Biomedical subjects

R W Frank

Publications and source records attributed to R W Frank.

At least 19 recordsLinked to original sources

PubMed1998

Disulfide analysis reveals a role for macrophage migration inhibitory factor (MIF) as thiol-protein oxidoreductase.

The molecular mechanism of action of macrophage migration inhibitory factor (MIF), a cytokine with a critical role in the immune and inflammatory response, has not yet been identified. Here we report that MIF can function as an enzyme exhibiting thiol-protein oxidoreductase activity. Using a decapeptide fragment of MIF (MF1) spanning the conserved cysteine sequence motif Cys57-Ala-Leu-Cys60 (CALC), Cys-->Ser mutants (C57S MIF, C60S MIF, and C57S/C60S MIF) of human MIF (wtMIF), and alkylated wtMIF, we show that this activity is mediated by the CALC region and is important for the macrophage-activating properties of MIF. Both wtMIF and MF1 were demonstrated to form an intramolecular disulfide bridge. Using two common oxidoreductase assays, MIF was shown to enzymatically catalyze the reduction of insulin and 2-hydroxyethyldisulfide (HED). Examination of wtMIF and the mutants by far-UV circular dichroism spectroscopy (CD) together with denaturation studies showed that substituting or reducing the cysteine residues of CALC led to a reduced conformational stability of MIF but did not significantly change its overall conformation. A functional role for the CALC region was revealed by subjecting the mutants and alkylated wtMIF to the enzymatic assays. Mutant C60S did not have any enzymatic activity while mutant C57S had a reduced activity. Thiol-modified wtMIF that was alkylated under oxidizing conditions was found to have full enzymatic activity, whereas alkylation of wtMIF under reducing conditions completely eliminated MIF-mediated redox activity. Importantly, further physiological relevance of the disulfide motif was obtained by examining the mutants and alkylated MIF in an immunological assay that involved the macrophage-activating properties of MIF. In this test, mutant C60S was essentially inactive and mutant C57S was partly active, indicating together that at least some of the cytokine-like biological activities of MIF are dependent on the presence of cysteine 57 and 60. Again, use of the alkylated MIF species confirmed the role of the cysteine motif for this MIF activity. In conclusion, our results argue (a) that MIF exhibits enzymatic oxidoreductase activity, (b) that this activity is dependent on the presence of the catalytic center that is formed by cysteine residues 57 and 60, and (c) that certain MIF-mediated immune processes are due to the cysteine-mediated redox mechanism.

Animals↗

PubMed1998

Contribution of advanced glycosylation to the amyloidogenicity of islet amyloid polypeptide.

The formation of amyloid within the islets of Langerhans is associated with the development of type II diabetes mellitus and occurs by the aggregation and insolubilization of islet amyloid polypeptide (IAPP). Recent in vitro studies suggest that amyloid formation follows a nucleation-dependent polymerization mechanism, i.e. aggregation is initiated by pre-formed aggregates or nucleation seeds. Modification of the Alzheimer's disease amyloid peptide by advanced glycosylation end products (AGEs), which form spontaneously by the non-enzymatic addition of glucose to protein amino groups, has been shown to enhance peptide aggregation in vitro. To explore the possibility that AGEs contribute to islet amyloid formation, we prepared AGE-modified IAPP (AGE-IAPP) in vitro and studied its properties by biochemical and biophysical techniques. AGE modification induced the formation of high-molecular-mass IAPP aggregates and amyloid formation was demonstrated by Congo red green-gold birefringence and by the presence of a characteristic fibrillar structure by electron microscopy. AGE-IAPP also showed an increase in cytotoxicity toward the astroglioma cell line HTB14. When added to soluble IAPP, AGE-IAPP seeds accelerated IAPP aggregation and abolished the nucleation period required for the polymerization of unseeded IAPP. Circular dichroism spectropolarimetry indicated that AGE-IAPP seeds may act as a template to stabilize the beta-sheet conformation of IAPP, thereby promoting its aggregation. Our studies demonstrate that AGE modification of IAPP results in high-molecular mass, fibrillar amyloid structures that nucleate IAPP amyloid formation and suggest a model for intra-islet amyloid deposition that may occur by the progressive advanced glycosylation of IAPP in vivo.

Amyloid↗

PubMed1998

Understanding the mechanism of the zinc-ion stains of biomacromolecules in electrophoresis gels: generalization of the reverse-staining technique.

We have recently shown that a few nanograms of protein separated by electrophoresis in sodium dodecyl sulfate-polyacrylamide gels can be detected by reverse-staining, exploiting the precipitation reaction between zinc(II) and imidazole. Modifications of this method have also been generated to detect gel-isolated nucleic acids and bacterial glycolipids. Because there is no recourse to chemical modifiers, the reverse-staining technique has been valuable when micropreparing these biomacromolecules for later use or characterization. The mechanism underlying the reverse-staining effect, however, remains incompletely understood and this has prevented a further generalization of the technique. Here, we have conducted physicochemical experiments and identified zinc imidazolate (ZnIm2) as the main component of the precipitate that forms along the surface of zinc-imidazole reverse-stained gels. Many staining effects observed when gels containing electrophoretically separated biopolymers are subjected to zinc-imidazole stains have been rationalized. The reverse-staining method has been vastly generalized, now allowing the detection of proteins and glycolipids as well as complexes of these macromolecules in native gels. We demonstrate the application of the reverse-staining technique in situations where Coomassie blue or silver staining was inappropriate or failed to produce detection of the species of interest. The present generalization of the reverse-staining method facilitated the characterization of biomacromolecular interaction partners in mixtures of bacterial glycolipids and human tears.

Bacteria↗

PubMed1997

The interaction of HIV-1 Tat(32-72) with its target RNA: a fluorescence and nuclear magnetic resonance study.

We performed intrinsic peptide fluorescence experiments to characterize the interaction between variants of the HIV-1 Tat(32-72) peptide BP1 and TAR RNA. Kd values for wild-type BP1 and cysteine-modified BP1 were found to be in the range of 60 to 70 nM for both peptides, indicating that free sulfhydryl groups of the cysteines within the peptide are not required for high affinity TAR binding. Thus, the mutant peptide BP1 (C34S, C37W) (BP1SW) was used to further investigate peptide RNA interaction by fluorescence studies. Titration of BP1SW with TAR resulted in a dissociation constant (Kd = 9 nM) nearly an order of magnitude lower than that of the wild-type peptide. The change of the BP1SW fluorescence intensity on TAR binding was used to investigate the kinetics of this interaction by stopped-flow experiments. The results can be explained in terms of a two-step binding model, with a rapid diffusion-limited initial formation of a tight, but unspecific peptide RNA complex, followed by a relatively slow structural rearrangement step (k approximately 60 s-1) in order to form the specific BP1SW-TAR complex. Comparison of heteronuclear two-dimensional NMR spectra of BP1SW and BP1SW bound to TAR shows that only resonances from amino acid residues of the core and basic sequence regions are shifted on TAR binding.

Amino Acid Sequence↗

PubMed1997

Spectroscopic investigations of HIV-1 trans-activator and related peptides in aqueous solutions.

The 86 amino acid trans-activator (Tat) protein of human immunodeficiency virus type 1 (HIV-1) is an RNA-binding transcriptional regulator. HIV-1 Tat proteins (wild type and Thr40Lys mutant) and the HIV-1 Tat peptide fragments Tat(32-48) and Tat(32-72) were chemically synthesized. One- and two-dimensional nuclear magnetic resonance spectroscopy experiments were performed to elucidate the structural features of these proteins. In fluorescence quenching studies of the full-length Tat protein (Thr40Lys), Trp11 was found to be only partially protected against solvent accessibility. Circular dichroism melting studies monitored a slight cooperative change in the conformation of the Tat with increasing temperature. Backbone NH protons of amino acids located in the main core element of the protein are partially protected against exchange.

Amino Acid Sequence↗

PubMed1996

Structural studies of the equine infectious anemia virus trans-activator protein.

Trans-activator (tat) proteins are necessary components for the completion of the T replication cycle of lentiviruses. The three-dimensional structure of the equine infectious anemia virus (EIAV) tat protein (e-tat) was studied with CD spectroscopy, NMR spectroscopy, and restrained molecular-dynamics calculations. No stable elements of regular secondary structure were detected, but the sequence regions responsible for nucleic acid binding showed helix-forming tendency, e-tat exhibits a flexible tertiary structure, and only the amino acids comprising the core sequence region form a well-defined tertiary fold. The three-dimensional structure allows discussion of biochemical data as well as data from molecular biological investigations of lentiviral tat proteins.

Amino Acid Sequence↗

PubMed1996

Secondary structure and tertiary fold of the birch pollen allergen Bet v 1 in solution.

Bet v 1 is the major birch pollen allergen and therefore the main cause of type I allergies observed in early spring. It is composed of 159 amino acid residues adding up to a molecular mass of 17 kDa. We determined the secondary structure and tertiary fold of full-length Bet v 1 by NMR spectroscopy. Two- and three-dimensional NMR measurements suggest that Bet v 1 is a globular monomer in solution with a high content of well defined secondary structure. Of the total of 159 residues, 135 could be sequentially assigned, using an improved assignment strategy based mainly on heteronuclear experiments. An improved strategy for structure calculation revealed three helices and two beta-sheets as major elements of secondary structure. The globular tertiary structure is mainly stabilized by two antiparallel beta-sheets. The two helices at the C terminus are in accordance with the results from the solution structure of the chemically synthesized peptide Bet v 1-(125-154). This peptide is composed of two helices connected by a hinge. The structural features of Bet v 1 are highly similar to those found in the Ambrosia allergen Amb t V.

Allergens↗

PubMed1996

Structural rearrangements on HIV-1 Tat (32-72) TAR complex formation.

Expression of the early genes of the human immunodeficiency virus type-I (HIV-1) genome is under the control of a trans-activator (Tat) protein. HIV-1 Tat action requires binding to TAR (trans-activation responsive element), an RNA sequence located at the 5'-end of all lentiviral mRNAs. We used various spectroscopic methods to investigate conformational changes on HIV-1 TAR binding to the HIV-1 (32-72) Tat peptide BP1. It comprises the RNA binding region and binds specifically to TAR. We conclude from our experiments that the regular A-form of the TAR RNA is slightly distorted towards the B-form when bound to BP1. Thus, the major groove is widened and the binding of BP1 facilitated. BP1 presumably adopts an extended conformation when binding to TAR and may fit well into the TAR major groove.

Amino Acid Sequence↗

PubMed1996

Photoaffinity labeling of the head-activator receptor from hydra.

A photoaffinity ligand for the head-activator (HA) receptor from hydra was synthesized using solid-phase peptide synthesis and coupling of two HA peptides over their epsilon-amino groups of Lys7 with succinimidyl esters. The new ligand, Bpa-HA-HA bipeptide, contains one normal HA peptide and another where p-benzoylphenylalanine (Bpa) was added at the amino terminus to allow ultraviolet activation and Tyr11 instead of Phe11 for radioiodination. The 125I-Bpa-HA-HA bipeptide bound with nanomolar affinity to the HA receptor from the multiheaded mutant of Chlorohydra viridissima as measured in a filter assay. After photoaffinity labeling of the hydra membrane fraction, a 200-kDa band was detected using reducing or non-reducing SDS/PAGE and autoradiography. Unlabeled HA derivatives, but no other neuropeptides, inhibited the labeling. Competition experiments with HA-HA homobipeptide in the nanomolar range indicate that predominantly the low-affinity and not the high-affinity HA receptor was photolabeled. Further evidence that the labeled molecule is the HA receptor comes from specific photoaffinity labeling with a second ultraviolet-activatable ligand containing p-nitrophenylalanine. The HA receptor could be functionally solubilized with Triton X-100 or Chaps. In the solubilizate the 200-kDa HA receptor was photolabeled specifically by both ligands. Liquid-phase isoelectric focussing of the solubilizate indicated a pI of about 5.4 of the photolabeled molecule. After chemical deglycosylation with trifluoromethanesulfonic acid, the apparent molecular mass of the labeled molecule was decreased to 180 kDa, indicating that the receptor is glycosylated.

Affinity Labels↗

PubMed1996

Cloning, high-yield expression in Escherichia coli, and purification of biologically active HIV-1 Tat protein.

We have established an expression system for full-length HIV-1 transactivator (Tat) protein in Escherichia coli. By constructing a synthetic gene for high level expression in enteric bacteria, the recombinant protein can be obtained in high yield. Fusion of the Tat sequence to an N-terminal histidine tag allows the rapid purification of the fusion protein through a single chromatographic step. After cleavage of the fusion protein with CNBr, pure Tat can be obtained through the use of a MonoS column. Reduction of the protein with Tris(2-carboxyethyl)phosphine-HCl and subsequent stepwise refolding yields biologically active Tat. Sample purity and the identity of the protein mass with the mass expected from the amino acid sequence was demonstrated by mass spectrometry. Nuclear magnetic resonance spectroscopy showed the identity of bacterially expressed and chemically synthesized Tat protein (P. Bayer et al., 1995, J. Mol. Biol. 247, 529-535). The expression of Tat in E. coli enables isotope labeling as a prerequisite for multidimensional NMR experiments toward the elucidation of the structure of the Tat-trans-activation response element complex.

Amino Acid Sequence↗

PubMed1995

Structure-activity relationship of the leucine-based sorting motifs in the cytosolic tail of the major histocompatibility complex-associated invariant chain.

The cytosolic tail of the major histocompatibility complex-associated invariant chain protein contains two Leu-based motifs that both mediate efficient sorting to the endocytic pathway. Nuclear magnetic resonance data on a peptide of 27 residues corresponding to the cytosolic tail of human invariant chain indicate that in water at pH 7.4 the membrane distal motif Leu7-Ile8 lies within a nascent helix, while the membrane proximal motif Met16-Leu17 is part of a turn. The presence of a small amount of methanol stabilizes an alpha helix from Gln4 to Leu17 with a kink on Pro15. Point mutations of the cytosolic tail of the protein suggest that amino-terminal residues located in spatial proximity to the Leu motifs contribute to efficient internalization and targeting to endosomes in transfected COS cells. Residues on the spatially opposite side of the Leu motifs were, on the other hand, mutated with no measurable effect on targeting. Structural and biological data thus suggest that the signals are not continuous but consist of "signal patches" formed by the three-dimensional structure of the cytosolic tail of invariant chain.

Amino Acid Sequence↗

PubMed1995

Structure of amyloid A4-(1-40)-peptide of Alzheimer's disease.

One of the principle peptide components of the amyloid plaque deposits of Alzheimer's disease in humans is the 40-amino-acid peptide beta-amyloid A4-(1-40)-peptide. The full-length A4-(1-40)-peptide was chemically synthesized and the solution structure determined by two-dimensional nuclear magnetic resonance spectroscopy and restrained molecular-dynamics calculations. Synthetic human A4-(1-40)-peptide was soluble and non-aggregating for several days in 40% (by vol.) trifluoroethanol/water. All spin systems could be unambiguously assigned, and a total of 203 sequential and medium-range cross-peaks were found in the NOESY (nuclear Overhauser enhancement spectroscopy) spectrum. Long-range NOE cross-peaks that would indicate tertiary structure of the peptide were absent. The main secondary-structure elements found by chemical-shift analysis, sequential and medium-range NOESY data, and NOE-based restrained molecular-dynamics calculations were two helices, Gln15-Asp23 and Ile31-Met35, whereas the rest of the peptide was in random-coil conformation. A similar secondary structure is suggested for the aggregation part of prions, the postulated causative agents of the transmissible spongiform encephalopathy. The sequence of the helical part of prion proteins was observed to be remarkably similar to the sequence of the helical part of human A4-(1-40)-peptide.

Alzheimer Disease↗

PubMed1994

Pheromones trigger filamentous growth in Ustilago maydis.

Cell recognition and mating in the smut fungus Ustilago maydis have been proposed to involve specific pheromones and pheromone receptors. The respective structural genes are located in the a mating type locus that exists in the alleles a1 and a2. We demonstrate that binding of pheromone to the receptor can induce a morphological switch from yeast-like to filamentous growth in certain strains. Using this as biological assay we were able to purify both the a1 and a2 pheromone. The structure of the secreted pheromones was determined to be 13 amino acids for a1 and nine amino acids for a2. Both pheromones are post-translationally modified by farnesylation and carboxyl methyl esterification of the C-terminal cysteine. An unmodified a1 peptide exhibits dramatically reduced activity. The pheromone alone is able to induce characteristic conjugation tubes in cells of opposite mating type and confers mating competence; even cells of the same mating type undergo fusion. We discuss the role of pheromones in initiating filamentous growth and pathogenic development.

Amino Acid Sequence↗

PubMed1994

Human immunodeficiency virus type 1 Tat upregulates interleukin-2 secretion in activated T cells.

Dysregulation of cytokines secreted by T cells may play an important role in the pathogenesis of AIDS. To investigate the effects of human immunodeficiency virus type 1 (HIV-1) Tat on interleukin-2 (IL-2) expression, we used IL-2 promoter-chloramphenicol acetyltransferase constructs and IL-2-secreting Jurkat T cells as a model system. Transient expression of HIV-1 Tat induced a five- to eightfold increase in IL-2 promoter activity in Jurkat T cells stimulated with phytohemagglutinin and phorbol myristate acetate. IL-2 secretion was increased more than twofold in both Jurkat T cells and primary T cells stimulated by extracellular HIV-1 Tat protein. Analysis of mRNA suggested that Tat exerts its effect on IL-2 primarily at the transcriptional level. The NF-kappa B site at positions -206 to -195 of the IL-2 promoter was required but not sufficient for the Tat effect. The Tat-mediated increase in IL-2 promoter activity could selectively be blocked by antisense tat or-unlike the analogous effect of human T-cell lymphotropic virus type 1 Tax-by cyclosporin A. The observed increase in IL-2 levels might facilitate virus spread from or to T cells. Furthermore, it might contribute to the hypergammaglobulinemia or, together with other cytokines found to be dysregulated, the T-helper cell dysfunctions observed in AIDS patients.

Base Sequence↗

PubMed1992

Purification and properties of prostaglandin 9-ketoreductase from pig and human kidney. Identity with human carbonyl reductase.

Prostaglandin 9-ketoreductase (PG-9-KR) was purified from pig kidney to homogeneity, as judged by SDS/PAGE using an improved procedure. The enzyme is pro-S stereoselective with regard to hydrogen transfer from NADPH with prostaglandin E2 as substrate and reduces its 9-keto group with approximately 90% stereoselectivity to form prostaglandin F2 alpha. Approximately 8% of the prostaglandin F formed has the beta-configuration. In addition to catalyzing the interconversion of prostaglandin E2 to F2 alpha, PG-9-KR also oxidizes prostaglandin E2, F2 alpha and D2 to their corresponding, biologically inactive, 15-keto metabolites. Incubation of PG-9-KR with prostaglandin F2 alpha and NAD+ leads to the preferential formation of 15-keto prostaglandin F2 alpha rather than prostaglandin E2. This suggests that the prostaglandin E2/prostaglandin F2 alpha ratio is not determined by the NADP+/NADPH redox couple. The enzyme also reduces various other carbonyl compounds (e.g. 9,10-phenanthrenequinone) with high efficiency. The catalytic properties measured for PG-9-KR suggest that its in vivo function is unlikely to be to catalyze formation of prostaglandin F2 alpha. The monomeric enzyme has a molecular mass of 32 kDa and exists as four isoforms, as judged by isoelectric focusing. PG-9-KR contains 1.9 mol Zn2+/mol enzyme and no other cofactors. Human kidney PG-9-KR was also purified to homogeneity. The human enzyme has a molecular mass of 34 kDa and also exists as four isoforms. Polyclonal antibodies raised against pig kidney PG-9-KR cross-react with human kidney PG-9-KR and also with human brain carbonyl reductase, as demonstrated by Western blot analysis. Sequence data of tryptic peptides from pig kidney PG-9-KR show greater than 90% identity with human placenta carbonyl reductase. From comparison of several properties (catalytical, structural and immunological properties), it is concluded that PG-9-KR and carbonyl reductase are identical enzymes.

Alcohol Oxidoreductases↗

PubMed1992

The rat poly pyrimidine tract binding protein (PTB) interacts with a single-stranded DNA motif in a liver-specific enhancer.

We characterized and purified a protein from rat liver which specifically binds to a DNA motif present in a liver-specific enhancer of the rat tyrosine aminotransferase (TAT) gene, when offered as single-stranded DNA. Binding is highly sequence-specific and coincides with a region known to be essential for function of the enhancer. Microsequencing revealed that this protein is the rat homologue of the mouse and human poly Pyrimidine Tract binding protein (PTB), which has been shown to bind to premRNA and may participate in RNA splicing. This finding was corroborated by subsequent Western blot experiments using a PTB-specific antibody. These findings indicate a possible dual role for this protein in RNA processing and transcription.

Amino Acid Sequence↗

PubMed1992

Immunological characterization of an immunomodulatory epitope in S-antigen/arrestin with a sequence motif common to tumor necrosis factor alpha.

Some monoclonal antibodies (mAbs) to retinal S-antigen recognize a phylogenetically conserved epitope (S2) in the N-terminal part of the protein. These antibodies have been shown to inhibit the induction of experimental autoimmune uveoretinitis by S-antigen in rats. Using Pepscan method, we localized this epitope on the amino acid (aa) residues 40-50, i.e., PVDGVVLVDPE (peptide S2). MAb binding was confirmed by ELISA, competition-ELISA and dot blot. Other S-antigen peptides with homologies to epitope S2 and peptides exhibiting the pathogenic and T-cell proliferation inducing sites did not bind these mAbs. Epitope S2 displays an immunological crossreactivity with human tumor necrosis factor (TNF) alpha. Recent results indicate that both peptide S2 and a peptide from human TNF alpha (aa residues 31-53) containing the common sequence motif GVxLxD induce TNF alpha production in monocytes. We analyzed the fine structure of the common epitope by studying mAb binding in an amino acid residue exchange experiment.

Amino Acid Sequence↗

PubMed1992

A common epitope on human tumor necrosis factor alpha and the autoantigen 'S-antigen/arrestin' induces TNF-alpha production.

A common epitope on S-antigen (arrestin), a potent autoantigen inducing experimental autoimmune uveoretinitis (EAU), and on human tumor necrosis factor alpha (hTNF alpha) was revealed using two monoclonal antibodies to S-antigen which inhibit EAU induction. The minimal common sequence for monoclonal antibody recognition is GVxLxD in the S-antigen/hTNF alpha amino acid sequences. Peptides containing this sequence motif exhibited monocyte activating capacity similar to the autocrine stimulatory capacity of hTNF alpha itself. In the S-antigen this activity was located from residue 40 to 50, corresponding to the peptide PVDGVVLVDPE (epitope S2). In hTNF alpha, the monocyte activating capacity correlated to residue 31 to 53, corresponding to the peptide RRANALLANGVELRDNQLVVPSE (peptide RRAN). The identified regions define common functional structures in the autoantigen and in the hTNF alpha molecule. The data suggest a regulatory function of this particular structure in TNF alpha expression and in autoimmunity.

Amino Acid Sequence↗