Interkeratin peptide-protein interactions that promote HPV16 E7 gene expression.

Human papillomavirus (HPV) 16 E7 gene product encodes the major transforming activity of the virus so as to induce neoplastic transformation. Continued expression of HPV16 E7 protein is required for both the establishment and maintenance of the transformed cellular phenotype. Therefore, understanding of the molecular and biochemical factors leading to the expression of E7 protein is important in relation to HPV-associated diseases. Previously, we identified a rare codon usage and a specific interaction between cytokeratin (CK) 7 and HPV16 E7 mRNA as factors modulating HPV16 E7 expression. In the present study we report that CK19, a biochemical marker of squamous oral and cervical cancer carcinogenesis, promotes the expression of HPV16 E7 oncoprotein by binding to the CK792-97SEQIKA peptide. These findings shed light on the dynamic functionality of the intermediate filament cytoskeleton, open new perspectives for investigating the role of CKs in controlling HPV16 E7 expression, and suggest new therapeutic avenues for HPV-associated carcinomas.

Sequence uniqueness as a molecular signature of HIV-1-derived B-cell epitopes.

The complex pathophysiology of human immunodeficiency virus (HIV) infection and the relatively high mutation rate of the retrovirus make it challenging to design effective anti-HIV vaccines. Several attempts have been made during the last decades to elucidate the enigmatic immunology of HIV infection and to predict potential immunogenic peptides for active vaccination using bioinformatic analysis methods. The results obtained to date to address this important problem are scarce. In this study, we exploit available HIV databases and analyse previously characterized HIV-encoded linear B-cell epitopes for their amino acid sequence similarity to the human or murine host proteome. We obtained further documentation that the HIV-derived antibody-targeted sequences mostly coincide with peptide areas rarely shared with the host proteins. In toto, our past and present data give clear-cut support to the statement that low-similarity to the host proteome is a major mechanism in defining viral peptide immunogenicity and indicate a possible way for inducing effective, high-titer, and non-crossreactive antibodies to be used in anti-HIV vaccine therapy.

Correlating low-similarity peptide sequences and allergenic epitopes.

Although a high number of allergenic peptide epitopes has been experimentally identified and defined, the molecular basis and the precise mechanisms underlying peptide allergenicity are unknown. This issue was analyzed exploring the relationship between peptide allergenicity and sequence similarity to the human proteome. The structured analysis of the data reported in literature put into evidence that the most part of IgE-binding epitopes are (or harbor) pentapeptide unit(s) with no/low similarity to the human proteome, this way suggesting that no or low sequence similarity to the host proteome might represent a minimum common denominator identifying allergenic peptides. The present literature analysis might be of relevance in devising and designing short amino acid modules to be used for blocking pathogenic IgE.

Mapping the human proteome for non-redundant peptide islands.

We describe immune-proteome structures using libraries of protein fragments that define a structural immunological alphabet. We propose and validate such an alphabet as i) composed of letters of five consecutive amino acids, pentapeptide units being sufficient minimal antigenic determinants in a protein, and ii) characterized by low-similarity to human proteins, so representing structures unknown to the host and potentially able to evoke an immune response. In this context, we have thoroughly sifted through the entire human proteome searching for non-redundant protein motifs. Here, for the first time, a complete sequence redundancy dissection of the human proteome has been conducted. The non-redundant peptide islands in the human proteome have been quantified and catalogued according to the amino acid length. The library of uniquely occurring n-peptide sequences that was obtained is characterized by a logarithmic decrease of the number of non-redundant peptides as a function of the peptide length. This library represents a highly specific catalogue of molecular protein signatures, the possible use of which in cancer/autoimmunity research is discussed, with a major focus on non-redundant dodecamer sequences.

Sub-epitopic dissection of HCV E1315-328HRMAWDMMMNWSPT sequence by similarity analysis.

Our labs are focused on identifying amino acid sequences having the ability to react specifically with the functional binding site of a complementary antibody. Our epitopic definition is based on the analysis of the similarity level of antigenic amino acid sequences to the host proteome. Here, the similarity profile to the human proteome of an HCV E1 immunodominant epitope, i.e. the HCV E1(315-328)HRMAWDMMMNWSPT sequence, led to i) characterizing the immunoreactive HCV E1 315-328 region as a sequence endowed with a low level of similarity to human proteins; ii) defining 2 contiguous immunodominant linear determinants respectively located at the NH(2) and COOH terminus of the conserved viral antigenic sequence. This study supports the hypothesis that low sequence similarity to the host's proteome modulates the pool of epitopic amino acid sequences in a viral antigen, and appears of potential value in defining immunogenic viral peptide sequences to be used in immunotherapeutic approaches for HCV treatment.

Peptidology: short amino acid modules in cell biology and immunology.

Short amino acid motifs, either linear sequences or discontinuous amino acid groupings, can interact with specific protein domains, so exerting a central role in cell adhesion, signal transduction, hormone activity, regulation of transcript expression, enzyme activity, and antigen-antibody interaction. Here, we analyze the literature for such critical short amino acid motifs to determine the minimal peptide length involved in biologically important interactions. We report the pentapeptide unit as a common minimal amino acid sequence critically involved in peptide-protein interaction and immune recognition. The present survey may have implications in defining the dimensional module for peptide-based therapeutical approaches such as the development of novel antibiotics, enzyme inhibitors/activators, mimetic agonists/antagonists of neuropeptides, thrombolitic agents, specific anti-viral agents, etc. In such a therapeutical context, it is of considerable interest that low molecular weight peptides can easily cross biological barriers, are less susceptible to protease attacks, and can be administered at high concentrations. In addition, small peptides are a rational target for strategies aimed at antigen-specific immunotherapeutical intervention. As an example, specific short peptide fragments might be used to elicit antibodies capable of reacting with the full-length proteins containing the peptide fragment's amino acid sequence, so abolishing the risk of cross-reactivity.

Peptide mimotopes of oncoproteins as therapeutic agents in breast cancer.

Generation of an immune response to oncoproteins can lead to a cancer specific protective immunity. Several such oncoproteins are being examined as tumor targets with mixed results. We are evaluating the clinical utility of synthetic peptides that would mimic the antigen immunologically and elicit a tumor specific immune response. HER-2/neu, an oncoprotein whose expression in breast cancer is associated with poor prognosis, lower disease free-survival and a propensity for metastases was chosen as a model. Antibodies, Ab2, Ab4 and Ab5 directed towards the extracellular domain of HER-2/neu were reacted to peptides from two synthetic phage display peptide libraries, LX-8 (12-mer peptide library containing disulfide bridge) and X-15 (linear 15-mer). The isolated peptides were sequenced and characterized for ability to produce high titer antibodies and cross-reactivity. The peptides isolated did not show any sequence homology to protein databases but did show a hierarchy of immunogenic epitopes. Antibodies generated against peptides selected against the same antibody Ab2 or Ab4 showed affinity variation. Phages selected against Ab2 were also able to compete with binding of Ab2 to HER-2/neu. These results validate our hypothesis that synthetic peptides that mimic the antigenic epitope of oncoprotein can be generated and their clinical utility rests on devising a screening mechanism to identify peptides that can elicit an immune response directed to the oncoprotein and if possible its antigenic variants.

Individuation of monoclonal anti-HPV16 E7 antibody linear peptide epitope by computational biology.

We applied computational biology to identify the linear amino acid sequence recognized by a mouse monoclonal antibody raised against the full length HPV16 E7 oncoprotein. Computer-assisted search for the epitopic peptide used two parameters: the capability of E7 peptides to bind to MHC class II molecules, and the similarity level of the oncoprotein sequence to the mouse proteome. We report that anti-E7 mAb recognized the peptide having both high binding potential to MHC II molecules and low level of molecular mimicry to mouse proteome. Peptide ability to bind to MHC II molecules appears a necessary but not sufficient condition to determine peptide immunodominance, by needing to be supported by a low degree of peptide similarity to the host's proteome.

Computer-assisted analysis of molecular mimicry between human papillomavirus 16 E7 oncoprotein and human protein sequences.

The immunology of human papillomavirus (HPV) infections has peculiar characteristics. The long latency for cervical cancer development after primary viral infection suggests mechanisms that may aid the virus in avoiding the host immunosurveillance and establishing persistent infections. In order to understand whether molecular mimicry phenomena might explain the ability of HPV to avoid a protective immune response by the host cell, sequence similarity between HPV16 E7 oncoprotein and human self-proteins was examined by computer-assisted analysis. Data were obtained showing that the HPV16 E7 protein has high and widespread similarity to several human proteins involved in a number of critical regulatory processes. In addition, multiple identical and different E7 peptide motifs are present in the same human protein. Thus, sharing of common motifs between viral oncoproteins and molecules of normal cells may be one cause underlying the scarce immunogenicity of HPV infections. The hypothesis is advanced that synthetic peptides harbouring viral motifs not and/or scarcely represented in the host's cellular proteins may represent a valuable immunotherapeutic approach for cervical cancer treatment.

A critical perspective in cancer research (Review).

Over the past few decades, there has been a tremendous increase in cancer biology data and treatment. Cancer research has opened exciting new areas of cellular and molecular biology. Month by month, new genes which regulate the carcinogenesis process are being discovered. The result is an incredible knowledge of cancer: what makes a cancer cell a cancer cell, what cancer cells need to develop, and how cancer cells behave, interact, overgrow and die. In parallel, gene manipulation within cells lets us foresee future possibilities of new cancer treatments. On the other hand, this combination of increased knowledge and powerful new techniques has provided no effective cancer therapy. As it has been quoted during the meeting held in New York, August 1999: <. The success in treating Hodgkin's disease means that patients now live enough to develop complications related to the treatment>. Thus, after dedicated decades of excellent research, cancer remains a significant human, clinical, and economical burden. The purpose of this review is 2-fold. First, to analyze areas of basic cancer research that still await adequate scientific explanations. Second, to stress that, for its continuing advancement, cancer research is dependent upon close relationships among many disciplines; an intimate alignment of oncologists with biochemists, geneticists, immunologists, experimental pathologists, and pharmacologists is needed. In light of the great success registered at the basic science level but lack of effective therapies, it would be wise to establish human and economical resources addressed to a multidisciplinary collaborative effort in cancer research.

Molecular mimicry of phage displayed peptides mimicking GD3 ganglioside.

We have analyzed sequence homologies between nonimmunogenic phage displayed peptides mimicking GD3 ganglioside and human/mouse self-proteins. The GD3 ganglioside phagotopes showed homology to proteins involved in carbohydrate metabolism/transport. Besides this expected homology, molecular mimicry of critical regulatory proteins was found. These data contribute to our understanding of the structural relatedness of antigenic determinants defined by specific anti-GD3 monoclonal antibodies and, in addition, suggest that molecular mimicry might explain the nonimmunogenicity of these peptides otherwise characterized by specificity to the mAb counterpart. We conclude that construction of peptides harboring motifs absent or scarcely represented in endogenous self-proteins might be a useful approach in melanoma immunotherapy.

Modulation of HPV16 E7 translation by tRNAs in eukaryotic cell-free translation systems.

Translational regulation of HPV16 E7 mRNA is of particular interest since the viral E7 protein has oncogenic activity. Here we report that additional supplementation of cell-free reticulocyte/wheat-germ translation systems with rat liver tRNA pool favors the expression of the viral oncoprotein E7 which otherwise is scarcely translatable. The translational activation is explained by the positive correlation between the frequency of the glutamic and aspartic codons in E7 mRNA, and the increased concentration of the corresponding tRNAs following tRNA supplementation. The present in vitro data suggest a link between E7 expression and cell conditions conductive to tRNA changes such as development, cell proliferation and aging.

Changes of tRNA population during compensatory cell proliferation: differential expression of methionine-tRNA species.

Changes of tRNA species, as both relative percentage of total tRNA and absolute concentration, occur during liver cell proliferation induced by partial hepatectomy. Transfer RNAs which are abundant under quiescence are found to decrease during hepatocyte proliferation, and vice versa. One consequence of these changes is the differential expression of methionine-isoaccepting tRNA species. Initiator tRNA(Met) is present in scarce amounts under quiescent conditions and increases during cell-cycle progression. Elongator tRNA(Met) shows the opposite behavior. Both the quantitative and qualitative tRNA changes return to control levels as the liver returns to resting conditions. These changes might have mechanistic implications in modulating the protein synthesis required by cell proliferation. Moreover, the increase of initiator tRNA(Met) species might be necessary to translate protooncogene, growth factor, and receptor mRNAs, the translation of which is hindered by inhibitory AUG triplets upstream from the coding sequence. Thus the tRNA changes described herein could be involved in regulating translation of transcripts encoding cell-cycle associated proteins.

Enhanced expression of initiator TRNA(Met) in human gastric and colorectal carcinoma.

Transfer RNA isoaccepting species are differentially expressed at different times during development, differentiation, growth, aging, and carcinogenesis processes. It has been suggested that alterations in tRNA patterns might be mechanistically important in modulating gene expression during the various physiological/pathological cellular stages. As part of a study to investigate the possible mechanisms by which alterations of translational machinery can start and/or sustain carcinogenic cell proliferation, in this communication we report analysis of tRNA distribution in two gastro-intestinal human tumors. The qualitative and quantitative data obtained for cellular tRNA distribution put into evidence a shift in the tRNA population with increased level of initiator tRNA(Met) in the malignant tissues. This observation confirms previous data obtained on experimental carcinogenesis models and suggests the possibility of specific involvement of tRNA changes in protein synthesis initiation during tumorigenesis.

Changes in tRNA pattern in ethionine-induced rat putative preneoplastic hepatocyte nodules.

As part of a study to explore further the biochemical pathology of chemical hepatocarcinogenesis, cytoplasmic tRNA patterns have been studied in ethionine-induced rat putative preneoplastic nodules in comparison to surrounding non-nodular liver and control liver. A new h.p.l.c. methodology, able to resolve contemporaneously the numerous components in a tRNA population, has been used. The results obtained indicate the presence of marked differences in the chromatographic profiles of hepatocyte nodules, non-nodular surrounding liver and control liver. The differences are both quantitative and qualitative, with some chromatographic peaks showing increases and some decreases or absences. Also, new peaks are seen reproducibly in the nodules. The data show major changes in the tRNA population during hepatocarcinogenesis that might have mechanistic implications.

Modulation of in vitro kidney cell growth by hepatic transfer RNAs.

The quiescent and proliferating status of the cell appears characterized by two different tRNA populations. The tRNA species changed both as relative percentage of total tRNA and in absolute concentration. The changes seem causally related to the proliferation status of the cell as tRNAs from proliferating hepatocytes were able to stimulate in vitro kidney cell growth. Moreover, tRNAs from quiescent hepatocytes inhibited kidney cell growth. It was concluded that the composition of tRNA population can modulate the proliferative behaviour of the cell.

N-methyl-N-nitrosourea evidences cell cycle associated transient sequences in hepatic replicating DNA.

Experiments of molecular hybridization were carried out with the aim of singling out DNA sequences which might be important in the hepatocarcinogenic action of N-methyl-N-nitrosourea. Results were obtained suggesting the synthesis of transitory DNA sequences during compensatory cell proliferation.

Fractionation of rat liver tRNA by reversed-phase high performance liquid chromatography: isolation of Iso-tRNAs(Pro).

This paper illustrates the fractionation of cytoplasmic transfer ribonucleic acid from rat liver by reversed-phase high performance liquid chromatography using a gradient of acetonitrile/ammonium acetate. The procedure is fast, highly reproducible, and gives an excellent resolution of the numerous tRNA population: about 50 peaks with area peak percentages ranging from 0.001 to 5 can be monitored. Uncharged tRNA preparations exhibited a chromatographic profile different from aminoacylated tRNA, thus suggesting a possible strategy to distinguish between aminoacylated and nonacylated tRNA species. Moreover, a first approach to map the HPLC peaks was attempted by chromatographing preparations of tRNA which had been aminoacylated with individual 3H-labeled aminoacids. Here is reported the case of tRNA(Pro), which gave three well separated radioactive peaks, most likely corresponding to tRNA(Pro) isoacceptor species.

Hypermethylation of replicating hepatic DNA following N-methyl-N-nitrosourea administration.

Changes in the degree of methylation of cytosine in DNA are considered to be mechanistically important in modulating gene expression. To gain a better understanding of the relationship(s) linking onco-proliferative processes and enzymatic DNA methylation, a study has been carried out on the hepatic DNA methylation pattern during DNA replication following partial hepatectomy (PH), mitogen treatment and N-methyl-N-nitrosourea (MNU) administration in rats. The following results were obtained: (i) DNA hypomethylation was seen during DNA synthesis, with each of the 3 stimuli, namely MNU administration, partial hepatectomy, and hepatomitogen treatment; (ii) the level of DNA hypomethylation was not quantificatively related to the extent of DNA replication as measured by incorporation of [3H]thymidine into hepatic DNA; (iii) MNU administration under conditions conducive to carcinogenic development, i.e. during the S phase of compensatory cell proliferation, caused hypermethylation of replicating hepatic DNA, as shown by HpaII and MspI restriction patterns.

Sequential alterations in tRNA population of 2-acetylaminofluorene-induced hepatocyte nodules.

In order to investigate the possible mechanisms by which cellular alterations can start an altered onco-developmental gene expression, we studied tRNA distribution profiles during the early steps of 2-acetylaminofluorene-induced hepatocarcinogenesis. The finding of progressive and sequential alterations appears to support the hypothesis of a causal connection between tRNA changes and nodular cell proliferation, possibly through the disruption of the mechanism which regulates tRNA functional adaptation.