Bax transmembrane domain interacts with prosurvival Bcl-2 proteins in biological membranes.

The Bcl-2 (B-cell lymphoma 2) protein Bax (Bcl-2 associated X, apoptosis regulator) can commit cells to apoptosis via outer mitochondrial membrane permeabilization. Bax activity is controlled in healthy cells by prosurvival Bcl-2 proteins. C-terminal Bax transmembrane domain interactions were implicated recently in Bax pore formation. Here, we show that the isolated transmembrane domains of Bax, Bcl-xL (B-cell lymphoma-extra large), and Bcl-2 can mediate interactions between Bax and prosurvival proteins inside the membrane in the absence of apoptotic stimuli. Bcl-2 protein transmembrane domains specifically homooligomerize and heterooligomerize in bacterial and mitochondrial membranes. Their interactions participate in the regulation of Bcl-2 proteins, thus modulating apoptotic activity. Our results suggest that interactions between the transmembrane domains of Bax and antiapoptotic Bcl-2 proteins represent a previously unappreciated level of apoptosis regulation.

Comparative proteomic analysis of melon phloem exudates in response to viral infection.

Phloem vasculature is the route that most plant viruses use to spread widely around the plant. In addition, phloem sap transports signals that trigger systemic defense responses to infection. We investigated the proteome-level changes that occur in phloem sap during virus infection using the 2D-DIGE technique. Total proteins were extracted from phloem exudates of healthy and Melon necrotic spot virus infected melon plants and analyzed by 2D-DIGE. A total of 1046 spots were detected but only 25 had significant changes in abundance. After mass spectrometry, 19 different proteins corresponding to 22 spots were further identified (13 of them up-accumulated and 9 down-accumulated). Most of them were involved in controlling redox balance and cell death. Only two of the differentially altered proteins had never been described to be present in the phloem before: a carboxylesterase and the fumarylacetoacetate hydrolase 1, both considered negative regulators of cell death. RT-PCR analysis of phloem sap RNAs revealed that the transcripts corresponding to some of the identified protein could be also loaded into the sieve elements. The impact of these proteins in the host response against viral infections and the potential involvement in regulating development, growth and stress response in melon plants is discussed. BIOLOGICAL SIGNIFICANCE: Despite the importance of phloem as an integrative pathway for resource distribution, signaling and plant virus transport little is known about the modifications induced by these pathogens in phloem sap proteome. Only one previous study has actually examined the phloem sap proteome during viral infection using conventional two-dimensional electrophoresis. Since the major limitation of this technique has been its low sensitivity, the authors only identified five phloem proteins with altered abundance. To circumvent this issue we use two-dimensional difference in-gel electrophoresis (2D DIGE) technique, which combined with DeCyder Differential Analysis Software allows a more accurate and sensitive quantitative analysis than with conventional 2D PAGE. We identified 19 different proteins which accumulation in phloem sap was altered during a compatible plant virus infection including redox and hypersensitivity response-related proteins. Therefore, this work would help to understand the basic processes that occur in phloem during plant-virus interaction.

Peptides derived from the transmembrane domain of Bcl-2 proteins as potential mitochondrial priming tools.

The Bcl-2 family of proteins is crucial for apoptosis regulation. Members of this family insert through a specific C-terminal anchoring transmembrane domain (TMD) in the mitochondrial outer membrane where they hierarchically interact to determine cell fate. While the mitochondrial membrane has been proposed to actively participate in these protein-protein interactions, the influence of the TMD in the membrane-mediated interaction is poorly understood. Synthetic peptides (TMD-pepts) corresponding to the putative TMD of antiapoptotic (Bcl-2, Bcl-xL, Bcl-w, and Mcl-1) and pro-apoptotic (Bax, Bak) members were synthesized and characterized. TMD-pepts bound more efficiently to mitochondria-like bilayers than to plasma membrane-like bilayers, and higher binding correlated with greater membrane perturbation. The Bcl-2 TMD peptides promoted mitochondrial outer membrane permeabilization (MOMP) and cytochrome c release from isolated mitochondria and different cell lines. TMD-pepts exhibited nonapoptotic pro-death activity when apoptosis stimuli were absent. In addition, the peptides enhanced the apoptotic pathway induced by chemotherapeutic agents in cotreatment. Overall, the membrane perturbation effects of the TMD-pepts observed in the present study open the way for their use as new chemical tools to sensitize tumor cells to chemotherapeutic agents, in accordance with the concept of mitochondria priming.

BH3-mimetics- and cisplatin-induced cell death proceeds through different pathways depending on the availability of death-related cellular components.

BACKGROUND: Owing to their important function in regulating cell death, pharmacological inhibition of Bcl-2 proteins by dubbed BH3-mimetics is a promising strategy for apoptosis induction or sensitization to chemotherapy. However, the role of Apaf-1, the main protein constituent of the apoptosome, in the process has yet not been analyzed. Furthermore as new chemotherapeutics develop, the possible chemotherapy-induced toxicity to rapidly dividing normal cells, especially sensitive differentiated cells, has to be considered. Such undesirable effects would probably be ameliorated by selectively and locally inhibiting apoptosis in defined sensitive cells. METHODOLOGY AND PRINCIPAL FINDINGS: Mouse embryonic fibroblasts (MEFS) from Apaf-1 knock out mouse (MEFS KO Apaf-1) and Bax/Bak double KO (MEFS KO Bax/Bak), MEFS from wild-type mouse (MEFS wt) and human cervix adenocarcinoma (HeLa) cells were used to comparatively investigate the signaling cell death-induced pathways of BH3-mimetics, like ABT737 and GX15-070, with DNA damage-inducing agent cisplatin (cis-diammineplatinum(II) dichloride, CDDP). The study was performed in the absence or presence of apoptosis inhibitors namely, caspase inhibitors or apoptosome inhibitors. BH3-mimetic ABT737 required of Apaf-1 to exert its apoptosis-inducing effect. In contrast, BH3-mimetic GX15-070 and DNA damage-inducing CDDP induced cell death in the absence of both Bax/Bak and Apaf-1. GX15-070 induced autophagy-based cell death in all the cell lines analyzed. MEFS wt cells were protected from the cytotoxic effects of ABT737 and CDDP by chemical inhibition of the apoptosome through QM31, but not by using general caspase inhibitors. CONCLUSIONS: BH3-mimetic ABT737 not only requires Bax/Bak to exert its apoptosis-inducing effect, but also Apaf-1, while GX15-070 and CDDP induce different modalities of cell death in the absence of Bax/Bak or Apaf-1. Inclusion of specific Apaf-1 inhibitors in topical and well-localized administrations, but not in systemic ones, to avoid interferences with chemotherapeutics would be of interest to prevent chemotherapeutic-induced unwanted cell death which could improve cancer patient care.

The Intra- and intercellular movement of Melon necrotic spot virus (MNSV) depends on an active secretory pathway.

Plant viruses hijack endogenous host transport machinery to aid their intracellular spread. Here, we study the localization of the p7B, the membrane-associated viral movement protein (MP) of the Melon necrotic spot virus (MNSV), and also the potential involvement of the secretory pathway on the p7B targeting and intra- and intercellular virus movements. p7B fused to fluorescent proteins was located throughout the endoplasmic reticulum (ER) at motile Golgi apparatus (GA) stacks that actively tracked the actin microfilaments, and at the plasmodesmata (PD). Hence, the secretory pathway inhibitor, Brefeldin A (BFA), and the overexpression of the GTPase-defective mutant of Sar1p, Sar1[H74L], fully retained the p7B within the ER, revealing that the protein is delivered to PD in a BFA-sensitive and COPII-dependent manner. Disruption of the actin cytoskeleton with latrunculin B led to the accumulation of p7B in the ER, which strongly suggests that p7B is also targeted to the cell periphery in an actin-dependent manner. Remarkably, the local spread of the viral infection was significantly restricted either with the presence of BFA or under the overexpression of Sar1[H74L], thus revealing the involvement of an active secretory pathway in the intracellular movement of MNSV. Overall, these findings support a novel route for the intracellular transport of a plant virus led by the GA.

A self-interacting carmovirus movement protein plays a role in binding of viral RNA during the cell-to-cell movement and shows an actin cytoskeleton dependent location in cell periphery.

The p7A of Melon necrotic spot virus has been described to be a RNA-binding movement protein essential for cell-to-cell movement but its role in this process is still unknown. Here, we found that primary and secondary structure elements on p7A appear to form a composite RNA-binding site required for both RNA interaction and cell-to-cell movement in plants indicating a direct correlation between these activities. Furthermore, we found that fluorescent-tagged p7A was distributed in punctuate structures at the cell periphery but also in motile cytoplasmic inclusion bodies which were in close association with the actin MFs and most likely generated by self-interacting p7A molecules as shown by BiFC assays. Consistently, the p7A subcellular distribution was revealed to be sensitive to the actin inhibitor, latrunculin B. The involvement of the RNA-binding capabilities and the subcellular location of the p7A in the intracellular and intercellular virus movement is discussed.

Distribution and pathway for phloem-dependent movement of Melon necrotic spot virus in melon plants.

The translocation of Melon necrotic spot virus (MNSV) within tissues of inoculated and systemically infected Cucumis melo L. 'Galia' was studied by tissue-printing and in situ hybridization techniques. The results were compatible with the phloem vascular components being used to spread MNSV systemically by the same assimilate transport route that runs from source to sink organs. Virus RNAs were shown to move from the inoculated cotyledon toward the hypocotyl and root system via the external phloem, whereas the upward spread through the stem to the young tissues took place via the internal phloem. Virus infection was absent from non-inoculated source tissues as well as from both shoot and root apical meristems, but active sink tissues such as the young leaves and root system were highly infected. Finally, our results suggest that the MNSV invasion of roots is due to virus replication although a destination-selective process is probably necessary to explain the high levels of virus accumulation in roots. This efficient invasion of the root system is discussed in terms of natural transmission of MNSV by the soil-borne fungal vector.

A multifunctional bicupin serves as precursor for a chromosomal protein of Pisum sativum seeds.

The fact that the psp54 gene codes for p16, a seed chromatin protein of Pisum sativum, has been described previously. In the present paper it is shown that p54, the p16 precursor, also exists as a free polypeptide in pea and that it also yields p38, a second polypeptide from the N-terminal region of p54, which is co-localized at a subcellular level with p16. By using antibodies against pea p16 and p38, it was found that these proteins are present in the members of the tribe Viciae examined. Sequence analysis and 3D modelling indicates that p54 proteins belong to the cupin superfamily, and that they are related to sucrose binding proteins and, to a lesser extent, to vicilin-type seed storage proteins. Nevertheless, several distinctive characteristics of psp54 expression have been found: (i) the gene is differentially induced by ABA and several stress situations, in accordance with the presence of putative separate ABA and stress responsive elements in its promoter; (ii) the proteins are present in pods and seed coats, tissues of maternal origin; and (iii) p54 mRNA accumulates in the dry seeds. In view of both the functional properties of p54-derived proteins and the features of the psp54 gene expression, it is concluded that p54 represents a novel class within the cupin superfamily.