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1.
Sci Rep ; 10(1): 5661, 2020 Mar 24.
Artigo em Inglês | MEDLINE | ID: mdl-32205847

RESUMO

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

2.
Front Plant Sci ; 10: 873, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31379892

RESUMO

Phytaspases belong to the family of plant subtilisin-like proteases and are distinct from other family members, as they have strict and rarely occurring aspartate cleavage specificity and unusual localization dynamics. After being secreted into the apoplast of healthy plant tissues, phytaspases are able to return back into cells that have been committed to cell death due to a variety of biotic and abiotic stresses. It was recently discovered that retrograde transport of phytaspases involves clathrin-mediated endocytosis. Here, consequences of phytaspase internalization were studied. Proteolytic activity of phytaspases in the apoplast and intracellular protein fractions obtained from Nicotiana benthamiana leaves containing either endogenous phytaspase only or transiently producing Nicotiana tabacum phytaspase-EGFP protein (NtPhyt-EGFP) was determined. We demonstrated that triggering phytaspase internalization by antimycin A-induced oxidative stress is accompanied by re-distribution of phytaspase activity from the apoplast to the cell interior. Inhibition of clathrin-mediated endocytosis by co-production of the Hub protein prevented phytaspase internalization and phytaspase activity re-localization. Specificity of endocytic uptake of phytaspases was demonstrated by the co-production of an apoplast-targeted mRFP protein marker, which retained its apoplastic localization when phytaspase internalization was essentially complete. Overproduction of NtPhyt-EGFP, but not of the proteolytically inactive phytaspase mutant, per se caused moderate damage in young Nicotiana benthamiana seedlings, whereas antimycin A treatment induced a pronounced loss of cell viability independent of the NtPhyt-EGFP overproduction. Interestingly, inhibition of clathrin-mediated endocytosis abrogated cell death symptoms in both cases. In contrast to stress-induced internalization of tobacco phytaspase, Arabidopsis thaliana phytaspase-EGFP protein (AtPhyt-EGFP) was spontaneously internalized when transiently produced in N. benthamiana leaves. The AtPhyt-EGFP uptake was dependent on clathrin-mediated endocytosis as well, the internalized protein being initially visualized within the membranous vesicles. At later time points, the EGFP tag was cleaved off from AtPhyt, though the elevated level of intracellular AtPhyt proteolytic activity persisted. Our data, therefore, point to clathrin-mediated endocytosis as a means to deliver proteolytically active phytaspases into plant cells. It would be interesting to learn whether or not phytaspases are unique among the large family of plant subtilisin-like proteases in their ability to utilize retrograde trafficking.

4.
Sci Rep ; 8(1): 10531, 2018 Jul 12.
Artigo em Inglês | MEDLINE | ID: mdl-30002392

RESUMO

Phytaspases are Asp-specific subtilisin-like plant proteases that have been likened to animal caspases with respect to their regulatory function in programmed cell death (PCD). We identified twelve putative phytaspase genes in tomato that differed widely in expression level and tissue-specific expression patterns. Most phytaspase genes are tandemly arranged on tomato chromosomes one, four, and eight, and many belong to taxon-specific clades, e.g. the P69 clade in the nightshade family, suggesting that these genes evolved by gene duplication after speciation. Five tomato phytaspases (SlPhyts) were expressed in N. benthamiana and purified to homogeneity. Substrate specificity was analyzed in a proteomics assay and with a panel of fluorogenic peptide substrates. Similar to animal caspases, SlPhyts recognized an extended sequence motif including Asp at the cleavage site. Clear differences in cleavage site preference were observed implying different substrates in vivo and, consequently, different physiological functions. A caspase-like function in PCD was confirmed for five of the seven tested phytaspases. Cell death was triggered by ectopic expression of SlPhyts 2, 3, 4, 5, 6 in tomato leaves by agro-infiltration, as well as in stably transformed transgenic tomato plants. SlPhyts 3, 4, and 5 were found to contribute to cell death under oxidative stress conditions.


Assuntos
Caspases/metabolismo , Lycopersicon esculentum/metabolismo , Proteínas de Plantas/metabolismo , Apoptose/fisiologia , Caspases/genética , Morte Celular , Expressão Ectópica do Gene , Duplicação Gênica , Genes de Plantas/genética , Lycopersicon esculentum/genética , Estresse Oxidativo/fisiologia , Folhas de Planta/genética , Folhas de Planta/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/isolamento & purificação , Plantas Geneticamente Modificadas/genética , Plantas Geneticamente Modificadas/metabolismo , Proteômica , Especificidade por Substrato , Tabaco/genética , Tabaco/metabolismo
5.
New Phytol ; 218(3): 901-915, 2018 05.
Artigo em Inglês | MEDLINE | ID: mdl-28467631

RESUMO

Contents Summary 901 I. Introduction 901 II. Biochemistry and structure of plant SBTs 902 III. Phylogeny of plant SBTs and family organization 903 IV. Physiological roles of plant SBTs 905 V. Conclusions and outlook 911 Acknowledgements 912 References 912 SUMMARY: Subtilases (SBTs) are serine peptidases that are found in all three domains of life. As compared with homologs in other Eucarya, plant SBTs are more closely related to archaeal and bacterial SBTs, with which they share many biochemical and structural features. However, in the course of evolution, functional diversification led to the acquisition of novel, plant-specific functions, resulting in the present-day complexity of the plant SBT family. SBTs are much more numerous in plants than in any other organism, and include enzymes involved in general proteolysis as well as highly specific processing proteases. Most SBTs are targeted to the cell wall, where they contribute to the control of growth and development by regulating the properties of the cell wall and the activity of extracellular signaling molecules. Plant SBTs affect all stages of the life cycle as they contribute to embryogenesis, seed development and germination, cuticle formation and epidermal patterning, vascular development, programmed cell death, organ abscission, senescence, and plant responses to their biotic and abiotic environments. In this article we provide a comprehensive picture of SBT structure and function in plants.


Assuntos
Plantas/enzimologia , Subtilisinas/química , Subtilisinas/metabolismo , Morte Celular , Filogenia , Fenômenos Fisiológicos Vegetais
6.
New Phytol ; 218(3): 1167-1178, 2018 05.
Artigo em Inglês | MEDLINE | ID: mdl-28407256

RESUMO

Peptide hormones are implicated in many important aspects of plant life and are usually synthesized as precursor proteins. In contrast to animals, data for plant peptide hormone maturation are scarce and the specificity of processing enzyme(s) is largely unknown. Here we tested a hypothesis that processing of prosystemin, a precursor of tomato (Solanum lycopersicum) wound hormone systemin, is performed by phytaspases, aspartate-specific proteases of the subtilase family. Following the purification of phytaspase from tomato leaves, two tomato phytaspase genes were identified, the cDNAs were cloned and the recombinant enzymes were obtained after transient expression in Nicotiana benthamiana. The newly identified tomato phytaspases hydrolyzed prosystemin at two aspartate residues flanking the systemin sequence. Site-directed mutagenesis of the phytaspase cleavage sites in prosystemin abrogated not only the phytaspase-mediated processing of the prohormone in vitro, but also the ability of prosystemin to trigger the systemic wound response in vivo. The data show that the prohormone prosystemin requires processing for signal biogenesis and biological activity. The identification of phytaspases as the proteases involved in prosystemin maturation provides insight into the mechanisms of wound signaling in tomato. Our data also suggest a novel role for cell death-related proteases in mediating defense signaling in plants.


Assuntos
Ácido Aspártico Endopeptidases/metabolismo , Lycopersicon esculentum/metabolismo , Peptídeos/metabolismo , Reguladores de Crescimento de Planta/metabolismo , Precursores de Proteínas/metabolismo , Processamento de Proteína Pós-Traducional , Hidrólise , Transdução de Sinais
7.
J Biol Chem ; 290(41): 24806-15, 2015 Oct 09.
Artigo em Inglês | MEDLINE | ID: mdl-26283788

RESUMO

Plants lack aspartate-specific cell death proteases homologous to animal caspases. Instead, a subtilisin-like serine-dependent plant protease named phytaspase shown to be involved in the accomplishment of programmed death of plant cells is able to hydrolyze a number of peptide-based caspase substrates. Here, we determined the substrate specificity of rice (Oryza sativa) phytaspase by using the positional scanning substrate combinatorial library approach. Phytaspase was shown to display an absolute specificity of hydrolysis after an aspartic acid residue. The preceding amino acid residues, however, significantly influence the efficiency of hydrolysis. Efficient phytaspase substrates demonstrated a remarkable preference for an aromatic amino acid residue in the P3 position. The deduced optimum phytaspase recognition motif has the sequence IWLD and is strikingly hydrophobic. The established pattern was confirmed through synthesis and kinetic analysis of cleavage of a set of optimized peptide substrates. An amino acid motif similar to the phytaspase cleavage site is shared by the human gastrointestinal peptide hormones gastrin and cholecystokinin. In agreement with the established enzyme specificity, phytaspase was shown to hydrolyze gastrin-1 and cholecystokinin at the predicted sites in vitro, thus destroying the active moieties of the hormones.


Assuntos
Oryza/enzimologia , Subtilisina/metabolismo , Sequência de Aminoácidos , Sítios de Ligação , Morte Celular , Colecistocinina/metabolismo , Gastrinas/metabolismo , Humanos , Hidrólise , Interações Hidrofóbicas e Hidrofílicas , Cinética , Oryza/citologia , Ligação Proteica , Especificidade por Substrato , Subtilisina/química
8.
Physiol Plant ; 145(1): 77-84, 2012 May.
Artigo em Inglês | MEDLINE | ID: mdl-22182311

RESUMO

Proteases with an aspartate cleavage specificity are known to contribute to programmed cell death (PCD) in animals and plants. In animal cells this proteolytic activity belongs to caspases, a well-characterized family of cysteine-dependent death proteases. Plants, however, lack caspase homologs and thus the origin of this type of proteolytic activity in planta was poorly understood. Here, we review recent data demonstrating that a plant serine-dependent protease, phytaspase, shares cleavage specificity and a role in PCD analogous to that of caspases. However, unlike caspases, regulation of phytaspase-mediated cleavage of intracellular target proteins appears to be attained not at the level of proenzyme processing/activation, which occurs, in the case of phytaspase, autocatalytically and constitutively. Rather, the mature phytaspase is excluded from healthy cells into the apoplast and is allowed to re-enter cells upon the induction of PCD. Thus, PCD-related proteases in animals and plants display both common features and important distinctions.


Assuntos
Caspases/metabolismo , Morte Celular , Proteínas de Plantas/metabolismo , Tabaco/enzimologia , Animais , Antivirais/química , Antivirais/farmacologia , Domínio Catalítico , Ativação Enzimática , Interações Hospedeiro-Patógeno , Doenças das Plantas/virologia , Proteólise , Especificidade da Espécie , Especificidade por Substrato , Subtilisinas/metabolismo , Tabaco/virologia , Vírus do Mosaico do Tabaco/efeitos dos fármacos , Vírus do Mosaico do Tabaco/patogenicidade
9.
Proc Natl Acad Sci U S A ; 107(29): 12828-33, 2010 Jul 20.
Artigo em Inglês | MEDLINE | ID: mdl-20566882

RESUMO

While many functions of the p53 tumor suppressor affect mitochondrial processes, the role of altered mitochondrial physiology in a modulation of p53 response remains unclear. As mitochondrial respiration is affected in many pathologic conditions such as hypoxia and intoxications, the impaired electron transport chain could emit additional p53-inducing signals and thereby contribute to tissue damage. Here we show that a shutdown of mitochondrial respiration per se does not trigger p53 response, because inhibitors acting in the proximal and distal segments of the respiratory chain do not activate p53. However, strong p53 response is induced specifically after an inhibition of the mitochondrial cytochrome bc1 (the electron transport chain complex III). The p53 response is triggered by the deficiency in pyrimidines that is developed due to a suppression of the functionally coupled mitochondrial pyrimidine biosynthesis enzyme dihydroorotate dehydrogenase (DHODH). In epithelial carcinoma cells the activation of p53 in response to mitochondrial electron transport chain complex III inhibitors does not require phosphorylation of p53 at Serine 15 or up-regulation of p14(ARF). Instead, our data suggest a contribution of NQO1 and NQO2 in stabilization of p53 in the nuclei. The results establish the deficiency in pyrimidine biosynthesis as the cause of p53 response in the cells with impaired mitochondrial respiration.


Assuntos
Mitocôndrias/metabolismo , Pirimidinas/biossíntese , Transdução de Sinais , Proteína Supressora de Tumor p53/metabolismo , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Respiração Celular/efeitos dos fármacos , Complexo III da Cadeia de Transporte de Elétrons/antagonistas & inibidores , Complexo III da Cadeia de Transporte de Elétrons/metabolismo , Humanos , Isoxazóis/farmacologia , Leflunomida , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Metacrilatos/farmacologia , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/enzimologia , NAD(P)H Desidrogenase (Quinona)/metabolismo , Oxirredutases atuantes sobre Doadores de Grupo CH-CH/antagonistas & inibidores , Oxirredutases atuantes sobre Doadores de Grupo CH-CH/deficiência , Cianeto de Potássio/farmacologia , Complexo de Endopeptidases do Proteassoma/metabolismo , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Quinona Redutases/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais/efeitos dos fármacos , Tiazóis/farmacologia , Regulação para Cima/efeitos dos fármacos
10.
EMBO J ; 29(6): 1149-61, 2010 Mar 17.
Artigo em Inglês | MEDLINE | ID: mdl-20111004

RESUMO

Caspases are cysteine-dependent proteases and are important components of animal apoptosis. They introduce specific breaks after aspartate residues in a number of cellular proteins mediating programmed cell death (PCD). Plants encode only distant homologues of caspases, the metacaspases that are involved in PCD, but do not possess caspase-specific proteolytic activity. Nevertheless, plants do display caspase-like activities indicating that enzymes structurally distinct from classical caspases may operate as caspase-like proteases. Here, we report the identification and characterisation of a novel PCD-related subtilisin-like protease from tobacco and rice named phytaspase (plant aspartate-specific protease) that possesses caspase specificity distinct from that of other known caspase-like proteases. We provide evidence that phytaspase is synthesised as a proenzyme, which is autocatalytically processed to generate the mature enzyme. Overexpression and silencing of the phytaspase gene showed that phytaspase is essential for PCD-related responses to tobacco mosaic virus and abiotic stresses. Phytaspase is constitutively secreted into the apoplast before PCD, but unexpectedly is re-imported into the cell during PCD providing insights into how phytaspase operates.


Assuntos
Caspases/metabolismo , Peptídeo Hidrolases/metabolismo , Proteínas de Plantas/metabolismo , Caspases/química , Caspases/genética , Morte Celular , Células Cultivadas , Oryza/genética , Oryza/metabolismo , Peptídeo Hidrolases/análise , Peptídeo Hidrolases/genética , Proteínas de Plantas/química , Plantas Geneticamente Modificadas , Especificidade por Substrato , Tabaco/genética , Tabaco/metabolismo
11.
Plant Cell Rep ; 26(8): 1215-9, 2007 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-17370074

RESUMO

Agrobacterium tumefaciens VirD2 protein is one of the key elements of Agrobacterium-mediated plant transformation, a process of transfer of T-DNA sequence from the Agrobacterium tumour inducing plasmid into the nucleus of infected plant cells and its integration into the host genome. The VirD2 protein has been shown to be a substrate for a plant caspase-like protease activity (PCLP) in tobacco. We demonstrate here that mutagenesis of the VirD2 protein to prevent cleavage by PCLP increases the efficiency of reporter gene transfer and expression. These results indicate that PCLP cleavage of the Agrobacterium VirD2 protein acts to limit the effectiveness of T-DNA transfer and is a novel resistance mechanism that plants utilise to combat Agrobacterium infection.


Assuntos
Agrobacterium tumefaciens/genética , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Caspases/metabolismo , Expressão Gênica , Plantas/genética , Plantas/metabolismo , DNA Bacteriano/genética , DNA Complementar/genética , Regulação Bacteriana da Expressão Gênica , Regulação da Expressão Gênica de Plantas
12.
Virology ; 331(2): 292-306, 2005 Jan 20.
Artigo em Inglês | MEDLINE | ID: mdl-15629772

RESUMO

In several cell types, poliovirus activates the apoptotic program, implementation of which is suppressed by viral antiapoptotic functions. In such cells, productive infection leads to a necrotic cytopathic effect (CPE), while abortive reproduction, associated with inadequate viral antiapoptotic functions, results in apoptosis. Here, we describe two other types of cell response to poliovirus infection. Murine L20B cells expressing human poliovirus receptor responded to the infection by both CPE and apoptosis concurrently. Interruption of productive infection decreased rather than increased the proportion of apoptotic cells. Productive infection was accompanied by the early efflux of cytochrome c from the mitochondria in a proportion of cells and by activation of DEVD-specific caspases. Inactivation of caspase-9 resulted in a marked, but incomplete, prevention of the apoptotic response of these cells to viral infection. Thus, the poliovirus-triggered apoptotic program in L20B cells was not completely suppressed by the viral antiapoptotic functions. In contrast, human rhabdomyosarcoma RD cells did not develop appreciable apoptosis during productive or abortive infection, exhibiting inefficient efflux of cytochrome c from mitochondria and no marked activation of DEVD-specific caspases. The cells were also refractory to several nonviral apoptosis inducers. Nevertheless, typical caspase-dependent signs of apoptosis in a proportion of RD cells were observed after cessation of viral reproduction. Such "late" apoptosis was also observed in productively infected HeLa cells. In addition, a tiny proportion of all studied cells were TUNEL positive even in the presence of a caspase inhibitor. Degradation of DNA in such cells appeared to be a postmortem phenomenon. Biological relevance of variable host responses to viral infection is discussed.


Assuntos
Apoptose , Efeito Citopatogênico Viral , Poliovirus/fisiologia , Inibidores de Caspase , Caspases/metabolismo , Fragmentação do DNA , Células HeLa , Humanos , Poliovirus/genética , Poliovirus/patogenicidade , Transdução de Sinais , Células Tumorais Cultivadas
13.
Mol Cell Biol ; 25(3): 1089-99, 2005 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-15657435

RESUMO

Animal cells counteract oxidative stress and electrophilic attack through coordinated expression of a set of detoxifying and antioxidant enzyme genes mediated by transcription factor Nrf2. In unstressed cells, Nrf2 appears to be sequestered in the cytoplasm via association with an inhibitor protein, Keap1. Here, by using the yeast two-hybrid screen, human Keap1 has been identified as a partner of the nuclear protein prothymosin alpha. The in vivo and in vitro data indicated that the prothymosin alpha-Keap1 interaction is direct, highly specific, and functionally relevant. Furthermore, we showed that Keap1 is a nuclear-cytoplasmic shuttling protein equipped with a nuclear export signal that is important for its inhibitory action. Prothymosin alpha was able to liberate Nrf2 from the Nrf2-Keap1 inhibitory complex in vitro through competition with Nrf2 for binding to the same domain of Keap1. In vivo, the level of Nrf2-dependent transcription was correlated with the intracellular level of prothymosin alpha by using prothymosin alpha overproduction and mRNA interference approaches. Our data attribute to prothymosin alpha the role of intranuclear dissociator of the Nrf2-Keap1 complex, thus revealing a novel function for prothymosin alpha and adding a new dimension to the molecular mechanisms underlying expression of oxidative stress-protecting genes.


Assuntos
Proteínas de Ligação a DNA/metabolismo , Estresse Oxidativo/genética , Precursores de Proteínas/metabolismo , Proteínas/metabolismo , Timosina/análogos & derivados , Timosina/metabolismo , Transativadores/metabolismo , Ativação Transcricional/genética , Transporte Ativo do Núcleo Celular/genética , Transporte Ativo do Núcleo Celular/fisiologia , Regulação Neoplásica da Expressão Gênica/genética , Regulação Neoplásica da Expressão Gênica/fisiologia , Células HeLa , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Proteína 1 Associada a ECH Semelhante a Kelch , Fator 2 Relacionado a NF-E2 , Estresse Oxidativo/fisiologia , Ligação Proteica , RNA Interferente Pequeno/metabolismo , Ativação Transcricional/fisiologia , Células Tumorais Cultivadas , Técnicas do Sistema de Duplo-Híbrido
14.
Gastroenterology ; 127(2): 582-94, 2004 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-15300590

RESUMO

BACKGROUND & AIMS: The emergence of oxyntic atrophy and metaplastic cell lineages in response to chronic Helicobacter pylori infection predisposes to gastric neoplasia. We have described a trefoil factor family 2 (TFF2; spasmolytic polypeptide) expressing metaplasia (SPEM) associated with gastric neoplasia in both rodent and human fundus. To examine the relationship of SPEM to the neoplastic process in the H. felis -infected C57BL/6 mouse, we have now studied the association of SPEM-related transcripts with preneoplasia. METHODS: SPEM-related transcripts were identified by microarray analysis of amplified cRNA from SPEM, and surface mucous cells were isolated by laser capture microdissection from the same gastric sections from male C57BL/6 mice infected with H. felis for 6 months. Expression of SPEM-related transcripts was assessed by in situ hybridization and quantitative RT-PCR, as well as immunohistochemistry for prothymosin alpha. RESULTS: Eleven SPEM-related transcripts were identified as detectable only in SPEM. The expression of the SPEM-related transcripts was validated by in situ hybridization and quantitative PCR. One transcript, the noncoding RNA Xist, was only identified in SPEM cells from the infected male mice. Ten of the 11 transcripts as well as TFF2 were also expressed in regions of gastritis cystica profunda. Immunocytochemistry for one of the identified proteins, prothymosin alpha, demonstrated prominent nuclear staining in SPEM and gastritis cystica profunda. CONCLUSIONS: The expression of SPEM-related transcripts in regions of gastritis cystica profunda suggests that SPEM represents a precursor lineage for the development of dysplasia in this animal model of gastric carcinogenesis.


Assuntos
Infecções por Helicobacter/fisiopatologia , Helicobacter felis , Mucinas , Proteínas Musculares , Neuropeptídeos , Peptídeos/genética , Lesões Pré-Cancerosas/fisiopatologia , Neoplasias Gástricas/fisiopatologia , Timosina/análogos & derivados , Animais , Infecções por Helicobacter/complicações , Infecções por Helicobacter/patologia , Imuno-Histoquímica , Hibridização In Situ , Peptídeos e Proteínas de Sinalização Intercelular , Masculino , Metaplasia , Camundongos , Camundongos Endogâmicos C57BL , Análise de Sequência com Séries de Oligonucleotídeos , Peptídeos/metabolismo , Reação em Cadeia da Polimerase , Lesões Pré-Cancerosas/microbiologia , Lesões Pré-Cancerosas/patologia , Precursores de Proteínas/metabolismo , RNA Mensageiro/análise , Neoplasias Gástricas/microbiologia , Neoplasias Gástricas/patologia , Timosina/metabolismo , Fator Trefoil-2 , Fator Trefoil-3
15.
Exp Cell Res ; 298(1): 197-206, 2004 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-15242774

RESUMO

Interferons (IFNs) play critical roles in host defense by modulating the expression of various genes via tyrosine phosphorylation of STAT transcription factors. Many cytokines including IFNs induce tyrosine phosphorylation of the STAT3 transcription factor, which regulates acute phase gene expression. Using the yeast two-hybrid interaction trap, in which a tyrosine kinase is introduced into the yeast to allow tyrosine phosphorylation of bait proteins, prothymosin-alpha (ProTalpha) was identified to interact with the amino terminal half of tyrosine-phosphorylated STAT3. ProTalpha is a small, acidic, extremely abundant, and essential protein that may play a role in chromatin remodeling, and has been implicated in regulating the growth and survival of mammalian cells. Besides the interaction of tyrosine-phosphorylated STAT3 with ProTalpha in yeast cells, IFN induced the interaction of ProTalpha with STAT3 in mammalian cells, and this interaction was dependent on the tyrosine phosphorylation of STAT3. Moreover, IFNalpha induces the translocation of STAT3 and ProTalpha from the cytoplasm to the nucleus where these proteins colocalize. Since ProTalpha has an extremely strong nuclear localization and STAT proteins apparently lack any nuclear localization signals, the association of STAT3 with ProTalpha may provide a mechanism to result in STAT localization in the nucleus.


Assuntos
Núcleo Celular/metabolismo , Proteínas de Ligação a DNA/metabolismo , Interferons/fisiologia , Precursores de Proteínas/metabolismo , Timosina/análogos & derivados , Timosina/metabolismo , Transativadores/metabolismo , Transporte Ativo do Núcleo Celular/efeitos dos fármacos , Transporte Ativo do Núcleo Celular/fisiologia , Animais , Células COS , Núcleo Celular/efeitos dos fármacos , Interferon-alfa/farmacologia , Interferons/farmacologia , Substâncias Macromoleculares , Fosforilação , Estrutura Terciária de Proteína/fisiologia , Transporte Proteico/efeitos dos fármacos , Transporte Proteico/fisiologia , Fator de Transcrição STAT3 , Técnicas do Sistema de Duplo-Híbrido , Tirosina/metabolismo , Leveduras/metabolismo
16.
Plant Cell ; 16(1): 157-71, 2004 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-14660804

RESUMO

To test the hypothesis that caspase-like proteases exist and are critically involved in the implementation of programmed cell death (PCD) in plants, a search was undertaken for plant caspases activated during the N gene-mediated hypersensitive response (HR; a form of pathogen-induced PCD in plants) in tobacco plants infected with Tobacco mosaic virus (TMV). For detection, characterization, and partial purification of a tobacco caspase, the Agrobacterium tumefaciens VirD2 protein, shown here to be cleaved specifically at two sites (TATD and GEQD) by human caspase-3, was used as a target. In tobacco leaves, specific proteolytic processing of the ectopically produced VirD2 derivatives at these sites was found to occur early in the course of the HR triggered by TMV. A proteolytic activity capable of specifically cleaving the model substrate at TATD was partially purified from these leaves. A tetrapeptide aldehyde designed and synthesized on the basis of the elucidated plant caspase cleavage site prevented fragmentation of the substrate protein by plant and human caspases in vitro and counteracted TMV-triggered HR in vivo. Therefore, our data provide a characterization of caspase-specific protein fragmentation in apoptotic plant cells, with implications for the importance of such activity in the implementation of plant PCD.


Assuntos
Endopeptidases/metabolismo , Tabaco/enzimologia , Agrobacterium tumefaciens/crescimento & desenvolvimento , Apoptose/efeitos dos fármacos , Apoptose/fisiologia , Caspase 3 , Inibidores de Caspase , Caspases/metabolismo , Inibidores de Cisteína Proteinase/farmacologia , Endopeptidases/genética , Ativação Enzimática , Proteínas de Fluorescência Verde , Humanos , Imunidade Inata/genética , Imunidade Inata/fisiologia , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Doenças das Plantas/genética , Doenças das Plantas/virologia , Tabaco/genética , Tabaco/virologia , Vírus do Mosaico do Tabaco/efeitos dos fármacos , Vírus do Mosaico do Tabaco/crescimento & desenvolvimento
17.
Exp Cell Res ; 284(2): 211-23, 2003 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-12651154

RESUMO

Human prothymosin alpha is a proliferation-related nuclear protein undergoing caspase-mediated fragmentation in apoptotic cells. We show here that caspase-3 is the principal executor of prothymosin alpha fragmentation in vivo. In apoptotic HeLa cells as well as in vitro, caspase-3 cleaves prothymosin alpha at one major carboxy terminal (DDVD(99)) and several suboptimal sites. Prothymosin alpha cleavage at two amino-terminal sites (AAVD(6) and NGRD(31)) contributes significantly to the final pattern of prothymosin alpha fragmentation in vitro and could be detected to occur in apoptotic cells. The major caspase cleavage at D(99) disrupts the nuclear localization signal of prothymosin alpha, which leads to a profound alteration in subcellular localization of the truncated protein. By using a set of anti-prothymosin alpha monoclonal antibodies, we were able to observe nuclear escape and cell surface exposure of endogenous prothymosin alpha in apoptotic, but not in normal, cells. We demonstrate also that ectopic production of human prothymosin alpha and its mutants with nuclear or nuclear-cytoplasmic localization confers increased resistance of HeLa cells toward the tumor necrosis factor-induced apoptosis.


Assuntos
Apoptose/fisiologia , Caspases/metabolismo , Células Eucarióticas/metabolismo , Fragmentos de Peptídeos/metabolismo , Precursores de Proteínas/biossíntese , Transporte Proteico/fisiologia , Timosina/análogos & derivados , Timosina/biossíntese , Transporte Ativo do Núcleo Celular/efeitos dos fármacos , Transporte Ativo do Núcleo Celular/fisiologia , Sequência de Aminoácidos/fisiologia , Anticorpos Monoclonais , Apoptose/efeitos dos fármacos , Caspase 3 , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/metabolismo , Citoplasma/efeitos dos fármacos , Citoplasma/metabolismo , Exocitose/efeitos dos fármacos , Exocitose/fisiologia , Células HeLa , Humanos , Mutação/genética , Precursores de Proteínas/antagonistas & inibidores , Precursores de Proteínas/genética , Estrutura Terciária de Proteína/efeitos dos fármacos , Estrutura Terciária de Proteína/fisiologia , Transporte Proteico/efeitos dos fármacos , Timosina/antagonistas & inibidores , Timosina/genética
18.
Biochim Biophys Acta ; 1557(1-3): 109-17, 2003 Mar 06.
Artigo em Inglês | MEDLINE | ID: mdl-12615354

RESUMO

Many apoptotic signals are known to induce release to cytosol of cytochrome c, a small mitochondrial protein with positively charged amino acid residues dominating over negatively charged ones. On the other hand, in this group, it was shown that prothymosin alpha (PT), a small nuclear protein where 53 of 109 amino acid residues are negatively charged, is truncated to form a protein of 99 amino acid residues which accumulates in cytosol during apoptosis [FEBS Lett. 467 (2000) 150]. It was suggested that positively charged cytochrome c and negatively charged truncated prothymosin alpha (tPT), when meeting in cytosol, can interact with each other. In this paper, such an interaction is shown. (1) Formation of cytochrome cz.ccirf;tPT complex is demonstrated by a blot-overlay assay. (2) Analytical centrifugation of solution containing cytochrome c and tPT reveals formation of complexes of molecular masses higher than those of these proteins. The masses increase when the cytochrome c/tPT ratio increases. High concentration of KCl prevents the complex formation. (3) In the complexes formed, cytochrome c becomes autoxidizable; its reduction by superoxide or ascorbate as well as its operation as electron carrier between the outer and inner mitochondrial membranes appear to be inhibited. (4) tPT inhibits cytochrome c oxidation by H(2)O(2), catalyzed by peroxidase. Thus, tPT abolishes all antioxidant functions of cytochrome c which, in the presence of tPT, becomes in fact a pro-oxidant. A possible role of tPT in the development of reactive oxygen species- and cytochrome c-mediated apoptosis is discussed.


Assuntos
Grupo dos Citocromos c/química , Precursores de Proteínas/química , Timosina/análogos & derivados , Timosina/química , Animais , Caspase 3 , Caspases , Grupo dos Citocromos c/metabolismo , Escherichia coli/metabolismo , Peróxido de Hidrogênio , Mitocôndrias Hepáticas/metabolismo , Oxirredução , Peroxidase/antagonistas & inibidores , Ácido Poliglutâmico , Precursores de Proteínas/biossíntese , Precursores de Proteínas/metabolismo , Ratos , Superóxidos , Timosina/biossíntese , Timosina/metabolismo , Zinco
19.
J Immunol Methods ; 266(1-2): 185-96, 2002 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-12133636

RESUMO

To overcome poor immunogenicity of prothymosin alpha, a small and highly acidic nuclear protein involved in cell proliferation, production of anti-prothymosin alpha antibodies in mice immunized with free human prothymosin alpha, with prothymosin alpha coupled to different carriers and with prothymosin alpha fused to green fluorescent protein was assessed. Fusing prothymosin alpha to green fluorescent protein turned out to be the superior approach resulting in production of high titer anti-prothymosin alpha antibodies. From these studies, two highly specific anti-prothymosin alpha monoclonal antibodies recognizing epitopes within the amino terminal (2F11) and middle (4F4) portions of the human prothymosin alpha molecule were obtained and characterized. As expected, the 2F11 antibody displayed broad species specificity, whereas the 4F4 antibody appeared to be species-specific permitting discrimination of human versus rat protein. Furthermore, a combination of point mutations in prothymosin alpha that alter the properties of the protein precluded recognition by the 4F4 antibody. Intramolecular masking of the 4F4 epitope in prothymosin alpha fused to the Tat transduction peptide of human immunodeficiency virus type 1 was observed. The anti-prothymosin alpha antibodies obtained were suitable for precipitation of human prothymosin alpha from HeLa cell lysates and for immunolocalization of the endogenous prothymosin alpha within the cells. Fusion with green fluorescent protein may thus be helpful in raising antibodies against 'problematic' proteins.


Assuntos
Anticorpos Monoclonais/imunologia , Precursores de Proteínas/imunologia , Timosina/análogos & derivados , Timosina/imunologia , Animais , Especificidade de Anticorpos , Mapeamento de Epitopos , Produtos do Gene tat/genética , Proteínas de Fluorescência Verde , Células HeLa , Humanos , Proteínas Luminescentes/genética , Camundongos , Camundongos Endogâmicos BALB C , Mutação Puntual , Conformação Proteica , Precursores de Proteínas/química , Precursores de Proteínas/genética , Proteínas Recombinantes de Fusão/imunologia , Especificidade da Espécie , Timosina/química , Timosina/genética
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