Modeling the Binding of Anticancer Peptides and Mcl-1.

Mcl-1 (myeloid cell leukemia 1), a member of the Bcl-2 family, is upregulated in various types of cancer. Peptides representing the BH3 (Bcl-2 homology 3) region of pro-apoptotic proteins have been demonstrated to bind the hydrophobic groove of anti-apoptotic Mcl-1, and this interaction is responsible for regulating apoptosis. Structural studies have shown that, while there is high overall structural conservation among the anti-apoptotic Bcl-2 (B-cell lymphoma 2) proteins, differences in the surface groove of these proteins facilitates binding specificity. This binding specificity is crucial for the mechanism of action of the Bcl-2 family in regulating apoptosis. Bim-based peptides bind specifically to the hydrophobic groove of Mcl-1, emphasizing the importance of these interactions in the regulation of cell death. Molecular docking was performed with BH3-like peptides derived from Bim to identify high affinity peptides that bind to Mcl-1 and to understand the molecular mechanism of their interactions. The interactions of three identified peptides, E2gY, E2gI, and XXA1_F3dI, were further evaluated using 250 ns molecular dynamics simulations. Conserved hydrophobic residues of the peptides play an important role in their binding and the structural stability of the complexes. Understanding the molecular basis of interaction of these peptides will assist in the development of more effective Mcl-1 specific inhibitors.

Structural and biochemical analyses of Bcl-xL in complex with the BH3 domain of peroxisomal testis-specific 1.

Antiapoptotic B-cell lymphoma-2 (Bcl-2) proteins suppress apoptosis by interacting with proapoptotic regulators. They commonly contain a hydrophobic groove where the Bcl-2 homology 3 (BH3) domain of Bcl-2 family members or BH3 domain-containing non-Bcl-2 family proteins can be accommodated. Peroxisomal testis-specific 1 (Pxt1) was previously identified as a male germ cell-specific protein whose overexpression causes germ cell apoptosis and infertility in male mice. Sequence and biochemical analyses also showed that human Pxt1, which is composed of 134 amino acids and is longer than mouse Pxt1 consisting of only 51 amino acids, has a BH3 domain that interacts with antiapoptotic Bcl-2 proteins, including Bcl-2 and Bcl-xL. In this study, we determined the crystal structure of Bcl-xL bound to the human Pxt1 BH3 domain. The five BH3 consensus residues are well conserved in the human Pxt1 BH3 domain and make a critical contribution to the complex formation in a canonical manner. Structural and biochemical analyses also demonstrated that Bcl-xL interacts with the BH3 domain of human Pxt1 but not with that of mouse Pxt1, and that residues 76-83 of human Pxt1, absent in mouse Pxt1, play a pivotal role in the intermolecular binding to Bcl-xL. While Bcl-xL consistently colocalized with human Pxt1 in mitochondria, it did not do so with mouse Pxt1, when expressed in HeLa cells. Collectively, these data verified that human and mouse Pxt1 differ in their binding ability to the antiapoptotic regulator Bcl-xL, which might affect their functionality in controlling apoptosis.

The Structural Biology of Bcl-xL.

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-xL. The first high-resolution structure of any Bcl-2 family member was of Bcl-xL, which revealed the conserved topology amongst all family members. Subsequent structures of Bcl-xL complexes with pro-apoptotic ligands demonstrated the general features of all pro-survival:pro-apoptotic complexes. Structural studies involving Bcl-xL 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-xL and how this has impacted our understanding of the molecular details of the intrinsic apoptotic pathway.

[Effects of Bcl-2 family on the thyroid cell apoptosis of experimental autoimmune thyroiditis mice induced by iodine].

OBJECTIVE: To study the effect of Bcl-2 family members on experimental autoimmune thyroiditis(EAT) and explore the pathogenesis of autoimmune thyroiditis. METHODS: Twenty-four female 4-5 week old NOD-SCID mice were randomly divided into four groups(six mice in each group): control group, high iodine group, poly(I:C) group and high iodine combined with poly(I:C) group. Control group and poly(I:C) group were fed with distilled water, while the high iodine group and high iodine combined with poly(I:C) group were supplied with 0. 05% NaI in their drinking water for 16 weeks. Poly(I:C) group and high iodine combined with poly(I:C) group received intraperitoneal injection of 100 µL poly(I:C)(1 µg/µL) at monday, wednesday and friday of the 11 th and 15 th week. Serum and thyroid were obtained at the last day of the 16 th week. The EAT model was confirmed by ELISA method and pathological HE staining, the apoptosis of thyroid cell were detected by TUNEL method and Cyt-C immunocytochemistry assay, and the mRNA levels of Bcl-2 family members in thyroid were determined by real-time qPCR method. RESULTS: EAT model was established using NOD-SCID mice through high-iodine feeding combined with poly(I:C) intraperitoneal injection. The degree of cell apoptosis and the Cyt-C expression levels were positively correlated with inflammation in thyroid follicular epithelial cells. The mRNA levels of Noxa, PUMA and Bid of high iodine group and high iodine combined with poly(I:C) group were higher than those in control and poly(I:C) groups(P<0. 05). CONCLUSION: Mitochondrial apoptosis pathway is involved in the thyroid cell apoptosis of EAT induced by high iodine, and the apoptosis may be regulated by the up-regulation of Noxa, PUMA and Bid, which belong to the pro-apoptotic members of Bcl-2 family.

Characterisation of the conformational preference and dynamics of the intrinsically disordered N-terminal region of Beclin 1 by NMR spectroscopy.

Beclin 1 is a 450 amino acid protein that plays critical roles in the early stages of autophagosome formation. We recently reported the successful expression, purification and structural characterisation of the entire N-terminal region of Beclin 1 (residues 1-150), including its backbone NMR chemical shift assignments. Based on assigned backbone NMR chemical shifts, it has been established that the N-terminal region of Beclin 1 (1-150), including the BH3 domain (112-123), is intrinsically disordered in the absence of its interaction partners. Here, a detailed study of its conformational preference and backbone dynamics obtained from an analysis of its secondary structure populations using the δ2D method, and the measurements of effective hydrodynamic radius as well as (1)H temperature coefficients, (1)H solvent exchange rates, and (15)N relaxation parameters of backbone amides using NMR spectroscopy is reported. These data provide further evidence for the intrinsically disordered nature of the N-terminal region of Beclin 1 and support the view that the helical conformation adopted by the Beclin 1 BH3 domain upon interaction with binding partners such as BCL-2 pro-survival proteins is likely induced rather than pre-existing.

Role of single disulfide linkages in the folding and activity of scyllatoxin-based BH3 domain mimetics.

Anti-apoptotic Bcl-2 proteins are implicated in pathogenic cell survival and have attracted considerable interest as therapeutic targets. We recently developed a class of synthetic peptide based on scyllatoxin (ScTx) designed to mimic the helical BH3 interaction domain of the pro-apoptotic Bcl-2 protein Bax. In this communication, the contribution of single disulfides in the folding and function of ScTx-Bax peptides was investigated. We synthesized five ScTx-Bax variants, each presenting a different combination of native disulfide linkage and evaluated their ability to directly bind Bcl-2 in vitro. It was determined that the position of the disulfide linkage had significant implications on the structure and function of ScTx-Bax peptides. This study underscores the importance of structural dynamics in BH3:Bcl-2 interactions and further validates ScTx-based ligands as potential modulators of anti-apoptotic Bcl-2 function. Copyright © 2017 European Peptide Society and John Wiley & Sons, Ltd.

Structural studies of the protein endostatin in fusion with BAX BH3 death domain, a hybrid that presents enhanced antitumoral activity.

Endostatin (ES) is an antiangiogenic protein that exhibits antitumor activity in animal models. However, the activity observed in animals was not observed in human clinical trials. ES-BAX is a fusion protein composed of two functional domains: ES, which presents specificity and is internalized by activated endothelial cells and the proapoptotic BH3 domain of the protein BAX, a peptide inductor of cellular death when internalized. We have previously shown (Chura-Chambi et al., Cell Death Dis, 5, e1371, 2014) that ES-BAX presents improved antitumor activity in relation to wild-type ES. Secondary and tertiary structures of ES-BAX are similar to ES, as indicated by homology-modeling studies and molecular dynamics simulations. Tryptophan intrinsic fluorescence and circular dichroism spectroscopy corroborate these data. 15 N HSQC NMR indicates that ES-BAX is structured, but some ES residues have suffered chemical shift perturbations, suggesting that the BH3 peptide interacts with some parts of the ES protein. ES and ES-BAX present similar stability to thermal denaturation. The production of stable hybrid proteins can be a new approach to the development of therapeutic agents presenting specificity for tumoral endothelium and improved antitumor effect.

Intrinsically disordered regions in autophagy proteins.

Autophagy is an essential eukaryotic pathway required for cellular homeostasis. Numerous key autophagy effectors and regulators have been identified, but the mechanism by which they carry out their function in autophagy is not fully understood. Our rigorous bioinformatic analysis shows that the majority of key human autophagy proteins include intrinsically disordered regions (IDRs), which are sequences lacking stable secondary and tertiary structure; suggesting that IDRs play an important, yet hitherto uninvestigated, role in autophagy. Available crystal structures corroborate the absence of structure in some of these predicted IDRs. Regions of orthologs equivalent to the IDRs predicted in the human autophagy proteins are poorly conserved, indicating that these regions may have diverse functions in different homologs. We also show that IDRs predicted in human proteins contain several regions predicted to facilitate protein-protein interactions, and delineate the network of proteins that interact with each predicted IDR-containing autophagy protein, suggesting that many of these interactions may involve IDRs. Lastly, we experimentally show that a BCL2 homology 3 domain (BH3D), within the key autophagy effector BECN1 is an IDR. This BH3D undergoes a dramatic conformational change from coil to α-helix upon binding to BCL2s, with the C-terminal half of this BH3D constituting a binding motif, which serves to anchor the interaction of the BH3D to BCL2s. The information presented here will help inform future in-depth investigations of the biological role and mechanism of IDRs in autophagy proteins.

Canonical and Noncanonical Functions of the BH3 Domain Protein Bid in Apoptosis, Oncogenesis, Cancer Therapeutics, and Aging.

Effective cancer therapy with limited adverse effects is a major challenge in the medical field. This is especially complicated by the development of acquired chemoresistance. Understanding the mechanisms that underlie these processes remains a major effort in cancer research. In this review, we focus on the dual role that Bid protein plays in apoptotic cell death via the mitochondrial pathway, in oncogenesis and in cancer therapeutics. The BH3 domain in Bid and the anti-apoptotic mitochondrial proteins (Bcl-2, Bcl-XL, mitochondrial ATR) it associates with at the outer mitochondrial membrane provides us with a viable target in cancer therapy. We will discuss the roles of Bid, mitochondrial ATR, and other anti-apoptotic proteins in intrinsic apoptosis, exploring how their interaction sustains cellular viability despite the initiation of upstream death signals. The unexpected upregulation of this Bid protein in cancer cells can also be instrumental in explaining the mechanisms behind acquired chemoresistance. The stable protein associations at the mitochondria between tBid and anti-apoptotic mitochondrial ATR play a crucial role in maintaining the viability of cancer cells, suggesting a novel mechanism to induce cancer cell apoptosis by freeing tBid from the ATR associations at mitochondria.

Structure-guided rational design of α/ß-peptide foldamers with high affinity for BCL-2 family prosurvival proteins.

We have used computational methods to improve the affinity of a foldamer ligand for its target protein. The effort began with a previously reported α/ß-peptide based on the BH3 domain of the proapoptotic protein Puma; this foldamer binds tightly to Bcl-x(L) but weakly to Mcl-1. The crystal structure of the Puma-derived α/ß-peptide complexed to Bcl-x(L) was used as the basis for computational design of variants intended to display improved binding to Mcl-1. Molecular modelling suggested modification of three α residues of the original α/ß backbone. Individually, each substitution caused only a modest (4- to 15-fold) gain in affinity; however, together the three substitutions led to a 250-fold increase in binding to Mcl-1. These modifications had very little effect on affinity for Bcl-x(L). Crystal structures of a number of the new α/ß-peptides bound to either Mcl-1 or Bcl-x(L) validated the selection of each substitution. Overall, our findings demonstrate that structure-guided rational design can be used to improve affinity and alter partner selectivity of peptidic ligands with unnatural backbones that bind to specific protein partners.

Engineered Polymeric Nanovector for Intracellular Peptide Delivery in Antitumor Therapy.

Objective: This study aimed to deliver a polypeptide from the Bax-BH3 domain (BHP) through the synthesis of self-assembled amphiphile nanovectors (NVs) and to assess their potential for cancer therapeutic applications and biological safety in vitro and in vivo. These findings provide valuable options for cancer intervention and a novel approach for the rational design of therapeutics. Methods: We studied the antitumor activity of BHP by preparing RGDfK-PHPMA-b-Poly (MMA-alt-(Rhob-MA)) (RPPMMRA) and encapsulating it in BHP-NV. We also performed a series of characterizations and property analyses of RPPMMRA, including its size, stability, and drug-carrying capacity. The biocompatibility of RPPMMRA was evaluated in terms of cytotoxicity and hemolytic effects. The pro-apoptotic capacity of BHP was evaluated in vitro using mitochondrial membrane potential, flow cytometry, and apoptosis visualization techniques. The potential therapeutic effects of BHP on tumors were explored using reverse molecular docking. We also investigated the in vivo proapoptotic effect of BHP-NV in a nude mouse tumor model. Results: NVs were successfully prepared with hydrated particle sizes ranging from 189.6 nm to 256.6 nm, spherical overall, and were able to remain stable in different media for 72 h with drug loading up to 15.2%. The NVs were be successfully internalized within 6 h with good biocompatibility. Neither BHP nor NV showed significant toxicity when administered alone, however, BHP-NV demonstrated significant side effects in vitro and in vivo. The apoptosis rate increased significantly from 14.13% to 66.34%. Experiments in vivo showed that BHP-NV exhibited significant apoptotic and tumor-suppressive effects. Conclusion: A targeted fluorescent NV with high drug delivery efficiency and sustained release protected the active center of BHP, constituting BHP-NV for targeted delivery. RPPMMRA demonstrated excellent biocompatibility, stability, and drug loading ability, whereas and BHP-NV demonstrated potent antitumor effects in vivo and in vitro.

Isolation, Characterization, and Autophagy Function of BECN1-Splicing Isoforms in Cancer Cells.

Alternative splicing allows the synthesis of different protein variants starting from a single gene. Human Beclin 1 (BECN1) is a key autophagy regulator that acts as haploinsufficient tumor suppressor since its decreased expression correlates with tumorigenesis and poor prognosis in cancer patients. Recent studies show that BECN1 mRNA undergoes alternative splicing. Here, we report on the isolation and molecular and functional characterization of three BECN1 transcript variants (named BECN1-α, -ß and -γ) in human cancer cells. In ovarian cancer NIHOVCAR3, these splicing variants were found along with the canonical wild-type. BECN1-α lacks 143 nucleotides at its C-terminus and corresponds to a variant previously described. BECN1-ß and -γ lack the BCL2 homology 3 domain and other regions at their C-termini. Following overexpression in breast cancer cells MDA-MB231, we found that BECN1-α stimulates autophagy. Specifically, BECN1-α binds to Parkin and stimulates mitophagy. On the contrary, BECN1-ß reduces autophagy with a dominant negative effect over the endogenous wild-type isoform. BECN1-γ maintains its ability to interact with the vacuolar protein sorting 34 and only has a slight effect on autophagy. It is possible that cancer cells utilize the alternative splicing of BECN1 for modulating autophagy and mitophagy in response to environmental stresses.

Synthesis of Scyllatoxin-Based BH3 Domain Mimetics with Diverse Patterns of Native Disulfide Bonds.

This article outlines the design and development of scyllatoxin (ScTx)-based BH3 domain mimetics with diverse patterns of native disulfide bonds. More specifically, this method summarizes the total chemical synthesis of ScTx-based peptides that contain zero, one, two, or three disulfide linkages, including techniques to generate variants with any combination of native disulfides. Each peptide reported herein is generated on solid-phase support using microwave-assisted coupling procedures, and all reaction parameters related to the peptide synthesis are described in detail. The various disulfide patterns of the ScTx-based constructs are established during peptide synthesis and are ultimately verified by mass analysis of trypsin-digested fragments. The BH3 domain mimetics developed herein were generated by transposing residues from the helical BH3 domain of the pro-apoptotic BCL2 protein Bax to the α-helix of wild-type ScTx. Interestingly, we found that the relative binding affinities of ScTx-Bax peptides for the anti-apoptotic BCL2 protein Bcl-2 (proper) were heavily influenced by the number and position of disulfide linkages within the ScTx-Bax sequence. As a consequence, we were able to utilize ScTx-Bax BH3 domain mimetics with varied patterns of disulfide bonds to survey how structural rigidity within the helical Bax BH3 domain affects binding to promiscuous anti-apoptotic BCL2 proteins. More broadly, the ability to generate ScTx-based molecules that contain any combination of native disulfide bonds expands the utility of such constructs as tools to study the molecular nature of protein-protein interactions. © 2022 Wiley Periodicals LLC. Basic Protocol 1: Synthesis and characterization of ScTx-based Bax BH3 domain mimetics Basic Protocol 2: Oxidation of ScTx-Bax BH3 domain mimetics containing one, two, or three disulfide linkages Support Protocol: Mapping of disulfide linkages in oxidized ScTx-Bax BH3 domain mimetics.

Design BH3 domain fusion protein as targeting pro-apoptotic self-assembling nanoparticles.

Cancer is a serious global health issue, and apoptosis is a logical and practical cancer therapeutic strategy. Apoptosis responses to internal and external signals. Both BH3 domain in the pro-apoptotic proteins and truncated BH3 domain can stimulate cell apoptosis. However, the faults of peptides in systemic administration restrict the applications of truncated BH3 domain. Ferritin, as an attractive nanoparticle with the capacity of self-assemble to unique hollow spherical structure, could display truncated BH3 domain an N-terminal. Thus, in this study, we designed a pro-apoptosis self-assembling protein nanoparticle by BH3 domain fusion at N-terminal of ferritin. We evaluated the size, cytotoxicity and pro-apoptosis effect of these nanoparticles. The results showed that RGD-BH3-HFn, BH3-HFn and HFn had uniformly spherical structure with sizes at 26.08 ± 0.11 nm, 22.07 ± 0.67 nm, and 16.81 ± 0.88 nm, respectively; RGD-BH3-HFn has stronger cytotoxicity against tumor cells than BH3-HFn and HFn. The total apoptosis ratios (including necrosis) of C6 cells induced by RGD-BH3-HFn, BH3-HFn, and HFn proteins were 15.24%, 10.13% and 2.14%, respectively; those of bEnd.3 cells were 15.47%, 7.33% and 1.70%, respectively; while the total apoptosis rate (including necrosis) of MCF-7 cells were 3.24%, 4.9% and - 1.68%, respectively. The results suggested self-assembling RGD-BH3-HFn could target to C6 cells and bEnd.3 cells, and enhance tumor cells apoptosis, its apoptosis effect against C6 cells was 7.11-fold that of HFn, and apoptosis effect against bEnd.3 cells was 9.08-fold that of HFn. These results indicated BH3 domain can be designed as targeting pro-apoptotic nanoparticles.

Discovery of Small Molecule Bak Activator for Lung Cancer Therapy.

Rationale: Bak is a major proapoptotic Bcl2 family member and a required molecule for apoptotic cell death. High levels of endogenous Bak were observed in both small cell lung cancer (SCLC) and non-small cell lung cancer (NSCLC) cell lines. Increased Bak expression was correlated with poor prognosis of NSCLC patients, suggesting that Bak protein is an attractive target for lung cancer therapy. The BH3 domain functions as death domain and is required for Bak to initiate apoptotic cell death. Thus, the BH3 domain is attractive target for discovery of Bak agonist. Methods: The BH3 death domain binding pocket (aa75-88) of Bak was chosen as a docking site for screening of small molecule Bak activators using the UCSF DOCK 6.1 program suite and the NCI chemical library (300,000 small molecules) database. The top 500 compounds determined to have the highest affinity for the BH3 domain were obtained from the NCI and tested for cytotoxicity for further screening. We identified a small molecule Bak activator BKA-073 as the lead compound. The binding affinity of BKA-073 with Bak protein was analyzed by isothermal titration calorimetry (ITC) assay. BKA-073-mediated Bak activation via oligomerization was analyzed by a cross-linking with Bis (maleimido) hexane (BMH). Sensitivity of BKA-073 to lung cancer cells in vitro was evaluated by dynamic BH3 profiling (DBP) and apoptotic cell death assay. The potency of BKA-073 alone or in combination with radiotherapy or Bcl2 inhibitor was evaluated in animal models. Results: We found that BKA-073 binds Bak at BH3 domain with high affinity and selectivity. BKA-073/Bak binding promotes Bak oligomerization and mitochondrial priming that activates its proapoptotic function. BKA-073 potently suppresses tumor growth without significant normal tissue toxicity in small cell lung cancer (SCLC) and NSCLC xenografts, patient-derived xenografts, and genetically engineered mouse models of mutant KRAS-driven cancer. Bak accumulates in radioresistant lung cancer cells and BKA-073 reverses radioresistance. Combination of BKA-073 with Bcl-2 inhibitor venetoclax exhibits strong synergy against lung cancer in vivo. Conclusions: Development of small molecule Bak activator may provide a new class of anticancer agents to treat lung cancer.

Crosstalk between apoptosis and autophagy signaling pathways.

The fate of a cell is determined by multiple signaling pathways in response to a range of stimuli. Probably the most prominent cell death mechanism is apoptosis which can be triggered by both internal stresses, as well as extracellular stimuli, and is executed by two well-characterized pathways, the intrinsic and the extrinsic apoptosis pathways. Although autophagy can also lead to cell death under certain circumstances, its major function is as a cell survival process. Given that the life of a cell is at stake, it is not surprising that there is significant molecular crosstalk between these pathways. The nature of these interconnections is diverse and ranges from protein-protein interactions and post-translational modifications through to the degradation of molecular components by different proteins and organelles. In this chapter we review these mechanisms in detail.

Liposomal Permeabilization Assay to Study the Functional Interactions of the BCL-2 Family.

Apoptosis, a form of programmed cell death that is important for development and homeostasis, is regulated by the BCL-2 family of proteins. Over twenty BCL-2 family members have been classified in three groups based on structural homology and function. The multidomain antiapoptotic proteins promote survival, whereas the multidomain and the BH3-only proapoptotic members induce cell death. Because the interaction among the BCL-2 family members occurs primarily at the mitochondrial outer membrane, biochemical assays using artificial liposomes have been developed to study the functional relationship between these proteins. The liposomal permeabilization assay is a cell-free system that relies on the ability of multidomain pro-apoptotic members to promote membrane permeabilization upon activation. By encapsulating a fluorophore and a quencher into liposomes, the degree of permeabilization can be quantified by the increase in fluorescence intensity as the fluorophore and quencher dissociate. The liposomal permeabilization assay has been used to delineate interactions among BCL-2 family members as well as to characterize peptides, small molecules, and lipids that modulate the function of BCL-2 family of proteins. Here, we describe in detail the permeabilization of liposomes induced by the interaction between BAX and BH3-only activator tBID.

Structural insights into BCL2 pro-survival protein interactions with the key autophagy regulator BECN1 following phosphorylation by STK4/MST1.

BECN1/Beclin 1 is a critical protein in the initiation of autophagosome formation. Recent studies have shown that phosphorylation of BECN1 by STK4/MST1 at threonine 108 (T108) within its BH3 domain blocks macroautophagy/autophagy by increasing BECN1 affinity for its negative regulators, the anti-apoptotic proteins BCL2/Bcl-2 and BCL2L1/Bcl-xL. It was proposed that this increased binding is due to formation of an electrostatic interaction with a conserved histidine residue on the anti-apoptotic molecules. Here, we performed biophysical studies which demonstrated that a peptide corresponding to the BECN1 BH3 domain in which T108 is phosphorylated (p-T108) does show increased affinity for anti-apoptotic proteins that is significant, though only minor (<2-fold). We also determined X-ray crystal structures of BCL2 and BCL2L1 with T108-modified BECN1 BH3 peptides, but only showed evidence of an interaction between the BH3 peptide and the conserved histidine residue when the histidine flexibility was restrained due to crystal contacts. These data, together with molecular dynamics studies, indicate that the histidine is highly flexible, even when complexed with BECN1 BH3. Binding studies also showed that detergent can increase the affinity of the interaction. Although this increase was similar for both the phosphorylated and non-phosphorylated peptides, it suggests factors such as membranes could impact on the interaction between BECN1 and BCL2 proteins, and therefore, on the regulation of autophagy. Hence, we propose that phosphorylation of BECN1 by STK4/MST1 can increase the affinity of the interaction between BECN1 and anti-apoptotic proteins and this interaction can be stabilized by local environmental factors. Abbreviations: asu: asymmetric unit; BH3: BCL2/Bcl-2 homology 3; DAPK: death associated protein kinase; MD: molecular dynamics; MST: microscale thermophoresis; NMR: nuclear magnetic resonance; PDB: protein data bank; p-T: phosphothreonine; SPR: surface plasmon resonance; STK4/MST1: serine/threonine kinase 4.

Synthesis and Biological Activity of Scyllatoxin-Based BH3 Domain Mimetics Containing Two Disulfide Linkages.

The B cell lymphoma 2 (BCL2) proteins are a family of evolutionarily related proteins that act as positive or negative regulators of the intrinsic apoptosis pathway. Overexpression of anti-apoptotic BCL2 proteins in cells is associated with apoptotic resistance, which can result in cancerous phenotypes and pathogenic cell survival. Consequently, anti-apoptotic BCL2 proteins have attracted considerable interest as therapeutic targets. We recently reported the development of a novel class of synthetic protein based on scyllatoxin (ScTx) designed to mimic the helical BH3 interaction domain of the pro-apoptotic BCL2 protein Bax. These studies showed that the number and position of native disulfide linkages contained within the ScTx-Bax structure significantly influences the ability for these constructs to target anti-apoptotic BCL2 proteins in vitro. The goal of the present study is to investigate the contribution of two disulfide linkages in the folding and biological activity of ScTx-Bax proteins. Here, we report the full chemical synthesis of three ScTx-Bax sequence variants, each presenting two native disulfide linkages at different positions within the folded structure. It was observed that two disulfide linkages were sufficient to fold ScTx-Bax proteins into native-like architectures reminiscent of wild-type ScTx. Furthermore, we show that select (bis)disulfide ScTx-Bax variants can target Bcl-2 (proper) in vitro and that the position of the disulfide bonds significantly influences binding affinity. Despite exhibiting only modest binding to Bcl-2, the successful synthesis of ScTx-Bax proteins containing two disulfide linkages represents a viable route to ScTx-based BH3 domain mimetics that preserve native-like conformations. Finally, structural models of ScTx-Bax proteins in complex with Bcl-2 indicate that these helical mimetics bind in similar configurations as wild-type Bax BH3 domains. Taken together, these results suggest that ScTx-Bax proteins may serve as potent lead compounds that expand the repertoire of "druggable" protein-protein interactions.

Prosurvival AMBRA1 turns into a proapoptotic BH3-like protein during mitochondrial apoptosis.

Autophagy and apoptosis are 2 stress-response mechanisms that are closely interconnected. However, the molecular interplays between these 2 pathways remain to be clarified. Here we report that the crucial proautophagic factor AMBRA1 can act as a positive mediator of mitochondrial apoptosis. Indeed, we show that, in a proapoptotic positive feedback loop, the C-terminal part of AMBRA1, generated by CASP/CASPASE cleavage upon apoptosis induction, inhibits the antiapoptotic factor BCL2 by a direct binding through its BH3-like domain. The mitochondrial AMBRA1-BCL2 complex is thus at the crossroad between autophagy and cell death and may represent a novel target in development of therapeutic approaches in clinical diseases.