RESUMO
KEY MESSAGE: The green - revertible yellow79 mutant resulting from a single-base mutation suggested that the GRY79 gene encoding a putative metallo-ß-lactamase-trihelix chimera is involved in chloroplast development at early seedling stage of rice. Functional studies of metallo-ß-lactamases and trihelix transcription factors in higher plants remain very sparse. In this study, we isolated the green-revertible yellow79 (gry79) mutant in rice. The mutant developed yellow-green leaves before the three-leaf stage but recovered to normal green at the sixth-leaf stage. Meanwhile, the mutant exhibited reduced level of chlorophylls and arrested development of chloroplasts in the yellow leaves. Genetic analysis suggested that the mutant phenotype was controlled by a single recessive nuclear gene on rice chromosome 2. Map-based cloning revealed that the candidate gene was Os02g33610 encoding a putative metallo-ß-lactamase-trihelix chimera. In the gry79 mutant, a single-base mutation occurred in coding region of the gene, resulting in an amino acid change in the encoded protein. Furthermore, the mutant phenotype was rescued by transformation with the wild-type gene. Therefore, we have confirmed that the gry79 mutant phenotype resulted from a single-base mutation in GRY79 (Os02g33610) gene, suggesting that the gene encoding a putative metallo-ß-lactamase-trihelix chimera is involved in chloroplast development at early seedling stage of rice. In addition, we considered that the gry79 mutant gene could be applicable as a leaf-color marker gene for efficient identification and elimination of false hybrids in commercial hybrid rice production.
Assuntos
Cloroplastos/fisiologia , Genes de Plantas/fisiologia , Proteínas Mutantes Quiméricas/genética , Oryza/crescimento & desenvolvimento , Plântula/crescimento & desenvolvimento , Fatores de Transcrição/genética , beta-Lactamases/genética , Substituição de Aminoácidos/genética , Substituição de Aminoácidos/fisiologia , Genes de Plantas/genética , Proteínas Mutantes Quiméricas/fisiologia , Oryza/genética , Frações Subcelulares/química , Fatores de Transcrição/fisiologia , beta-Lactamases/fisiologiaAssuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/fisiologia , Metiltransferases/fisiologia , Proteínas Mutantes Quiméricas/fisiologia , Neoplasias da Próstata/metabolismo , Proteínas Repressoras/fisiologia , Idoso , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Linhagem Celular Tumoral , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Rearranjo Gênico , Histona-Lisina N-Metiltransferase , Humanos , Masculino , Metiltransferases/genética , Metiltransferases/metabolismo , Pessoa de Meia-Idade , Proteínas Mutantes Quiméricas/genética , Proteínas Mutantes Quiméricas/metabolismo , Neoplasias da Próstata/patologia , Neoplasias da Próstata/cirurgia , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Análise de Sequência de DNARESUMO
Photoreceptor phosphodiesterase-6 (PDE6) is a peripheral membrane protein synthesized in the inner segment of photoreceptor cells. Newly synthesized PDE6 is transported to the outer segment (OS) where it serves as a key effector enzyme in the phototransduction cascade. Proper localization of PDE6 in photoreceptors is critically important to the function and survival of photoreceptor cells. The mechanism of PDE6 transport to the OS remains largely unknown. In this study, we investigated potential OS targeting signals of PDE6 by constructing cGMP-binding, cGMP-specific phosphodiesterase-5/PDE6 chimeric proteins and analyzing their localization in rods of transgenic Xenopus laevis. We found that efficient OS localization of chimeric isoprenylated PDE enzymes required the presence of a targeting motif within the PDE6 GAFa domain. Furthermore, the GAFa-dependent localization signal was sufficient to target GAFa fusion protein to the OS. Our results support the idea that effective trafficking of the peripheral membrane proteins to the OS of photoreceptor cells requires a sorting/targeting motif in addition to a membrane-binding signal.
Assuntos
Nucleotídeo Cíclico Fosfodiesterase do Tipo 6/fisiologia , Segmento Externo da Célula Bastonete/fisiologia , Animais , Animais Geneticamente Modificados , Western Blotting , Clonagem Molecular , Olho/química , Olho/metabolismo , Humanos , Larva , Fígado/citologia , Fígado/metabolismo , Microscopia Confocal , Proteínas Mutantes Quiméricas/fisiologia , Mutação/fisiologia , Inibidores de Fosfodiesterase/farmacologia , Células Fotorreceptoras Retinianas Bastonetes/efeitos dos fármacos , Células Fotorreceptoras Retinianas Bastonetes/fisiologia , Transdução de Sinais/fisiologia , Xenopus laevisRESUMO
The vesicular glutamate transporters (VGLUTs) package glutamate into synaptic vesicles, and the two principal isoforms VGLUT1 and VGLUT2 have been suggested to influence the properties of release. To understand how a VGLUT isoform might influence transmitter release, we have studied their trafficking and previously identified a dileucine-like endocytic motif in the C terminus of VGLUT1. Disruption of this motif impairs the activity-dependent recycling of VGLUT1, but does not eliminate its endocytosis. We now report the identification of two additional dileucine-like motifs in the N terminus of VGLUT1 that are not well conserved in the other isoforms. In the absence of all three motifs, rat VGLUT1 shows limited accumulation at synaptic sites and no longer responds to stimulation. In addition, shRNA-mediated knockdown of clathrin adaptor proteins AP-1 and AP-2 shows that the C-terminal motif acts largely via AP-2, whereas the N-terminal motifs use AP-1. Without the C-terminal motif, knockdown of AP-1 reduces the proportion of VGLUT1 that responds to stimulation. VGLUT1 thus contains multiple sorting signals that engage distinct trafficking mechanisms. In contrast to VGLUT1, the trafficking of VGLUT2 depends almost entirely on the conserved C-terminal dileucine-like motif: without this motif, a substantial fraction of VGLUT2 redistributes to the plasma membrane and the transporter's synaptic localization is disrupted. Consistent with these differences in trafficking signals, wild-type VGLUT1 and VGLUT2 differ in their response to stimulation.
Assuntos
Leucina/genética , Leucina/fisiologia , Proteína Vesicular 1 de Transporte de Glutamato/fisiologia , Complexo 2 de Proteínas Adaptadoras/metabolismo , Motivos de Aminoácidos/fisiologia , Sequência de Aminoácidos , Animais , Células Cultivadas , Clatrina/metabolismo , Endocitose/fisiologia , Hipocampo/citologia , Hipocampo/metabolismo , Concentração de Íons de Hidrogênio , Imuno-Histoquímica , Dados de Sequência Molecular , Proteínas Mutantes Quiméricas/genética , Proteínas Mutantes Quiméricas/fisiologia , Reação em Cadeia da Polimerase , Interferência de RNA , Ratos , Proteína Vesicular 1 de Transporte de Glutamato/genética , Proteína Vesicular 2 de Transporte de Glutamato/genética , Proteína Vesicular 2 de Transporte de Glutamato/fisiologiaRESUMO
Antitumor alkylphospholipids initiate apoptosis in transformed HL-60 and Jurkat cells while sparing their progenitors. 1-O-Alkyl-2-carboxymethyl-sn-glycero-3-phosphocholine (Edelfosine) like other short-chained phospholipids--inflammatory platelet-activating factor (PAF) and apoptotic oxidatively truncated phospholipids--are proposed to have intracellular sites of action, yet a conduit for these choline phospholipids into mammalian cells is undefined. Edelfosine is also accumulated by Saccharomyces cerevisiae in a process requiring the membrane protein Lem3p, and the human genome contains a Lem3p homolog TMEM30a. We show that import of choline phospholipids into S. cerevisiae ΔLem3 is partially reconstituted by human TMEM30a and by Lem3p-TMEM30a chimeras, showing the proteins are orthologous. TMEM30a-GFP chimeras expressed in mammalian cells localized in plasma membranes, as well as internal organelles, and ectopic TMEM30a expression promoted uptake of exogenous choline and ethanolamine phospholipids. Short hairpin RNA knockdown of TMEM30a reduced fluorescent choline phospholipid and [(3)H]PAF import. This knockdown also reduced mitochondrial depolarization from exogenous Edelfosine or the mitotoxic oxidatively truncated phospholipid azelaoyl phosphatidylcholine, and the knockdown reduced apoptosis in response to these two phospholipids. These results show that extracellular choline phospholipids with short sn-2 residues can have intracellular roles and sites of metabolism because they are transport substrates for a TMEM30a phospholipid import system. Variation in this mechanism could limit sensitivity to short chain choline phospholipids such as Edelfosine, PAF, and proapoptotic phospholipids.
Assuntos
Antineoplásicos/metabolismo , Colina/metabolismo , Proteínas de Membrana/fisiologia , Fosfolipídeos/metabolismo , Sequência de Aminoácidos , Animais , Transporte Biológico Ativo/genética , Transporte Biológico Ativo/imunologia , Células CHO , Linhagem Celular Transformada , Cricetinae , Cricetulus , Resistencia a Medicamentos Antineoplásicos/genética , Resistencia a Medicamentos Antineoplásicos/imunologia , Células HL-60 , Células Hep G2 , Humanos , Células Jurkat , Proteínas de Membrana/antagonistas & inibidores , Proteínas de Membrana/genética , Proteínas de Membrana Transportadoras/genética , Proteínas de Membrana Transportadoras/fisiologia , Dados de Sequência Molecular , Proteínas Mutantes Quiméricas/biossíntese , Proteínas Mutantes Quiméricas/genética , Proteínas Mutantes Quiméricas/fisiologia , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/fisiologia , Homologia de Sequência de AminoácidosRESUMO
Large-conductance, Ca(2+)- and voltage-sensitive K(+) (BK) channels regulate neuronal functions such as spike frequency adaptation and transmitter release. BK channels are composed of four Slo1 subunits, which contain the voltage-sensing and pore-gate domains in the membrane and Ca(2+) binding sites in the cytoplasmic domain, and accessory ß subunits. Four types of BK channel ß subunits (ß1-ß4) show differential tissue distribution and unique functional modulation, resulting in diverse phenotypes of BK channels. Previous studies show that both the ß1 and ß2 subunits increase Ca(2+) sensitivity, but different mechanisms may underline these modulations. However, the structural domains in Slo1 that are critical for Ca(2+)-dependent activation and targeted by these ß subunits are not known. Here, we report that the N termini of both the transmembrane (including S0) and cytoplasmic domains of Slo1 are critical for ß2 modulation based on the study of differential effects of the ß2 subunit on two orthologs, mouse Slo1 and Drosophila Slo1. The N terminus of the cytoplasmic domain of Slo1, including the AC region (ßA-αC) of the RCK1 (regulator of K(+) conductance) domain and the peptide linking it to S6, both of which have been shown previously to mediate the coupling between Ca(2+) binding and channel opening, is specifically required for the ß2 but not for the ß1 modulation. These results suggest that the ß2 subunit modulates the coupling between Ca(2+) binding and channel opening, and, although sharing structural homology, the BK channel ß subunits interact with structural domains in the Slo1 subunit differently to enhance channel activity.
Assuntos
Membrana Celular/química , Membrana Celular/fisiologia , Citoplasma/fisiologia , Ativação do Canal Iônico/fisiologia , Subunidades alfa do Canal de Potássio Ativado por Cálcio de Condutância Alta/química , Subunidades alfa do Canal de Potássio Ativado por Cálcio de Condutância Alta/fisiologia , Subunidades beta do Canal de Potássio Ativado por Cálcio de Condutância Alta/fisiologia , Sequência de Aminoácidos , Animais , Membrana Celular/genética , Citoplasma/química , Drosophila , Feminino , Humanos , Subunidades alfa do Canal de Potássio Ativado por Cálcio de Condutância Alta/genética , Subunidades beta do Canal de Potássio Ativado por Cálcio de Condutância Alta/química , Canais de Potássio Ativados por Cálcio de Condutância Alta/química , Canais de Potássio Ativados por Cálcio de Condutância Alta/fisiologia , Camundongos , Dados de Sequência Molecular , Proteínas Mutantes Quiméricas/química , Proteínas Mutantes Quiméricas/genética , Proteínas Mutantes Quiméricas/fisiologia , Estrutura Terciária de Proteína/genética , Xenopus laevisRESUMO
Saccharomyces cerevisiae Pex3p has been shown to act at the ER during de novo peroxisome formation. However, its steady state is at the peroxisomal membrane, where its role is debated. Here we show that Pex3p has a dual function: one in peroxisome formation and one in peroxisome segregation. We show that the peroxisome retention factor Inp1p interacts physically with Pex3p in vitro and in vivo, and split-GFP analysis shows that the site of interaction is the peroxisomal membrane. Furthermore, we have generated PEX3 alleles that support peroxisome formation but fail to support recruitment of Inp1p to peroxisomes, and as a consequence are affected in peroxisome segregation. We conclude that Pex3p functions as an anchor for Inp1p at the peroxisomal membrane, and that this function is independent of its role at the ER in peroxisome biogenesis.
Assuntos
Proteínas de Membrana/fisiologia , Peroxissomos/genética , Peroxissomos/metabolismo , Proteínas de Saccharomyces cerevisiae/fisiologia , Saccharomyces cerevisiae/fisiologia , Membranas Intracelulares/metabolismo , Membranas Intracelulares/fisiologia , Proteínas de Membrana/antagonistas & inibidores , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Proteínas Mutantes Quiméricas/genética , Proteínas Mutantes Quiméricas/fisiologia , Peroxinas , Peroxissomos/fisiologia , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crescimento & desenvolvimento , Proteínas de Saccharomyces cerevisiae/antagonistas & inibidores , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMO
Receptor-protein tyrosine phosphatases (RPTPs), like receptor tyrosine kinases, regulate neuronal differentiation. While receptor tyrosine kinases are dimerized and activated by extracellular ligands, the extent to which RPTPs dimerize, and the effects of dimerization on phosphatase activity, are poorly understood. We have examined a neuronal type III RPTP, PTPRO; we find that PTPRO can form dimers in living cells, and that disulfide linkages in PTPROs intracellular domain likely regulate dimerization. Dimerization of PTPROs transmembrane and intracellular domains, achieved by ligand binding to a chimeric fusion protein, decreases activity toward artificial peptides and toward a putative substrate, tropomyosin-related kinase C (TrkC). Dephosphorylation of TrkC by PTPRO may be physiologically relevant, as it is efficient, and TrkC and PTPRO can be co-precipitated from transfected cells. Inhibition of PTPROs phosphatase activity by dimerization is interesting, as dimerization of a related RPTP, CD148/PTPRJ, increases activity. Thus, our results suggest a complex relationship between dimerization and activity in type III RPTPs.
Assuntos
Multimerização Proteica , Receptor trkC/antagonistas & inibidores , Receptor trkC/metabolismo , Proteínas Tirosina Fosfatases Classe 3 Semelhantes a Receptores/fisiologia , Animais , Células COS , Galinhas , Chlorocebus aethiops , Dissulfetos/química , Dissulfetos/metabolismo , Ativação Enzimática/genética , Humanos , Ligação de Hidrogênio , Camundongos , Camundongos Knockout , Mutagênese Sítio-Dirigida , Proteínas Mutantes Quiméricas/química , Proteínas Mutantes Quiméricas/genética , Proteínas Mutantes Quiméricas/fisiologia , Multimerização Proteica/genética , Estrutura Terciária de Proteína/genética , Proteínas Tirosina Fosfatases Classe 3 Semelhantes a Receptores/química , Proteínas Tirosina Fosfatases Classe 3 Semelhantes a Receptores/genéticaRESUMO
The superfamily of globins has emerged some 4000 Myr from a common ancestor, which was among the basic protein components required for life. Globins are present in the three kingdoms of life. From a structure point of view, these molecules are defined by the presence of a characteristic protein fold, rich in alpha-helix, surrounding a heme group. Depending on the species or organs, they may be physiologically active as monomers, tetramers or large size polymers. Their function varies from the classical reversible binding of oxygen for transport and storage to cytoprotection against reactive oxygen species, NO scavenging, signaling in oxygen dependent metabolic pathways, or possibly other specific properties involving ligand or electron transfer. All these aspects are discussed in this review.
Assuntos
Evolução Biológica , Globinas/química , Globinas/fisiologia , Adaptação Fisiológica , Animais , Globinas/genética , Hemoglobinas/química , Hemoglobinas/genética , Hemoglobinas/fisiologia , Humanos , Modelos Moleculares , Proteínas Mutantes Quiméricas/química , Proteínas Mutantes Quiméricas/genética , Proteínas Mutantes Quiméricas/fisiologia , Relação Estrutura-AtividadeRESUMO
BACKGROUND: FUS-DDIT3 is a chimeric protein generated by the most common chromosomal translocation t(12;16)(q13;p11) linked to liposarcomas, which are characterized by the accumulation of early adipocytic precursors. Current studies indicate that FUS-DDIT3- liposarcoma develops from uncommitted progenitors. However, the precise mechanism whereby FUS-DDIT3 contributes to the differentiation arrest remains to be elucidated. METHODOLOGY/PRINCIPAL FINDINGS: Here we have characterized the adipocyte regulatory protein network in liposarcomas of FUS-DITT3 transgenic mice and showed that PPARgamma2 and C/EBPalpha expression was altered. Consistent with in vivo data, FUS-DDIT3 MEFs and human liposarcoma cell lines showed a similar downregulation of both PPARgamma2 and C/EBPalpha expression. Complementation studies with PPARgamma but not C/EBPalpha rescued the differentiation block in committed adipocytic precursors expressing FUS-DDIT3. Our results further show that FUS-DDIT3 interferes with the control of initiation of translation by upregulation of the eukaryotic translation initiation factors eIF2 and eIF4E both in FUS-DDIT3 mice and human liposarcomas cell lines, explaining the shift towards the truncated p30 isoform of C/EBPalpha in liposarcomas. Suppression of the FUS-DDIT3 transgene did rescue this adipocyte differentiation block. Moreover, eIF4E was also strongly upregulated in normal adipose tissue of FUS-DDIT3 transgenic mice, suggesting that overexpression of eIF4E may be a primary event in the initiation of liposarcomas. Reporter assays showed FUS-DDIT3 is involved in the upregulation of eIF4E in liposarcomas and that both domains of the fusion protein are required for affecting eIF4E expression. CONCLUSIONS/SIGNIFICANCE: Taken together, this study provides evidence of the molecular mechanisms involve in the disruption of normal adipocyte differentiation program in liposarcoma harbouring the chimeric gene FUS-DDIT3.
Assuntos
Adipócitos/citologia , Proteína alfa Estimuladora de Ligação a CCAAT/antagonistas & inibidores , Fator de Iniciação 4E em Eucariotos/metabolismo , Lipossarcoma/metabolismo , Proteínas Mutantes Quiméricas/fisiologia , Proteínas de Fusão Oncogênica/fisiologia , PPAR gama/antagonistas & inibidores , Animais , Linhagem Celular , Humanos , Lipossarcoma/patologia , Camundongos , Camundongos Transgênicos , Proteínas Mutantes Quiméricas/genética , Proteínas de Fusão Oncogênica/genética , Células-Tronco/metabolismo , Regulação para CimaRESUMO
Extracellular pH, especially in relatively inaccessible microdomains between cells, affects transport membrane protein activity and might have an intercellular signaling role. We have developed a genetically encoded extracellular pH sensor capable of detecting pH changes in basolateral spaces of epithelial cells. It consists of a chimerical membrane protein displaying concatenated enhanced variants of cyan fluorescence protein (ECFP) and yellow fluorescence protein (EYFP) at the external aspect of the cell surface. The construct, termed pHCECSensor01, was targeted to basolateral membranes of Madin-Darby canine kidney (MDCK) cells by means of a sequence derived from the aquaporin AQP4. The fusion of pH-sensitive EYFP with pH-insensitive ECFP allows ratiometric pH measurements. The titration curve of pHCECSensor01 in vivo had a pK (a) value of 6.5 +/- 0.04. Only minor effects of extracellular chloride on pHCECSensor01 were observed around the physiological concentrations of this anion. In MDCK cells, the sensor was able to detect changes in pH secondary to H(+) efflux into the basolateral spaces elicited by an ammonium prepulse or lactate load. This genetically encoded sensor has the potential to serve as a noninvasive tool for monitoring changes in extracellular pH microdomains in epithelial and other tissues in vivo.
Assuntos
Membrana Celular/fisiologia , Células Epiteliais/fisiologia , Proteínas Recombinantes/genética , Animais , Aquaporina 4/metabolismo , Aquaporina 4/fisiologia , Proteínas de Bactérias/química , Linhagem Celular , Cloretos/metabolismo , Interpretação Estatística de Dados , Cães , Vetores Genéticos , Proteínas de Fluorescência Verde/química , Hidrogênio/metabolismo , Concentração de Íons de Hidrogênio , Ácido Láctico/farmacologia , Proteínas Luminescentes/química , Proteínas Mutantes Quiméricas/metabolismo , Proteínas Mutantes Quiméricas/fisiologia , Plasmídeos , Compostos de Amônio Quaternário/farmacologiaRESUMO
AIM: The molecular mechanisms of the affinity regulation of alphavbeta3 integrin are important in tumor development, wound repairing, and angiogenesis. It has been established that the cytoplasmic domains of alphavbeta3 integrin play an important role in integrin-ligand affinity regulation. However, the relationship of structure-function within these domains remains unclear. METHODS: The extracellular and transmembrane domain of alphaIIb was fused to the alphav integrin cytoplasmic domain, and the chimeric alpha subunit was coexpressed in Chinese hamster ovary (CHO) cells with the wild-type beta3 subunit or with 3 mutant beta3 sequences bearing truncations at the positions of T741, Y747, and F754, respectively. The CHO cells expressing these recombinant integrins were tested for soluble fibrinogen binding and the cell adhesion and spreading on immobilized fibrinogen. RESULTS: All 4 types of integrins bound soluble fibrinogen in the absence of agonist stimulation, and only the cells expressing the chimeric alpha subunit with the wild-type beta3 subunit, but not those with truncated beta3, could adhere to and spread on immobilized fibrinogen. CONCLUSION: The substitution alphaIIb at the cytoplasmic domain with the alphav cytoplasmic sequence rendered the extracellular alphaIIbbeta3 a constitutively activated conformation for ligands without the need of pinside-outq signals. Our results also indicated that the COOH-terminal sequence of beta3 might play a key role in integrin alphaIIb/alphavbeta3-mediated cell adhesion and spreading on immobilized fibrinogen. The cells expressing alphaIIb/alphavbeta3 have enormous potential for facilitating drug screening for antagonists either to alphavbeta3 intracellular interactions or to alphaIIbbeta3 receptor functions.
Assuntos
Fibrinogênio/metabolismo , Integrina alfaVbeta3/genética , Integrina alfaVbeta3/fisiologia , Animais , Células CHO , Cricetinae , Cricetulus , Fibrinogênio/química , Humanos , Proteínas Mutantes Quiméricas/genética , Proteínas Mutantes Quiméricas/fisiologia , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologiaRESUMO
BACKGROUND: The male-specific region of the mouse Y chromosome long arm (MSYq) contains three known highly multi-copy X-Y homologous gene families, Ssty1/2, Sly and Asty. Deletions on MSYq lead to teratozoospermia and subfertility or infertility, with a sex ratio skew in the offspring of subfertile MSYqdel males RESULTS: We report the highly unusual genomic structure of a novel MSYq locus, Orly, and a diverse set of spermatid-specific transcripts arising from copies of this locus. Orly is composed of partial copies of Ssty1, Asty and Sly arranged in sequence. The Ssty1- and Sly-derived segments are in antisense orientation relative to each other, leading to bi-directional transcription of Orly. Genome search and phylogenetic tree analysis is used to determine the order of events in mouse Yq evolution. We find that Orly is the most recent gene to arise on Yq, and that subsequently there was massive expansion in copy number of all Yq-linked genes. CONCLUSION: Orly has an unprecedented chimeric structure, and generates both "forward" (Orly) and "reverse" (Orlyos) transcripts arising from the promoters at each end of the locus. The region of overlap of known Orly and Orlyos transcripts is homologous to Sly intron 2. We propose that Orly may be involved in an intragenomic conflict between mouse X and Y chromosomes, and that this process underlies the massive expansion in copy number of the genes on MSYq and their X homologues.
Assuntos
Evolução Molecular , Genes Ligados ao Cromossomo Y , Proteínas Mutantes Quiméricas/genética , Cromossomo Y/genética , Processamento Alternativo , Animais , Sequência de Bases , Mapeamento de Sequências Contíguas , Retrovirus Endógenos/genética , Componentes do Gene/genética , Dosagem de Genes , Masculino , Camundongos , Dados de Sequência Molecular , Família Multigênica , Proteínas Mutantes Quiméricas/fisiologia , Proteínas Nucleares/genética , Proteínas/genética , Alinhamento de Sequência , Homologia de Sequência do Ácido Nucleico , Testículo/crescimento & desenvolvimento , Testículo/metabolismo , Transcrição Gênica , Cromossomo Y/ultraestruturaRESUMO
The hypersensitive response (HR) involves programmed cell death (PCD) in response to pathogen infection. To investigate the pathogen resistance signaling pathway, we previously identified the Arabidopsis mutant cpr22, which displays constitutive activation of multiple defense responses including HR like cell death. The cpr22 mutation has been identified as a 3 kb deletion that fuses two cyclic nucleotide-gated ion channel (CNGC)-encoding genes, ATCNGC11 and ATCNGC12, to generate a novel chimeric gene, ATCNGC11/12. In this study, we conducted a characterization of cell death induced by transient expression of ATCNGC11/12 in Nicotiana benthamiana. Electron microscopic analysis of this cell death showed similar characteristics to PCD, such as plasma membrane shrinkage and vesicle formation. The hallmark of animal PCD, fragmentation of nuclear DNA, was also observed in ATCNGC11/12-induced cell death. The development of cell death was significantly suppressed by caspase-1 inhibitors, suggesting the involvement of caspases in this process. Recently, vacuolar processing enzyme (VPE) was isolated as the first plant caspase-like protein, which is involved in HR development. In VPE-silenced plants development of cell death induced by ATCNGC11/12 was much slower and weaker compared to control plants, suggesting the involvement of VPE as a caspase in ATCNGC11/12-induced cell death. Complementation analysis using a Ca2+ uptake deficient yeast mutant demonstrated that the ATCNGC11/12 channel is permeable to Ca2+. Additionally, calcium channel blockers such as GdCl3 inhibited ATCNGC11/12-induced HR formation, whereas potassium channel blockers did not. Taken together, these results indicate that the cell death that develops in the cpr22 mutant is indeed PCD and that the chimeric channel, ATCNGC11/12, is at the point of, or up-stream of the calcium signal necessary for the development of HR.
Assuntos
Apoptose/fisiologia , Proteínas de Arabidopsis/genética , Canais de Cálcio/genética , Sinalização do Cálcio , Canais de Cátion Regulados por Nucleotídeos Cíclicos/genética , Proteínas Mutantes Quiméricas/fisiologia , Apoptose/efeitos dos fármacos , Arabidopsis/citologia , Arabidopsis/metabolismo , Proteínas de Arabidopsis/análise , Proteínas de Arabidopsis/fisiologia , Canais de Cálcio/análise , Canais de Cálcio/fisiologia , Inibidores de Caspase , Caspases/fisiologia , Canais de Cátion Regulados por Nucleotídeos Cíclicos/análise , Canais de Cátion Regulados por Nucleotídeos Cíclicos/fisiologia , Cisteína Endopeptidases/fisiologia , Inibidores de Cisteína Proteinase/farmacologia , Fragmentação do DNA , Proteínas Mutantes Quiméricas/análise , Plantas Geneticamente Modificadas/citologia , Plantas Geneticamente Modificadas/efeitos dos fármacos , Plantas Geneticamente Modificadas/metabolismo , Nicotiana/genéticaRESUMO
During serial passaging of rubella virus (RUB) in cell culture, the dominant species of defective-interfering RNA (DI) generated contains an in-frame deletion between the capsid protein (C) gene and E1 glycoprotein gene resulting in production of a C-E1 fusion protein that is necessary for the maintenance of the DI [Tzeng, W.P., Frey, T.K. (2006). C-E1 fusion protein synthesized by rubella virus DI RNAs maintained during serial passage. Virology 356 198-207.]. A BHK cell line stably expressing the RUB structural proteins was established which was used to package DIs into virus particles following transfection with in vitro transcripts from DI infectious cDNA constructs. Packaging of a DI encoding an in-frame C-GFP-E1 reporter fusion protein corresponding to the C-E1 fusion protein expressed in a native DI was only marginally more efficient than packaging of a DI encoding GFP, indicating that the C-E1 fusion protein did not function by enhancing packaging. However, infection with the DI encoding the C-GFP-E1 fusion protein (in the absence of wt RUB helper virus) resulted in formation of clusters of GFP-positive cells and the percentage of GFP-positive cells in the culture following infection remained relatively constant. In contrast, a DI encoding GFP did not form GFP-positive clusters and the percentage of GFP-positive cells declined by roughly half from 2 to 4 days post-infection. Cluster formation and sustaining the percentage of infected (GFP-positive) cells required the C part of the fusion protein, including the downstream but not the upstream of two arginine clusters (both of which are associated with RNA binding and association with mitochondrial p32 protein) and the E1 part through the transmembrane sequence, but not the C-terminal cytoplasmic tail. Among a collection of mutant DI constructs, cluster formation and sustaining infected cell percentage correlated with maintenance during serial passage with wt RUB. We hypothesize that cluster formation and sustaining infected cell percentage increase the likelihood of co-infection by a DI and wt RUB during serial passage thus enhancing maintenance of the DI. Cluster formation and sustaining infected cell percentage were found to be due to a combination of attenuated cytopathogenicity of DIs that express the C-E1 fusion protein and cell-to-cell movement of the DI. In infected cells, the C-GFP-E1 fusion protein was localized to potentially novel vesicular structures that appear to originate from ER-Golgi transport vacuoles. This species of DI expressing a C-E1 fusion protein that exhibits attenuated cytopathogenicity and the ability to increase the number of infected cells through cell-to-cell movement could be the basis for development of an attractive vaccine vector.
Assuntos
Vírus Defeituosos/crescimento & desenvolvimento , Fusão Gênica/fisiologia , Proteínas Mutantes Quiméricas/fisiologia , Vírus da Rubéola/crescimento & desenvolvimento , Proteínas do Core Viral/fisiologia , Proteínas do Envelope Viral/fisiologia , Animais , Linhagem Celular , Cricetinae , Efeito Citopatogênico Viral , Vírus Defeituosos/genética , Vírus Defeituosos/patogenicidade , Fusão Gênica/genética , Genes Reporter , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Proteínas Mutantes Quiméricas/análise , Proteínas Mutantes Quiméricas/genética , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Vírus da Rubéola/patogenicidade , Inoculações Seriadas , Vesículas Transportadoras/química , Proteínas do Core Viral/genética , Proteínas do Envelope Viral/genética , Proteínas Estruturais Virais/análise , Proteínas Estruturais Virais/genética , Proteínas Estruturais Virais/fisiologia , Montagem de Vírus/genética , Montagem de Vírus/fisiologiaRESUMO
Lipoxygenases induce malignant tumor progression and lipoxygenase inhibitors have been considered as promising anti-tumor agents. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is one of the most promising candidates for new cancer therapeutics. Combined treatment with nordihydroguaiaretic acid (NDGA), a lipoxygenase inhibitor, and TRAIL markedly induced apoptosis in Jurkat T-cell leukemia cells at suboptimal concentrations for each agent. The combined treatment efficiently activated caspase-3, -8 and -10, and Bid. The underling mechanism by which NDGA enhanced TRAIL-induced apoptosis was examined. NDGA did not change the expression levels of anti-apoptotic factors, Bcl-x(L), Bcl-2, cIAP-1, XIAP and survivin. The expression of death receptor-related genes was investigated and it was found that NDGA specifically up-regulated the expression of death receptor 5 (DR5) at mRNA and protein levels. Down-regulation of DR5 by small interfering RNA prevented the sensitizing effect of NDGA on TRAIL-induced apoptosis. Furthermore, NDGA sensitized prostate cancer and colorectal cancer cells to TRAIL-induced apoptosis. In contrast, NDGA neither enhanced TRAIL-induced apoptosis nor up-regulated DR5 expression in normal peripheral blood mononuclear cells. Another lipoxygenase inhibitor, AA861, also up-regulated DR5 and sensitized Jurkat and DU145 cells to TRAIL. These results indicate that lipoxygenase inhibitors augment the apoptotic efficiency of TRAIL through DR5 up-regulation in malignant tumor cells, and raise the possibility that the combination of lipoxygenase inhibitor and TRAIL is a promising strategy for malignant tumor treatment.
Assuntos
Apoptose/efeitos dos fármacos , Leucemia de Células T/metabolismo , Leucemia de Células T/patologia , Inibidores de Lipoxigenase/farmacologia , Receptores do Ligante Indutor de Apoptose Relacionado a TNF/biossíntese , Receptores do Ligante Indutor de Apoptose Relacionado a TNF/genética , Ligante Indutor de Apoptose Relacionado a TNF/fisiologia , Apoptose/genética , Apoptose/fisiologia , Proteínas Reguladoras de Apoptose/genética , Proteínas Reguladoras de Apoptose/fisiologia , Benzoquinonas/farmacologia , Linhagem Celular Tumoral , Regulação para Baixo/efeitos dos fármacos , Sinergismo Farmacológico , Humanos , Fragmentos Fc das Imunoglobulinas/genética , Células Jurkat , Leucemia de Células T/enzimologia , Masoprocol/farmacologia , Proteínas Mutantes Quiméricas/fisiologia , Receptores do Ligante Indutor de Apoptose Relacionado a TNF/antagonistas & inibidores , Receptores do Ligante Indutor de Apoptose Relacionado a TNF/fisiologia , Regulação para Cima/efeitos dos fármacos , Regulação para Cima/genéticaRESUMO
The electrogenic Na+-HCO3- cotransporter (NBCe1) plays a central role in intracellular pH (pHi) regulation as well as HCO3- secretion by pancreatic ducts and HCO3- reabsorption by renal proximal tubules. To understand the structural requirements for the electrogenicity of NBCe1, we constructed chimeras of NBCe1-A and the electroneutral NBCn1-B, and used two-electrode voltage clamp to measure electrogenic transporter current in Xenopus oocytes exposed to 5% CO2-26 mm HCO3- (pH 7.40). The chimera consisting of NBCe1-A (i.e. NBCe1-A 'background') with the cytoplasmic N-terminal domain (Nt) of NBCn1-B had a reversal potential of -156.3 mV (compared with a membrane potential Vm of -43.1 mV in a HCO3(-)-free solution) and a slope conductance of 3.0 microS (compared with 12.5 microS for NBCe1-A). Also electrogenic were chimeras with an NBCe1-A background but with NBCn1-B contributing the extracellular loop (L) between transmembrane segment (TM) 5 and 6 (-140.9 mV/11.1 microS), the cytoplasmic C-terminal domain (Ct; -123.8 mV/9.7 microS) or Nt + L + Ct (-120.9 mV/3.7 microS). Reciprocal chimeras (with an NBCn1 background but with NBCe1 contributing Nt, L, Ct or Nt + L + Ct) produced no measurable electrogenic transporter currents in the presence of CO2-HCO3-. pHi recovered from an acid load, but without the negative shift of Vm that is characteristic of electrogenic Na+-HCO3- cotransporters. Thus, these chimeras were electroneutral, as were two others consisting of NBCe1(Nt-L)/NBCn1(TM6-Ct) and NBCn1(Nt-L)/NBCe1(TM6-Ct). We propose that the electrogenicity of NBCe1 requires interactions between TM1-5 and TM6-13.
Assuntos
Potenciais da Membrana/fisiologia , Simportadores de Sódio-Bicarbonato/fisiologia , Animais , Bicarbonatos/metabolismo , Citoplasma/metabolismo , Eletrofisiologia , Espaço Extracelular/fisiologia , Concentração de Íons de Hidrogênio , Dados de Sequência Molecular , Proteínas Mutantes Quiméricas/fisiologia , Oócitos/metabolismo , Técnicas de Patch-Clamp , Ratos , Simportadores de Sódio-Bicarbonato/genética , XenopusRESUMO
The ninaE-encoded Rh1 rhodopsin is the major light-sensitive pigment expressed in Drosophila R1-6 photoreceptor cells. Rh1 rhodopsin localizes to and is essential for the development and maintenance of the rhabdomere, the specialized membrane-rich organelle that serves as the site of phototransduction. We showed previously that the vertebrate bovine rhodopsin (Rho) is expressed and properly localized in Drosophila photoreceptor cells. Drosophila photoreceptors expressing only Rho have normal rhabdomere structure at young ages, but the rhabdomeres are not maintained and show extensive disorganization by 7-10 days of age. A series of Rho-Rh1 opsin chimeric rhodopsins were used to identify Rh1 domains required for maintenance of rhabdomeric structure. The results show that the Rh1 rhodopsin cytoplasmic tail domain, positioned to interact with cytoplasmic structural components, plays a major role in promoting rhabdomeric organization.
Assuntos
Proteínas de Drosophila/fisiologia , Células Fotorreceptoras de Invertebrados/metabolismo , Rodopsina/fisiologia , Opsinas de Bastonetes/fisiologia , Motivos de Aminoácidos , Sequência de Aminoácidos , Animais , Animais Geneticamente Modificados , Western Blotting , Bovinos , Proteínas de Drosophila/química , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Proteínas do Olho/química , Proteínas do Olho/genética , Proteínas do Olho/fisiologia , Imunofluorescência , Microscopia Eletrônica de Transmissão , Dados de Sequência Molecular , Proteínas Mutantes Quiméricas/química , Proteínas Mutantes Quiméricas/genética , Proteínas Mutantes Quiméricas/fisiologia , Células Fotorreceptoras de Invertebrados/fisiologia , Células Fotorreceptoras de Invertebrados/ultraestrutura , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Rodopsina/química , Rodopsina/genética , Opsinas de Bastonetes/química , Opsinas de Bastonetes/genéticaRESUMO
OBJECTIVE: In Old World monkeys, the tripartite motif 5alpha (TRIM5alpha) protein confers resistance to HIV-1 infection following virus entry into host cells. However, the pig-tailed macaque (Macaca nemestrina) is an exception and is susceptible to HIV-1 infection. This study dissects the molecular mechanism of the pig-tailed macaque's susceptibility to HIV-1 infection. METHODS: Genomic sequencing and expression analysis of the TRIM5alpha gene was conducted in the pig-tailed macaque. A novel TRIM5-Cyclophilin A fusion gene isoform was identified and subsequently cloned into the pcDNA3.1(+) expression vector. This construct was transfected into HeLa-T4 or HeLa cells which were then infected with the HIV-1IIIB or HIV-GFP-VSVG pseudotyped virus, to examine the effects of the TRIM5-Cyclophilin A fusion protein on HIV-1 infection. RESULTS: A novel TRIM5-Cyclophilin A fusion gene (mnTRIMCyp) in the pig-tailed macaque was found and its fusion pattern is different from the known fusion gene in the owl monkey (owlTRIMCyp). TRIMCyp protein expression in transfected cells was confirmed by western blotting. The tests using HIV-1IIIB and HIV-GFP-VSVG pseudotyped virus indicated that mnTRIMCyp did not inhibit HIV-1 replication at various multiplicities of infection. CONCLUSIONS: The mnTRIMCyp fusion protein does not restrict replication of HIV-1, which provides a potential molecular mechanism that might explain why the pig-tailed macaque is prone to HIV-1 infection, the only known exception in Old World monkeys.
Assuntos
Infecções por HIV/veterinária , HIV-1/fisiologia , Macaca nemestrina/genética , Doenças dos Macacos/genética , Proteínas Mutantes Quiméricas/genética , Sequência de Aminoácidos , Animais , Aotidae/genética , Sequência de Bases , Suscetibilidade a Doenças , Infecções por HIV/genética , Células HeLa/virologia , Humanos , Imunidade Inata/genética , Macaca nemestrina/virologia , Dados de Sequência Molecular , Mutagênese Insercional , Proteínas Mutantes Quiméricas/fisiologia , Retroelementos , Especificidade da Espécie , Internalização do Vírus , Replicação ViralRESUMO
Small heat shock proteins (sHSPs) are a ubiquitous class of molecular chaperones that interacts with substrates to prevent their irreversible insolubilization during denaturation. How sHSPs interact with substrates remains poorly defined. To investigate the role of the conserved C-terminal alpha-crystallin domain versus the variable N-terminal arm in substrate interactions, we compared two closely related dodecameric plant sHSPs, Hsp18.1 and Hsp16.9, and four chimeras of these two sHSPs, in which all or part of the N-terminal arm was switched. The efficiency of substrate protection and formation of sHSP-substrate complexes by these sHSPs with three different model substrates, firefly luciferase, citrate synthase, and malate dehydrogenase (MDH) provide new insights into sHSP/substrate interactions. Results indicate that different substrates have varying affinities for different domains of the sHSP. For luciferase and citrate synthase, the efficiency of substrate protection was determined by the identity of the N-terminal arm in the chimeric proteins. In contrast, for MDH, efficient protection clearly required interactions with the alpha-crystallin domain in addition to the N-terminal arm. Furthermore, we show that sHSP-substrate complexes with varying stability and composition can protect substrate equally, and substrate protection is not correlated with sHSP oligomeric stability for all substrates. Protection of MDH by the dimeric chimera composed of the Hsp16.9 N-terminal arm and Hsp18.1 alpha-crystallin domain supports the model that a dimeric form of the sHSP can bind and protect substrate. In total, results demonstrate that sHSP-substrate interactions are complex, likely involve multiple sites on the sHSP, and vary depending on substrate.