RESUMO
Mitochondrial ribosomal small subunit (MRPS) group of proteins is structural constituents of the small subunit of mitoribosomes involved in translation. Recent studies indicate role in tumourigenic process, however, unlike cytosolic ribosomal proteins, knowledge on the role of MRPS proteins in alternate cellular processes is very limited. Mapping protein-protein interactions (PPIs) onto known cellular processes can be a valuable tool to identify novel protein functions. In this study, to identify PPIs of MRPS proteins, we have constructed 31 glutathione-S-transferase (GST)/MRPS fusion clones. GST/MRPS fusion proteins were confirmed by MALDI-TOF analysis. GST pull-downs were performed using eight GST/MRPS proteins (MRPS9, MRPS10, MRPS11, MRPS18B, MRPS31, MRPS33, MRPS38 and MRPS39), GST alone as pull-down control and HEK293 cell lysate as the source for anchor proteins followed by nLC/MS/MS analysis and probable PPIs of eight MRPS proteins were identified. Three PPIs from GST pull-downs and interaction between six MRPS proteins and p53 previously reported in PPI database were validated. The PPI network analysis revealed putative role in cellular processes with implications for tumourigenesis. Gene expression screening of a cancer cell line panel indicated overexpression of MRPS10 and MRPS31 in breast cancer. Co-expression module identification tool analysis of breast cancer gene expression and MRPS10 and MRPS31 PPIs revealed putative role for PPI with acyl-CoA dehydrogenase in fatty acid oxidation process regulated by brain-derived neurotrophic factor signalling pathway.
Assuntos
Neoplasias da Mama/patologia , Proteínas Mitocondriais/metabolismo , Mapas de Interação de Proteínas , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Ribossômicas/metabolismo , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Cromatografia de Afinidade , Feminino , Glutationa Transferase/genética , Glutationa Transferase/metabolismo , Humanos , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/isolamento & purificação , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/isolamento & purificação , Proteínas Ribossômicas/genética , Proteínas Ribossômicas/isolamento & purificação , Células Tumorais CultivadasRESUMO
Alpha-momorcharin (Alpha-MMC) from the seed of bitter melon is a type I ribosome inactivating protein (RIP) that removes a specific adenine from 28S rRNA and inhibits protein biosynthesis. Here, we report seven crystal complex structures of alpha-MMC with different substrate analogs (adenine, AMP, cAMP, dAMP, ADP, GMP, and xanthosine) at 1.08 Å to 1.52 Å resolution. These structures reveal that not only adenine, but also guanine and their analogs can effectively bind to alpha-MMC. The side chain of Tyr93 adopts two conformations, serving as a switch to open and close the substrate binding pocket of alpha-MMC. Although adenine, AMP, GMP, and guanine are located in a similar active site in different RIPs, residues involved in the interaction between RIPs and substrate analogs are slightly different. Complex structures of alpha-MMC with different substrate analogs solved in this study provide useful information on its enzymatic mechanisms and may enable the development of new inhibitors to treat the poisoning of alpha-MMC.
Assuntos
Biossíntese de Proteínas , Proteínas Inativadoras de Ribossomos/química , Proteínas Inativadoras de Ribossomos/metabolismo , Difosfato de Adenosina/química , Difosfato de Adenosina/metabolismo , Fracionamento Químico , Modelos Moleculares , Momordica charantia/química , Conformação Proteica , Proteínas Ribossômicas/química , Proteínas Ribossômicas/isolamento & purificação , Proteínas Ribossômicas/metabolismo , Proteínas Ribossômicas/ultraestrutura , Proteínas Inativadoras de Ribossomos/isolamento & purificação , Proteínas Inativadoras de Ribossomos/ultraestrutura , Ribossomos/metabolismo , Sementes/química , Relação Estrutura-AtividadeRESUMO
Translation of mitochondrial encoded mRNAs by mitochondrial ribosomes is thought to play a major role in regulating the expression of mitochondrial proteins. However, the structure and function of plant mitochondrial ribosomes remains poorly understood. To study mitochondrial ribosomes, it is necessary to separate them from plastidic and cytosolic ribosomes that are generally present at much higher concentrations. Here, a straight forward protocol for the preparation of fractions highly enriched in mitochondrial ribosomes from plant cells is described. The method begins with purification of mitochondria followed by mitochondrial lysis and ultracentrifugation of released ribosomes through sucrose cushions and gradients. Dark-grown Arabidopsis cells were used in this example because of the ease with which good yields of pure mitochondria can be obtained from them. However, the steps for isolation of ribosomes from mitochondria could be applied to mitochondria obtained from other sources. Proteomic analyses of resulting fractions have confirmed strong enrichment of mitochondrial ribosomal proteins.
Assuntos
Proteínas de Arabidopsis/isolamento & purificação , Fracionamento Celular/métodos , Centrifugação com Gradiente de Concentração/métodos , Mitocôndrias/química , Proteínas Mitocondriais/isolamento & purificação , Proteínas Ribossômicas/isolamento & purificação , Ribossomos/química , Sulfato de Amônio/química , Arabidopsis/química , Arabidopsis/metabolismo , Proteínas de Arabidopsis/química , Técnicas de Cultura de Células , Fracionamento Celular/instrumentação , Centrifugação com Gradiente de Concentração/instrumentação , Meios de Cultura/química , Escuridão , Mitocôndrias/metabolismo , Células Vegetais/química , Células Vegetais/metabolismo , Povidona/química , Biossíntese de Proteínas , Dióxido de Silício/química , Ultracentrifugação/instrumentação , Ultracentrifugação/métodosRESUMO
Human YVH1 (hYVH1), also known as dual specificity phosphatase 12 (DUSP12), is a poorly characterized atypical dual specificity phosphatase widely conserved throughout evolution. Recent findings have demonstrated that hYVH1 expression affects cellular DNA content and is a novel cell survival phosphatase preventing both thermal and oxidative stress-induced cell death, whereas studies in yeast have established YVH1 as a novel 60S ribosome biogenesis factor. In this study, we have isolated novel hYVH1-associating proteins from human U2OS osteosarcoma cells using affinity chromatography coupled to mass spectrometry employing ion mobility separation. Numerous ribosomal proteins were identified, confirming the work done in yeast. Furthermore, proteins known to be present on additional RNP particles were identified, including Y box-binding protein 1 (YB-1) and fragile X mental retardation protein, proteins that function in translational repression and stress granule regulation. Follow-up studies demonstrated that hYVH1 co-localizes with YB-1 and fragile X mental retardation protein on stress granules in response to arsenic treatment. Interestingly, hYVH1-positive stress granules were significantly smaller, whereas knocking down hYVH1 expression attenuated stress granule breakdown during recovery from arsenite stress, indicating a possible role for hYVH1 in stress granule disassembly. These results propagate a role for dual specificity phosphatases at RNP particles and suggest that hYVH1 may affect a variety of fundamental cellular processes by regulating messenger ribonucleoprotein (mRNP) dynamics.
Assuntos
Grânulos Citoplasmáticos/metabolismo , Fosfatase 1 de Especificidade Dupla/metabolismo , Ribonucleoproteínas/metabolismo , Arsenitos/farmacologia , Linhagem Celular Tumoral , Grânulos Citoplasmáticos/química , Fosfatase 1 de Especificidade Dupla/química , Fosfatase 1 de Especificidade Dupla/isolamento & purificação , Humanos , Ribonucleoproteínas/química , Ribonucleoproteínas/isolamento & purificação , Proteínas Ribossômicas/química , Proteínas Ribossômicas/isolamento & purificação , Proteínas Ribossômicas/metabolismo , Estresse Fisiológico/efeitos dos fármacos , Proteína 1 de Ligação a Y-Box/química , Proteína 1 de Ligação a Y-Box/isolamento & purificação , Proteína 1 de Ligação a Y-Box/metabolismoRESUMO
The paper reports an inexpensive and efficient procedure for the removal of protein S1 from E. coli ribosomes. It comprises incubation of ribosomes in a pyrimidine polyribonucleotide solution followed by centrifugation of the sample through a sucrose cushion. To avoid co-sedimentation of the S1-bound polypyrimidine with the ribosomes, its length should not exceed several hundred nucleotides. Unlike popular affinity chromatography through a poly(U) Sepharose or poly(U) cellulose column, the method tolerates limited polyribonucleotide degradation by eventual traces of ribonucleases, and can readily be incorporated into standard protocols for the isolation of ribosomes by centrifugation.
Assuntos
Proteínas de Escherichia coli/isolamento & purificação , Escherichia coli/química , Proteínas Ribossômicas/isolamento & purificação , Ribossomos/química , Centrifugação com Gradiente de Concentração/métodos , Cromatografia de Afinidade , Poli U/química , Sacarose/análogos & derivados , Sacarose/químicaRESUMO
Ribosome biogenesis is a highly complex process in eukaryotes, involving temporally and spatially regulated ribosomal protein (r-protein) binding and ribosomal RNA remodelling events in the nucleolus, nucleoplasm and cytoplasm. Hundreds of assembly factors, organized into sequential functional groups, facilitate and guide the maturation process into productive assembly branches in and across different cellular compartments. However, the precise mechanisms by which these assembly factors function are largely unknown. Here we use cryo-electron microscopy to characterize the structures of yeast nucleoplasmic pre-60S particles affinity-purified using the epitope-tagged assembly factor Nog2. Our data pinpoint the locations and determine the structures of over 20 assembly factors, which are enriched in two areas: an arc region extending from the central protuberance to the polypeptide tunnel exit, and the domain including the internal transcribed spacer 2 (ITS2) that separates 5.8S and 25S ribosomal RNAs. In particular, two regulatory GTPases, Nog2 and Nog1, act as hub proteins to interact with multiple, distant assembly factors and functional ribosomal RNA elements, manifesting their critical roles in structural remodelling checkpoints and nuclear export. Moreover, our snapshots of compositionally and structurally different pre-60S intermediates provide essential mechanistic details for three major remodelling events before nuclear export: rotation of the 5S ribonucleoprotein, construction of the active centre and ITS2 removal. The rich structural information in our structures provides a framework to dissect molecular roles of diverse assembly factors in eukaryotic ribosome assembly.
Assuntos
Microscopia Crioeletrônica , Proteínas Ribossômicas/metabolismo , Proteínas Ribossômicas/ultraestrutura , Subunidades Ribossômicas Maiores de Eucariotos/química , Subunidades Ribossômicas Maiores de Eucariotos/ultraestrutura , Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/ultraestrutura , Transporte Ativo do Núcleo Celular , Sequência de Bases , Domínio Catalítico , Núcleo Celular/química , Núcleo Celular/metabolismo , Núcleo Celular/ultraestrutura , Citoplasma/metabolismo , DNA Espaçador Ribossômico/química , DNA Espaçador Ribossômico/genética , DNA Espaçador Ribossômico/metabolismo , DNA Espaçador Ribossômico/ultraestrutura , GTP Fosfo-Hidrolases/química , GTP Fosfo-Hidrolases/metabolismo , GTP Fosfo-Hidrolases/ultraestrutura , Proteínas de Ligação ao GTP/química , Proteínas de Ligação ao GTP/metabolismo , Proteínas de Ligação ao GTP/ultraestrutura , Modelos Moleculares , Dados de Sequência Molecular , Proteínas Nucleares/química , Proteínas Nucleares/metabolismo , Proteínas Nucleares/ultraestrutura , Ligação Proteica , RNA Fúngico/genética , RNA Fúngico/metabolismo , RNA Fúngico/ultraestrutura , RNA Ribossômico/genética , RNA Ribossômico/metabolismo , RNA Ribossômico/ultraestrutura , Ribonucleoproteínas/química , Ribonucleoproteínas/metabolismo , Ribonucleoproteínas/ultraestrutura , Proteínas Ribossômicas/química , Proteínas Ribossômicas/isolamento & purificação , Subunidades Ribossômicas Maiores de Eucariotos/metabolismo , Rotação , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/isolamento & purificação , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/ultraestruturaRESUMO
Blackleg is a highly fatal disease of cattle and sheep, caused by Clostridium chauvoei, a Gram positive, anaerobic, spore forming bacteria. Cell surface-associated proteins play a major role in inducing the protective immunity. However, the identity of a majority of cell surface-associated proteins of C. chauvoei is not known. In the present investigation, we have used SDS-PAGE, 2D-gel electrophoresis and Western blotting followed by mass spectrometry to identify cell surface-associated proteins of C. chauvoei. Among the identified proteins, which have shown to offer protective antigencity in other bacteria, Enolase, Chaperonin, Ribosomal protein L10, Glycosyl Hydrolase and Flavoprotein were characterized by sequencing and their overexpression in Escherichia coli. In conclusion, cell surface-associated proteins were identified using proteomic approach and the genes for the immunoreactive proteins were expressed, which may prove to be potential diagnostic or vaccine candidates.
Assuntos
Antígenos de Bactérias/isolamento & purificação , Proteínas de Bactérias/isolamento & purificação , Clostridium chauvoei/genética , Proteínas de Membrana/isolamento & purificação , Animais , Antígenos de Bactérias/genética , Antígenos de Bactérias/imunologia , Proteínas de Bactérias/genética , Proteínas de Bactérias/imunologia , Western Blotting , Chaperoninas/genética , Chaperoninas/imunologia , Chaperoninas/isolamento & purificação , Clonagem Molecular , Clostridium chauvoei/imunologia , Eletroforese em Gel Bidimensional , Escherichia coli/genética , Escherichia coli/metabolismo , Flavoproteínas/genética , Flavoproteínas/imunologia , Flavoproteínas/isolamento & purificação , Expressão Gênica , Soros Imunes/química , Soros Imunes/isolamento & purificação , Proteínas de Membrana/genética , Proteínas de Membrana/imunologia , Fosfopiruvato Hidratase/genética , Fosfopiruvato Hidratase/imunologia , Fosfopiruvato Hidratase/isolamento & purificação , Proteômica , Coelhos , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/isolamento & purificação , Proteína Ribossômica L10 , Proteínas Ribossômicas/genética , Proteínas Ribossômicas/imunologia , Proteínas Ribossômicas/isolamento & purificação , Análise de Sequência de DNARESUMO
Exosomes are 30-120 nm-sized membrane vesicles of endocytic origin that are released into the extracellular environment and play roles in cell-cell communication. Tumor-associated macrophages (TAMs) are important constituents of the tumor microenvironment; thus, it is critical to study the features and complex biological functions of TAM-derived exosomes. Here, we constructed a TAM cell model from a mouse macrophage cell line, Ana-1, and performed comparative proteomics on exosomes, exosome-free media, and cells between TAMs and Ana-1. Proteomic analysis between exosome and exosome-free fractions indicated that the functions of exosome dominant proteins were mainly enriched in RNA processing and proteolysis. TAM status dramatically affected the abundances of 20S proteasome subunits and ribosomal proteins in their exosomes. The 20S proteasome activity assay strongly indicated that TAM exosomes possessed higher proteolytic activity. In addition, Ana-1- and TAM-derived exosomes have different RNA profiles, which may result from differential RNA processing proteins. Taken together, our comprehensive proteomics study provides novel views for understanding the complicated roles of macrophage-derived exosomes in the tumor microenvironment.
Assuntos
Exossomos/metabolismo , Macrófagos/metabolismo , Proteoma/isolamento & purificação , Processamento Pós-Transcricional do RNA , Proteínas Ribossômicas/isolamento & purificação , Animais , Comunicação Celular , Linhagem Celular Tumoral , Neoplasias Colorretais/genética , Neoplasias Colorretais/metabolismo , Neoplasias Colorretais/patologia , Ensaios Enzimáticos , Exossomos/química , Macrófagos/química , Macrófagos/patologia , Camundongos , Anotação de Sequência Molecular , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteólise , Proteoma/genética , Proteoma/metabolismo , Proteômica/métodos , Proteínas Ribossômicas/genética , Proteínas Ribossômicas/metabolismo , Espectrometria de Massas em Tandem , Microambiente Tumoral/genéticaRESUMO
Finkel-Biskis-Reilly murine sarcoma virus (FBR-MuSV) ubiquitously expressed (FAU) gene is down-regulated in human prostate, breast and ovarian cancers. Moreover, its dysregulation is associated with poor prognosis in breast cancer. Sponges (Porifera) are animals without tissues which branched off first from the common ancestor of all metazoans. A large majority of genes implicated in human cancers have their homologues in the sponge genome. Our study suggests that FAU gene from the sponge Suberites domuncula reflects characteristics of the FAU gene from the metazoan ancestor, which have changed only slightly during the course of animal evolution. We found pro-apoptotic activity of sponge FAU protein. The same as its human homologue, sponge FAU increases apoptosis in human HEK293T cells. This indicates that the biological functions of FAU, usually associated with "higher" metazoans, particularly in cancer etiology, possess a biochemical background established early in metazoan evolution. The ancestor of all animals possibly possessed FAU protein with the structure and function similar to evolutionarily more recent versions of the protein, even before the appearance of true tissues and the origin of tumors and metastasis. It provides an opportunity to use pre-bilaterian animals as a simpler model for studying complex interactions in human cancerogenesis.
Assuntos
Proteínas Ribossômicas/isolamento & purificação , Suberites/genética , Animais , Apoptose/efeitos dos fármacos , Evolução Biológica , DNA/genética , DNA/isolamento & purificação , Células HEK293/efeitos dos fármacos , Células HeLa/efeitos dos fármacos , Humanos , Pequeno RNA não Traduzido/genética , Pequeno RNA não Traduzido/isolamento & purificação , Proteínas Ribossômicas/genética , Proteínas Ribossômicas/farmacologia , Alinhamento de Sequência , Frações Subcelulares/química , Suberites/químicaRESUMO
AIM: Study the effect of ribosomal proteins of respiratory bacteria composing the basis of the immune-modulating preparation ribomunil on adhesive properties of buccal epithelium of healthy donors, and carry out comparison of this parameter during use of other bacterial products. MATERIALS AND METHODS: Various amounts of bacterial ribosomal proteins, Escherichia coli (serotype O127:B8) and one-day Staphylococcus aureus (strain 5983) culture supernatant were added to "buccal epitheliocytes--candida" system and incubated. Buccal cells were washed after the incubation from non-bound candida and differentiated microscopically by the amount of cells with various levels of candida adhesion. Separate effect of ribosomal proteins on buccal cells and candida was studied, as well as their impact on the production of secretory products of buccal cells. RESULTS: Buccal epitheliocytes in control adhered on average 14.6 candidiasis cells. After incubation with bacterial ribosomal proteins the index decreased by 2.3 +/- 0.2 times. During separate addition of ribomunil to buccal cells and candida, ribosomal bacterial proteins were shown to have effect only on epitheliocytes. Activity of ribosomal proteins had a selective character, as shown by the lack of effect under the influence of S. aureus supernatant on buccal cells as well as an increase of adhesion under the influence of lipopolysaccharide on epitheliocytes. Viability of cells in all the cases remained at a level of 90 - 98%. Buccal cells during contact with ribomunil produced a complex of soluble mediators that took part in its blocking effect. CONCLUSION: The increase of stability of mucosal tract to microbial adhesion is an element of innate immunity and may be one of the components of immune-protecting effect of bacterial ribosomal proteins.
Assuntos
Proteínas de Bactérias/farmacologia , Candida albicans/fisiologia , Células Epiteliais/efeitos dos fármacos , Mucosa Bucal/efeitos dos fármacos , Proteínas Ribossômicas/farmacologia , Adolescente , Adulto , Proteínas de Bactérias/isolamento & purificação , Adesão Celular , Técnicas de Cocultura , Células Epiteliais/citologia , Células Epiteliais/imunologia , Escherichia coli/química , Feminino , Humanos , Imunidade Inata , Lipopolissacarídeos/isolamento & purificação , Lipopolissacarídeos/farmacologia , Masculino , Mucosa Bucal/citologia , Mucosa Bucal/imunologia , Cultura Primária de Células , Proteínas Ribossômicas/isolamento & purificação , Staphylococcus aureus/químicaRESUMO
The L10 ribosomal protein (RPL10) plays a role in the binding of the 60 S and 40 S ribosomal subunits and in mRNA translation. The evidence indicates that RPL10 also has multiple extra-ribosomal functions, including tumor suppression. Recently, the presence of RPL10 in prostate and ovarian cancers was evaluated, and it was demonstrated to be associated with autistic disorders and premature ovarian failure. In the present work, we successfully cloned and expressed full-length human RPL10 (hRPL10) protein and isolated inclusion bodies containing this protein that had formed under mild growth conditions. The culture produced 376mg of hRPL10 protein per liter of induced bacterial culture, of which 102.4mg was present in the soluble fraction, and 25.6mg was recovered at approximately 94% purity. These results were obtained using a two-step process of non-denaturing protein extraction from pelleted inclusion bodies. We studied the characteristics of this protein using circular dichroism spectroscopy and by monitoring the changes induced by the presence or absence of zinc ions using fluorescence spectrometry. The results demonstrated that the protein obtained using these non-conventional methods retained its secondary and tertiary structure. The conformational changes induced by the incorporation of zinc suggested that this protein could interact with Jun or the SH3 domain of c-yes. The results suggested that the strategy used to obtain hRPL10 is simple and could be applied to obtaining other proteins that are susceptible to degradation.
Assuntos
Corpos de Inclusão/metabolismo , Proteínas Recombinantes/isolamento & purificação , Proteínas Ribossômicas/isolamento & purificação , Proteínas Supressoras de Tumor/isolamento & purificação , Sequência de Aminoácidos , Dicroísmo Circular , Clonagem Molecular , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Humanos , Dados de Sequência Molecular , Conformação Proteica , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteína Ribossômica L10 , Proteínas Ribossômicas/genética , Proteínas Ribossômicas/metabolismo , Alinhamento de Sequência , Espectrometria de Fluorescência , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/metabolismo , Zinco/químicaRESUMO
The ribosomal protein L22 (RPL22) protein belongs to the L22E family of ribosomal proteins. It is located in the cytoplasm. The purpose of this paper was to explore the structure and anti-cancer function of RPL22 of the Giant Panda (Ailuropoda melanoleuca). The cDNA of RPL22 was cloned successfully from the Giant Panda using RT-PCR technology. We constructed a recombinant expression vector containing RPL22 cDNA and over-expressed it in Escherichia coli using pET28a plasmids. The expression product obtained was purified by using Ni chelating affinity chromatography. The result indicated that the length of the fragment cloned is 414 bp, and it contains an open-reading frame of 387 bp encoding 128 amino acids. Primary structure analysis revealed that the molecular weight of the putative RPL22 protein is 14.74 kDa with a theoretical pI 9.21. The RPL22 gene can be really expressed in E. coli and the RPL22 protein, fusioned with the N-terminally His-tagged protein, gave rise to the accumulation of an expected 20.1 kDa polypeptide. The data showed that the recombinant protein RPL22 had a time- and dose-dependency on the cell growth inhibition rate. The human laryngeal carcinoma Hep-2 cells treated with 0.05-6 µg/ml of RPL22 for 24 h displayed significant cell growth inhibition (p<0.05, n=8) in assayed using MTT compared to the control (untreated) cells. The data indicate that the effect at low concentrations is better than high concentrations, and the concentration of 1.5 µg/ml has the best rate of growth inhibition of 47.70%. The inhibitory rate in mice treated with 1.5 µg/ml RPL22 protein can reach 43.75%. Histology of tumor organs shows that the tissues arranged looser in RPL22 group than those in control group. Meanwhile, there is no obvious damage to other organs, such as heart, lung and kidney. Further research is on going to determine the bioactive principle(s) of recombinant protein RPL22 responsible for its anticancer activity.
Assuntos
Proteínas Recombinantes/genética , Proteínas Ribossômicas/genética , Ursidae/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Linhagem Celular Tumoral , DNA Complementar/química , DNA Complementar/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Genes Essenciais , Humanos , Masculino , Camundongos , Dados de Sequência Molecular , Filogenia , Conformação Proteica , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/farmacologia , Proteínas Ribossômicas/isolamento & purificação , Carga Tumoral/efeitos dos fármacos , Ursidae/metabolismo , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Ribosome profiling is a technique used to separate ribosomal subunits, 80S ribosomes (monosomes), and polyribosomes (polysomes) from other RNA-protein complexes. It is traditionally performed in sucrose gradients. In this study, we used asymmetric flow field-flow fractionation (AsFlFFF) to characterize ribosome profiles of Nicotiana benthamiana plants. With the optimized running conditions, we were able to separate free molecules from ribosomal subunits and intact ribosomes. We used various chemical and enzymatic treatments to validate the positions of subunits, monosomes, and polysomes in the AsFlFFF fractograms. We also characterized the protein and RNA content of AsFlFFF fractions by gel electrophoresis and western blotting. The reverse transcription polymerase chain reaction (RT-PCR) analysis showed that ribosomes remained bound to messenger RNAs (mRNAs) during the analysis. Therefore, we conclude that AsFlFFF can be used for ribosome profiling to study the mRNAs that are being translated. It can also be used to study the protein composition of ribosomes that are active in translation at that particular moment.
Assuntos
Fracionamento por Campo e Fluxo/métodos , Nicotiana/química , Ribossomos/química , Triticum/química , Proteínas de Plantas/isolamento & purificação , RNA de Plantas/isolamento & purificação , RNA Ribossômico/isolamento & purificação , Proteínas Ribossômicas/isolamento & purificação , Nicotiana/citologia , Triticum/citologiaRESUMO
The ribosomal protein L24 (RPL24) belongs to the L24E family of ribosomal proteins and is located in the cytoplasm. The purpose of this study was to investigate the structure and anti-cancer function of RPL24 of the giant panda (Ailuropoda melanoleuca). The complementary DNA of RPL24 was cloned successfully using reverse transcription-polymerase chain reaction technology. We constructed a recombinant expression vector containing RPL24 complementary DNA and overexpressed it in Escherichia coli using pET28a plasmids. The expression product obtained was purified using Ni-chelating affinity chromatography. The results indicated that the length of the fragment cloned is 509 bp, and it contains an open-reading frame of 474 bp encoding 157 amino acids. Primary structure analysis revealed that the molecular weight of the putative RPL24 protein is 17.78 kDa with a theoretical isoelectric point of 11.86. The RPL24 gene is readily expressed in E. coli, and the RPL24 fused with the N-terminal histidine-tagged protein to give rise to the accumulation of an expected 23.51-kDa polypeptide. The inhibitory rate in mice treated with 0.1 mg/mL RPL24, the highest of 3 doses administered, can reach 67.662%, which may be comparable to the response to mannatide. The histology of organs with tumors showed that the tissues in the RPL24 group displayed a looser arrangement compared with that in the control group. Furthermore, no obvious damage was apparent in other organs, such as heart, lung, and kidney. The data showed that the recombinant RPL24 had time and dose dependency on the cell growth inhibition rate. Human laryngeal carcinoma Hep-2 cells treated with 0.3125-10 µg/mL RPL24 for 24 h displayed significant cell growth inhibition (P < 0.05; N = 6) in assays using 3-(4,5-dimethylthiazol- 2-yl)-2,5-diphenyltetrazolium bromide compared with that in control (untreated) cells. By contrast, human hepatoma Hep G-2 cells displayed no significant change (P > 0.05; N = 6) from control (untreated) cells. RPL24 has time and dose dependency on Hep-2 cell growth inhibition. The data indicate that the effect at low concentrations is better than that at high concentrations, and the concentration of 0.625 µg/mL provides the best rate of growth inhibition. Further research is ongoing to determine the bioactive principles of recombinant RPL24 protein that are responsible for its anticancer activity.
Assuntos
Antineoplásicos/farmacologia , Proteínas Ribossômicas/farmacologia , Ursidae/genética , Sequência de Aminoácidos , Animais , Antineoplásicos/química , Antineoplásicos/isolamento & purificação , Sequência de Bases , Forma Celular/efeitos dos fármacos , Clonagem Molecular , Expressão Gênica , Células Hep G2 , Humanos , Masculino , Camundongos , Modelos Moleculares , Dados de Sequência Molecular , Filogenia , Conformação Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/farmacologia , Proteínas Ribossômicas/química , Proteínas Ribossômicas/isolamento & purificação , Proteínas Ribossômicas/fisiologia , Análise de Sequência de DNA , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Cisplatin is widely used as an antineoplastic drug, but its ototoxic and nephrotoxic side-effects, as well as the inherent or acquired resistance of some cancers to cisplatin, remain significant clinical problems. Cisplatin's selectivity in killing rapidly proliferating cancer cells is largely dependent on covalent binding to DNA via cisplatin's chloride sites that had been aquated. We hypothesized that cisplatin's toxicity in slowly proliferating or terminally differentiated cells is primarily due to drug-protein interactions, instead of drug-DNA binding. To identify proteins that bind to cisplatin, we synthesized two different platinum-agarose conjugates, one with two amino groups and another with two chlorides attached to platinum that are available for protein binding, and conducted pull-down assays using cochlear and kidney cells. Mass spectrometric analysis on protein bands after gel electrophoresis and Coomassie blue staining identified several proteins, including myosin IIA, glucose-regulated protein 94 (GRP94), heat shock protein 90 (HSP90), calreticulin, valosin containing protein (VCP), and ribosomal protein L5, as cisplatin-binding proteins. Future studies on the interaction of these proteins with cisplatin will elucidate whether these drug-protein interactions are involved in ototoxicity and nephrotoxicity, or contribute to tumor sensitivity or resistance to cisplatin treatment.
Assuntos
Antineoplásicos/química , Extratos Celulares/química , Cisplatino/química , Glicoconjugados/química , Sefarose/química , Adenosina Trifosfatases/química , Adenosina Trifosfatases/isolamento & purificação , Animais , Antineoplásicos/síntese química , Calreticulina/química , Calreticulina/isolamento & purificação , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/isolamento & purificação , Linhagem Celular , Cisplatino/análogos & derivados , Cisplatino/síntese química , Células Epiteliais/química , Células Epiteliais/citologia , Glicoconjugados/síntese química , Proteínas de Choque Térmico HSP90/química , Proteínas de Choque Térmico HSP90/isolamento & purificação , Túbulos Renais Proximais/química , Túbulos Renais Proximais/citologia , Glicoproteínas de Membrana/química , Glicoproteínas de Membrana/isolamento & purificação , Camundongos , Miosina não Muscular Tipo IIA/química , Miosina não Muscular Tipo IIA/isolamento & purificação , Órgão Espiral/química , Órgão Espiral/citologia , Ligação Proteica , Proteínas Ribossômicas/química , Proteínas Ribossômicas/isolamento & purificação , Proteína com ValosinaRESUMO
The regulation of transcription and translation by specific cell types is essential to generate the cellular diversity that typifies complex multicellular organisms. Tagging and purification of ribosomal proteins has been shown to be an innovative and effective means of characterizing the ribosome bound transcriptome of highly specific cell populations in vivo. To test the feasibility of using translating ribosome affinity purification (TRAP) in zebrafish, we have generated both a ubiquitous TRAP line and a melanocyte-specific TRAP line using the native zebrafish rpl10a ribosomal protein. We have demonstrated the capacity to capture mRNA transcripts bound to ribosomes, and confirmed the expected enrichment of melanocyte specific genes and depletion of non-melanocyte genes when expressing the TRAP construct with a cell specific promoter. We have also generated a generic EGFP-rpl10a Tol2 plasmid construct (Tol2-zTRAP) that can be readily modified to target any additional cell populations with characterized promoters in zebrafish.
Assuntos
Engenharia Genética/métodos , Polirribossomos/química , Proteínas de Ligação a RNA/isolamento & purificação , Proteínas Ribossômicas/isolamento & purificação , Proteínas de Peixe-Zebra/isolamento & purificação , Peixe-Zebra/genética , Animais , Animais Geneticamente Modificados , Proteínas de Fluorescência Verde/genética , Melanócitos/metabolismo , Regiões Promotoras Genéticas , Ligação Proteica , Biossíntese de Proteínas , RNA Mensageiro/isolamento & purificação , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Proteína Ribossômica L10 , Proteínas Ribossômicas/genética , Proteínas Ribossômicas/metabolismo , Peixe-Zebra/metabolismo , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismoRESUMO
In order to investigate the immune role of ribosomal protein L10 (RPL10/QM-like gene) in marine fish, we challenged the large yellow croaker Pseudosciaena (= Larimichthys) crocea, the most important marine fish culture species in China, by injection with a mixture of the bacteria Vibrio harveyi and V. parahaemolyticus (3:1 in volume). Microarray analysis and real-time PCR were performed 24 and 48 h post-challenge to isolate and identify the QM-like gene from the gill P. crocea (designated PcQM). The expression level of the PcQM gene did not changed significantly at 24 h post-challenge, but was significantly downregulated at 48 h post-challenge, suggesting that the gene had an immune-modulatory effect in P. crocea. Full-length PcQM cDNA and genomic sequences were obtained by rapid amplification of cDNA ends (RACE)-PCR. The sequence of the PcQM gene clustered together with those of other QM-like genes from other aquatic organisms, indicating that the QM-like gene is highly conserved in teleosts.
Assuntos
Imunidade/genética , Perciformes/genética , Proteínas Ribossômicas/genética , Proteínas Ribossômicas/isolamento & purificação , Sequência de Aminoácidos , Animais , Sequência de Bases , DNA Complementar/genética , Regulação da Expressão Gênica , Genoma/genética , Brânquias/metabolismo , Brânquias/microbiologia , Dados de Sequência Molecular , Análise de Sequência com Séries de Oligonucleotídeos , Perciformes/microbiologia , Filogenia , Proteína Ribossômica L10 , Proteínas Ribossômicas/química , Proteínas Ribossômicas/metabolismo , Alinhamento de Sequência , Vibrio/fisiologia , Vibrioses/genética , Vibrioses/imunologiaRESUMO
Human nonintegrin laminin receptor is a multifunctional protein acting as an integral component of the ribosome and a cell surface receptor for laminin-1. The laminin receptor is overexpressed in several human cancers and is also the cell surface receptor for several viruses and pathogenic prion proteins, making it a pathologically significant protein. This study focused on the proteomic characterization of laminin receptor interacting proteins from Mus musculus. The use of affinity chromatography with immobilized recombinant laminin receptor coupled with mass spectrometry analysis identified 45 proteins with high confidence. Following validation through coimmunoprecipitation, the proteins were classified based on predicted function into ribosomal, RNA processing, signal transduction/metabolism, protein processing, cytoskeleton/cell anchorage, DNA/chromatin, and unknown functions. A significant portion of the identified proteins is related to functions or localizations previously described for laminin receptor. This work represents a comprehensive proteomic approach to studying laminin receptor and provides an essential stepping stone to a better mechanistic understanding of this protein's diverse functions.
Assuntos
Proteoma/química , Animais , Chaperonina com TCP-1/química , Chaperonina com TCP-1/isolamento & purificação , Cromatografia de Afinidade , Proteínas do Citoesqueleto/química , Proteínas do Citoesqueleto/isolamento & purificação , Hexosiltransferases , Histonas/química , Histonas/isolamento & purificação , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/química , Peptídeos e Proteínas de Sinalização Intracelular/isolamento & purificação , Camundongos , Células NIH 3T3 , Complexo de Endopeptidases do Proteassoma/química , Complexo de Endopeptidases do Proteassoma/isolamento & purificação , Ligação Proteica , Proteoma/isolamento & purificação , Proteômica , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/isolamento & purificação , Receptores de Laminina/química , Proteínas Ribossômicas/química , Proteínas Ribossômicas/isolamento & purificação , Fatores de Transcrição/química , Fatores de Transcrição/isolamento & purificaçãoRESUMO
Ribosomal protein L31 gene is a component of the 60S large ribosomal subunit encoded by RPL31 gene, while ribosomal protein L31 (RPL31) is an important constituent of peptidyltransferase center. In our research, the cDNA and the genomic sequence of RPL31 were cloned successfully from the giant panda (Ailuropoda melanoleuca) using RT-PCR technology respectively, following sequencing and analyzing preliminarily. We constructed a recombinant expression vector contained RPL31 cDNA and over-expressed it in Escherichia coli using pET28a plasmids. The expression product was purified to obtain recombinant protein of RPL31 from the giant panda. Recombinant protein of RPL31 obtained from the experiment acted on human laryngeal carcinoma Hep-2 and human hepatoma HepG-2 cells for study of its anti-cancer activity by MTT [3-(4, 5-dimehyl-2-thiazolyl)-2, 5-diphenyl-2H-tetrazolium bromide] method. Then observe these cells growth depressive effect. The result indicated that the cDNA fragment of the RPL31 cloned from the giant panda is 419 bp in size, containing an open reading frame of 378 bp, and deduced protein was composed of 125 amino acids with an estimated molecular weight of 14.46-kDa and PI of 11.21. The length of the genomic sequence is 8,091 bp, which was found to possess four exons and three introns. The RPL31 gene can be readily expressed in E.coli, expecting 18-kDa polypeptide that formed inclusion bodies. Recombinant protein RPL31 from the giant panda consists of 157 amino acids with an estimated molecular weight of 17.86 kDa and PI of 10.77. The outcomes showed that the cell growth inhibition rate in a time- and dose-dependent on recombinant protein RPL31. And also indicated that the effect at low concentrations was better than high concentrations on Hep-2 cells, and the concentration of 0.33 µg/mL had the best rate of growth inhibition, 44 %. Consequently, our study aimed at revealing the recombinant protein RPL31 anti-cancer function from the giant panda, providing scientific basis and resources for the research and development of cancer protein drugs anti-cancer mechanism research. Further studies of the mechanism and the signal transduction pathways are in progress.
Assuntos
Antineoplásicos/farmacologia , Proteínas Recombinantes/farmacologia , Proteínas Ribossômicas/farmacologia , Proteínas Ribossômicas/fisiologia , Ursidae , Sequência de Aminoácidos , Animais , Antineoplásicos/isolamento & purificação , Sequência de Bases , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sequência Consenso , DNA Complementar/química , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Células Hep G2 , Humanos , Modelos Moleculares , Dados de Sequência Molecular , Estrutura Terciária de Proteína , Proteínas Recombinantes/isolamento & purificação , Proteínas Ribossômicas/isolamento & purificação , Homologia de SequênciaRESUMO
Bacterial identification is important for the proper treatment of infected patients hospitalized with serious infections especially in critical care units. Identification by conventional methods used in microbiology laboratories takes at least 16 hours since a culture is positive. The introduction of mass spectrometry, specifically MALDI-TOF MS (matrix-assisted laser desorption/ ionization time-of-flight mass spectrometer) in the microbiology laboratory could mean a radical change in the identification accuracy, turn around time (6 minutes per bacteria) and cost (about 5 times cheaper than conventional identification). Since its introduction in clinical microbiology laboratories in 2008, many reports about its usefulness in identifying microorganisms from colonies, as well as directly from positive blood cultures and urine samples have been published. This review describes MALDI-TOF MS methodology, its identification performance for bacteria (aerobic and anaerobic), mycobacterium and yeasts, its future applications in microbiology and its main disadvantages.
La identificación bacteriana es muy importante en el manejo adecuado de los pacientes infectados, especialmente aquellos con infecciones graves hospitalizados en unidades de pacientes críticos. La identificación por los métodos convencionales utilizados en los laboratorios de microbiología clínica demora al menos 16 horas desde que un cultivo es positivo. La introducción de la espectrometría de masas, específicamente del espectrómetro de masas por tiempo de migración (tiempo de vuelo) con desorción/ionización laser asistida por una matriz (MALDI-TOF MS, por su sigla en inglés matrix-assisted laser desorption/ionization time-of-flight mass spectrometer), en el laboratorio de microbiología podría significar un cambio radical en la precisión de la identificación, el tiempo de detección (6 minutos por bacterias) y el costo (aproximadamente 5 veces más económico que la identificación convencional). Desde su introducción en los laboratorios de microbiología clínica en el año 2008, se han escrito numerosas publicaciones sobre su utilidad en la identificación de microorganismos desde colonias, así como directamente desde hemocultivos positivos y de muestras de orina. Esta revisión describe la metodología de MALDI-TOF MS, su rendimiento en la identificación de bacterias aerobias, anaerobias, micobacterias y levaduras, sus futuras aplicaciones en microbiología y sus principales desventajas.