Identification of an immunodominant and highly immunopathogenic determinant in the retinal interphotoreceptor retinoid-binding protein (IRBP).

Interphotoreceptor retinoid-binding protein (IRBP), a glycoprotein specific for the retina and pineal gland, induces inflammatory changes in these two organs in immunized animals. We report here on the identification of an immunodominant determinant of bovine IRBP that is highly immunogenic and immunopathogenic in the Lewis rat. The peptide, which comprises the sequence 1169-1191 of bovine IRBP, was shown to be immunodominant by its capacity to stimulate lymphocytes sensitized against whole IRBP. A comparison was made between peptide 1169-1191 and another peptide, 1158-1180, which is nondominant but is immunogenic and immunopathogenic in the Lewis rat. Peptide 1169-1191 was found to be superior in its immunological capacities; the minimal dose of 1169-1191 needed to induce cellular immune response or disease in Lewis rats (0.02-0.1 nmol/rat) is congruent to 1,000 times smaller than that of 1158-1180. In addition, unlike the ocular disease induced by 1158-1180, the disease produced by 1169-1191 resembled that induced by whole IRBP in its kinetics and histopathological features. The immunological activity of 1169-1191 in the Lewis rat was localized to the 10 residues at the COOH terminus; no such activity was exhibited by the truncated peptide 1169-1188, which comprises the 20 residues at the NH2 terminus of the full peptide. The usefulness of this unique experimental system in analyzing the role of immunodominance in peptide immunogenicity and immunopathogenicity is underscored.

Construction of synthetic immunogen: use of new T-helper epitope on malaria circumsporozoite protein.

The circumsporozoite (CS) protein of Plasmodium falciparum is the focus of intense efforts to develop an antisporozoite malaria vaccine. Localization of sites for T-cell recognition on this molecule is critical for vaccine design. By using an algorithm designed to predict T-cell sites and a large panel of H-2 congenic mice, a major nonrepetitive T-cell site was located. When a synthetic peptide corresponding to this site was covalently linked to the major B-cell site on the molecule, an immunogen capable of eliciting a high-titer antibody response was formed. This peptide sequence could prime helper T cells for a secondary response to the intact CS protein. The new helper T-cell site is located outside the repetitive region of the CS protein and appears to be the immunodominant T site on the molecule. This approach should be useful in the rational design and construction of vaccines.

Novel small RNA-encoding genes in the intergenic regions of Escherichia coli.

BACKGROUND: Small, untranslated RNA molecules were identified initially in bacteria, but examples can be found in all kingdoms of life. These RNAs carry out diverse functions, and many of them are regulators of gene expression. Genes encoding small, untranslated RNAs are difficult to detect experimentally or to predict by traditional sequence analysis approaches. Thus, in spite of the rising recognition that such RNAs may play key roles in bacterial physiology, many of the small RNAs known to date were discovered fortuitously. RESULTS: To search the Escherichia coli genome sequence for genes encoding small RNAs, we developed a computational strategy employing transcription signals and genomic features of the known small RNA-encoding genes. The search, for which we used rather restrictive criteria, has led to the prediction of 24 putative sRNA-encoding genes, of which 23 were tested experimentally. Here we report on the discovery of 14 genes encoding novel small RNAs in E. coli and their expression patterns under a variety of physiological conditions. Most of the newly discovered RNAs are abundant. Interestingly, the expression level of a significant number of these RNAs increases upon entry into stationary phase. CONCLUSIONS: Based on our results, we conclude that small RNAs are much more widespread than previously imagined and that these versatile molecules may play important roles in the fine-tuning of cell responses to changing environments.

Harnessing the cellular immune system to the gene-prediction cart.

Prediction of genes and verification of their bona fide expression in the cell are major challenges of the post-genomic era. Here, we demonstrate how information from the apparently unrelated field of cellular immunology can be recruited for these challenging tasks. The cellular immune system presents short peptides that are the degradation products of both foreign and self-proteins expressed in the cell. We carried out a comprehensive search comparing these peptides to all accumulated human sequence data. Our findings illustrate how these 'presented self-peptides' are informative for the identification of new genes, for hypothetical gene verification, for verifying gene expression at the protein level and for supporting splice junctions.

PromEC: An updated database of Escherichia coli mRNA promoters with experimentally identified transcriptional start sites.

PromEC is an updated compilation of Escherichia coli mRNA promoter sequences. It includes documentation on the location of experimentally identified mRNA transcriptional start sites on the E. coli chromosome, as well as the actual sequences in the promoter region. The database was updated as of July 2000 and includes 472 entries. PromEC is accessible at http://bioinfo.md.huji.ac. il/marg/promec

Correlated sequence-signatures as markers of protein-protein interaction.

As protein-protein interaction is intrinsic to most cellular processes, the ability to predict which proteins in the cell interact can aid significantly in identifying the function of newly discovered proteins, and in understanding the molecular networks they participate in. Here we demonstrate that characteristic pairs of sequence-signatures can be learned from a database of experimentally determined interacting proteins, where one protein contains the one sequence-signature and its interacting partner contains the other sequence-signature. The sequence-signatures that recur in concert in various pairs of interacting proteins are termed correlated sequence-signatures, and it is proposed that they can be used for predicting putative pairs of interacting partners in the cell. We demonstrate the potential of this approach on a comprehensive database of experimentally determined pairs of interacting proteins in the yeast Saccharomyces cerevisiae. The proteins in this database have been characterized by their sequence-signatures, as defined by the InterPro classification. A statistical analysis performed on all possible combinations of sequence-signature pairs has identified those pairs that are over-represented in the database of yeast interacting proteins. It is demonstrated how the use of the correlated sequence-signatures as identifiers of interacting proteins can reduce significantly the search space, and enable directed experimental interaction screens.

Sequence signals for generation of antigenic peptides by the proteasome: implications for proteasomal cleavage mechanism.

Proteasomal cleavage of proteins is the first step in the processing of most antigenic peptides that are presented to cytotoxic T cells. Still, its specificity and mechanism are not fully understood. To identify preferred sequence signals that are used for generation of antigenic peptides by the proteasome, we performed a rigorous analysis of the residues at the termini and flanking regions of naturally processed peptides eluted from MHC class I molecules. Our results show that both the C terminus (position P1 of the cleavage site) and its immediate flanking position (P1') possess significant signals. The N termini of the peptides show these signals only weakly, consistent with previous findings that antigenic peptides may be cleaved by the proteasome with N-terminal extensions. Nevertheless, we succeed to demonstrate indirectly that the N-terminal cleavage sites contain the same preferred signals at position P1'. This reinforces previous findings regarding the role of the P1' position of a cleavage site in determining the cleavage specificity, in addition to the well-known contribution of position P1. Our results apply to the generation of antigenic peptides and bare direct implications for the mechanism of proteasomal cleavage. We propose a model for proteasomal cleavage mechanism by which both ends of cleaved fragments are determined by the same cleavage signals, involving preferred residues at both P1 and P1' positions of a cleavage site. The compatibility of this model with experimental data on protein degradation products and generation of antigenic peptides is demonstrated.

Conservation of salt bridges in protein families.

A detailed computational analysis is presented that focuses on the relationship between structural attributes and the degree and mode of salt bridge conservation. A data set of conserved and non-conserved salt bridges was constructed from eight protein families, based on the structural alignment of family members. Salt bridges were defined at the secondary structure level rather than at the residue level, implying different possible modes of conservation: preservation (same charges at the same residue positions), compensation (reversal of charges), and complementation (maintenance of a salt bridge between two segments of secondary structures, not involving the same residue positions). Structural attributes such as the surface accessibility, distance from the active site, or type of secondary structures involved, were studied. No significant differences were found between conserved and non-conserved salt bridges, except for the surface accessibility. Conserved salt bridges were shown to be less exposed than non-conserved ones. Moreover, within the set of conserved salt bridges, the degree of conservation was shown to negatively correlate with surface exposure; however, not to an extent that could indicate a general role for electrostatic interactions in the protein interior. Examination of the most conserved salt bridge in each family showed a variety of typical features: Some involved the terminal segments of the protein, some were buried and one involved the catalytic site of the protein. Hence, the role of salt bridges is more specific, probably in fine tuning of a specific structure through the folding process or in determining the functional site. As for the conservation mode, preservations were found to predominate in the conserved interactions, while complementations were of secondary importance. Compensations occurred only rarely and mostly in exposed salt bridges, suggesting that this mechanism is not utilized frequently and especially not in important interactions.

Ranking potential binding peptides to MHC molecules by a computational threading approach.

In this paper, an approach developed to address the inverse protein folding problem is applied to prediction of potential binding peptides to a specific major histocompatibility complex (MHC) molecule. Overlapping peptides, spanning the entire protein sequence, are threaded through the backbone coordinates of a known peptide fold in the MHC groove, and their interaction energies are evaluated using statistical pairwise contact potentials. With currently available tables for pairwise potentials, promising results are obtained for MHC-peptide complexes where hydrophobic interactions predominate. By ranking the peptides in an ascending order according to their energy values, it is demonstrated that, in most cases, known antigenic peptides are highly ranked. Furthermore, predicted hierarchies are consistent with experimental binding results. Currently, predictions of potential binding peptides to a specific MHC molecule are based on the identification of allele-specific binding motifs. However, it has been demonstrated that these motifs are neither sufficient nor strictly required to ensure binding. The computational procedure presented here succeeds in determining the MHC binding potential of peptides along a protein amino acid sequence, without relying on binding motifs. The proposed scheme may significantly reduce the number of peptides to be tested, identify good binders that do not necessarily show the known allele-specific binding motifs, and identify the best candidates among those with the motifs. In general, when structural information about a protein-peptide complex is available, the current application of the threading approach can be used to screen a large library of peptides for selection of the best binders to the target protein.

Comprehensive analysis of hydrogen bonds in regulatory protein DNA-complexes: in search of common principles.

A systematic analysis of hydrogen bonds between regulatory proteins and their DNA targets is presented, based on 28 crystallographically solved complexes. All possible hydrogen bonds were screened and classified into different types: those that involve the amino acid side-chains and DNA base edges and those that involve the backbone atoms of the molecules. For each interaction type, all bonds were characterized and a statistical analysis was performed to reveal significant amino acid-base interdependence. The interactions between the amino acid side-chains and DNA backbone constitute about half of the interactions, but did not show any amino acid-base correlation. Interactions via the protein backbone were also observed, predominantly with the DNA backbone. As expected, the most significant pairing preference was demonstrated for interactions between the amino acid side-chains and the DNA base edges. The statistically significant relationships could mostly be explained by the chemical nature of the participants. However, correlations that could not be trivially predicted from the hydrogen bonding potential of the residues were also identified, like the preference of lysine for guanine over adenine, or the preference of glutamic acid for cystosine over adenine. While Lys x G interactions were very frequent and spread over various families, the Glu x C interactions were found mainly in the basic helix-loop-helix family. Further examination of the side-chain-base edge contacts at the atomic level revealed a trend of the amino acids to contact the DNA by their donor atoms, preferably at position W2 in the major groove. In most cases it seems that the interactions are not guided simply by the presence of a required atom in a specific position in the groove, but that the identity of the base possessing this atom is crucial. This may have important implications in molecular design experiments.

A role for CH...O interactions in protein-DNA recognition.

The concept of CH...O hydrogen bonds has recently gained much interest, with a number of reports indicating the significance of these non-classical hydrogen bonds in stabilizing nucleic acid and protein structures. Here, we analyze the CH...O interactions in the protein-DNA interface, based on 43 crystal structures of protein-DNA complexes. Surprisingly, we find that the number of close intermolecular CH...O contacts involving the thymine methyl group and position C5 of cytosine is comparable to the number of protein-DNA hydrogen bonds involving nitrogen and oxygen atoms as donors and acceptors. A comprehensive analysis of the geometries of these close contacts shows that they are similar to other CH...O interactions found in proteins and small molecules, as well as to classical NH...O hydrogen bonds. Thus, we suggest that C5 of cytosine and C5-Met of thymine form relatively weak CH...O hydrogen bonds with Asp, Asn, Glu, Gln, Ser, and Thr, contributing to the specificity of recognition. Including these interactions, in addition to the classical protein-DNA hydrogen bonds, enables the extraction of simple structural principles for amino acid-base recognition consistent with electrostatic considerations.

Hydrophobicity scales and computational techniques for detecting amphipathic structures in proteins.

Protein segments that form amphipathic alpha-helices in their native state have periodic variation in the hydrophobicity values of the residues along the segment, with a 3.6 residue per cycle period characteristic of the alpha-helix. The assignment of hydrophobicity values to amino acids (hydrophobicity scale) affects the display of periodicity. Thirty-eight published hydrophobicity scales are compared for their ability to identify the characteristic period of alpha-helices, and an optimum scale for this purpose is computed using a new eigenvector method. Two of the published scales are also characterized by eigenvectors. We compare the usual method for detecting periodicity based on the discrete Fourier transform with a method based on a least-squares fit of a harmonic sequence to a sequence of hydrophobicity values. The two become equivalent for very long sequences, but, for shorter sequences with lengths commonly found in alpha-helices, the least-squares procedure gives a more reliable estimate of the period. The analog to the usual Fourier transform power spectrum is the "least-squares power spectrum", the sum of squares accounted for in fitting a sinusoid of given frequency to a sequence of hydrophobicity values. The sum of the spectra of the alpha-helices in our data base peaks at 97.5 degrees, and approximately 50% of the helices can account for this peak. Thus, approximately 50% of the alpha-helices appear to be amphipathic, and, of those that are, the dominant frequency at 97.5 degrees rather than 100 degrees indicates that the helix is slightly more open than previously thought, with the number of residues per turn closer to 3.7 than 3.6. The extra openness is examined in crystallographic data, and is shown to be associated with the C terminus of the helix. The alpha amphipathic index, the key quantity in our analysis, measures the fraction of the total spectral area that is under the 97.5 degrees peak, and is a characteristic of hydrophobicity scales that is consistent for different sets of helices. Our optimized scale maximizes the amphipathic index and has a correlation of 0.85 or higher with nine previously published scales. The most surprising feature of the optimized scale is that arginine tends to behave as if it were hydrophobic; i.e. in the crystallographic data base it has a tendency to be on the hydrophobic face of teh amphipathic helix. Although the scale is optimal only for predicting alpha-amphipathicity, it also ranks high in identifying beta-amphipathicity and in distinguishing interior from exterior residues in a protein.(ABSTRACT TRUNCATED AT 400 WORDS)

Sequence features that correlate with MHC restriction.

Identification of common sequence motifs in antigenic peptides restricted to a specific class II molecule has not been easy due to the large variation in length and sequence that is observed in these peptides. The goal of this study is to develop an automated computerized method for the identification of sequence features and structural determinants that play a role in the MHC restriction of helper T-cell antigenic peptides. For this, we compiled an extended database of helper T-cell sites, including the information on MHC restriction, when available. Two groups of peptides are assigned to each MHC type: (1) peptides that bind to that MHC molecule to elicit a T-cell response, and (2) peptides that were shown experimentally either not to bind to or not to elicit a T-cell proliferative response in association with that MHC molecule. We search for common motifs in the group of binding peptides, and identify significant motifs that are frequent among these peptides but almost absent in the group of non-binding peptides. A motif consists of physical-chemical and structural properties that may be responsible for binding specificity and can be extracted from sequence data, such as, hydrophobicity, charge, hydrogen bonding capability, etc. The first search is performed on the non-aligned binding peptides. Next, the sequences are aligned according to an identified motif and a search for additional, conserved, properties is performed. The statistical significance of the motifs is evaluated as well as their compatibility with published experimental results on substitution effects. Here we demonstrate the general scheme of the analysis and results for I-Ek and I-Ak associated peptides.

Evaluation of PSI-BLAST alignment accuracy in comparison to structural alignments.

The PSI-BLAST algorithm has been acknowledged as one of the most powerful tools for detecting remote evolutionary relationships by sequence considerations only. This has been demonstrated by its ability to recognize remote structural homologues and by the greatest coverage it enables in annotation of a complete genome. Although recognizing the correct fold of a sequence is of major importance, the accuracy of the alignment is crucial for the success of modeling one sequence by the structure of its remote homologue. Here we assess the accuracy of PSI-BLAST alignments on a stringent database of 123 structurally similar, sequence-dissimilar pairs of proteins, by comparing them to the alignments defined on a structural basis. Each protein sequence is compared to a nonredundant database of the protein sequences by PSI-BLAST. Whenever a pair member detects its pair-mate, the positions that are aligned both in the sequential and structural alignments are determined, and the alignment sensitivity is expressed as the percentage of these positions out of the structural alignment. Fifty-two sequences detected their pair-mates (for 16 pairs the success was bi-directional when either pair member was used as a query). The average percentage of correctly aligned residues per structural alignment was 43.5+/-2.2%. Other properties of the alignments were also examined, such as the sensitivity vs. specificity and the change in these parameters over consecutive iterations. Notably, there is an improvement in alignment sensitivity over consecutive iterations, reaching an average of 50.9+/-2.5% within the five iterations tested in the current study.

Structure-based prediction of binding peptides to MHC class I molecules: application to a broad range of MHC alleles.

Specific binding of antigenic peptides to major histocompatibility complex (MHC) class I molecules is a prerequisite for their recognition by cytotoxic T-cells. Prediction of MHC-binding peptides must therefore be incorporated in any predictive algorithm attempting to identify immunodominant T-cell epitopes, based on the amino acid sequence of the protein antigen. Development of predictive algorithms based on experimental binding data requires experimental testing of a very large number of peptides. A complementary approach relies on the structural conservation observed in crystallographically solved peptide-MHC complexes. By this approach, the peptide structure in the MHC groove is used as a template upon which peptide candidates are threaded, and their compatibility to bind is evaluated by statistical pairwise potentials. Our original algorithm based on this approach used the pairwise potential table of Miyazawa and Jernigan (Miyazawa S, Jernigan RL, 1996, J Mol Biol 256:623-644) and succeeded to correctly identify good binders only for MHC molecules with hydrophobic binding pockets, probably because of the high emphasis of hydrophobic interactions in this table. A recently developed pairwise potential table by Betancourt and Thirumalai (Betancourt MR, Thirumalai D, 1999, Protein Sci 8:361-369) that is based on the Miyazawa and Jernigan table describes the hydrophilic interactions more appropriately. In this paper, we demonstrate how the use of this table, together with a new definition of MHC contact residues by which only residues that contribute exclusively to sequence specific binding are included, allows the development of an improved algorithm that can be applied to a wide range of MHC class I alleles.

Identification of T-cell epitopes and use in construction of synthetic vaccines.

The T cell is central to the immune system response to foreign antigens, and understanding the mechanism of T cell response to antigen is crucial for vaccine development. Short subpeptides of foreign antigen can prime the T cells to respond to the whole antigen, in some cases as well as or better than immunization with the whole antigen itself. Antigenic sites located first in the murine model are also antigenic in the human, suggesting that the structural features of antigenic sites are species-independent. The amphipathic helix hypothesis has proven useful in developing an algorithm that has successfully located immunodominant sites in important proteins, thus reducing substantially the experimental time and effort required to locate those sites. Other algorithms have also been used successfully, but in all cases there are proven T-cell sites not accounted for by the algorithm. A data base showing T-cell response to collections of peptides uniformly distributed along protein antigens would be very useful in subsequent efforts to characterize the physical and chemical properties of T-cell antigenic sites.

A structure-based algorithm to predict potential binding peptides to MHC molecules with hydrophobic binding pockets.

Binding of peptides to MHC class I molecules is a prerequisite for their recognition by cytotoxic T cells. Consequently, identification of peptides that will bind to a given MHC molecule must constitute a central part of any algorithm for prediction of T-cell antigenic peptides based on the amino acid sequence of the protein. Binding motifs, defined by anchor positions only, have proven to be insufficient to ensure binding, suggesting that other positions along the peptide sequence also affect peptide-MHC interaction. The second phase of prediction schemes therefore take into account the effect of all positions along the peptide sequence, and are based on position-dependent-coefficients that are used in the calculation of a peptide score. These coefficients can be extracted from a large ensemble of binding sequences that were tested experimentally, or derived from structural considerations, as in the algorithm developed by us recently. This algorithm uses the coordinates of solved complexes to evaluate the interactions of peptide amino acids with MHC contact residues, and results in a peptide score that reflects its binding energy. Here we present our analysis for peptide binding to four MHC alleles (HLA-A2, HLA-A68, HLA-B27 and H-2Kb), and compare the predictions of the algorithm to experimental binding data. The algorithm performs successfully in predicting peptide binding to MHC molecules with hydrophobic binding pockets but not when MHC molecules with hydrophilic, charged pockets are considered. For MHC molecules with hydrophobic pockets it is demonstrated how the algorithm succeeds in distinguishing binding from non-binding peptides, and in high ranking of immunogenic peptides within all overlapping same-length peptides spanning their respective protein sequences. The latter property of the algorithm makes it a useful tool in the rational design of peptide vaccines aimed at T-cell immunity.

Molecular analysis of HLA class II genes in primary Sjögren's syndrome. A study of Israeli Jewish and Greek non-Jewish patients.

In an attempt to define the role of HLA class II genes in predisposition to primary Sjögren's syndrome, patients of two different ethnic groups (Israeli Jews and Greeks of non-Jewish origin) suffering from this disorder were studied. Oligonucleotide genotyping revealed the majority in both groups to carry either DRB1*1101 or DRB1*1104, alleles that are in linkage disequilibrium with DQB1*0301 and DQA1*0501. The high frequency of the two alleles in these SS patients is in contrast with the accepted association of primary SS with HLA-DR3 in Italian and American individuals. Molecular analysis of DQB1 and DQA1 alleles found in American Caucasian and American black SS (or SLE) patients demonstrated high frequencies of DQB1*0201 and DQA1*0501. The fact that the majority of SS patients, across racial and ethnic boundaries, carry a common allele, DQA1*0501, implies its involvement in the predisposition to primary SS. Based on sequence analysis and the computer imaging of the HLA class II molecule structure, a hypothetical model for the role of the DQ molecule in promoting primary SS is proposed.

Zinc fingers: conserved properties that can distinguish between spurious and actual DNA-binding motifs.

The zinc finger, one of the major structural motifs used for sequence specific DNA binding, has been identified in many regulatory proteins. By analogy, involvement in nucleic acid recognition has been implied for proteins that contain two cysteines and two histidines, spaced in accordance with the zinc finger motif. In this study we identify sequence dependent characteristics that are conserved in the DNA-binding zinc fingers and are probably required for a functional DNA-binding zinc finger. Examination of the conserved properties in view of the solved three dimensional structure of a zinc finger, confirms the importance of most of these properties. The absence of the identified physical-chemical characteristics from CCHH containing sequences of non-DNA binding proteins suggest that they can be used to distinguish between spurious and actual DNA binding zinc fingers.

Synthetic peptides derived from IRBP induce EAU and EAP in Lewis rats.

In an earlier study we isolated three cyanogen bromide cleavage fragments of bovine IRBP that exhibited high levels of immunopathogenicity, producing inflammatory changes in the eyes (EAU) and pineal gland (EAP) of Lewis rats. These fragments have been localized within the IRBP sequence. In order to identify these putative immunopathogenic epitopes of IRBP, nine selected peptide sequences were synthesized and tested for the induction of disease in Lewis rats. Seven of the peptides were found inactive in producing disease while two closely related peptides, designated R4 (23-mer) and R9 (27-mer) were found to reproducibly induce EAU and EAP in immunized rats. No good correlation was found between the immunopathogenicity of the nine tested peptides and their amphipathicity: peptides R4 and R9 were not predicted to form strong amphipathic helices, while peptides selected for their high predicted helical amphipathicity were not immunopathogenic. EAU induced by peptides R4 and R9 was less severe and had a longer onset time than the disease induced by whole IRBP. In addition, the inflammatory changes induced by R4 and R9 in the posterior segment of the eye were less acute than those induced by whole IRBP and included granuloma formation and perivasculitis, features which are not generally seen in rats immunized with whole IRBP. Thus, the changes induced by R4 and R9 more closely resemble those which are characteristically found in human eyes affected by certain uveitic diseases than do changes produced by the intact protein.