Selective inhibition of Alzheimer disease-like tau aggregation by phenothiazines.

In Alzheimer disease (AD) the microtubule-associated protein tau is redistributed exponentially into paired helical filaments (PHFs) forming neurofibrillary tangles, which correlate with pyramidal cell destruction and dementia. Amorphous neuronal deposits and PHFs in AD are characterized by aggregation through the repeat domain and C-terminal truncation at Glu-391 by endogenous proteases. We show that a similar proteolytically stable complex can be generated in vitro following the self-aggregation of tau protein through a high-affinity binding site in the repeat domain. Once started, tau capture can be propagated by seeding the further accumulation of truncated tau in the presence of proteases. We have identified a nonneuroleptic phenothiazine previously used in man (methylene blue, MB), which reverses the proteolytic stability of protease-resistant PHFs by blocking the tau-tau binding interaction through the repeat domain. Although MB is inhibitory at a higher concentration than may be achieved clinically, the tau-tau binding assay was used to identify desmethyl derivatives of MB that have Ki values in the nanomolar range. Neuroleptic phenothiazines are inactive. Tau aggregation inhibitors do not affect the tau-tubulin interaction, which also occurs through the repeat domain. Our findings demonstrate that biologically selective pharmaceutical agents could be developed to facilitate the proteolytic degradation of tau aggregates and prevent the further propagation of tau capture in AD.

Mutations in the small subunit of the Drosophila U2AF splicing factor cause lethality and developmental defects.

The essential eukaryotic pre-mRNA splicing factor U2AF (U2 small nuclear ribonucleoprotein auxiliary factor) is required to specify the 3' splice at an early step in spliceosome assembly. U2AF binds site-specifically to the intron polypyrimidine tract and recruits U2 small nuclear ribonucleoprotein to the branch site. Human U2AF (hU2AF) is a heterodimer composed of a large (hU2AF65) and small (hU2AF35) subunit. Although these proteins associate in a tight complex, the biochemical requirement for U2AF activity can be satisfied solely by the large subunit. The requirement for the small subunit in splicing has remained enigmatic. No biochemical activity has been found for hU2AF35 and it has been implicated in splicing only indirectly by its interaction with known splicing factors. In the absence of a biochemical assay, we have taken a genetic approach to investigate the function of the small subunit in the fruit fly Drosophila melanogaster. A cDNA clone encoding the small subunit of Drosophila U2AF (dU2AF38) has been isolated and sequenced. The dU2AF38 protein is highly homologous to hU2AF35 containing a conserved central arginine- and serine-rich (RS) domain. A recessive P-element insertion mutation affecting dU2AF38 causes a reduction in viability and fertility and morphological bristle defects. Consistent with a general role in splicing, a null allele of dU2AF38 is fully penetrant recessive lethal, like null alleles of the Drosophila U2AF large subunit.

Apoptosis mediated by HIV protease is preceded by cleavage of Bcl-2.

Expression of the human immunodeficiency virus type 1 (HIV) protease in cultured cells leads to apoptosis, preceded by cleavage of bcl-2, a key negative regulator of cell death. In contrast, a high level of bcl-2 protects cells in vitro and in vivo from the viral protease and prevents cell death following HIV infection of human lymphocytes, while reducing the yields of viral structural proteins, infectivity, and tumor necrosis factor alpha. We present a model for HIV replication in which the viral protease depletes the infected cells of bcl-2, leading to oxidative stress-dependent activation of NF kappa B, a cellular factor required for HIV transcription, and ultimately to cell death. Purified bcl-2 is cleaved by HIV protease between phenylalanine 112 and alanine 113. The results suggest a new option for HIV gene therapy; bcl-2 muteins that have noncleavable alterations surrounding the HIV protease cleavage site.

Fibroblast growth factor (FGF) homologous factors: new members of the FGF family implicated in nervous system development.

Four new members of the fibroblast growth factor (FGF) family, referred to as fibroblast growth factor homologous factors (FHFs), have been identified by a combination of random cDNA sequencing, data base searches, and degenerate PCR. Pairwise comparisons between the four FHFs show between 58% and 71% amino acid sequence identity, but each FHF shows less than 30% identity when compared with other FGFs. Like FGF-1 (acidic FGF) and FGF-2 (basic FGF), the FHFs lack a classical signal sequence and contain clusters of basic residues that can act as nuclear localization signals. In transiently transfected 293 cells FHF-1 accumulates in the nucleus and is not secreted. Each FHF is expressed in the developing and adult nervous systems, suggesting a role for this branch of the FGF family in nervous system development and function.

Identification of potential target genes for the neuron-restrictive silencer factor.

The neuron-restrictive silencer factor (NRSF) represses transcription of several neuronal genes in nonneuronal cells by binding to a 21-bp element called the neuron-restrictive silencer element (NRSE). We have performed data base searches with a composite NRSE to identify additional candidate NRSF target genes. Twenty-two more genes, 17 of which are expressed mainly in neurons, were found to contain NRSE-like sequences. Many of these putative NRSEs bound NRSF in vitro and repressed transcription in vivo. Most of the neuronal genes identified contribute to the basic structural or functional properties of neurons. However, two neuronal transcription factor genes contain NRSEs, suggesting that NRSF may repress neuronal differentiation both directly and indirectly. Functional NRSEs were also found in several nonneuronal genes, implying that NRSF may play a broader role than originally anticipated.

Mammalian dwarfins are phosphorylated in response to transforming growth factor beta and are implicated in control of cell growth.

The dwarfin protein family has been genetically implicated in transforming growth factor beta (TGF-beta)-like signaling pathways in Drosophila and Caenorhabditis elegans. To investigate the role of these proteins in mammalian signaling pathways, we have isolated and studied two murine dwarfins, dwarfin-A and dwarfin-C. Using antibodies against dwarfin-A and dwarfin-C, we show that these two dwarfins and an immunogenically related protein, presumably also a dwarfin, are phosphorylated in a time- and dose-dependent manner in response to TGF-beta. Bone morphogenetic protein 2, a TGF-beta superfamily ligand, induces phosphorylation of only the related dwarfin protein. Thus, TGF-beta superfamily members may use overlapping yet distinct dwarfins to mediate their intracellular signals. Furthermore, transient overexpression of either dwarfin-A or dwarfin-C causes growth arrest, implicating the dwarfins in growth regulation. This work provides strong biochemical and preliminary functional evidence that dwarfin-A and dwarfin-C represent prototypic members of a family of mammalian proteins that may serve as mediators of signaling pathways for TGF-beta superfamily members.

A human homologue of the Drosophila sex determination factor transformer-2 has conserved splicing regulatory functions.

Regulation of gene expression through alternative pre-mRNA splicing appears to occur in all metazoans, but most of our knowledge about splicing regulators derives from studies on genetically identified factors from Drosophila. Among the best studied of these is the transformer-2 (TRA-2) protein which, in combination with the transformer (TRA) protein, directs sex-specific splicing of pre-mRNA from the sex determination gene doublesex (dsx). Here we report the identification of htra-2 alpha, a human homologue of tra-2. Two alternative types of htra-2 alpha cDNA clones were identified that encode different protein isoforms with striking organizational similarity to Drosophila tra-2 proteins. When expressed in flies, one hTRA-2 alpha isoform partially replaces the function of Drosophila TRA-2, affecting both female sexual differentiation and alternative splicing of dsx pre-mRNA. Like Drosophila TRA-2, the ability of hTRA-2 alpha to regulate dsx is female-specific and depends on the presence of the dsx splicing enhancer. These results demonstrate that htra-2 alpha has conserved a striking degree of functional specificity during evolution and leads us to suggest that, although they are likely to serve different roles in development, the tra-2 products of flies and humans have similar molecular functions.

Hepatocyte growth factor is a coupling factor for osteoclasts and osteoblasts in vitro.

Hepatocyte growth factor (HGF), also known as scatter factor, is a powerful motogen, mitogen, and morphogen produced by cells of mesodermal origin, acting on epithelial and endothelial cells. Its receptor is the tyrosine kinase encoded by the c-MET protooncogene. We show that the HGF receptor is expressed by human primary osteoclasts, by osteoclast-like cell lines, and by osteoblasts. In both cell lineages, HGF stimulation triggers the receptor kinase activity and autophosphorylation. In osteoclasts, HGF receptor activation is followed by increase in intracellular Ca2+ concentration and by activation of the pp60c-Src kinase. HGF induces changes in osteoclast shape and stimulates chemotactic migration and DNA replication. Osteoblasts respond to HGF by entering the cell cycle, as indicated by stimulation of DNA synthesis. Interestingly, osteoclasts were found to synthesize and secrete biologically active HGF. These data strongly suggest the possibility of an autocrine regulation of the osteoclast by HGF and a paracrine regulation of the osteoblast by the HGF produced by the osteoclast.

Isolation of the human PC6 gene encoding the putative host protease for HIV-1 gp160 processing in CD4+ T lymphocytes.

Production of infectious HIV-1 virions is dependent on the processing of envelope glycoprotein gp160 by a host cell protease. The protease in human CD4+ T lymphocytes has not been unequivocally identified, yet members of the family of mammalian subtilisin-like protein convertases (SPCs), which are soluble or membrane-bound proteases of the secretory pathway, best fulfill the criteria. These proteases are required for proprotein maturation and cleave at paired basic amino acid motifs in numerous cellular and viral glycoprotein precursors, both in vivo and in vitro. To identify the gp160 processing protease, we have used reverse transcription-PCR and Northern blot analyses to ascertain the spectrum of SPC proteases in human CD4+ T cells. We have cloned novel members of the SPC family, known as the human PC6 genes. Two isoforms of the hPC6 protease are expressed in human T cells, hPC6A and the larger hPC6B. The patterns of SPC gene expression in human T cells has been compared with the furin-defective LoVo cell line, both of which are competent in the production of infectious HIV virions. This comparison led to the conclusion that the hPC6 gene products are the most likely candidates for the host cell protease responsible for HIV-1 gp160 processing in human CD4+ T cells.

Bioactivation of Müllerian inhibiting substance during gonadal development by a kex2/subtilisin-like endoprotease.

During male gonadal development Müllerian duct regression is mediated by the actions of the hormone Müllerian inhibiting substance (MIS), a member of the transforming growth factor beta superfamily. MIS is considered to be unique among members of this superfamily because bioactivation of MIS via proteolytic processing is hypothesized to occur at its target organ, the Müllerian duct. We find instead that the majority of MIS is processed and secreted from the embryonic testes as a complex in which the mature region remains noncovalently associated with the prodomain. In addition, we have identified two candidate endoproteases that are expressed in the testes and that may be capable of processing MIS in vivo. These kex2/subtilisin-like enzymes, PC5 and furin, are members of the proprotein convertase family that have been implicated in hormone bioactivation via proteolytic processing after dibasic amino acid cleavage recognition sites. Coexpression of PC5 and MIS in transfected mammalian cells results in efficient processing and bioactivation of MIS. Our results suggest that MIS is a natural substrate for PC5, thereby supporting a role for prohormone convertases in the activation of transforming growth factor beta-related hormones during development.

Induction of intestinal epithelial proliferation by glucagon-like peptide 2.

Injury, inflammation, or resection of the small intestine results in severe compromise of intestinal function. Nevertheless, therapeutic strategies for enhancing growth and repair of the intestinal mucosal epithelium are currently not available. We demonstrate that nude mice bearing subcutaneous proglucagon-producing tumors exhibit marked proliferation of the small intestinal epithelium. The factor responsible for inducing intestinal proliferation was identified as glucagon-like peptide 2 (GLP-2), a 33-aa peptide with no previously ascribed biological function. GLP-2 stimulated crypt cell proliferation and consistently induced a marked increase in bowel weight and villus growth of the jejunum and ileum that was evident within 4 days after initiation of GLP-2 administration. These observations define a novel biological role for GLP-2 as an intestinal-derived peptide stimulator of small bowel epithelial proliferation.

A family of lysozyme-like virulence factors in bacterial pathogens of plants and animals.

We describe a conserved family of bacterial gene products that includes the VirB1 virulence factor encoded by tumor-inducing plasmids of Agrobacterium spp., proteins involved in conjugative DNA transfer of broad-host-range bacterial plasmids, and gene products that may be involved in invasion by Shigella spp. and Salmonella enterica. Sequence analysis and structural modeling show that the proteins in this group are related to chicken egg white lysozyme and are likely to adopt a lysozyme-like structural fold. Based on their similarity to lysozyme, we predict that these proteins have glycosidase activity. Iterative data base searches with three conserved sequence motifs from this protein family detect a more distant relationship to bacterial and bacteriophage lytic transglycosylases, and goose egg white lysozyme. Two acidic residues in the VirB1 protein of Agrobacterium tumefaciens form a putative catalytic dyad, Each of these residues was changed into the corresponding amide by site-directed mutagenesis. Strains of A. tumefaciens that express mutated VirB1 proteins have a significantly reduced virulence. We hypothesize that many bacterial proteins involved in export of macromolecules belong to a widespread class of hydrolases and cleave beta-1,4-glycosidic bonds as part of their function.

The human multidrug resistance-associated protein functionally complements the yeast cadmium resistance factor 1.

A Saccharomyces cerevisiae strain with a disrupted yeast cadmium resistance factor (YCF1) gene (DTY168) is hypersensitive to cadmium. YCF1 resembles the human multidrug resistance-associated protein MRP (63% amino acid similarity), which confers resistance to various cytotoxic drugs by lowering the intracellular drug concentration. Whereas the mechanism of action of YCF1 is not known, MRP was recently found to transport glutathione S-conjugates across membranes. Here we show that expression of the human MRP cDNA in yeast mutant DTY168 cells restores cadmium resistance to the wild-type level. Transport of S-(2,4-dinitrobenzene)-glutathione into isolated yeast microsomal vesicles is strongly reduced in the DTY168 mutant and this transport is restored to wild-type level in mutant cells expressing MRP cDNA. We find in cell fractionation experiments that YCF1 is mainly localized in the vacuolar membrane in yeast, whereas MRP is associated both with the vacuolar membrane and with other internal membranes in the transformed yeast cells. Our results indicate that yeast YCF1 is a glutathione S-conjugate pump, like MRP, and they raise the possibility that the cadmium resistance in yeast involves cotransport of cadmium with glutathione derivatives.

HslV-HslU: A novel ATP-dependent protease complex in Escherichia coli related to the eukaryotic proteasome.

We have isolated a new type of ATP-dependent protease from Escherichia coli. It is the product of the heat-shock locus hslVU that encodes two proteins: HslV, a 19-kDa protein similar to proteasome beta subunits, and HslU, a 50-kDa protein related to the ATPase ClpX. In the presence of ATP, the protease hydrolyzes rapidly the fluorogenic peptide Z-Gly-Gly-Leu-AMC and very slowly certain other chymotrypsin substrates. This activity increased 10-fold in E. coli expressing heat-shock proteins constitutively and 100-fold in cells expressing HslV and HslU from a high copy plasmid. Although HslV and HslU could be coimmunoprecipitated from cell extracts of both strains with an anti-HslV antibody, these two components were readily separated by various types of chromatography. ATP stimulated peptidase activity up to 150-fold, whereas other nucleoside triphosphates, a nonhydrolyzable ATP analog, ADP, or AMP had no effect. Peptidase activity was blocked by the anti-HslV antibody and by several types of inhibitors of the eukaryotic proteasome (a threonine protease) but not by inhibitors of other classes of proteases. Unlike eukaryotic proteasomes, the HslVU protease lacked tryptic-like and peptidyl-glutamyl-peptidase activities. Electron micrographs reveal ring-shaped particles similar to en face images of the 20S proteasome or the ClpAP protease. Thus, HslV and HslU appear to form a complex in which ATP hydrolysis by HslU is essential for peptide hydrolysis by the proteasome-like component HslV.

The multidomain protein Trio binds the LAR transmembrane tyrosine phosphatase, contains a protein kinase domain, and has separate rac-specific and rho-specific guanine nucleotide exchange factor domains.

rho-like GTP binding proteins play an essential role in regulating cell growth and actin polymerization. These molecular switches are positively regulated by guanine nucleotide exchange factors (GEFs) that promote the exchange of GDP for GTP. Using the interaction-trap assay to identify candidate proteins that bind the cytoplasmic region of the LAR transmembrane protein tyrosine phosphatase (PT-Pase), we isolated a cDNA encoding a 2861-amino acid protein termed Trio that contains three enzyme domains: two functional GEF domains and a protein serine/threonine kinase (PSK) domain. One of the Trio GEF domains (Trio GEF-D1) has rac-specific GEF activity, while the other Trio GEF domain (Trio GEF-D2) has rho-specific activity. The C-terminal PSK domain is adjacent to an Ig-like domain and is most similar to calcium/calmodulin-dependent kinases, such as smooth muscle myosin light chain kinase which similarly contains associated Ig-like domains. Near the N terminus, Trio has four spectrin-like repeats that may play a role in intracellular targeting. Northern blot analysis indicates that Trio has a broad tissue distribution. Trio appears to be phosphorylated only on serine residues, suggesting that Trio is not a LAR substrate, but rather that it forms a complex with LAR. As the LAR PTPase localizes to the ends of focal adhesions, we propose that LAR and the Trio GEF/PSK may orchestrate cell-matrix and cytoskeletal rearrangements necessary for cell migration.