Deciphering the BH3 code for the neutralization of pro-survival Bcl-2 proteins in membranes

The proteins of Bcl-2 family, the pro-survival and the pro-apoptotic tightly regulates the process of apoptosis. The pro-survival proteins show a specific interaction pattern with BH3 domain of BH3 only proteins, determining the cellular fate during apoptotic stress. This interaction specificity is pivotal in designing BH3 mimetics, a class of anticancer drug molecules based on the BH3 domain of BH3 only proteins showing promising results in clinical trials. The role of the mitochondrial outer membrane in exhibiting Bcl2 complex interactome is extensively studied recently. Overall most studies addressed so far on the interactions of BH3 peptides and the truncated Bcl-2 proteins are reported in the solution / cytosolic environment while the quantitative interactions in membranes are still missing. To tackle this, we systematically quantified the library of BH3 peptides using two-color fluorescence correlation spectroscopy in solution and in the model membrane. We further extended our investigations to isolated yeast mitochondria using ensemble FRET and in mammalian cancer cell lines using a high throughput screening called BH3 profiling. We show that BH3 peptides derived from Hrk and Bim are the most effective in disrupting cBid/Bcl-xL complexes, which correlates with their response in mitochondria and in cells. Moreover, to understand the activation process of pro-apoptotic effector protein Bax on membranes, we designed an ​in-vitro system to investigate its autoactivation by the recruitment of inactive cytosolic Bax molecules by active membrane-bound Bax. Furthermore, ​in vitro studies also showed active membrane-bound Bax recruits Bcl-xL to the membrane, which retrotranslocates active Bax back into the cytosol, thereby maintaining membrane integrity. Quantitative analysis showed that Bax retrotranslocation activity potentiates Bcl-xL antiapoptotic activity by at least 10 fold. Overall, these findings highlights the importance of the membrane in Bcl-2 family interactions and thereby screening peptides that can disrupt specific interactions of these proteins in the membrane and can improve cancer therapies.Der mitochondriale Apoptosesignalweg wird durch die anti- und pro-apoptotischen Vertreter der Bcl-2 Proteinfamilie kontrolliert. Die anti-apoptotischen Proteine spielen eine zentrale Rolle, in dem sie durch die selektive Interaktion mit der BH3 Domäne der BH3-only Proteinen den apoptotischen Prozess regulieren. Basierend auf dieser Interaktion und der BH3 Domäne werden BH3-Mimetika, eine Gruppe von anti-Krebs Medikamenten, entworfen. BH3-Mimetika zeigen in klinischen Studien eine signifikante Aktivierung von Apoptose, indem sie diese Interaktion stören. Der Einfluss der äußeren Mitochondrienmembran auf die Interaktion der Bcl-2 Proteinen wird zur Zeit detailliert untersucht. Bisher wurde ausschließlich die Interaktion der BH3 Peptiden mit den verkürzten Formen der Bcl-2 Proteinen nur in Lösung und im Zytoplasma untersucht, während quantitative Interaktionen in Membranen komplett außer Acht gelassen wurden. Um diese Fragestellung anzugehen, haben wir systematisch die Interaktion bestehender BH3 Peptiden mit Hilfe der zwei Farben Fluoreszenz-Korrelations- Spektroskopie, sowohl in Lösung als auch in Modell Membranen, quantifiziert. Wir erweiterten unsere Untersuchungen auf isolierte Hefe Mitochondrien mit Hilfe der FRET Analyse und auf Säugetier Krebszelllinien mit Hilfe des Hochdurchsatz-Screenings BH3 Profiling. Wir zeigen, dass die BH3 Peptide, die von Hrk und Bim abgeleitet wurden, die effektivesten in der Störung des cBid / Bcl-xL Komplexes seien. Diese Daten spiegeln die Antwort in Mitochondrien und in Zellen wieder. Um den Aktivierungsprozess des pro-apoptotischen Effektorproteins Bax an Membranen zu verstehen, haben wir ein in-vitro Assay entwickelt, das erlaubt, die Autoaktivierung von Bax zu untersuchen. Das aktive, membrangebundene Bax rekrutiert dabei das inaktive, zytoplasmatische Bax zu der Membran. Des Weiteren haben in-vitro Analysen gezeigt, dass membrangebundenes Bax, Bcl-xL zu der Membran rekrutiert. Bcl-xL retrotranloziert aktives, nicht oligomerisiertes Bax zurück in das Zytoplasma, um die Membranintegrität aufrecht zu erhalten. Quantitative Analysen zeigen, dass die Bax-retrotranslokationsaktivität die anti-apoptotische Bcl-xL-Aktivität um mindestens das 10fache verstärkt. Unsere Ergebnisse zeigen die Wichtigkeit der Membran für die Interaktion der Bcl-2 Proteinen auf, deshalb ist das Screening von Peptiden, die die Interaktion dieser Proteine an der Membran stört von großer Bedeutung für die Krebstherapie

Electrical Characteristics of a Turmeric Dye-Based Organic Thin Film Device and the Effect of Light on Barrier Height

Herbal (turmeric) dye-based organic thin film is fabricated and characterized to prepare thin film electronic devices (i.e., Al/turmeric/Cu diodes) for optoelectronic applications. The temperature-dependent current–voltage (I-V) characteristics were evaluated considering different conditions. The values of the ideality factor (n), series resistance (RS) and shunt resistance (Rsh) of the Al/turmeric/Cu diode were calculated using thermionic emission theory. The rectification ratio was found to be very high, and the current increased with the temperature. At 65 °C, RS was 56 KΩ, and at 28 °C, RS was 1.06 MΩ. The values of n, RS and Rsh were found to be 6.56, 8 KΩ and 100 KΩ, respectively. An interesting result obtained from the present work was that the series resistance decreased with the temperature, indicating the semiconducting behavior of the present dye material

Quantitative interactome of a membrane Bcl-2 network identifies a hierarchy of complexes for apoptosis regulation

The Bcl-2 proteins form a complex interaction network that controls mitochondrial permeabilization and apoptosis. The relative importance of different Bcl-2 complexes and their spatio-temporal regulation is debated. Using fluorescence cross-correlation spectroscopy to quantify the interactions within a minimal Bcl-2 network, comprised by cBid, Bax, and Bcl-xL, we show that membrane insertion drastically alters the pattern of Bcl-2 complexes, and that the C-terminal helix of Bcl-xL determines its binding preferences. At physiological temperature, Bax can spontaneously activate in a self-amplifying process. Strikingly, Bax also recruits Bcl-xL to membranes, which is sufficient to retrotranslocate Bax back into solution to secure membrane integrity. Our study disentangles the hierarchy of Bcl-2 complex formation in relation to their environment: Bcl-xL association with cBid occurs in solution and in membranes, where the complex is stabilized, whereas Bcl-xL binding to Bax occurs only in membranes and with lower affinity than to cBid, leading instead to Bax retrotranslocation.The permeabilization of the mitochondrial outer membrane to induce apoptosis is regulated by complex interactions between Bcl-2 family members. Here the authors develop a quantitative interactome of a membrane Bcl-2 network and identify a hierarchy of protein complexes in apoptosis induction.publishe

Determinants of BH3 Sequence Specificity for the Disruption of Bcl-xL/cBid Complexes in Membranes

The prosurvival Bcl-2 proteins exhibit a specific pattern of interactions with BH3-only proteins that determines the cellular dependence on apoptotic stress. This specificity is crucial for the development of BH3 mimetics, a class of anticancer molecules based on the BH3 domain with promising activity in clinical trials. Although complex formation mainly takes place in the mitochondrial outer membrane, most studies so far addressed the interaction between BH3 peptides and truncated Bcl-2 proteins in solution. As a consequence, quantitative understanding of the sequence specificity determinants of BH3 peptides in the membrane environment is missing. Here, we tackle this issue by systematically quantifying the ability of BH3 peptides to compete for the complexes between cBid and Bcl-xL in giant unilamellar vesicles and compare it with solution and mitochondria. We show that the BH3 peptides derived from Hrk, Bim, Bid, and Bad are the most efficient in disrupting cBid/Bcl-xL complexes in the membrane, which correlates with their activity in mitochondria. Our findings support the targeting to the membrane of small molecules that bind Bcl-2 proteins as a strategy to improve their efficiency