Relationship between anti-insulin antibodies and albuminuria or proteinuria in human insulin-treated type 2 diabetes mellitus.

In order to examine the relationship between anti-insulin antibodies (AIA) caused by extrinsic human insulin and albuminuria or proteinuria, 53 human insulin-treated type 2 diabetics were divided into two groups: (AIA(+) group) 27 patients with a titer of AIA greater than 7.6% and (AIA(-) group) 26 patients with a titer of AIA less than 7.5%. Although no significant difference was found between the two groups for age, gender, body mass index, duration of diabetes, duration of insulin treatment, blood pressure, serum creatinine, glycosylated hemoglobin (HbA1c), daily dose of insulin, daily insulin injection times, or treatment of hypertension, the AIA(+) group had a significantly higher urinary albumin to creatinine ratio and urinary protein to creatinine ratio than the AIA(-) group (p < 0.05). It is suggested that AIA in type 1 diabetics might be insulin autoantibodies, which is not the case with type 2 diabetics. To our knowledge this is the first study demonstrating the relationship between AIA induced not by porcine or bovine insulin, but by human insulin and albuminuria or proteinuria in type 2 diabetics.

Structure of the C-terminal RNA-binding domain of hnRNP D0 (AUF1), its interactions with RNA and DNA, and change in backbone dynamics upon complex formation with DNA.

Heterogeneous nuclear ribonucleoprotein (hnRNP) D0 has two ribonucleoprotein (RNP) -type RNA-binding domains (RBDs), each of which can specifically bind to the UUAG-sequence. hnRNP D0 also binds specifically to single-stranded d(TTAGGG)(n), the human telomeric DNA repeat. We have already reported the structure and interactions with RNA of the N-terminal RBD (RBD1). Here, the structure of the C-terminal RBD (RBD2) determined by NMR is presented. It folds into a compact alpha beta structure comprising an antiparallel beta-sheet packed against two alpha-helices, which is characteristic of RNP-type RBDs. In addition to the four beta-strands commonly found in RNP-type RBDs, an extra beta-strand, termed beta 4(-), was found just before the fourth beta-strand, yielding a five-stranded beta-sheet. Candidate residues of RBD2 involved in the interactions with RNA were identified by chemical shift perturbation analysis. Perturbation was detected on the beta-sheet side, not on the opposite alpha-helix side, as observed for RBD1. It is notable that the beta 4(-) to beta 4 region of RBD2 is involved in the interactions in contrast to the case of RBD1. The chemical shift perturbation analysis also showed that RBD2 interacts with DNA in essentially the same way as with RNA. Changes in the backbone dynamics upon complex formation with DNA were examined by means of model free analysis of relaxation data. In free RBD2, the beta 4(-) to beta 4 region exhibits slow conformational exchange on the milli- to microsecond time scale. The exchange is quenched upon complex formation. The flexibility of free RBD2 may be utilized in the recognition process by allowing different conformational states to be accessed and facilitating induced fit. Additionally, faster flexibility on the nano- to picosecond time scale was observed for loop 3 located between beta 2 and beta 3 in free RBD2, which is retained by the complex as well.

Interaction of the C-terminal domain of the E. coli RNA polymerase alpha subunit with the UP element: recognizing the backbone structure in the minor groove surface.

The C-terminal domain of the alpha-subunit of Escherichia coli RNA polymerase (alphaCTD) is responsible for transcriptional activation through interaction with both activator proteins and UP element DNA. Previously, we determined the solution structure of alphaCTD. Here, we investigated the interaction between alphaCTD and UP element DNA by NMR. DNA titration curves and intermolecular NOE measurements indicate that alphaCTD can bind to multiple sites on the UP element DNA. Unlike many transcription factors, alphaCTD does not have a strict base sequence requirement for binding. There is a good correlation between the strength of the interaction and the extent of intrinsic bending of the DNA oligomer estimated from the gel retardation assay. We propose that alphaCTD recognizes the backbone structure of DNA oligomers responsible for the intrinsic bending. Moreover, NMR studies and drug competition experiments indicated that alphaCTD interacts with the UP element on the minor groove side of the DNA. The C-terminal end of helix-1, the N-terminal end of helix-4, and the loop between helices 3 and 4 are used for the interaction. Based on these observations, we propose a model for the UP element-alphaCTD complex.

New quadruplex structure of GGA triplet repeat DNA--an intramolecular quadruplex composed of a G:G:G:G tetrad and G(:A):G(:A):G(:A):G heptad, and its dimerization.

The structure of d(GGAGGAGGAGGA) containing four tandem repeats of a GGA triplet sequence has been determined under physiological K+ conditions by NMR. d(GGAGGAGGAGGA) folds into an intramolecular quadruplex composed of a G:G:G:G tetrad and a G(:A):G(:A):G(:A):G heptad. Four G-G segments of d(GGAGGAGGAGGA) are aligned parallel to each other due to seven successive turns of the main chain at each of the GGA and GAGG segments. Two quadruplexes form a dimer stabilized through a stacking interaction between the heptads of the two quadruplexes. On the basis of these results, the biological implications of naturally occurring GGA triplet repeat DNA are discussed.

An intramolecular quadruplex of (GGA)(4) triplet repeat DNA with a G:G:G:G tetrad and a G(:A):G(:A):G(:A):G heptad, and its dimeric interaction.

The structure of d(GGAGGAGGAGGA) containing four tandem repeats of a GGA triplet sequence has been determined under physiological K(+) conditions. d(GGAGGAGGAGGA) folds into an intramolecular quadruplex composed of a G:G:G:G tetrad and a G(:A):G(:A):G(:A):G heptad. Four G-G segments of d(GGAGGAGGAGGA) are aligned parallel with each other due to six successive turns of the main chain at each of the GGA and GAGG segments. Two quadruplexes form a dimer stabilized through a stacking interaction between the heptads of the two quadruplexes. Comparison of the structure of d(GGAGGAGGAGGA) with the reported structure of d(GGAGGAN) (N=G or T) containing two tandem repeats of the GGA triplet revealed that although the two structures resemble each other to some extent, the extension of the repeats of the GGA triplet leads to distinct structural differences: intramolecular quadruplex for 12-mer versus intermolecular quadruplex for 7-mer; heptad versus hexad in the quadruplex; and three sheared G:A base-pairs versus two sheared G:A base-pairs plus one A:A base-pair per quadruplex. It was also suggested that d(GGAGGAGGAGGA) forms a similar quadruplex under low salt concentration conditions. This is in contrast to the case of d(GGAGGAN) (N=G or T), which forms a duplex under low salt concentration conditions. On the basis of these results, the structure of naturally occurring GGA triplet repeat DNA is discussed.

A novel RNA motif that binds efficiently and specifically to the Ttat protein of HIV and inhibits the trans-activation by Tat of transcription in vitro and in vivo.

BACKGROUND: To find a novel RNA that would bind efficiently and specifically to Tat protein but not to other cellular factors, we used an in vitro selection method and isolated a novel aptamer RNATat, a 37-mer RNA oligomer, that binds efficiently to the Tat protein of HIV-1. In the present study, we analysed various properties of aptamer RNATat, including binding kinetics, identification of functional groups for Tat binding, and inhibition of Tat function. RESULTS: The binding affinity of the isolated aptamer RNATat to Tat-1 was 133 times higher than that of authentic TAR-1 RNA. RNATat is composed of inverted repeats of two TAR-like motifs, and even though RNATat had two Tat-binding core elements, the interaction with Tat took place at a molar ratio of 1 : 1. Several functional groups of aptamer RNATat responsible for Tat binding were identified. The selected aptamer RNATat competed effectively for binding to Tat even in the presence of a large excess of TAR-1 or TAR-2 RNA in vitro, and specifically prevented Tat-dependent trans-activation both in vitro and in vivo. CONCLUSIONS: Our results indicate that a novel aptamer, RNATat, retained strong affinity for Tat even in the presence of a large excess of HIV TAR. RNATat binds efficiently to Tat proteins or peptides derived from either HIV-1 or HIV-2. Unlike TAR RNA, RNATat affinity does not depend upon cellular proteins such as cyclin T1, thus RNATat has the potential for use as a molecular recognition element in biosensors.

NMR study of a novel RNA quadruplex structure.

The structure of an RNA oligomer, r (GGAGGUUUUGGAGG) (R14-2) whose G-G steps are separated by adenine and uracil residues has been investigated by NMR. In the presence of 20 mM K+, a novel dimeric multiplex architecture is adopted by two strands of R14-2. In each strand a UUUU loop and two A residues connect four parallel G-G steps that pair-align into two tetrads. One of the tetrads is further pair-aligned by two A residues through the sheared mismatch and a novel hexad is subsequently formed. Two hexads coming from two different strands stack to make a dimeric multiplex. All of the guanosine and adenosine residues take an anti conformation.

NMR analysis of tertiary interactions in HDV ribozymes.

Three variants of minimized hepatitis delta virus (HDV) RNA ribozyme systems designed on the basis of the "pseudoknot" model were synthesized and their tertiary interactions were analyzed by NMR spectroscopy. Rz-1 is a cis-acting ribozyme system (the cleaved form, 56-mer) in which stem IV is deleted from the active domain of genomic HDV RNA. Rz-1 was uniformly labeled with stable isotopes, 13C and 15N. Rz-2 is a trans-acting ribozyme system (substrate: 8-mer, the cytidine residue at the cleavage site is replaced by 2'-O-methylcytidine; enzyme: 16-mer plus 35-mer). Rz-2 was partially labeled with stable isotopes in guanosine residues of enzyme 35mer. Rz-4 is a trans-acting ribozyme system (substrate: 8mer, the cytidine residue at the cleavage site is replaced by 2'-O-methylcytidine; enzyme 53mer) which was designed by Perrotta and Been. Rz-4 has the same sequence and an extra loop closing stem IV. From 2D-NOESY and 2D-HSQC (except for Rz-4) spectra, it was suggested each ribozyme forms "pseudoknot" type structure in solution. Additionally, it was found that G38 of Rz-1, G28 and G29 of Rz-2 and Rz-4 form base-pairs. These novel base-pairs are observed in the crystal structure of a modified genomic HDV RNA. From temperature change experiment of Rz-2, the imino proton signal of G28 disappeared at 50 degrees C earlier than the other corresponding signals. Upon MgCl2 titration of Rz-2, this signal showed the largest shift.

Intramolecular quadruplex formation of the G-rich strand of the mouse hypervariable minisatellite Pc-1.

The minisatellite Pc-1, isolated from the mouse genome consisting of a tandem repeat of d(GGCAG), is hypervariable with a mutation rate of 0.15/generation. Here we describe a structural characterization of the G-rich strand of Pc-1 by biochemical and physicochemical methods. It was found to be comparatively resistant to both single-stranded DNA-binding protein binding and digestion by single-stranded DNA-specific nuclease and to cause arrest of DNA synthesis. The guanine imino proton NMR signals observed on the Pc-1 G-rich strand and their slow (1)H/(2)H exchange profiles pointed to a quadruplex structure with guanine quartets. The melting temperature of the quadruplex determined by CD was not dependent on DNA concentration. These results indicate that the G-rich strand of Pc-1 forms an intramolecular folded-back quadruplex structure under physiological conditions. Possible mechanisms of the Pc-1 mutations implicated with the formation of the quadruplex structure are discussed.

Structure and interactions with RNA of the N-terminal UUAG-specific RNA-binding domain of hnRNP D0.

Heterogeneous nuclear ribonucleoprotein (hnRNP) D0 has two ribonucleoprotein (RNP)-type RNA-binding domains (RBDs), each of which can bind solely to the UUAG sequence specifically. The structure of the N-terminal RBD (RBD1) determined by NMR is presented here. It folds into a compact alphabeta structure comprising a four-stranded antiparallel beta-sheet packed against two alpha-helices, which is characteristic of the RNP-type RBDs. Special structural features of RBD1 include N-capping boxes for both alpha-helices, a beta-bulge in the second beta-strand, and an additional short antiparallel beta-sheet coupled with a beta-turn-like structure in a loop. Two hydrogen bonds which restrict the positions of loops were identified. Backbone resonance assignments for RBD1 complexed with r(UUAGGG) revealed that the overall folding is maintained in the complex. The candidate residues involved in the interactions with RNA were identified by chemical shift perturbation analysis. They are located in the central and peripheral regions of the RNA-binding surface composed of the four-stranded beta-sheet, loops, and the C-terminal region. It is suggested that non-specific interactions with RNA are performed by the residues in the central region of the RNA-binding surface, while specific interactions are performed by those in the peripheral regions. It was also found that RBD1 has the ability to inhibit the formation of the quadruplex structure.

Structure, backbone dynamics and interactions with RNA of the C-terminal RNA-binding domain of a mouse neural RNA-binding protein, Musashi1.

Musashi1 is an RNA-binding protein abundantly expressed in the developing mouse central nervous system. Its restricted expression in neural precursor cells suggests that it is involved in the regulation of asymmetric cell division. Musashi1 contains two ribonucleoprotein (RNP)-type RNA-binding domains (RBDs), RBD1 and RBD2. Our previous studies showed that RBD1 alone binds to RNA, while the binding of RBD2 is not detected under the same conditions. Joining of RBD2 to RBD1, however, increases the affinity to greater than that of RBD1 alone, indicating that RBD2 contributes to RNA-binding. We have determined the three-dimensional solution structure of the C-terminal RBD (RBD2) of Musashi1 by NMR. It folds into a compact alpha beta structure comprising a four-stranded antiparallel beta-sheet packed against two alpha-helices, which is characteristic of RNP-type RBDs. Special structural features of RBD2 include a beta-bulge in beta2 and a shallow twist of the beta-sheet. The smaller 1H-15N nuclear Overhauser enhancement values for the residues of loop 3 between beta2 and beta3 suggest that this loop is flexible in the time-scale of nano- to picosecond order. The smaller 15N T2 values for the residues around the border between alpha2 and the following loop (loop 5) suggest this region undergoes conformational exchange in the milli- to microsecond time-scale. Chemical shift perturbation analysis indicated that RBD2 binds to an RNA oligomer obtained by in vitro selection under the conditions for NMR measurements, and thus the nature of the weak RNA-binding of RBD2 was successfully characterized by NMR, which is otherwise difficult to assess. Mainly the residues of the surface composed of the four-stranded beta-sheet, loops and C-terminal region are involved in the interaction. The appearance of side-chain NH proton resonances of arginine residues of loop 3 and imino proton resonances of RNA bases upon complex formation suggests the formation of intermolecular hydrogen bonds. The structural arrangement of the rings of the conserved aromatic residues of beta2 and beta3 is suitable for stacking interaction with RNA bases, known to be one of the major protein-RNA interactions, but a survey of the perturbation data suggested that the stacking interaction is not ideally achieved in the complex, which may be related to the weaker RNA-binding of RBD2.

Design and NMR analysis of HDV ribozymes for structural investigation.

Three variants of minimized hepatitis delta virus (HDV) RNA ribozyme systems (Rz-1 to approximately Rz-3) (Fig. 1) were designed on the basis of the "pseudoknot" structure model and synthesized. Rz-1 is a cis-acting ribozyme system (a cleaved form, 56-mer) in which stem IV is deleted from the active domain of genomic HDV RNA. Rz-1 was uniformly labeled with stable isotopes, 13C and 15N. The 2D-NOESY and 2D-HSQC data for Rz-1 suggest that Rz-1 forms the pseudoknot structure and G38 which is opposite to the cleavage site makes a base-pair. Rz-2 is a trans-acting ribozyme system which consists of three RNA oligomer strands (substrate: 8-mer, the cytidine residue at the cleavage site is replaced by 2'-O-methylcytidine; enzyme: 16-mer plus 35-mer). Rz-3 is a ribozyme in which the three RNA strands of Rz-2 are connected. It turns out that Rz-3 forms an inactive structure with low cleavage activity (k(obs) = 0.009) and final cleavage yield (6%). Rz-3 has the highest cleavage activity at pH 5.5. The optimal activity at acidic pH is similar to that of the wild type ribozyme. We also synthesized and examined the activity and structure of Rz-4 (designed by Perrotta and Been) which consists of two RNA strands (1).

Structural study of an RNA aptamer for a Tat protein complexed with ligands.

An RNA aptamer for an HIV Tat protein has been isolated by the in vitro SELEX method. The RNA aptamer binds to the Tat protein 50-100 times more strongly than native TAR RNA does. Here, we have investigated the structure of the RNA aptamer complexed with ligands, partial peptide fragments of the Tat protein or argininamide, by multidimensional 1H/13C/15N NMR. It is strongly suggested that two U:A:U base triples are formed in the RNA aptamer upon binding of ligands. Specific hydrogen bonds between arginine side chains of ligands and guanine bases located adjacent to the base triples are identified. On the basis of many intramolecular and intermolecular NOEs, a structural model of the complex has been constructed.

Two metal-binding sites in a lead ribozyme bound to competitively by Pb2+ and Mg2+--induced structural changes as revealed by NMR.

We reported recently that a lead ribozyme with modified bases cleaved at an additional site at high Pb2+ concentrations (>0.1 mM), and that the cleavage at a canonical site was enhanced nearly fourfold at the optimum combination of Pb2+ and Mg2+ concentrations [Kim, M. H., Katahira, M., Sugiyama, T. & Uesugi, S. (1997) J. Biochem. (Tokyo) 122, 1062-1067]. Here we have identified two metal-binding sites (sites 1 and 2) of the lead ribozyme at the residue level by NMR. Both sites are located in an asymmetric internal loop of the lead ribozyme. Site 1 is composed of residues of an enzyme strand and site 2 of residues of a substrate strand. The two sites are bound to competitively by Pb2+ and Mg2+. It was revealed that at certain Pb2+ and Mg2+ concentrations, site 1 is occupied by Pb2+ and site 2 is occupied by Mg2+. The dependency of the cleavage at the canonical and other sites on the Pb2+ and Mg2+ concentrations is interpreted by considering the species of metal ions bound to the two sites. It is suggested that the addition of the two metal ions produces similar and different effects on the structure of the lead ribozyme, and the two metal ions have a synergistic effect on the structure.

Structural characterization of a DNA duplex modeled on a DNA:RNA hybrid of the polypurine tract recognized by a reverse transcriptase.

The structure of d(TTAAAAGAAAAGGG):d(CCCTTTTCTTTTAA) has been characterized by NMR. The minor grooves of the two dA-tracts are suggested to be rather narrow, and the portion linking the two dA tracts exhibits a slightly deviated structure from a standard B DNA, in order to maintain the narrowness of the minor groove. The structure of the dG-tract is also slightly deviated. Additionally, specific broadening of resonances is observed for the residues at or near the junction between the dA-tract and the dG-tract, suggesting local structural polymorphology.

Cytokine regulation of the rat proopiomelanocortin gene expression in AtT-20 cells.

Although cytokines are known to be involved in the regulation of ACTH secretion, their effects, along with the molecular mechanisms, on POMC gene expression are not thoroughly characterized. In this study we examined the effects of representative cytokines on transcription of the POMC gene in corticotrophs in vitro using AtT20PL, a clone of the AtT20 cell line stably transfected with approximately 0.7 kilobase of the rat POMC 5'-promoter-luciferase fusion gene. In each experiment, cells were incubated with the cytokine tested, and the changes in POMC 5'-promoter activity were determined by a luciferase assay. The results showed that interleukin-1beta (IL-1beta) stimulated promoter activity in a biphasic manner [weak short term effects (2-3 h) followed by potent long term effects (>12-16 h)]. Tumor necrosis factor-alpha had similar effects, but much less potency. IL-6 showed a profound stimulatory, but only a long term (>20 h), effect. IL-2 did not influence POMC expression. In contrast, interferon-alpha (IFN alpha) and IFN-gamma showed acute stimulatory effects (approximately 4 h) followed by marked inhibitory effects (>8 h). Although the acute effects of IL-1beta, IL-6, and tumor necrosis factor-alpha alone were minimal, they significantly potentiated the stimulatory effect of CRH on POMC expression. Finally, pretreatment of the cells with a broad spectrum tyrosine kinase inhibitor, genistein, abolished or significantly diminished the effects of all cytokines except IFNs. Our results suggest that 1) each cytokine tested has a distinct effect on POMC gene expression; 2) there are positive cross-talk effects between CRH and cytokines at the corticotroph level; and 3) tyrosine phosphorylation cascades are involved in the intracellular signaling mechanisms of some cytokines.

Solution structure of the novel antitumor drug UCH9 complexed with d(TTGGCCAA)2 as determined by NMR.

Aureolic acid group compounds, such as chromomycin A3(CHM) and mithramycin (MIT), are known as antitumor drugs. Recently we isolated a novel aureolic acid group antitumor drug, UCH9, from Streptomyces sp. The chemical structure of UCH9 is unique in that mono- (A ring) and tetrasaccharide (B-E rings) segments and a longer hydrophobic sidechain are attached to the chromophore, while di- and trisaccharide segments and a methyl group are attached to it in the cases of CHM and MIT. It has been shown by two-dimensional agarose gel electrophoresis that the three drugs cause DNA unwinding, UCH9 causing less than the others. A photo-CIDNP experiment has revealed that UCH9 binds to the minor groove of DNA. The structure of the UCH9-d(TTGGCCAA)2 complex has been determined by 1H NMR and simulated annealing calculations. The obtained structure indicates that UCH9 binds as a dimer to the minor groove of d(TTGGCCAA)2, like CHM and MIT, but that the structural change in DNA induced on binding of UCH9 is moderate in comparison with those on binding of the other two drugs. It turns out that the dimer structure of UCH9, stabilized presumably through a hydrophobic interaction involving the A, D and E rings and the hydrophobic sidechain is different from that of CHM and thus DNA can interact with UCH9 in the minor groove with a moderate structural change.

Dimerization and DNA binding facilitate alpha-helix formation of Max in solution.

Max is a basic region/helix-loop-helix/leucine zipper (b/HLH/Z) protein that forms a hetero-complex with the Myc family proteins Myc, Mad, and Mxi1, and a homo-complex with itself. These complexes specifically bind to double-stranded DNA containing CACGTG sequences. Here, we report on the structural properties in aqueous solution of a 109-amino-acid protein, Max110, corresponding to the N-terminal domain of Max containing the b/HLH/Z motif (residues 2-110), as characterized by combined use of circular dichroism (CD) and sedimentation equilibrium experiments. The results showed that the alpha-helical content of Max110 increases with increasing protein concentration. The sedimentation equilibrium data indicated that Max110 exists as a monomer at low protein concentration, and forms a dimer at high protein concentration. Further increases in the alpha-helical content of Max110 occur upon addition of DNA with the CACGTG recognition sequence. Thus, dimerization and binding to DNA of Max both favor an increase of the alpha-helical content.