Characterization of cholyl-leu-val-phe-phe-ala-OH as an inhibitor of amyloid beta-peptide polymerization.

Cholyl-LVFFA-OH (1, PPI-368) is an organic-modified peptide based on the sequence of amyloid beta-peptide (A beta). It is a potent and selective inhibitor of A beta polymerization that blocks the formation of neurotoxic species of A beta. In a nucleation-dependent polymerization assay of 50 microM A beta(1-40), equimolar concentrations of PPI-368 block polymerization based on turbidity and electron microscopy. Monomeric A beta(1-40) and A beta(1-42) are non-toxic when incubated with neuronal cell lines, but become toxic during polymerization. PPI-368 coordinately delays the onset of polymerization and the formation of neurotoxic A beta species for both peptides. In a polymerization extension assay seeded with pre-formed A beta polymer, similar inhibition and dose-dependency phenomena are observed with PPI-368. Radiolabeled PPI-368 is incorporated into fibrils during polymerization demonstrating binding to A beta peptide within afibrillar structure. Gel-filtration studies show progressive disappearance of A beta monomer and concomitant appearance of soluble higher molecular weight oligomers. In the presence of submolar concentrations of PPI-368, monomeric A beta is still present and oligomers are not observed PPI-368 does not inhibit the polymerization of other amyloidogenic proteins such as transthyretin (TTR) or islet amyloid polypeptide (IAPP(20-29).

The use of retroviruses as pharmaceutical tools for target discovery and validation in the field of functional genomics.

Retrovirally mediated functional genomics enables identification of physiologically relevant cellular therapeutic targets. Unique properties of retroviruses make them ideal tools for the introduction of large and diverse libraries of potential genetic effectors to a variety of cell types. The identification and recovery of intracellular library elements responsible for altered disease responses establishes a direct basis for pharmaceutical development. Recent innovations in retroviral infection efficiency and expression control have broadened application of the methodology to include libraries of mutagenized cDNAs, peptides and ribozyme genetic effectors.

Modified-peptide inhibitors of amyloid beta-peptide polymerization.

Cellular toxicity resulting from nucleation-dependent polymerization of amyloid beta-peptide (Abeta) is considered to be a major and possibly the primary component of Alzheimer's disease (AD). Inhibition of Abeta polymerization has thus been identified as a target for the development of therapeutic agents for the treatment of AD. The intrinsic affinity of Abeta for itself suggested that Abeta-specific interactions could be adapted to the development of compounds that would bind to Abeta and prevent it from polymerizing. Abeta-derived peptides of fifteen residues were found to be inhibitory of Abeta polymerization. The activity of these peptides was subsequently enhanced through modification of their amino termini with specific organic reagents. Additional series of compounds prepared to probe structural requirements for activity allowed reduction of the size of the inhibitors and optimization of the Abeta-derived peptide portion to afford a lead compound, cholyl-Leu-Val-Phe-Phe-Ala-OH (PPI-368), with potent polymerization inhibitory activity but limited biochemical stability. The corresponding all-D-amino acyl analogue peptide acid (PPI-433) and amide (PPI-457) retained inhibitory activity and were both stable in monkey cerebrospinal fluid for 24 h.

Molecular cloning and pro-apoptotic activity of ICErelII and ICErelIII, members of the ICE/CED-3 family of cysteine proteases.

Cysteine proteases related to mammalian interleukin-1 beta-converting enzyme (ICE) and the nematode cell death abnormal ced-3 gene product have been implicated in the effector mechanism of apoptotic cell death. Two novel members of this new family of ICE/CED-3-related proteases, designated ICErel-II and ICErel-III, were cloned from human monocytic cells. Both were highly homologous to human ICE (52% identical) and CED-3 (25% identical) and both contained the absolutely conserved pentapeptide sequence Gln-Ala-Cys-Arg-Asp containing the catalytic cysteine residue. Other structural motifs that were comparable with ICE suggest that ICErel-II and ICErel-III are also synthesized as larger proenzymes which are proteolytically processed to form heterodimeric active enzymes. Pro-interleukin-1 beta processing activity could not be detected in cells transfected with ICErel-II or ICErel-III, but pro-domain-less truncated forms of ICErel-II and ICErel-III were capable of effectively inducing fibroblast apoptosis. ICErel-II and ICErel-III may, therefore, participate in proteolytic events culminating in the apoptotic death of human cells.

Interleukin-1 beta converting enzyme: a novel cysteine protease required for IL-1 beta production and implicated in programmed cell death.

Interleukin-1 beta converting enzyme is the first member of a new class of cysteine proteases. The most distinguishing feature of this family is a nearly absolute specificity for cleavage at aspartic acid. This enzyme has been the subject of intense research because of its role in the production of IL-1 beta, a key mediator of inflammation. These studies have culminated in the design of potent inhibitors and determination of its crystal structure. The structure secures the relationship of the enzyme to CED-3, the product of a gene required for programmed cell death in Caenorhabditis elegans, suggesting that members of this family function in cell death in vertebrates.

The structure and complete nucleotide sequence of the murine gene encoding interleukin-1 beta converting enzyme (ICE).

Interleukin-1 beta (IL-1 beta) converting enzyme (ICE) processes the precursor of the cytokine IL-1 beta to a mature, biologically active form in monocytes and macrophages. To further understand the role of ICE in regulating IL-1 beta-mediated biological functions, we have isolated several genomic clones encoding the full-length murine ICE gene. Southern blot comparison of murine genomic DNA and the clones indicates that ICE is a compact, single-copy gene 8616 bp in size. We sequenced the entire gene as well as 1.0-kb segment upstream of the coding region and determined that the gene consists of 10 exons whose organization parallels the functional organization of the ICE proenzyme in that the prodomain and p20 and p10 subunits of ICE are encoded by three clusters of exons. Two initiation sites, 37 and 32 nucleotides upstream of the initiator methionine, were identified by primer extension analysis. The 5' region of the ICE gene lacks a TATA box, a CAAT box, and SP1 sites. However, the presence of a completely conserved 14-bp sequence spanning the transcription initiation site of both the murine and the human ICE genes suggests that this sequence plays a role in transcription.

Interleukin 1 beta (IL-1 beta) processing in murine macrophages requires a structurally conserved homologue of human IL-1 beta converting enzyme.

Murine interleukin 1 beta (IL-1 beta) convertase (mICE) was identified in cytosolic extracts of peritoneal exudate cells (PECs) and macrophage cell lines. mICE cleaves both the human and mouse IL-1 beta precursors (pIL-1 beta) at sites 1 and 2 but fails to cleave a human pIL-1 beta (Asp116 to Ala) mutant at site 2, indicating that Asp is required to the left of the scissile bond. Ac-Tyr-Val-Ala-Asp-amino-4-methyl coumarin, patterned after site 2 of human pIL-1 beta, is a fluorogenic substrate for mICE, while the tetrapeptide aldehyde Ac-Tyr-Val-Ala-Asp-CHO is a potent inhibitor (Ki = 3 nM) that prevents generation and release of mature IL-1 beta by PECs (IC50 = 7 microM). Cloning of a full-length 1.4-kb cDNA shows that mICE is encoded as a 402-aa proenzyme (p45) that can be divided into a prodomain (Met1-Asp122), followed by a p20 subunit (Gly123-Asp296), a connecting peptide (Ser297-Asp314), and a p10 subunit (Gly315-His402). At the amino acid level, p45, p20, and p10 are 62%, 60%, and 81% identical with human IL-1 beta convertase (hICE). The active site Cys284 lies within a completely conserved stretch of 18 residues; however, Ser289 in hICE, which aligns with the catalytic region of serine and viral cysteinyl proteases, is absent from mICE. Expression in Escherichia coli of a truncated cDNA encoding Asn119-His402 generated active enzyme, which was autocatalytically processed at three internal Asp-Xaa bonds to generate a p20 subunit (Asn119-Asp296) complexed with either p11 (Ala309-His402) or p10. Recombinant mICE cleaves murine pIL-1 beta accurately at the Asp117-Val118 bond. The striking similarities of the human and murine enzymes will make it possible to assess the therapeutic potential of hICE inhibitors in murine models of disease.

A novel heterodimeric cysteine protease is required for interleukin-1 beta processing in monocytes.

Interleukin-1 beta (IL-1 beta)-converting enzyme cleaves the IL-1 beta precursor to mature IL-1 beta, an important mediator of inflammation. The identification of the enzyme as a unique cysteine protease and the design of potent peptide aldehyde inhibitors are described. Purification and cloning of the complementary DNA indicates that IL-1 beta-converting enzyme is composed of two nonidentical subunits that are derived from a single proenzyme, possibly by autoproteolysis. Selective inhibition of the enzyme in human blood monocytes blocks production of mature IL-1 beta, indicating that it is a potential therapeutic target.

5-HT1c receptor is a prominent serotonin receptor subtype in the central nervous system.

Neurons in rat central nervous system (CNS) that express 5-HT1c receptor mRNA have been localized by in situ hybridization histochemistry. The 5-HT1c receptor is expressed in a wide variety of cortical and subcortical neurons including hippocampal pyramidal neurons, neurons within most of the central monoaminergic cell groups, neurons in thalamic sensory relay nuclei, and neurons involved in the central processing and regulation of nociceptive transmission. Therefore, the 5-HT1c receptor is a prominent but poorly characterized central subclass of serotonin (5-HT) receptor. The distribution of the 5-HT1c receptor within the CNS is considerably more widespread than that of the structurally and functionally related 5-HT2 receptor.

Structure and expression of the human and mouse T4 genes.

The T4 molecule may serve as a T-cell receptor recognizing molecules on the surface of specific target cells and also serves as the receptor for the human immunodeficiency virus. To define the mechanisms of interaction of T4 with the surface of antigen-presenting cells as well as with human immunodeficiency virus, we have further analyzed the sequence, structure, and expression of the human and mouse T4 genes. T4 consists of an extracellular segment comprised of a leader sequence followed by four tandem variable-joining (VJ)-like domains, a transmembrane domain, and a cytoplasmic segment. The structural domains of the T4 protein deduced from amino acid sequence are precisely reflected in the intron-exon organization of the gene. Analysis of the expression of the T4 gene indicates that T4 RNA is expressed not only in T lymphocytes, but in B cells, macrophages, and granulocytes. T4 is also expressed in a developmentally regulated manner in specific regions of the brain. It is, therefore, possible that T4 plays a more general role in mediating cell recognition events that are not restricted to the cellular immune response.

Recombination within the myelin basic protein gene created the dysmyelinating shiverer mouse mutation.

Shiverer (shi) is an autosomal recessive mutation in the mouse characterized by an almost total lack of central nervous system myelin. While small amounts of other myelin components are present in the brain of the shi mouse, the four forms of myelin basic protein (MBP) are not detectable. Previous investigations by us and others indicate that the MBP gene has undergone a major rearrangement in the shi mutant. Herein, we report in detail the nature and extent of the rearrangement: a 20-kilobase region within the MBP gene is missing in the mutant. We map the 5' breakpoint of the deletion to the second intron and the 3' breakpoint to a site 2 kilobases beyond the last MBP exon. The junction of the upstream and downstream portions of the gene contains only one nucleotide not accounted for by the wild-type MBP gene sequence. The 3' side of the deletion occurs in the 3rd of 11 tandem repeats of a 31-base-pair sequence. This region is rich in alternating purine and pyrimidine stretches, sequences that have been associated with both Z-DNA structures and gene rearrangements. The recombination junction shares several features with the junctions characterized by Anderson et al. [Anderson, R., Kato, S. & Camerini-Otero, D. (1984) Proc. Natl. Acad. Sci. USA 81, 206-210] in mouse L cells and is consistent with their model for a partially homologous recombination event. The structure of the shi recombination junction suggests that the donor DNA molecules were aligned in a partially homologous region before staggered cutting and joining occurred.

Genetic variation among lentiviruses: homology between visna virus and caprine arthritis-encephalitis virus is confined to the 5' gag-pol region and a small portion of the env gene.

Visna virus of sheep and arthritis-encephalitis virus of goats are serologically related but genetically distinct retroviruses which cause slowly progressive diseases in their natural hosts. To localize homologous regions of the DNAs of these two viruses, we constructed a physical map of caprine arthritis-encephalitis virus DNA and aligned it with the viral RNA. Cloned probes of visna virus DNA were then used to localize regions of homology with the caprine arthritis-encephalitis virus DNA. These studies showed homology in the 5' region of the genome encompassing U5 and the gag and pol genes and also in a small region in the env gene. These findings correlate with biological data suggesting that the regions of the DNA which are homologous may be responsible for virus group characteristics such as the closely related virus core antigens. Regions which did not show homology such as large sections in the env gene may represent unique sequences which control highly strain-specific characteristics such as the neutralization antigen and specific cell tropisms.