DRP1 interacts directly with BAX to induce its activation and apoptosis.

The apoptotic executioner protein BAX and the dynamin-like protein DRP1 co-localize at mitochondria during apoptosis to mediate mitochondrial permeabilization and fragmentation. However, the molecular basis and functional consequences of this interplay remain unknown. Here, we show that BAX and DRP1 physically interact, and that this interaction is enhanced during apoptosis. Complex formation between BAX and DRP1 occurs exclusively in the membrane environment and requires the BAX N-terminal region, but also involves several other BAX surfaces. Furthermore, the association between BAX and DRP1 enhances the membrane activity of both proteins. Forced dimerization of BAX and DRP1 triggers their activation and translocation to mitochondria, where they induce mitochondrial remodeling and permeabilization to cause apoptosis even in the absence of apoptotic triggers. Based on this, we propose that DRP1 can promote apoptosis by acting as noncanonical direct activator of BAX through physical contacts with its N-terminal region.

Type IV Coupling Proteins as Potential Targets to Control the Dissemination of Antibiotic Resistance.

The increase of infections caused by multidrug-resistant bacteria, together with the loss of effectiveness of currently available antibiotics, represents one of the most serious threats to public health worldwide. The loss of human lives and the economic costs associated to the problem of the dissemination of antibiotic resistance require immediate action. Bacteria, known by their great genetic plasticity, are capable not only of mutating their genes to adapt to disturbances and environmental changes but also of acquiring new genes that allow them to survive in hostile environments, such as in the presence of antibiotics. One of the major mechanisms responsible for the horizontal acquisition of new genes (e.g., antibiotic resistance genes) is bacterial conjugation, a process mediated by mobile genetic elements such as conjugative plasmids and integrative conjugative elements. Conjugative plasmids harboring antibiotic resistance genes can be transferred from a donor to a recipient bacterium in a process that requires physical contact. After conjugation, the recipient bacterium not only harbors the antibiotic resistance genes but it can also transfer the acquired plasmid to other bacteria, thus contributing to the spread of antibiotic resistance. Conjugative plasmids have genes that encode all the proteins necessary for the conjugation to take place, such as the type IV coupling proteins (T4CPs) present in all conjugative plasmids. Type VI coupling proteins constitute a heterogeneous family of hexameric ATPases that use energy from the ATP hydrolysis for plasmid transfer. Taking into account their essential role in bacterial conjugation, T4CPs are attractive targets for the inhibition of bacterial conjugation and, concomitantly, the limitation of antibiotic resistance dissemination. This review aims to compile present knowledge on T4CPs as a starting point for delving into their molecular structure and functioning in future studies. Likewise, the scientific literature on bacterial conjugation inhibitors has been reviewed here, in an attempt to elucidate the possibility of designing T4CP-inhibitors as a potential solution to the dissemination of multidrug-resistant bacteria.

Conjugative Coupling Proteins and the Role of Their Domains in Conjugation, Secondary Structure and in vivo Subcellular Location.

Type IV Coupling Proteins (T4CPs) are essential elements in many type IV secretion systems (T4SSs). The members of this family display sequence, length, and domain architecture heterogeneity, being the conserved Nucleotide-Binding Domain the motif that defines them. In addition, most T4CPs contain a Transmembrane Domain (TMD) in the amino end and an All-Alpha Domain facing the cytoplasm. Additionally, a few T4CPs present a variable domain at the carboxyl end. The structural paradigm of this family is TrwBR388, the T4CP of conjugative plasmid R388. This protein has been widely studied, in particular the role of the TMD on the different characteristics of TrwBR388. To gain knowledge about T4CPs and their TMD, in this work a chimeric protein containing the TMD of TraJpKM101 and the cytosolic domain of TrwBR388 has been constructed. Additionally, one of the few T4CPs of mobilizable plasmids, MobBCloDF13 of mobilizable plasmid CloDF13, together with its TMD-less mutant MobBΔTMD have been studied. Mating studies showed that the chimeric protein is functional in vivo and that it exerted negative dominance against the native proteins TrwBR388 and TraJpKM101. Also, it was observed that the TMD of MobBCloDF13 is essential for the mobilization of CloDF13 plasmid. Analysis of the secondary structure components showed that the presence of a heterologous TMD alters the structure of the cytosolic domain in the chimeric protein. On the contrary, the absence of the TMD in MobBCloDF13 does not affect the secondary structure of its cytosolic domain. Subcellular localization studies showed that T4CPs have a unipolar or bipolar location, which is enhanced by the presence of the remaining proteins of the conjugative system. Unlike what has been described for TrwBR388, the TMD is not an essential element for the polar location of MobBCloDF13. The main conclusion is that the characteristics described for the paradigmatic TrwBR388 T4CP should not be ascribed to the whole T4CP family. Specifically, it has been proven that the mobilizable plasmid-related MobBCloDF13 presents different characteristics regarding the role of its TMD. This work will contribute to better understand the T4CP family, a key element in bacterial conjugation, the main mechanism responsible for antibiotic resistance spread.

BFL1 modulates apoptosis at the membrane level through a bifunctional and multimodal mechanism showing key differences with BCLXL.

BFL1 is a relatively understudied member of the BCL2 protein family which has been implicated in the pathogenesis and chemoresistance of a variety of human cancers, including hematological malignancies and solid tumours. BFL1 is generally considered to have an antiapoptotic function, although its precise mode of action remains unclear. By quantitatively analyzing BFL1 action in synthetic membrane models and in cells, we found that BFL1 inhibits apoptosis through three distinct mechanisms which are similar but not identical to those of BCLXL, the paradigmatic antiapoptotic BCL2 family protein. Strikingly, alterations in lipid composition during apoptosis activate a prodeath function of BFL1 that is based on noncanonical oligomerization of the protein and breaching of the permeability barrier of the outer mitochondrial membrane (OMM). This lipid-triggered prodeath function of BFL1 is absent in BCLXL and also differs from that of the apoptotic effector BAX, which sets it apart from other BCL2 family members. Our findings support a new model in which BFL1 modulates apoptosis through a bifunctional and multimodal mode of action that is distinctly regulated by OMM lipids compared to BCLXL.

Drp1 polymerization stabilizes curved tubular membranes similar to those of constricted mitochondria.

Dynamin-related protein 1 (Drp1), an 80 kDa mechanochemical GTPase of the dynamin superfamily, is required for mitochondrial division in mammals. Despite the role of Drp1 dysfunction in human disease, its molecular mechanism remains poorly understood. Here, we examined the effect of Drp1 on membrane curvature using tubes pulled from giant unilamellar vesicles (GUVs). We found that GTP promoted rapid rearrangement of Drp1 from a uniform distribution to discrete foci, in line with the assembly of Drp1 scaffolds at multiple nucleation sites around the lipid tube. Polymerized Drp1 preserved the membrane tube below the protein coat, also in the absence of pulling forces, but did not induce spontaneous membrane fission. Strikingly, Drp1 polymers stabilized membrane curvatures similar to those of constricted mitochondria against pressure changes. Our findings support a new model for mitochondrial division whereby Drp1 mainly acts as a scaffold for membrane curvature stabilization, which sets it apart from other dynamin homologs.

Does Ceramide Form Channels? The Ceramide-Induced Membrane Permeabilization Mechanism.

Ceramide is a sphingolipid involved in several cellular processes, including apoptosis. It has been proposed that ceramide forms large and stable channels in the mitochondrial outer membrane that induce cell death through direct release of cytochrome c. However, this mechanism is still debated because the membrane permeabilizing activity of ceramide remains poorly understood. To determine whether the mechanism of ceramide-induced membrane leakage is consistent with the hypothesis of an apoptotic ceramide channel, we have used here assays of calcein release from liposomes. When assaying liposomes containing sphingomyelin and cholesterol, we observed an overall gradual phenomenon of contents release, together with some all-or-none leakage (at low ceramide concentrations or short times). The presence of channels in the bilayer should cause only an all-or-none leakage. When liposomes poor in sphingomyelin/cholesterol or mimicking the lipid composition of the mitochondrial outer membrane were tested, we did not detect any leakage. In consequence, the hypothesis of formation of large ceramide channels in the membrane is not consistent with our results. Instead we propose that the presence of ceramide in one of the membrane monolayers causes a surface area mismatch between both monolayers, which leads to vesicle collapse. The gradual phenomenon of calcein release would be due to a competition between two ceramide effects; namely, lateral segregation that facilitates permeabilization, and at longer times, trans-bilayer flip-flop that opposes asymmetric lateral segregation and causes a mismatch.

Apoptotic foci at mitochondria: in and around Bax pores.

The permeabilization of the mitochondrial outer membrane by Bax and Bak during apoptosis is considered a key step and a point of no return in the signalling pathway. It is always closely related to the reorganization of mitochondrial cristae that frees cytochrome c to the intermembrane space and to massive mitochondrial fragmentation mediated by the dynamin-like protein Drp1. Despite multiple evidence in favour of a functional link between these processes, the molecular mechanisms that connect them and their relevance for efficient apoptosis signalling remain obscure. In this review, we discuss recent progress on our understanding of how Bax forms pores in the context of Drp1-stabilized signalling platforms at apoptotic foci in mitochondria.This article is part of the themed issue 'Membrane pores: from structure and assembly, to medicine and technology'.

Lipid-modified oligonucleotide conjugates: Insights into gene silencing, interaction with model membranes and cellular uptake mechanisms.

The ability of oligonucleotides to silence specific genes or inhibit the biological activity of specific proteins has generated great interest in their use as research tools and therapeutic agents. Unfortunately, their biological applications meet the limitation of their poor cellular accessibility. Developing an appropriate delivery system for oligonucleotides is essential to achieve their efficient cellular uptake. In the present work a series of phosphorothioate lipid-oligonucleotide hybrids were synthesized introducing covalently single or double lipid tails at both 3'- and 5'-termini of an antisense oligonucleotide. Gene transfections in cultured cells showed antisense luciferase inhibition without the use of a transfecting agent for conjugates modified with the double-lipid tail at 5'-termini. The effect of the double lipid-tailed modification was further studied in detail in several model membrane systems as well as in cellular uptake experiments. During these studies the spontaneous formation of self-assembled microstructures is clearly observed. Lipidation allowed the efficient incorporation of the oligonucleotide in HeLa cells by a macropinocytosis mechanism without causing cytotoxicity in cells or altering the binding properties of the oligonucleotide conjugates. In addition, both single- and double-tailed compounds showed a similar behavior in lipid model membranes, making them useful in nucleotide-based technologies.

Dynamin-related protein 1 (Drp1) promotes structural intermediates of membrane division.

Drp1 is a dynamin-like GTPase that mediates mitochondrial and peroxisomal division in a process dependent on self-assembly and coupled to GTP hydrolysis. Despite the link between Drp1 malfunction and human disease, the molecular details of its membrane activity remain poorly understood. Here we reconstituted and directly visualized Drp1 activity in giant unilamellar vesicles. We quantified the effect of lipid composition and GTP on membrane binding and remodeling activity by fluorescence confocal microscopy and flow cytometry. In contrast to other dynamin relatives, Drp1 bound to both curved and flat membranes even in the absence of nucleotides. We also found that Drp1 induced membrane tubulation that was stimulated by cardiolipin. Moreover, Drp1 promoted membrane tethering dependent on the intrinsic curvature of the membrane lipids and on GTP. Interestingly, Drp1 concentrated at membrane contact surfaces and, in the presence of GTP, formed discrete clusters on the vesicles. Our findings support a role of Drp1 not only in the formation of lipid tubes but also on the stabilization of tightly apposed membranes, which are intermediate states in the process of mitochondrial fission.

Membranes in motion: mitochondrial dynamics and their role in apoptosis.

Mitochondrial dynamics is crucial for cell survival, development and homeostasis and impairment of these functions leads to neurologic disorders and metabolic diseases. The key components of mitochondrial dynamics have been identified. Mitofusins and OPA1 mediate mitochondrial fusion, whereas Drp1 is responsible for mitochondrial fission. In addition, an interplay between the proteins of the mitochondrial fission/fusion machinery and the Bcl-2 proteins, essential mediators in apoptosis, has been also described. Here, we review the molecular mechanisms regarding mitochondrial dynamics together with their role in apoptosis.

Subcellular location of the coupling protein TrwB and the role of its transmembrane domain.

Conjugation is the most important mechanism for horizontal gene transfer and it is the main responsible for the successful adaptation of bacteria to the environment. Conjugative plasmids are the DNA molecules transferred and a multiprotein system encoded by the conjugative plasmid itself is necessary. The high number of proteins involved in the process suggests that they should have a defined location in the cell and therefore, they should be recruited to that specific point. One of these proteins is the coupling protein that plays an essential role in bacterial conjugation. TrwB is the coupling protein of R388 plasmid that is divided in two domains: i) The N-terminal domain referred as transmembrane domain and ii) a large cytosolic domain that contains a nucleotide-binding motif similar to other ATPases. To investigate the role of these domains in the subcellular location of TrwB, we constructed two mutant proteins that comprised the transmembrane (TrwBTM) or the cytoplasmic (TrwBΔN70) domain of TrwB. By immunofluorescence and GFP-fusion proteins we demonstrate that TrwB and TrwBTM mutant protein were localized to the cell pole independently of the remaining R388 proteins. On the contrary, a soluble mutant protein (TrwBΔN70) was localized to the cytoplasm in the absence of R388 proteins. However, in the presence of other R388-encoded proteins, TrwBΔN70 localizes uniformly to the cell membrane, suggesting that interactions between the cytosolic domain of TrwB and other membrane proteins of R388 plasmid may happen. Our results suggest that the transmembrane domain of TrwB leads the protein to the cell pole.

The transmembrane domain of the T4SS coupling protein TrwB and its role in protein-protein interactions.

Bacteria use type IV secretion systems to transfer genetic material and proteins from donor to recipient cells, using proteins encoded by conjugative plasmids. Among those proteins the so-called Type IV Coupling Protein plays a central role in the process. One of the best studied members of this family is TrwB, the conjugative coupling protein of R388 plasmid. Previous studies indicated that the transmembrane domain of TrwB plays a role beyond the mere anchoring of the protein to the membrane. TrwB has also been shown to interact with other conjugative proteins, such as the VirB10-like protein of R388 TrwE. The goal of this study is to elucidate the role of the different domains of TrwB and TrwE in their biological function, and in both self- and TrwB-TrwE interactions. To this aim, a series of TrwB and TrwE deletion mutant proteins were constructed. Conjugation and interaction studies revealed that the transmembrane domain of TrwB, and particularly its second transmembrane helix, is needed for TrwB self-interaction and for R388 conjugative transfer and that there are contacts between TrwB and TrwE in the membrane. On the contrary, the lack of the TMD of TrwE does not completely abolish R388 conjugation although the interaction between TrwE-TrwB is lost. These results identify protein-protein interactions inside the membrane needed for T4SS function.

Double-tailed lipid modification as a promising candidate for oligonucleotide delivery in mammalian cells.

BACKGROUND: The potential use of nucleic acids as therapeutic drugs has triggered the quest for oligonucleotide conjugates with enhanced cellular permeability. To this end, the biophysical aspects of previously reported potential lipid oligodeoxyribonucleotide conjugates were studied including its membrane-binding properties and cellular uptake. METHODS: These conjugates were fully characterized by MALDI-TOF mass spectrometry and HPLC chromatography. Their ability to insert into lipid model membrane systems was evaluated by Langmuir balance and confocal microscopy followed by the study of the internalization of a lipid oligodeoxyribonucleotide conjugate bearing a double-tail lipid modification (C28) into different cell lines by confocal microscopy and flow cytometry. This compound was also compared with other lipid containing conjugates and with the classical lipoplex formulation using Transfectin as transfection reagent. RESULTS: This double-tail lipid modification showed better incorporation into both lipid model membranes and cell systems. Indeed, this lipid conjugation was capable of inserting the oligodeoxyribonucleotide into both liquid-disordered and liquid-ordered domains of model lipid bilayer systems and produced an enhancement of oligodeoxyribonucleotide uptake in cells, even better than the effect caused by lipoplexes. In addition, in ß2 integrin (CR3) expressing cells this receptor was directly involved in the enhanced internalization of this compound. CONCLUSIONS: All these features confirm that the dual lipid modification (C28) is an excellent modification for enhancing nucleic acid delivery without altering their binding properties. GENERAL SIGNIFICANCE: Compared to the commercial lipoplex approach, oligodeoxyribonucleotide conjugation with C28 dual lipid modification seems to be promising to improve oligonucleotide delivery in mammalian cells.

Synthesis, cell-surface binding, and cellular uptake of fluorescently labeled glucose-DNA conjugates with different carbohydrate presentation.

Oligonucleotide conjugates carrying carbohydrates at the 5'-end have been prepared. Glucose, fucose, and saccharides containing glucose at the nonreducing end were attached to DNA strands using the classical phosphoramidite chemistry. Two types of spacers and a dendron scaffold helped to obtain a diversity of sugar presentations in the DNA conjugates. Cellular surface adsorption and cellular uptake of carbohydrate oligonucleotide antisense sequences were measured using flow cytometric analysis. Conjugates with the glucose moiety linked through long spacers (15 to 18 atom distances) were internalized better than those with short linkers (4 atom distance) and than DNA control strands without sugar modification. Conjugates with tetravalent presentation of glucose did not improve cell uptake.

Reconstitution in liposome bilayers enhances nucleotide binding affinity and ATP-specificity of TrwB conjugative coupling protein.

Bacterial conjugative systems code for an essential membrane protein that couples the relaxosome to the DNA transport apparatus, called type IV coupling protein (T4CP). TrwB is the T4CP of the conjugative plasmid R388. In earlier work we found that this protein, purified in the presence of detergents, binds preferentially purine nucleotides trisphosphate. In contrast a soluble truncated mutant TrwBΔN70 binds uniformly all nucleotides tested. In this work, TrwB has been successfully reconstituted into liposomes. The non-membranous portion of the protein is almost exclusively oriented towards the outside of the vesicles. Functional analysis of TrwB proteoliposomes demonstrates that when the protein is inserted into the lipid bilayer the affinity for adenine and guanine nucleotides is enhanced as compared to that of the protein purified in detergent or to the soluble deletion mutant, TrwBΔN70. The protein specificity for adenine nucleotides is also increased. No ATPase activity has been found in TrwB reconstituted in proteoliposomes. This result suggests that the N-terminal transmembrane segment of this T4CP interferes with its ATPase activity and can be taken to imply that the TrwB transmembrane domain plays a regulatory role in its biological activity.