Corresponding authorCommunicated by E.NeherThe interaction of the presynaptic membrane proteins SNAP-25 and syntaxin with the synaptic vesicle protein synaptobrevin (VAMP) plays a key role in the regulated exocytosis of neurotransmitters. Clostridial neurotoxins, which proteolyze these polypeptides, are potent inhibitors of neurotransmission. The cytoplasmic domains of the three membrane proteins join into a tight SDS-resistant complex .Here, we show that this reconstituted complex, as well as heterodimers composed of syntaxin and SNAP-25, can be disassembled by the concerted action of the Nethylmaleimide-sensitive factor, NSF, and the soluble NSF attachment protein, a-SNAP. a-SNAP binds to predicted a-helical coiled-coil regions of syntaxin and SNAP-25, shown previously to be engaged in their direct interaction. Synaptobrevin, although incapable of binding a-SNAP individually, induced a third a-SNAP binding site when associated with syntaxin and SNAP-25 into heterotrimers. NSF released prebound a-SNAP from full-length syntaxin but not from a syntaxin derivative truncated at the N-terminus. Disassembly of complexes containing this syntaxin mutant was impaired, indicating a critical role for the Nterminal domain in the a-SNAP/NSF-mediated dissociation process. Complexes containing C-terminally deleted SNAP-25 derivatives, as generated by botulinal toxins type A and E, were dissociated more efficiently. In contrast, the N-terminal fragment generated from synaptobrevin by botulinal toxin type F produced an SDS-sensitive complex that was poorly dissociated.
Abstract:Tetanus toxin and the seven serologically distinct botulinal neurotoxins (BoNT/A to BoNT/G) abrogate synaptic transmission at nerve endings through the action of their light chains (L chains), which proteolytically cleave VAMP (vesicle-associated membrane protein)/ synaptobrevin, SNAP-25 (synaptosome-associated protein of 25 kDa), or syntaxin. BoNT/C was reported to proteolyze both syntaxin and SNAP-25. Clostridia produce several powerful neurotoxins; tetanus toxin and botulinal neurotoxins BoNT/A to BoNT/G cause the clinical manifestations of tetanus and botulism in a large variety of animal species and humans. The toxins are synthesized as single-chain polypeptides with molar masses of ϳ150 kDa. On lysis of the bacteria and activation by proteolytic cleavage, the light chains (L chains; 50 kDa) remain disulfide bound to the heavy chains (H chains; 100 kDa). The extreme neurotoxicity is largely ascribed to the H chains, which bind to neuronal receptors that internalize the holotoxins, and translocate the L chains into the cytosol. Here, the L chains block fusion of synaptic vesicles with the presynaptic membrane (Simpson, 1989).The genes of the eight known clostridial neurotoxins have been cloned and characterized (for review, see Niemann et al., 1994). The L chains contain a Zn 2ϩ -binding motif, His-Glu-X-X-His, also found in an increasing number of zinc-dependent metalloproteases (Jongeneel et al., 1989). Soon after demonstration of cleavage of VAMP (vesicle-associated membrane protein)/synaptobrevin (Trimble et al., 1988) by tetanus toxin and BoNT/B at the same peptide bond (Link et al., 1992;Schiavo et al., 1992), substrates and scissile bonds were identified for all other botulinum serotypes. These studies revealed that BoNT/D, BoNT/F, and BoNT/G also hydrolyze synaptobrevin, although each at a different peptide bond. BoNT/A and BoNT/E cleave SNAP-25 (synaptosome-associated protein of 25 kDa), again at distinct sites close to the C-terminus (for review, see Received March 20, 1998; revised manuscript received July 20, 1998; accepted July 23, 1998. Address correspondence and reprint requests to Dr. T. Binz at Department of Biochemistry, Medizinische Hochschule Hannover, Carl-Neuberg-Straße 1, D-30623, Hannover, Germany.The present address of Dr. V. V. Vaidyanathan is Experimental Immunology Branch, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892, U.S.A.Abbreviations used: BoNT, botulinal neurotoxin; GST, glutathione S-transferase; GT, glutathione; L chain, light chain; NSF, Nethylmaleimide sensitive fusion protein; PAGE, polyacrylamide gel electrophoresis; SDS, sodium dodecyl sulfate; SNAP, soluble NSF attachment protein; SNAP-23, 23-kDa isoform of SNAP-25; SNAP-25, synaptosome-associated protein of 25 kDa; hSNAP-23 and mSNAP-23, human and murine SNAP-23, respectively; SNARE, soluble NSF attachment protein receptor; VAMP, vesicle-associated membrane protein.
Changes in the cellular carbohydrate metabolism are a hallmark of malignant transformation and represent one of the earliest discernible events in tumorigenesis. In the early stages of certain epithelial cancers, a metabolic switch is regularly observed, in which slowly growing glycogenotic cells are converted to highly proliferating basophilic cells. This step is accompanied by a rapid depletion of the intracellular glycogen stores, which in liver carcinogenesis results from the activation of the enzyme acid ␣-glucosidase by an as yet unknown mechanism. We show here that acid ␣-glucosidase is a target for the E7 protein encoded by human papillomavirus type 16, a human tumor virus that plays a key role in the genesis of cervical carcinoma. We show that expression of E7 induces the catalytic activity of acid ␣-glucosidase in vivo and wild type E7, but not transformation-deficient mutants bind directly to acid ␣-glucosidase and increase the catalytic activity of the enzyme in vitro. The data suggest that the E7 protein encoded by human papillomavirus type 16 can act as an allosteric activator of acid ␣-glucosidase.It has long been known that tumor cells display characteristic alterations of the carbohydrate metabolism (for review see Ref. 1), which represent one of the earliest discernible events in tumorigenesis (for recent review see Ref.2). In certain epithelial cancers, such as liver cancer or kidney cancer, one of the first detectable alterations is a metabolic switch, in which slowly growing glycogenotic cells, also referred to as clear cells, are converted to highly proliferating basophilic cells (for review see Ref. 3). This step is accompanied by a rapid depletion of the intracellular glycogen stores, which in liver carcinogenesis results from the activation of acid ␣-glucosidase (4), whereas the activity of glycogen phosphorylase, the other cellular glycogendegrading enzyme, is reduced throughout hepatocarcinogenesis (5). The actual content of glycogen in a cell is controlled through the balance of glycogen-synthesizing enzymes and glycogen-degrading enzymes, which themselves are under allosteric control by various metabolites (6). It is unknown at present how the activity of these enzymes is modulated in early carcinogenesis, to first build up the clear cell phenotype and then trigger its disappearance at later stages. According to the current concept (7), the deregulation of metabolic enzymes in early tumor cells reflects changes in cellular signal transduction which lead to tumor-specific alterations of the metabolic apparatus.Reduced glycogen storage is regularly observed in early lesions of the cervix, a finding that has been used for clinical diagnosis of cervical dysplasia for more than 60 years (8). Cervical neoplasia is tightly linked to infection by human papillomaviruses (HPV)1 of the high risk group, e.g. HPV-16 (9), and two viral genes, E6 and E7, are required for papillomavirus-associated carcinogenesis (for review see Refs. 9 and 10). The E7 oncogene of HPV-16 cooperates with HPV-16 E6 to ...
Human papillomaviruses (HPV) of the high-risk type are causally involved in human tumors, in particular cervical carcinoma. Expression of the viral oncogenes E6 and E7 is maintained in HPV-positive tumors, and it was shown that E6 and E7 of HPV-16 can immortalize human keratinocytes, the natural host cells of the virus. Expression of the viral genes is also required for maintenance of the transformed phenotype. The oncogenic activity of the E6 and E7 oncoproteins is mediated by their physical and functional interaction with cellular regulatory proteins. To knock out the function of the E7 protein in living cells, we have developed peptide aptamers with high specific binding activity for the E7 protein of HPV-16. We show here that E7-binding peptide aptamers induce programmed cell death (apoptosis) in E7-expressing cells, whereas E7-negative cells are not affected. Furthermore, E7-binding peptide aptamers induce apoptosis in HPV-16-positive tumor cells derived from cervical carcinoma. The data suggest that E7-binding peptide aptamers may be useful tools to specifically eliminate HPV-positive tumors.
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