Tumor necrosis factor-alpha (TNF, cachectin), a protein secreted by activated macrophages, participates in inflammatory responses and in infectious and neoplastic disease states. The mechanisms by which TNF exerts cytotoxic, hormonal, and other specific effects are obscure. Structural studies of the TNF trimer have revealed a central pore-like region. Although several amino acid side chains appear to preclude an open channel, the ability of TNF to insert into lipid vesicles raised the possibility that opening might occur in a bilayer milieu. Acidification of TNF promoted conformational changes concordant with increased surface hydrophobicity and membrane insertion. Furthermore, TNF formed pH-dependent, voltage-dependent, ion-permeable channels in planar lipid bilayer membranes and increased the sodium permeability of human U937 histiocytic lymphoma cells. Thus, some of the physiological effects of TNF may be elicited through its intrinsic ion channel-forming activity.
Low pH enhances tumor necrosis factor a (TNF)-induced cytolysis of cancer cells and TNF-membrane interactions that include binding, insertion, and ion-channel formation. We have also found that TNF increases Na+ influx in cells. Here, we examined the structural features of the TNF-membrane interaction pathway that lead to channel formation. Fluorometric studies link TNF's acid-enhanced membrane interactions to rapid but reversible acquisition of hydrophobic surface properties. Intramembranous photolabeling shows that (i) protonation of TNF promotes membrane insertion, (ii) the physical state of the target bilayer affects the kinetics and efficiency of TNF insertion, and (iii) binding and insertion of TNF are two distinct events. Acidification relaxes the trimeric structure of soluble TNF so that the cryptic carboxyl termini, centrally located at the base of the trimer cone, become susceptible to carboxypeptidase Y. After membrane insertion, TNF exhibits a trimeric configuration in which the carboxyl termini are no longer exposed; however, the proximal salt-bridged Lys-li residues as well as regional surface amino acids are notably more accessible to proteases. The sequenced cleavage products bear the membrane-restricted photoreactive probe, proof that surface-cleaved TNF has an intramembranous disposition. In summary, the trimer's structural plasticity is a major determinant of its channel-forming ability. Channel formation occurs when cracked or partially splayed trimers bind and penetrate the bilayer. Reannealing leads to a slightly relaxed trimeric structure. The directionality of bilayer penetration conforms with x-ray data showing that receptor binding to the monomer interfaces of TNF poises the tip of the trimeric cone directly above the target cell membrane.
There has been much speculation about the mechanism by which cholera toxin exerts its effect on the cytoplasmic side of the membranes with which it interacts. After the pentamer of B subunits (5B) binds to membrane receptors, particularly the monosialylganglioside GM1, the disulphide-linked dimer A1SSA2 (which together with 5B constitutes the complete toxin) is thought to penetrate the membrane, perhaps through a channel formed by 5B and become reduced so that A1SH units reach the cytoplasm and stimulate adenylate cyclase. Evidence for this mechanism is circumstantial. If it is correct, a compound which will specifically label intramembranous sections of the toxin should label the channel-forming B subunits but not the channel-contained A1 subunit. We have tested this prediction with a photoreactive glycolipid compound and have obtained the opposite result. Therefore, we propose that only the A1 subunit enters the membrane and we provide here data on the kinetics of that process.
Diphtheria toxin (DTx) is an extremely potent inhibitor of protein synthesis. It While fragment A is an active inhibitor of cell-free protein synthesis, it requires fragment B for entry into intact cells. Fragment B is the binding-recognition subunit. The receptor for B has been identified as a glycoprotein in several cell types (2). However, toxin has been shown to bind to proteinfree membranes under low pH conditions (3) and the functional insertion of A has been detected in liposome targets (J. J. Donovan, personal communication). Conductance studies with a segment of DTx called cross-reacting material 45 (CRM45) led to the proposal that B forms a membrane channel through which A travels to the cytosol (4, 5). CRM45 is produced by C. diphtheriae cells lysogenic for the mutant phage /345 and unlike DTx has an exposed hydrophobic domain on a shortened B segment (5). CRM45 is relatively nontoxic to intact cells, apparently lacking the cell surface recognition domain (6). Kagan and co-workers observed that at low pH the CRM45 B segment as well as CRM45 itself formed cation-selective channels in planar lipid membranes and 18-A pores in liposomes (4, 7). In contrast, Donovan et al. (8) found that native DTx forms anion-selective channels under acidic conditions and concluded these pores were too small (5 A) to allow A to cross the membrane. While the calculated channel size has been questioned, to our knowledge no other pore size determinations have been performed with native DTx.Because direct information about the nature of toxinmembrane interactions is critical to solving the entry pathway, we used a photoreactive glycolipid probe (9, 10) to ascertain which of the two domains of the toxin penetrates the membrane bilayer. These experiments were carried out with simple biological and artificial targets and both cleaved and uncleaved forms of DTx. The effect of low pH on toxin entry was also monitored in light of previous models of lysosomal involvement in the toxin entry process (4, 11-13). Sendai virus served as the biological target. Our preliminary studies showed that DTx binds to paramyxoviruses (Sendai and Newcastle disease virus) and the photolabeling of viral envelope proteins is independent of temperature and pH in the ranges employed. Parallel studies of the rates of diffusion of different-sized solutes through liposomes containing toxin were performed to assess the relative effectiveness of cleaved and uncleaved toxin at channel formation. Use of a well-established liposomal swelling assay (14, 15) enabled us to delineate the size and structure of the resultant channels.
Diphtheria toxin (DTx) provokes extensive internucleosomal degradation of DNA before cell lysis. The possibility that DNA cleavage stems from direct chromosomal attack by intracellular toxin molecules was tested by in vitro assays for a DTx-associated nuclease activity. DTx incubated with DNA in solution or in a DNA-gel assay showed Ca2+- and Mg2+-stimulated nuclease activity. This activity proved susceptible to inhibition by specific antitoxin and migrated with fragment A of the toxin. Assays in which supercoiled double-stranded DNA was used revealed rapid endonucleolytic attack. Discovery of a DTx-associated nuclease activity lends support to the model that DTx-induced cell lysis is not a simple consequence of protein synthesis inhibition.
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