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A new mass-spectrometry method has been developed to obtain high-resolution spectra of folded proteins bound to lipids; using this technique as well as X-ray crystallography provides evidence for membrane protein conformational change as a result of lipid–protein interaction. Lipid bound to influence protein structure Many of the high-resolution membrane protein structures published recently are notable for the presence of lipids closely associated with the protein, prompting the question, how are these lipids influencing membrane complex structure? Carol Robinson and colleagues have developed a new ion mobility mass spectrometry (IM-MS) method that enabled them to obtain mass spectra of folded protein conformations bound to lipids. Using this method they identified lipids that altered the stability of MscL (mechanosensitive channel of large conductance), aquaporin Z and the ammonia channel. They then determined the X-ray crystal structure of the ammonia channel bound to one of these lipids (phosphatidylglycerol), which revealed how a conformational change in a specific loop led to the formation of a phosphatidylglycerol-binding site. The major conclusion from this work is that an individual lipid-binding event can change the stability of a membrane complex. On the cover, IM-MS captures a native membrane protein complex emerging from an ion mobility cell. Shown is the ammonia channel in apo, one- and two-lipid bound states. Previous studies have established that the folding, structure and function of membrane proteins are influenced by their lipid environments 1 , 2 , 3 , 4 , 5 , 6 , 7 and that lipids can bind to specific sites, for example, in potassium channels 8 . Fundamental questions remain however regarding the extent of membrane protein selectivity towards lipids. Here we report a mass spectrometry approach designed to determine the selectivity of lipid binding to membrane protein complexes. We investigate the mechanosensitive channel of large conductance (MscL) from Mycobacterium tuberculosis and aquaporin Z (AqpZ) and the ammonia channel (AmtB) from Escherichia coli , using ion mobility mass spectrometry (IM-MS), which reports gas-phase collision cross-sections. We demonstrate that folded conformations of membrane protein complexes can exist in the gas phase. By resolving lipid-bound states, we then rank bound lipids on the basis of their ability to resist gas phase unfolding and thereby stabilize membrane protein structure. Lipids bind non-selectively and with high avidity to MscL, all imparting comparable stability; however, the highest-ranking lipid is phosphatidylinositol phosphate, in line with its proposed functional role in mechanosensation 9 . AqpZ is also stabilized by many lipids, with cardiolipin imparting the most significant resistance to unfolding. Subsequently, through functional assays we show that cardiolipin modulates AqpZ function. Similar experiments identify AmtB as being highly selective for phosphatidylglycerol, prompting us to obtain an X-ray structure in this lipid membrane-like environment. The 2.3 Å resolution structure, when compared with others obtained without lipid bound, reveals distinct conformational changes that re-position AmtB residues to interact with the lipid bilayer. Our results demonstrate that resistance to unfolding correlates with specific lipid-binding events, enabling a distinction to be made between lipids that merely bind from those that modulate membrane protein structure and/or function. We anticipate that these findings will be important not only for defining the selectivity of membrane proteins towards lipids, but also for understanding the role of lipids in modulating protein function or drug binding.

Membrane lipids such as cardiolipin act as molecular glue to preserve the oligomeric states of membrane proteins with low oligomeric stability. Membrane-protein stabilization by lipid binding It is well established that lipid binding leads to the oligomerization of membrane proteins, and hence the activation of many signalling pathways, but it is not clear how the lipid bilayer affects the structure and function of membrane–protein complexes. Carol Robinson and colleagues have developed a mass-spectroscopy-based technique that allows the observation of oligomeric membrane–protein complexes with sufficient resolution to characterize their bound lipids. Using this technique, they evaluate the strength of oligomer formation for some 125 α-helical membrane proteins, including G-protein-coupled receptors. They find that lipid binding modulates protein interfaces, perturbing their monomer–oligomer equilibria, and show that manipulation of lipid binding can modify oligomer stability. And they use modelling to investigate possible binding sites for lipid molecules at the interfaces involved in oligomerization. These findings could aid the optimization of membrane–protein complexes for structural analysis. Oligomerization of membrane proteins in response to lipid binding has a critical role in many cell-signalling pathways 1 but is often difficult to define 2 or predict 3 . Here we report the development of a mass spectrometry platform to determine simultaneously the presence of interfacial lipids and oligomeric stability and to uncover how lipids act as key regulators of membrane-protein association. Evaluation of oligomeric strength for a dataset of 125 α-helical oligomeric membrane proteins reveals an absence of interfacial lipids in the mass spectra of 12 membrane proteins with high oligomeric stability. For the bacterial homologue of the eukaryotic biogenic transporters (LeuT 4 , one of the proteins with the lowest oligomeric stability), we found a precise cohort of lipids within the dimer interface. Delipidation, mutation of lipid-binding sites or expression in cardiolipin-deficient Escherichia coli abrogated dimer formation. Molecular dynamics simulation revealed that cardiolipin acts as a bidentate ligand, bridging across subunits. Subsequently, we show that for the Vibrio splendidus sugar transporter SemiSWEET 5 , another protein with low oligomeric stability, cardiolipin shifts the equilibrium from monomer to functional dimer. We hypothesized that lipids are essential for dimerization of the Na + /H + antiporter NhaA from E. coli , which has the lowest oligomeric strength, but not for the substantially more stable homologous Thermus thermophilus protein NapA. We found that lipid binding is obligatory for dimerization of NhaA, whereas NapA has adapted to form an interface that is stable without lipids. Overall, by correlating interfacial strength with the presence of interfacial lipids, we provide a rationale for understanding the role of lipids in both transient and stable interactions within a range of α-helical membrane proteins, including G-protein-coupled receptors.

Cardiolipins (CLs) are tetra-acylated diphosphatidylglycerols found in bacteria, yeast, plants, and animals. In healthy mammals, CLs are unsaturated, whereas saturated CLs are found in blood cells from Barth syndrome patients and in some Gram-positive bacteria. Here, we show that unsaturated but not saturated CLs block LPS-induced NF-κB activation, TNF-α and IP-10 secretion in human and murine macrophages, as well as LPS-induced TNF-α and IL-1β release in human blood mononuclear cells. Using HEK293 cells transfected with Toll-like receptor 4 (TLR4) and its co-receptor Myeloid Differentiation 2 (MD2), we demonstrate that unsaturated CLs compete with LPS for binding TLR4/MD2 preventing its activation, whereas saturated CLs are TLR4/MD2 agonists. As a consequence, saturated CLs induce a pro-inflammatory response in macrophages characterized by TNF-α and IP-10 secretion, and activate the alternative NLRP3 inflammasome pathway in human blood-derived monocytes. Thus, we identify that double bonds discriminate between anti- and pro-inflammatory properties of tetra-acylated molecules, providing a rationale for the development of TLR4 activators and inhibitors for use as vaccine adjuvants or in the treatment of TLR4-related diseases.Graphical abstract

Neuronal loss in Parkinson’s disease (PD) is associated with aberrant mitochondrial function and impaired proteostasis. Identifying the mechanisms that link these pathologies is critical to furthering our understanding of PD pathogenesis. Using human pluripotent stem cells (hPSCs) that allow comparison of cells expressing mutant SNCA (encoding α-synuclein (α-syn)) with isogenic controls, or SNCA -transgenic mice, we show that SNCA -mutant neurons display fragmented mitochondria and accumulate α-syn deposits that cluster to mitochondrial membranes in response to exposure of cardiolipin on the mitochondrial surface. Whereas exposed cardiolipin specifically binds to and facilitates refolding of α-syn fibrils, prolonged cardiolipin exposure in SNCA -mutants initiates recruitment of LC3 to the mitochondria and mitophagy. Moreover, we find that co-culture of SNCA -mutant neurons with their isogenic controls results in transmission of α-syn pathology coincident with mitochondrial pathology in control neurons. Transmission of pathology is effectively blocked using an anti-α-syn monoclonal antibody (mAb), consistent with cell-to-cell seeding of α-syn. Cardiolipin is a phospholipid component of the inner mitochondrial membrane. Here the authors demonstrate that cardiolipin interacts with mutant α-synuclein, and that impaired cardiolipin function can lead to spread of α-synuclein between neurons.

Regulation of apoptosis is tightly linked with the targeting of numerous Bcl-2 proteins to the mitochondrial outer membrane (MOM), where their activation or inhibition dictates cell death or survival. According to the traditional view of apoptotic regulation, BH3-effector proteins are indispensable for the cytosol-to-MOM targeting and activation of proapoptotic and antiapoptotic members of the Bcl-2 protein family. This view is challenged by recent studies showing that these processes can occur in cells lacking BH3 effectors by as yet to be determined mechanism(s). Here, we exploit a model membrane system that recapitulates key features of MOM to demonstrate that the proapoptotic Bcl-2 protein BAX and antiapoptotic Bcl-xL have an inherent ability to interact with membranes in the absence of BH3 effectors, but only in the presence of cellular concentrations of Mg2+/Ca2+. Under these conditions, BAX and Bcl-xL are selectively targeted to membranes, refolded, and activated in the presence of anionic lipids especially the mitochondrial-specific lipid cardiolipin. These results provide a mechanistic explanation for the mitochondrial targeting and activation of Bcl-2 proteins in cells lacking BH3 effectors. At cytosolic Mg2+ levels, the BH3-independent activation of BAX could provide localized amplification of apoptotic signaling at regions enriched in cardiolipin (e.g., contact sites between MOM and mitochondrial inner membrane). Increases in MOM cardiolipin, as well as cytosolic [Ca2+] during apoptosis could further contribute to its MOM targeting and activity. Meanwhile, the BH3-independent targeting and activation of Bcl-xL to the MOM is expected to counter the action of proapoptotic BAX, thereby preventing premature commitment to apoptosis.

Alzheimer’s disease (AD) is characterized by chronic neuroinflammation, which is partially mediated by dysregulated functions of glial cells. Cardiolipin (CL) is a phospholipid normally confined to the inner mitochondrial membrane; however, it has been detected in human sera, indicating that it can exist in the extracellular space where it may interact with nearby cells. Although CL has been shown to modulate several functions of microglia in a toll-like receptor (TLR) 4-dependent manner, the effects of extracellular CL on astrocytes are unknown. In addition to their homeostatic functions, astrocytes participate in neuroimmune responses of the brain and express TLR 4. Therefore, we hypothesized that extracellular CL (1) modulates the secretion of cytokines and cytotoxins by astrocytes, as well as their phagocytic activity, and (2) acts by interacting with astrocyte TLR 4. We demonstrate that CL inhibits the lipopolysaccharide- (LPS-) induced secretion of cytotoxins and expression of glial fibrillary acidic protein (GFAP) by human U118 MG astrocytic cells. CL alone upregulates the phagocytic activity of human astrocytic cells and primary murine astrocytes. CL in combination with LPS upregulates secretion of interleukin (IL)-1β by astrocytic cells. Furthermore, CL alone increases the secretion of monocyte chemoattractant protein (MCP)-1 by astrocytic cells, which is blocked by the TLR 4-specific antagonist TAK-242. We demonstrate that CL upregulates MCP-1 secretion in the absence of its natural carrier protein, β2-glycoprotein 1, indicating that CL may be bioactive in the brain where this protein is not present. Lastly, we show that CL downregulates the expression of astrocytic TLR 4, implying that CL engages this receptor, as its activation has been shown to lead to its degradation. Overall, our study extends the list of cell type functions of which CL modulates and provides evidence that CL, or liposomes containing this phospholipid can be used to modulate specific neuroimmune functions of astrocytes.

Purpose The effect of existing anti-cancer therapies is based mainly on the stimulation of apoptosis in cancer cells. Here, we have demonstrated the ability of a catalytically-reactive nanoparticle-based complex of cytochrome c with cardiolipin (Cyt-CL) to induce the apoptosis and killing of cancer cells in a monolayer cell culture. Methods Cyt-CL nanoparticles were prepared by complexing CytC with different molar excesses of CL. Following characterization, cytotoxicity and apoptosis inducing effects of nanoparticles were investigated. In an attempt to identify the anticancer activity mechanism of Cyt-CL, pseudo-lipoxygenase and lipoperoxidase reaction kinetics were measured by chemiluminescence. Results Using chemiluminescence, we have demonstrated that the Cyt-CL complex produces lipoperoxide radicals in two reactions: by decomposition of lipid hydroperoxides, and by lipid peroxidation under the action of H 2 O 2 . Antioxidants inhibited the formation of lipid radicals. Cyt-CL nanoparticles, but not the CytC alone, dramatically enhanced the level of apoptosis and cell death in two cell lines: drug-sensitive (A2780) and doxorubicin-resistant (A2780-Adr). The proposed mechanism of the cytotoxic action of Cyt-CL involves either penetration through the cytoplasm and outer mitochondrial membrane and catalysis of lipid peroxidation reactions at the inner mitochondrial membrane, or/and activation of lipid peroxidation within the cytoplasmic membrane. Conclusions Here we propose a new type of anticancer nano-formulation, with an action based on the catalytic action of Cyt-CL nanoparticles on the cell membrane and and/or mitochondrial membranes that results in lipid peroxidation reactions, which give rise to activation of apoptosis in cancer cells, including multidrug resistant cells.

Curcumin (CRM) and nerve growth factor (NGF) were entrapped in liposomes (LIP) with surface wheat germ agglutinin (WGA) to downregulate the phosphorylation of kinases in Alzheimer's disease (AD) therapy. Cardiolipin (CL)-conjugated LIP carrying CRM (CRM-CL/LIP) and also carrying NGF (NGF-CL/LIP) were used with AD models of SK-N-MC cells and Wistar rats after an insult with β-amyloid peptide (Aβ). We found that CRM-CL/LIP inhibited the expression of phosphorylated p38 (p-p38), phosphorylated c-Jun N-terminal kinase (p-JNK), and p-tau protein at serine 202 and prevented neurodegeneration of SK-N-MC cells. In addition, NGF-CL/LIP could enhance the quantities of p-neurotrophic tyrosine kinase receptor type 1 and p-extracellular signal-regulated kinase 5 for neuronal rescue. Moreover, WGA-grafted CRM-CL/LIP and WGA-grafted NGF-CL/LIP significantly improved the permeation of CRM and NGF across the blood-brain barrier, reduced Aβ plaque deposition and the malondialdehyde level, and increased the percentage of normal neurons and cholinergic activity in the hippocampus of AD rats. Based on the marker expressions and in vivo evidence, current LIP carriers can be promising drug delivery systems to protect nervous tissue against Aβ-induced apoptosis in the brain during the clinical management of AD.

Liposomes with cardiolipin (CL) and wheat germ agglutinin (WGA) were developed to permeate the blood-brain barrier and treat Alzheimer's disease. WGA-conjugated and CL-incorporated liposomes (WGA-CL-liposomes) were used to transport nerve growth factor (NGF) and curcumin (CUR) across a monolayer of human brain-microvascular endothelial cells regulated by human astrocytes and to protect SK-N-MC cells against apoptosis induced by β-amyloid1-42 (Aβ(1-42)) fibrils. An increase in the CL mole percentage in lipids increased the liposomal diameter, absolute zeta potential value, entrapment efficiency of NGF and CUR, release of NGF, biocompatibility, and viability of SK-N-MC cells with Aβ(1-42), but decreased the atomic ratio of nitrogen to phosphorus and release of CUR. In addition, an increase in the WGA concentration for grafting enhanced the liposomal diameter, atomic ratio of nitrogen to phosphorus, and permeability of NGF and CUR across the blood-brain barrier, but reduced the absolute zeta potential value and biocompatibility. WGA-CL-liposomes carrying NGF and CUR could be promising colloidal delivery carriers for future clinical application in targeting the blood-brain barrier and inhibiting neurotoxicity.

Cardiolipin (CL) was shown to bound to the dimer interface of NhaA Na + /H + antiporter. Here, we explore the cardiolipin-NhaA interaction both in vitro and in vivo . Using a novel and straightforward in-vitro assay in which n-dodecyl β-D maltoside (DDM) detergent is used to delipidate the dimer interface and to split the dimers into monomers; the monomers are subsequently exposed to cardiolipin or the other E. coli phospholipids. Most efficient reconstitution of dimers is observed by cardiolipin. This assay is likely to be applicable to future studies of protein–lipid interactions. In-vivo experiments further reveal that cardiolipin is necessary for NhaA survival. Although less efficient phosphatidyl-glycerol (PG) can also reconstitute NhaA monomers to dimers. We also identify a putative cardiolipin binding site. Our observations may contribute to drug design, as human NhaA homologues, which are involved in severe pathologies, might also require specific phospholipids.