Cytoskeleton-mediated death receptor and ligand concentration in lipid rafts forms apoptosis-promoting clusters in cancer chemotherapy

While investigating the mechanism of action of the novel antitumor drug Aplidin, we have discovered a potent and novel cell-killing mechanism that involves the formation of Fas/CD95-driven scaffolds in membrane raft clusters housing death receptors and apoptosis-related molecules. Fas, tumor necrosis factor-receptor 1, and tumor necrosis factor-related apoptosis-inducing ligand receptor 2/death receptor 5 were clustered into lipid rafts in leukemic Jurkat cells following Aplidin treatment, the presence of Fas being essential for apoptosis. Preformed membrane-bound Fas ligand (FasL) as well as downstream signaling molecules, including Fas-associated death domain-containing protein, procaspase-8, procaspase-10, c-Jun amino-terminal kinase, and Bid, were also translocated into lipid rafts, connecting death receptor extrinsic and mitochondrial intrinsic apoptotic pathways. Blocking Fas/FasL interaction partially inhibited Aplidin-induced apoptosis. Aplidin was rapidly incorporated into membrane rafts, and drug uptake was inhibited by lipid raft disruption. Actin-linking proteins ezrin, moesin, RhoA, and RhoGDI were conveyed into Fas-enriched rafts in drug-treated leukemic cells. Disruption of lipid rafts and interference with actin cytoskeleton prevented Fas clustering and apoptosis. Thus, Aplidin-induced apoptosis involves Fas activation in both a FasL-independent way and, following Fas/FasL interaction, an autocrine way through the concentration of Fas, membrane-bound FasL, and signaling molecules in membrane rafts. These data indicate a major role of actin cytoskeleton in the formation of Fas caps and highlight the crucial role of the clusters of apoptotic signaling molecule-enriched rafts in apoptosis, acting as concentrators of death receptors and downstream signaling molecules and as the linchpin from which a potent death signal is launched.     

Formation of lipid raft nanodomains in homogeneous ternary lipid mixture of POPC/DPSM/cholesterol: Theoretical insights

Lipid rafts, in biological membranes, are cholesterol-rich nanodomains that regulate many protein activities and cellular processes. Understanding the formation of the lipid-raft nanodomains helps us elucidate many complex interactions in the cell. In this study, the formation of lipid-raft nanodomains in a ternary palmitoyl-oleoyl-phosphatidylcholine/stearoyl-sphingomyelin/cholesterol (POPC/DPSM/Chol) lipid mixture, the most realistic surrogate model for biological membranes, has been successfully observed for the first time in-silico using microsecond timescale molecular dynamics simulations. The model reveals the formation of cholesterol-induced nanodomains with raft-like characteristics and their underlying mechanism: the cholesterol molecules segregate themselves into cholesterol nanodomains and then enrich the cholesterol-rich domain with sphingomyelin molecules to form a lipid raft thanks to the weak bonding of cholesterol with sphingomyelin. Besides, it is found that the increase in cholesterol concentration enhances the biophysical properties (e.g., bilayer thickness, area per lipid headgroup, and order parameter) of the lipid raft nanodomains. Such findings suggest that the POPC/DPSM/Chol bilayer is a suitable model to fundamentally extend the nanodomain evolution to investigate their lifetime and kinetics as well as to study protein-lipid interaction, protein-protein interaction, and selection of therapeutic molecules in the presence of lipid rafts.     

跨膜蛋白CD3ε与磷脂酰肌醇二磷酸在脂筏域体系中蛋白质-脂质相互作用的粗粒化模拟研究

细胞膜局部区域可形成富含饱和脂质、胆固醇、鞘脂的脂筏域作为其信号转导调控平台。传统实验手段在研究脂筏及其功能时受到系统复杂度高及区域结构瞬时性强等制约。近年来,分子动力学模拟技术为细胞膜的组织原则提供了重要的理论支撑,从简单的单一组分模型到多组分系统转变,最终形成了越来越多的细胞膜仿真模型。其中,粗粒化模拟由于其简化模型,可大副拓展模拟体系的复杂程度与模拟时间,在细胞膜以及蛋白质-脂质相互作用相关研究中得到了广泛应用。本文采用MARTINI粗粒化力场模拟,构建了一种含有阴离子脂质磷脂酰肌醇二磷酸(phosphatidylinositol diphosphate, PIP2)的混合膜体系。模拟结果表明,该体系在适当温度及饱和度条件下,能自发分层形成脂筏域;膜厚度、膜组分分布、膜组分流动性等多种参数均表明,脂筏结构形成且符合其结构特征;少量PIP2添加不影响分层特性且PIP2对脂筏具有显著亲和性。此外,利用该模型以跨膜信号蛋白CD3ε为例研究了脂筏域体系中蛋白质-脂质相互作用。结果表明,PIP2-CD3ε胞内区相互作用可能是脂筏招募CD3ε的驱动力,且该过程可受钙离子调控。本工作体现了粗粒化模拟在仿真膜相关研究中的巨大优势及良好应用前景。     

Suprachiasmatic nucleus circadian oscillatory protein,a novel binding partner of K-Ras in the membrane rafts,negatively regulates MAPK pathway

Suprachiasmatic nucleus circadian oscillatory protein (SCOP) is a member of the leucine-rich repeat (LRR)-containing protein family. In addition to circadian expression in the rat hypothalamic suprachiasmatic nucleus, SCOP is constitutively expressed in neurons throughout the rat brain. Here we found that a substantial amount of SCOP was localized in the brain membrane rafts, in which only K-Ras was abundant among Ras isoforms. SCOP interacted directly through its LRR domain with a subset of K-Ras in the guanine nucleotide-free form that was present in the raft fraction. This interaction interfered with the binding of added guanine nucleotide to K-Ras in vitro. A negative regulatory role of SCOP for K-Ras function was examined in PC12 cell lines stably overexpressing SCOP or its deletion mutants. Overexpression of full-length SCOP markedly down-regulated ERK1/ERK2 activation induced by depolarization or phorbol ester stimulation, and this inhibitory effect of overexpressed SCOP was dependent on its LRR domain. These results strongly suggest that SCOP negatively regulates K-Ras signaling in the membrane rafts, identifying a novel mechanism for regulation of the Ras-MAPK pathway.     

TRIP: a novel double stranded RNA binding protein which interacts with the leucine rich repeat of flightless I.

A northwestern screen of a CHO-K1 cell line cDNA library with radiolabelled HIV-1 TAR RNA identified a novel TAR RNA interacting protein, TRIP. The human trip cDNA was also cloned and its expression is induced by phorbol esters. The N-terminus of TRIP shows high homology to the coiled coil domain of FLAP, a protein which binds the leucine-rich repeat (LRR) of Flightless I (FLI) and the interaction of TRIP with the FLI LRR has been confirmed in vitro . TRIP does not bind single stranded DNA or RNA significantly and binds double stranded DNA weakly. In contrast, TRIP binds double stranded RNA with high affinity and two molecules of TRIP bind the TAR stem. The RNA binding domain has been identified and encompasses a lysine-rich motif. A TRIP-GFP fusion is localised in the cytoplasm and excluded from the nucleus. FLI has a C-terminal gelsolin-like domain which binds actin and therefore the association of TRIP with the FLI LRR may provide a link between the actin cytoskeleton and RNA in mammalian cells.     

The 213-amino-acid leucine-rich repeat region of the Listeria monocytogenes InlB protein is sufficient for entry into mammalian cells, stimulation of PI 3-kinase and membrane ruffling

The Listeria monocytogenes InlB protein is a 630-amino-acid surface protein that mediates entry of the bacterium into a wide variety of cell types, including hepatocytes, fibroblasts and epithelial cells such as Vero, HEp-2 and HeLa cells. Invasion stimulates host proteins tyrosine phosphorylation, PI 3-kinase activity and rearrangements in the actin cytoskeleton. We previously showed that InlB is sufficient for entry of InlB-coated latex beads into cells and recent results indicate that purified InlB can stimulate PI 3-kinase activity and is thus the first bacterial agonist of this lipid kinase. In this study, we identified the region of InlB responsible for entry and stimulation of signal transduction events. Eight monoclonal antibodies directed against InlB were raised and, of those, five inhibited bacterial entry. These five antibodies recognized epitopes within the leucine-rich repeat (LRR) region and/or the inter-repeat (IR) region. InlB-staphylococcal protein A (SPA) fusion proteins and recombinant InlB derivatives were generated and tested for their capacity to mediate entry into cultured mammalian cells. All the InlB derivatives that carried the amino-terminal 213-amino-acid LRR region conferred invasiveness to the normally non-invasive bacterium L. innocua or to inert latex beads and the corresponding purified polypeptides inhibited bacterial entry. In addition, the 213-amino-acid LRR region was able to stimulate PI 3-kinase activity and changes in the actin cytoskeleton (membrane ruffling). These properties were not detected with purified internalin, another invasion protein of L. monocytogenes that displays LRRs similar to those of InlB. Taken together, these results show that the first 213 amino acids of InlB are critical for its specific properties.     

Attribution of the various inhibitory actions of the streptococcal inhibitor of complement (SIC) to regions within the molecule

Some strains of Streptococcus pyogenes secrete a virulence factor called the streptococcal inhibitor of complement (SIC) function. SIC is a polyfunctional protein that interacts with a number of host proteins and peptides, especially with those that are involved in host defense systems. In addition to inhibiting the complement-mediated lysis of cells, SIC inhibits lysozyme, secretory leukocyte proteinase inhibitor, and beta-defensins. SIC also binds to proteins associated with the cytoskeleton and thereby may cause cytoskeletal derangement. The SIC molecule has three distinct structural domains constituting the N-proximal short repeat region (SRR), the central long repeat region (LRR), and the C-proximal proline-rich region (PRR). To map various functions to the structural domains, we have analyzed recombinant subclones expressing various parts of SIC and elastase-generated discrete fragments of SIC for binding to various ligands and for determining their biological properties. The results demonstrate the following. (a) SRR alone was sufficient to confer inhibition of complement function. (b) Anti-defensin and anti-lysozyme activities were mapped to the SRR plus LRR. (c) The LRR plus PRR harbored ezrin binding activity.     

Distinct domains in the ARC region of the potato resistance protein Rx mediate LRR binding and inhibition of activation

Plant nucleotide binding and leucine-rich repeat (NB-LRR) proteins contain a region of homology known as the ARC domain located between the NB and LRR domains. Structural modeling suggests that the ARC region can be subdivided into ARC1 and ARC2 domains. We have used the potato (Solanum tuberosum) Rx protein, which confers resistance to Potato virus X (PVX), to investigate the function of the ARC region. We demonstrate that the ARC1 domain is required for binding of the Rx N terminus to the LRR domain. Domain-swap experiments with Rx and a homologous disease resistance gene, Gpa2, showed that PVX recognition localized to the C-terminal half of the LRR domain. However, inappropriate pairings of LRR and ARC2 domains resulted in autoactive molecules. Thus, the ARC2 domain is required to condition an autoinhibited state in the absence of elicitor as well as for the subsequent elicitor-induced activation. Our data suggest that the ARC region, through its interaction with the LRR, translates elicitor-induced modulations of the C terminus into a signal initiation event. Furthermore, we demonstrate that physical disruption of the LRR-ARC interaction is not required for signal initiation. We propose instead that this activity can lead to multiple rounds of elicitor recognition, providing a means of signal amplification.     

Expression, purification and preliminary biochemical and structural characterization of the leucine rich repeat namesake domain of leucine rich repeat kinase 2

Mutations in leucine-rich repeat kinase 2 (LRRK2) are the most common cause of familial Parkinson's disease. Much research effort has been directed towards the catalytic core region of LRRK2 composed of GTPase (ROC, Ras of complex proteins) and kinase domains and a connecting COR (C-terminus of ROC) domain. In contrast, the precise functions of the protein-protein interaction domains, such as the leucine-rich repeat (LRR) domain, are not known. In the present study, we modeled the LRRK2 LRR domain (LRR(LRRK2)) using a template assembly approach, revealing the presence of 14 LRRs. Next, we focused on the expression and purification of LRR(LRRK2) in Escherichia coli. Buffer optimization revealed that the protein requires the presence of a zwitterionic detergent, namely Empigen BB, during solubilization and the subsequent purification and characterization steps. This indicates that the detergent captures the hydrophobic surface patches of LRR(LRRK2) thereby suppressing its aggregation. Circular dichroism (CD) spectroscopy measured 18% α-helices and 21% β-sheets, consistent with predictions from the homology model. Size exclusion chromatography (SEC) and dynamic light scattering measurements showed the presence of a single species, with a Stokes radius corresponding to the model dimensions of a protein monomer. Furthermore, no obvious LRR(LRRK2) multimerization was detected via cross-linking studies. Finally, the LRR(LRRK2) clinical mutations did not influence LRR(LRRK2) secondary, tertiary or quaternary structure as determined via SEC and CD spectroscopy. We therefore conclude that these mutations are likely to affect putative LRR(LRRK2) inter- and intramolecular interactions.     

A novel role of microtubular cytoskeleton in the dynamics of caspase-dependent Fas/CD95 death receptor complexes during apoptosis

The activation of cysteine-aspartic proteases or caspases and the dynamic arrangement of cytoskeletal components are crucial during apoptosis. Here we describe the fate of Fas downstream of the FasL-induced internalization step, including formation of caspase-dependent SDS-stable Fas complexes, which is mediated by cytoskeleton integrity. We show, in particular, that following FasL treatment, the Fas lower aggregate complex can be co-immunoprecipitated with tubulin and an active form of caspase-8 and that this interaction contributes to the propagation of FasL-induced cell death. The importance of cytoskeletal components during FasL-induced apoptosis is highlighted by our detection of a pool of microtubule-associated Fas complexes.     

Role of GD3-CLIPR-59 Association in Lymphoblastoid T Cell Apoptosis Triggered by CD95/Fas

We previously found that a directional movement of the raft component GD3 towards mitochondria, by its association with microtubules, was mandatory to late apoptogenic events triggered by CD95/Fas. Since CLIPR-59, CLIP-170-related protein, has recently been identified as a microtubule binding protein associated with lipid rafts, we analyzed the role of GD3-CLIPR-59 association in lymphoblastoid T cell apoptosis triggered by CD95/Fas. To test whether CLIPR-59 could play a role at the raft-microtubule junction, we performed a series of experiments by using immunoelectron microscopy, static or flow cytometry and biochemical analyses. We first assessed the presence of CLIPR-59 molecule in lymphoblastoid T cells (CEM). Then, we demonstrated that GD3-microtubule interaction occurs via CLIPR-59 and takes place at early time points after CD95/Fas ligation, preceding the association GD3-tubulin. GD3-CLIPR-59 association was demonstrated by fluorescence resonance energy transfer (FRET) analysis. The key role of CLIPR-59 in this dynamic process was clarified by the observation that silencing CLIPR-59 by siRNA affected the kinetics of GD3-tubulin association, spreading of GD3 towards mitochondria and apoptosis execution. We find that CLIPR-59 may act as a typical chaperone, allowing a prompt interaction between tubulin and the raft component GD3 during cell apoptosis triggered by CD95/Fas. On the basis of the suggested role of lipid rafts in conveying pro-apoptotic signals these results disclose new perspectives in the understanding of the mechanisms by which raft-mediated pro-apoptotic signals can directionally reach their target, i.e. the mitochondria, and trigger apoptosis execution.     

Palmitoylation is required for efficient Fas cell death signaling

Localization of the death receptor Fas to specialized membrane microdomains is crucial to Fas-mediated cell death signaling. Here, we report that the post-translational modification of Fas by palmitoylation at the membrane proximal cysteine residue in the cytoplasmic region is the targeting signal for Fas localization to lipid rafts, as demonstrated in both cell-free and living cell systems. Palmitoylation is required for the redistribution of Fas to actin cytoskeleton-linked rafts upon Fas stimulation and for the raft-dependent, ezrin-mediated cytoskeleton association, which is necessary for the efficient Fas receptor internalization, death-inducing signaling complex assembly and subsequent caspase cascade leading to cell death.     

Crystal structure of a plant leucine rich repeat protein with two island domains

Leucine rich repeat(LRR)domain,characterized by a repetitive sequence pattern rich in leucine residues,is a universal protein-protein interaction motif present in all life forms.LRR repeats interrupted by sequences of 30 70 residues(termed island domain,ID)have been found in some plant LRR receptor-like kinases(RLKs)and animal Toll-like receptors(TLR7-9).Recent studies provide insight into how a single ID is structurally integrated into an LRR protein.However,structural information on an LRR protein with two IDs is lacking.The receptor-like protein kinase 2(RPK2)is an LRR-RLK and has important roles in controlling plant growth and development by perception and transduction of hormone signal.Here we present the crystal structure of the extracellular LRR domain of RPK2(RPK2-LRR)containing two IDs from Arabidopsis.The structure reveals that both of the IDs are helical and located at the central region of the single RPK2-LRR solenoid.One of them binds to the inner surface of the solenoid,whereas the other one mainly interacts with the lateral side.Unexpectedly,a long loop immediately following the N-terminal capping domain of RPK2-LRR is presented toward and sandwiched between the two IDs,further stabilizing their embedding to the LRR solenoid.A potential ligand binding site formed by the two IDs and the solenoid is located at the C-terminal side of RPK2-LRR.The structural information of RPK2-LRR broadens our understanding toward the large family of LRR proteins and provides insight into RPK2-mediated signaling.     

Membrane rafts: a potential gateway for bacterial entry into host cells

Pathogenic bacteria have developed various mechanisms to evade host immune defense systems. Invasion of pathogenic bacteria requires interaction of the pathogen with host receptors, followed by activation of signal transduction pathways and rearrangement of the cytoskeleton to facilitate bacterial entry. Numerous bacteria exploit specialized plasma membrane microdomains, commonly called membrane rafts, which are rich in cholesterol, sphingolipids and a special set of signaling molecules which allow entry to host cells and establishment of a protected niche within the host. This review focuses on the current understanding of the raft hypothesis and the means by which pathogenic bacteria subvert membrane microdomains to promote infection.     

Palmitoylation of human FasL modulates its cell death-inducing function

Fas ligand (FasL) is a transmembrane protein that regulates cell death in Fas-bearing cells. FasL-mediated cell death is essential for immune system homeostasis and the elimination of viral or transformed cells. Because of its potent cytotoxic activity, FasL expression at the cell surface is tightly regulated, for example, via processing by ADAM10 and SPPL2a generating soluble FasL and the intracellular fragments APL (ADAM10-processed FasL form) and SPA (SPPL2a-processed APL). In this study, we report that FasL processing by ADAM10 counteracts Fas-mediated cell death and is strictly regulated by membrane localization, interactions and modifications of FasL. According to our observations, FasL processing occurs preferentially within cholesterol and sphingolipid-rich nanodomains (rafts) where efficient Fas–FasL contact occurs, Fas receptor and FasL interaction is also required for efficient FasL processing, and FasL palmitoylation, which occurs within its transmembrane domain, is critical for efficient FasL-mediated killing and FasL processing.     

The internal region leucine-rich repeat 6 of decorin interacts with low density lipoprotein receptor-related protein-1, modulates transforming growth factor (TGF)-β-dependent signaling, and inhibits TGF-β-dependent fibrotic response in skeletal muscles

Decorin is a small proteoglycan, composed of 12 leucine-rich repeats (LRRs) that modulates the activity of transforming growth factor type β (TGF-β) and other growth factors, and thereby influences proliferation and differentiation in a wide array of physiological and pathological processes, such as fibrosis, in several tissues and organs. Previously we described two novel modulators of the TGF-β-dependent signaling pathway: LDL receptor-related protein (LRP-1) and decorin. Here we have determined the regions in decorin that are responsible for interaction with LRP-1 and are involved in TGF-β-dependent binding and signaling. Specifically, we used decorin deletion mutants, as well as peptides derived from internal LRR regions, to determine the LRRs responsible for these decorin functions. Our results indicate that LRR6 and LRR5 participate in the interaction with LRP-1 and TGF-β as well as in its dependent signaling. Furthermore, the internal region (LRR6i), composed of 11 amino acids, is responsible for decorin binding to LRP-1 and subsequent TGF-β-dependent signaling. Furthermore, using an in vivo approach, we also demonstrate that the LRR6 region of decorin can inhibit TGF-β mediated action in response to skeletal muscle injury.     

The death-inducing signalling complex is recruited to lipid rafts in Fas-induced apoptosis

Membrane microdomains known as lipid rafts have been shown recently to be involved in Fas signalling and apoptosis in T and B cell lines. Here, we have investigated further the role of lipid rafts in Fas-induced apoptosis in non-transformed human CD4 T cells. We show that Fas-induced apoptosis in CD4 T cells was inhibited by the lipid raft disrupter methyl-beta-cyclodextrin. When lipid rafts were isolated from control and Fas ligand treated cells, we found that a small proportion of Fas was present in the raft fraction in untreated cells and that this was greatly increased upon Fas ligation. The other components of the Death Inducing Signalling Complex (DISC), FADD, and procaspase 8, were also present at higher levels in the raft fraction isolated from Fas ligand treated cells. We conclude that formation of the DISC occurs in lipid rafts and that these membrane microdomains are required for efficient Fas signalling and apoptosis.     

Receptor Displacement in the Cell Membrane by Hydrodynamic Force Amplification through Nanoparticles

We introduce an intrinsically multiplexed and easy to implement method to apply an external force to a biomolecule and thus probe its interaction with a second biomolecule or, more generally, its environment (for example, the cell membrane). We take advantage of the hydrodynamic interaction with a controlled fluid flow within a microfluidic channel to apply a force. By labeling the biomolecule with a nanoparticle that acts as a kite and increases the hydrodynamic interaction with the fluid, the drag induced by convection becomes important. We use this approach to track the motion of single membrane receptors, the Clostridium perfringens ε-toxin (CPεT) receptors that are confined in lipid raft platforms, and probe their interaction with the environment. Under external force, we observe displacements over distances up to 10 times the confining domain diameter due to elastic deformation of a barrier and return to the initial position after the flow is stopped. Receptors can also jump over such barriers. Analysis of the receptor motion characteristics before, during, and after a force is applied via the flow indicates that the receptors are displaced together with their confining raft platform. Experiments before and after incubation with latrunculin B reveal that the barriers are part of the actin cytoskeleton and have an average spring constant of 2.5 ± 0.6 pN/μm before vs. 0.6 ± 0.2 pN/μm after partial actin depolymerization. Our data, in combination with our previous work demonstrating that the ε-toxin receptor confinement is not influenced by the cytoskeleton, imply that it is the raft platform and its constituents rather than the receptor itself that encounters and deforms the barriers formed by the actin cytoskeleton.     

Cutting edge: negative regulation of immune synapse formation by anchoring lipid raft to cytoskeleton through Cbp-EBP50-ERM assembly.

Ag recognition by T lymphocytes induces immune synapse formation and recruitment of signaling molecules into a lipid raft. Cbp/PAG is a Csk-associated membrane adapter protein exclusively localized in a lipid raft. We identified NHERF/EBP50 as a Cbp-binding molecule, which connects the membrane raft and cytoskeleton by binding to both Cbp through its PDZ domain and ezrin-radixin-moesin through the C terminus. Overexpression of Cbp reduced the mobility of the raft on the cell surface of unstimulated T cells and prevented synapse formation and subsequent T cell activation, whereas a mutant incapable of EBP50 binding restored both synapse formation and activation. These results suggest that anchoring of lipid raft to the cytoskeleton through Cbp-EBP50-ezrin-radixin-moesin assembly regulates membrane dynamism for synapse formation and T cell activation.     

Interaction of Daxx, a Fas binding protein, with sentrin and Ubc9

Sentrin is a ubiquitin-like protein that can covalently modify cellular proteins, and is a Fas binding protein that protects cells against anti-Fas induced cell death. However, the mechanism by which sentrin exerts its effect upon Fas-mediated apoptosis is not well known. Thus, this study examined the interaction of sentrin with Daxx. Sentrin interacted with Daxx but not with FADD when analyzed by yeast two-hybrid assay. In vitro translated Daxx bound to GST-sentrin fusion protein. FLAG-sentrin fusion protein was also coimmunoprecipitated with Daxx in BOSC23 cells. Also, Daxx interacted with Ubc9, an essential protein as a key conjugating enzyme. Amino acids 625-740 of Daxx, known as Fas binding region, was also mapped as sentrin and Ubc9 binding region. Colocalization of Fas, sentrin, and Ubc9 binding regions suggests the importance of that region upon the regulation of Daxx. Our data also demonstrated that sentrin could homooligomerize by protein-protein interaction.