Systematic simulation of the interactions of Pleckstrin homology domains with membranes

Pleckstrin homology (PH) domains can recruit proteins to membranes by recognition of phosphatidylinositol phosphates (PIPs). Here we report the systematic simulation of the interactions of 100 mammalian PH domains with PIP containing model membranes. Comparison with crystal structures of PH domains bound to PIP analogues demonstrates that our method correctly identifies interactions at known canonical and non-canonical sites, while revealing additional functionally important sites for interaction not observed in the crystal structure, such as for P-Rex1 and Akt1. At the family level, we find that the β1 and β2 strands and their connecting loop constitute the primary PIP interaction site for the majority of PH domains, but we highlight interesting exceptional cases. Simultaneous interaction with multiple PIPs and clustering of PIPs induced by PH domain binding are also observed. Our findings support a general paradigm for PH domain membrane association involving multivalent interactions with anionic lipids.

Characterization of oxidation of glutathione by cytochrome c

Cytochrome c is a member of the respiratory chain of the mitochondria. Non-membrane-bound (free) cytochrome c can be reduced by gluthatione as well as ascorbic acid. We investigated the effect of pH, Ca2+, Mg2+ and anionic phospholipids on the reduction of cytochrome c by glutathione.The reduction of cytochrome c by thiols was measured using photometry. Mitochondrial oxygen consumption was detected by use of oxygen electrode. Glutathione does not reduce cytochrome c at pH = 7.0 in the absence of Ca2+ and Mg2+. The reduction of cytochrome c by glutathione is inhibited by anionic lipids, especially cardiolipin. The typical conditions of apoptosis—elevated pH, Ca2+ level and Mg2+—increases the reduction of cytochrome c. Glutathione (5 mM) causes increased mitochondrial O2 consumption at pH = 8.0, in the presence of ADP either 1 mM Mg2+ or 1 mM Ca2+. Our results suggest that membrane bound cyt c does not oxidize glutathione. Free (not membrane bound) cytochrome c can oxidize glutathione. In mitochondria, O2 is depleted only in the presence of ADP, so the O2 depletion observed in the presence of glutathione can be related to the respiratory chain. Decreased glutathione levels play a role in apoptosis. Therefore, membrane unbound cyt c can contribute to apoptosis by oxidation of glutathione.

Ion currents through Kir potassium channels are gated by anionic lipids

Ion currents through potassium channels are gated. Constriction of the ion conduction pathway at the inner helix bundle, the textbook ‘gate’ of Kir potassium channels, has been shown to be an ineffective permeation control, creating a rift in our understanding of how these channels are gated. Here we present the first evidence that anionic lipids act as interactive response elements sufficient to gate potassium conduction. We demonstrate the limiting barrier to K+ permeation lies within the ion conduction pathway and show that this ‘gate’ is operated by the fatty acyl tails of lipids that infiltrate the conduction pathway via fenestrations in the walls of the pore. Acyl tails occupying a surface groove extending from the cytosolic interface to the conduction pathway provide a potential means of relaying cellular signals, mediated by anionic lipid head groups bound at the canonical lipid binding site, to the internal gate.

Origin of Cyanobacteria thylakoids via a non-vesicular glycolipid phase transition and impact on the Great Oxygenation Event

Appearance of oxygenic photosynthesis in Cyanobacteria is a major event in the evolution of Life. It had an irreversible impact on our planet, promoting the Great Oxygenation Event (GOE), ~2.4 b.y.a. Ancient Cyanobacteria predating the GOE were Gloeobacter-type cells, having no thylakoids. They hosted photosystems in their cytoplasmic membrane. The driver of the GOE was proposed to be the transition from unicellular to filamentous Cyanobacteria. However, the appearance of thylakoids expanded the photosynthetic surface by multiple logs: this multiplier effect would be more coherent with an impact on the atmosphere. Primitive thylakoids self-organize as concentric parietal uninterrupted multilayers. The quest for their origin resists vesicular-based scenarios. This review reports studies supporting that Hexagonal II-forming gluco- and galactolipids at the periphery of the cytosolic membrane could be turned within nanoseconds and without any external source of energy into membrane multilayers. Comparison of lipid biosynthetic pathways further shows that ancient Cyanobacteria contained only one anionic Lamellar-forming lipid, phosphatidylglycerol. Acquisition of sulfoquinovosyldiacylglycerol biosynthesis correlates with thylakoid emergence, possibly enabling a sufficient provision of anionic lipids to trigger an Hexagonal II-to-Lamellar phase transition. With this non-vesicular lipid-phase transition, a framework is also available to reexamine the role of companion proteins in thylakoid biogenesis processes.

Lipids modulate the BH3-independent membrane targeting and activation of BAX and Bcl-xL

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.

Sphingolipids mediate polar sorting of PIN2 through phosphoinositide consumption at the trans-Golgi network

The lipid composition of organelles acts as a landmark to define membrane identity and specify subcellular function. Phosphoinositides are anionic lipids acting in protein sorting and trafficking at the trans-Golgi network (TGN). In animal cells, sphingolipids control the turnover of phosphoinositides through lipid exchange mechanisms at endoplasmic reticulum/TGN contact sites. In this study, we discover a mechanism for how sphingolipids mediate phosphoinositide homeostasis at the TGN in plant cells. Using multiple approaches, we show that a reduction of the acyl-chain length of sphingolipids results in an increased level of phosphatidylinositol-4-phosphate (PtdIns(4)P or PI4P) at the TGN but not of other lipids usually coupled to PI4P during exchange mechanisms. We show that sphingolipids mediate Phospholipase C (PLC)-driven consumption of PI4P at the TGN rather than local PI4P synthesis and that this mechanism is involved in the polar sorting of the auxin efflux carrier PIN2 at the TGN. Together, our data identify a mode of action of sphingolipids in lipid interplay at the TGN during protein sorting.

Insights into the Action Mechanism of the Antimicrobial Peptide Lasioglossin III

Lasioglossin III (LL-III) is a cationic antimicrobial peptide derived from the venom of the eusocial bee Lasioglossum laticeps. LL-III is extremely toxic to both Gram-positive and Gram-negative bacteria, and it exhibits antifungal as well as antitumor activity. Moreover, it shows low hemolytic activity, and it has almost no toxic effects on eukaryotic cells. However, the molecular basis of the LL-III mechanism of action is still unclear. In this study, we characterized by means of calorimetric (DSC) and spectroscopic (CD, fluorescence) techniques its interaction with liposomes composed of a mixture of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and 1-palmitoyl-2-oleoyl-sn-glycero-3-rac-phosphoglycerol (POPG) lipids as a model of the negatively charged membrane of pathogens. For comparison, the interaction of LL-III with the uncharged POPC liposomes was also studied. Our data showed that LL-III preferentially interacted with anionic lipids in the POPC/POPG liposomes and induces the formation of lipid domains. Furthermore, the leakage experiments showed that the peptide could permeabilize the membrane. Interestingly, our DSC results showed that the peptide-membrane interaction occurs in a non-disruptive manner, indicating an intracellular targeting mode of action for this peptide. Consistent with this hypothesis, our gel-retardation assay experiments showed that LL-III could interact with plasmid DNA, suggesting a possible intracellular target.