Structural changes of tumor necrosis factor alpha associated with membrane insertion and channel formation.

Abstract

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.

Differential acute and chronic responses of tumor necrosis factor-deficient mice to experimental brain injury

“…In their study, Bruce et al (16) observed similar injury-induced increases in TNF protein in both wt and TNF receptor-deficient mice. The possibility exists that the prolonged presence of unbound TNF may induce pathologic cellular changes in a receptor-independent fashion (71,72). Alternatively, the differences between our observation and those of Bruce et al may be due, in part, to differences in the injury models, the time course of the pathological changes, and/or the strain of mice used.…”

A Novel Tumor Necrosis Factor–mediated Mechanism of Direct Epithelial Sodium Channel Activation

“…TNF can, however, increase ALC in mice lacking both TNF receptors (24), strongly suggesting TNF receptor-independent actions of the cytokine. Moreover, TNF has a membrane insertion capacity (54). Also, intra-alveolar catabolism is an important mechanism of protein removal, which is particularly important during recovery from pulmonary edema (55).…”

Acute bacterial pneumonia in rats increases alveolar epithelial fluid clearance by a tumor necrosis factor-alpha-dependent mechanism.

“…However, interestingly, TNF␣ does regulate G proteins (55), and recently G proteins have been reported to regulate amiloride-sensitive sodium uptake in fetal alveolar type II epithelial cells (56). There is also recent evidence that TNF-␣ can increase uptake of sodium into cells by inserting an ion channel into cell membranes (57). In summary, acute P. aeruginosa pneumonia in rats results in a marked upregulation of the rate of net alveolar epithelial sodium and fluid clearance.…”