Conformational Switching in Bcl-xL: Enabling Non-Canonic Inhibition of Apoptosis Involves Multiple Intermediates and Lipid Interactions.

The inhibition of mitochondrial permeabilization by the anti-apoptotic protein Bcl-xL is crucial for cell survival and homeostasis. Its inhibitory role requires the partitioning of Bcl-xL to the mitochondrial outer membrane from an inactive state in the cytosol, leading to its extensive refolding. The molecular mechanisms behind these events and the resulting conformations in the bilayer are unclear, and different models have been proposed to explain them.

Lipid-modulation of membrane insertion and refolding of the apoptotic inhibitor Bcl-xL.

Bcl-xL is a member of the Bcl-2 family of apoptotic regulators, responsible for inhibiting the permeabilization of the mitochondrial outer membrane, and a promising anti-cancer target. Bcl-xL exists in the following conformations, each believed to play a role in the inhibition of apoptosis: (a) a soluble folded conformation, (b) a membrane-anchored (by its C-terminal α8 helix) form, which retains the same fold as in solution and (c) refolded membrane-inserted conformations, for which no structural data are available. Previous studies established that in the cell Bcl-xL exists in a dynamic equilibrium between soluble and membranous states, however, no direct evidence exists in support of either anchored or inserted conformation of the membranous state in vivo.

Cellular Entry of the Diphtheria Toxin Does Not Require the Formation of the Open-Channel State by Its Translocation Domain.

Cellular entry of diphtheria toxin is a multistage process involving receptor targeting, endocytosis, and translocation of the catalytic domain across the endosomal membrane into the cytosol. The latter is ensured by the translocation (T) domain of the toxin, capable of undergoing conformational refolding and membrane insertion in response to the acidification of the endosomal environment. While numerous now classical studies have demonstrated the formation of an ion-conducting conformation-the Open-Channel State (OCS)-as the final step of the refolding pathway, it remains unclear whether this channel constitutes an in vivo translocation pathway or is a byproduct of the translocation.

Effect of nordihydroguaiaretic acid on cell viability and glucose transport in human leukemic cell lines.

The polyphenol nordihydroguaiaretic acid (NDGA) has antineoplastic properties, hence it is critical to understand its action at the molecular level. Here, we establish that NDGA inhibits glucose uptake and cell viability in leukemic HL-60 and U-937 cell lines. We monitored hexose uptake using radio-labeled 2-deoxyglucose (2DG) and found that the inhibition by NDGA followed a noncompetitive mechanism.

Role of acidic residues in helices TH8-TH9 in membrane interactions of the diphtheria toxin T domain.

The pH-triggered membrane insertion of the diphtheria toxin translocation domain (T domain) results in transferring the catalytic domain into the cytosol, which is relevant to potential biomedical applications as a cargo-delivery system. Protonation of residues is suggested to play a key role in the process, and residues E349, D352 and E362 are of particular interest because of their location within the membrane insertion unit TH8-TH9. We have used various spectroscopic, computational and functional assays to characterize the properties of the T domain carrying the double mutation E349Q/D352N or the single mutation E362Q.

Membrane Association of the Diphtheria Toxin Translocation Domain Studied by Coarse-Grained Simulations and Experiment.

Diphtheria toxin translocation (T) domain inserts in lipid bilayers upon acidification of the environment. Computational and experimental studies have suggested that low pH triggers a conformational change of the T-domain in solution preceding membrane binding. The refolded membrane-competent state was modeled to be compact and mostly retain globular structure.

Thermodynamics of Membrane Insertion and Refolding of the Diphtheria Toxin T-Domain.

The diphtheria toxin translocation (T) domain inserts into the endosomal membrane in response to the endosomal acidification and enables the delivery of the catalytic domain into the cell. The insertion pathway consists of a series of conformational changes that occur in solution and in the membrane and leads to the conversion of a water-soluble state into a transmembrane state. In this work, we utilize various biophysical techniques to characterize the insertion pathway from the thermodynamic perspective.

Comparison of membrane insertion pathways of the apoptotic regulator Bcl-xL and the diphtheria toxin translocation domain.

The diphtheria toxin translocation domain (T-domain) and the apoptotic repressor Bcl-xL are membrane proteins that adopt their final topology by switching folds from a water-soluble to a membrane-inserted state. While the exact molecular mechanisms of this transition are not clearly understood in either case, the similarity in the structures of soluble states of the T-domain and Bcl-xL led to the suggestion that their membrane insertion pathways will be similar, as well. Previously, we have applied an array of spectroscopic methods to characterize the pH-triggered refolding and membrane insertion of the diphtheria toxin T-domain.

pH-triggered conformational switching of the diphtheria toxin T-domain: the roles of N-terminal histidines.

pH-induced conformational switching is essential for functioning of diphtheria toxin, which undergoes a membrane insertion/translocation transition triggered by endosomal acidification as a key step of cellular entry. In order to establish the sequence of molecular rearrangements and side-chain protonation accompanying the formation of the membrane-competent state of the toxin's translocation (T) domain, we have developed and applied an integrated approach that combines multiple techniques of computational chemistry [e.g.

Crucial role of H322 in folding of the diphtheria toxin T-domain into the open-channel state.

The translocation (T) domain plays a key role in the entry of diphtheria toxin into the cell. Upon endosomal acidification, the T-domain undergoes a series of conformational changes that lead to its membrane insertion and formation of a channel. Recently, we have reported that the triple replacement of C-terminal histidines H322, H323, and H372 with glutamines prevents the formation of open channels in planar lipid bilayers.

Resolution of the direct interaction with and inhibition of the human GLUT1 hexose transporter by resveratrol from its effect on glucose accumulation.

Resveratrol acts as a chemopreventive agent for cancer and as a potential antiobesity and antidiabetic compound, by leading to reduced body fat and improved glucose homeostasis. The exact mechanisms involved in improving hyperglycemic state are not known, but most of the glucose uptake into mammalian cells is facilitated by the GLUT hexose transporters. Resveratrol is structurally similar to isoflavones such as genistein, which inhibit the glucose uptake facilitated by the GLUT1 hexose transporter.

[Rhabdomyomatous mesenchymal hamartoma].

Rhabdomyomatous mesenchymal hamartoma is a rare congenital lesion which consists of randomly arranged striated muscle fibers interspersed with mesenchymal elements. We describe the clinical and histopathological features of a rhabdomyomatous mesenchymal hamartoma in a one year-old patient presenting a bilobulated lesion in the mid-cervical line. No associated congenital malformations were observed.

Noncompetitive blocking of human GLUT1 hexose transporter by methylxanthines reveals an exofacial regulatory binding site.

Glucose transporter (GLUT)1 has become an attractive target to block glucose uptake in malignant cells since most cancer cells overexpress GLUT1 and are sensitive to glucose deprivation. Methylxanthines are natural compounds that inhibit glucose uptake; however, the mechanism of inhibition remains unknown. Here, we used a combination of binding and glucose transport kinetic assays to analyze in detail the effects of caffeine, pentoxifylline, and theophylline on hexose transport in human erythrocytes.

Replacement of C-terminal histidines uncouples membrane insertion and translocation in diphtheria toxin T-domain.

The translocation (T) domain plays a key role in the action of diphtheria toxin and is responsible for transferring the N-terminus-attached catalytic domain across the endosomal membrane into the cytosol in response to acidification. The T-domain undergoes a series of pH-triggered conformational changes that take place in solution and on the membrane interface, and ultimately result in transbilayer insertion and N-terminus translocation. Structure-function studies along this pathway have been hindered because the protein population occupies multiple conformations at the same time.

Folding of diphtheria toxin T-domain in the presence of amphipols and fluorinated surfactants: Toward thermodynamic measurements of membrane protein folding.

Solubilizing membrane proteins for functional, structural and thermodynamic studies is usually achieved with the help of detergents, which, however, tend to destabilize them. Several classes of non-detergent surfactants have been designed as milder substitutes for detergents, most prominently amphipathic polymers called 'amphipols' and fluorinated surfactants. Here we test the potential usefulness of these compounds for thermodynamic studies by examining their effect on conformational transitions of the diphtheria toxin T-domain.