Biomedical subjects
Publications and source records attributed to S J Horvath.
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Two-dimensional NMR (2DNMR) studies of several different zinc finger peptides have yielded a picture of the three-dimensional structure of this small DNA-binding motif. Details of the differences among fingers with different sequences may provide some insight into how these domains interact with DNA. Toward this end, we have reanalyzed the 2DNMR spectra of the C-terminal zinc finger sequence from the yeast transcriptional factor ADR1. Although this was the sequence on which our original report describing the overall fold of zinc fingers was based, complete spectral assignments (reported here) were needed to compare this sequence in detail with that of ADR1b, for which we have reported an atomic level structure. In the process of analyzing the spectra of ADR1a and a mutant of ADR1a, it was noted that the peptides give two sets of NMR lines, indicating that this sequence, unlike the other ADR1 zinc finger sequence, exists in two slowly interconverting folded conformations in solution. Residues that exhibit peak doubling are located in the Cys loop, the alpha-helix, and the extreme C-terminus of the peptide. Differences in NOEs observed for the two forms indicate that there are detectable conformational differences in the Zn2+ cluster and in the fingertip region. This conformational flexibility, which has not been observed for other zinc finger peptides, may stem from the presence of an additional residue between the histidine ligands (His-X4-His versus His-X3-His).
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Incorporation of the DNA-cleaving moiety EDTA.Fe at discrete amino acid residues along a DNA-binding protein allows the positions of these residues relative to DNA bases, and hence the organization of the folded protein, to be mapped by high-resolution gel electrophoresis. A 52-residue protein, based on the sequence-specific DNA-binding domain of Hin recombinase (139-190), with EDTA at the NH2 terminus cleaves DNA at Hin recombination sites. The cleavage data for EDTA-Hin(139-190) reveal that the NH2 terminus of Hin(139-190) is bound in the minor groove of DNA near the symmetry axis of Hin-binding sites [Sluka, J. P., Horvath, S. J., Bruist, M. F., Simon, M. I., & Dervan, P. B. (1987) Science 238, 1129]. Six proteins, varying in length from 49 to 60 residues and corresponding to the DNA-binding domain of Hin recombinase, were synthesized by solid-phase methods: Hin(142-190), Hin(141-190), Hin(140-190), Hin(139-190), Hin(135-190), and Hin(131-190) were prepared with and without EDTA at the NH2 termini in order to test the relative importance of the residues Gly139-Arg140-Pro141-Arg142, located near the minor groove, for sequence-specific recognition at five imperfectly conserved 12-base-pair binding sites. Footprinting and affinity cleaving reveal that deletion of Gly139 results in a protein with affinity and specificity similar to those of Hin(139-190) but that deletion of Gly139-Arg140 affords a protein with altered affinities and sequence specificities for the five binding sites. It appears that Arg140 in the DNA-binding domain of Hin is important for recognition of the 5'-AAA-3' sequence in the minor groove of DNA. Our results indicate modular DNA and protein interactions with two adjacent DNA sites (major and minor grooves, respectively) bound on the same face of the helix by two separate parts of the protein.
The "zinc finger" is a 30-residue repeating motif that has been identified in a variety of eukaryotic transcription factors. Each domain is capable of binding a Zn2+ ion through invariant Cys and His residues. We have determined the three-dimensional structure of a synthetic peptide that corresponds to one of the two zinc finger domains in the yeast transcription factor ADR1, using two-dimensional nuclear magnetic resonance spectroscopy. The Zn2(+)-bound structure of the peptide consists of a loop containing the two Cys residues, a "fingertip," a 12- to 13-residue alpha-helix containing the two His residues, and a C-terminal tail. A majority of the interresidue contacts observed involve the seven conserved residues of the prototypic zinc finger (i.e., the four zinc ligands and the three hydrophobic residues), indicating that these residues are largely responsible for the three-dimensional structure of the domain and that all the zinc finger domains of the TFIIIA class will have similar structures. Potential DNA-binding residues are found throughout the structure, with the highest concentration of such residues on the external face of the alpha-helix.
Myelin basic protein (MBP) or helper T cells reactive against MBP induce an autoimmune disease, experimental allergic encephalomyelitis, in B10.PL and PL/J inbred mice. In both strains, virtually the entire repertoire of MBP-specific T cells recognize an N-terminal peptide fragment in the context of the I-Au molecule encoded by the major histocompatibility complex (MHC) and utilize a very limited set of T-cell receptor genes. To delineate the nature of the trimolecular complex, consisting of the T-cell receptor, MBP-peptide fragment, and MHC molecule (I-Au), we have synthesized 13 variants of the 9-mer N-terminal immunodominant peptide differing at residue 4 and studied their immune recognition in vitro and in vivo. These substitutions have a striking range of effects on T-cell activation, ability to bind to the MHC molecule, and initiation of immune responses in vivo. An understanding of the autoimmune peptide/MHC/T-cell receptor interactions allowed us to design variant 9-mer peptides that have high affinity for an MHC molecule and are effective in blocking experimental allergic encephalomyelitis, possibly through two distinct mechanisms, peripheral T-cell tolerance and the inhibition of binding of the encephalitogenic peptide to the MHC molecules.
Pulmonary hypertension occurs after the intravascular activation of complement. However, it is unclear which activated complement fragments are responsible for the pulmonary vascular constriction. We investigated the 21-carboxy-terminal peptide of C3a (C3a57-77) to see if it would cause pulmonary vascular constriction when infused into isolated buffer-perfused rat lungs. Injection of C3a57-77 (225 to 450 micrograms) caused mean pulmonary arterial pressure (Ppa) to rapidly increase. However, the response was transient, with Ppa returning to baseline within 10 min of its administration. C3a57-77 also resulted in an increase in lung effluent thromboxane B2 (TXB2), concomitant with the peak increase in Ppa. C3a57-77 did not affect the amount of 6-keto-PGF1 alpha in the same effluent samples. Indomethacin inhibited the C3a57-77-induced pulmonary artery pressor response and the associated TXB2 production. Indomethacin also decreased lung effluent 6-keto-PGF1 alpha. The thromboxane synthetase inhibitors CGS 13080 and U63,357 inhibited the C3a57-77-induced pulmonary artery pressor response and TXB2 production without affecting 6-keto-PGF1 alpha. These inhibitors did not inhibit pulmonary artery pressor responses to angiotensin II. Tachyphylaxis to C3a57-77 occurred because a second dose of C3a57-77 administered to the same lung failed to cause a pulmonary artery pressor response or TXB2 production. The loss of the pressor response was not due to a C3a57-77-induced decrease in pulmonary vascular responsiveness because pressor responses elicited by angiotensin II were not altered by lung contact with C3a57-77. Thus, C3a57-77 caused thromboxane-dependent pulmonary vascular constriction in isolated buffer perfused rat lungs.
Experimental autoimmune encephalomyelitis (EAE) results from T helper (TH) cell recognition of myelin basic protein (MBP). We have characterized TH cell reactivity in B10.PL and PL/J (H-2u) mice to 39 N-terminal MBP peptide derivatives of different lengths and with individual amino acid substitutions. The peptide determinant of murine MBP can be divided into a minimal stimulatory core region (residues 1-6) and a tail region (residues 7-20) that alters the structure of the core region to affect both T cell recognition and MHC binding. Core recognition by B10.PL and PL/J mice is highly similar but in one case strain dependent. Peptide analogs that do not stimulate MBP-specific TH cells but bind to the I-Au molecule competitively inhibit T cell reactivity to MBP in vitro and prevent the induction of EAE in vivo.
In the proposed "zinc finger" DNA-binding motif, each repeat unit binds a zinc metal ion through invariant Cys and His residues and this drives the folding of each 30-residue unit into an independent nucleic acid-binding domain. To obtain structural information, we synthesized single and double zinc finger peptides from the yeast transcription activator ADR1, and assessed the metal-binding and DNA-binding properties of these peptides, as well as the solution structure of the metal-stabilized domains, with the use of a variety of spectroscopic techniques. A single zinc finger can exist as an independent structure sufficient for zinc-dependent DNA binding. An experimentally determined model of the single finger is proposed that is consistent with circular dichroism, one- and two-dimensional nuclear magnetic resonance, and visual spectroscopy of the single-finger peptide reconstituted in the presence of zinc.
Experimental allergic encephalomyelitis (EAE) is a paralytic autoimmune disease induced in susceptible animals by active immunization with myelin basic protein (MBP) or by passive transfer of MBP-specific T helper (TH) lymphocytes. We have analyzed the T cell receptor genes of 33 clonally distinct TH cells specific for a nonapeptide of MBP inducing EAE in B10.PL (H-2u) mice. All 33 TH cells used two alpha variable gene segments (V alpha 2.3, 61%; V alpha 4.2, 39%), the same alpha joining gene segment (J alpha 39), and two V beta and J beta gene segments (V beta 8.2-J beta 2.6, 79%; V beta 13-J beta 2.2, 21%). The anti-V beta 8 monoclonal antibody F23.1 was found to block completely recognition of the nonapeptide by V beta 8 TH cells in vitro and to reduce significantly the susceptibility of B10.PL mice to peptide-induced EAE.
Experimental allergic encephalomyelitis (EAE) is an autoimmune demyelinating disease of the central nervous system (CNS) that occurs after immunization of animals with myelin basic protein (MBP). The disease is a prototype model for the study of antigen-specific T helper cell-mediated autoimmune disease. In SJL/J mice, EAE is mediated by T helper cells directed against a 40-amino acid COOH-terminal peptic fragment of mouse small MBP. To identify the minimal T cell epitopes of MBP responsible for EAE, overlapping peptides completely encompassing the epitopes within this region were synthesized. A 28-residue peptide of mouse MBP spanning residues 87-114 (pM87-114) was able to elicit both a strong T cell response and chronic relapsing disease. To better localize the T cell epitopes, shorter peptides within this region were synthesized and two overlapping peptides, pM87-98 and pM91-104, were able to induce EAE. T cell clones and bulk lymph node cell populations reactive with pM87-98 did not respond to pM91-104. However, lymph node cells reactive with pM91-104 also reacted with pM87-98, thus showing that these two peptides represent contiguous, but distinct encephalitogenic epitopes and that both these epitopes may be contained within pM87-98. In addition, pM87-114 and pM87-98 were found to be minor determinants of the total T cell response to rat and rabbit MBP. The restricted response to MBP in SJL/J mice is similar to that of the PL/J mice in that each appears to have only a single peptide region in MBP that elicits encephalitogenic T cells. However, within the region studied, there were two if not more T cell epitopes. This differs from the single encephalitogenic PL/J epitope. These findings of a single encephalitogenic peptide region with multiple T cell epitopes and the fact that encephalitogenic T cell epitopes may be subdominant have implications for the design of treatments directed at the T cell receptor-MHC-peptide epitope complex in autoimmune disease.
We have shown earlier that a mitochondrial presequence peptide can form an amphiphilic helix. However, the importance of amphiphilicity for mitochondrial presequence function became doubtful when an artificial presequence, designed to be non-amphiphilic, proved to be active as a mitochondrial import signal. We now show experimentally that this 'non-amphiphilic' presequence peptide is, in fact, highly amphiphilic as measured by its ability to insert into phospholipid monolayers and to disrupt phospholipid vesicles. This result, and similar tests on three additional artificial presequences (two functionally active and one inactive), revealed that all active presequences were amphiphilic whereas the inactive presequence was non-amphiphilic. One of the active presequence peptides was non-helical in solution and in the presence of detergent micelles. We conclude that amphiphilicity is necessary for mitochondrial presequence function whereas a helical structure may not be essential.
A synthetic 52-residue peptide based on the sequence-specific DNA-binding domain of Hin recombinase (139-190) has been equipped with ethylenediaminetetraacetic acid (EDTA) at the amino terminus. In the presence of Fe(II), this synthetic EDTA-peptide cleaves DNA at Hin recombination sites. The cleavage data reveal that the amino terminus of Hin(139-190) is bound in the minor groove of DNA near the symmetry axis of Hin recombination sites. This work demonstrates the construction of a hybrid peptide combining two functional domains: sequence-specific DNA binding and DNA cleavage.
The Hin recombinase binds to specific sites on DNA and mediates a recombination event that results in DNA inversion. In order to define the DNA-binding domain of the Hin protein two peptides 31 and 52 amino acids long were synthesized. Even though the 31mer encompassed the sequence encoding the putative helix-coil-helix-binding domain, it was not sufficient for binding to the 26-base pair DNA crossover site. However, the 52mer specifically interacted with the site and also effectively inhibited the Hin-mediated recombination reaction. The 52mer bound effectively to both the 26-base pair complete site and to a 14-base pair "half site." Nuclease and chemical protection studies with the 52mer helped to define the DNA base pairs that contributed to the specificity of binding. The synthetic peptide provides opportunities for new approaches to the study of the nature of protein-DNA interaction.
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Subunit IV of yeast cytochrome oxidase is made in the cytoplasm with a transient pre-sequence of 25 amino acids which is removed upon import of the protein into mitochondria. To study the function of this cleavable pre-sequence in mitochondrial protein import, three peptides representing 15, 25 or 33 amino-terminal residues of the subunit IV precursor were chemically synthesized. All three peptides were freely soluble in aqueous buffers, yet inserted spontaneously from an aqueous subphase into phospholipid monolayers up to an extrapolated limiting monolayer pressure of 40-50 mN/m. The two longer peptides also caused disruption of unilamellar liposomes. This effect was increased by a diffusion potential, negative inside the liposomes, and decreased by a diffusion potential of opposite polarity. The peptides, particularly the two longer ones, also uncoupled respiratory control of isolated yeast mitochondria. The 25-residue peptide had little secondary structure in aqueous buffer but became partly alpha-helical in the presence of detergent micelles. Based on the amino acid sequence of the peptides, a helical structure would have a highly asymmetric distribution of charged and apolar residues and would be surface active. Amphiphilic helicity appears to be a general feature of mitochondrial pre-sequences. We suggest that this feature plays a crucial role in transporting proteins into mitochondria.
A leucine transfer RNA has been transformed into a serine transfer RNA by changing 12 nucleotides. This result indicates that a limited set of residues determine tRNA identity.