RESUMO
Human mitochondrial chaperonin mHsp60 is essential for mitochondrial function by assisting folding of mitochondrial proteins. Unlike the double-ring bacterial GroEL, mHsp60 exists as a heptameric ring that is unstable and dissociates to subunits. The structural dynamics has been implicated for a unique mechanism of mHsp60. We purified active heptameric mHsp60, and determined a cryo-EM structure of mHsp60 heptamer at 3.4 Å. Of the three domains, the equatorial domains contribute most to the inter-subunit interactions, which include a four-stranded ß sheet. Our structural comparison with GroEL shows that mHsp60 contains several unique sequences that directly decrease the sidechain interactions around the ß sheet and indirectly shorten ß strands by disengaging the backbones of the flanking residues from hydrogen bonding in the ß strand conformation. The decreased inter-subunit interactions result in a small inter-subunit interface in mHsp60 compared to GroEL, providing a structural basis for the dynamics of mHsp60 subunit association. Importantly, the unique sequences are conserved among higher eukaryotic mitochondrial chaperonins, suggesting the importance of structural dynamics for eukaryotic chaperonins. Our structural comparison with the single-ring mHsp60-mHsp10 shows that upon mHsp10 binding the shortened inter-subunit ß sheet is restored and the overall inter-subunit interface of mHsp60 increases drastically. Our structural basis for the mHsp10 induced stabilization of mHsp60 subunit interaction is consistent with the literature that mHsp10 stabilizes mHsp60 quaternary structure. Together, our studies provide structural bases for structural dynamics of the mHsp60 heptamer and for the stabilizing effect of mHsp10 on mHsp60 subunit association.
Assuntos
Chaperonina 10/química , Chaperonina 10/metabolismo , Chaperonina 60/química , Chaperonina 60/metabolismo , Proteínas Mitocondriais/química , Proteínas Mitocondriais/metabolismo , Proteínas da Gravidez/química , Proteínas da Gravidez/metabolismo , Fatores Supressores Imunológicos/química , Fatores Supressores Imunológicos/metabolismo , Sítios de Ligação , Microscopia Crioeletrônica , Humanos , Ligação de Hidrogênio , Modelos Moleculares , Ligação Proteica , Multimerização Proteica , Estrutura Secundária de ProteínaRESUMO
Human Chaperonin 10 (hCpn10) was utilised as a novel scaffold for presenting peptides of therapeutic and diagnostic significance. Molecular dynamic simulations and protein sizing analyses identified a peptide linker (P1) optimal for the formation of the quarternary hCpn10 heptamer structure. hCpn10 scaffold displaying peptides targeting Factor VIIa (CE76-P1) and CD44 (CP7) were expressed in E. coli. Functional studies of CE76-P1 indicated nanomolar affinity for Factor VIIa (3 nM) similar to the E-76 peptide (6 nM), with undetectable binding to Factor X. CE76-P1 was a potent inhibitor of FX activity (via inhibition of Factor VIIa) and prolonged clot formation 4 times longer than achieved by E-76 peptide as determined by prothrombin time (PT) assays. This improvement in clotting function by CE76-P1, highlights the advantages of a heptamer-based scaffold for improving avidity by multiple peptide presentation. In another example of hCPn10 utility as a scaffold, CP7 bound to native CD44 overexpressed on cancer cells and bound rCD44 with high affinity (KD 9.6 nM). The ability to present various peptides through substitution of the hCpn10 mobile loop demonstrates its utility as a novel protein scaffold.
Assuntos
Chaperonina 10/química , Fator VIIa/farmacologia , Receptores de Hialuronatos/metabolismo , Peptídeos/química , Peptídeos/farmacologia , Proteínas da Gravidez/química , Fatores Supressores Imunológicos/química , Sítios de Ligação , Coagulação Sanguínea/efeitos dos fármacos , Humanos , Modelos Moleculares , Simulação de Dinâmica Molecular , Biblioteca de Peptídeos , Ligação Proteica , Estrutura Quaternária de ProteínaRESUMO
MNSFß, a ubiquitin-like protein, covalently binds to various target proteins including proapoptotic Bcl-G. During the course of isolation of MNSFß-conjugating enzyme(s), we identified a novel target protein for MNSFß. MALDI-TOF MS fingerprinting revealed that the MNSFß-interacting protein is HSPA8 (heat shock 70-kDa protein 8). We observed that MNSFß noncovalently binds to HSPA8 in the presence of ATP in vitro. Double knockdown of MNSFß and HSPA8 strongly inhibited RANKL-induced osteoclastogenesis from Raw264.7 macrophage-like cells. The same treatment inhibited RANKL-induced ERK1/2 and p38 phosphorylation and TNFα production, suggesting that the association of MNSFß with HSPA8 may promote RANKL-induced osteoclastogenesis. This is the first report that MNSFß binds to a protein substrate via the noncovalent association and exerts biological effects.
Assuntos
Proteínas de Choque Térmico HSC70/metabolismo , Sistema de Sinalização das MAP Quinases , Osteoclastos/metabolismo , Fatores Supressores Imunológicos/metabolismo , Trifosfato de Adenosina/química , Trifosfato de Adenosina/metabolismo , Animais , Linhagem Celular , Proteínas de Choque Térmico HSC70/química , Camundongos , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Ligação Proteica , Ligante RANK/metabolismo , Fatores Supressores Imunológicos/químicaRESUMO
A previously studied immunosuppressive cytokine, Soluble Immune Response Suppressor (SIRS), may have relevance to current studies of immune suppression in a variety of human disease states. Despite extensive efforts using experimental models, mainly in mice, much remains to be discovered as to how autoimmune cells in mice and humans escape normal regulation and, conversely, how tumor cells evade evoking an immune response. It is the contention of this commentary that the literature pre-2000 contain results that might inform current studies. The broadly immunosuppressive protein, SIRS, was studied extensively from the 1970s to 1990s and culminated in the determination of the n-terminal 21mer sequence of this 15kDa protein which had high homology to the short neurotoxins from sea snakes, that are canonical members of the three finger neurotoxin superfamily (3FTx). It was not until 2007 that the prophylactic administration of the synthetic N-terminal peptide of the SIRS 21mer, identical to the published sequence, was reported to inhibit or delay the development of two autoimmune diseases in mice: experimental allergic encephalomyelitis (EAE) and type I diabetes (T1D). These findings were consistent with other studies of the 3FTx superfamily as important probes in the study of mammalian pharmacology. It is the perspective of this commentary that SIRS, SIRS peptide and the anti-peptide mAb, represent useful, pharmacologically-active probes for the study of the immune response as well as in the potential treatment of autoimmune, inflammatory diseases and cancer.
Assuntos
Doenças Autoimunes/tratamento farmacológico , Imunossupressores/uso terapêutico , Modelos Moleculares , Fatores Supressores Imunológicos/uso terapêutico , Algoritmos , Animais , Anticorpos Monoclonais/farmacologia , Anticorpos Monoclonais/uso terapêutico , Doenças Autoimunes/imunologia , Doenças Autoimunes/terapia , Biologia Computacional , Sistemas Inteligentes , Humanos , Imunomodulação/efeitos dos fármacos , Terapia de Imunossupressão/métodos , Imunossupressores/antagonistas & inibidores , Imunossupressores/química , Imunossupressores/farmacologia , Neurotoxinas/química , Neurotoxinas/toxicidade , Fragmentos de Peptídeos/antagonistas & inibidores , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/farmacologia , Fragmentos de Peptídeos/uso terapêutico , Conformação Proteica , Homologia de Sequência de Aminoácidos , Venenos de Serpentes/química , Venenos de Serpentes/toxicidade , Fatores Supressores Imunológicos/antagonistas & inibidores , Fatores Supressores Imunológicos/química , Fatores Supressores Imunológicos/farmacologiaRESUMO
Disease and death caused by bacterial infections are global health problems. Effective bacterial strategies are required to promote survival and proliferation within a human host, and it is important to explore how this adaption occurs. However, the detection and quantification of bacterial virulence factors in complex biological samples are technically demanding challenges. These can be addressed by combining targeted affinity enrichment of antibodies with the sensitivity of liquid chromatography-selected reaction monitoring mass spectrometry (LC-SRM MS). However, many virulence factors have evolved properties that make specific detection by conventional antibodies difficult. We here present an antibody format that is particularly well suited for detection and analysis of immunoglobulin G (IgG)-binding virulence factors. As proof of concept, we have generated single chain fragment variable (scFv) antibodies that specifically target the IgG-binding surface proteins M1 and H of Streptococcus pyogenes. The binding ability of the developed scFv is demonstrated against both recombinant soluble protein M1 and H as well as the intact surface proteins on a wild-type S. pyogenes strain. Additionally, the capacity of the developed scFv antibodies to enrich their target proteins from both simple and complex backgrounds, thereby allowing for detection and quantification with LC-SRM MS, was demonstrated. We have established a workflow that allows for affinity enrichment of bacterial virulence factors.
Assuntos
Antígenos de Bactérias/química , Proteínas da Membrana Bacteriana Externa/química , Proteínas de Bactérias/química , Proteínas de Transporte/química , Proteínas de Ligação a DNA/química , Linfocinas/química , Biblioteca de Peptídeos , Anticorpos de Cadeia Única/química , Fatores Supressores Imunológicos/química , Fatores de Virulência/química , Sequência de Aminoácidos , Afinidade de Anticorpos , Especificidade de Anticorpos , Antígenos de Bactérias/genética , Antígenos de Bactérias/imunologia , Proteínas da Membrana Bacteriana Externa/genética , Proteínas da Membrana Bacteriana Externa/imunologia , Proteínas de Bactérias/genética , Proteínas de Bactérias/imunologia , Sítios de Ligação , Proteínas de Transporte/genética , Proteínas de Transporte/imunologia , Cromatografia Líquida , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/imunologia , Mapeamento de Epitopos , Expressão Gênica , Humanos , Imunoglobulina G/química , Imunoglobulina G/genética , Imunoglobulina G/imunologia , Linfocinas/genética , Linfocinas/imunologia , Dados de Sequência Molecular , Ligação Proteica , Kit de Reagentes para Diagnóstico , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Anticorpos de Cadeia Única/genética , Anticorpos de Cadeia Única/imunologia , Streptococcus pyogenes/química , Streptococcus pyogenes/imunologia , Fatores Supressores Imunológicos/genética , Fatores Supressores Imunológicos/imunologia , Espectrometria de Massas em Tandem/métodos , Fatores de Virulência/genética , Fatores de Virulência/imunologiaRESUMO
MNSFß is a ubiquitously expressed member of the ubiquitin-like family that has been involved in various biological functions. Previous studies have demonstrated that MNSFß covalently binds to various target proteins including Bcl-G, a proapoptotic protein. In this study, we purified a 115 kDa MNSFß adduct from murine liver lysates by sequential chromatography on DEAE and anti-MNSFß IgG-conjugated Sepharose in the presence of ATP. MALDI-TOF MS fingerprinting revealed that this MNSFß adduct consists of an 8.5 kDa MNSFß and 10-formyltetrahydrofolate dehydrogenase (FDH), an abundant enzyme of folate metabolism. Interestingly, MNSFß preferably binds to cytosolic but not mitochondrial FDH. Fingerprinting analysis of the MNSFß adduct demonstrate that MNSFß conjugates to cytosolic FDH with a linkage between the C-terminal Gly74 and Lys72. The 115 kDa MNSFß/FDH complex was not expressed in any of the tissues examined, indicating that this adduct formation is not ubiquitous. We found that MNSFß/FDH complex formation was induced by dexamethasone in thymocytes. Double knockdown of MNSFß and FDH strongly reduced dexamethasone-induced apoptosis. Collectively, MNSFß/FDH complex formation may positively regulate apoptosis in thymocytes.
Assuntos
Oxirredutases atuantes sobre Doadores de Grupo CH-NH/metabolismo , Fatores Supressores Imunológicos/metabolismo , Timócitos/citologia , Timócitos/metabolismo , Ubiquitinas/metabolismo , Animais , Apoptose/fisiologia , Células Cultivadas , Citosol/enzimologia , Camundongos , Oxirredutases atuantes sobre Doadores de Grupo CH-NH/química , Ligação Proteica , Fatores Supressores Imunológicos/química , Ubiquitinas/químicaRESUMO
Receptor-ligand binding is an essential interaction for biological function. Oxidative stress can modify receptors and/or membrane lipid dynamics, thus altering cell physiological functions. The aim of this study is to analyze how oxidative stress may alter receptor-ligand binding and lipid domain distribution in the case of progesterone-induced blocking factor/progesterone-induced blocking factor-receptor. For membrane fluidity regionalization analysis of MEC-1 lymphocytes, two-photon microscopy was used in individual living cells. Lymphocytes were also double stained with AlexaFluor647/progesterone-induced blocking factor and Laurdan to evaluate -induced blocking factor/progesterone-induced blocking factor-receptor distribution in the different membrane domains, under oxidative stress. A new procedure has been developed which quantitatively analyzes the regionalization of a membrane receptor among the lipid domains of different fluidity in the plasma membrane. We have been able to establish a new tool which detects and evaluates lipid raft clustering from two-photon microscopy images of individual living cells. We show that binding of progesterone-induced blocking factor to progesterone-induced blocking factor-receptor causes a rigidification of plasma membrane which is related to an increase of lipid raft clustering. However, this clustering is inhibited under oxidative stress conditions. In conclusion, oxidative stress decreases membrane fluidity, impairs receptor-ligand binding and reduces lipid raft clustering.
Assuntos
Linfócitos B/efeitos dos fármacos , Peróxido de Hidrogênio/farmacologia , Microdomínios da Membrana/efeitos dos fármacos , Microscopia de Fluorescência por Excitação Multifotônica/métodos , Proteínas da Gravidez/química , Receptores de Citocinas/química , Análise de Célula Única/métodos , Fatores Supressores Imunológicos/química , 2-Naftilamina/análogos & derivados , 2-Naftilamina/química , Linfócitos B/citologia , Linfócitos B/metabolismo , Carbocianinas/química , Linhagem Celular Tumoral , Corantes Fluorescentes/química , Humanos , Lauratos/química , Fluidez de Membrana/efeitos dos fármacos , Microdomínios da Membrana/química , Microdomínios da Membrana/metabolismo , Estresse Oxidativo , Proteínas da Gravidez/metabolismo , Ligação Proteica , Receptores de Citocinas/metabolismo , Coloração e Rotulagem/métodos , Fatores Supressores Imunológicos/metabolismoRESUMO
MNSFbeta (Monoclonal nonspecific suppressor factor beta) is a natural immunosuppressive factor which has been reported to be involved in various biological processes, such as immune responses, cell division, stress response, cell apoptosis, and nuclear transport. However, study on porcine MNSFbeta has been rarely reported. In this study, the full-length sequence of porcine MNSFbeta (GenBank accession number: KF77642500) was predicted in silicon and its cDNA sequence was obtained through RT-PCR from porcine spleen. The nucleic acid and protein sequences were analyzed. Then, the gene was subcloned into pEGFP-C1 to construct a recombinant plasmid pEGFP-MNSFbeta which was transfected into swine umbilical vein endothelial cells (SUVECs) using Lipofectamine 2000. The expression of GFP was detected by fluorescence microscopy, Western blot, and laser confocal fluorescence microscopy. The spatial expression patterns of porcine MNSFbeta were detected by real-time qPCR. Results showed that the full length of porcine MNSFbeta was 402 bp encoding 133 amino acids with only one exon. Bioinformatics analysis showed that porcine MNSFbeta protein was a stable protein consisting of a ubiquitin-like domain fused to the ribosomal protein S30 with no signal peptide. The analyses of homology and phylogenetic tree of porcine MNSFbeta and its homologs in other 18 species showed that the identities of MNSFbeta protein sequence were higher than 91% among different species and the evolutionary distance was less than 0.05. It indicates that MNSFbeta is highly conserved in the process of evolution. Fluorescence signal showed that the fusion protein GFP-MNSFbeta was successfully expressed in SUVECs which was then confirmed by Western blot. Laser confocal fluorescence microscopy showed that MNSFbeta was expressed in both nucleus and cytoplasm. Analysis of spatial expression patterns showed that procine MNSFbeta was widely expressed in immune tissues, but not in lung, suggesting that MNSFbeta may play an important role in immune response.
Assuntos
Fatores Supressores Imunológicos/metabolismo , Animais , Western Blotting , Biologia Computacional , Masculino , Estrutura Secundária de Proteína , Fatores Supressores Imunológicos/química , Fatores Supressores Imunológicos/genética , SuínosRESUMO
In experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis, apoptosis of T cells is mainly seen at inflammation sites of the central nervous system (CNS). Cumulative data suggests that astrocytes might render T cells susceptible to induction of apoptotic cell death. We observed that apoptotic cell death of proteolipid protein (PLP)-reactive T cells was induced by an interferon (IFN)-γ-treated astrocyte cell line. In this study, we have identified and cloned the genes derived from the IFN-γ-treated astrocyte cell line that induce apoptosis of autoreactive T cells. We created subtraction cDNA libraries from the IFN-γ-treated astrocyte cell line and obtained 100 positive clones. After screening of subtracted cDNAs, we found two candidate genes that induced apoptosis of the PLP-reactive T cell line. The first is a previously unknown gene of 726 base pairs that we named astrocyte-derived immune suppressor factor (AdIF). It contained an open reading frame encoding a polypeptide of 228 amino acids. The second was SPARC/osteonectin, a multifunctional glycoprotein secreted in the extracellular matrix. AdIF protein was found at the inflammatory sites of the EAE brain, and bound to the surface of CD4(+) T cells. Purified recombinant AdIF protein inhibited the proliferation of activated PLP-reactive CD4(+) T cells and induced their apoptosis in vitro. Intravenous administration of recombinant AdIF protein to mice with in which acute EAE was induced prevented the incidence of EAE and suppressed the symptoms. The newly discovered molecule AdIF may render auto-reactive T cells susceptible to the induction of apoptotic cell death and could potentially be a new therapeutic agent for multiple sclerosis.
Assuntos
Proteínas Reguladoras de Apoptose/fisiologia , Apoptose/imunologia , Astrócitos/imunologia , Encefalomielite Autoimune Experimental/imunologia , Encefalomielite Autoimune Experimental/patologia , Fatores Supressores Imunológicos/fisiologia , Subpopulações de Linfócitos T/imunologia , Sequência de Aminoácidos , Animais , Apoptose/efeitos dos fármacos , Proteínas Reguladoras de Apoptose/química , Proteínas Reguladoras de Apoptose/genética , Astrócitos/efeitos dos fármacos , Astrócitos/metabolismo , Sequência de Bases , Células CHO , Linhagem Celular , Cricetinae , Cricetulus , Encefalomielite Autoimune Experimental/metabolismo , Terapia de Imunossupressão/métodos , Camundongos , Camundongos Endogâmicos C57BL , Dados de Sequência Molecular , Fatores Supressores Imunológicos/química , Fatores Supressores Imunológicos/genética , Subpopulações de Linfócitos T/efeitos dos fármacos , Subpopulações de Linfócitos T/patologiaRESUMO
Monoclonal nonspecific suppressor factor beta (MNSFbeta) is a ubiquitously expressed member of the ubiquitin-like family that has been implicated in various biological functions. Previous studies have demonstrated that MNSFbeta covalently binds to the intracellular proapoptotic protein Bcl-G in cells of the macrophage cell line Raw264.7, suggesting involvement of this ubiquitin-like protein in apoptosis. In this study, we purified a 62 kDa MNSFbeta adduct from murine liver lysates by sequential chromatography on DEAE and anti-MNSFbeta IgG-conjugated Sepharose. MALDI-TOF MS fingerprinting revealed that this MNSFbeta adduct consists of an 8.5 kDa MNSFbeta and endophilin II, a member of the endophilin A family. MNSFbeta may conjugate to endophilin II with a linkage between the C-terminal Gly74 and Lys294. We confirmed this result by immunoprecipitation/western blot studies. Endophilin II was ubiquitously expressed in various tissues, although a truncated form was observed in liver. The 62 kDa MNSFbeta-endophilin II was specifically expressed in liver and macrophages. Small interfering RNA-mediated knockdown of endophilin II and/or MNSFbeta promoted phagocytosis of zymosan in Raw264.7 cells. Conversely, cotransfection of endophilin II and MNSFbeta cDNAs inhibited the phagocytosis of zymosan. Such inhibition was not observed in cells expressing a mutant of endophilin II in which Lys294 was replaced by arginine. These results suggest that the post-translational modification of endophilin II by MNSFbeta might be implicated in phagocytosis by macrophages.
Assuntos
Peptídeos e Proteínas de Sinalização Intracelular/fisiologia , Fagocitose , Fatores Supressores Imunológicos/fisiologia , Sequência de Aminoácidos , Animais , Linhagem Celular , Imunoprecipitação , Peptídeos e Proteínas de Sinalização Intracelular/química , Fígado/química , Camundongos , Dados de Sequência Molecular , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Fatores Supressores Imunológicos/química , Fatores Supressores Imunológicos/isolamento & purificaçãoRESUMO
The yeast Paf1 complex consists of Paf1, Rtf1, Cdc73, Ctr9, and Leo1 and regulates histone H2B ubiquitination, histone H3 methylation, RNA polymerase II carboxy-terminal domain (CTD) Ser2 phosphorylation, and RNA 3' end processing. We provide structural insight into the Paf1 complex with the NMR structure of the conserved and functionally important Plus3 domain of human Rtf1. A predominantly beta-stranded subdomain displays structural similarity to Dicer/Argonaute PAZ domains and to Tudor domains. We further demonstrate that the highly basic Rtf1 Plus3 domain can interact in vitro with single-stranded DNA via residues on the rim of the beta sheet, reminiscent of siRNA binding by PAZ domains, but did not detect binding to double-stranded DNA or RNA. We discuss the potential role of Rtf1 Plus3 ssDNA binding during transcription elongation.
Assuntos
Fatores Supressores Imunológicos/química , Fatores Supressores Imunológicos/metabolismo , Fatores de Transcrição/química , Fatores de Transcrição/genética , Sequência de Aminoácidos , Sítios de Ligação , Sequência Conservada , DNA de Cadeia Simples/química , DNA de Cadeia Simples/genética , DNA de Cadeia Simples/metabolismo , Humanos , Modelos Moleculares , Dados de Sequência Molecular , Ácidos Nucleicos/química , Ácidos Nucleicos/metabolismo , Conformação Proteica , Alinhamento de Sequência , Fatores Supressores Imunológicos/genética , Transcrição GênicaRESUMO
OBJECTIVE: Ingested type I IFN inhibits clinical attacks, relapses and inflammation in murine chronic relapsing EAE by inhibiting Th1-like cytokines. Type I IFN activates human suppressor T cells that produce SIRS. METHODS: We examined whether oral (ingested) SIRS peptide inhibits EAE by decreasing Th1-like cytokines. RESULTS: Parenteral SIRS peptide 1-21 showed a significant inhibition of disease severity in murine EAE. Ingested SIRS peptide at 10 and 100 microg SIRS peptide showed a significant inhibition of disease severity but also a prolonged delay in the onset of disease compared to placebo. There were significantly less inflammatory foci in the SIRS peptide fed group compared to the control mock fed group. Splenocytes from SIRS peptide 1-21 fed mice showed increased production of Th2-like CD30L, IL-13, TCA-3 cytokines/chemokines and decreased production of Th1-like cytokine lymphotactin. INTERPRETATION: Ingested (oral) SIRS peptide significantly inhibits both clinical EAE and inflammation predominately via counter-regulatory type 2-like cytokines/chemokines IL-13, CD30L and TCA-3.
Assuntos
Citocinas/metabolismo , Encefalomielite Autoimune Experimental/tratamento farmacológico , Regulação da Expressão Gênica/efeitos dos fármacos , Peptídeos/administração & dosagem , Fatores Supressores Imunológicos/química , Administração Oral , Transferência Adotiva/métodos , Animais , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Relação Dose-Resposta Imunológica , Feminino , Camundongos , Camundongos Endogâmicos C57BL , Fatores Supressores Imunológicos/administração & dosagem , Fatores de TempoRESUMO
In an attempt to identify new immunoreceptor tyrosine-based activation motif (ITAM)-containing human molecules that may regulate hitherto unknown immune cell functions, we BLAST searched the National Center for Biotechnology Information database for ITAM-containing sequences. A human expressed sequence tag showing partial homology to the murine TJ6 (mTJ6) gene and encoding a putative ITAM sequence has been identified and used to clone the human TJ6 (hTJ6) gene from an HL-60-derived cDNA library. hTJ6 was found to encode a protein of 856 residues with a calculated mass of 98 155 Da. Immunolocalization and sequence analysis revealed that hTJ6 is a membrane protein with predicted six transmembrane-spanning regions, typical of ion channels, and a single putative ITAM (residues 452-466) in a juxtamembrane or hydrophobic intramembrane region. hTJ6 is highly homologous to Bos taurus 116-kDa subunit of the vacuolar proton-translocating ATPase. Over-expression of hTJ6 in HEK 293 cells increased H+ uptake into intracellular organelles, an effect that was sensitive to inhibition by bafilomycin, a selective inhibitor of vacuolar H+ pump. Northern blot analysis demonstrated three different hybridizing mRNA transcripts corresponding to 3.2, 5.0 and 7.3 kb, indicating the presence of several splice variants. Significant differences in hTJ6 mRNA levels in human tissues of different origins point to possible tissue-specific function. Although hTJ6 was found to be a poor substrate for tyrosine-phosphorylating enzymes, suggesting that its ITAM sequence is non-functional in protein tyrosine kinase-mediated signaling pathways, its role in organellar H+ pumping suggests that hTJ6 function may participate in protein trafficking/processing.
Assuntos
Proteínas da Gravidez/química , Proteínas da Gravidez/genética , Subunidades Proteicas/química , Subunidades Proteicas/genética , Fatores Supressores Imunológicos/química , Fatores Supressores Imunológicos/genética , Tirosina/química , ATPases Vacuolares Próton-Translocadoras/fisiologia , Motivos de Aminoácidos/imunologia , Sequência de Aminoácidos , Animais , Northern Blotting , Bovinos , Linhagem Celular , Clonagem Molecular , Sequência Conservada , Humanos , Células Jurkat , Dados de Sequência Molecular , Proteínas da Gravidez/biossíntese , Proteínas da Gravidez/fisiologia , Processamento de Proteína Pós-Traducional/imunologia , Subunidades Proteicas/fisiologia , Transporte Proteico/imunologia , Receptores Imunológicos/genética , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Fatores Supressores Imunológicos/biossíntese , Fatores Supressores Imunológicos/fisiologia , Tirosina/genética , Tirosina/metabolismo , ATPases Vacuolares Próton-Translocadoras/biossíntese , ATPases Vacuolares Próton-Translocadoras/genéticaRESUMO
Monoclonal nonspecific suppressor factor (MNSF), a lymphokine produced by murine T cell hybridoma, possesses pleiotrophic antigen-nonspecific suppressive functions. A cDNA clone encoding MNSFbeta, an isoform of the MNSF, has been isolated and characterized. MNSFbeta cDNA encodes a fusion protein consisting of a ubiquitin-like segment (Ubi-L) and ribosomal protein S30. Most recently, we observed that Ubi-L covalently conjugates to Bcl-G, a novel pro-apoptotic protein. In this study, we observed that Ubi-L noncovalently and specifically binds to histone 2A. The maximum binding was observed at a molar ratio equal to 1 for GST-Ubi-L and 2 for histone 2A. Ubi-L formed complex with histone 2A in the presence of 1% Triton X-100. Free Ubi-L was detected in nuclei from unstimulated murine helper T cell line, D10. The increased amounts of free Ubi-L and some Ubi-L adducts were observed in nuclei from mitogen-activated D10 cells. Interestingly, two Ubi-L adducts were unique to the chromatin fraction of nuclei from the activated D10 cells.
Assuntos
Histonas/metabolismo , Fatores Supressores Imunológicos/química , Fatores Supressores Imunológicos/metabolismo , Ubiquitinas/química , Animais , Cinética , Camundongos , Ligação Proteica , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , Fatores Supressores Imunológicos/genéticaRESUMO
PROBLEM: Early pregnancy factor (EPF) is an immunosuppressive protein detected in the serum in early pregnancy. We have already reported the development of the rosette inhibition test for mare EPF and have detected EPF in thoroughbreds and ponies. Here, we attempted to purify equine EPF from pregnant mare serum. METHODS OF STUDY: Mare EPF was purified by ultrafiltration and ion-exchange chromatography. Purified EPF was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), immunoblotting and a neutralization test. EPF activity was estimated as the rosette inhibition titer (RIT) by the rosette inhibition test. RESULTS: Purified EPF bound to carboxymethyl (CM) sepharose and did not adsorb to diethylaminoethyl (DEAE) sepharose. SDS-PAGE revealed that in the final purified fraction there were many proteins. In the immunoblotting analysis, a protein band of 25.8 kDa was detected as the pregnancy-specific band. Further, antibody gained from the 20 to 30 kDa protein band of the final purified fraction neutralized the RIT activity of pregnant mare serum. CONCLUSIONS: Mare EPF was detected in the final purified fraction and had a molecular weight of 25.8 kDa. EPF in the mare is similar to that obtained from the serum of pregnant cows.
Assuntos
Cavalos , Peptídeos/isolamento & purificação , Proteínas da Gravidez/isolamento & purificação , Fatores Supressores Imunológicos/isolamento & purificação , Animais , Chaperonina 10 , Cromatografia em Gel , Cromatografia por Troca Iônica , Eletroforese em Gel de Poliacrilamida , Feminino , Peptídeos/química , Gravidez , Proteínas da Gravidez/química , Testes de Gravidez , Coelhos , Formação de Roseta , Fatores Supressores Imunológicos/químicaRESUMO
Previous studies from our laboratory showed that the immunomodulatory effects of progesterone are mediated by a 34-kDa protein, named the progesterone-induced blocking factor (PIBF). Lymphocytes of women with threatened abortion fail to produce this factor. Via inducing a Th2 biased cytokine production and blocking of NK activity, PIBF prevents induced pregnancy loss in mice, suggesting that substitution therapy with PIBF could be useful as an alternative treatment of certain forms of recurrent spontaneous abortions. Our study was aimed at mapping the sequence and structure of PIBF coding cDNA and characterizing the encoded protein product. Screening of a human liver cDNA library revealed a 2765-bp clone with a 2271-bp open reading frame. The PIBF1 cDNA encodes a protein of 757 amino acid residues with an 89-kDa predicted molecular mass, which shows no significant amino acid sequence homology with any known protein. PIBF produced via recombinant technique is recognized by the Ab specific for the secreted lymphocyte PIBF Ab, and possesses the biological activities of the secreted lymphocyte PIBF. The full-length PIBF is associated with the nucleus, whereas secretion of shorter forms, such a 34-kDa protein is induced by activation of the cell. The 48-kDa N-terminal part of PIBF is biologically active, and the part of the molecule, responsible for modulating NK activity is encoded by exons 2-4. These data provide an initial step for exploiting the possible diagnostic and therapeutic potential of this immunomodulatory molecule.
Assuntos
Adjuvantes Imunológicos/química , Adjuvantes Imunológicos/genética , Clonagem Molecular/métodos , DNA Complementar/química , DNA Complementar/imunologia , Proteínas da Gravidez/química , Proteínas da Gravidez/genética , Adjuvantes Imunológicos/isolamento & purificação , Adjuvantes Imunológicos/metabolismo , Sequência de Aminoácidos , Especificidade de Anticorpos , Linhagem Celular Tumoral , Citocinas/biossíntese , Testes Imunológicos de Citotoxicidade , DNA Complementar/isolamento & purificação , Feminino , Humanos , Soros Imunes/metabolismo , Imuno-Histoquímica , Líquido Intracelular/imunologia , Líquido Intracelular/metabolismo , Células K562 , Células Matadoras Naturais/imunologia , Células Matadoras Naturais/metabolismo , Dados de Sequência Molecular , Gravidez , Proteínas da Gravidez/imunologia , Proteínas da Gravidez/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/farmacologia , Fatores Supressores Imunológicos/química , Fatores Supressores Imunológicos/genética , Fatores Supressores Imunológicos/imunologia , Fatores Supressores Imunológicos/metabolismoAssuntos
Plasmodium falciparum/química , Plasmodium falciparum/metabolismo , Alinhamento de Sequência/métodos , Análise de Sequência de Proteína/métodos , Fatores Supressores Imunológicos/química , Fatores Supressores Imunológicos/metabolismo , Linfócitos T/metabolismo , Sequência de Aminoácidos , Animais , Biomarcadores , Perfilação da Expressão Gênica/métodos , Dados de Sequência Molecular , Plasmodium falciparum/genética , Fatores Supressores Imunológicos/genética , Especificidade do Receptor de Antígeno de Linfócitos T/genética , Linfócitos T/química , Linfócitos T/efeitos dos fármacos , Linfócitos T/imunologiaRESUMO
AIM: To purify a protein in pig spleens, which was similar to immune suppressive protein of stress (ISPS), and characterize its properties and functions. METHODS: 1) Pig spleen was extracted in dilute hydrochloric acid. 2) The extract was ultra-filtrated for having high molecular weight proteins (Mr>30 000). 3) The filtrates were purified with FPLC affinity chromatography. 4) The elute from FPLC was used for T-lymphocyte proliferation and ELISA test. 5) Lastly, SDS-PAGE was used to determine the molecular weight and purity of the final product. RESULTS: A protein purified from pig spleen (the pig ISPS homologue) inhibited concanavalin A (Con A)-induced mouse lymphocyte proliferation. The molecular weight of this protein was about Mr 190 000. It has a stronger selectivity against T-lymphocyte line such as Jurkat cell line and mastocyte line (P8l5) and has a weaker inhibitory activity on macrophage line (U937). CONCLUSION: A protein similar to rat/mouse ISPS was found in pig spleen. This may provide an opportunity to study its roles in tumors and autoimmune diseases.
Assuntos
Baço/química , Estresse Fisiológico/metabolismo , Fatores Supressores Imunológicos/isolamento & purificação , Animais , Divisão Celular/efeitos dos fármacos , Linhagem Celular , Humanos , Células Jurkat/efeitos dos fármacos , Linfócitos/citologia , Linfócitos/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos BALB C , Peso Molecular , Fatores Supressores Imunológicos/química , Fatores Supressores Imunológicos/farmacologia , SuínosRESUMO
A coordinated effort combining bioinformatic tools with high-throughput cell-based screening assays was implemented to identify novel factors involved in T-cell biology. We generated a unique library of cDNAs encoding predicted secreted and transmembrane domain-containing proteins generated by analyzing the Human Genome Sciences cDNA database with a combination of two algorithms that predict signal peptides. Supernatants from mammalian cells transiently transfected with this library were incubated with primary T cells and T-cell lines in several high-throughput assays. Here we describe the discovery of a T cell factor, TIP (T cell immunomodulatory protein), which does not show any homology to proteins with known function. Treatment of primary human and murine T cells with TIP in vitro resulted in the secretion of IFN-gamma, TNF-alpha, and IL-10, whereas in vivo TIP had a protective effect in a mouse acute graft-versus-host disease (GVHD) model. Therefore, combining functional genomics with high-throughput cell-based screening is a valuable and efficient approach to identifying immunomodulatory activities for novel proteins.
Assuntos
Doença Enxerto-Hospedeiro/tratamento farmacológico , Análise de Sequência de Proteína/métodos , Fatores Supressores Imunológicos/administração & dosagem , Fatores Supressores Imunológicos/química , Linfócitos T/metabolismo , Adjuvantes Imunológicos/química , Adjuvantes Imunológicos/genética , Adjuvantes Imunológicos/metabolismo , Animais , Linhagem Celular , Perfilação da Expressão Gênica/métodos , Doença Enxerto-Hospedeiro/imunologia , Doença Enxerto-Hospedeiro/metabolismo , Humanos , Rim/química , Rim/embriologia , Rim/imunologia , Camundongos/genética , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/imunologia , Proteínas Recombinantes de Fusão/metabolismo , Fatores Supressores Imunológicos/genética , Fatores Supressores Imunológicos/imunologia , Especificidade do Receptor de Antígeno de Linfócitos T/genética , Linfócitos T/química , Linfócitos T/efeitos dos fármacos , Linfócitos T/imunologia , Transfecção/métodosRESUMO
Monoclonal nonspecific suppressor factor (MNSF), a lymphokine produced by murine T cell hybridoma, possesses a pleiotropic antigen-nonspecific suppressive function. We have shown that 70 kDa MNSF comprises an 8 kDa ubiquitin-like polypeptide (Ubi-L) and 62 kDa T cell receptor (TCR) alpha-like molecule. Ubi-L binds specifically to its 82 kDa receptor protein on target cells. In the current study, we have further characterized the biochemical nature of the TCR(alpha)-like molecule. The 62 kDa protein was separated into two species of 46 kDa and 16 kDa on reverse-phase HPLC. Anti-TCR(alpha) monoclonal antibody recognized the 46 kDa, but not the 16 kDa protein. Anti-TCRbeta monoclonal antibody failed to recognize these proteins. Ubi-L conjugated to the 46 kDa protein, whereas Ubi-L lacking its C-terminal Gly-Gly did not. Although Ubi-L was labile both to heating at 56 degrees C and to acidification to pH 4, the Ubi-L-46 kDa protein complex was unaffected by these treatments. In addition, the 46 kDa protein elongated the Ubi-L-induced protein tyrosine phosphorylation in a concanavalin A-activated murine T helper type 2 clone, D10 cells. One of the four tryptic peptide sequences derived from the 46 kDa protein was in alignment with a related sequence found in the J(alpha) region of the TCR(alpha), including the highly conserved motif F-G-X-G-T-X-L.