ABSTRACT
A hypoxic microenvironment induces resistance to alkylating agents by activating targets in the mammalian target of rapamycin (mTOR) pathway. The molecular mechanisms involved in this mTOR-mediated hypoxia-induced chemoresistance, however, are unclear. Here we identify the mTOR target N-myc downstream regulated gene 1 (NDRG1) as a key determinant of resistance toward alkylating chemotherapy, driven by hypoxia but also by therapeutic measures such as irradiation, corticosteroids, and chronic exposure to alkylating agents via distinct molecular routes involving hypoxia-inducible factor (HIF)-1alpha, p53, and the mTOR complex 2 (mTORC2)/serum glucocorticoid-induced protein kinase 1 (SGK1) pathway. Resistance toward alkylating chemotherapy but not radiotherapy was dependent on NDRG1 expression and activity. In posttreatment tumor tissue of patients with malignant gliomas, NDRG1 was induced and predictive of poor response to alkylating chemotherapy. On a molecular level, NDRG1 bound and stabilized methyltransferases, chiefly O(6)-methylguanine-DNA methyltransferase (MGMT), a key enzyme for resistance to alkylating agents in glioblastoma patients. In patients with glioblastoma, MGMT promoter methylation in tumor tissue was not more predictive for response to alkylating chemotherapy in patients who received concomitant corticosteroids.
Subject(s)
Antineoplastic Agents, Alkylating/pharmacology , Brain Neoplasms/drug therapy , Cell Cycle Proteins/metabolism , Drug Resistance, Neoplasm , Gene Expression Regulation, Neoplastic , Glioblastoma/drug therapy , Glioma/drug therapy , Intracellular Signaling Peptides and Proteins/metabolism , O(6)-Methylguanine-DNA Methyltransferase/pharmacology , TOR Serine-Threonine Kinases/metabolism , Animals , Brain Neoplasms/metabolism , DNA Repair , Glioblastoma/metabolism , Glioma/metabolism , Humans , Hypoxia , Immunoblotting , Lentivirus/metabolism , Mice , Mice, Nude , Neoplasm Transplantation , Plasmids/metabolism , Time FactorsABSTRACT
The R2TP is a recently identified Hsp90 co-chaperone, composed of four proteins as follows: Pih1D1, RPAP3, and the AAA(+)-ATPases RUVBL1 and RUVBL2. In mammals, the R2TP is involved in the biogenesis of cellular machineries such as RNA polymerases, small nucleolar ribonucleoparticles and phosphatidylinositol 3-kinase-related kinases. Here, we characterize the spaghetti (spag) gene of Drosophila, the homolog of human RPAP3. This gene plays an essential function during Drosophila development. We show that Spag protein binds Drosophila orthologs of R2TP components and Hsp90, like its yeast counterpart. Unexpectedly, Spag also interacts and stimulates the chaperone activity of Hsp70. Using null mutants and flies with inducible RNAi, we show that spaghetti is necessary for the stabilization of snoRNP core proteins and target of rapamycin activity and likely the assembly of RNA polymerase II. This work highlights the strong conservation of both the HSP90/R2TP system and its clients and further shows that Spag, unlike Saccharomyces cerevisiae Tah1, performs essential functions in metazoans. Interaction of Spag with both Hsp70 and Hsp90 suggests a model whereby R2TP would accompany clients from Hsp70 to Hsp90 to facilitate their assembly into macromolecular complexes.
Subject(s)
Drosophila Proteins/metabolism , HSP70 Heat-Shock Proteins/metabolism , Heat-Shock Proteins/metabolism , Models, Biological , Molecular Chaperones/metabolism , Ribonucleoproteins, Small Nucleolar/metabolism , Animals , Anti-Bacterial Agents/pharmacology , Apoptosis Regulatory Proteins , Carrier Proteins/genetics , Carrier Proteins/metabolism , Drosophila Proteins/genetics , Drosophila melanogaster , HSP70 Heat-Shock Proteins/genetics , Heat-Shock Proteins/genetics , Humans , Molecular Chaperones/genetics , RNA Polymerase II/genetics , RNA Polymerase II/metabolism , Ribonucleoproteins, Small Nucleolar/genetics , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/metabolism , Saccharomyces cerevisiae Proteins/genetics , Saccharomyces cerevisiae Proteins/metabolism , Sirolimus/pharmacologyABSTRACT
Mapping of proteins involved in normal eye functions is a prerequisite to identify pathological changes during eye disease processes. We therefore analysed the proteome of human vitreous by applying in-depth proteomic screening technologies. For ethical reasons human vitreous samples were obtained by vitrectomy from "surrogate normal patients" with epiretinal gliosis that is considered to constitute only negligible pathological vitreoretinal changes. We applied different protein prefractionation strategies including liquid phase isoelectric focussing, 1D SDS gel electrophoresis and a combination of both and compared the number of identified proteins obtained by the respective method. Liquid phase isoelectric focussing followed by SDS gel electrophoresis increased the number of identified proteins by a factor of five compared to the analysis of crude unseparated human vitreous. Depending on the prefractionation method proteins were subjected to trypsin digestion either in-gel or in solution and the resulting peptides were analysed on a UPLC system coupled online to an LTQ Orbitrap XL mass spectrometer. The obtained mass spectra were searched against the SwissProt database using the Mascot search engine. Bioinformatics tools were used to annotate known biological functions to the detected proteins. Following this strategy we examined the vitreous proteomes of three individuals and identified 1111 unique proteins. Besides structural, transport and binding proteins, we detected 261 proteins with known enzymatic activity, 51 proteases, 35 protease inhibitors, 35 members of complement and coagulation cascades, 15 peptide hormones, 5 growth factors, 11 cytokines, 47 receptors, 30 proteins of visual perception, 91 proteins involved in apoptosis regulation and 265 proteins with signalling activity. This highly complex mixture strikingly differs from the human plasma proteome. Thus human vitreous fluid seems to be a unique body fluid. 262 unique proteins were detected which are present in all three patient samples indicating that these might represent the constitutive protein pattern of human vitreous. The presented catalogue of human vitreous proteins will enhance our understanding of physiological processes in the eye and provides the groundwork for future studies on pathological vitreous proteome changes.
ABSTRACT
Signals elicited by TLRs following the detection of microbes are integrated and diversified by a group of four cytoplasmic adaptor molecules featuring an evolutionarily conserved Toll/IL-1R signaling domain. Single nucleotide polymorphisms (SNPs) in TLRs and their adaptor molecules have been shown to influence susceptibility to a range of infectious and other diseases. The adaptor MyD88 adaptor-like (Mal)/Toll/IL-1R-containing adaptor protein is involved in TLR2 and 4 signal transduction by recruiting another adaptor molecule, MyD88, to the plasma membrane. In this study, we used naturally occurring variants of Mal as tools to study the molecular biology of Mal in more detail in cellular model systems and to thereby identify functionally interesting variants whose corresponding nonsynonymous SNPs might be of further epidemiological interest. Of seven reported variants for Mal, we found Mal D96N associated with reduced NF-kappaB signaling and cytokine production after overexpression in HEK293 and Huh-7 cells. The D96N mutation prevented Mal from recruiting its signaling partner MyD88 to the plasma membrane and altered posttranslational modification of Mal. These findings led us to investigate the frequency of heterozygosity for the corresponding SNP rs8177400 in a Caucasian case-control study on the etiology of lymphoma, a disease in which TLRs have been implicated. Although rs8177400 did not modify lymphoma risk in general, its frequency of heterozygosity was accurately determined to 0.97%. Our data add rs8177400 (D96N) to the list of functionally important variants of Mal and warrant further research into its immunological, epidemiological, and diagnostic relevance.
Subject(s)
Genetic Variation/immunology , Membrane Glycoproteins/deficiency , Membrane Glycoproteins/genetics , Myeloid Differentiation Factor 88 , Protein Processing, Post-Translational/immunology , Receptors, Interleukin-1/deficiency , Receptors, Interleukin-1/genetics , Signal Transduction/genetics , Signal Transduction/immunology , Adolescent , Adult , Aged , Aged, 80 and over , Asparagine/genetics , Aspartic Acid/genetics , Case-Control Studies , Cell Line, Transformed , Cell Line, Tumor , Genetic Predisposition to Disease/epidemiology , Humans , Immunophenotyping , Membrane Glycoproteins/physiology , Middle Aged , Multicenter Studies as Topic , Myeloid Differentiation Factor 88/physiology , NF-kappa B/metabolism , Point Mutation/immunology , Protein Processing, Post-Translational/genetics , Protein Transport/genetics , Protein Transport/immunology , Receptors, Interleukin-1/physiologyABSTRACT
Progression through mitosis requires the coordinated regulation of Cdk1 kinase activity. Activation of Cdk1 is a multistep process comprising binding of Cdk1 to cyclin B, relocation of cyclin-kinase complexes to the nucleus, activating phosphorylation of Cdk1 on Thr(161) by the Cdk-activating kinase (CAK; Cdk7 in metazoans), and removal of inhibitory Thr(14) and Tyr(15) phosphorylations. This dephosphorylation is catalyzed by the dual specific Cdc25 phosphatases, which occur in three isoforms in mammalian cells, Cdc25A, -B, and -C. We find that expression of Cdc25A leads to an accelerated G(2)/M phase transition. In Cdc25A-overexpressing cells, Cdk1 exhibits high kinase activity despite being phosphorylated on Tyr(15). In addition, Tyr(15)-phosphorylated Cdk1 binds more cyclin B in Cdc25A-overexpressing cells compared with control cells. Consistent with this observation, we demonstrate that in human transformed cells, Cdc25A and Cdc25B, but not Cdc25C phosphatases have an effect on timing and efficiency of cyclin-kinase complex formation. Overexpression of Cdc25A or Cdc25B promotes earlier assembly and activation of Cdk1-cyclin B complexes, whereas repression of these phosphatases by short hairpin RNA has a reverse effect, leading to a substantial decrease in amounts of cyclin B-bound Cdk1 in G(2) and mitosis. Importantly, we find that Cdc25A overexpression leads to an activation of Cdk7 and increase in Thr(161) phosphorylation of Cdk1. In conclusion, our data suggest that complex assembly and dephosphorylation of Cdk1 at G(2)/M is tightly coupled and regulated by Cdc25 phosphatases.
Subject(s)
CDC2 Protein Kinase/metabolism , Cell Division , Cyclin B/metabolism , G2 Phase , cdc25 Phosphatases/metabolism , Base Sequence , Cell Cycle , Cell Line, Tumor , Cyclin-Dependent Kinases/metabolism , Flow Cytometry/methods , Humans , Molecular Sequence Data , Phosphorylation , Tyrosine/chemistry , Cyclin-Dependent Kinase-Activating KinaseABSTRACT
Our studies aim to elucidate the functions carried out by the very long, and in its length highly conserved, C-terminal cytoplasmic domain (Env-CT) of the HIV-1 glycoprotein. Mass spectrometric analysis of cellular proteins bound to a tagged version of the HIV Env-CT led to the identification of the prohibitin 1 and 2 proteins (Phb1 and Phb2). These ubiquitously expressed proteins, which exist as stable heterodimers, have been shown to have multiple functions within cells and to localize to multiple cellular and extracellular compartments. The specificity of binding of the Phb1/Phb2 complex to the Env-CT was confirmed in various manners, including coimmunoprecipitation with authentic provirally encoded, full-length Env. Strong binding was dependent on Env residues 790 to 800 and could be severely inhibited by the double mutation L799R/L800Q but not by mutation of these amino acids individually. Analysis of the respective mutant virions revealed that their different abilities to bind Phb1/Phb2 correlated with their replicative properties. Thus, mutated virions with single mutations [HIV-Env-(L799R) and HIV-Env-(L800Q)] replicated similarly to wild-type HIV, but HIV-Env-(L799R/L800Q) virions, which cannot bind Phb1/Phb2, exhibited a cell-dependent replicative phenotype similar to that of HIV-Env-Tr712, lacking the entire Env-CT domain. Thus, replicative spread was achieved, although somewhat delayed, in "permissive" MT-4 cells but failed to occur in "nonpermissive" H9 T cells. These results point to binding of the Phb1/Phb2 complex to the Env-CT as being of importance for replicative spread in nonpermissive cells, possibly by modulating critical Phb-dependent cellular process(es).
Subject(s)
Cytoplasm/metabolism , Gene Products, env/metabolism , HIV-1/metabolism , Repressor Proteins/metabolism , Amino Acid Sequence , Cell Line , Chromatography, Affinity , Dimerization , Electrophoresis, Polyacrylamide Gel , Fluorescent Antibody Technique , Gene Products, env/chemistry , Gene Products, env/genetics , Gene Products, env/isolation & purification , HIV-1/pathogenicity , Humans , Immunoprecipitation , Mutation , Prohibitins , Protein Binding , Repressor Proteins/chemistry , Spectrometry, Mass, Matrix-Assisted Laser Desorption-IonizationABSTRACT
By comparing newly available cDNA sequences of the human intermediate filament protein lamin B(2) with published sequences, we have identified an additional translation initiation codon 60 nucleotides upstream of the previously assumed translation start. In addition, corresponding sequences were identified in the chimpanzee, mouse, rat and bovine genes and cDNAs, respectively. Therefore, we generated antibodies against these potential 20 new amino acids of the human sequence. By immunoblot analysis and immunofluorescence microscopy we show that human lamin B(2) is indeed synthesized as a longer version than previously reported, because it contains these additional 20 amino acids. Notably, the sequence homology to mouse, rat and bovine lamin B(2) is significantly lower in this segment than in that between the second methionine codon and the start of the alpha-helical rod indicating that the tip of the "head" is engaged in more species-specific functions. Forced expression of the GFP-tagged authentic "long" and the 20 amino acid shorter version of lamin B(2) in human cultured SW-13 cells demonstrated that both the longer and the shorter version are properly integrated into the nuclear lamina, although the shorter version exhibited a tendency to disturb envelope architecture at higher expression levels.
Subject(s)
Lamin Type B/chemistry , Nuclear Proteins/chemistry , Amino Acid Sequence , Animals , Cell Line , Codon, Initiator , Humans , Lamin Type B/biosynthesis , Molecular Sequence Data , Nuclear Lamina , Nuclear Proteins/biosynthesis , Sequence Homology , Transfection , VertebratesABSTRACT
Tetraspanins function as molecular organizers of multi-protein complexes by assembling primary complexes of a relatively low mass into extensive networks involved in cellular signalling. In this paper, we summarize our studies performed on the tetraspanin D6.1A/CO-029/TM4SF3 expressed by rat carcinoma cells. Primary complexes of D6.1A are almost indistinguishable from complexes isolated with anti-CD9 antibody. Indeed, both tetraspanins directly associate with each other and with a third tetraspanin, CD81. Moreover, FPRP (prostaglandin F2alpha receptor-regulatory protein)/EWI-F/CD9P-1), an Ig superfamily member that has been described to interact with CD9 and CD81, is also a prominent element in D6.1A complexes. Primary complexes isolated with D6.1A-specific antibody are clearly different from complexes containing the tetraspanin CD151. CD151 is found to interact only with D6.1A if milder conditions, i.e. lysis with LubrolWX instead of Brij96, are applied to disrupt cellular membranes. CD151 probably mediates the interaction of D6.1A primary complexes with alpha3beta1 integrin. In addition, two other molecules were identified to be part of D6.1A complexes at this higher level of association: type II phosphatidylinositol 4-kinase and EpCAM, an epithelial marker protein overexpressed by many carcinomas. The characterization of the D6.1A core complex and additional more indirect interactions will help to elucidate the role in tumour progression and metastasis attributed to D6.1A.
Subject(s)
Membrane Glycoproteins/metabolism , Neoplasm Proteins/metabolism , Animals , Antigens, CD/metabolism , Cell Line, Tumor , Detergents/pharmacology , Multiprotein Complexes/chemistry , Multiprotein Complexes/metabolism , Pancreatic Neoplasms/metabolism , Protein Binding/drug effects , Rats , Solubility/drug effects , Tetraspanin 28 , Tetraspanin 29 , Tetraspanins , Urinary Bladder Neoplasms/metabolismABSTRACT
OBJECTIVES: The lack of specific biochemical markers is a major drawback for the diagnosis of autoimmune pancreatitis (AIP). The aims were to characterize the autoantibody profiles in AIP and pancreatic ductal adenocarcinoma (PDAC) and to identify circulating autoantibodies that could be diagnostic markers differentiating PDAC and the AIP subtypes. METHODS: Tissue lysates obtained from the resected pancreas of patients with AIP and patients with PDAC were separated by 2-dimensional polyacrylamide gel electrophoresis subsequently immunoblotted with autologous sera. The immunoreactive spots were subjected to nanoscale liquid chromatography-electrospray ionization tandem mass spectrometry to identify serum autoantibodies to tissue-derived autoantigens associated with AIP and PDAC. Autoantibody concentrations for selected autoantigens were assessed by enzyme-linked immunosorbent assays. RESULTS: A total of 115 immunoreactive spots were identified by 2-dimensional polyacrylamide gel electrophoresis/immunobloting. Nanoscale liquid chromatography-electrospray ionization tandem mass spectrometry-based analysis revealed 68 autoantigens in AIP, 26 in PDAC, and 21 present in both diseases. Assessment of 13 selected AIP autoantibody serum levels revealed that 7 of them had significantly higher titers in AIP versus PDAC. IgG-directed against transaldolase could significantly differentiate between the 2 AIP subtypes. CONCLUSIONS: The novel panel of AIP autoantibodies is promising to supplement the predictive tests for AIP of the currently known autoantigens and represent a basis for a combined blood test to differentiate AIP from PDAC in the future.
Subject(s)
Carcinoma, Pancreatic Ductal , Pancreatic Neoplasms , Pancreatitis , Autoantibodies , Autoimmune Diseases , Diagnosis, Differential , HumansABSTRACT
Autoimmune pancreatitis (AIP) is defined by characteristic lymphoplasmacytic infiltrate, ductal strictures and a pancreatic enlargement or mass that can mimic pancreatic cancer (PaCa). The distinction between this benign disease and pancreatic cancer can be challenging. However, an accurate diagnosis may pre-empt the misdiagnosis of cancer, allowing the appropriate medical treatment of AIP and, consequently, decreasing the number of unnecessary pancreatic resections. Mass spectrometry (MS) and two-dimensional differential gel electrophoresis (2D-DIGE) have been applied to analyse serum protein alterations associated with AIP and PaCa, and to identify protein signatures indicative of the diseases. Patients' sera were immunodepleted from the 20 most prominent serum proteins prior to further 2D-DIGE and image analysis. The identity of the most-discriminatory proteins detected, was performed by MS and ELISAs were applied to confirm their expression. Serum profiling data analysis with 2D-DIGE revealed 39 protein peaks able to discriminate between AIP and PaCa. Proteins were purified and further analysed by MALDI-TOF-MS. Peptide mass fingerprinting led to identification of eleven proteins. Among them apolipoprotein A-I, apolipoprotein A-II, transthyretin, and tetranectin were identified and found as 3.0-, 3.5-, 2-, and 1.6-fold decreased in PaCa sera, respectively, whereas haptoglobin and apolipoprotein E were found to be 3.8- and 1.6-fold elevated in PaCa sera. With the exception of haptoglobin the ELISA results of the identified proteins confirmed the 2D-DIGE image analysis characteristics. Integration of the identified serum proteins as AIP markers may have considerable potential to provide additional information for the diagnosis of AIP to choose the appropriate treatment.
Subject(s)
Autoimmune Diseases/blood , Autoimmune Diseases/diagnosis , Blood Proteins/metabolism , Pancreatic Neoplasms/blood , Pancreatic Neoplasms/diagnosis , Pancreatitis, Chronic/blood , Pancreatitis, Chronic/diagnosis , Autoimmune Diseases/immunology , Autoimmune Diseases/microbiology , Biomarkers/blood , Biometry , Diagnosis, Differential , Helicobacter Infections/complications , Helicobacter Infections/immunology , Helicobacter pylori/immunology , Humans , Immunoglobulin G/blood , Immunoglobulin G/immunology , Pancreatitis, Chronic/immunology , Pancreatitis, Chronic/microbiology , Proteomics/methodsABSTRACT
PURPOSE: Recent work points out a role of B7H3, a member of the B7-family of costimulatory proteins, in conveying immunosuppression and enforced invasiveness in a variety of tumor entities. Glioblastoma is armed with effective immunosuppressive properties resulting in an impaired recognition and ineffective attack of tumor cells by the immune system. In addition, extensive and diffuse invasion of tumor cells into the surrounding brain tissue limits the efficacy of local therapies. Here, 4IgB7H3 is assessed as diagnostic and therapeutic target for glioblastoma. EXPERIMENTAL DESIGN: To characterize B7H3 in glioblastoma, we conduct analyses not only in glioma cell lines and glioma-initiating cells but also in human glioma tissue specimens. RESULTS: B7H3 expression by tumor and endothelial cells correlates with the grade of malignancy in gliomas and with poor survival. Both soluble 4IgB7H3 in the supernatant of glioma cells and cell-bound 4IgB7H3 are functional and suppress natural killer cell-mediated tumor cell lysis. Gene silencing showed that membrane and soluble 4IgB7H3 convey a proinvasive phenotype in glioma cells and glioma-initiating cells in vitro. These proinvasive and immunosuppressive properties were confirmed in vivo by xenografted 4IgB7H3 gene silenced glioma-initiating cells, which invaded significantly less into the surrounding brain tissue in an orthotopic model and by subcutaneously injected LN-229 cells, which were more susceptible to natural killer cell-mediated cytotoxicity than unsilenced control cells. CONCLUSIONS: Because of its immunosuppressive and proinvasive function, 4IgB7H3 may serve as a therapeutic target in the treatment of glioblastoma.
Subject(s)
B7 Antigens/metabolism , Cell Movement/immunology , Cytotoxicity, Immunologic/immunology , Glioblastoma/immunology , Glioblastoma/pathology , Killer Cells, Natural/immunology , Animals , B7 Antigens/genetics , Blotting, Western , Brain Neoplasms/immunology , Brain Neoplasms/metabolism , Brain Neoplasms/mortality , Brain Neoplasms/pathology , Cell Proliferation , Gene Expression Regulation, Neoplastic , Glioblastoma/metabolism , Glioblastoma/mortality , Humans , Immunoenzyme Techniques , Mice , Mice, Nude , Neoplasm Invasiveness , Phenotype , RNA, Messenger/genetics , Real-Time Polymerase Chain Reaction , Survival Rate , Tumor Cells, CulturedABSTRACT
As an acute-phase protein, human C-reactive protein (CRP) is clinically important. CRPs were purified from several samples in six different pathological conditions, where their levels ranged from 22 to 342 microg/ml. Small, but significant, variations in electrophoretic mobilities on native PAGE suggested differences in molecular mass, charge and/or shape. Following separation by SDS/PAGE, they showed single subunits with some differences in their molecular masses ranging between 27 and 30.5 kDa, but for a particular disease, the mobility was the same for CRPs purified from multiple individuals or pooled sera. Isoelectric focusing (IEF) also indicated that the purified CRPs differed from each other. Glycosylation was demonstrated in these purified CRPs by Digoxigenin kits, neuraminidase treatment and binding with lectins. The presence of N-linked sugar moiety was confirmed by N-glycosidase F digestion. The presence of sialic acid, glucose, galactose and mannose has been demonstrated by gas liquid chromatography, mass spectroscopic and fluorimetric analysis. Matrix-assisted laser-desorption ionization analysis of the tryptic digests of three CRPs showed systematic absence of two peptide fragments, one at the N-terminus and the other near the C-terminus. Model-building suggested that the loss of these fragments exposed two potential glycosylation sites on a cleft floor keeping the protein-protein interactions in pentraxins and calcium-dependent phosphorylcholine-binding qualitatively unaffected. Thus we have convincingly demonstrated that human CRP is glycosylated in some pathological conditions.
Subject(s)
C-Reactive Protein/metabolism , Lupus Erythematosus, Systemic/pathology , Osteosarcoma/pathology , Precursor Cell Lymphoblastic Leukemia-Lymphoma/pathology , Amino Acid Sequence , C-Reactive Protein/chemistry , Electrophoresis, Polyacrylamide Gel , Galactose/metabolism , Glucose/metabolism , Glycosylation , Humans , Isoelectric Focusing , Lectins/metabolism , Lupus Erythematosus, Systemic/metabolism , Mannose/metabolism , Models, Molecular , Molecular Sequence Data , N-Acetylneuraminic Acid/metabolism , Osteosarcoma/metabolism , Phosphorylcholine/metabolism , Precursor Cell Lymphoblastic Leukemia-Lymphoma/metabolism , Spectrometry, Mass, Matrix-Assisted Laser Desorption-IonizationABSTRACT
The molecular biology of spuma or foamy retroviruses is different from that of the other members of the Retroviridae. Among the distinguishing features, the N-terminal domain of the foamy virus Env glycoprotein, the 16-kDa Env leader protein Elp, is a component of released, infectious virions and is required for particle budding. The transmembrane protein Elp specifically interacts with N-terminal Gag sequences during morphogenesis. In this study, we investigate the mechanism of Elp release from the Env precursor protein. By a combination of genetic, biochemical, and biophysical methods, we show that the feline foamy virus (FFV) Elp is released by a cellular furin-like protease, most likely furin itself, generating an Elp protein consisting of 127 amino acid residues. The cleavage site fully conforms to the rules for an optimal furin site. Proteolytic processing at the furin cleavage site is required for full infectivity of FFV. However, utilization of other furin proteases and/or cleavage at a suboptimal signal peptidase cleavage site can partially rescue virus viability. In addition, we show that FFV Elp carries an N-linked oligosaccharide that is not conserved among the known foamy viruses.
Subject(s)
Furin/metabolism , Gene Products, env/metabolism , Protein Precursors/metabolism , Protein Sorting Signals/physiology , Spumavirus/metabolism , Amino Acid Sequence , Animals , Cats , Cell Line , Consensus Sequence , Gene Products, env/chemistry , Gene Products, env/genetics , Humans , Molecular Sequence Data , Protein Precursors/chemistry , Protein Precursors/genetics , Protein Sorting Signals/genetics , Spumavirus/genetics , Virion/metabolismABSTRACT
Human xylosyltransferase I (XT-I) catalyzes the transfer of xylose from UDP-xylose to consensus serine residues of proteoglycan core proteins. Expression of a soluble form of recombinant histidine-tagged XT-I (rXT-I-HIS) was accomplished at a high level with High Five/pCG255-1 insect cells in suspension culture. The recombinant protein was purified to homogeneity by a combination of heparin affinity chromatography and metal (Ni(2+)) chelate affinity chromatography. Using the modern technique of perfusion chromatography, a rapid procedure for purification of the rXT-I-HIS from insect cell culture supernatant was developed. The purified, biologically active enzyme was homogeneous on SDS-PAGE, was detected with anti-XT-I-antibodies, and had the expected tryptic fragment mass spectrum. N-terminal amino acid sequencing demonstrated that the N-terminal signal sequence of the expressed protein was quantitatively cleaved. The total yield of the enzyme after purification was 18% and resulted in a specific XT-I activity of 7.9mU/mg. The K(m) of the enzyme for recombinant [Val(36),Val(38)](delta1),[Gly(92),Ile(94)](delta2)bikunin was 0.8microM. About 5mg purified enzyme could be obtained from 1L cell culture supernatant. The availability of substantial quantities of active, homogeneous enzyme will be of help in future biochemical and biophysical characterization of XT-I and for the development of a immunological XT-I assay.
Subject(s)
Moths/metabolism , Pentosyltransferases/biosynthesis , Pentosyltransferases/isolation & purification , Protein Engineering/methods , Amino Acid Sequence , Animals , Cells, Cultured , Cloning, Molecular , Enzyme Activation , Gene Expression Regulation, Enzymologic/physiology , Humans , Molecular Weight , Pentosyltransferases/chemistry , Pentosyltransferases/genetics , Quality Control , Recombinant Proteins/biosynthesis , Recombinant Proteins/genetics , Recombinant Proteins/isolation & purification , UDP Xylose-Protein XylosyltransferaseABSTRACT
The anucleate prismoid fiber cells of the eye lens are densely packed to form a tissue in which the plasma membranes and their associated cytoplasmic coat form a single giant cell-cell adhesive complex, the cortex adhaerens. Using biochemical and immunoprecipitation methods in various species (cow, pig, rat), in combination with immunolocalization microscopy, we have identified two different major kinds of cortical complex. In one, the transmembrane glycoproteins N-cadherin and cadherin-11 [which also occur in heterotypic ('mixed') complexes] are associated with alpha- and beta-catenin, plakoglobin (proportions variable among species), p120ctn and vinculin. The other complex contains ezrin, periplakin, periaxin and desmoyokin (and so is called the EPPD complex), usually together with moesin, spectrin(s) and plectin. In sections through lens fiber tissue, the short sides of the lens fiber hexagons appear to be enriched in the cadherin-based complexes, whereas the EPPD complexes also occur on the long sides. Moreover, high resolution double-label fluorescence microscopy has revealed, on the short sides, a finer, almost regular mosaicism of blocks comprising the cadherin-based, catenin-containing complexes, alternating with patches formed by the EPPD complexes. The latter, a new type of junctional plaque ensemble of proteins hitherto known only from certain other cell types, must be added to the list of major lens cortex proteins. We here discuss its possible functional importance for the maintenance of lens structure and functions, notably clear and sharp vision.