Allosteric Regulation of BH3 Proteins in Bcl-xL Complexes Enables Switch-like Activation of Bax.

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-xL 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-xL complexes changes from an inactive to an active form while bound to a Bcl-xL complex only when Bad is also bound. Bcl-xL complexes enable Bad to function as a non-competitive inhibitor of Bcl-xL and allosterically activate cBid, dramatically enhancing the pro-apoptotic potency of Bad.

Shedding light on mitochondrial outer-membrane permeabilization and membrane potential: State of the art methods and biosensors.

Membrane structural integrity is essential for optimal mitochondrial function. These organelles produce the energy needed for all vital processes, provided their outer and inner membranes are intact. This prevents the release of mitochondrial apoptogenic factors into the cytosol and ensures intact mitochondrial membrane potential (ΔΨm) to sustain ATP production. Cell death by apoptosis is generally triggered by outer mitochondrial membrane permeabilization (MOMP), tightly coupled with loss of ΔΨ m. As these two processes are essential for both mitochondrial function and cell death, researchers have devised various techniques to assess them. Here, we discuss current methods and biosensors available for detecting MOMP and measuring ΔΨ m, focusing on their advantages and limitations and discuss what new imaging tools are needed to improve our knowledge of mitochondrial function.

Expanding roles of BCL-2 proteins in apoptosis execution and beyond.

The proteins of the BCL-2 family are known as key regulators of apoptosis, with interactions between family members determining permeabilisation of the mitochondrial outer membrane (MOM) and subsequent cell death. However, the exact mechanism through which they form the apoptotic pore responsible for MOM permeabilisation (MOMP), the structure and specific components of this pore, and what roles BCL-2 proteins play outside of directly regulating MOMP are incompletely understood. Owing to the link between apoptosis dysregulation and disease, the BCL-2 proteins are important targets for drug development. With the development and clinical use of drugs targeting BCL-2 proteins showing success in multiple haematological malignancies, enhancing the efficacy of these drugs, or indeed developing novel drugs targeting BCL-2 proteins is of great interest to treat cancer patients who have developed resistance or who suffer other disease types. Here, we review our current understanding of the molecular mechanism of MOMP, with a particular focus on recently discovered roles of BCL-2 proteins in apoptosis and beyond, and discuss what implications these functions might have in both healthy tissues and disease.

Efficacy of a novel affitoxin targeting MOMP against Chlamydia trachomatis in vitro and in vivo.

Targeted therapy is an attractive approach for treating infectious diseases. Affibody molecules have similar capability to antibodies that facilitate molecular recognition in both diagnostic and therapeutic applications. Targeting major outer membrane protein (MOMP) for treating infection of Chlamydia trachomatis, one of the most common sexually transmitted pathogens, is a promising therapeutic approach. Previously, we have reported a MOMP-specific affibody (ZMOMP:461) from phage display library. Here, we first fused it with modified Pseudomonas Exotoxin (PE38KDEL) and a cell-penetrating peptide (CPP) to develop an affitoxin, Z461X-CPP. We then verified the addition of both toxin and CPPs that did not affect the affinitive capability of ZMOMP:461 to MOMP. Upon uptake by C.trachomatis-infected cells, Z461X-CPP induced cell apoptosis in vitro. In animal model, Z461X significantly shortened the duration of C. trachomatis infection and prevented pathological damage in mouse reproductive system. These findings provide compelling evidence that the MOMP-specific affitoxin has great potential for targeting therapy of C. trachomatis infection.

A non-death function of the mitochondrial apoptosis apparatus in immunity.

Apoptosis is a frequent form of programmed cell death, but the apoptotic signaling pathway can also be engaged at a low level, in the absence of cell death. We here report that such sub-lethal engagement of mitochondrial apoptosis signaling causes the secretion of cytokines from human epithelial cells in a process controlled by the Bcl-2 family of proteins. We further show that sub-lethal signaling of the mitochondrial apoptosis pathway is initiated by infections with all tested viral, bacterial, and protozoan pathogens and causes damage to the genomic DNA. Epithelial cells infected with these pathogens secreted cytokines, and this cytokine secretion upon microbial infection was substantially reduced if mitochondrial sub-lethal apoptosis signaling was blocked. In the absence of mitochondrial pro-apoptotic signaling, the ability of epithelial cells to restrict intracellular bacterial growth was impaired. Triggering of the mitochondrial apoptosis apparatus thus not only causes apoptosis but also has an independent role in immune defense.

Promising Strategy of mPTP Modulation in Cancer Therapy: An Emerging Progress and Future Insight.

Cancer has been progressively a major global health concern. With this developing global concern, cancer determent is one of the most significant public health challenges of this era. To date, the scientific community undoubtedly highlights mitochondrial dysfunction as a hallmark of cancer cells. Permeabilization of the mitochondrial membranes has been implicated as the most considerable footprint in apoptosis-mediated cancer cell death. Under the condition of mitochondrial calcium overload, exclusively mediated by oxidative stress, an opening of a nonspecific channel with a well-defined diameter in mitochondrial membrane allows free exchange between the mitochondrial matrix and the extra mitochondrial cytosol of solutes and proteins up to 1.5 kDa. Such a channel/nonspecific pore is recognized as the mitochondrial permeability transition pore (mPTP). mPTP has been established for regulating apoptosis-mediated cancer cell death. It has been evident that mPTP is critically linked with the glycolytic enzyme hexokinase II to defend cellular death and reduce cytochrome c release. However, elevated mitochondrial Ca2+ loading, oxidative stress, and mitochondrial depolarization are critical factors leading to mPTP opening/activation. Although the exact mechanism underlying mPTP-mediated cell death remains elusive, mPTP-mediated apoptosis machinery has been considered as an important clamp and plays a critical role in the pathogenesis of several types of cancers. In this review, we focus on structure and regulation of the mPTP complex-mediated apoptosis mechanisms and follow with a comprehensive discussion addressing the development of novel mPTP-targeting drugs/molecules in cancer treatment.

Apoptosis Regulation in Osteoarthritis and the Influence of Lipid Interactions.

Osteoarthritis (OA) is one of the most common chronic diseases in human and animal joints. The joints undergo several morphological and histological changes during the development of radiographically visible osteoarthritis. The most discussed changes include synovial inflammation, the massive destruction of articular cartilage and ongoing joint destruction accompanied by massive joint pain in the later stadium. Either the increased apoptosis of chondrocytes or the insufficient apoptosis of inflammatory macrophages and synovial fibroblasts are likely to underly this process. In this review, we discuss the current state of research on the pathogenesis of OA with special regard to the involvement of apoptosis.

Mitochondrial outer membrane permeabilization at the single molecule level.

Apoptotic cell death is essential for development, immune function or tissue homeostasis, and its mis-regulation is linked to various diseases. Mitochondrial outer membrane permeabilization (MOMP) is a central event in the intrinsic apoptotic pathway and essential to control the execution of cell death. Here we review current concepts in regulation of MOMP focusing on the interaction network of the Bcl-2 family proteins as well as further regulatory elements influencing MOMP. As MOMP is a complex spatially and temporally controlled process, we point out the importance of single-molecule techniques to unveil processes which would be masked by ensemble measurements. We report key single-molecule studies applied to decipher the composition, assembly mechanism and structure of protein complexes involved in MOMP regulation.

Apoptotic Features in Non-Apoptotic Processes.

Apoptosis is the most thoroughly studied type of regulated cell death. Certain events, such as externalization of phosphatidylserine (PS) into the outer leaflet of plasma membrane, mitochondrial outer membrane permeabilization, caspase cascade activation, DNA fragmentation and blebbing, are widely considered to be hallmarks of apoptosis as well as being traditionally viewed as irreversible. This review shows that under particular circumstances these events can also participate in physiological processes not associated with initiation of apoptosis, such as cell differentiation, division, and motility, as well as non-apoptotic types of cell death. Moreover, these events may often be reversible. This review focuses on three processes: phosphatidylserine externalization, blebbing, and activation of apoptotic caspases. Mitochondrial outer membrane permeabilization and DNA fragmentation are not discussed.

Antimicrobial Peptides Mediate Apoptosis by Changing Mitochondrial Membrane Permeability.

Changes in mitochondrial membrane permeability are closely associated with mitochondria-mediated apoptosis. Antimicrobial peptides (AMPs), which have been found to enter cells to exert physiological effects, cause damage to the mitochondria. This paper reviews the molecular mechanisms of AMP-mediated apoptosis by changing the permeability of the mitochondrial membrane through three pathways: the outer mitochondrial membrane (OMM), inner mitochondrial membrane (IMM), and mitochondrial permeability transition pore (MPTP). The roles of AMPs in inducing changes in membrane permeability and apoptosis are also discussed. Combined with recent research results, the possible application prospects of AMPs are proposed to provide a theoretical reference for the development of AMPs as therapeutic agents for human diseases.

Mitochondrial residence of the apoptosis inducer BAX is more important than BAX oligomerization in promoting membrane permeabilization.

Permeabilization of the mitochondrial outer membrane is a key step in the intrinsic apoptosis pathway, triggered by the release of mitochondrial intermembrane space proteins into the cytoplasm. The BCL-2-associated X apoptosis regulator (BAX) protein critically contributes to this process by forming pores in the mitochondrial outer membrane. However, the relative roles of the mitochondrial residence of BAX and its oligomerization in promoting membrane permeabilization are unclear. To this end, using both cell-free and cellular experimental systems, including membrane permeabilization, size-exclusion chromatography-based oligomer, and retrotranslocation assays, along with confocal microscopy analysis, here we studied two BAX C-terminal variants, T182I and G179P. Neither variant formed large oligomers when activated in liposomes. Nevertheless, the G179P variant could permeabilize liposome membranes, suggesting that large BAX oligomers are not essential for the permeabilization. However, when G179P was transduced into BAX/BCL2 agonist killer (BAK) double-knockout mouse embryonic fibroblasts, its location was solely cytoplasmic, and it then failed to mediate cell death. In contrast, T182I was inefficient in both liposome insertion and permeabilization. Yet, when transduced into cells, BAXT182I resided predominantly on mitochondria, because of its slow retrotranslocation and mediated apoptosis as efficiently as WT BAX. We conclude that BAX's mitochondrial residence in vivo, regulated by both targeting and retrotranslocation, is more significant for its pro-apoptotic activity than its ability to insert and to form higher-order oligomers in model membranes. We propose that this finding should be taken into account when developing drugs that modulate BAX activity.

Mechanisms of mitochondrial cell death.

Mitochondria are double-membrane bound organelles that not only provide energy for intracellular metabolism, but also play a key role in the regulation of cell death. Mitochondrial outer membrane permeabilization (MOMP), allowing the release of intermembrane space proteins like cytochrome c, is considered a point of no return in apoptosis. MOMP is controlled by the proteins of the B-cell lymphoma 2 (BCL-2) family, including pro-and anti-apoptotic members, whose balance determines the decision between cell death and survival. Other factors such as membrane lipid environment, membrane dynamics, and inter-organelle communications are also known to influence this process. MOMP and apoptosis have been acknowledged as immunologically silent. Remarkably, a growing body of evidence indicates that MOMP can engage in various pro-inflammatory signaling functions. In this mini-review, we discuss about our current knowledge on the mechanisms of mitochondrial apoptosis, as well as the involvement of mitochondria in other kinds of programmed cell death pathways.

Generation of a novel affibody molecule targeting Chlamydia trachomatis MOMP.

Chlamydia trachomatis (C. trachomatis) is the leading cause of preventable blindness worldwide and the most prevalent cause of bacterial sexually transmitted diseases. At present, there is no available vaccine, and recurrences after antibiotics treatment are substantial problems. Major outer membrane protein (MOMP) accounts for 60% of the outer mass of C. trachomatis, functioning as trimeric porin, and it is highly antigenic. Therefore, MOMP is the most promising candidate for vaccine developing and target therapy of Chlamydia. Affibody, a new class of affinity ligands derived from the Z-domain in the binding region of Staphylococcus aureus protein A, has been the focus of researchers as a viable alternative to antibodies. In this study, the MOMP-targeted affibody molecule (ZMOMP:461) was screened by phage-displayed peptide library. Further, the affinity and specificity were characterized by surface plasmon resonance (SPR) and Western blot. Immunofluorescence assay (IFA) indicated that the MOMP-binding affibody could recognize native MOMP in HeLa229 cells infected C. trachomatis. Immunoprecipitation assay confirmed further that ZMOMP:461 molecule specifically recognizes the epitope on relaxed trimer MOMP. Our findings provide strong evidence that affibody molecule (ZMOMP:461) serves as substitute for MOMP antibody for biological applications and has a great potential for delivering drugs for target therapy. KEY POINTS : • We screened a novel affibody molecule ZMOMP:461 targeting Chlamydia trachomatis MOMP. • ZMOMP:461 recognizes the recombinant and native MOMP with high affinity and specificity. • ZMOMP:461 could be internalized into live target cells.

The retinoblastoma protein induces apoptosis directly at the mitochondria.

The retinoblastoma protein gene RB-1 is mutated in one-third of human tumors. Its protein product, pRB (retinoblastoma protein), functions as a transcriptional coregulator in many fundamental cellular processes. Here, we report a nonnuclear role for pRB in apoptosis induction via pRB's direct participation in mitochondrial apoptosis. We uncovered this activity by finding that pRB potentiated TNFα-induced apoptosis even when translation was blocked. This proapoptotic function was highly BAX-dependent, suggesting a role in mitochondrial apoptosis, and accordingly, a fraction of endogenous pRB constitutively associated with mitochondria. Remarkably, we found that recombinant pRB was sufficient to trigger the BAX-dependent permeabilization of mitochondria or liposomes in vitro. Moreover, pRB interacted with BAX in vivo and could directly bind and conformationally activate BAX in vitro. Finally, by targeting pRB specifically to mitochondria, we generated a mutant that lacked pRB's classic nuclear roles. This mito-tagged pRB retained the ability to promote apoptosis in response to TNFα and also additional apoptotic stimuli. Most importantly, induced expression of mito-tagged pRB in Rb(-/-);p53(-/-) tumors was sufficient to block further tumor development. Together, these data establish a nontranscriptional role for pRB in direct activation of BAX and mitochondrial apoptosis in response to diverse stimuli, which is profoundly tumor-suppressive.

RB goes mitochondrial.

The retinoblastoma tumor suppressor RB is well known for its capacity to restrict cell cycle progression at the G1/S transition of the cell cycle by controlling the transcription of cell cycle genes. In this issue of Genes & Development, Hilgendorf and colleagues (pp. 1003-1015) have identified a novel tumor suppressor function for RB independent of its role as a transcriptional regulator, in which RB directly activates the apoptosis regulator Bax at the mitochondria to promote cell death.

Intrinsically Connected: Therapeutically Targeting the Cathepsin Proteases and the Bcl-2 Family of Protein Substrates as Co-regulators of Apoptosis.

Taken with the growing importance of cathepsin-mediated substrate proteolysis in tumor biology and progression, the focus and emphasis placed on therapeutic design and development is coming into fruition. Underpinning this approach is the invariable progression from the direction of fully characterizing cathepsin protease members and their substrate targets, towards targeting such an interaction with tangible therapeutics. The two groups of such substrates that have gained much attention over the years are the pro- and anti- apoptotic protein intermediates from the extrinsic and intrinsic signaling arms of the apoptosis pathway. As proteins that are central to determining cellular fate, some of them present themselves as very favorable candidates for therapeutic targeting. However, considering that both anti- and pro- apoptotic signaling intermediates have been reported to be downstream substrates for certain activated cathepsin proteases, therapeutic targeting approaches based on greater selectivity do need to be given greater consideration. Herein, we review the relationships shared by the cathepsin proteases and the Bcl-2 homology domain proteins, in the context of how the topical approach of adopting 'BH3-mimetics' can be explored further in modulating the relationship between the anti- and pro- apoptotic signaling intermediates from the intrinsic apoptosis pathway and their upstream cathepsin protease regulators. Based on this, we highlight important future considerations for improved therapeutic design.

A comprehensive review on avian chlamydiosis: a neglected zoonotic disease.

Avian chlamydiosis is one of the important neglected diseases with critical zoonotic potential. Chlamydia psittaci, the causative agent, affects most categories of birds, livestock, companion animals, and humans. It has many obscured characters and epidemiological dimensions, which makes it unique among other bacterial agents. Recent reports on transmission from equine to humans alarmed the public health authorities, and it necessitates the importance of routine screening of this infectious disease. High prevalence of spill-over infection in equines was associated with reproductive losses. Newer avian chlamydial species are being reported in the recent years. It is a potential biological warfare agent and the disease is an occupational hazard mainly to custom officers handling exotic birds. Prevalence of the disease in wild birds, pet birds, and poultry causes economic losses to the poultry industry and the pet bird trade. Interestingly, there are speculations on the 'legal' and 'illegal' bird trade that may be the global source of some of the most virulent strains of this pathogen. The mortality rate generally ranges from 5 to 40% in untreated cases, but it can sometimes be higher in co-infection. The intracellular lifestyle of this pathogen makes the diagnosis more complicated and there is also lack of accurate diagnostics. Resistance to antibiotics is reported only in some pathogens of the Chlamydiaceae family, but routine screening may assess the actual situation in all pathogens. Due to the diverse nature of the pathogen, the organism necessitates the One Health partnerships to have complete understanding. The present review focuses on the zoonotic aspects of avian chlamydiosis with its new insights into the pathogenesis, transmission, treatment, prevention, and control strategies. The review also briefs on the basic understandings and complex epidemiology of avian chlamydiosis, highlighting the need for research on emerging one health perspectives.

A Recombinant Chlamydia trachomatis MOMP Vaccine Elicits Cross-serogroup Protection in Mice Against Vaginal Shedding and Infertility.

BACKGROUND: Chlamydia trachomatis is the most common sexually transmitted bacterial pathogen worldwide. Here, we determined the ability of a C. trachomatis recombinant major outer membrane protein (rMOMP) vaccine to elicit cross-serogroup protection. METHODS: Female C3H/HeN mice were vaccinated by mucosal and systemic routes with C. trachomatis serovar D (UW-3/Cx) rMOMP and challenged in the ovarian bursa with serovars D (UW-3/Cx), D (UCI-96/Cx), E (IOL-43), or F (N.I.1). CpG-1826 and Montanide ISA 720 were used as adjuvants. RESULTS: Immune responses following vaccination were more robust against the most closely related serovars. Following a genital challenge (as determined by number of mice with positive vaginal cultures, number of positive cultures, number of inclusion forming units recovered, and number of days with positive cultures) mice challenged with C. trachomatis serovars of the same complex were protected but not those challenged with serovar F (N.I.1) from a different subcomplex. Females were caged with male mice. Based on fertility rates, number of embryos, and hydrosalpinx formation, vaccinated mice were protected against challenges with serovars D (UW-3/Cx), D (UCI-96/Cx), and E (IOL-43) but not F (N.I.1). CONCLUSIONS: This is the first subunit vaccine shown to protect mice against infection, pathology, and infertility caused by different C. trachomatis serovars.

Imaging of morphological and biochemical hallmarks of apoptosis with optimized optogenetic tools.

Creation of optogenetic switches for specific activation of cell death pathways can provide insights into apoptosis and could also form a basis for noninvasive, next-generation therapeutic strategies. Previous work has demonstrated that cryptochrome 2 (Cry2)/cryptochrome-interacting ß helix-loop-helix (CIB), a blue light-activated protein-protein dimerization module from the plant Arabidopsis thaliana, together with BCL2-associated X apoptosis regulator (BAX), an outer mitochondrial membrane-targeting pro-apoptotic protein, can be used for light-mediated initiation of mitochondrial outer membrane permeabilization (MOMP) and downstream apoptosis. In this work, we further developed the original light-activated Cry2-BAX system (hereafter referred to as OptoBAX) by improving the photophysical properties and light-independent interactions of this optogenetic switch. The resulting optogenetic constructs significantly reduced the frequency of light exposure required for membrane permeabilization activation and also decreased dark-state cytotoxicity. We used OptoBAX in a series of experiments in Neuro-2a and HEK293T cells to measure the timing of the dramatic morphological and biochemical changes occurring in cells after light-induced MOMP. In these experiments, we used OptoBAX in tandem with fluorescent reporters to image key events in early apoptosis, including membrane inversion, caspase cleavage, and actin redistribution. We then used these data to construct a timeline of biochemical and morphological events in early apoptosis, demonstrating a direct link between MOMP-induced redistribution of actin and apoptosis progression. In summary, we created a next-generation Cry2/CIB-BAX system requiring less frequent light stimulation and established a timeline of critical apoptotic events, providing detailed insights into key steps in early apoptosis.

Cardiac Mitochondrial PTEN-L determines cell fate between apoptosis and survival during chronic alcohol consumption.

Chronic alcohol consumption induces myocardial damage and a type of non-ischemic cardiomyopathy termed alcoholic cardiomyopathy, where mitochondrial ultrastructural damages and suppressed fusion activity promote cardiomyocyte apoptosis. The aim of the present study is to determine the role of mitochondrial fission proteins and/or other proteins that localise on cardiac mitochondria for apoptosis upon ethanol consumption. In vivo and in vitro chronic alcohol exposure increased mitochondrial Drp1 levels but knockdown of the same did not confer cardioprotection in H9c2 cells. These cells displayed downregulated expression of MFN2 and OPA1 for Bak-mediated cytochrome c release and apoptosis. Dysregulated PTEN/AKT cell survival signal in both ethanol treated and Drp1 knockdown cells augmented oxidative stress by promoting  mitochondrial PTEN-L and MFN1 interaction. Inhibiting this interaction with VO-OHpic, a reversible PTEN inhibitor, prevented Bak insertion into the mitochondria and release of cytochrome c to cytoplasm. Thus, our study provides evidence that Drp1-mediated mitochondrial fission is dispensable for ethanol-induced cardiotoxicity and that stress signals induce mitochondrial PTEN-L accumulation for structural and functional dyshomeostasis. Our in vivo results also demonstrates the therapeutic potential of VO-OHpic for habitual alcoholics developing myocardial dysfunction.