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In this study, molecular dynamics simulations were used to systematically explore the reason why BH3-only protein BAD binds to anti-apoptotic protein BCL- x L but not to MCL-1 to give more theoretical hints for the design of BAD mimetic inhibitors for the dual-targeting of BCL- x L and MCL-1. Starting with the difference in residue-based binding energy contributions, a series of analyses were conducted to identify the hotspot residues in MCL-1 that significantly affect the interaction with BAD. Among them, the insertion of the T residue in the loop between α4 and α5 domains of MCL-1 is considered to be the main cause of BAD selective binding. The inserted T residue reduces the stability of the loop and weakens the hydrogen bond interactions that originally bound E19 of BAD in BCL- x L/BAD, and the freed E19 severely interferes with the salt bridge between D16 and Arg53 by electrostatic repulsion. This salt-bridge is believed to be critical for maintaining the binding between BCL- x L and BAD. By clarifying the reasons for differential binding, we can more specifically optimize the BAD sequence to target both BCL- x L and MCL-1.

Hepatitis B virus X protein (HBx) plays a crucial role in the development of hepatocellular carcinoma (HCC) associated with hepatitis B virus (HBV) infection. The full-length HBx protein interacts with Bcl-x L and is involved in the HBV replication and cell death processes. The three hydrophobic residues Trp120, Leu123, and Ile127 of the HBx BH3-like motif are essential for the Bcl-x L -binding. On the other hand, various lengths of C-terminal-truncated HBx mutants are frequently detected in HCC tissues, and these mutants, rather than the full-length HBx, appear to be responsible for HCC development. Notably, the region spanning residues 1-120 of HBx [HBx(1 and 120)] has been strongly associated with an increased risk of HCC development. However, the mode of interaction between HBx(1-120) and Bcl-x L remains unclear. HBx(1-120) possesses only Trp120 among the three hydrophobic residues essential for the Bcl-x L -binding. To elucidate this interaction mode, we employed a C-terminal-deleted HBx BH3-like motif peptide composed of residues 101-120. Here, we present the NMR complex structure of Bcl-x L and HBx(101-120). Our results demonstrate that HBx(101-120) binds to Bcl-x L in a weaker manner. Considering the high expression of Bcl-x L in HCC cells, this weak interaction, in conjunction with the overexpression of Bcl-x L in HCC cells, may potentially contribute to HCC development through the interaction between C-terminal-truncated HBx and Bcl-x L .

Anti-apoptotic proteins such as BCL-X L promote cell survival by sequestering pro-apoptotic BCL-2 family members, an activity that frequently contributes to tumorigenesis. Thus, the development of small-molecule inhibitors for anti-apoptotic proteins, termed BH3-mimetics, is revolutionizing how we treat cancer. BH3 mimetics kill cells by displacing sequestered pro-apoptotic proteins to initiate tumor-cell death. Recent evidence has demonstrated that in live cells the BH3-only proteins PUMA and BIM resist displacement by BH3-mimetics, while others like tBID do not. Analysis of the molecular mechanism by which PUMA resists BH3-mimetic mediated displacement from full-length anti-apoptotic proteins (BCL-X L , BCL-2, BCL-W, and MCL-1) reveals that both the BH3-motif and a novel binding site within the carboxyl-terminal sequence (CTS) of PUMA contribute to binding. Together these sequences bind to anti-apoptotic proteins, which effectively 'double-bolt locks' the proteins to resist BH3-mimetic displacement. The pro-apoptotic protein BIM has also been shown to double-bolt lock to anti-apoptotic proteins however, the novel binding sequence in PUMA is unrelated to that in the CTS of BIM and functions independent of PUMA binding to membranes. Moreover, contrary to previous reports, we find that when exogenously expressed, the CTS of PUMA directs the protein primarily to the endoplasmic reticulum (ER) rather than mitochondria and that residues I175 and P180 within the CTS are required for both ER localization and BH3-mimetic resistance. Understanding how PUMA resists BH3-mimetic displacement will be useful in designing more efficacious small-molecule inhibitors of anti-apoptotic BCL-2 proteins., Competing Interests: JP, DN, EO, WS, JP, NH, BL, DA No competing interests declared, (© 2023, Pemberton et al.)

Anti-apoptotic and anti-autophagic Bcl-2 homologues commonly contain a hydrophobic groove in which the BH3 domain is accommodated. The BH3 domain is usually considered a feature of Bcl-2 family members; however, it has also been found in various non-Bcl-2 family proteins. Although interactions among Bcl-2 family members have been extensively investigated and highlighted, those mediated by the BH3 domain of non-Bcl-2 family proteins have not been the focus of substantial research. In this review, the author conducted a structural analysis of Bcl-xL complexed with the BH3 domain of four non-Bcl-2 family proteins, Beclin 1, SOUL, TCTP, and Pxt1, at an atomic level. Although the overall Bcl-xL-binding modes are similar among these proteins, they are characterized by limited sequence conservation of the BH3 consensus motif and differences in residues involved in complex formation. Based on the structural analysis, the author suggests that more "undiscovered" BH3 domain-containing proteins might exist, which have been unidentified due to their limited sequence conservation but can bind to Bcl-2 family proteins and control apoptosis, autophagy, or other biological processes., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

Chronic hepatitis B virus (HBV) infection is a major risk factor for the development of liver diseases including fibrosis, cirrhosis, and hepatocellular carcinoma (HCC). HBV has the multifunctional protein, HBV X protein (HBx, 154 residues), which plays key roles in HBV replication and liver disease development. Interaction of HBx through its BH3-like motif with the anti-apoptotic protein Bcl-x L leads to HBV replication and induction of apoptosis, resulting in HCC development. Our previous nuclear magnetic resonance (NMR) study revealed that the HBx BH3-like motif peptide (residues 101-136) binds to the common BH3-binding groove of Bcl-x L . Importantly, a C-terminal-truncated HBx, e.g., residues 1-120 of HBx, is strongly associated with the increased risk of HBV-related HCC development. However, the interaction mode between the C-terminal-truncated HBx and Bcl-x L remains unclear. To elucidate this interaction mode, the C-terminal-deleted HBx BH3-like motif peptide (residues 101-120) was used as a model peptide in this study. To facilitate the NMR analysis, we prepared a fusion protein of HBx (101-120) and Bcl-x L connected with five repeats of the glycine-serine dipeptide as a linker. Here, we report the 1 H, 13 C, and 15 N resonance assignments of the fusion protein. This is the first step for the elucidation of the pathogenesis of liver diseases caused by the interaction between the C-terminal-truncated HBx and Bcl-x L ., (© 2022. The Author(s), under exclusive licence to Springer Nature B.V.)

The BCL-XL protein is among the most important members of the anti-apoptotic subfamily of the BCL-2 protein family, and is currently a promising new target for anti-tumor drug research. However, the BCL-XL/2 proteins have similar structures and functions, which could lead to undesirable side effects because of inhibitors that can bind to both BCL-XL and BCL-2. Therefore, it is crucial to expound on the structural basis of the selective mechanism towards BCL-XL/2 inhibition. In the current study, we employed hybrid computational methods including molecular docking and dynamics simulation, MM/GBSA energy calculation, alanine scanning mutagenesis and Hirshfeld surface analysis to comprehensively reveal the selectivity mechanism towards BCL-XL/2 from multiple perspectives, revealing the significant effects of the BCL-XL residues SER106 and LEU108 as well as the BCL-2 residue ASP103 on the inhibitory selectivity. Overall, our findings provide useful references for the rational design of BCL-XL/2 selective inhibitors with better affinity.

Drug development for cancer treatment is a complex process that requires special efforts. Targeting crucial proteins is the most essential purpose of drug design in cancers. Bcl-xl is an anti-apoptotic protein that binds to pro-apoptotic proteins and interrupts their signals. Pro-apoptotic Bcl-xl effectors are short BH3 sequences that form an alpha helix and bind to anti-apoptotic proteins to inhibit their activity. Computational systematic evolution of ligands by exponential enrichment (SELEX) is an exclusive approach for developing peptide aptamers as potential effectors. Here, the amino acids with a high tendency for constructing an alpha-helical structure were selected. Due to the enormous number of pentapeptides, Taguchi method was used to study a selected number of peptides. The binding affinity of the peptides to Bcl-xl was assessed using molecular docking, and after analysis of the obtained results, a final set of optimized peptides was arranged and constructed. For a better comparison, three chemical compounds with approved anti-Bcl-xl activity were selected for comparison with the top-ranked 5mer peptides. The optimized peptides showed considerable binding affinity to Bcl-xl. The molecular dynamics (MD) simulation indicated that the designed peptide (PO5) could create considerable interactions with the BH3 domain of Bcl-xl. The MM/GBSA calculations revealed that these interactions were even stronger than those created by chemical compounds. In silico SELEX is a novel approach to design and evaluate peptide-aptamers. The experimental design improves the SELEX process considerably. Finally, PO5 could be considered a potential inhibitor of Bcl-xl and a potential candidate for cancer treatment., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Systematic incorporation of ring-constrained β- and γ-amino acid residues into α-helix mimetics engenders stable helical secondary structures. In this paper, functional α/β/γ-helical peptidomimetics were explored for mimicry of BH3 helical domains, Bim as a pioneering study. The Bim-based α/β/γ-peptides in an αγααβα-hexad repeat with five helical turns inhibited the interaction between Bak and Bcl-x L with excellent resistance towards proteolytic digestion. Further optimization of the α/β/γ-backbone strategy will considerably expand the utility of functional α/β/γ-peptidomimetics, in particular due to its prominent stability against proteolysis.

Apoptosis plays an essential role in maintaining cellular homeostasis and preventing cancer progression. Bcl-xL, an anti-apoptotic protein, is an important modulator of the mitochondrial apoptosis pathway and is a promising target for anticancer therapy. In this study, we identified octenidine as a novel Bcl-xL inhibitor through structural feature-based deep learning and molecular docking from a library of approved drugs. The NMR experiments demonstrated that octenidine binds to the Bcl-2 homology 3 (BH3) domain-binding hydrophobic region that consists of the BH1, BH2, and BH3 domains in Bcl-xL. A structural model of the Bcl-xL/octenidine complex revealed that octenidine binds to Bcl-xL in a similar manner to that of the well-known Bcl-2 family protein antagonist ABT-737. Using the NanoBiT protein-protein interaction system, we confirmed that the interaction between Bcl-xL and Bak-BH3 domains within cells was inhibited by octenidine. Furthermore, octenidine inhibited the proliferation of MCF-7 breast and H1299 lung cancer cells by promoting apoptosis. Taken together, our results shed light on a novel mechanism in which octenidine directly targets anti-apoptotic Bcl-xL to trigger mitochondrial apoptosis in cancer cells., Competing Interests: Declaration of competing interest The authors declare no conflict of interest., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Bcl-x L is an antiapoptotic mitochondrial trans-membrane protein, which is known to play a crucial role in the survival of tumor cells. The deamidation of Bcl-x L is a pivotal switch that regulates its biological function. The potential impact of deamidation on the structure and dynamics of Bcl-x L is directly linked to the intrinsically disordered region (IDR), which is the main site for post-translational modifications (PTMs). In this study, we explored deamidation-induced conformational changes in Bcl-x L to gain insight into its loss of function by performing microsecond-long molecular dynamics (MD) simulations. MD simulation outcomes showed that the IDR motion and interaction patterns have changed notably upon deamidation. Principal component analysis (PCA) demonstrates significant differences between wild-type and deamidated Bcl-x L and suggests that deamidation affects the structure and dynamics of Bcl-x L . The combination of clustering analysis, H-bond analysis, and PCA revealed changes in conformation, interaction, and dynamics upon deamidation. Differences in contact patterns and essential dynamics that lead to a narrowing in the binding groove (BG) are clear indications of deamidation-induced allosteric effects. In line with previous studies, we show that the IDR plays a very important role in the loss of apoptotic functions of Bcl-x L while providing a unique perspective on the underlying mechanism of Bcl-x L deamidation-induced cell death.

PROteolysis-TArgeting Chimeras (PROTACs) have emerged as an innovative drug development platform. However, most PROTACs have been generated empirically because many determinants of PROTAC specificity and activity remain elusive. Through computational modelling of the entire NEDD8-VHL Cullin RING E3 ubiquitin ligase (CRL VHL )/PROTAC/BCL-xL/UbcH5B(E2)-Ub/RBX1 complex, we find that this complex can only ubiquitinate the lysines in a defined band region on BCL-xL. Using this approach to guide our development of a series of ABT263-derived and VHL-recruiting PROTACs, we generate a potent BCL-xL and BCL-2 (BCL-xL/2) dual degrader with significantly improved antitumor activity against BCL-xL/2-dependent leukemia cells. Our results provide experimental evidence that the accessibility of lysines on a target protein plays an important role in determining the selectivity and potency of a PROTAC in inducing protein degradation, which may serve as a conceptual framework to guide the future development of PROTACs., (© 2021. The Author(s).)

Hepatitis B virus (HBV) is the world's most prevalent chronic viral infection. More than 350 million individuals are chronic carriers of the virus, with an estimated 2 billion infected persons. For instance, the role of HBx protein in attachment and infection is very obvious and consequently deemed as an important druggable target. Targeting the interface and discovering novel drugs greatly advanced the field of therapeutics development. Therefore, in the current study, HBx to Bcl-xL is abrogated on high-affinity carbon nanotubes using computational structural biology tools. Our analysis revealed that among the total 62 carbon fullerenes, only 13 compounds exhibited inhibitory activity against HBx, which was further confirmed through IFD-based rescoring. Structural dynamics investigation revealed stable binding, compactness, and hydrogen bonds reprogramming. Moreover, the binding free energy calculation results revealed that the top hits1-4 possess the total binding energy of -54.36 kcal/mol (hit1), -50.81 kcal/mol (hit2), -47.09 kcal/mol (hit3), and -45.59 kcal/mol for hit4. In addition, the predicted K D values and bioactivity scores further validated the inhibitory potential of these top hits. The identified compounds need further in vitro and in vivo validation to aid the treatment process of HBV.

Permeabilization of the outer mitochondrial membrane by pore-forming Bcl2 proteins is a crucial step for the induction of apoptosis. Despite a large set of data suggesting global conformational changes within pro-apoptotic Bak during pore formation, high-resolution structural details in a membrane environment remain sparse. Here, we used NMR and HDX-MS (Hydrogen deuterium exchange mass spectrometry) in lipid nanodiscs to gain important high-resolution structural insights into the conformational changes of Bak at the membrane that are dependent on a direct activation by BH3-only proteins. Furthermore, we determined the first high-resolution structure of the Bak transmembrane helix. Upon activation, α-helix 1 in the soluble domain of Bak dissociates from the protein and adopts an unfolded and dynamic potentially membrane-bound state. In line with this finding, comparative protein folding experiments with Bak and anti-apoptotic BclxL suggest that α-helix 1 in Bak is a metastable structural element contributing to its pro-apoptotic features. Consequently, mutagenesis experiments aimed at stabilizing α-helix 1 yielded Bak variants with delayed pore-forming activity. These insights will contribute to a better mechanistic understanding of Bak-mediated membrane permeabilization., (© 2021 The Authors. Published under the terms of the CC BY NC ND 4.0 license.)

A series of novel hybrid chalcone N-ethyl-piperazinyl amide derivatives of oleanonic and ursonic acids were synthesized, and their cytotoxic potential was evaluated in vitro against the NCI-60 cancer cell line panel. Compounds 4 and 6 exhibited the highest overall anticancer activity, with GI 50 values in some cases reaching nanomolar values. Thus, the two compounds were further assessed in detail in order to identify a possible apoptosis- and antiangiogenic-based mechanism of action induced by the assessed compounds. DAPI staining revealed that both compounds induced nuclei condensation and overall cell morphological changes consistent with apoptotic cell death. rtPCR analysis showed that up-regulation of pro-apoptotic Bak gene combined with the down-regulation of the pro-survival Bcl-XL and Bcl-2 genes caused altered ratios between the pro-apoptotic and anti-apoptotic proteins' levels, leading to overall induced apoptosis. Molecular docking analysis revealed that both compounds exhibited high scores for Bcl-XL inhibition, suggesting that compounds may induce apoptotic cell death through targeted anti-apoptotic protein inhibition, as well. Ex vivo determinations showed that both compounds did not significantly alter the angiogenesis process on the tested cell lines.

B-cell lymphoma/leukemia gene-2(Bcl-2) protein family known for regulating cell cycle arrest and subsequent cell death is highly expressed in a variety of cancers. Among them, the Bcl-xL and Bcl-2 are two essential proteins in the Bcl-2 family. In the present work, the differences in binding modes as between the two proteins and two ligands ABT-263/43b were investigated and compared. And the computational alanine scanning combined with the recently developed interaction entropy (AS-IE) method was employed for predicting their binding free energies and finding those amino acids that were more critical during the binding process. The result showed that the binding free energy calculated by the AS-IE method was more in line with experimental values than the molecular mechanics/Poisson-Boltzmann surface area (MM/PBSA) method. Besides, no significant difference was found between Bcl-xL and ABT-263/43b in the binding free energy, which Bcl-xL showed slightly weaker binding free energy to 43b because of the fewer number of key residues with interactions. Nonetheless, compared with the Bcl-2 and 43b complex, the Bcl-2 and ABT-263 system had greater number of key residues interacting with ABT-263, in particular, contribute favorably, resulting in a stronger binding ability for the Bcl-2 and ABT-263 systems. The van der Waals and hydrogen bond contributions were significant in the four protein-ligand complexes. Overall, Tyr108 was found to be the common key residues in the Bcl-xL-ligand complex, while Tyr105, Glu100, and Glu143 were established as the common key residue in the Bcl-2-ligand systems. We hope that the predicted hot spot residues and their energy distributions can guide the design of peptide and small-molecule drugs targeting Bcl-xL and Bcl-2., (© 2021. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

The anti-apoptotic protein Bcl-xL regulates apoptosis by preventing the permeation of the mitochondrial outer membrane by pro-apoptotic pore-forming proteins, which release apoptotic factors into the cytosol that ultimately lead to cell death. Two different membrane-integrated Bcl-xL constructs have been identified: a membrane-anchored and a membrane-inserted conformation. Here, we use molecular dynamics simulations to study the effect of the mitochondrial specific lipid cardiolipin and the protein protonation state on the conformational dynamics of membrane-anchored Bcl-xL. The analysis reveals that the protonation state of the protein and cardiolipin content of the membrane modulate the orientation of the soluble head region (helices α1 through α7) and hence the exposure of its BH3-binding groove, which is required for its interaction with pro-apoptotic proteins.

Chemical biologists use chemical tools to answer biological questions. The translational application of these principles has led to an explosion in the discovery and druggability of new protein targets, including protein-protein interactions (PPIs). Proteins tend to interact with other macromolecules using relatively large and featureless binding surfaces, which has hampered traditional drug discovery efforts, particularly for interactions with weaker affinity. In this article, I discuss several emerging strategies for targeting PPIs, including computational and structural methods and novel screening approaches. In particular, I focus on hijacking intrinsic protein allosteric pathways for the discovery and design of small-molecule and peptide ligands., (© 2020 Wiley-VCH GmbH.)

Bcl-2 proteins orchestrate the mitochondrial pathway of apoptosis, pivotal for cell death. Yet, the structural details of the conformational changes of pro- and antiapoptotic proteins and their interactions remain unclear. Pulse dipolar spectroscopy (double electron-electron resonance [DEER], also known as PELDOR) in combination with spin-labeled apoptotic Bcl-2 proteins unveils conformational changes and interactions of each protein player via detection of intra- and inter-protein distances. Here, we present the synthesis and characterization of pro-apoptotic BimBH3 peptides of different lengths carrying cysteines for labeling with nitroxide or gadolinium spin probes. We show by DEER that the length of the peptides modulates their homo-interactions in the absence of other Bcl-2 proteins and solve by X-ray crystallography the structure of a BimBH3 tetramer, revealing the molecular details of the inter-peptide interactions. Finally, we prove that using orthogonal labels and three-channel DEER we can disentangle the Bim-Bim, Bcl-xL-Bcl-xL, and Bim-Bcl-xL interactions in a simplified interactome., Competing Interests: Declaration of Interests The authors declare no conflict of interest., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

The anti-apoptotic proteins B-cell lymphoma-extra large (Bcl-xL) and B-cell lymphoma/leukemia-2 (Bcl-2) are members of the Bcl-2 protein family, and they play important roles in regulating apoptosis and cell cycle retardation. However, the binding mechanisms of Bcl-xL/Bcl-2 with their associated agonists, including Bcl-2-associated death promoter (Bad) and Bcl-2-associated X protein (Bax), are not well understood. In the present study, the recently developed interaction entropy approach was employed for the calculation of entropic contribution, and the computational alanine scanning method was used to identify the hot spot in the protein-protein interactions between Bcl-xL/Bcl-2 and Bad/Bax. The calculated binding free energies and their ranks for the four systems were in good agreement with the experimental results. Computational analysis shows that there are more hot-spot residues in the Bcl-xL/Bad complex than that in the Bcl-xL/Bax complex, leading to a stronger binding affinity in the former. It is interesting to find that the reason for the stronger binding affinity of Bcl-2 to Bad than to Bax is different for the Bcl-xL system. Although there are more hot-spot residues in the Bcl-2/Bax system than in the Bcl-2/Bad complex, there are also more negatively contributing residues in the Bcl-2/Bax. Our study identified Arg104, Tyr105, Leu116, and Leu134 to be the common key residues in the Bcl-xL complexes, and Arg107, Tyr108, Phe112, Gln118, Leu137, Arg146, and Tyr202 are common key residues in the Bcl-2 complexes. These results would provide valuable information for the design of potent inhibitors of Bcl-xL/Bcl-2.

In silico rational drug design is one of the major pylons in the drug discovery process. Drugs usually act on specific targets such as proteins, DNA, and lipid bilayers. Thus, molecular docking is an essential part of the rational drug design process. Molecular docking uses specific algorithms and scoring functions to reveal the strength of the interaction of the ligand to its target. AutoDock is a molecular docking suite that offers a variety of algorithms to tackle specific problems. These algorithms include Monte Carlo Simulated Annealing (SA), a Genetic Algorithm (GA), and a hybrid local search GA, also known as the Lamarckian Genetic Algorithm (LGA). This chapter aims to acquaint the reader with the docking process using AutoDockTools (GUI of AutoDock). Furthermore, herein is described the docking process of calf thymus DNA with three metal complexes, as a potential metallo-therapeutics as also the docking process of the plant flavonoid quercetin to the antiapoptotic protein BcL-xL.

Members of the B-cell lymphoma (BCL-2) protein family regulate mitochondrial outer membrane permeabilization (MOMP), a phenomenon in which mitochondria become porous and release death-propagating complexes during the early stages of apoptosis. Pro-apoptotic BCL-2 proteins oligomerize at the mitochondrial outer membrane during MOMP, inducing pore formation. Of current interest are endogenous factors that can inhibit pro-apoptotic BCL-2 mitochondrial outer membrane translocation and oligomerization. A mitochondrial-derived peptide, Humanin (HN), was reported being expressed from an alternate ORF in the mitochondrial genome and inhibiting apoptosis through interactions with the pro-apoptotic BCL-2 proteins. Specifically, it is known to complex with BAX and BID. We recently reported the fibrillation of HN and BAX into β-sheets. Here, we detail the fibrillation between HN and BID. These fibers were characterized using several spectroscopic techniques, protease fragmentation with mass analysis, and EM. Enhanced fibrillation rates were detected with rising temperatures or pH values and the presence of a detergent. BID fibers are similar to those produced using BAX; however, the structures differ in final conformations of the BCL-2 proteins. BID fibers display both types of secondary structure in the fiber, whereas BAX was converted entirely to β-sheets. The data show that two distinct segments of BID are incorporated into the fiber structure, whereas other portions of BID remain solvent-exposed and retain helical structure. Similar analyses show that anti-apoptotic BCL-x L does not form fibers with humanin. These results support a general mechanism of sequestration of pro-apoptotic BCL-2 proteins into fibers by HN to inhibit MOMP., Competing Interests: Conflict of interest— The authors declare that they have no conflicts of interest with the contents of this article.

Interactions between pro- and anti-apoptotic Bcl-2 proteins decide the fate of the cell. The BH3 domain of pro-apoptotic Bcl-2 proteins interacts with the exposed hydrophobic groove of their anti-apoptotic counterparts. Through their design and development, BH3 mimetics that target the hydrophobic groove of specific anti-apoptotic Bcl-2 proteins have the potential to become anticancer drugs. We have developed a novel computational method for designing sequences with BH3 domain features that can bind specifically to anti-apoptotic Mcl-1 or Bcl-X L . In this method, we retained the four highly conserved hydrophobic and aspartic residues of wild-type BH3 sequences and randomly substituted all other positions to generate a large number of BH3-like sequences. We modeled 20000 complex structures with Mcl-1 or Bcl-X L using the BH3-like sequences derived from five wild-type pro-apoptotic BH3 peptides. Peptide-protein interaction energies calculated from these models for each set of BH3-like sequences resulted in negatively skewed extreme value distributions. The selected BH3-like sequences from the extreme negative tail regions have highly favorable interaction energies with Mcl-1 or Bcl-X L . They are enriched in acidic and basic residues when they bind to Mcl-1 and Bcl-X L , respectively. With the charged residues often away from the binding interface, the overall electric field generated by the charged residues results in strong long-range electrostatic interaction energies between the peptide and the protein giving rise to high specificity. Cell viability studies of representative BH3-like peptides further validated the predicted specificity. This study has revealed the importance of non-hot spot residues in BH3-mimetic peptides in providing specificity to a particular anti-apoptotic protein.

Bcl-xL, a member of the Bcl-2 family, is a pro-survival protein involved in apoptosis regulation. We have previously reported the ability of Bcl-xL to form various types of fibers, from native to amyloid conformations. Here, we have mimicked the effect of apoptosis-induced caspase activity on Bcl-xL by limited proteolysis using trypsin. We show that cleaved Bcl-xL (ΔN-Bcl-xL) forms fibers that exhibit the features of amyloid structures (BclxLcf37). Moreover, three monoclonal antibodies (mAbs), produced by mouse immunization and directed against ΔN-Bcl-xL or Bcl-xL fibers, were selected and characterized. Our results show that these mAbs specifically target ΔN-Bcl-xL in amyloid fibers in vitro. Upon metal-stress-induced apoptosis, these mAbs are able to detect the presence of Bcl-xL in amyloid aggregates in neuroblastoma SH-SY5Y cell lines. In conclusion, these specific mAbs directed against amyloidogenic conformations of Bcl-xL constitute promising tools for studying, in vitro and in cellulo, the contribution of Bcl-xL in apoptosis. These mAbs may further help in developing new diagnostics and therapies, considering Bcl-xL as a strategic target for treating brain lesions relevant to stroke and neurodegenerative diseases.

A novel set of 2-cyanoacrylamides linked to ethyl 1,3-diphenylpyrazole-4-carboxylates moiety were synthesized and elucidated by different spectroscopic tools. In vitro cytotoxic assay was carried out against different cell lines (Hct 116 , A 549 , MDA-MB 231 , and HFB 4 ). Ethyl 5-(2-cyano-3-(furan-2-yl)acrylamido)-1,3-diphenylpyrazole-4-carboxylate 5 achieved the potent cytotoxic effect toward all tested cancer cell lines especially colon cancer (HCT 116 ) with IC 50 value (30.6 µg/ml) relative to the lead compound 3 and the standard positive control 5-FU. Additionally, it exhibited less toxic effect toward the normal human melanocytes (HFB4) cell line. Compound 5 was theoretically investigated and compared for its binding affinity to a model of protein markers relative to the lead compound 3 using two different molecular docking programs. More investigations were performed in an attempt to find out the molecular mechanism of this novel compound inside colon cancer cells, as real time PCR analysis, Elisa assay, flow cytometry, and morphological characterizations using TEM and SEM tools.Herein, we showed that compound 5 interferes with the intrinsic pathway of apoptosis at the mitochondrial level in response to an apoptogenic stimulus as cytochromec, caspase-9 and caspases-3 which were triggered by our novel compound 5. All molecular investigations proved that intrinsic apoptotic pathway of colorectal carcinoma was strongly initiated by the effect of compound 5 through upregulation of mitochondrial apoptosis related genes as (Caspase-3, caspase-9, BAX, P 53 , and cytochrome-c) and down-regulated anti-apoptotic proteins (BCL2, MMP1, CDK4, and VEGFR). Further studies proved cell cycle arrest of HCT 116 cell lines at G2/M phase after treatment. In addition, our data revealed that our novel efficiently damage the genomic DNA of colorectal cells involving P 53 dependent mechanism using DPA assay. Sever morphological and ultrastructural changes were detected in colorectal cells treated by compound 5 compared to control using both scanning electron microscopy (SEM) and transmission electron microscopy (TEM)., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Inc. All rights reserved.)

The Bcl-2 family of proteins, such as Bcl-xL and Bcl-2, play key roles in cancer cell survival. Structural studies of Bcl-xL formed the foundation for the development of the first Bcl-2 family inhibitors and FDA approved drugs. Recently, Pro teolysis Ta rgeting C himeras (PROTACs) that degrade Bcl-xL have been proposed as a therapeutic modality with the potential to enhance potency and reduce toxicity versus antagonists. However, no ternary complex structures of Bcl-xL with a PROTAC and an E3 ligase have been successfully determined to guide this approach. Herein, we report the design, characterization, and X-ray structure of a VHL E3 ligase-recruiting Bcl-xL PROTAC degrader. The 1.9 Å heterotetrameric structure, composed of (ElonginB:ElonginC:VHL):PROTAC:Bcl-xL, reveals an extensive network of neo-interactions, between the E3 ligase and the target protein, and between noncognate parts of the PROTAC and partner proteins. This work illustrates the challenges associated with the rational design of bifunctional molecules where interactions involve composite interfaces.

A membrane protein's oligomeric state modulates its functionality in various cellular processes. Since membrane proteins have to be solubilized in an appropriate membrane mimetic, the use of classical biophysical methods to analyze protein oligomers is challenging. We here present a method to determine the number of membrane proteins inserted into lipid nanodiscs. It is based on the ability to selectively quantify the amount of a small and robust fusion protein that can be proteolytically cleaved off from a membrane protein after incorporation into lipid nanodiscs. A detailed knowledge of the number of membrane proteins per nanodisc at defined assembly conditions is essential to estimate the tendency for oligomerization, but also for guiding sample optimization for structural investigations that require the presence of a homogenous oligomeric state. We show that this method can efficiently be used to determine the number of VDAC1 channels in nanodiscs at various assembly conditions, as confirmed by negative stain EM. The presented method is suitable in particular for membrane proteins that cannot be probed easily by other methods such as single span transmembrane helices. This assay can be applied to any membrane protein that can be incorporated into a nanodisc without the requirement for special instrumentation and will thus be widely applicable and complementary to other methods that quantify membrane protein insertion in lipid nanodiscs., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier B.V. All rights reserved.)

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. In the most recently proposed non-canonical model, the active form of Bcl-xL employs its N-terminal BH4 helix to bind and block its pro-apoptotic target. Here, we used a combination of various spectroscopic techniques to study the release of the BH4 helix (α1) during the membrane insertion of Bcl-xL. This refolding was characterized by a gradual increase in helicity due to the lipid-dependent partitioning-coupled folding and formation of new helix αX (presumably in the originally disordered loop between helices α1 and α2). Notably, a comparison of various fluorescence and circular dichroism measurements suggested the presence of multiple Bcl-xL conformations in the bilayer. This conclusion was explicitly confirmed by single-molecule measurements of Fӧrster Resonance Energy Transfer from Alexa-Fluor-488-labeled Bcl-xL D189C to a mCherry fluorescent protein attached at the N-terminus. These measurements clearly indicated that the refolding of Bcl-xL in the bilayer is not a two-state transition and involves multiple membranous intermediates of variable compactness., Competing Interests: The authors declare no conflict of interest.

Current models of apoptosis regulation by the Bcl-2 family of proteins postulate that heterodimeric interactions between family members determine whether Bax and Bak are activated to trigger cell death. Thus, the relative abundance and binding affinities between pro- and anti-apoptotic proteins determines the outcome of these interactions. Examination of these interactions using purified mitochondria and liposomes with full-length recombinant proteins revealed that Bcl-x L inhibits apoptosis as a higher-order complex that binds multiple BH3 proteins. Allosteric regulation of this complex by the BH3 sensitizer Bad confers switch-like activity to the indirect activation of Bax. The BH3 activator cBid sequestered by Bcl-x L complexes changes from an inactive to an active form while bound to a Bcl-x L complex only when Bad is also bound. Bcl-x L complexes enable Bad to function as a non-competitive inhibitor of Bcl-x L and allosterically activate cBid, dramatically enhancing the pro-apoptotic potency of Bad., Competing Interests: Declaration of Interests The authors declare no competing interests., (Copyright © 2019 Elsevier Inc. All rights reserved.)

B-Cell Lymphoma-extra-large (BCL-xL) is involved in longevity and successful aging, which indicates a role for BCL-xL in cell survival pathway regulation. Beyond its well described role as an inhibitor of apoptosis by preventing cytochrome c release, BCL-xL has also been related, indirectly, to autophagy and senescence pathways. Although in these latter cases, BCL-xL has dual roles, either activating or inhibiting, depending on the cell type and the specific conditions. Taken together, all these findings suggest a precise mechanism of action for BCL-xL, able to regulate the crosstalk between apoptosis, autophagy, and senescence, thus promoting cell survival or cell death. All three pathways can be both beneficial or detrimental depending on the circumstances. Thus, targeting BCL-xL would in turn be a "double-edge sword" and therefore, additional studies are needed to better comprehend this dual and apparently contradictory role of BCL-XL in longevity.

Hepatitis B virus X protein (HBx) possesses a BH3-like motif that directly interacts with the anti-apoptotic proteins, Bcl-2 and Bcl-x L . Here we report the interaction between the HBx BH3-like motif and Bcl-x L , as revealed by nuclear magnetic resonance spectroscopy. Our results showed that this motif binds to the common BH3-binding hydrophobic groove on the surface of Bcl-x L , with a binding affinity of 89 μM. Furthermore, we examined the role of the tryptophan residue (Trp120) in this motif in Bcl-x L binding using three mutants. The W120A mutant showed weaker binding affinity (294 μM) to Bcl-x L , whereas the W120L and W120F mutants exhibited almost equivalent binding affinity to the wild-type. These results indicate that the bulky hydrophobic residues are important for Bcl-x L binding. The findings will be helpful in understanding the apoptosis networks between viral proteins and host factors., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Supercritical fluid technologies offer an innovative method for food industry and drug discovery from natural sources. The aim of the study is to investigate the anti-tumor activity of piperine rich extract by supercritical fluid (SFE) from black pepper (Piper nigrum). In silico docking simulations predicted anti-tumor molecular mechanism and protein-piperine hydrophobic interactions, showing hydrogen bonds between piperine and residue Ser5 inside the ATP binding site in CDK2. Moreover, piperine interacts with peptide substrate residue Lys8 inside its binding site in Cyclin A molecule. Other predicted interaction showed piperine inside the hydrophobic groove of Bcl-xL. Confirming the docking simulation, in vitro assays with SFE (40 °C/30 MPa) showed cytotoxicity to MCF-7 cells (IC50 = 27.8 ± 6.8 μg/ml) correlated to increased apoptosis. Balb/c mice-bearing Ehrlich Ascites Carcinoma (EAC) group that received the SFE (100 mg/kg/day) showed tumor growth inhibition (60%) and increased mice survival (50%), probably related to cell cycle arrest (G2/M) and increased apoptosis. In vivo treatments with SFE increased the expression of pro-apoptotic proteins (p53 and Bax), inhibited cell cycle proteins (CDK2, Cyclin A) and anti-apoptotic protein (Bcl-xL). Thus, confirming in silico predicted inhibitory interactions. These results clearly showed promising performance of the piperine-rich fraction recovered from black pepper, drawing attention to its use as complementary therapy for cancer., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Hepatitis B virus (HBV) X protein, HBx, interacts with anti-apoptotic Bcl-2 and Bcl-xL proteins through its BH3-like motif to promote HBV replication and cytotoxicity. Here we report the crystal structure of HBx BH3-like motif in complex with Bcl-xL where the BH3-like motif adopts a short α-helix to snuggle into a hydrophobic pocket in Bcl-xL via its noncanonical Trp120 residue and conserved Leu123 residue. This binding pocket is ~2 Å away from the canonical BH3-only binding pocket in structures of Bcl-xL with proapoptotic BH3-only proteins. Mutations altering Trp120 and Leu123 in HBx impair its binding to Bcl-xL in vitro and HBV replication in vivo, confirming the importance of this motif to HBV. A HBx BH3-like peptide, HBx-aa113-135, restores HBV replication from a HBx-null HBV replicon, while a shorter peptide, HBx-aa118-127, inhibits HBV replication. These results provide crucial structural and functional insights into drug designs for inhibiting HBV replication and treating HBV patients.

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. In this in vitro study, we employed a combination of fluorescence and EPR spectroscopy to characterize structural features of the bilayer-inserted conformation of Bcl-xL and the lipid modulation of its membrane insertion transition. Our results indicate that the core hydrophobic helix α6 inserts into the bilayer without adopting a transmembrane orientation. This insertion disrupts the packing of Bcl-xL and releases the regulatory N-terminal BH4 domain (α1) from the rest of the protein structure. Our data demonstrate that both insertion and refolding of Bcl-xL are modulated by lipid composition, which brings the apparent pK a of insertion to the threshold of physiological pH. We hypothesize that conformational rearrangements associated with the bilayer insertion of Bcl-xL result in its switching to a so-called non-canonical mode of apoptotic inhibition. Presented results suggest that the alteration in lipid composition before and during apoptosis can serve as an additional factor regulating the permeabilization of the mitochondrial outer membrane., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Irinotecan is a strong anticancer drug whose mechanism of action has been reported only for the inhibition of DNA topoisomerase I (Topo I) through its active metabolite SN-38. In this study, we present a new mechanism of Irinotecan which inhibits the activities of MDM2, an E3 ligase of tumour suppressor p53, and Bcl-xL, an anti-apoptotic protein, through direct binding. In our structure modelling study, Irinotecan could fit to the binding sites of MDM2 and Bcl-xL for their known drugs, Nutlin-3 and ABT-737, with a better binding affinity than to Topo I. The direct binding of Irinotecan to both proteins was confirmed through a NMR study. We further showed that Irinotecan increased the amount of p53 only in the presence of MDM2 and inhibited the physical interaction of Bcl-xL with Bim, a core pro-apoptotic protein. In addition, we demonstrated that Irinotecan induced the down regulation of proliferation and strong G2/M arrest in HCT116 colon cancer cells shortly after treatment. Collectively, we suggest a new mechanism of action for Irinotecan as a dual target inhibitor of MDM2 and Bcl-xL facilitating the anticancer activities mediated by p53 and Bcl-xL interaction partners., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Interactions between the pro-survival and pro-apoptotic members of the Bcl-2 family of proteins dictate whether a cell lives or dies. Much of our knowledge of the molecular details of these interactions has come from biochemical and structural studies on the pro-survival protein Bcl-x L . The first high-resolution structure of any Bcl-2 family member was of Bcl-x L , which revealed the conserved topology amongst all family members. Subsequent structures of Bcl-x L complexes with pro-apoptotic ligands demonstrated the general features of all pro-survival:pro-apoptotic complexes. Structural studies involving Bcl-x L were also the basis for the discovery of the first small-molecule pro-survival protein inhibitors, leading ultimately to the development of a new class of drugs now successfully used for cancer treatment in the clinic. This article will review our current knowledge of the structural biology of Bcl-x L and how this has impacted our understanding of the molecular details of the intrinsic apoptotic pathway.

Tumor initiation, progression and resistance to chemotherapy rely on cancer cells bypassing programmed cell death by apoptosis. We report that unlike other pro-apoptotic proteins, Bim contains two distinct binding sites for the anti-apoptotic proteins Bcl-XL and Bcl-2. These include the BH3 sequence shared with other pro-apoptotic proteins and an unexpected sequence located near the Bim carboxyl-terminus (residues 181-192). Using automated Fluorescence Lifetime Imaging Microscopy - Fluorescence Resonance Energy Transfer (FLIM-FRET) we show that the two binding interfaces enable Bim to double-bolt lock Bcl-XL and Bcl-2 in complexes resistant to displacement by BH3-mimetic drugs currently in use or being evaluated for cancer therapy. Quantifying in live cells the contributions of individual amino acids revealed that residue L185 previously thought involved in binding Bim to membranes, instead contributes to binding to anti-apoptotic proteins. This double-bolt lock mechanism has profound implications for the utility of BH3-mimetics as drugs. ​., Competing Interests: QL, EO, XC, JP, BL, DA No competing interests declared, (© 2019, Liu et al.)

Selective inhibition is a key focus in the design of chemotherapeutic compounds that can abrogate the oncogenic activities of anti-apoptotic Bcl-2 proteins. Although recent efforts have led to the development of highly selective BH3 mimetics, setbacks such as toxicities have limited their use in cancer therapy. Epigallocatechingallate (EGCG) has been widely reported to selectively inhibit Bcl-2 and Bcl-xL compared to other green tea phenols due to its gallate group. Herein, we investigate the interaction dynamics of EGCG at the hydrophobic grooves of Bcl-2 and Bcl-xL and the consequential effects on their BH4 domains. Arg143 and Asp108 (Bcl-2), and Glu96 and Tyr195 (Bcl-xL) formed high-affinity hydrogen interactions with the gallate group while non-gallate groups of EGCG formed weak interactions. EGCG-bound proteins showed systemic perturbations of BH4 domains coupled with the burial of crucial surface-exposed residues such as Lys17 (Bcl-2) and Asp11 (Bcl-xL); hence, a distortion of non-canonical domain interactions. Interactions of gallate group of EGCG with key hydrophobic groove residues underlie EGCG selectivity while concurrent BH4 domain perturbations potentiate EGCG inhibitory activities. Findings will aid the optimization and design of selective inhibitors that could suppress anti-apoptotic activities of Bcl2-family proteins with minimal toxicities.

Networks of biological molecules are key to cellular function, governing processes ranging from signal cascade propagation to metabolic pathway regulation. Genetic duplication processes give rise to sets of regulatory proteins that have evolved from a common ancestor, leading to interactomes whose dysregulation is often associated with disease. A better understanding of the determinants of specificity at interfaces shared by functionally related proteins is crucial to the rational design of novel pharmacotherapeutic agents. To this end, a comprehensive data set of drug and drug-like binders was assembled for the Bcl-xL and Bcl-2 antiapoptotic proteins-archetypal examples of regulatory systems governed by evolutionarily conserved protein-protein interactions. These were first used to derive a two-dimensional quantitative structure-activity relationship (2D QSAR) model, predicting ligand specificity for these homologous proteins. The strengths and weaknesses of high-throughput 2D QSAR were then compared and contrasted to those of theoretically rigorous thermodynamic integration calculations performed on 14 complexes of Bcl-xL-specific, Bcl-2-specific, and potent dual binders bound to the Bcl-xL and Bcl-2 proteins. We demonstrate that free energy calculations provide an added layer of essential information, which traditional QSAR cannot capture. Moreover, we show that protein energetic responses to different ligands, expressed as per-residue energy values, can be used to fingerprint the protein-ligand interaction, extending the framework of four-dimensional molecular dynamics/quantitative structure-activity relationships (4D-MD/QSAR) toward the facilitation of future drug design strategies.

Progress in drug delivery is hampered by a lack of efficient strategies to target drugs with high specificity and precise spatiotemporal regulation. The remote control of nanoparticles and drugs with light allows regulation of their action site and dosage. Peptide-based drugs are highly specific, non-immunogenic, and can be designed to cross the plasma membrane. In order to combine target specificity and remote control of drug action, here we describe a versatile strategy based on a generalized template to design nanoswitchable peptides that modulate protein-protein interactions upon light activation. This approach is demonstrated to promote photomodulation of two important targets involved in apoptosis (the interactions Bcl-xL-Bak and MDM2-p53), but can be also applied to a large pool of therapeutically relevant protein-protein interactions mediated by α-helical motifs. The template can be adjusted using readily available information about hot spots (residues contributing most to the binding energy) at the protein-protein interface of interest., (© 2019 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The C-terminal helix of the Bcl-xl is known to initiate the membrane insertion of the protein by anchoring into the mitochondrial outer membrane. The C-terminal charged residues of that helix, R232 and K233, are reported to have an important structural role in the process of that insertion. The present work provides a quantitative understanding of the thermodynamic contribution of these residues on the membrane insertion energy-profile, calculated from the Adaptive Biasing Force based MD simulations of 2.67 μs altogether. Interestingly, the effect of the single neutralizing mutations at the C-terminus, i.e. K233A or R232A, is easily tolerated by the peptide without impacting the nature of insertion energy-profile, indicating the efficiency of one positively charged residue to drive the insertion. Whereas a double mutant, i.e. R232A and K233A, makes a significant impact on the energy-profile by destabilizing the membrane-associated states, as well as the membrane-embedded states. The finding provides molecular-level mechanistic insight. The water-mediated interaction formed by the peptide polar side chains within the bilayer core is found to modulate the membrane response during peptide insertion and that subsequently regulates the insertion mechanism. Mutation of the C-terminal residues eventually alters such a cascade of interactions that results in an insertion through energetically more expensive pathway. Since any one of the positively charged residues at the terminal is critical to ensure the membrane insertion, it appears that the natural selection of 'two' instead of 'one' charged residue is redundant in the context of membrane anchoring but may be important for other biochemical events., (Copyright © 2018 Elsevier B.V. All rights reserved.)

The proteins of Bcl-2 family, which are promising anti-cancer-drug targets, have substantial similarity in primary sequence and share homologous domains as well as similar structural folds. In spite of similarities in sequence and structures, the members of its pro- and anti- apoptotic subgroups form complexes with different type of partners with discriminating binding affinities. Understanding the origin of this discrimination is very important for designing ligands that can either selectively target a protein or could be made broad ranged as necessary. Using principal component analysis (PCA) of the available structures and from the analysis of the evolution of the binding pocket residues, the correlation has been investigated considering two important anti-apoptotic protein Bcl-xl and Mcl-1, which serve as two ideal representatives of this family. The flexibility of the receptor enables them to discriminate between the ligands or the binding partners. It has been observed that although Bcl-xl and Mcl-1 are classified as homologous proteins, through the course of evolution the binding pocket residues are highly conserved for Bcl-xl; whereas they have been substituted frequently in Mcl-1. The investigation has revealed that the Bcl-xl can adjust the backbone conformation of the binding pocket residues to a larger extent to complement with the shape of different binding partners whereas the Mcl-1 shows more variation in the side chain conformation of binding pocket residues for the same purpose., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Chronic hypoxic stress results in deposition of lipofuscin granules in the CA3 region of hippocampal neurons which contributes to neurodegeneration and accelerated neuronal aging. Oxidative stress and mitophagy during hypoxia are crucial to cause aggregation of these lipofuscin granules in hypoxic neurons. Salidroside, a glucoside derivative of β-Tyrosol, has been reported to protect hypoxic neurons through maintenance of mitochondrial activity. The present study is aimed at investigating the potential of Salidroside in preventing mitophagy during chronic hypoxia and identification of the molecular targets and underlying signaling mechanisms. In-silico analysis for interaction of salidroside with Bcl-x L was carried out using VLife MDS software. The prophylactic efficacy of Salidroside for amelioration of global hypoxia induced neuronal aging was studied in adult male Sprague-Dawley rats exposed to hypobaric hypoxia simulating an altitude of 7600 m for 21 days. Salidroside was supplemented at a daily dose of 25 mg kg -1 b.w. p.o. during hypoxic exposure. Ultra-structural and immune-histological studies were conducted to study lipofuscin aggregation and mitophagy. In-silico findings on salidroside mediated stabilization of Bcl-x L were validated by investigating its effect on downstream signaling molecules involved in mitophagy. Administration of Salidroside reduced deposition of lipofuscin in hypoxic CA3 hippocampal neurons and prevented mitophagy. Salidroside stabilizes Bcl-x L in hypoxic neurons resulting in inhibition of PGAM5 phosphatase activity and maintenance of FUNDC1 in phosphorylated state. Salidroside mediated inhibition of pFUNDC1 dephosphorylation prevents FUNDC1-LC3 II interaction which is crucial for mitophagy. The present study demonstrates potential of Salidroside in preventing lipofuscin deposition during chronic hypoxic stress., (Copyright © 2018. Published by Elsevier Inc.)

In susceptible tumor cells, DNA-damaging antineoplastic agents induce an increase in intracellular pH during the premitochondrial stage of apoptosis. The rate of nonenzymatic deamidation of two asparagines in the anti-apoptotic protein Bcl-x L is accelerated by this increase in pH. Deamidation of these asparagines is a signal for the degradation of Bcl-x L , which is a component of the apoptotic response to DNA damage. It has previously been shown that the increase in pH is mediated by the ion transporter Na + /H + exchanger 1 in some cells. Here we demonstrate that one or more additional ion transporters also have a role in the regulation of Bcl-x L deamidation in at least some tumor cell lines and fibroblasts. As a second, independent finding, we report that there are histidines in close proximity to the Bcl-x L deamidation sites that are highly conserved in land-dwelling species and we present evidence that deamidation of human Bcl-x L is intramolecularly catalyzed in a manner that is dependent upon these histidines. Further, we present evidence that these histidines act as a pH-sensitive switch that enhances the effect of the increase in pH on the rate of Bcl-x L deamidation. The conservation of such histidines implies that human Bcl-x L is in essence "designed" to be deamidated, which provides further evidence that deamidation serves as a bona fide regulatory post-translational modification of Bcl-x L ., (Copyright © 2018 Elsevier B.V. All rights reserved.)

E2F1 is the main pro-apoptotic effector of the pRB-regulated tumor suppressor pathway by promoting the transcription of various pro-apoptotic proteins. We report here that E2F1 partly localizes to mitochondria, where it favors mitochondrial outer membrane permeabilization. E2F1 interacts with BCL-xL independently from its BH3 binding interface and induces a stabilization of BCL-xL at mitochondrial membranes. This prevents efficient control of BCL-xL over its binding partners, in particular over BAK resulting in the induction of cell death. We thus identify a new, non-BH3-binding regulator of BCL-xL localization dynamics that influences its anti-apoptotic activity., (© 2017 The Authors.)

B-cell lymphoma 2 (Bcl-2) family proteins are potential drug targets in cancer and have a relatively flat and flexible binding site. ABT-199 is one of the most promising selective Bcl-2 inhibitors, and A-1155463 selectively inhibits Bcl-XL. Although the amino acid sequences of the binding sites of these two inhibitors are similar, the inhibitors selectively bind the target protein. In order to determine the origin of the selectivity of these inhibitors, we conducted molecular dynamics simulations using protein-inhibitor modeling. We confirmed that ASP103 of Bcl-2 is a key residue and that hydrogen bonding between ASP103 and ABT-199 confers the Bcl-2 selectivity of this inhibitor. For Bcl-XL selectivity, the secondary structure of α-helix 3 is a key factor. PHE105, SER106, and LEU108 in the loose α-helix 3 interact with A-1155463 to confer Bcl-XL selectivity. These findings provide important insights into the molecular mechanisms of selective inhibitors of Bcl-2 family proteins., (Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.)

DJ-1 is a multifunctional protein associated with Parkinson's disease (PD) and tumorigenesis. In response to ultraviolet B (UVB) irradiation, DJ-1 is translocated into the mitochondria, and its interaction with the mitochondrial protein Bcl-X L protects cells against death. In this study, we characterized the molecular interaction between DJ-1 and Bcl-X L by NMR spectroscopy. The NMR chemical shift perturbation data demonstrated that the oxidized but not the reduced form of DJ-1 binds to the predominantly hydrophobic groove surrounded by the BH1-BH3 domains in Bcl-X L . In addition, our results showed that the C-terminal α8-helix peptide (Cpep) of DJ-1 binds to the pro-apoptotic BH3 peptide-binding hydrophobic groove in Bcl-X L and, thus, acts as a Bcl-X L -binding motif. In combination with the NMR chemical shift perturbation data, a refined structural model of the Bcl-X L /DJ-1 Cpep complex revealed that the binding mode is remarkably similar to that of other Bcl-X L /pro-apoptotic BH3 peptide complexes. Taken together, our results provide a structural basis for the binding mechanism between DJ-1 and Bcl-X L , which will contribute to molecular understanding of the role of mitochondrial DJ-1 in Bcl-X L regulation in response to oxidative stress., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Antiapoptotic Bcl-xL plays central roles in regulating programed cell death. Partial unfolding of Bcl-xL has been observed at the interface upon specific binding to the pro-apoptotic BH3-only protein PUMA, which in turn disrupts the interaction of Bcl-xL with tumor suppressor p53 and promotes apoptosis. Previous analysis of existing Bcl-xL structures and atomistic molecular dynamics (MD) simulations have suggested that substantial intrinsic structure heterogeneity exists at the BH3-only protein binding interface of Bcl-xL to facilitate its conformational transitions upon binding. In this study, enhanced sampling is applied to further characterize the interfacial conformations of unbound Bcl-xL in explicit solvent. Extensive replica exchange with solute tempering (REST) simulations, with a total accumulated time of 16 μs, were able to cover much wider conformational spaces for the interfacial region of Bcl-xL. The resulting structural ensembles are much better converged, with local and long-range structural features that are highly consistent with existing NMR data. These simulations further demonstrate that the BH3-only protein binding interface of Bcl-xL is intrinsically disordered and samples many rapidly interconverting conformations. Intriguingly, all previously observed conformers are well represented in the unbound structure ensemble. Such intrinsic structural heterogeneity and flexibility may be critical for Bcl-xL to undergo partial unfolding induced by PUMA binding, and likely provide a robust basis that allows Bcl-xL to respond sensitively to binding of various ligands in cellular signaling and regulation.

Bcl-xl protein has a long unstructured loop attached to its structured region which joins two helices. The necessity to have this unstructured segment in Bcl-xl is not yet well understood. To what extent the unstructured segment can influence the dynamics of the structured region of protein, with potential to influence the function, has been investigated in this work. Molecular dynamics simulation and principal component analysis show how the loop affects the internal motions of the protein, particularly its ligand binding pocket. Generally an unstructured region in the structure would promote flexibility resulting entropic stability but in contrary, here it narrows down the conformational space of the structured region of protein that could be hypothesized to impact the functional precision. Effects of the loop propagate to the binding pocket through structural rearrangements of polar side chains. The immediate suspicion of possible impact of phosphorylation to modulate the function of the protein is proven to be a fact, as the phosphorylated S49 and S62 located on the large unstructured region are seen to perturb the electrostatic network of the structure; an observation that validates and clarifies the role of loop as a modulator through biophysical and biochemical mechanisms. Proteins 2017; 85:1567-1579. © 2017 Wiley Periodicals, Inc., (© 2017 Wiley Periodicals, Inc.)

An interesting feature of Bcl-xL protein is the presence of an unstructured loop domain between α1 and α2 helices, a domain not essential for its anti-apoptotic function and absent in CED-9 protein. Within this domain, Bcl-xL undergoes dynamic phosphorylation and dephosphorylation at Ser49 and Ser62 during G2 and mitosis in human cells. Studies have revealed that when these residues are mutated, cells harbour mitotic defects, including chromosome mis-attachment, lagging, bridging and mis-segregation with, ultimately, chromosome instability and aneuploidy. We undertook genetic experiments in Caenorhabditis elegans to understand the importance of Bcl-xL (Ser49) and (Ser62) in vivo. Transgenic worms carrying single-site S49A, S62A, S49D, S62D and dual site S49/62A mutants were generated and their effects were analyzed in germlines of young adult worms. Worms expressing Bcl-xL variants showed decreased egg-laying and hatching potency, variations in the length of their mitotic regions but not of their transition zones, appearance of chromosomal abnormalities at their diplotene stages, and increased germline apoptosis, with the exception of the S62D variants. Some of these transgenic strains, particularly the Ser to Ala variants, also showed slight modulations of lifespan compared to their controls. In addition, RNAi experiments silencing expression of the various Bcl-xL variants reversed their effects in vivo. Our in vivo observations confirmed the importance of Ser49 and Ser62 within Bcl-xL loop domain in maintaining chromosome stability.

The limited efficacy of marketed anticancer agents demands the design of novel target-specific hybrid molecules incorporating multiple bioactive pharmacores to combat cancer. In the present study, a one-pot simple and efficient T3P ® mediated procedure for the preparation of twelve new 3-(substituted- [1,2,4]triazolo[3,4-b] [1,3,4]thiadiazolo)-1H-indoles with short reaction times, easy workup procedure, good yields, and purity of products is described. Cytotoxicity assay (MTT), flow-cytometric univariate cell cycle analysis, Annexin V-FITC staining and DNA fragmentation for cell death mechanism suggested that compound 3d with chloro-substituted phenyl ring induced enhanced cytotoxicity by an apoptotic pathway with high differential toxicity to breast adenocarcinoma cells (MCF-7) when compared with normal human dermal fibroblast cells. Additionally, the interaction between the BH3 domain of anti-apoptotic proteins Bcl-2 and Bcl-xL with the pharmacophore 3d was examined by molecular docking simulations to assess its potential to induce apoptosis. The docking solutions were proposed to explain the observed selectivity of 3d to Bcl-xL protein. From the present findings, the lead compound, 3d exhibited better anticancer activity when related to the other synthesized molecules with specific action on MCF-7 cells and hence can be considered as a plausible candidate chemo-therapeutic agent, although this warrants further experimentation., (Copyright © 2017 Elsevier B.V. All rights reserved.)