. Moreover, inhibition of TRAIL-mediated cytochrome c release and caspase 7 activation by Bcl2 correlated with the inability of PARP to be cleaved and the inability of the Bcl2 transfectants to undergo apoptosis. Thus, these results suggest that Bcl2 can serve an anti-apoptotic function during TRAIL-dependent apoptosis by inhibiting the release of cytochrome c and activation of caspase 7, thereby blocking caspase 7-dependent cleavage of cellular substrates. 相似文献