Fas is a cell surface receptor that can transmit signals for programmed cell death. Using a retroviral expression system, we have demonstrated that a short peptide derived from the cleavage site in a cellular target of a pro-apoptotic cysteine protease can be expressed within intact cells with sufficient activity to inhibit Fas-mediated apoptosis. In vitro analysis demonstrates that this retrovirally-expressed peptide is as potent as 150uM levels of the chemically synthesized peptide. Furthermore, using retroviral peptide library-based functional cloning we identified variants of this peptide with apparent anti-apoptotic activity. This approach is likely to lead to the identification of peptide variants with activity against a variety of signaling processes, both normal and pathological.

Abstract: Multiple biologically active peptides arising from a common prohormone are sorted into distinct classes of dense core vesicles within the bag cell neurons of Aplysia californica. In this study, pulse‐chase analysis, combined with subcellular fractionation on Percoll gradients, are used to define the location of the prohormone processing events within the secretory pathway. Initial cleavage of the prohormone occurs in a light cellular compartment associated with the Golgi apparatus. The amino‐terminal processing intermediate then accumulates in a denser compartment containing small dense cores enclosed in membranous sacs, as well as larger immature vesicles. After 4 h, amino‐terminai products are found primarily in a much denser compartment which consists of large and small dense core vesicles. These large and small vesicles can be separated from each other using Percoll gradient centrifugation and are found to be enriched in amino‐ and carboxy‐terminal products, respectively. Lastly, membrane association experiments suggest differential binding to membranes, or integral membrane proteins, as a possible mechanism for sorting of amino‐ and carboxy‐terminal products.

The dioxin-responsive enhancer upstream of the CYP1A1 gene contains four copies of the recognition motif for the liganded Ah receptor. The results of deletion analyses, linker-scanning analyses, and the analysis of individual enhancer subdomains reveal that each copy of the motif contributes to the response of the enhancer to 2,3,7,8-tetrachlorodibenzo-p-dioxin. In the context of the dioxin-responsive enhancer, a GC box, representing the DNA binding site for Sp1 (or a related transcription factor), has no detectable intrinsic activity but enhances gene expression when linked to a recognition motif for the liganded Ah receptor, thereby producing a synergistic effect on enhancer function.

Bioactive peptides cleaved from the egg-laying hormone precursor in the bag cell neurons of Aplysia are sorted into distinct dense core vesicle classes (DCVs). Bag cell prohormone processing can be divided into two stages, an initial cleavage occurring in a late Golgi compartment, which is not blocked by monensin, and later cleavages that occur within DCVs and are blocked by monensin. Prohormone intermediates are sorted in the trans-Golgi network. The large soma-specific DCVs turn over, while the small DCVs are transported to processes for regulated release. Thus, protein trafficking differentially regulates the levels and localization of multiple biologically active peptides derived from a common prohormone.

Gel retardation analyses reveal a cluster of six binding sites for the liganded Ah receptor within a 700-base pair DNA domain upstream of the mouse CYP1A1 gene. The nucleotide sequences of the binding sites define a consensus recognition motif for the liganded receptor. The consensus motif is not symmetric. Alteration of the consensus motif produces a decrease in the receptor-DNA interaction. The ligand receptor binds as a monomer to its recognition motif and preferentially binds to double-stranded DNA. These observations reveal apparent differences between 2,3,7,8-tetrachlorodibenzo-p-dioxin and steroid hormones in their respective mechanisms of action.

The bag cell neurons in the marine snail Aplysia synthesize large amounts of the egg-laying hormone (ELH) prohormone. The ELH precursor is proteolytically processed into 9 peptides making this a useful system for studying prohormone processing and the sorting of proteins destined for the secretory pathway. The peptides derived from the ELH prohormone are differentially packaged into four distinct classes of dense cored vesicles (DVCs). Dense cored vesicles in the large class are greater than 250 nm in diameter, contain the 6 peptides derived from the aminoterminus of the prohormone and are localized to the cell soma and not the neuronal processes. Here we demonstrate that the large DCVs are enriched in prohormone processing intermediates. In addition, many of the large DCVs do not contain acid phosphatase activity suggesting they are an organelle distinct from the lysosomes and that different classes of DCVs may subserve unique functions within the secretory pathway.

The halogenated aromatic hydrocarbon 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD, dioxin) is a persistent, widespread, potentially toxic environmental contaminant, which is a potent inducer of aryl hydrocarbon hydroxylase activity in the liver and other tissues. TCDD induces hydroxylase activity by increasing the rate of transcription of the CYP1A1 gene. Activation of CYP1A1 transcription requires the binding of TCDD to an intracellular protein, the Ah receptor, followed by the binding of the liganded receptor to a dioxin-responsive enhancer that is located upstream from the CYP1A1 gene. The liganded receptor recognizes a specific DNA sequence, which is present in multiple copies within the enhancer. The receptor-enhancer interaction occurs within the major groove of the DNA helix. DNA methylation in vitro interferes with the receptor-enhancer interaction and, therefore, has the potential to inhibit the biological response to TCDD.

Egg laying in Aplysia is mediated by a battery of neuropeptides released from the bag cell neurons. Predominant intermediates in the proteolytic processing of the Aplysia egg-laying hormone neuropeptide precursor were characterized using biochemical and immunological techniques. Following removal of the signal peptide, a rapid cleavage at the tetrabasic sequence Arg-Arg-Lys-Arg separates the amino and carboxyl regions of the prohormone. Processing of the carboxyl-terminal portion of the precursor then proceeds rapidly via two further cleavages at dibasic residues, resulting in a well defined product mixture within 4 h of chase. By contrast, processing of the amino-terminal side of the molecule proceeds only partially to completion after 20 h of chase and a well defined set of intermediates is not observed. Molecular genetic, physiological, and behavioral studies in conjunction with the biochemical investigations presented here are defining the information flow which governs the egg-laying behavior of Aplysia.

We have identified in mouse hepatoma cells a third cis-acting dioxin-responsive element (DRE) within the 5' flanking region of the cytochrome P1-450 gene, which is transcriptionally activated by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). The DRE can activate a heterologous promoter and functions in either orientation; therefore, it has the properties of a transcriptional enhancer. The DRE fails to activate transcription in receptor-defective cells; therefore, it requires TCDD-receptor complexes for its function. By using a gel retardation assay, we show that nuclear extracts contain a protein that binds to the DRE in TCDD-inducible, receptor-dependent, and DNA sequence-specific fashion. The protein-DNA interaction occurs within 10 min of exposure of the cell to TCDD and does not require ongoing protein synthesis. Our results imply that the TCDD-receptor complex interacts specifically with the DRE and demonstrate a relationship between protein-DNA interaction in vitro and function in vivo. Our findings also suggest that the affinity of the TCDD-receptor complex for the DRE may be relatively high in comparison to analogous protein-DNA interactions at other inducible enhancers.

The environmental contaminant 2,3,7,8-tetra-chlorodibenzo-p-dioxin produces its biological effects by binding to an intracellular protein (the Ah receptor). The dioxin-Ah receptor complex activates cytochrome P1-450 gene transcription by interacting with dioxin-responsive enhancers. Here, we report that the dioxin-Ah receptor complex binds to DNA containing the "core" sequence 5'-TA/TGCGTG-3', which is present in each of three receptor-dependent enhancers. Functional analyses indicate that binding of the liganded Ah receptor to the core sequence fails to generate an active enhancer and that nucleotides flanking the core sequence must contribute to enhancer function and dioxin action.

Aplysia abdominal ganglion neuron L5 is immunoreactive with an antiserum generated against the tetrapeptide Phe-Met-Arg-Phe-amide (FMRFamide); however, the specificity of this immune reagent is limited to the sequence Arg-Phe-amide. We isolated cDNA clones homologous to mRNAs specifically expressed in L5 and demonstrated that these clones do not hybridize to a previously characterized gene encoding FMRFamide. The nucleotide sequence of one of these clones, L5-67, does not encode any FMRFamide peptides but does reveal a Gly-Lys-Arg cleavage site following the amino acids Arg-Phe. This data predicts that neuron L5 expresses a peptide ending in Arg-Phe-amide, consistent with the FMRFamide immunoreactivity.

The environmental contaminant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) may produce its effects by altering gene expression in susceptible cells. In mouse hepatoma cells, TCDD induces the transcription of the cytochrome P1-450 gene, whose product, aryl hydrocarbon hydroxylase, contributes both to the detoxification and to the metabolic activation of carcinogenic polycyclic aromatic hydrocarbons. A DNA fragment containing sequences flanking the 5' end of the cytochrome P1-450 gene was isolated and analyzed. This DNA fragment contains a cis-acting control element with at least three functional domains: a putative promoter, an inhibitory domain upstream from the promoter that blocks its function, and a TCDD-responsive domain still farther (1265 to 1535 base pairs) upstream of the promoter. These findings, together with results from earlier studies, imply that transcription of the cytochrome P1-450 gene is under both positive and negative control by at least two trans-acting regulatory factors.