Paolo Monaci

The liver-specific transcription factor LF-B1 contains a highly diverged homeobox DNA binding domain

The nuclear protein LF-B1 (also referred to as HNF-1) is a transcription activator required for the expression of several liver-specific genes. LF-B1 has been purified to homogeneity from rat liver nuclear extracts. The sequence of the protein has been partially determined and, subsequently, overlapping cDNA clones containing the entire open reading frame of LF-B1 were isolated. The full-length cDNA encodes a 628 amino acid protein and directs the synthesis in vitro of a protein capable of binding DNA with the same specificity as LF-B1. The cDNA was recombined into a vaccinia virus vector and active LF-B1 was obtained from infected HeLa cells. Addition of the vaccinia recombinant protein to rat spleen extracts results in activation of transcription of an LF-B1-dependent promoter. The DNA binding domain of LF-B1 is located in the amino-terminal part of the protein and displays distant structural similarity to the homeobox domain. The distribution of LF-B1 mRNA is restricted to liver, which correlates with the tissue-specific expression of its target genes.

A general strategy to identify mimotopes of pathological antigens using only random peptide libraries and human sera.

A strategy to identify disease‐specific epitopes from phage‐displayed random peptide libraries using human sera is described. Peptides on phage (phagotopes) that react with antibodies present in patient sera are purified from > 10(7) different sequences by affinity selection and immunological screening of plaques. Disease‐specific phagotopes can be identified out of this pool through an ‘antigen independent’ procedure which avails itself only of patient and normal human sera. Using this strategy, we have selected antigenic mimics (mimotopes) of two different epitopes from the human hepatitis B virus envelope protein (HBsAg). We could show that a humoral response to these mimotopes is widespread in the immunized population, suggesting that the strategy identifies phagotopes that have a potential role as diagnostic reagents. Immunization of mice with the selected phagotopes elicited a strong specific response against the HBsAg. These results open new inroads into disease‐related epitope discovery and provide the potential for vaccine development without a requirement for the use of, or even information about, the aetiological agent or its antigens.

Epitope discovery using peptide libraries displayed on phage

Peptides displayed on phage, which mimic continuous and discontinuous epitopes, can be selected using purified antibodies or preparations of polyclonal serum. This review describes recent advances in this field, discusses the application of phage-display technology to the diagnosis of human diseases, and presents new ideas for the preparation of vaccines directed against specific epitopes on a pathogen.

Selection of biologically active peptides by phage display of random peptide libraries.

Random peptide libraries displayed on phage are used as a source of peptides for epitope mapping, for the identification of critical amino acids responsible for protein-protein interactions and as leads for the discovery of new therapeutics. Efficient and simple procedures have been devised to select peptides binding to purified proteins, to monoclonal and polyclonal antibodies and to cell surfaces in vivo and in vitro.

LFB3, a heterodimer-forming homeoprotein of the LFB1 family, is expressed in specialized epithelia.

We have cloned and characterized a mouse cDNA coding for LFB3, a DNA binding protein containing an extra‐large homeodomain. The first 315 amino acids of LFB3 are highly homologous to the DNA binding domain of LFB1, a regulatory protein involved in the expression of several liver‐specific genes. LFB3 is a transcriptional activator which binds to DNA as a dimer and forms heterodimers with LFB1 both in vitro and in vivo. However, LFB3 expression seems not to be directly correlated with the liver‐specific phenotype, since it is detected in dedifferentiated hepatoma cell lines which express neither LFB1 nor several liver‐specific genes. LFB3 expression starts before that of LFB1 during mouse and rat development, and is strongly increased upon retinoic acid induced differentiation of F9 embryonic carcinoma cells. LFB3 and LFB1 are expressed in the epithelial component of many organs of endodermal and mesodermal origin, suggesting that they may play a more general role associated with the differentiation of specialized epithelia.

Identification of biologically active peptides using random libraries displayed on phage

The construction of new and increasingly diverse libraries, as well as the implementation of more powerful selection schemes, has led to the identification of linear peptides that mimic complex epitopes. Phage display techniques are allowing the selection of disease-related peptides, which reproduce the antigenic and immunogenic properties of natural antigens, using whole sera from patients. The range of applications of phage technology has been extended to include the search for peptides binding to molecules other than antibodies, such as cell receptors and enzymes.

Two different liver-specific factors stimulate in vitro transcription from the human alpha 1-antitrypsin promoter.

The region from −137 to −2 of the human alpha 1‐antitrypsin (alpha 1AT) promoter directs liver‐specific in vitro transcription. Two cis‐acting elements, A and B, have been identified within this segment by site‐directed mutagenesis. Competition with synthetic oligonucleotides corresponding either to the A or to the B sequence inhibits transcription from the wild‐type promoter in vitro. Cis‐linked A and B elements mediate liver‐specific transcription from a truncated HSV‐TK promoter in vitro. Five different proteins, LF‐A1, LF‐A2, LF‐B1, LF‐B2 and LF‐C, bind to the alpha 1AT promoter in liver extracts. LF‐A1 and LF‐B1 are positive transcriptional factors which bind to the A and B elements respectively. Their absence in spleen provides an explanation for the liver specificity of transcription. A protein similar to LF‐B2 is present in spleen. Binding of LF‐B1 and LF‐B2 to the alpha 1AT promoter is mutually exclusive, suggesting that LF‐B2 might be a repressor.

A myosin-like dimerization helix and an extra-large homeodomain are essential elements of the tripartite DNA binding structure of LFB1.

The transcription activator LFB1 is a major determinant of hepatocyte-specific expression of many genes. To study the mechanisms underlying LFB1 transcriptional selectivity, we have initiated its biochemical characterization. By in vitro complementation assays we have defined two distinct regions required for high levels of transcription, which resemble previously described activation domains. In contrast, the region of LFB1 necessary for DNA binding displays several novel features. The DNA binding domain is tripartite, including a homeodomain of unusual length (81 amino acids) and an N-terminal helix similar to part of myosin. This helical region mediates dimerization, which is shown to be essential for DNA binding.

Synergistic trans-activation of the human C-reactive protein promoter by transcription factor HNF-1 binding at two distinct sites.

The promoter region of the human C‐reactive protein (CRP) gene comprises two distinct regions (APREs, for Acute Phase Responsive Elements) each one containing information necessary and sufficient for liver specific and IL‐6 inducible expression in human hepatoma Hep3B cells. In this paper we show that both APREs contain a low affinity binding site for the liver specific transcription factor HNF‐1/LF‐B1. The two sites are separated by approximately 80 bp. Mutations in either of the two sites abolish inducible expression. The same effect is specifically obtained in cotransfection competition experiments when the human albumin HNF‐1 site is used as competitor. However, HNF‐1 is not the intranuclear mediator of IL‐6 because synthetic promoters formed by multimerized copies of different HNF‐1 binding sites are not transcriptionally activated by this cytokine. An expression vector encoding full length HNF‐1 is capable of trans‐activating transcription from the wild‐type CRP promoter but not from mutants which have lost the ability to bind HNF‐1. Moreover, the level of trans‐activation observed with the natural promoter containing both HNF‐1 binding sites is far greater than the level of mutated variants containing only one of the two sites. This result strongly suggests that two HNF‐1 molecules bound simultaneously to sites distant from each other can act synergistically to activate gene expression.

Identification of tumor-associated antigens by screening phage-displayed human cDNA libraries with sera from tumor patients

Screening cDNA libraries from solid human tumors with sera of autologous patients (SEREX) has proven to be a powerful approach to identifying tumor antigens recognized by the humoral arm of the immune system. In many cases, application of this methodology has led to the discovery of novel tumor antigens as unknown gene products. We tried to improve the potency of the SEREX approach by combining it with phage‐display technology. We designed a new lambda vector to express protein fragments as N‐terminal fusions to the D capsid protein and generated high‐complexity cDNA libraries from human breast carcinoma cell lines and solid tumors. Screening these phage‐displayed libraries required limited amounts of sera from patients and efficiently identified several tumor antigens specifically reacting with sera from breast cancer patients.