Comparison of the anthrax toxin lethal factor (LF) amino acid sequence with sequences in the Swiss protein database revealed short regions of similarity with the consensus zinc-binding site, HEXXH, that is characteristic of metalloproteases. Several protease inhibitors, including bestatin and captopril, prevented intoxication of macrophages by lethal toxin. LF was fully inactivated by site-directed mutagenesis that substituted Ala for either of the residues (H-686 and H-690) implicated in zinc binding. Similarly, LF was inactivated by substitution of Cys for E-687, which is thought to be an essential part of the catalytic site. In contrast, replacement of E-720 and E-721 with Ala had no effect on LF activity. LF bound 65Zn both in solution and on protein blots. The 65Zn binding was reduced for several of the LF mutants. These data suggest that anthrax toxin LF is a zinc metallopeptidase, the catalytic function of which is responsible for the lethal activity observed in cultured cells and in animals.
These results may be used to help predict the outcome of surgical treatment of FAI in different patient populations and to assess the need for labral refixation.
Nonstructural protein 4B (NS4B) plays an essential role in the formation of the hepatitis C virus (HCV) replication complex. It is a relatively poorly characterized integral membrane protein predicted to comprise four transmembrane segments in its central portion. Here, we describe a novel determinant for membrane association represented by amino acids (aa) 40 to 69 in the N-terminal portion of NS4B. This segment was sufficient to target and tightly anchor the green fluorescent protein to cellular membranes, as assessed by fluorescence microscopy as well as membrane extraction and flotation analyses. Circular dichroism and nuclear magnetic resonance structural analyses showed that this segment comprises an amphipathic ␣-helix extending from aa 42 to 66. Attenuated total reflection infrared spectroscopy and glycosylation acceptor site tagging revealed that this amphipathic ␣-helix has the potential to traverse the phospholipid bilayer as a transmembrane segment, likely upon oligomerization. Alanine substitution of the fully conserved aromatic residues on the hydrophobic helix side abrogated membrane association of the segment comprising aa 40 to 69 and disrupted the formation of a functional replication complex. These results provide the first atomic resolution structure of an essential membrane-associated determinant of HCV NS4B.With 120 to 180 million chronically infected individuals worldwide, hepatitis C virus (HCV) infection represents a major cause of chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma (38). HCV contains a 9.6-kb positive-strand RNA genome that encodes a polyprotein of about 3,000 amino acids (reviewed in references 36 and 51). The polyprotein precursor is co-and posttranslationally processed by cellular and viral proteases to yield the mature structural and nonstructural proteins. The structural proteins include the core and the envelope glycoproteins E1 and E2. The nonstructural proteins include the p7 ion channel polypeptide, the NS2-3 and NS3-4A proteases, an RNA helicase located in the C-terminal twothirds of NS3, the NS4B and NS5A proteins, and the NS5B RNA-dependent RNA polymerase. HCV replication takes place in a membrane-associated complex, composed of viral proteins, replicating RNA, altered cellular membranes, and other host factors (7,18,31,43). Determinants for membrane association of the HCV nonstructural proteins have been mapped and a likely endoplasmic reticulum (ER)-derived membrane alteration, designated the membranous web, was found to harbor the HCV replication complex (7, 18; reviewed in reference 36).NS4B is a 27-kDa integral ER membrane protein (21). The expression of NS4B alone induces the formation of the membranous web (7). Thus, an essential function of NS4B is the induction of the specific membrane alteration that serves as a scaffold for the HCV replication complex. In addition, a nucleotide-binding motif has been proposed to reside in the middle of NS4B (8), and RNA binding properties have recently been reported for NS4B (9).Both the N and the C t...
Before intoxication can occur, anthrax toxin protective antigen (PA), Pseudomonas exotoxin A (PE), and diphtheria toxin (DT) must be activated by proteolytic cleavage at specific amino acid sequences. Previously, it was shown that PA and DT can be activated by furin. In Chinese hamster ovary (CHO) cells, wild-type (RKKR) and cleavage site mutants of PA, each administered with a modified form of anthrax toxin lethal factor (the N terminus of lethal factor fused to PE domain III), had the following potencies: RKKR (wild type) (concentration causing 50% cell death [EC 50 ] ؍ 12 ng/ml) Ն RAAR (EC 50 ؍ 18 ng/ml) > FTKR (EC 50 ؍ 24 ng/ml) > STRR (EC 50 ؍ 49 ng/ml). In vitro cleavage of PA and cleavage site mutants of PA by furin demonstrated that native PA (RKKR) and PA with the cleavage sequence RAAR are substrates for furin. To characterize eukaryotic proteases that play a role in activating bacterial toxins, furin-deficient CHO cells were selected after chemical mutagenesis. Furin-deficient cells were resistant to PE, whose cleavage site, RQPR, constitutes a furin recognition site and to all PA cleavage site mutants, but were sensitive to DT (EC 50 ؍ 2.9 ng/ml) and PA (EC 50 ؍ 23 ng/ml), whose respective cleavage sites, RKKR and RVRR, contain additional basic residues. Furin-deficient cells that were transfected with the furin gene regained sensitivity to PE and PA cleavage site mutants. These studies provide evidence that furin can activate the three toxins and that one or more additional proteases contribute to the activation of DT and PA.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
customersupport@researchsolutions.com
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
This site is protected by reCAPTCHA and the Google Privacy Policy and Terms of Service apply.
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.