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1.
Nat Methods ; 14(5): 487-490, 2017 May.
Article in English | MEDLINE | ID: mdl-28346450

ABSTRACT

Ribonucleoproteins (RNPs) are key regulators of cellular function. We established an efficient approach, crosslinking of segmentally isotope-labeled RNA and tandem mass spectrometry (CLIR-MS/MS), to localize protein-RNA interactions simultaneously at amino acid and nucleotide resolution. The approach was tested on polypyrimidine tract binding protein 1 and U1 small nuclear RNP. Our method provides distance restraints to support integrative atomic-scale structural modeling and to gain mechanistic insights into RNP-regulated processes.


Subject(s)
Heterogeneous-Nuclear Ribonucleoproteins/chemistry , Models, Molecular , Nucleic Acid Conformation , Polypyrimidine Tract-Binding Protein/chemistry , RNA/chemistry , Ribonucleoprotein, U1 Small Nuclear/chemistry , Binding Sites , Carbon Isotopes , Chromatography, High Pressure Liquid , Heterogeneous-Nuclear Ribonucleoproteins/genetics , Nitrogen Isotopes , Nuclear Magnetic Resonance, Biomolecular , Polypyrimidine Tract-Binding Protein/genetics , Protein Binding , Ribonucleoprotein, U1 Small Nuclear/genetics , Software , Tandem Mass Spectrometry , Ultraviolet Rays
2.
Cell Mol Life Sci ; 65(4): 516-27, 2008 Feb.
Article in English | MEDLINE | ID: mdl-17975705

ABSTRACT

The polypyrimidine tract binding protein (PTB) is a 58-kDa RNA binding protein involved in multiple aspects of mRNA metabolism including splicing regulation, polyadenylation, 3'end formation, internal ribosomal entry site-mediated translation, RNA localization and stability. PTB contains four RNA recognition motifs (RRMs) separated by three linkers. In this review we summarize structural information on PTB in solution that has been gathered during the past 7 years using NMR spectroscopy and small-angle X-ray scattering. The structures of all RRMs of PTB in their free state and in complex with short pyrimidine tracts, as well as a structural model of PTB RRM2 in complex with a peptide, revealed unusual structural features that provided new insights into the mechanisms of action of PTB in the different processes of RNA metabolism and in particular splicing regulation.


Subject(s)
Polypyrimidine Tract-Binding Protein/chemistry , Polypyrimidine Tract-Binding Protein/physiology , Animals , Binding Sites , Humans , Models, Molecular , Protein Binding , Protein Conformation , RNA, Messenger/metabolism , RNA-Binding Proteins/chemistry , RNA-Binding Proteins/physiology , Ribosomes/metabolism , Structure-Activity Relationship
3.
Rapid Commun Mass Spectrom ; 17(12): 1247-1255, 2003.
Article in English | MEDLINE | ID: mdl-12811747

ABSTRACT

The extraction of (135)Cs from high activity waste arising from reprocessing of spent fuel can be achieved by using calix[4]arene crown compounds. The radiolytic degradation of calix[4]arene crowns as well as their solvent, o-nitrophenyloctyl ether (NPOE), was studied using electrospray ionization mass spectrometry (ESI-MS) (that formed Cs(+) or Na(+) adducts) in nitric acid, as well as by chemical ionization tandem mass spectrometry (MS/MS) experiments. The structures of major degradation products were identified with MS and specifically labelled nitric acid. Although NPOE and calix[4]arene crowns alone are relatively stable, under simulated conditions resembling the real industrial processes involving radiolysis in the presence of nitric acid, several products resulting from nitration and oxidation were observed.

4.
J Mol Biol ; 309(3): 763-75, 2001 Jun 08.
Article in English | MEDLINE | ID: mdl-11397095

ABSTRACT

Nucleolin is an abundant nucleolar protein which is essential for ribosome biogenesis. The first two of its four tandem RNA-binding domains (RBD12) specifically recognize a stem-loop structure containing a conserved UCCCGA sequence in the loop called the nucleolin-recognition element (NRE). We have determined the structure of the consensus SELEX NRE (sNRE) by NMR spectroscopy. In both the free and bound RNA the top part of the stem forms a loop E (or S-turn) motif. In the absence of protein, the structure of the hairpin loop is not well defined due to conformational heterogeneity, and appears to be in equilibrium between two families of conformations. Titrations of RBD1, RBD2, and RBD12 with the sNRE show that specific binding requires RBD12. In complex with RBD12, the hairpin loop interacts specifically with the protein and adopts a well-defined structure which shares some of the features of the free form. The loop E motif also has specific interactions with the protein. Implications of these findings for the mechanism of recognition of RNA structures by modular proteins are discussed.


Subject(s)
Nucleic Acid Conformation , Phosphoproteins/chemistry , Phosphoproteins/metabolism , RNA, Ribosomal/chemistry , RNA, Ribosomal/metabolism , RNA-Binding Proteins/chemistry , RNA-Binding Proteins/metabolism , Animals , Base Pairing , Base Sequence , Binding Sites , Consensus Sequence/genetics , Humans , Mice , Models, Molecular , Mutation/genetics , Nuclear Magnetic Resonance, Biomolecular , Nucleotides/chemistry , Nucleotides/genetics , Nucleotides/metabolism , Pliability , Protein Binding , Protein Structure, Tertiary , RNA Precursors/chemistry , RNA Precursors/genetics , RNA Precursors/metabolism , RNA Stability , RNA, Ribosomal/genetics , Regulatory Sequences, Nucleic Acid/genetics , Substrate Specificity , Thermodynamics , Titrimetry , Nucleolin
5.
EMBO J ; 19(24): 6870-81, 2000 Dec 15.
Article in English | MEDLINE | ID: mdl-11118222

ABSTRACT

The structure of the 28 kDa complex of the first two RNA binding domains (RBDs) of nucleolin (RBD12) with an RNA stem-loop that includes the nucleolin recognition element UCCCGA in the loop was determined by NMR spectroscopy. The structure of nucleolin RBD12 with the nucleolin recognition element (NRE) reveals that the two RBDs bind on opposite sides of the RNA loop, forming a molecular clamp that brings the 5' and 3' ends of the recognition sequence close together and stabilizing the stem-loop. The specific interactions observed in the structure explain the sequence specificity for the NRE sequence. Binding studies of mutant proteins and analysis of conserved residues support the proposed interactions. The mode of interaction of the protein with the RNA and the location of the putative NRE sites suggest that nucleolin may function as an RNA chaperone to prevent improper folding of the nascent pre-rRNA.


Subject(s)
Phosphoproteins/chemistry , Phosphoproteins/metabolism , RNA Precursors/chemistry , RNA Precursors/metabolism , RNA-Binding Proteins/chemistry , RNA-Binding Proteins/metabolism , Amino Acid Sequence , Animals , Base Sequence , Binding Sites , Calorimetry , Cricetinae , Image Processing, Computer-Assisted , Models, Molecular , Molecular Sequence Data , Mutagenesis, Site-Directed , Nuclear Magnetic Resonance, Biomolecular , Nuclear Proteins/chemistry , Nuclear Proteins/metabolism , Nucleic Acid Conformation , Protein Conformation , Recombinant Proteins/chemistry , Recombinant Proteins/metabolism , Thermodynamics , Nucleolin
6.
J Mol Biol ; 303(2): 227-41, 2000 Oct 20.
Article in English | MEDLINE | ID: mdl-11023788

ABSTRACT

Nucleolin is an abundant 70 kDa nucleolar protein involved in many aspects of ribosomal RNA biogenesis. The central region of nucleolin contains four tandem consensus RNA-binding domains (RBD). The two most N-terminal domains (RBD12) bind with nanomolar affinity to an RNA stem-loop containing the consensus sequence UCCCGA in the loop. We have determined the solution structure of nucleolin RBD12 in its free form and have studied its interaction with a 22 nt RNA stem-loop using multidimensional NMR spectroscopy. The two RBDs adopt the expected beta alpha beta beta alpha beta fold, but the position of the beta 2 strand in both domains differs from what was predicted from sequence alignments. RBD1 and RBD2 are significantly different from each others and this is likely important in their sequence specific recognition of the RNA. RBD1 has a longer alpha-helix 1 and a shorter beta 2-beta 3 loop than RBD2, and differs from most other RBDs in these respects. The two RBDs are separated by a 12 amino acid flexible linker and do not interact with one another in the free protein. This linker becomes ordered when RBD12 binds to the RNA. Analysis of the observed NOEs between the protein and the RNA indicates that both RBDs interact with the RNA loop via their beta-sheet. Each domain binds residues on one side of the loop; specifically, RBD2 contacts the 5' side and RBD1 contacts the 3'.


Subject(s)
Nuclear Magnetic Resonance, Biomolecular , Phosphoproteins/chemistry , Phosphoproteins/metabolism , RNA-Binding Proteins/chemistry , RNA-Binding Proteins/metabolism , RNA/chemistry , RNA/metabolism , Amino Acid Sequence , Base Sequence , Binding Sites , Models, Molecular , Molecular Sequence Data , Nucleic Acid Conformation , Pliability , Protein Structure, Secondary , Protein Structure, Tertiary , RNA/genetics , Regulatory Sequences, Nucleic Acid/genetics , Sequence Alignment , Solutions , Substrate Specificity , Nucleolin
7.
Biochemistry ; 39(9): 2174-82, 2000 Mar 07.
Article in English | MEDLINE | ID: mdl-10694382

ABSTRACT

Cations play an important role in RNA folding and stabilization. The hairpin ribozyme is a small catalytic RNA consisting of two domains, A and B, which interact in the transition state in an ion-dependent fashion. Here we describe the interaction of mono-, di-, and trivalent cations with the domains of the ribozyme, as studied by homo- and heteronuclear NMR spectroscopy. Paramagnetic line broadening, chemical shift mapping, and intermolecular NOEs indicate that the B domain contains four to five metal binding sites, which bind Mn(2+), Mg(2+), and Co(NH(3))(6)(3+). There is no significant structural change in the B domain upon the addition of Co(NH(3))(6)(3+) or Mg(2+). No specific monovalent ion binding sites exist on the B domain, as determined by (15)NH(4)(+) binding studies. In contrast to the B domain, there are no observable metal ion interactions within the internal loop of the A domain. Model structure calculations of Mn(2+) interactions at two sites within the B domain indicate that the binding sites comprise major groove pockets lined with functional groups oriented so that multiple hydrogen bonds can be formed between the RNA and Mn(H(2)O)(6)(2+) or Co(NH(3))(6)(3+). Site 1 is very similar in geometry to a site within the P4-P6 domain of the Tetrahymena group I intron, while site 2 is unique among known ion binding sites. The site 2 ion interacts with a catalytically essential nucleotide and bridges two phosphates. Due to its location and geometry, this ion may play an important role in the docking of the A and B domains.


Subject(s)
Metals/chemistry , Metals/metabolism , Nucleic Acid Conformation , RNA, Catalytic/chemistry , RNA, Catalytic/metabolism , Binding Sites , Cations, Divalent , Cations, Monovalent , Manganese/chemistry , Manganese/metabolism , Models, Molecular , Nuclear Magnetic Resonance, Biomolecular , Solutions
8.
Protein Sci ; 9(12): 2386-93, 2000 Dec.
Article in English | MEDLINE | ID: mdl-11206060

ABSTRACT

The immunosuppressant drug cyclosporin A (CsA) inhibits T-cell function by blocking the phosphatase activity of calcineurin. This effect is mediated by formation of a complex between the drug and cyclophilin (CyP), which creates a composite surface able to make high-affinity contacts with calcineurin. In vitro, the CyPB/CsA complex is more effective in inhibiting calcineurin than the CyPA/CsA and CyPC/CsA complexes, pointing to fine structural differences in the calcineurin-binding region. To delineate the calcineurin-binding region of CyPB, we mutated several amino acids, located in two loops corresponding to CyPA regions known to be involved, as follows: R76A, G77H, D155R, and D158R. Compared to wild-type CyPB, the G77H, D155R, and D158R mutants had intact isomerase and CsA-binding activities, indicating that no major conformational changes had taken place. When complexed to CsA, they all displayed only reduced affinity for calcineurin and much decreased inhibition of calcineurin phosphatase activity. These results strongly suggest that the three amino acids G77, D155, and D158 are directly involved in the interaction of CyPB/CsA with calcineurin, in agreement with their exposed position. The G77, D155, and D158 residues are not maintained in CyPA and might therefore account for the higher affinity of the CyPB/CsA complex for calcineurin.


Subject(s)
Calcineurin/metabolism , Cyclophilins/chemistry , Amino Acid Sequence , Binding Sites/genetics , Cyclophilins/genetics , Cyclophilins/metabolism , Cyclosporine/metabolism , Cyclosporine/pharmacology , Humans , Models, Molecular , Mutagenesis, Site-Directed , Peptidylprolyl Isomerase , Phosphoric Monoester Hydrolases/antagonists & inhibitors , Point Mutation , Protein Binding/genetics
9.
Blood ; 94(3): 976-83, 1999 Aug 01.
Article in English | MEDLINE | ID: mdl-10419889

ABSTRACT

We have recently reported that cyclophilin B (CyPB), a secreted cyclosporine-binding protein, could bind to T lymphocytes through interactions with two types of binding sites. The first ones, referred to as type I, involve interactions with the conserved domain of CyPB and promote the endocytosis of surface-bound ligand, while the second type of binding sites, termed type II, are represented by glycosaminoglycans (GAG). Here, we further investigated the interactions of CyPB with blood cell populations. In addition to lymphocytes, CyPB was found to interact mainly with platelets. The binding is specific, with a dissociation constant (kd) of 9 +/- 3 nmol/L and the number of sites estimated at 960 +/- 60 per cell. Platelet glycosaminoglycans are not required for the interactions, but the binding is dramatically reduced by active cyclosporine derivatives. We then analyzed the biologic effects of CyPB and found a significant increase in platelet adhesion to collagen. Concurrently, CyPB initiates a transmembranous influx of Ca(2+) and induces the phosphorylation of the P-20 light chains of myosin. Taken together, the present results demonstrate for the first time that extracellular CyPB specifically interacts with platelets through a functional receptor related to the lymphocyte type I binding sites and might act by regulating the activity of a receptor-operated membrane Ca(2+) channel.


Subject(s)
Blood Platelets/metabolism , Blood Platelets/pathology , Calcium/metabolism , Cyclophilins , Immunophilins/metabolism , Platelet Adhesiveness , Collagen , Humans , Peptidylprolyl Isomerase , Protein Binding , Signal Transduction
10.
J Neurochem ; 73(1): 260-70, 1999 Jul.
Article in English | MEDLINE | ID: mdl-10386979

ABSTRACT

Cyclophilin B (CyPB) is a cyclosporin A (CsA)-binding protein mainly located in intracellular vesicles and secreted in biological fluids. In previous works, we demonstrated that CyPB interacts with T lymphocytes and enhances in vitro cellular incorporation and activity of CsA. In addition to its immunosuppressive activity, CsA is able to promote regeneration of damaged peripheral nerves. However, the crossing of the drug from plasma to neural tissue is restricted by the relative impermeability of the blood-brain barrier. To know whether CyPB might also participate in the delivery of CsA into the brain, we have analyzed the interactions of CyPB with brain capillary endothelial cells. First, we demonstrated that CyPB binds to two types of binding sites present at the surface of capillary endothelial cells from various species of tissues. The first type of binding sites (K(D) = 300 nM; number of sites = 3 x 10(6)) is related to interactions with negatively charged compounds such as proteoglycans. The second type of binding sites, approximately 50,000 per cell, exhibits a higher affinity for CyPB (K(D) = 15 nM) and is involved in an endocytosis process, indicating it might correspond to a functional receptor. Finally, the use of an in vitro model of blood-brain barrier allowed us to demonstrate that CyPB is transcytosed by a receptor-mediated pathway (flux = 16.5 fmol/cm2/h). In these conditions, CyPB did not significantly modify the passage of CsA, indicating that it is unlikely to provide a pathway for CsA brain delivery.


Subject(s)
Blood-Brain Barrier/physiology , Cyclophilins , Endothelium, Vascular/metabolism , Immunophilins/metabolism , Receptors, Peptide/physiology , Animals , Appendix/blood supply , Astrocytes/physiology , Biological Transport , Brain/blood supply , Capillaries , Cattle , Cell Line , Coculture Techniques , Cyclosporine/metabolism , Humans , Iodine Radioisotopes , Kinetics , Peptidylprolyl Isomerase , Umbilical Veins
11.
EMBO J ; 18(9): 2563-79, 1999 May 04.
Article in English | MEDLINE | ID: mdl-10228169

ABSTRACT

NHP6A is a chromatin-associated protein from Saccharomyces cerevisiae belonging to the HMG1/2 family of non-specific DNA binding proteins. NHP6A has only one HMG DNA binding domain and forms relatively stable complexes with DNA. We have determined the solution structure of NHP6A and constructed an NMR-based model structure of the DNA complex. The free NHP6A folds into an L-shaped three alpha-helix structure, and contains an unstructured 17 amino acid basic tail N-terminal to the HMG box. Intermolecular NOEs assigned between NHP6A and a 15 bp 13C,15N-labeled DNA duplex containing the SRY recognition sequence have positioned the NHP6A HMG domain onto the minor groove of the DNA at a site that is shifted by 1 bp and in reverse orientation from that found in the SRY-DNA complex. In the model structure of the NHP6A-DNA complex, the N-terminal basic tail is wrapped around the major groove in a manner mimicking the C-terminal tail of LEF1. The DNA in the complex is severely distorted and contains two adjacent kinks where side chains of methionine and phenylalanine that are important for bending are inserted. The NHP6A-DNA model structure provides insight into how this class of architectural DNA binding proteins may select preferential binding sites.


Subject(s)
DNA-Binding Proteins/chemistry , Fungal Proteins/chemistry , High Mobility Group Proteins/chemistry , Nuclear Proteins/chemistry , Saccharomyces cerevisiae Proteins , Amino Acid Sequence , Base Sequence , Binding Sites , Computer Simulation , DNA/chemistry , DNA/metabolism , DNA-Binding Proteins/genetics , DNA-Binding Proteins/metabolism , Fungal Proteins/genetics , Fungal Proteins/metabolism , HMGN Proteins , High Mobility Group Proteins/genetics , High Mobility Group Proteins/metabolism , Lymphoid Enhancer-Binding Factor 1 , Models, Molecular , Molecular Sequence Data , Mutation , Nuclear Magnetic Resonance, Biomolecular , Nuclear Proteins/genetics , Nuclear Proteins/metabolism , Protein Binding , Protein Conformation , Saccharomyces cerevisiae , Sequence Homology, Amino Acid , Sex-Determining Region Y Protein , Transcription Factors/chemistry
12.
J Biol Chem ; 274(16): 10990-8, 1999 Apr 16.
Article in English | MEDLINE | ID: mdl-10196180

ABSTRACT

Cyclophilin B is a cyclosporin A-binding protein exhibiting peptidyl-prolyl cis/trans isomerase activity. We have previously shown that it interacts with two types of binding sites on T lymphocytes. The type I sites correspond to specific functional receptors and the type II sites to sulfated glycosaminoglycans. The interactions of cyclophilin B with type I and type II sites are reduced in the presence of cyclosporin A and of a synthetic peptide mimicking the N-terminal part of cyclophilin B, respectively, suggesting that the protein possesses two distinct binding regions. In this study, we intended to characterize the areas of cyclophilin B involved in the interactions with binding sites present on Jurkat cells. The use of cyclophilin B mutants modified in the N-terminal region demonstrated that the 3Lys-Lys-Lys5 and 14Tyr-Phe-Asp16 clusters are probably solely required for the interactions with the type II sites. We further engineered mutants of the conserved central core of cyclophilin B, which bears the catalytic and the cyclosporin A binding sites as an approach to localize the binding regions for the type I sites. The enzymatic activity of cyclophilin B was dramatically reduced after substitution of the Arg62 and Phe67 residues, whereas the cyclosporin A binding activity was destroyed by mutation of the Trp128 residue and strongly decreased after modification of the Phe67 residue. Only the substitution of the Trp128 residue reduced the binding of the resulting cyclophilin B mutant to type I binding sites. The catalytic site of cyclophilin B therefore did not seem to be essential for cellular binding and the cyclosporin A binding site appeared to be partially involved in the binding to type I sites.


Subject(s)
Cyclophilins , Glycosaminoglycans/metabolism , Immunophilins/metabolism , T-Lymphocytes/metabolism , Amino Acid Sequence , Binding Sites , Humans , Immunophilins/chemistry , Immunophilins/genetics , Jurkat Cells , Models, Molecular , Molecular Sequence Data , Mutagenesis, Site-Directed , Peptidylprolyl Isomerase , Protein Binding , Protein Conformation , Sequence Homology, Amino Acid
13.
Nat Struct Biol ; 6(3): 212-6, 1999 Mar.
Article in English | MEDLINE | ID: mdl-10074938

ABSTRACT

The hairpin ribozyme is a small catalytic RNA with a unique two-domain structure. Here we present the solution structure of the loop B domain of the hairpin ribozyme, which contains most of the catalytically essential nucleotides. The 38-nucleotide domain contains a 16-nucleotide internal loop that forms one of the largest non-Watson-Crick segments of base pairing thus far determined by either NMR or crystallography. Since the solution structure of the smaller loop A domain has been previously solved, an NMR structure-based model of the 22,000 Mr hairpin ribozyme-substrate open complex emerges by joining the two domain structures. Strikingly, catalytically essential functional groups for the loop B domain are concentrated within an expanded minor groove, presenting a clear docking surface for the loop A domain.


Subject(s)
Nucleic Acid Conformation , RNA, Catalytic/chemistry , Base Sequence , Crystallography, X-Ray , Hydrogen Bonding , Magnetic Resonance Spectroscopy
14.
Biochem J ; 336 ( Pt 3): 689-97, 1998 Dec 15.
Article in English | MEDLINE | ID: mdl-9841882

ABSTRACT

Cyclophilin B (CyPB) is a cyclosporin A (CsA)-binding protein, mainly associated with the secretory pathway, and is released in biological fluids. We recently reported that CyPB specifically binds to T-lymphocytes and promotes enhanced incorporation of CsA. The interactions with cellular binding sites involved, at least in part, the specific N-terminal extension of the protein. In this study, we intended to specify further the nature of the CyPB-binding sites on peripheral blood T-lymphocytes. We first provide evidence that the CyPB binding to heparin-Sepharose is prevented by soluble sulphated glycosaminoglycans (GAG), raising the interesting possibility that such interactions may occur on the T-cell surface. We then characterized CyPB binding to T-cell surface GAG and found that these interactions involved the N-terminal extension of CyPB, but not its conserved CsA-binding domain. In addition, we determined the presence of a second CyPB binding site, which we termed a type I site, in contrast with type II for GAG interactions. The two binding sites exhibit a similar affinity but the expression of the type I site was 3-fold lower. The conclusion that CyPB binding to the type I site is distinct from the interactions with GAG was based on the findings that it was (1) resistant to NaCl wash and GAG-degrading enzyme treatments, (2) reduced in the presence of CsA or cyclophilin C, and (3) unmodified in the presence of either the N-terminal peptide of CyPB or protamine. Finally, we showed that the type I binding sites were involved in an endocytosis process, supporting the hypothesis that they may correspond to a functional receptor for CyPB.


Subject(s)
Cyclophilins , Immunophilins/metabolism , T-Lymphocytes/metabolism , Binding Sites , Chromatography, Affinity , Endocytosis , Glycosaminoglycans/metabolism , Humans , In Vitro Techniques , Ligands , Peptidylprolyl Isomerase , Recombinant Proteins/metabolism , Surface Properties
15.
Transplantation ; 65(8): 1076-84, 1998 Apr 27.
Article in English | MEDLINE | ID: mdl-9583869

ABSTRACT

BACKGROUND: Cyclophilin B (CyPB) is a cyclosporine (CsA)-binding protein, located within intracellular vesicles and secreted in biological fluids. In previous works, we reported that CyPB specifically interacts with the T-cell membrane and potentiates the ability of CsA to inhibit CD3-induced proliferation of T lymphocytes. METHODS: CyPB levels were measured in plasma from healthy donors and transplant patients. The role of extracellular CyPB on the distribution and activity of CsA was investigated first by studies on the uptake of free and CyPB-complexed drug by blood cells, and second by studies on the inhibitory effects of these two compounds on the CD3-induced proliferation of peripheral blood mononuclear cells. RESULTS: A significant increase in plasma CyPB level was observed for CsA-treated patients (13+/-6.4 nM, n=42) in comparison with untreated donors (4.3+/-2.1 nM, n=34). In vitro, extracellular CyPB dose dependently modified CsA distribution between plasma, erythrocyte, and lymphocyte contents, by both retaining the complexed drug extracellularly and promoting its specific accumulation within peripheral blood mononuclear cells. Moreover, the enhanced ability of CyPB-complexed CsA to suppress CD3-induced T-cell proliferation was preserved in the presence of other blood cells, implying specific targeting of the drug to sensitive cells. Furthermore, although a large interindividual variability of sensitivity to the drug was confirmed for 18 individuals, we found that CyPB potentiated the activity of CsA in restoring a high sensitivity to the immunosuppressant. CONCLUSION: These results suggest that plasma CyPB may contribute to the acceptance and the good maintenance of organ transplantation by enhancing the immunosuppressive activity of CsA through a receptor-mediated incorporation of CyPB-complexed CsA within peripheral blood lymphocytes.


Subject(s)
Amino Acid Isomerases/blood , Carrier Proteins/blood , Cyclophilins , Cyclosporine/therapeutic use , Heart Transplantation/immunology , Immunosuppressive Agents/therapeutic use , Kidney Transplantation/immunology , Adult , Amino Acid Isomerases/biosynthesis , Blotting, Western , Carrier Proteins/biosynthesis , Cyclosporine/blood , Enzyme-Linked Immunosorbent Assay , Erythrocytes/immunology , Erythrocytes/metabolism , Female , Humans , Immunosuppressive Agents/blood , Kinetics , Male , Middle Aged , Peptidylprolyl Isomerase , Reference Values
16.
J Biomol NMR ; 11(1): 59-84, 1998 Jan.
Article in English | MEDLINE | ID: mdl-9566313

ABSTRACT

RNA-protein recognition is critical to post-transcriptional regulation of gene expression, yet poorly understood at the molecular level. The relatively slow progress in understanding this important area of molecular biology is due to difficulties in obtaining good-quality crystals and derivatives, and in preparing samples suitable for NMR investigation. The determination of the structure of the complex between the human U1A protein and its polyadenylation inhibition element is described here. In this paper, we describe the sample preparation, spectral assignments, construction of the NOE-based distance constraints and methodology for calculating the structure of the complex. The structure was determined to an overall precision of 2.03 A (for all ordered regions), and 1.08 A for the protein-RNA interface. The patterns of hydrogen bonding and hydrophobic interactions at the interface were analysed statistically using the final ensemble of 31 structures.


Subject(s)
RNA, Messenger/chemistry , RNA-Binding Proteins/chemistry , Ribonucleoprotein, U1 Small Nuclear/chemistry , Amino Acid Sequence , Base Sequence , Binding Sites , Humans , Macromolecular Substances , Magnetic Resonance Spectroscopy , Models, Molecular , Molecular Sequence Data , Nucleic Acid Conformation , Protein Conformation , Thermodynamics
17.
EMBO J ; 16(18): 5764-72, 1997 Sep 15.
Article in English | MEDLINE | ID: mdl-9312034

ABSTRACT

The RNP domain is a very common eukaryotic protein domain involved in recognition of a wide range of RNA structures and sequences. Two structures of human U1A in complex with distinct RNA substrates have revealed important aspects of RNP-RNA recognition, but have also raised intriguing questions concerning the origin of binding specificity. The beta-sheet of the domain provides an extensive RNA-binding platform for packing aromatic RNA bases and hydrophobic protein side chains. However, many interactions between functional groups on the single-stranded nucleotides and residues on the beta-sheet surface are potentially common to RNP proteins with diverse specificity and therefore make only limited contribution to molecular discrimination. The refined structure of the U1A complex with the RNA polyadenylation inhibition element reported here clarifies the role of the RNP domain principal specificity determinants (the variable loops) in molecular recognition. The most variable region of RNP proteins, loop 3, plays a crucial role in defining the global geometry of the intermolecular interface. Electrostatic interactions with the RNA phosphodiester backbone involve protein side chains that are unique to U1A and are likely to be important for discrimination. This analysis provides a novel picture of RNA-protein recognition, much closer to our current understanding of protein-protein recognition than that of DNA-protein recognition.


Subject(s)
Nucleic Acid Conformation , Protein Structure, Secondary , RNA/chemistry , RNA/metabolism , Ribonucleoprotein, U1 Small Nuclear/chemistry , Ribonucleoprotein, U1 Small Nuclear/metabolism , Amino Acid Sequence , Base Sequence , Computer Simulation , Humans , Models, Molecular , Molecular Sequence Data , Nuclear Magnetic Resonance, Biomolecular , RNA-Binding Proteins/chemistry , RNA-Binding Proteins/metabolism , Substrate Specificity
18.
Immunology ; 91(4): 609-17, 1997 Aug.
Article in English | MEDLINE | ID: mdl-9378502

ABSTRACT

Cyclophilin B (CyPB) is a cyclosporin A (CsA)-binding protein, mainly associated with the secretory pathway and released in biological fluids. We have recently demonstrated that both free CyPB and CyPB-CsA complex specifically bind to peripheral blood T lymphocytes and are internalized. These results suggest that CyPB might promote the targeting of the drug into sensitive cells. Peripheral blood lymphocytes are subdivided in several populations according to their biological functions and sensitivity to CsA. We have investigated the binding of CyPB to these different subsets using a CyPB derivatized by fluorescein through its single cysteine which retains its binding properties. We have confirmed that only T cells were involved in the interaction with CyPB. The ligand binding was found to be heterogeneously distributed on the different T-cell subsets and surface-bound CyPB was mainly associated with the CD4-positive cells. No significant difference was noted between the CD45RA and CD45RO subsets, demonstrating that CyPB-binding sites were equally distributed between native and memory T cells. CD3 stimulation of T lymphocytes led to a decrease in the CyPB-binding capacity, that may be explained by a down-regulation of the CyPB-receptor expression upon T-cell activation. Finally, we demonstrated that CyPB-receptor-positive cells, isolated on CyPB sulphydryl-coupled affinity matrices, are more sensitive to CyPB-complexed CsA than mixed peripheral blood lymphocytes, suggesting that CyPB potentiates CsA activity through the binding of the complex. Taken together, our results demonstrate that CyPB-binding sites are mainly associated with resting cells of the helper T lymphocyte, and that CyPB might modulate the distribution of CsA through the drug targeting to sensitive cells.


Subject(s)
Amino Acid Isomerases/metabolism , Carrier Proteins/metabolism , Cyclophilins , T-Lymphocyte Subsets/metabolism , Binding Sites , Binding, Competitive , CD4-Positive T-Lymphocytes/metabolism , Cell Culture Techniques , Cyclosporine/pharmacology , Fluoresceins , Humans , Immunophenotyping , Immunosuppressive Agents/pharmacology , Lymphocyte Activation , Peptidylprolyl Isomerase , Recombinant Proteins/metabolism , T-Lymphocyte Subsets/drug effects
19.
J Mol Biol ; 267(2): 338-51, 1997 Mar 28.
Article in English | MEDLINE | ID: mdl-9096230

ABSTRACT

The great diversity of RNA biological functions has led to widespread interest in RNA structure. Advances in synthetic and spectroscopic techniques have recently allowed the extension of NMR methods of structure determination to RNA structures of significant size and increased biological significance. However, it has not yet been established how accurately and precisely RNA structure can be determined by NMR. The extensive simulations presented here establish credible limits on accuracy and precision of NMR-derived RNA structures and provide quantitative calibrations to evaluate new structures. Synthetic sets of NMR constraints were generated from a crystallographically-derived ribozyme structure. The target structure was then redetermined using approximations and computational protocols derived from our experimental work. The results demonstrate that the structure of RNA molecules of at least 15,000 Da can be determined with precision and accuracy of 1 to 1.5 A, comparable to values obtained for proteins of similar size. Most encouragingly, it is shown that larger, globular and biologically more important RNA structures can be determined with significantly better accuracy and precision than smaller, elongated structures investigated until now.


Subject(s)
Magnetic Resonance Spectroscopy , RNA/chemistry , Base Composition , Models, Molecular , Nucleic Acid Conformation , Proteins/chemistry , RNA, Catalytic/chemistry , Reproducibility of Results , Software
20.
Biochem J ; 317 ( Pt 2): 565-70, 1996 Jul 15.
Article in English | MEDLINE | ID: mdl-8713086

ABSTRACT

Cyclophilin B (CyPB) is a cyclosporin A (CsA)-binding protein located within intracellular vesicles and released in biological fluids. We recently reported the specific binding of this protein to T-cell surface receptor which is internalized even in the presence of CsA. These results suggest that CyPB might target the drug to lymphocytes and consequently modify its activity. To verify this hypothesis, we have first investigated the binding capacity and internalization of the CsA-CyPB complex in human peripheral blood T-lymphocytes and secondly compared the inhibitory effect of both free and CyPB-complexed CsA on the CD3-induced activation and proliferation of T-cells. Here, we present evidence that both the CsA-CyPB complex and free CyPB bind to the T-lymphocyte surface, with similar values of Kd and number of sites. At 37 degrees C, the complex is internalized but, in contrast to the protein, the drug is accumulated within the cell. Moreover, CyPB receptors are internalized together with the ligand and rapidly recycled to the cell surface. Finally, we demonstrate that CyPB-complexed CsA remains as efficient as uncomplexed CsA and that CyPB enhances the immunosuppressive activity of the drug. Taken together, our results support the hypothesis that surface CyPB receptors may be related to the selective and variable action of CsA, through specific binding and targeting of the CyPB-CsA complex to peripheral blood T-lymphocytes.


Subject(s)
Amino Acid Isomerases/metabolism , Carrier Proteins/metabolism , Cyclophilins , Cyclosporine/metabolism , Immunosuppressive Agents/metabolism , T-Lymphocytes/metabolism , Amino Acid Isomerases/pharmacology , Binding, Competitive , Biological Transport , Carrier Proteins/pharmacology , Cyclosporine/pharmacology , Humans , Ligands , Lymphocyte Activation/drug effects , Peptidylprolyl Isomerase , Protein Binding , T-Lymphocytes/enzymology
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