Mechanism Of Membrane Targeting Research Articles

Spontaneous Formation of an Ensemble of Structurally Diverse Membrane Channel Architectures from a Single Antimicrobial Peptide Maculatin

Antimicrobial peptides (AMPs) are an ancient, powerful, and ubiquitous component of the innate immune defence in all domains of life and play a key role in controlling the human microbiome. Many AMPs are known to selectively target and form pores in microbial membranes, killing a wide variety of pathogens at low micromolar concentrations. Fundamental questions remain, however, regarding the molecular mechanisms of membrane targeting, pore formation and function, and the extent to which poration contributes towards antimicrobial activity. Here we report an experimentally guided and validated unbiased long-timescale simulation methodology that yields the complete mechanism of spontaneous pore assembly in the membrane for the amphiphilic pore-forming AMP maculatin at atomic resolution. Rather than a single well defined pore, maculatin was found to form an ensemble of well defined temporarily functional channels that continuously form and dissociate in the membrane. All channels are formed from highly symmetric low-oligomeric assemblies of up to 8 membrane-spanning peptides that mimic integral membrane protein channels in structure. Each channel has a different architecture, functional lifetime, and ion conductivity and overall membrane permeabilization is dominated by higher order oligomers, which form ion channels that also conduct water at high rates. The highest order oligomers were also found to efficiently conduct small dyes. Channel architectures as well as their relative weighting in the ensemble are sensitive to minor mutations as well as variation of lipid tail length. All pores are formed spontaneously by the consecutive addition of individual helices to a transmembrane helix or helix bundle, in contrast to current poration models postulated for AMPs. The diversity of channel architectures formed by a single sequence is remarkable and could explain why no sequence-function relationships in AMP sequences have been discovered to date. Structural ensembles formed by AMP may be the key to preventing bacterial resistance.

Spontaneous formation of structurally diverse membrane channel architectures from a single antimicrobial peptide.

Many antimicrobial peptides (AMPs) selectively target and form pores in microbial membranes. However, the mechanisms of membrane targeting, pore formation and function remain elusive. Here we report an experimentally guided unbiased simulation methodology that yields the mechanism of spontaneous pore assembly for the AMP maculatin at atomic resolution. Rather than a single pore, maculatin forms an ensemble of structurally diverse temporarily functional low-oligomeric pores, which mimic integral membrane protein channels in structure. These pores continuously form and dissociate in the membrane. Membrane permeabilization is dominated by hexa-, hepta- and octamers, which conduct water, ions and small dyes. Pores form by consecutive addition of individual helices to a transmembrane helix or helix bundle, in contrast to current poration models. The diversity of the pore architectures—formed by a single sequence—may be a key feature in preventing bacterial resistance and could explain why sequence–function relationships in AMPs remain elusive.

Liprotides made of α-lactalbumin and cis fatty acids form core–shell and multi-layer structures with a common membrane-targeting mechanism

α-Lactalbumin (aLA) has been shown to form complexes with oleic acid (OA), which may target cancer cells. We recently showed that aLA and several other proteins all form protein–OA complexes called liprotides with a generic structure consisting of a micellar OA core surrounded by a shell of partially denatured protein. Here we report that a heat treatment and an alkaline treatment method both allow us to prepare liprotide complexes composed of aLA and a range of unsaturated fatty acids (FA), provided the FAs contain cis (but not trans) double bonds. All liprotides containing cis-FA form both small and large species, which all consist of partially denatured aLA, though the overall shape of the species differs. Small liprotides have a simple core–shell structure while the larger liprotides are multi-layered, i.e. they have an additional layer of both FA and aLA surrounding the outside of the core–shell structure. All liprotides can transfer their entire FA content to vesicles, releasing aLA as monomers and softening the lipid membrane. The more similar to OA, the more efficiently the different FAs induce hemolysis. We conclude that aLA can take up and transfer a wide variety of FA to membranes, provided they contain a cis-bond. This highlights liprotides as a general class of complexes where both protein and cis-FA component can be varied without departing from a generic (though sometimes multi-layered) core–shell structure.

Virucidal activity of Haemaphysalis longicornis longicin P4 peptide against tick-borne encephalitis virus surrogate Langat virus.

BackgroundLongicin is a defensin-like peptide, identified from the midgut epithelium of hard tick Haemaphysalis longicornis. Several studies have already shown the antimicrobial and parasiticidal activities of longicin peptide and one of its synthetic partial analogs, longicin P4. In this study, longicin peptides were tested for potential antiviral activity against Langat virus (LGTV), a tick-borne flavivirus.MethodsLongicin P1 and P4 peptides were chemically synthesized. Antiviral activity of the longicin peptides against LGTV was evaluated through in vitro virucidal assays, wherein the antiviral efficacy was determined by reduction in number of viral foci and virus yield. Additionally, longicin P4 was also tested for its activity against human adenovirus, a non-enveloped virus. Lastly, to assess the importance of longicin on the innate antiviral immunity of H. longicornis ticks, gene silencing through RNAi was performed.ResultsLongicin P4 produced significant viral foci reduction and lower virus yield against LGTV, while longicin P1 failed to demonstrate the same results. Conversely, both longicin partial analogs (P1 and P4) did not show significant antiviral activity when tested on adenovirus. In addition, longicin-silenced ticks showed significantly higher virus titer after 7 days post-infection but a significantly lower titer was detected after an additional 14 days of observation as compared to the Luc dsRNA-injected ticks. Mortality in both groups did not show any significant difference.ConclusionOur results suggest that longicin P4 has in vitro antiviral activity against LGTV but not against a non-enveloped virus such as adenovirus. Likewise, though most cationic antimicrobial peptides like longicin act directly on target membranes, the exact mechanism of membrane targeting of longicin P4 in enveloped viruses, such as LGTV, requires further investigation. Lastly, while the in vitro virucidal capacity of longicin P4 was confirmed in this study, the role of the endogenous tick longicin in the antiviral defense of H. longicornis against LGTV still remains to be demonstrated.Electronic supplementary materialThe online version of this article (doi:10.1186/s13071-016-1344-5) contains supplementary material, which is available to authorized users.

Structural Basis of Membrane Targeting by the Innate Immunity Adaptor TIRAP

Toll-like receptors (TLRs) are responsible for early immune system recognition and response to infection. Pathogen-activated TLRs trigger a cytoplasmic signaling cascade though adaptor proteins, starting with the TIR domain-containing adaptor protein (TIRAP). TIRAP contains a C-terminal TIR domain, which is responsible for association with TLRs and other adaptors including the myeloid differentiation primary response gene88 (MyD88) protein. Membrane recruitment of TIRAP is mediated by its N-terminal phosphoinositide (PIP)-binding motif (PBM). Upon ligand-mediated activation, TLRs are recruited to the PIP-enriched regions where TIRAP resides. At these sites, TIRAP recruits MyD88 to the membrane to activate the TLR signaling pathway. To understand the mechanism of TIRAP membrane targeting and the basis for its regulation, we functionally and structurally characterized TIRAP and its PBM using biophysical approaches. We show that TIRAP's PBM adopts a helical conformation in the presence of dodecylphosphocholine (DPC) micelles. NMR studies revealed that TIRAP PBM binds PIPs through its two conserved basic termini following a fast-exchange regime with moderate affinity. Mutation of these two basic regions abolishes PIP binding without distorting the helical structure of the peptide. Interestingly, association of TIRAP PBM to PIPs requires the acyl chains of the lipid. The solution NMR structure of TIRAP PBM exhibits a central relatively hydrophobic helix surrounded by the flexible N- and C-termini. Paramagnetic studies indicate that the helix is close to the micelle core, whereas the N- and C- termini are located on the micellar surface. Nuclear spin relaxation experiments indicate that this N- and C-termini of TIRAP PBM become more ordered when bound to PIPs. Thus, we propose that the central helix in PBM is responsible for membrane insertion, whereas the two sets of basic residues interact with PIPs to stabilize TIRAP's membrane interaction.

A conserved polybasic domain mediates plasma membrane targeting of Lgl and its regulation by hypoxia.

Lethal giant larvae (Lgl) plays essential and conserved functions in regulating both cell polarity and tumorigenesis in Drosophila melanogaster and vertebrates. It is well recognized that plasma membrane (PM) or cell cortex localization is crucial for Lgl function in vivo, but its membrane-targeting mechanisms remain poorly understood. Here, we discovered that hypoxia acutely and reversibly inhibits Lgl PM targeting through a posttranslational mechanism that is independent of the well-characterized atypical protein kinase C (aPKC) or Aurora kinase-mediated phosphorylations. Instead, we identified an evolutionarily conserved polybasic (PB) domain that targets Lgl to the PM via electrostatic binding to membrane phosphatidylinositol phosphates. Such PB domain-mediated PM targeting is inhibited by hypoxia, which reduces inositol phospholipid levels on the PM through adenosine triphosphate depletion. Moreover, Lgl PB domain contains all the identified phosphorylation sites of aPKC and Aurora kinases, providing a molecular mechanism by which phosphorylations neutralize the positive charges on the PB domain to inhibit Lgl PM targeting.

A novel antimicrobial peptide, scolopendin, from Scolopendra subspinipes mutilans and its microbicidal mechanism.

A novel antimicrobial peptide (AMP) was identified from the centipede Scolopendra subspinipes mutilans by RNA sequencing, and the amino acid sequences predicted from the sequenced mRNAs were compared with those of known AMPs. We named this peptide scolopendin, according to its origin, and investigated the molecular mechanisms underlying its antimicrobial activity. Our findings showed that scolopendin had antimicrobial activity against several pathogenic microorganisms, but did not produce hemolysis of human erythrocytes. In addition, disturbances in the cell membrane potential, induction of potassium release from the cytosol, and increased membrane permeability of the microbes Candida albicans and Escherichia coli O157 were detected by the use of 3,3'-dipropylthiacarbocyanine iodide [DiSC3(5)] dye, potassium leakage assay, and propidium iodide influx assay, respectively, following scolopendin treatment. Further evidence to support the membrane-targeted action of scolopendin was obtained using artificial liposomes as models of the cell membrane. Use of calcein and FITC-labeled dextran leakage assays from scolopendin-treated giant unilamellar vesicles and large unilamellar vesicles showed that scolopendin has a pore-forming action on microbial membrane, with an estimated pore radius of 2.3-3.3nm. In conclusion, scolopendin is a novel and potent AMP with a membrane-targeted mechanism of action.

NMR structure of the myristylated feline immunodeficiency virus matrix protein.

Membrane targeting by the Gag proteins of the human immunodeficiency viruses (HIV types-1 and -2) is mediated by Gag’s N-terminally myristylated matrix (MA) domain and is dependent on cellular phosphatidylinositol-4,5-bisphosphate [PI(4,5)P2]. To determine if other lentiviruses employ a similar membrane targeting mechanism, we initiated studies of the feline immunodeficiency virus (FIV), a widespread feline pathogen with potential utility for development of human therapeutics. Bacterial co-translational myristylation was facilitated by mutation of two amino acids near the amino-terminus of the protein (Q5A/G6S; myrMAQ5A/G6S). These substitutions did not affect virus assembly or release from transfected cells. NMR studies revealed that the myristyl group is buried within a hydrophobic pocket in a manner that is structurally similar to that observed for the myristylated HIV-1 protein. Comparisons with a recent crystal structure of the unmyristylated FIV protein [myr(-)MA] indicate that only small changes in helix orientation are required to accommodate the sequestered myr group. Depletion of PI(4,5)P2 from the plasma membrane of FIV-infected CRFK cells inhibited production of FIV particles, indicating that, like HIV, FIV hijacks the PI(4,5)P2 cellular signaling system to direct intracellular Gag trafficking during virus assembly.

Structural Basis of Phosphoinositide (PIP) Recognition by the TIRAP PIP-Binding Motif

Toll-like receptors (TLRs) provide early immune system recognition and response to infection. TLRs activated by pathogens consequentially initiate a cytoplasmic signaling cascade though adaptor proteins, the first being the modular TIR domain-containing adaptor protein (TIRAP). TIRAP contains a C-terminal TIR domain, which is responsible for association with TLRs and other adaptors including the myeloid differentiation primary response gene88 (MyD88) protein. Membrane recruitment of TIRAP is mediated by its N-terminal PIP-binding motif (PBM). Upon ligand-mediated activation, TLRs are recruited to the PIP-enriched regions where TIRAP resides. At these sites, TIRAP recruits MyD88 to the membrane by bridging MyD88 to activate the TLR signaling pathway. To understand the mechanism of membrane targeting of TIRAP and the basis for its regulation, we functionally and structurally characterized its PBM using experimental and computational studies. TIRAP PBM adopts a folded conformation in membrane mimics, such as dodecylphosphocholine micelles, and binds PIPs. Structural rearrangements of TIRAP PBM were influenced by membrane interaction, with monodispersed PIPs inducing helical structure in the peptide. In contrast, monodispersed phosphatidylinositol and inositol trisphosphate did not promote structural changes in TIRAP PBM. NMR spectra reveal that TIRAP PBM binds PIPs in a fast exchange regime with a moderate affinity through two conserved basic regions. Solution NMR structure of TIRAP PBM shows a central short helix, and paramagnetic studies indicate that this region is close to the micelle core. Molecular dynamics simulations studies indicated that TIRAP PBM diffused to and interacted with a model membrane composed of palmitoyl oleoyl phosphatidylcholine and phosphatidylinositol 4,5-bisphosphate. Thus, we propose that two sets of basic residues contact both the head group and acyl chains of PIPs, whereas the central helix is responsible for membrane insertion.

Fungicidal mechanisms of the antimicrobial peptide Bac8c

Bac8c (RIWVIWRR-NH2) is an analogue peptide derived through complete substitution analysis of the linear bovine host defense peptide variant Bac2A. In the present study, the antifungal mechanism of Bac8c against pathogenic fungi was investigated, with a particular focus on the effects of Bac8c on the cytoplasmic membrane. We used bis-(1,3-dibutylbarbituric acid) trimethine oxonol [DiBAC4(3)] staining and 3,3’-dipropylthiacarbocyanine iodide [DiSC3(5)] assays to show that Bac8c induced disturbances in the membrane potential of Candida albicans. An increase in membrane permeability and suppression of cell wall regeneration were also observed in Bac8c-treated C. albicans. We studied the effects of Bac8c treatment on model membranes to elucidate its antifungal mechanism. Using calcein and FITC-labeled dextran leakage assays from Bac8c-treated large unilamellar vesicles (LUVs) and giant unilamellar vesicles (GUVs), we found that Bac8c has a pore-forming action on fungal membranes, with an estimated pore radius of between 2.3 and 3.3nm. A membrane-targeted mechanism of action was also supported by the observation of potassium release from the cytosol of Bac8c-treated C. albicans. These results indicate that Bac8c is considered as a potential candidate to develop a novel antimicrobial agent because of its low-cost production characteristics and high antimicrobial activity via its ability to induce membrane perturbations in fungi.

An antifungal mechanism of curcumin lies in membrane-targeted action within Candida albicans.

The aim of this study is to investigate the antifungal mechanism of curcumin. This polyphenolic compound has been used traditionally in Asia for medicinal, culinary, and other purposes. Although antifungal effect of curcumin has been reported, this is the first study for its mode of action underlying disruption of plasma membrane in Candida albicans. The leakage of potassium ion from the fungal cytosol and dissipation in membrane potential was detected by bis-(1,3-dibutylbarbituric acid)trimethine oxonol [DiBAC4 ] staining. We also investigated an increase in membrane permeability in curcumin-treated C. albicans with influx of propidium iodide assay. Fluorescence analysis with 1,6-diphenyl-1,3,5-hexatriene supported the membrane-targeted mechanism of action indicating membrane disruption. On the basis of these results, we studied the effects of curcumin treatment on model membrane to elucidate its antifungal mechanism. Using calcein leakage assays from curcumin-treated large unilamellar vesicles and giant unilamellar vesicles, we found that curcumin has membrane-active mechanism inducing leakage of intracellular component through the flappy membrane. Therefore, this study suggests that curcumin exerts antifungal activity via inducing disruption of fungal plasma membrane.

Intracellular Membrane Association of the Aplysia cAMP Phosphodiesterase Long and Short Forms via Different Targeting Mechanisms

Phosphodiesterases (PDEs) play key roles in cAMP compartmentalization, which is required for intracellular signaling processes, through specific subcellular targeting. Previously, we showed that the long and short forms of Aplysia PDE4 (ApPDE4), which are localized to the membranes of distinct subcellular organelles, play key roles in 5-hydroxytryptamine-induced synaptic facilitation in Aplysia sensory and motor synapses. However, the molecular mechanism of the isoform-specific distinct membrane targeting was not clear. In this study, we further investigated the molecular mechanism of the membrane targeting of the ApPDE4 long and short forms. We found that the membrane targeting of the long form was mediated by hydrophobic interactions, mainly via 16 amino acids at the N-terminal region, whereas the short form was targeted solely to the plasma membrane, mainly by nonspecific electrostatic interactions between their N termini and the negatively charged lipids such as the phosphatidylinositol polyphosphates PI4P and PI(4,5)P2, which are embedded in the inner leaflet of the plasma membrane. Moreover, oligomerization of the long or short form by interaction of their respective upstream conserved region domains, UCR1 and UCR2, enhanced their plasma membrane targeting. These results suggest that the long and short forms of ApPDE4 are distinctly targeted to intracellular membranes through their direct association with the membranes via hydrophobic and electrostatic interactions, respectively.

Structure of the type VI secretion phospholipase effector Tle1 provides insight into its hydrolysis and membrane targeting.

A diverse superfamily of phospholipases consisting of the type VI lipase effectors Tle1-Tle5 secreted by the bacterial type VI secretion system (T6SS) have recently been identified as antibacterial effectors that hydrolyze membrane phospholipids. These effectors show no significant homology to known lipases, and their mechanism of membrane targeting and hydrolysis of phospholipids remains unknown. Here, the crystal structure of Tle1 (∼96.5 kDa) from Pseudomonas aeruginosa refined to 2.0 Å resolution is reported, representing the first structure of this superfamily. Its overall structure can be divided into two distinct parts, the phospholipase catalytic module and the putative membrane-anchoring module; this arrangement has not previously been observed in known lipase structures. The phospholipase catalytic module has a canonical α/β-hydrolase fold and mutation of any residue in the Ser-Asp-His catalytic triad abolishes its toxicity. The putative membrane-anchoring module adopts an open conformation composed of three amphipathic domains, and its partial folds are similar to those of several periplasmic or membrane proteins. A cell-toxicity assay revealed that the putative membrane-anchoring module is critical to Tle1 antibacterial activity. A molecular-dynamics (MD) simulation system in which the putative membrane-anchoring module embedded into a bilayer was stable over 50 ns. These structure-function studies provide insight into the hydrolysis and membrane-targeting process of the unique phospholipase Tle1.

Unraveling molecular mechanism of cell migration using novel perturbation tools

Complexity in signaling networks is often derived from co-opting particular sets of molecules for multiple operations. Understanding how cells achieve such sophisticated processing using a finite set of molecules within a confined space - what we call the "signaling paradox"- is critical to cell biology and bioengineering as well as the emerging field of synthetic biology. We have recently developed a series of chemical-molecular tools that allow for inducible, quick-onset and specific perturbation of various signaling molecules. The present technique has been employed to unravel several important, previously unresolved questions regarding the regulatory mechanisms of potassium ion channels, the membrane targeting mechanisms of small GTPases and positive feedback machinery in neutrophil migration. Using this novel technique in conjunction with conventional fluorescence imaging and biochemical analysis, we are currently further dissecting intricate signaling networks in living cells. Ultimately, we will generate completely orthogonal machinery in cells to achieve existing, as well as novel, cellular functions. Our synthetic, multidisciplinary approach will elucidate the signaling paradox in cells created by nature.

The role of the hypervariable C-terminal domain in Rab GTPases membrane targeting

Intracellular membrane trafficking requires correct and specific localization of Rab GTPases. The hypervariable C-terminal domain (HVD) of Rabs is posttranslationally modified by isoprenyl moieties that enable membrane association. A model asserting HVD-directed targeting has been contested in previous studies, but the role of the Rab HVD and the mechanism of Rab membrane targeting remain elusive. To elucidate the function of the HVD, we have substituted this region with an unnatural polyethylenglycol (PEG) linker by using oxime ligation. The PEGylated Rab proteins undergo normal prenylation, underlining the unique ability of the Rab prenylation machinery to process the Rab family with diverse C-terminal sequences. Through localization studies and functional analyses of semisynthetic PEGylated Rab1, Rab5, Rab7, and Rab35 proteins, we demonstrate that the role of the HVD of Rabs in membrane targeting is more complex than previously understood. The HVD of Rab1 and Rab5 is dispensable for membrane targeting and appears to function simply as a linker between the GTPase domain and the membrane. The N-terminal residues of the Rab7 HVD are important for late endosomal/lysosomal localization, apparently due to their involvement in interaction with the Rab7 effector Rab-interacting lysosomal protein. The C-terminal polybasic cluster of the Rab35 HVD is essential for plasma membrane (PM) targeting, presumably because of the electrostatic interaction with negatively charged lipids on the PM. Our findings suggest that Rab membrane targeting is dictated by a complex mechanism involving GEFs, GAPs, effectors, and C-terminal interaction with membranes to varying extents, and possibly other binding partners.

Molecular Basis of Phosphatidylinositol 4,5-Bisphosphate Recognition by the Adaptor Protein Tirap

Toll-like receptors (TLRs) provide a mechanism for host defense by activating innate immune responses. Activated TLRs [e.g., by bacterial lipopolysaccharide (LPS)] dimerize, and interact with adaptor proteins through their cytosolic TIR domains to trigger a signaling cascade that ultimately leads to the expression of proteins involved in pro-inflammatory responses. One such adaptor protein is the TIR domain-containing adaptor protein (TIRAP), which contains an N-terminal phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2)-binding region that is required for plasma membrane targeting and a C-terminal TIR domain, which mediates myeloid differentiation primary response gene 88 (MyD88) association. Upon ligand binding, the LPS-binding protein TLR4 is proposed to be recruited to PtdIns(4,5)P2-rich regions where TIRAP resides. At these sites, TIRAP recruits MyD88 to the plasma membrane via TIR-TIR domain interactions; thus TIRAP bridges MyD88 binding to activated TLR4. A conserved short stretch at the N-terminus of TIRAP has been shown to be sufficient to target the protein to the plasma membrane. We show that this region, which we named the PtdIns(4,5)P2 binding motif (PBM), folds in dodecylphosphocholine (DPC) micelles, and binds PtdIns(4,5)P2. The solution structure of the DPC-associated TIRAP PBM indicates that the peptide adopts a helical structure in micelles. Furthermore, we demonstrate that PtdIns(4,5)P2 can induce a helical structure in TIRAP PBM, a feature observed in other PtdIns(4,5)P2 modules. NMR data indicates that TIRAP PBM binds PIP2 in a fast exchange regime with a moderate affinity through two conserved basic regions. Thus, these studies will provide a basis for understanding the mechanism of TIRAP's membrane targeting and recruitment of TIR-containing proteins required to trigger pro-inflammatory signaling.

Molecular mechanism of junctional membrane-targeting of cardiac and skeletal muscle L-type calcium channels.

横紋筋のL 型カルシウムチャネル（LTCC）は，筋収縮において電気信号をカルシウム信号に変換する重要な役割を担っている．LTCC と筋小胞体膜上に存在するリアノジン受容体（RyR）は，形質膜と筋小胞体膜が近接する結合膜構造で，機能的複合体を形成している．LTCC が結合膜構造に集積してクラスターを形成することは，筋の正常な興奮収縮連関に必要不可欠であるが，その詳細なメカニズムには不明な点が多く残されている．近年，骨格筋芽細胞株であるGLTを用いた研究により，骨格筋型LTCC の結合膜への局在化には，LTCC のポアを形成するCaV1.1 サブユニットのC末端配列が重要な役割を果たしていることが明らかになった．一方，成熟心筋細胞の培養が困難なため，心筋型LTCC の結合膜局在化の分子機構については，全く解析が進んでいなかった．我々はGLT 細胞を用いて，心筋LTCC の結合膜局在化機構を明らかにするための検討を行った．心筋LTCC のCaV1.2 サブユニットの各細胞内ドメインを，結合膜非局在性である神経型CaV2.1 サブユニットに置換した変異体を作製し，GLT 細胞に発現させた．その結果，近位C 末端の配列が結合膜への集積に必須であることが明らかになった．さらに，この部位に変異を導入すると，局在のみでなく，興奮収縮連関の効率も大きく低下することが分かった．また，LTCC の結合膜局在化に関係する分子として，ジャンクトフィリンに注目した検討も行った．C2C12 細胞のジャンクトフィリンをノックダウンすると，LTCC の結合膜への局在の阻害や，カルシウムトランジェントの減弱が認められた．また，共免疫沈降法によってLTCC のCaV1.1 サブユニットとジャンクトフィリンが結合していることが確認された．これらの結果は，ジャンクトフィリンがLTCC の正常な局在や機能に関与することを示唆している．

The C2 domains of granuphilin are high-affinity sensors for plasma membrane lipids

Membrane-targeting proteins are crucial components of many cell signaling pathways, including the secretion of insulin. Granuphilin, also known as synaptotagmin-like protein 4, functions in tethering secretory vesicles to the plasma membrane prior to exocytosis. Granuphilin docks to insulin secretory vesicles through interaction of its N-terminal domain with vesicular Rab proteins; however, the mechanisms of granuphilin plasma membrane targeting and release are less clear. Granuphilin contains two C2 domains, C2A and C2B, that interact with the plasma membrane lipid phosphatidylinositol-(4,5)-bisphosphate [PI(4,5)P2]. The goal of this study was to determine membrane-binding mechanisms, affinities, and kinetics of both granuphilin C2 domains using fluorescence spectroscopic techniques. Results indicate that both C2A and C2B bind anionic lipids in a Ca2+-independent manner. The C2A domain binds liposomes containing a physiological mixture of lipids including 2% PI(4,5)P2 or PI(3,4,5)P3 with high affinity (apparent Kd,PIPx of 2–5nM), and binds nonspecifically with moderate affinity to anionic liposomes lacking phosphatidylinositol phosphate (PIPx) lipids. The C2B domain binds with sub-micromolar affinity to liposomes containing PI(4,5)P2 but does not have a measurable affinity for background anionic lipids. Both domains can be competed away from their target lipids by the soluble PIPx analog inositol-(1,2,3,4,5,6)-hexakisphosphate (IP6), which is a positive regulator of insulin secretion. Potential roles of these interactions in the docking and release of granuphilin from the plasma membrane are discussed.

Membrane-Targeting Approaches for Enhanced Cancer Cell Destruction with Irreversible Electroporation

Irreversible electroporation (IRE) is a promising technology to treat local malignant cancer using short, high-voltage electric pulses. Unfortunately, in vivo studies show that IRE suffers from an inability to destroy large volumes of cancer tissue without introduction of cytotoxic agents and/or increasing the applied electrical dose to dangerous levels. This research will address this limitation by leveraging membrane-targeting mechanisms that increase lethal membrane permeabilization. Methods that directly modify membrane properties or change the pulse delivery timing are proposed that do not rely on cytotoxic agents. This work shows that significant enhancement (67-75% more cell destruction in vitro and >100% treatment volume increase in vivo) can be achieved using membrane-targeting approaches for IRE cancer destruction. The methods introduced are surfactants (i.e., DMSO) and pulse timing which are low cost, non-toxic, and easy to be incorporated into existing clinical use. Moreover, when needed, these methods can also be combined with electrochemotherapy to further enhance IRE treatment efficacy.

Quantitative Control of Protein S-Palmitoylation Regulates Meiotic Entry in Fission Yeast

Author SummaryPalmitoylation is a fatty acid modification, which increases the hydrophobicity of proteins, known to control the membrane trafficking and function of many proteins important for cellular homeostasis and disease. Being a reversible process, palmitoylation is proposed to be a major cellular regulator. While changes in global protein palmitoylation patterns are often associated with different physiological states, the mechanisms that regulate this posttranslational modification and their functional consequences have been elusive. Using improved methods for protein palmitoylation analysis in the genetically tractable fission yeast Schizosaccharomyces pombe, we discovered that the global protein palmitoylation pattern is significantly altered and correlates with entry into meiosis, a major developmental transition. We further showed that the precise level of a palmitoylating enzyme is important to shape the subset of lipid-modified proteins and influence the start of this cellular transition. As palmitoylated proteins are involved in eukaryotic cellular pathways, our results provide a crucial insight into how the quantitative control of palmitoylating enzymes may impact human physiology and disease.