The low-density-lipoprotein receptor-related protein (LRP) is a multifunctional receptor involved in the clearance of a large number of diverse ligands, including proteases, proteaseinhibitor complexes and lipoproteins. The mature receptor is composed of a 515 kDa and a 85 kDa subunit generated by proteolytic cleavage from a 600 kDa precursor polypeptide in a trans-Golgi compartment. Proteolytic processing occurs C-terminal to the tetrabasic amino acid sequence RHRR, a consensus recognition site for precursor processing endoproteases or convertases. In this study we have identified furin, a subtilisintype protease, to be necessary for efficient processing of LRP in
Neural tube defects in infants of diabetic mothers constitute an important and frequent cause of neonatal mortality/morbidity and long-term chronic handicaps. The mechanism by which normal neural tube fusion occurs is not known. The failure of rostral neural tube fusion seen in mouse embryos incubated in the presence of excess-D-glucose can be significantly prevented by the supplementation of myo-inositol to the culture medium. This protective effect of myo-inositol is reversed by indomethacin, an inhibitor of arachidonic acid metabolism leading to prostaglandin synthesis. Prostaglandin E2 added to the culture medium completely protects against the glucose-induced neural tube defect. These data suggest that the failure of neural tube fusion seen in diabetic embryopathy is mediated through a mechanism involving abnormalities in both the myo-inositol and arachidonic acid pathways, resulting in a functional deficiency of prostaglandins at a critical time of neural tube fusion.
Diphthamide, a unique amino acid, is a post-translational derivative of histidine that exists in protein synthesis elongation factor 2 at the site of diphtheria toxin-catalyzed ADP-ribosylation of elongation factor 2. We investigated steps in the biosynthesis of diphthamide with mutants of Chinese hamster ovary cells that were altered in different steps of this complex post-translational modification. Biochemical evidence indicates that this modification requires a minimum of three steps, two of which we accomplished in vitro. We identified a methyltransferase activity that transfers methyl groups from S-adenosyl methionine to an unmethylated form of diphthine (the deamidated form of diphthamide), and we tentatively identified an ATP-dependent synthetase activity involved in the biosynthesis of diphthamide from diphthine. Our results are in accord with the proposed structure of diphthamide (B. G. VanNess, et al., J. Biol. Chem. 255:10710-10716, 1980).
We addressed the question of whether furin is the endoprotease primarily responsible for processing the human immunodeficiency virus type I (HIV-I) envelope protein gp160 in mammalian cells. The furin-deficient Chinese hamster ovary (CHO)-K1 strain RPE.40 processed gp160 as efficiently as wild-type CHO-K1 cells in vivo. Although furin can process gp160 in vitro, this processing is probably not physiologically relevent, because it occurs with very low efficiency. PACE4, a furin homologue, allowed processing of gp160 when both were expressed in RPE.40 cells. Further, PACE4 participated in the activation of a calcium-independent protease activity in RPE.40 cells, which efficiently processed the gp160 precursor in vitro. This calcium-independent protease activity was not found in another furin-deficient cell strain, 7.P15, selected from the monkey kidney cell line COS-7.
Like many physiological ligands, several viruses and toxins enter mammalian cells through receptor-mediated endocytosis. Once internalized, the nucleic acids of several viruses and the toxic subunit of diphtheria toxin gain access to the cytosol of the host cell through an acidic intracellular compartment. In this report, we present evidence that one class of mutants of Chinese hamster ovary (CHO)-K1 cells, which is "cross-resistant" to Pseudomonas exotoxin A, diphtheria toxin, and several animal viruses, has a defect in acidification of the endosome. Cells were allowed to internalize fluorescein isothiocyanate-conjugated dextran before subcellular fractionation. Fluorescence measurements on subcellular fractions permitted measurement of the internal pH of the isolated endosomes and lysosomes. Our results show that (i) endosomes and lysosomes from CHO-KI cells maintain an acidic pH, (ii) acidification of both endosomes and lysosomes is mediated by a Mg2"/ATP-dependent process, (iii) GTP can satisfy the ATP requirement for acidification of lysosomes but not of endosomes, and (iv) at leastone class of mutants thatis cross-resistant to toxins and animal viruses has a defect in the ATP-dependent acidification of their endosomes. These studies provide biochemical and genetic evidence that the mechanisms of acidification of endosomes and lysosomes are distinct and that a defect in acidification of endosomes is one biochemical basis for cross-resistance to toxins and viruses.Receptor-mediated endocytosis is a specific cell-directed mechanism by which extracellular substances can enter the intracellular vesicular system (1, 2). Steps in the entry pathway include (i) binding to cell surface receptors; (ii) internalization within coated vesicles, (iii) distribution of ligand to appropriate intracellular compartments; and (iv) in many-cases, return of the receptor to the cell surface. For some pathogenic viruses and toxins, step iii includes transfer of a portion of the ligand to the cytosol (3-8). Recently, it has become apparent that this transfer, which is critical for the biologic activity of these viruses and toxins, requires an acidic environment (7-13). Although lysosomes are known to be acidic (14,15), recent evidence (16) 5315The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
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