A New Inovirus from the Human Blood Encodes Proteins with Nuclear Subcellular Localization.

Viruses infecting bacteria (bacteriophages) represent the most abundant viral particles in the human body. They participate in the control of the human-associated bacterial communities and play an important role in the dissemination of virulence genes. Here, we present the identification of a new filamentous single-stranded DNA phage of the family Inoviridae, named Ralstonia Inoviridae Phage 1 (RIP1), in the human blood. Metagenomics and PCR analyses detected the RIP1 genome in blood serum, in the absence of concomitant bacterial infection or contamination, suggesting inovirus persistence in the human blood. Finally, we have experimentally demonstrated that the RIP1-encoded rolling circle replication initiation protein and serine integrase have functional nuclear localization signals and upon expression in eukaryotic cells both proteins were translocated into the nucleus. This observation adds to the growing body of data suggesting that phages could have an overlooked impact on the evolution of eukaryotic cells.

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.

Nrh L11R single nucleotide polymorphism, a new prediction biomarker in breast cancer, impacts endoplasmic reticulum-dependent Ca2+ traffic and response to neoadjuvant chemotherapy.

Overexpression of Bcl-2 proteins such as Bcl2L10, also referred to as Nrh, is associated with resistance to therapy and poor survival in various cancers, including breast cancer, lung cancer, and leukemia. The single nucleotide polymorphism (SNP) of BCL2L10 in its BH4 domain at position 11 (BCL2L10 Leu11Arg, rs2231292), corresponding to position 11 in the Nrh open reading frame, is reported to lower resistance towards chemotherapy, with patients showing better survival in the context of acute leukemia and colorectal cancer. Using cellular models and clinical data, we aimed to extend this knowledge to breast cancer. We report that the homozygous status of the Nrh Leu11Arg isoform (Nrh-R) is found in 9.7-11% percent of the clinical datasets studied. Furthermore, Nrh-R confers higher sensitivity towards Thapsigargin-induced cell death compared to the Nrh-L isoform, due to altered interactions with IP3R1 Ca2+ channels in the former case. Collectively, our data show that cells expressing the Nrh-R isoform are more prone to death triggered by Ca2+ stress inducers, compared to Nrh-L expressing cells. Analysis of breast cancer cohorts revealed that patients genotyped as Nrh-R/Nrh-R may have a better outcome. Overall, this study supports the notion that the rs2231292 Nrh SNP could be used as a predictive tool regarding chemoresistance, improving therapeutic decision-making processes. Moreover, it sheds new light on the contribution of the BH4 domain to the anti-apoptotic function of Nrh and identifies the IP3R1/Nrh complex as a potential therapeutic target in the context of breast cancer.

Mitochondrial Bcl-xL promotes brain synaptogenesis by controlling non-lethal caspase activation.

Non-lethal caspase activation (NLCA) has been linked to neurodevelopmental processes. However, how neurons control NLCA remains elusive. Here, we focused on Bcl-xL, a Bcl-2 homolog regulating caspase activation through the mitochondria. We generated a mouse model, referred to as ER-xL, in which Bcl-xL is absent in the mitochondria, yet present in the endoplasmic reticulum. Unlike bclx knockout mice that died at E13.5, ER-xL mice survived embryonic development but died post-partum because of altered feeding behavior. Enhanced caspase-3 activity was observed in the brain and the spinal cord white matter, but not the gray matter. No increase in cell death was observed in ER-xL cortical neurons, suggesting that the observed caspase-3 activation was apoptosis-independent. ER-xL neurons displayed increased caspase-3 activity in the neurites, resulting in impaired axon arborescence and synaptogenesis. Together, our findings suggest that mitochondrial Bcl-xL finely tunes caspase-3 through Drp-1-dependent mitochondrial fission, which is critical to neural network design.

Keeping Cell Death Alive: An Introduction into the French Cell Death Research Network.

Since the Nobel Prize award more than twenty years ago for discovering the core apoptotic pathway in C. elegans, apoptosis and various other forms of regulated cell death have been thoroughly characterized by researchers around the world. Although many aspects of regulated cell death still remain to be elucidated in specific cell subtypes and disease conditions, many predicted that research into cell death was inexorably reaching a plateau. However, this was not the case since the last decade saw a multitude of cell death modalities being described, while harnessing their therapeutic potential reached clinical use in certain cases. In line with keeping research into cell death alive, francophone researchers from several institutions in France and Belgium established the French Cell Death Research Network (FCDRN). The research conducted by FCDRN is at the leading edge of emerging topics such as non-apoptotic functions of apoptotic effectors, paracrine effects of cell death, novel canonical and non-canonical mechanisms to induce apoptosis in cell death-resistant cancer cells or regulated forms of necrosis and the associated immunogenic response. Collectively, these various lines of research all emerged from the study of apoptosis and in the next few years will increase the mechanistic knowledge into regulated cell death and how to harness it for therapy.

Bcl-xL and IP3R interaction: Intimate relationship with an uncertain outcome.

Bcl-2 family proteins are major apoptosis regulators. They control a key step in apoptosis execution referred to as the mitochondrial outer membrane permeabilization. Several Bcl-2 homologs were also reported to act at the level of the endoplasmic reticulum (ER) where they control intracellular Ca2+ trafficking. There is an increasing body of evidence that, in addition to their conventional role as MOMP regulators, several Bcl-2 family members, including Bcl-xL, are linked to Ca2+ -dependent processes, independent of cell death. Among them Bcl-xL has been proposed to promote IP3R1 channel opening and sustain mitochondrial bioenergetics. A recent article by Rosa and colleagues in Cell Death & Differentiation challenges this model and support the notion that Bcl-xL acts more as a repressor than as a sensitizer of IP3R1 opening. They suggest the existence of intrafamilial competition among the Bcl-2 family of protein with respect to their effect on IP3R Ca2+ permeability, which might be important regarding their respective non-canonical functions. In this regard, the results by Rosa and colleagues open exciting avenues regarding the biological process by which Bcl-xL affects Ca2+ trafficking through IP 3 R channels.

Caspases in the Developing Central Nervous System: Apoptosis and Beyond.

The caspase family of cysteine proteases represents the executioners of programmed cell death (PCD) type I or apoptosis. For years, caspases have been known for their critical roles in shaping embryonic structures, including the development of the central nervous system (CNS). Interestingly, recent findings have suggested that aside from their roles in eliminating unnecessary neural cells, caspases are also implicated in other neurodevelopmental processes such as axon guidance, synapse formation, axon pruning, and synaptic functions. These results raise the question as to how neurons regulate this decision-making, leading either to cell death or to proper development and differentiation. This review highlights current knowledge on apoptotic and non-apoptotic functions of caspases in the developing CNS. We also discuss the molecular factors involved in the regulation of caspase-mediated roles, emphasizing the mitochondrial pathway of cell death.

Bcl-2 Family of Proteins in the Control of Mitochondrial Calcium Signalling: An Old Chap with New Roles.

Bcl-2 family proteins are considered as one of the major regulators of apoptosis. Indeed, this family is known to control the mitochondrial outer membrane permeabilization (MOMP): a central step in the mitochondrial pathway of apoptosis. However, in recent years Bcl-2 family members began to emerge as a new class of intracellular calcium (Ca2+) regulators. At mitochondria-ER contacts (MERCs) these proteins are able to interact with major Ca2+ transporters, thus controlling mitochondrial Ca2+ homeostasis and downstream Ca2+ signalling pathways. Beyond the regulation of cell survival, this Bcl-2-dependent control over the mitochondrial Ca2+ dynamics has far-reaching consequences on the physiology of the cell. Here, we review how the Bcl-2 family of proteins mechanistically regulate mitochondrial Ca2+ homeostasis and how this regulation orchestrates cell death/survival decisions as well as the non-apoptotic process of cell migration.

Ancient and conserved functional interplay between Bcl-2 family proteins in the mitochondrial pathway of apoptosis.

In metazoans, Bcl-2 family proteins are major regulators of mitochondrially mediated apoptosis; however, their evolution remains poorly understood. Here, we describe the molecular characterization of the four members of the Bcl-2 family in the most primitive metazoan, Trichoplax adhaerens All four trBcl-2 homologs are multimotif Bcl-2 group, with trBcl-2L1 and trBcl-2L2 being highly divergent antiapoptotic Bcl-2 members, whereas trBcl-2L3 and trBcl-2L4 are homologs of proapoptotic Bax and Bak, respectively. trBax expression permeabilizes the mitochondrial outer membrane, while trBak operates as a BH3-only sensitizer repressing antiapoptotic activities of trBcl-2L1 and trBcl-2L2. The crystal structure of a trBcl-2L2:trBak BH3 complex reveals that trBcl-2L2 uses the canonical Bcl-2 ligand binding groove to sequester trBak BH3, indicating that the structural basis for apoptosis control is conserved from T. adhaerens to mammals. Finally, we demonstrate that both trBax and trBak BH3 peptides bind selectively to human Bcl-2 homologs to sensitize cancer cells to chemotherapy treatment.

Failed Apoptosis Enhances Melanoma Cancer Cell Aggressiveness.

Triggering apoptosis remains an efficient strategy to treat cancer. However, apoptosis is no longer a final destination since cancer cells can undergo partial apoptosis without dying. Recent evidence shows that partial mitochondrial permeabilization and non-lethal caspase activation occur under certain circumstances, although it remains unclear how failed apoptosis affects cancer cells. Using a cancer cell model to trigger non-lethal caspase activation, we find that melanoma cancer cells undergoing failed apoptosis have a particular transcriptomic signature associated with focal adhesions, transendothelial migration, and modifications of the actin cytoskeleton. In line with this, cancer cells surviving apoptosis gain migration and invasion properties in vitro and in vivo. We further demonstrate that failed apoptosis-associated gain in invasiveness is regulated by the c-Jun N-terminal kinase (JNK) pathway, whereas its RNA sequencing signature is found in metastatic melanoma. These findings advance our understanding of how cell death can both cure and promote cancer.

The apoptosis inhibitor Bcl-xL controls breast cancer cell migration through mitochondria-dependent reactive oxygen species production.

The Bcl-xL apoptosis inhibitor plays a major role in vertebrate development. In addition to its effect on apoptosis, Bcl-xL is also involved in cell migration and mitochondrial metabolism. These effects may favour the onset and dissemination of metastasis. However, the underlying molecular mechanisms remain to be fully understood. Here we focus on the control of cell migration by Bcl-xL in the context of breast cancer cells. We show that Bcl-xL silencing led to migration defects in Hs578T and MDA-MB231 cells. These defects were rescued by re-expressing mitochondria-addressed, but not endoplasmic reticulum-addressed, Bcl-xL. The use of BH3 mimetics, such as ABT-737 and WEHI-539 confirmed that the effect of Bcl-xL on migration did not depend on interactions with BH3-containing death accelerators such as Bax or BH3-only proteins. In contrast, the use of a BH4 peptide that disrupts the Bcl-xL/VDAC1 complex supports that Bcl-xL by acting on VDAC1 permeability contributes to cell migration through the promotion of reactive oxygen species production by the electron transport chain. Collectively our data highlight the key role of Bcl-xL at the interface between cell metabolism, cell death, and cell migration, thus exposing the VDAC1/Bcl-xL interaction as a promising target for anti-tumour therapy in the context of metastatic breast cancer.

Subcellular Localization and Dynamics of the Bcl-2 Family of Proteins.

Bcl-2 family proteins are recognized as major regulators of the mitochondrial pathway of apoptosis. They control the mitochondrial outer membrane permeabilization (MOMP) by directly localizing to this organelle. Further investigations demonstrated that Bcl-2 related proteins are also found in other intracellular compartments such as the endoplasmic reticulum, the Golgi apparatus, the nucleus and the peroxisomes. At the level of these organelles, Bcl-2 family proteins not only regulate MOMP in a remote fashion but also participate in major cellular processes including calcium homeostasis, cell cycle control and cell migration. With the advances of live cell imaging techniques and the generation of fluorescent recombinant proteins, it became clear that the distribution of Bcl-2 proteins inside the cell is a dynamic process which is profoundly affected by changes in the cellular microenvironment. Here, we describe the current knowledge related to the subcellular distribution of the Bcl-2 family of proteins and further emphasize on the emerging concept that this highly dynamic process is critical for cell fate determination.

Breast Cancer Targeting through Inhibition of the Endoplasmic Reticulum-Based Apoptosis Regulator Nrh/BCL2L10.

Drug resistance and metastatic relapse remain a top challenge in breast cancer treatment. In this study, we present preclinical evidence for a strategy to eradicate advanced breast cancers by targeting the BCL-2 homolog Nrh/BCL2L10, which we discovered to be overexpressed in >45% of a large cohort of breast invasive carcinomas. Nrh expression in these tumors correlated with reduced metastasis-free survival, and we determined it to be an independent marker of poor prognosis. Nrh protein localized to the endoplasmic reticulum. Mechanistic investigations showed that Nrh made BH4 domain-dependent interactions with the ligand-binding domain of the inositol-1,4,5-triphosphate receptor (IP3R), a type 1/3 Ca2+ channel, allowing Nrh to negatively regulate ER-Ca2+ release and to mediate antiapoptosis. Notably, disrupting Nrh/IP3R complexes by BH4 mimetic peptides was sufficient to inhibit the growth of breast cancer cells in vitro and in vivo Taken together, our results highlighted Nrh as a novel prognostic marker and a candidate therapeutic target for late stage breast cancers that may be addicted to Nrh.Significance: These findings offer a comprehensive molecular model for the activity of Nrh/BCL2L10, a little studied antiapoptotic molecule, prognostic marker, and candidate drug target in breast cancer. Cancer Res; 78(6); 1404-17. ©2018 AACR.

Mitochondrial Ca2+ uptake controls actin cytoskeleton dynamics during cell migration.

Intracellular Ca2+ signaling regulates cell migration by acting on cytoskeleton architecture, cell directionality and focal adhesions dynamics. In migrating cells, cytosolic Ca2+ pool and Ca2+ pulses are described as key components of these effects. Whereas the role of the mitochondrial calcium homeostasis and the Mitochondria Cacium Uniporter (MCU) in cell migration were recently highlighted in vivo using the zebrafish model, their implication in actin cystokeleton dynamics and cell migration in mammals is not totally characterized. Here, we show that mcu silencing in two human cell lines compromises their migration capacities. This phenotype is characterized by actin cytoskeleton stiffness, a cell polarization loss and an impairment of the focal adhesion proteins dynamics. At the molecular level, these effects appear to be mediated by the reduction of the ER and cytosolic Ca2+ pools, which leads to a decrease in Rho-GTPases, RhoA and Rac1, and Ca2+-dependent Calpain activites, but seem to be independent of intracellular ATP levels. Together, this study highlights the fundamental and evolutionary conserved role of the mitochondrial Ca2+ homeostasis in cytoskeleton dynamics and cell migration.

Viral communities associated with human pericardial fluids in idiopathic pericarditis.

Pericarditis is a common human disease defined by inflammation of the pericardium. Currently, 40% to 85% of pericarditis cases have no identified etiology. Most of these cases are thought to be caused by an infection of undetected, unsuspected or unknown viruses. In this work, we used a culture- and sequence-independent approach to investigate the viral DNA communities present in human pericardial fluids. Seven viral metagenomes were generated from the pericardial fluid of patients affected by pericarditis of unknown etiology and one metagenome was generated from the pericardial fluid of a sudden infant death case. As a positive control we generated one metagenome from the pericardial fluid of a patient affected by pericarditis caused by herpesvirus type 3. Furthermore, we used as negative controls a total of 6 pericardial fluids from 6 different individuals affected by pericarditis of non-infectious origin: 5 of them were sequenced as a unique pool and the remaining one was sequenced separately. The results showed a significant presence of torque teno viruses especially in one patient, while herpesviruses and papillomaviruses were present in the positive control. Co-infections by different genotypes of the same viral type (torque teno viruses) or different viruses (herpesviruses and papillomaviruses) were observed. Sequences related to bacteriophages infecting Staphylococcus, Enterobacteria, Streptococcus, Burkholderia and Pseudomonas were also detected in three patients. This study detected torque teno viruses and papillomaviruses, for the first time, in human pericardial fluids.

Nrz but not zBcl-xL antagonizes Bcl-wav pro-apoptotic activity in zebrafish.

We recently identified a new highly divergent Bcl-2 related protein, named Bcl-wav, with phylogenetic pattern restricted to aquatic anamniotes. In zebrafish gastrula, bclwav gene silencing resulted in calcium-dependent cytoskeleton remodeling leading to convergence and extension movements defaults and abnormal orientation of the larva notochord. Beyond its function in cell migration, Bcl-wav was found to act as a pro-apoptotic Bcl-2 member inducing Bax/Bak dependent cell death. Here we show that, in zebrafish, pro-apoptotic Bcl-wav activity is selectively counteracted by the anti-apoptotic Nrz protein but not by zBcl-xL. Indeed Nrz but not zBcl-xL was able to decrease Bcl-wav dependent embryo mortality. Furthermore Nrz was able to prevent apoptosis induced by Bcl-wav ectopic expression in the embryo's head and tail. Finally co-immunoprecipitation experiments in HeLa cells showed that Bcl-wav directly interacts with Nrz. Overall these results expand our current knowledge about Bcl-2 family proteins interactome during early zebrafish development.

The Bcl-2 homolog Nrz inhibits binding of IP3 to its receptor to control calcium signaling during zebrafish epiboly.

Members of the Bcl-2 protein family regulate mitochondrial membrane permeability and also localize to the endoplasmic reticulum where they control Ca(2+) homeostasis by interacting with inositol 1,4,5-trisphosphate (IP3) receptors (IP3Rs). In zebrafish, Bcl-2-like 10 (Nrz) is required for Ca(2+) signaling during epiboly and gastrulation. We characterized the mechanism by which Nrz controls IP3-mediated Ca(2+) release during this process. We showed that Nrz was phosphorylated during early epiboly, and that in embryos in which Nrz was knocked down, reconstitution with Nrz bearing mutations designed to prevent its phosphorylation disrupted cyclic Ca(2+) transients and the assembly of the actin-myosin ring and led to epiboly arrest. In cultured cells, wild-type Nrz, but not Nrz with phosphomimetic mutations, interacted with the IP3 binding domain of IP3R1, inhibited binding of IP3 to IP3R1, and prevented histamine-induced increases in cytosolic Ca(2+). Collectively, these data suggest that Nrz phosphorylation is necessary for the generation of IP3-mediated Ca(2+) transients and the formation of circumferential actin-myosin cables required for epiboly. Thus, in addition to their role in apoptosis, by tightly regulating Ca(2+) signaling, Bcl-2 family members participate in the cellular events associated with early vertebrate development, including cytoskeletal dynamics and cell movement.

Marseillevirus prevalence in multitransfused patients suggests blood transmission.

BACKGROUND: Emerging viral infections in humans are appearing at an increasing rate. Recently, we identified a new Marseillevirus, named Giant Blood Marseillevirus (GBM), by performing viral metagenomics on asymptomatic blood donors. OBJECTIVES: To study and compare the prevalence of Marseillevirus between asymptomatic blood donors and thalassemia patients. DESIGN: Here, we present a combined molecular and serological study on 174 asymptomatic blood donors and 22 patients with thalassemia who receive repeated blood transfusions to estimate the prevalence of Marseillevirus in these two populations. RESULTS: We identified Marseillevirus genomic DNA in 4% of donors, whereas 9.1% of the thalassemia patients were positive for this virus. Moreover, IgG seropositivity was detected in 22.7% of patients in the thalassemia group, whereas this seropositivity was observed in 12.6% of the blood donor population. CONCLUSION: These results suggest that Marseillevirus infection is not rare in healthy persons and may be transmitted by transfusion, thus raising speculation regarding the long-term consequences of this viral infection, particularly in patients requiring repeated blood transfusions.