Structural basis of BAK activation in mitochondrial apoptosis initiation

BCL-2 proteins regulate mitochondrial poration in apoptosis initiation. How the pore-forming BCL-2 Effector BAK is activated remains incompletely understood mechanistically. Here we investigate autoactivation and direct activation by BH3-only proteins, which cooperate to lower BAK threshold in membrane poration and apoptosis initiation. We define in trans BAK autoactivation as the asymmetric “BH3-in-groove” triggering of dormant BAK by active BAK. BAK autoactivation is mechanistically similar to direct activation. The structure of autoactivated BAK BH3-BAK complex reveals the conformational changes leading to helix α1 destabilization, which is a hallmark of BAK activation. Helix α1 is destabilized and restabilized in structures of BAK engaged by rationally designed, high-affinity activating and inactivating BID-like BH3 ligands, respectively. Altogether our data support the long-standing hit-and-run mechanism of BAK activation by transient binding of BH3-only proteins, demonstrating that BH3-induced structural changes are more important in BAK activation than BH3 ligand affinity.

Peptide Triazole Thiol Irreversibly Inactivates Metastable HIV-1 Env by Accessing Conformational Triggers Intrinsic to Virus–Cell Entry

KR13, a peptide triazole thiol previously established to inhibit HIV-1 infection and cause virus lysis, was evaluated by flow cytometry against JRFL Env-presenting cells to characterize induced Env and membrane transformations leading to irreversible inactivation. Transiently transfected HEK293T cells were preloaded with calcein dye, treated with KR13 or its thiol-blocked analogue KR13b, fixed, and stained for gp120 (35O22), MPER (10E8), 6-helix-bundle (NC-1), immunodominant loop (50-69), and fusion peptide (VRC34.01). KR13 induced dose-dependent transformations of Env and membrane characterized by transient poration, MPER exposure, and 6-helix-bundle formation (analogous to native fusion events), but also reduced immunodominant loop and fusion peptide exposure. Using a fusion peptide mutant (V504E), we found that KR13 transformation does not require functional fusion peptide for poration. In contrast, simultaneous treatment with fusion inhibitor T20 alongside KR13 prevented membrane poration and MPER exposure, showing that these events require 6-helix-bundle formation. Based on these results, we formulated a model for PTT-induced Env transformation portraying how, in the absence of CD4/co-receptor signaling, PTT may provide alternate means of perturbing the metastable Env-membrane complex, and inducing fusion-like transformation. In turn, the results show that such transformations are intrinsic to Env and can be diverted for irreversible inactivation of the protein complex.

Cholesterols Work as a Molecular Regulator of the Antimicrobial Peptide-Membrane Interactions

The existing cholesterols (Chols) in animal cell membranes play key roles in many fundamental cellular processes, which also promise the possibility to modulate the bioactivity of various membrane-active biomacromolecules. Here, combining dynamic giant unilamellar vesicle leakage experiments and molecular dynamics simulations, the inhibitory effect of Chols on the membrane poration activity of melittin (Mel), a typical natural antimicrobial peptide, is demonstrated. Molecular details of the Mel-Chol interactions in membrane show that, for a Chol-contained lipid membrane, Mel exposure would perturb the symmetric bilayer structure of the membrane and specifically influence the location and orientation distributions of Chol molecules to an asymmetric state between the two leaflets; moreover, the Mel-Chol interactions are significantly influenced by the membrane environment such as unsaturation degree of the lipid components. Such inhibitory effect is normally ascribed to an accumulation of Chol molecules around the membrane-bound peptide chains and formation of Chol-Mel complexes in the membrane, which hinder the further insertion of peptides into the membrane. This work clarifies the molecular interactions between membrane-active peptides and Chol-contained membranes, and suggest the possibility to develop targeted drugs due to the membrane component specificity between bacterial and animal cells.

Mechanically Induced Integrin Ligation Mediates Intracellular Calcium Signaling with Single Pulsating Cavitation Bubbles

Ultrasound or shockwave-induced cavitation is used therapeutically to stimulate neural and muscle tissue, but the mechanisms underlying this mechanotransduction are unclear. Intracellular Ca2+ signaling is one of the earliest events in mechanotransduction. In this study, we investigate the mechanism of Ca2+ signaling in individual HEK293T cells stimulated by single cavitation bubbles. Ca2+ responses are rare at cell-bubble distance that avoids membrane poration, even with overexpression of the mechanosensitive ion channel Piezo1, but could be increased in frequency to 42% of cells by attaching RGD beads to the apical surface of the cells. By using Piezo1 knockout and Piezo1-expressing cells, integrin-blocking antibodies, and inhibitors of P2X ion channels, key molecular players are identified in the RGD bead-enhanced Ca2+ response: increased integrin ligation by substrate ECM triggers ATP release and activation of P2X—but not Piezo1—ion channels. These molecular players have not been examined previously in cavitation-induced Ca2+ signaling. The resultant Ca2+ influx causes dynamic changes in cell spread area. This approach to eliciting a Ca2+ response with cavitation microbubbles without cell injury, and the uncovered mechanotransduction mechanism by which increased integrin-ligation mediates ATP release and Ca2+ signaling will inform new strategies to stimulate tissues with ultrasound and shockwaves.

Ultrasound neuromodulation through nanobubble-actuated sonogenetics

Ultrasound neuromodulation is a promising new method to manipulate brain activity noninvasively. Here, we detail a neurostimulation scheme using gas-filled nanostructures, gas vesicles (GVs), as actuators for improving the efficacy and precision of ultrasound stimuli. Sonicated primary neurons displayed dose-dependent, repeatable Ca2+ responses, closely synced to stimuli, and increased nuclear expression of the activation marker c-Fos only in the presence of GVs but not without. We identified mechanosensitive ion channels as important mediators of this effect, and neurons heterologously expressing the mechanosensitive MscL-G22S channel showed greater activation at lower acoustic pressure. This treatment scheme was also found not to induce significant cytotoxicity, apoptosis or membrane poration in treated cells. Altogether, we demonstrate a simple and effective method to achieve enhanced and more selective ultrasound neurostimulation.Graphical abstract

The Lipid-Chaperon Hypothesis: A Common Molecular Mechanism of Membrane Disruption by Intrinsically Disordered Proteins

<p>Increasing number of human diseases have been shown to be linked to aggregation and amyloid formation by intrinsically disordered proteins (IDPs). Amylin, amyloid-β, and α-synuclein are, indeed, involved in type-II diabetes, Alzheimer’s, and Parkinson’s, respectively. Despite the correlation of the toxicity of these proteins at early aggregation stages with membrane damage, the molecular events underlying the process is quite complex to understand. In this study, we demonstrate the crucial role of free lipids in the formation of lipid-protein complex, which enables an easy membrane insertion for amylin, amyloid-β, and α-synuclein. Experimental results from a variety of biophysical methods and molecular dynamics results reveal this common molecular pathway in membrane poration is shared by amyloidogenic (amylin, amyloid-β, and α-synuclein) and non-amyloidogenic (rat IAPP, β-synuclein) proteins. Based on these results, we propose a “lipid-chaperone” hypothesis as a unifying framework for protein-membrane poration.<b></b></p>

The Lipid-Chaperon Hypothesis: A Common Molecular Mechanism of Membrane Disruption by Intrinsically Disordered Proteins

<p>Increasing number of human diseases have been shown to be linked to aggregation and amyloid formation by intrinsically disordered proteins (IDPs). Amylin, amyloid-β, and α-synuclein are, indeed, involved in type-II diabetes, Alzheimer’s, and Parkinson’s, respectively. Despite the correlation of the toxicity of these proteins at early aggregation stages with membrane damage, the molecular events underlying the process is quite complex to understand. In this study, we demonstrate the crucial role of free lipids in the formation of lipid-protein complex, which enables an easy membrane insertion for amylin, amyloid-β, and α-synuclein. Experimental results from a variety of biophysical methods and molecular dynamics results reveal this common molecular pathway in membrane poration is shared by amyloidogenic (amylin, amyloid-β, and α-synuclein) and non-amyloidogenic (rat IAPP, β-synuclein) proteins. Based on these results, we propose a “lipid-chaperone” hypothesis as a unifying framework for protein-membrane poration.<b></b></p>