scholarly journals Characterization of heterogeneous nuclear RNA-protein complexes in vivo with monoclonal antibodies.

1984 ◽  
Vol 4 (6) ◽  
pp. 1104-1114 ◽  
Author(s):  
G Dreyfuss ◽  
Y D Choi ◽  
S A Adam
Keyword(s):  
Monoclonal Antibodies ◽  
Uv Light ◽  
Protein Complexes ◽  
Cross Linking ◽  
Intact Cells ◽  
Molecular Weights ◽  
Nuclear Rna ◽  
Nonionic Detergent ◽  
Sufficient Intensity

Exposure of cells to UV light of sufficient intensity brings about cross-linking of RNA to proteins which are in direct contact with it in vivo. The major [35S]methionine-labeled proteins which become cross-linked to polyadenylated heterogeneous nuclear RNA in HeLa cells have molecular weights of 120,000 (120K), 68K, 53K, 43K, 41K, 38K, and 36K. Purified complexes of polyadenylated RNA with proteins obtained by UV cross-linking in intact cells were used to immunize mice and generate monoclonal antibodies to several of these proteins. Some properties of three of the proteins, 41K, 43K, and 120K, were characterized with these antibodies. The 41K and 43K polypeptides are highly related. They were recognized by the same antibody (2B12) and have identical isoelectric points (pl = 6.0 +/- 0.2) but different partial peptide maps. The 41K and 43K polypeptides were part of the 40S heterogeneous nuclear ribonucleoprotein particle and appear to correspond to the previously described C proteins (Beyer et al., Cell II:127-138, 1977). A different monoclonal antibody (3G6) defined a new major heterogeneous ribonucleoprotein of 120K. The 41K, 43K, and 120K polypeptides were associated in vivo with both polyadenylated and non-polyadenylated nuclear RNA, and all three proteins were phosphorylated. The monoclonal antibodies recognized similar proteins in human and monkey cells but not in several other vertebrates. Immunofluorescence microscopy demonstrated that these proteins are segregated to the nucleus, where they are part of a fine particulate nonnucleolar structure. In cells extracted in situ with nonionic detergent, all of the 41K and 43K polypeptides were associated with the nucleus at salt concentrations up to 0.5 M NaCl, whereas the 120K polypeptide was completely extracted at this NaCl concentration. A substantial fraction of the 41K and 43K polypeptides (up to 40%) was retained with a nuclear matrix--a structure which is resistant to digestion with DNase I and to extraction by 2 M NaCl, but the 41K and 43K polypeptides were quantitatively removed at 0.5 M NaCl after digestion with RNase.

Characterization of heterogeneous nuclear RNA-protein complexes in vivo with monoclonal antibodies

1984 ◽  
Vol 4 (6) ◽  
pp. 1104-1114
Author(s):  
G Dreyfuss ◽  
Y D Choi ◽  
S A Adam
Keyword(s):  
Monoclonal Antibodies ◽  
Uv Light ◽  
Protein Complexes ◽  
Cross Linking ◽  
Intact Cells ◽  
Molecular Weights ◽  
Nuclear Rna ◽  
Nonionic Detergent ◽  
Sufficient Intensity

Exposure of cells to UV light of sufficient intensity brings about cross-linking of RNA to proteins which are in direct contact with it in vivo. The major [35S]methionine-labeled proteins which become cross-linked to polyadenylated heterogeneous nuclear RNA in HeLa cells have molecular weights of 120,000 (120K), 68K, 53K, 43K, 41K, 38K, and 36K. Purified complexes of polyadenylated RNA with proteins obtained by UV cross-linking in intact cells were used to immunize mice and generate monoclonal antibodies to several of these proteins. Some properties of three of the proteins, 41K, 43K, and 120K, were characterized with these antibodies. The 41K and 43K polypeptides are highly related. They were recognized by the same antibody (2B12) and have identical isoelectric points (pl = 6.0 +/- 0.2) but different partial peptide maps. The 41K and 43K polypeptides were part of the 40S heterogeneous nuclear ribonucleoprotein particle and appear to correspond to the previously described C proteins (Beyer et al., Cell II:127-138, 1977). A different monoclonal antibody (3G6) defined a new major heterogeneous ribonucleoprotein of 120K. The 41K, 43K, and 120K polypeptides were associated in vivo with both polyadenylated and non-polyadenylated nuclear RNA, and all three proteins were phosphorylated. The monoclonal antibodies recognized similar proteins in human and monkey cells but not in several other vertebrates. Immunofluorescence microscopy demonstrated that these proteins are segregated to the nucleus, where they are part of a fine particulate nonnucleolar structure. In cells extracted in situ with nonionic detergent, all of the 41K and 43K polypeptides were associated with the nucleus at salt concentrations up to 0.5 M NaCl, whereas the 120K polypeptide was completely extracted at this NaCl concentration. A substantial fraction of the 41K and 43K polypeptides (up to 40%) was retained with a nuclear matrix--a structure which is resistant to digestion with DNase I and to extraction by 2 M NaCl, but the 41K and 43K polypeptides were quantitatively removed at 0.5 M NaCl after digestion with RNase.

scholarly journals In Vitro and In Vivo Neutralization of the Relapsing Fever Agent Borrelia hermsii with Serotype-Specific Immunoglobulin M Antibodies

2001 ◽  
Vol 69 (2) ◽  
pp. 1009-1015 ◽  
Author(s):  
Alan G. Barbour ◽  
Virgilio Bundoc
Keyword(s):  
Monoclonal Antibodies ◽  
Antibody Response ◽  
Immunoglobulin M ◽  
Enzyme Linked Immunosorbent Assay ◽  
Relapsing Fever ◽  
Borrelia Hermsii ◽  
Intact Cells ◽  
Whole Cells

ABSTRACT The antigenic variation of the relapsing fever agent Borrelia hermsii is associated with changes in the expression of the Vlp and Vsp outer membrane lipoproteins. To investigate whether these serotype-defining proteins are the target of a neutralizing and protective antibody response, monoclonal antibodies were produced from spleens of infected mice just after clearance of serotype 7 cells from the blood. Two immunoglobulin M monoclonal antibodies, H7-7 and H7-12, were studied in detail. Both antibodies specifically agglutinated serotype 7 cells and inhibited their growth in vitro. Administered to mice before or after infection, both antibodies provided protection against infection or substantially reduced the number of spirochetes in the blood of mice after infection. Whereas antibody H7-12 bound to Vlp7 in Western blotting, enzyme-linked immunosorbent assay, and immunoprecipitation assays, as well as to whole cells in other immunoassays, antibody H7-7 only bound to wet, intact cells of serotype 7. Antibody H7-7 selected against cells expressing Vlp7 in vitro and in vivo, an indication that Vlp7 was a conformation-sensitive antigen for the antibody. Vaccination of mice with recombinant Vlp7 with adjuvant elicited antibodies that bound to fixed whole cells of serotype 7 and to Vlp7 in Western blots, but these antibodies did not inhibit the growth of serotype 7 in vitro and did not provide protection against an infectious challenge with serotype 7. The study established that a Vlp protein was the target of a neutralizing antibody response, and it also indicated that the conformation and/or the native topology of Vlp were important for eliciting that immunity.

NAB2: a yeast nuclear polyadenylated RNA-binding protein essential for cell viability

1993 ◽  
Vol 13 (5) ◽  
pp. 2730-2741 ◽  
Author(s):  
J T Anderson ◽  
S M Wilson ◽  
K V Datar ◽  
M S Swanson
Keyword(s):  
Rna Binding ◽  
Uv Light ◽  
Polyclonal Antibodies ◽  
Yeast Cells ◽  
Binding Studies ◽  
Single Stranded Dna ◽  
Binding Motifs ◽  
Polyadenylated Rna ◽  
Nuclear Rna

A variety of nuclear ribonucleoproteins are believed to associate directly with nascent RNA polymerase II transcripts and remain associated during subsequent nuclear RNA processing reactions, including pre-mRNA polyadenylation and splicing as well as nucleocytoplasmic mRNA transport. To investigate the functions of these proteins by using a combined biochemical and genetic approach, we have isolated nuclear polyadenylated RNA-binding (NAB) proteins from Saccharomyces cerevisiae. Living yeast cells were irradiated with UV light to covalently cross-link proteins intimately associated with RNA in vivo. Polyadenylated RNAs were then selectively purified, and the covalent RNA-protein complexes were used to elicit antibodies in mice. Both monoclonal and polyclonal antibodies which detect a variety of NAB proteins were prepared. Here we characterize one of these proteins, NAB2. NAB2 is one of the major proteins associated with nuclear polyadenylated RNA in vivo, as detected by UV light-induced cross-linking. Cellular immunofluorescence, using both monoclonal and polyclonal antibodies, demonstrates that the NAB2 protein is localized within the nucleus. The deduced primary structure of NAB2 indicates that it is composed of at least two distinct types of RNA-binding motifs: (i) an RGG box recently described in a variety of heterogeneous nuclear RNA-, pre-rRNA-, mRNA-, and small nucleolar RNA-binding proteins and (ii) CCCH motif repeats related to the zinc-binding motifs of the largest subunit of RNA polymerases I, II, and III. In vitro RNA homopolymer/single-stranded DNA binding studies indicate that although both the RGG box and CCCH motifs bind poly(G), poly(U), and single-stranded DNA, the CCCH motifs also bind to poly(A). NAB2 is located on chromosome VII within a cluster of ribonucleoprotein genes, and its expression is essential for cell growth.

scholarly journals In vivo protein-DNA interactions at the c-jun promoter: preformed complexes mediate the UV response.

1993 ◽  
Vol 13 (9) ◽  
pp. 5490-5499 ◽  
Author(s):  
D Rozek ◽  
G P Pfeifer
Keyword(s):  
Cpg Island ◽  
Uv Light ◽  
Dimethyl Sulfate ◽  
Related Factor ◽  
Dna Interactions ◽  
Intact Cells ◽  
Response Elements ◽  
Protein Dna Interactions ◽  
Uv Response

Irradiation of cells with UV light triggers a genetic response, called the UV response, which results in induction of a set of genes containing AP-1-binding sites. The c-jun gene itself, which codes for AP-1-binding activity, is strongly (> 100-fold) and rapidly activated by UV. The UV induction of c-jun is mediated by two UV response elements consisting of AP-1-like sequences within its 5' control region. We have analyzed protein-DNA interactions in vivo at the c-jun promoter in noninduced and UV-irradiated HeLa cells. In vivo footprint analysis was performed by using dimethyl sulfate on intact cells and DNase I on lysolecithihin-permeabilized cells in conjunction with ligation-mediated polymerase chain reaction to cover about 450 bp of the c-jun promoter, including the transcription start sites. We find that this region does not contain methylated cytosines and is thus a typical CpG island. In uninduced cells, in vivo protein-DNA interactions were localized to an AP-1-like sequence (nucleotides [nt] -71 to -64), a CCAAT box element (nt -91 to -87), two SP1 sequences (nt -115 to -110 and -123 to -118), a nuclear factor jun site (nt -140 to -132), and a second AP-1-like sequence (nt -190 to -183). These results indicate that complex protein-DNA interactions exist at the c-jun promoter prior to induction by an external stimulus. Surprisingly, after stimulation of c-jun expression by UV irradiation, all in vivo protein-DNA contacts remained essentially unchanged, including the two UV response elements located at the AP-1-like sequences. The UV-induced signalling cascade leads to phosphorylation of c-Jun on serines 63 and 73 (Y. Devary, R.A. Gottlieb, T. Smeal, and M. Karin, Cell 71:1081-1091, 1992). Taken together, these data suggest that modification of the transactivating domain of DNA-bound c-Jun or a closely related factor may trigger the rapid induction of the c-jun gene.

scholarly journals Driving integrative structural modeling with serial capture affinity purification

2020 ◽  
Vol 117 (50) ◽  
pp. 31861-31870
Author(s):  
Xingyu Liu ◽  
Ying Zhang ◽  
Zhihui Wen ◽  
Yan Hao ◽  
Charles A. S. Banks ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Structural Model ◽  
Protein Complexes ◽  
Affinity Purification ◽  
Quantitative Imaging ◽  
Resonance Energy ◽  
Correlation Spectroscopy ◽  
Cross Linking ◽  
Affinity Enrichment

Streamlined characterization of protein complexes remains a challenge for the study of protein interaction networks. Here we describe serial capture affinity purification (SCAP), in which two separate proteins are tagged with either the HaloTag or the SNAP-tag, permitting a multistep affinity enrichment of specific protein complexes. The multifunctional capabilities of this protein-tagging system also permit in vivo validation of interactions using acceptor photobleaching Förster resonance energy transfer and fluorescence cross-correlation spectroscopy quantitative imaging. By coupling SCAP to cross-linking mass spectrometry, an integrative structural model of the complex of interest can be generated. We demonstrate this approach using the Spindlin1 and SPINDOC protein complex, culminating in a structural model with two SPINDOC molecules docked on one SPIN1 molecule. In this model, SPINDOC interacts with the SPIN1 interface previously shown to bind a lysine and arginine methylated sequence of histone H3. Our approach combines serial affinity purification, live cell imaging, and cross-linking mass spectrometry to build integrative structural models of protein complexes.

Characterization of monoclonal antibodies recognizing each extracellular loop domain of occludin

2019 ◽  
Vol 166 (4) ◽  
pp. 297-308 ◽  
Author(s):  
Yoshimi Shimizu ◽  
Yoshitaka Shirasago ◽  
Takeru Suzuki ◽  
Tomoyuki Hata ◽  
Masuo Kondoh ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Transmembrane Protein ◽  
Site Directed Mutagenesis ◽  
Enzyme Linked Immunosorbent Assay ◽  
Intact Cells ◽  
Flow Cytometric ◽  
Junction Protein ◽  
Fcm Analysis

Abstract The tight junction protein occludin (OCLN) is a four-pass transmembrane protein with two extracellular loops (ELs), and also functions as a co-receptor for hepatitis C virus (HCV). Recently, we reported the establishment of monoclonal antibodies (mAbs) recognizing each intact EL domain of OCLN that can strongly prevent HCV infection in vitro and in vivo, and these mAbs were applicable for flow cytometric (FCM) analysis, immunocytochemistry (ICC) and cell-based enzyme-linked immunosorbent assay. In the present study, we further examined the application of these anti-OCLN mAbs and characterized their binding properties. All four mAbs were available for immunoprecipitation. The three first EL (EL1)-recognizing mAbs were applicable for immunoblotting, but the second EL (EL2)-recognizing one was not. Using site-directed mutagenesis, we also determined residues of OCLN critical for recognition by each mAb. Our findings showed that the small loop between two cysteines of the EL2 domain is essential for the binding to one EL2-recognizing mAb and that the recognition regions by three EL1-recognizing mAbs overlap, but are not the same sites of EL1. To obtain a deeper understanding of OCLN biology and its potential as a therapeutic target, specific mAbs to detect or target OCLN in intact cells should be powerful tools for future studies.

scholarly journals Current Trends on Protein Driven Bioinks for 3D Printing

Pharmaceutics ◽  
2021 ◽  
Vol 13 (9) ◽  
pp. 1444
Author(s):  
Anabela Veiga ◽  
Inês V. Silva ◽  
Marta M. Duarte ◽  
Ana L. Oliveira
Keyword(s):  
Protein Complexes ◽  
Technological Development ◽  
Three Dimensional ◽  
Extraction Methods ◽  
Polymeric Systems ◽  
Molecular Weights ◽  
Decellularized Extracellular Matrix ◽  
Methodological Guidelines

In the last decade, three-dimensional (3D) extrusion bioprinting has been on the top trend for innovative technologies in the field of biomedical engineering. In particular, protein-based bioinks such as collagen, gelatin, silk fibroin, elastic, fibrin and protein complexes based on decellularized extracellular matrix (dECM) are receiving increasing attention. This current interest is the result of protein’s tunable properties, biocompatibility, environmentally friendly nature and possibility to provide cells with the adequate cues, mimicking the extracellular matrix’s function. In this review we describe the most relevant stages of the development of a protein-driven bioink. The most popular formulations, molecular weights and extraction methods are covered. The different crosslinking methods used in protein bioinks, the formulation with other polymeric systems or molecules of interest as well as the bioprinting settings are herein highlighted. The cell embedding procedures, the in vitro, in vivo, in situ studies and final applications are also discussed. Finally, we approach the development and optimization of bioinks from a sequential perspective, discussing the relevance of each parameter during the pre-processing, processing, and post-processing stages of technological development. Through this approach the present review expects to provide, in a sequential manner, helpful methodological guidelines for the development of novel bioinks.

In vivo protein-DNA interactions at the c-jun promoter: preformed complexes mediate the UV response

1993 ◽  
Vol 13 (9) ◽  
pp. 5490-5499
Author(s):  
D Rozek ◽  
G P Pfeifer
Keyword(s):  
Cpg Island ◽  
Uv Light ◽  
Dimethyl Sulfate ◽  
Related Factor ◽  
Dna Interactions ◽  
Intact Cells ◽  
Response Elements ◽  
Protein Dna Interactions ◽  
Uv Response

Irradiation of cells with UV light triggers a genetic response, called the UV response, which results in induction of a set of genes containing AP-1-binding sites. The c-jun gene itself, which codes for AP-1-binding activity, is strongly (> 100-fold) and rapidly activated by UV. The UV induction of c-jun is mediated by two UV response elements consisting of AP-1-like sequences within its 5' control region. We have analyzed protein-DNA interactions in vivo at the c-jun promoter in noninduced and UV-irradiated HeLa cells. In vivo footprint analysis was performed by using dimethyl sulfate on intact cells and DNase I on lysolecithihin-permeabilized cells in conjunction with ligation-mediated polymerase chain reaction to cover about 450 bp of the c-jun promoter, including the transcription start sites. We find that this region does not contain methylated cytosines and is thus a typical CpG island. In uninduced cells, in vivo protein-DNA interactions were localized to an AP-1-like sequence (nucleotides [nt] -71 to -64), a CCAAT box element (nt -91 to -87), two SP1 sequences (nt -115 to -110 and -123 to -118), a nuclear factor jun site (nt -140 to -132), and a second AP-1-like sequence (nt -190 to -183). These results indicate that complex protein-DNA interactions exist at the c-jun promoter prior to induction by an external stimulus. Surprisingly, after stimulation of c-jun expression by UV irradiation, all in vivo protein-DNA contacts remained essentially unchanged, including the two UV response elements located at the AP-1-like sequences. The UV-induced signalling cascade leads to phosphorylation of c-Jun on serines 63 and 73 (Y. Devary, R.A. Gottlieb, T. Smeal, and M. Karin, Cell 71:1081-1091, 1992). Taken together, these data suggest that modification of the transactivating domain of DNA-bound c-Jun or a closely related factor may trigger the rapid induction of the c-jun gene.

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