Faculty Bibliography

The viral transactivator Rev is essential for HIV replication, since it allows the nuclear export of unspliced and partially spliced viral mRNAs that encode the structural proteins. Rev is an RNA binding protein that interacts with a highly structured RNA element, the RRE, found within the envelope sequences. This viral protein also interacts with cellular proteins, termed nucleoporins, and acts as an adaptor between the viral mRNAs and the cellular nuclear export machinery. Both interactions are specific, and required for Rev function. Because of its crucial role in the HIV replication cycle, and its novel mechanism of action, Rev represents an ideal target for therapeutic intervention. This review describes the efforts towards Rev inhibition. Gene therapy approaches, including the expression of trans-dominant mutants and RNA decoys, as well as antisense therapies and small molecule inhibitors of Rev-RRE binding or Rev interaction with the cellular machinery will be discussed

We have described a protein (Hep27) [Donadel, G., Garzelli, C., Frank, R. & Gabrielli, F. (1991) Eur. J. Biochem. 195, 723-729] which is synthesized and accumulated in the nucleus of human hepatoblastoma (HepG2) cells, following growth arrest induced by butyrate treatment. The synthesis of Hep27 is inhibited in cells that, released from the butyrate block, have resumed DNA synthesis. This report describes the cloning and the characterization of the cDNA coding for the Hep27 protein. The translation of the Hep27 cDNA predicts an amino acid sequence that can be aligned with those of the known short-chain alcohol dehydrogenase enzymes (SCAD) family. Both the recognition of enzymic functional domains and the similarity with the SCAD family of proteins of several amino acid blocks throughout the molecule, strongly suggest that this protein is a new member of the SCAD family. In agreement with its nuclear localization Hep27 has a region similar to the bipartite nuclear-targeting sequence. The study of Hep27 mRNA expression and protein synthesis suggests the existence of a regulation at the post-transcriptional level. The possible nuclear role of the Hep27 protein is discussed.

Over the last 7 years we have carried out a major research effort focused on gene transcription as a novel approach to drug discovery. The goal is to identify small molecular weight compounds that modulate the expression of a target gene in a specific manner, thereby either increasing or decreasing the concentration of the corresponding protein product. Transcriptional modulation not only provides a potential means to replace recombinant proteins as drugs, but also provides a novel approach to manipulate key gene targets in many therapeutic areas. This article describes some of the features and advantages of transcription-based pharmaceuticals and illustrates how this approach can be applied to drug discovery with a program we are pursuing to identify new treatments for sickle cell disease and beta-thalassemia.

T-cell antigen receptor triggering results in activation of protein kinase C and mobilization of calcium. These two signals are necessary and sufficient to activate the T-cell specific transcription factor NF-AT, which cooperates with other transcription factors activated by accessory signals to initiate expression of interleukin 2 and its receptor. The protein kinase C mediated pathway involves activation of ras proteins. In a Jurkat cell model of T-cell activation, treatment with antigen receptor agonists results in induction of expression of a reporter gene under the control of a NF-AT dependent promoter. Overexpression of the ras GTPase activating protein p120GAP in these cells caused a significant inhibition of T-cell antigen receptor mediated induction, suggesting a role for p120GAP in regulation of ras. The inhibition was overcome by expression of a valine-12 mutant ras which lacks GTPase activity.

A reporter gene under the control of a T-cell antigen receptor element was activated in Jurkat cells by antigen receptor triggering or by a combination of phorbol myristate acetate, which activates protein kinase C, and a calcium ionophore. Both these signals were necessary for expression of the reporter gene. When co-transfected with a construct capable of overexpressing the tyrosine kinase p59fyn, the reporter gene was activated by PMA alone. Thus p59fyn could replace the calcium ionophore but not activation of protein kinase C. The activation by p59fyn plus PMA was blocked by EGTA and by the immunosuppressant drug cyclosporin A.

The type I interleukin-1 receptor (IL-1R) is capable of transducing a signal resulting in promoter activation in T cells. This signal transduction is dependent on the cytoplasmic domain, which consists of 213 amino acids. In contrast to the type I receptor, the type II IL-1R has a small cytoplasmic tail, and it is not clear whether this receptor is a signal-transducing or a regulatory molecule. Here we report that the type II IL-1R does not mediate gene activation in Jurkat cells. However, a hybrid receptor composed of the extracellular and transmembrane regions of the human type II interleukin-1 fused to the cytoplasmic domain of the human type I IL-1R was capable of transducing a signal across the membrane resulting in a pattern of gene activation identical to that mediated by the type I IL-1R. Our results indicated that the extracellular domain of the type II IL-1R was capable of functionally interacting with interleukin-1 and transmitting the resulting signal to a heterologous cytoplasmic domain.

T-cell antigen receptor engagement results in suboptimal activation of protein kinase C and a prolonged increase in intracellular free calcium concentration. These signals, in combination with stimulation via accessory molecules usually supplied by the antigen presenting cell, activate expression of interleukin-2 (IL-2) and initiate autocrine growth. The lymphocyte-specific tyrosine kinase p56lck is physically associated with CD4 and is brought into close proximity of the intracellular domain of the antigen receptor by CD4 recognition of the major histocompatibility complex during antigen presentation. p56lck activation enhances and may be essential for antigen receptor signaling. We report that a constitutively active form of p56lck delivers a signal which contributes to IL-2 promoter activation. The signal substituted for a calcium-mobilizing signal in a Jurkat cell model of T-cell activation. The activation was sensitive to EGTA and cyclosporin A, indicating that p56lck functions at an early stage of the calcium-mediated pathway. The transcription factor NF-AT mediated, at least in part, the p56lck activation of IL-2 expression. In addition, activated p56lck synergized with constitutively active p21Ha-ras, which can replace protein kinase C activation, resulting in activation of NF-AT in the absence of external signals.

In a Jurkat cell model of T-cell activation an interleukin-2 promoter/reporter gene construct was activated by antigen receptor agonism in combination with the lymphokine interleukin-1. Antigen receptor signals could be mimicked by suboptimal activation of protein kinase C (PKC) with phorbol esters in combination with calcium mobilization by an ionophore. In cotransfection experiments, oncogenic rats obviated the need for PKC stimulation but did not replace either the calcium signal or interleukin-1. Activated ras expression also replaced the requirement for PKC stimulation in activation of the T-cell transcription factor NF-AT. A dominant inhibitory ras mutant specifically blocked antigen receptor agonism, indicating that ras activity is required for antigen receptor signaling. In addition, an inhibitor of PKC blocked both activated ras and phorbol ester stimulation, suggesting a role for ras upstream of PKC.

The evolutionary conservation of a sequence or part of it can help to identify the essential functional and structural domains within a protein. We have cloned and characterised a cDNA coding for the type-I interleukin-1 receptor (IL-1R) of chick (ch) embryo fibroblasts. The comparison of the amino acid (aa) sequences of the avian with that of murine (m) and human (h) IL-1Rs shows a 60% homology. The intracellular domain is the most conserved region of the chIL-1R, showing 76-79% homology to the murine and human sequences, respectively. The striking conservation of the cytoplasmic region of the receptor is confirmed by its homology with the Toll receptor protein of Drosophila melanogaster. The alignment between the chicken and D. melanogaster proteins shows the presence of four aa blocks with more than 80% homology. The possible functional significance of this homology is discussed. The extracellular binding region of the receptor has a clearly recognisable immunoglobulin-like structure although the sequence divergence is higher than in the cytoplasmic domain.

The cytoplasmic domain of the human T cell-type interleukin-1 receptor (hIL-1R) is not involved in the binding, internalization, or nuclear localization of interleukin-1 (IL-1), but is essential for signal transduction. We have previously localized a 50-amino acid region (residues 477-527) critical for IL-1-mediated activation of the interleukin-2 promoter in T cells. This region displays a striking degree of amino acid conservation in human, murine, and chicken IL-1Rs. Here we report the results of a site-directed mutational analysis of the cytoplasmic domain of the hIL-1R. We have introduced single-amino acid substitutions at positions conserved in all three receptors and at nonconserved positions and identified key amino acids for IL-1R function in signal transduction. Three basic (Arg431, Lys515, and Arg518) and 3 aromatic (Phe513, Trp514, and Tyr519) amino acids that are conserved in human, murine, and chicken IL-1Rs could not be replaced without abolishing IL-1R-mediated signal transduction. A substitution at another conserved position (Pro521) reduces significantly the ability of the IL-1R to transmit the IL-1 signal. Nonconserved residues could be replaced without affecting signal transduction. The cytoplasmic domain of the IL-1R is related to that of the Drosophila Toll protein, with a 26% identity and a 43% similarity in amino acid sequence. The amino acids shown to be essential for IL-1R function are conserved in the Toll protein. Our experimental data indicate that the amino acid sequence similarity between the IL-1R and the Drosophila toll protein reflects a functional homology between the two proteins.