Global analysis of dynamic changes in lipid raft proteins during T-cell activation

Lipid rafts are considered as specialized microdomains within the plasma membrane with unique lipid compositions different from surrounding membranes. Following T-cell receptor (TCR) stimulation, lipid rafts assemble in T-cell/antigen-presenting cell (APC) contact site known as the immunological synapse, inner leaflets of which serve as activation or docking sites for downstream signaling components. To understand the signaling events occurring in lipid rafts, we globally analyzed dynamic changes in lipid raft proteins during TCR/CD28 costimulation using 2-D fluorescence difference gel electrophoresis. We detected multiple spots whose intensities were enhanced after costimulation, and identified proteins in these spots by PMF. Identified proteins include Src family tyrosine kinases, tyrosine phosphatase, phosphatidylinositol 3-kinase (PI3-kinase), actin-binding proteins, and regulators for small GTPases. Of particular interest, a number of pleckstrin homology (PH) domain-containing proteins were identified. Biochemical and histochemical analyses confirmed the translocation of these proteins from cytosol to lipid rafts. We also demonstrated that these proteins assembled at the T-cell/APC interface. These results indicate the efficacy of our system to systematically analyze dynamics of lipid raft proteins during extracellular stimulation.     

Methods of analysis for chemicals that promote/disrupt cellular signaling.

Methods of analysis were presented for chemicals that promote or disrupt cellular signaling pathways. The developed analytical methods are based not only on receptor binding, but also on the following known molecular-level processes involved in signal transduction along signaling pathways, reconstituted in vitro or taken in part in living cells. The methods were discussed in relation to receptor binding assay and/or bioassay. Examples include: (1) Insulin signaling pathways; (1-i) Chemical selectivity of agonists for insulin signaling pathways based on agonist-induced phosphorylation of a target peptide; (1-ii) An SPR-based screening method for agonist selectivity for insulin signaling pathways based on the binding of phosphotyrosine to its specific binding protein; (1-iii) A fluorescent indicator for tyrosine phosphorylation-based insulin signaling pathways; (2) An optical method for evaluating ion selectivity for calcium signaling pathways in the cell; (3) Assay and screening of chemicals that disrupt cellular signaling pathways, potential endocrine disruptors in particular; (4) Protein conformational changes, and (5) A screening method for antigen-specific IgE using mast cells, based on intracellular calcium signaling.     

A human mutant CD4 molecule resistant to HIV-1 binding restores helper T-lymphocyte functions in murine CD4-deficient mice

CD4 is a cell surface glycoprotein that acts as a co-receptor for the T cell antigen receptor by binding to a non-polymorphic portion of MHC molecules. CD4 also functions as a receptor for human immunodeficiency virus type-I (HIV-1) because the viral envelope glycoprotein gp120 binds to CD4 with a high affinity. We have previously demonstrated that introduction of mutations into CD4 abolished the binding of gp120 and prevented HIV-1 from entering cells and spreading. However, whether introduction of such mutations into CD4 causes decreased binding to MHC and loss of function is yet to be determined. We generated transgenic mouse lines by injecting a mutant human CD4 (muthCD4) gene under a murine CD4 enhancer/promoter to ensure tissue and stage specific expression. To exclude the influence of endogenous murine CD4, transgenic mice were crossed with murine CD4-targeted mice to produce muthCD4 transgenic mice lacking endogenous CD4 (muthCD4TG/KO mice). In these mice, T lymphocytes expressing muthCD4 expanded and matured in the thymus and were present in the spleen and lymph nodes. They also activated B cells to mount an antibody response to a T-dependent antigen. The results from this study suggest that a human variant of CD4 modified to be resistant to HIV-1 binding can rescue the signaling for T cell development in the thymus in vivo, having helper T cell functions. Thus, further characterization of muthCD4 molecules should open the way to new HIV treatment modalities.     

Small multivalent architectures mimicking homotrimers of the TNF superfamily member CD40L: delineating the relationship between structure and effector function

Synthetic multivalent ligands, owing to the presence of multiple copies of a recognition motif attached to a central scaffold, can mediate clustering of cell surface receptors and thereby function as effector molecules. This paper dissects the relationship between structure and effector function of synthetic multivalent ligands targeting CD40, a cell surface receptor of the tumor necrosis factor receptor (TNF-R) superfamily. Triggering CD40 signaling in vivo can be used to enhance immunity against intracellular pathogens or tumors. A series of multimeric molecules has been prepared by systematically varying the shape and the valency of the central scaffold, the nature and the length of the linker as well as the sequence of the receptor binding motif. The data reported here (i) suggest that radial distribution of CD40-binding units and C3-symmetry are preferred for optimal binding to CD40 and signaling, (ii) underscore the importance of choosing an appropriate linker to connect the receptor binding motif to the central scaffold, and (iii) show the versatility of planar cyclic alpha- and beta-peptides as templates for the design of CD40L mimetics. In particular, the (Ahx)3-B trimeric scaffold-linker combination equally accommodated binding elements derived from distinct CD40L hot-spot regions including AA" loop and beta-strand E. The use of miniCD40Ls such as those reported here is complementary to other approaches (recombinant ligands, agonistic anti-receptor antibodies) and may find interesting therapeutic applications. Furthermore, the results disclosed in this paper provide the basis for future design of other TNF family member mimetics.     

TERS Detection of αVβ3 Integrins in Intact Cell Membranes

Integrins are important membrane receptors that form focal adhesions with the extracellular matrix and are transmembrane signaling proteins. We demonstrate that nanoparticles functionalized with c‐RGDfC ligands bind to intact cell membranes and selectively enhance the amino acid signals of the integrin receptor when coupled with tip‐enhanced Raman scattering (TERS) detection. Controlling the plasmonic interaction between the functionalized nanoparticle and the TERS tip provides a clear Raman signal from α_Vβ₃ integrins in the cell membrane that matches the signal of the purified integrin receptor. Random aggregation of nanoparticles on the cell does not provide the same spectral information. Chemical characterization of membrane receptors in intact cellular membranes is important for understanding membrane signaling and drug targeting. These results provide a new method to investigate the chemical interactions associated with ligand binding to membrane receptors in cells.     

Cross-linking of CD4 induces cytoskeletal association of CD4 and p56lck

A membrane glycoprotein CD4 functions as a co-receptor of a T lymphocyte. The co-receptor function has been attributed to a protein tyrosine kinase, p56lck, which is activated upon CD4 binding to MHC molecule. In this study, we present evidences that one of the pathways through which CD4 transmits its signal is cytoskeleton association of p56lck tyrosine kinase as well as CD4 itself. Cytoskeletal association of both proteins is inhibited by a tyrosine kinase inhibitor, genistein, indicating that tyrosine protein kinase activation is important for cytoskeletal association of CD4 and p56lck. Cytoskeletal association of these proteins by CD4 cross-linking is not affected by inhibitors of protein kinase C nor PI3-kinase. Taken together, these results suggest that CD4 cross-linking activates a tyrosine kinase which then induces the simultaneous association of CD4 and p56lck with cytoskeleton.     

Functionalized surface arrays for spatial targeting of immune cell signaling

The effect of surface topography and chemistry on cellular response is of fundamental importance, especially where living systems encounter device surfaces as in medical implants, tissue engineering, and cell-based sensors. To understand these biological processes on surfaces, there is a widespread interest in tailored surface-active materials produced by a combination of surface chemistry coupled to advanced patterning processes. We utilize self-assembled monolayers (SAMs) as molecular templates with submicrometer-scale spatial resolution to engage and cluster IgE receptors on rat basophilic leukemia (RBL) mast cells. Bioactive templates consisted of gold arrays on silicon with patterns from 1 mum down to 45 nm. These gold arrays served as molecular tethering sites, enabling covalent binding of functionalized self-assembled monolayers of alkanethiols. The free ends of the monolayers were functionalized with 2,4-dinitrophenyl(DNP)-caproate-based ligands which interact specifically with anti-DNP IgE bound to its high affinity cell surface receptor, FcepsilonRI on RBL mast cells. Present results on structures 1 mum down to 600 nm in size indicate that these ligand-immobilized patterned arrays can function as a powerful tool for visualization and systematic characterization of cell membrane involvement in IgE receptor-mediated immune cell signaling.     

Characterization of the transmembrane serine receptor by capillary zone electrophoresis

Summary Capillary zone electrophoresis (CZE) was applied to the characterization of the transmembrane serine receptor in biosynthetic samples. The serine receptor, otherwise known as Tsr (taxis to serine and repellents), is a ∼ 60,000 Dalton intrinsic membrane protein whose periplasmic domain (ligand binding domain) reversibly binds the amino acid serine. In general, the electrophoresis of intrinsic membrane proteins is difficult due to severe solubility problems and adsorption which occurs during the electrophoretic run. This is due to the tendency of these types of proteins to undergo aggregation, self-aggregation and precipitation in aqueous environments. The addition of percentage levels of the surfactant, sodium dodecyl sulfate (SDS), to a tetraborate run buffer was shown to be effective both in enhancing the solubility of intact Tsr and in preventing the adsorption of intact Tsr to the fused-silica capillary wall during electrophoretic analysis. Critical separation parameters such as run buffer concentration, surfactant concentration and surfactant type were optimized to give the best separation profiles.     

Characterization of the early stages of programmed cell death in maize root cells by using comet assay and the combination of cell electrophoresis with annexin binding

An emerging topic in plant biology is whether plant cells display similar elements of programmed cell death (PCD) as animal cells do. We have studied cell death in maize roots exposed to cold stress by using fluorescence microscopy, terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end labeling (TUNEL), DNA gel electrophoresis, single cell gel electrophoresis (SCGE), cell electrophoresis, and annexin binding techniques. The results showed that cell death in maize root cells triggered by cold stress was accompanied by a subset of features characteristic of animal PCD such as nuclear condensation and fragmentation, and oligonucleosomal DNA fragmentation. In addition to DNA laddering and TUNEL positivity, a "comet" pattern indicative of DNA breakage appeared as short as after one day of treatment. The maize root cell PCD process was also accompanied by an increase in negative surface charge of the dying cells due to exposure of phosphatiolylserine (PS) from inner to outer membrane. After annexin binding, however, the enhanced electrophoretic mobility (EPM) of the dying cells decreased nearly to normal values. This result suggests that the combination between cell electrophoresis and annexin binding provides a quantitative method for monitoring PS exposure during plant PCD.     

Multi-transmembrane protein K15 of Kaposi's sarcoma-associated herpesvirus targets Lyn kinase in the membrane raft and induces NFAT/AP1 activities

Viral proteins of gamma-2 herpesviruses, such as LMP2A of Epstein Barr virus (EBV) and Tip of herpesvirus saimiri (HVS) dysregulate lymphocyte signaling by interacting with Src family kinases. K15 open reading frame of Kaposi's sarcoma associated herpesvirus (KSHV), located at the right end of the viral genome, encodes several splicing variants differing in numbers of transmembrane domains. Previously, we demonstrated that the cytoplasmic tail of the K15 protein interfered with B cell receptor signal transduction to cellular tyrosine phosphorylation and calcium mobilization. However, the detailed mechanism underlying this phenomenon was not understood. In the C-terminal cytoplasmic region of K15, putative binding domains for Src-SH2 and -SH3 were identified. In this study, we attempted to characterize these modular elements and cellular binding protein(s) by GST pull down and co-immunoprecipitation assays. These studies revealed that K15 interacted with the major B cell tyrosine kinase Lyn. In vitro kinase and transient co-expression assays showed that the expression of K15 protein resulted in activation of Lyn kinase activity. In addition, GST pull down assay suggested that the SH2 domain of Lyn alone was necessary for interaction with the C-terminal SH2B (YEEV) of K15, but the addition of Lyn SH3 to the SH2 domain increases the binding affinity to K15 protein. The data from luciferase assays indicate that K15 expression in BJAB cells induced NFAT and AP1 activities. The tyrosine residue in the C-terminal end of K15 required for the Lyn interaction appeared to be essential for NFAT/AP1 activation, highlighting the significance of the C-terminal SH2B of K15 as a modular element in interfering with B lymphocyte signaling through interaction with Lyn kinase.     

Molecular Alliance—From Orthosteric and Allosteric Ligands to Dualsteric/Bitopic Agonists at G Protein Coupled Receptors

Cell‐membrane‐spanning G protein coupled receptors (GPCRs) belong to the most important therapeutic target structures. Endogenous transmitters bind from the outer side of the membrane to the “orthosteric” binding site either deep in the binding pocket or at the extracellular N‐terminal end of the receptor protein. Exogenous modulators that utilize a different, “allosteric”, binding site unveil a pathway to receptor subtype‐selectivity. However, receptor activation through the orthosteric area is often more powerful. Recently there has been evidence that orthosteric/allosteric, in other words “dualsteric”, hybrid compounds unite subtype selectivity and receptor activation. These “bitopic” modulators channelreceptor activation and subsequent intracellular signaling into a subset of possible routes. This concept offers access to GPCR modulators with an unprecedented receptor‐subtype and signaling selectivity profile and, as a consequence, to drugs with fewer side effects.     

High-affinity small molecule-phospholipid complex formation: binding of siramesine to phosphatidic acid

Siramesine (SRM) is a sigma-2 receptor agonist which has been recently shown to inhibit growth of cancer cells. Fluorescence spectroscopy experiments revealed two distinct binding sites for this drug in phospholipid membranes. More specifically, acidic phospholipids retain siramesine on the bilayer surface due to a high-affinity interaction, reaching saturation at an apparent 1:1 drug-acidic phospholipid stoichiometry, where after the drug penetrates into the hydrocarbon core of the membrane. This behavior was confirmed using Langmuir films. Of the anionic phospholipids, the highest affinity, comparable to the affinities for the binding of small molecule ligands to proteins, was measured for phosphatidic acid (PA, mole fraction of X(PA) = 0.2 in phosphatidylcholine vesicles), yielding a molecular partition coefficient of 240 +/- 80 x 10(6). An MD simulation on the siramesine:PA interaction was in agreement with the above data. Taking into account the key role of PA as a signaling molecule promoting cell growth our results suggest a new paradigm for the development of anticancer drugs, viz. design of small molecules specifically scavenging phospholipids involved in the signaling cascades controlling cell behavior.     

Single-molecule force spectroscopy study of interaction between transforming growth factor beta1 and its receptor in living cells

Transforming growth factor beta1 (TGF-beta1) regulates many important cellular processes such as cell proliferation, differentiation, and apoptosis, etc. Its signaling is initiated by binding to and bringing together TGF-beta type II receptor (TbetaRII) and type I receptor (TbetaRI). However, it is not fully understood how the TGF-beta1 ligand-receptor interaction occurs in living cells and what is the molecular mechanism of the signaling complex TGF-beta1/TbetaRII/TbetaRI formation. In this study, we have investigated the interaction between TGF-beta1 and its receptors in living cells with single-molecule force spectroscopy for the first time. By positioning TGF-beta1-modified atomic force microscope (AFM) tips on the cells expressing fluorescent protein tagged TGF-beta receptors, the living-cell force measurement was realized with a combined fluorescence microscope and AFM. We found that coexpression of TbetaRI with TbetaRII enhanced the binding force of TGF-beta1 with its receptors, whereas the expressed TbetaRI itself exhibited no binding affinity to TGF-beta1. Moreover, the unbinding dynamics of TGF-beta1/TbetaRII and TGF-beta1/TbetaRI/TbetaRII were investigated with dynamic force spectroscopy under different AFM loading rates. The dissociation rate constants of TGF-beta1 with its receptors as well as other parameters characterizing their dissociation pathways were obtained. The results suggested a more stable binding of TGF-beta1 with the receptor after TbetaRI is recruited and the important contribution of TbetaRI to the signaling complex formation during TGF-beta1 signaling.     

Enhanced integrin mediated signaling and cell cycle progression on fibronectin mimetic peptide amphiphile monolayers

In recent years, a variety of biomimetic constructs have emerged which mimic the bioactive sequences found in the natural extracellular matrix (ECM) proteins such as fibronectin (FN) that promote cell adhesion as well as proliferation on artificially functionalized interfaces. Much interest lies in investigating the ability of the ECM mimetic materials in regulating a number of vital cell functions including differentiation, gene expression, migration, and proliferation. A peptide amphiphile PR_b containing both the cell adhesive GRGDSP and synergistic PHSRN peptide sequences was developed in our group that was shown to support enhanced cell proliferation and ECM FN secretion as compared to GRGDSP and FN functionalized interfaces. In this study, we have investigated the binding affinity of the PR_b peptide ligand with the FN cell surface receptor, the α(5)β(1) integrin. We compared PR_b functionalized surfaces with FN and BSA coated surfaces and GRGDSP functionalized surfaces in terms of promoting intracellular signaling cascades that are essential for enhanced cellular activity. Specifically, we studied the phosphorylation of focal adhesion kinase (FAK) at tyrosine residues Y397 and Y576 and the formation of cyclin D1, both of which are intracellular markers of integrin mediated attachment of cells, signaling pathways, and progression of cell cycle. FAK and cyclin D1 encourage enhanced cell proliferation, differentiation, and gene expression. Our results show that the PR_b peptide ligand has a specific and strong binding affinity for the α(5)β(1) integrin with a dissociation constant of 76.3 ± 6.3 nM. The PR_b peptide ligands supported enhanced FAK phosphorylation activity and increased cyclin D1 formation as compared to the widely used GRGDSP ligand, the native protein FN (positive control), and BSA nonadhesive surfaces (negative control). These results encourage the use of the FN mimetic PR_b peptide in functionalizing biomaterials for potential tissue engineering and therapeutic applications.     

Activation of G-protein-coupled receptors in cell-derived plasma membranes supported on porous beads

G-protein-coupled receptors (GPCRs) are ubiquitous mediators of signal transduction across cell membranes and constitute a very important class of therapeutic targets. In order to study the complex biochemical signaling network coupling to the intracellular side of GPCRs, it is necessary to engineer and control the downstream signaling components, which is difficult to realize in living cells. We have developed a bioanalytical platform enabling the study of GPCRs in their native membrane transferred inside-out from live cells to lectin-coated beads, with both membrane sides of the receptor being accessible for molecular interactions. Using heterologously expressed adenosine A(2A) receptor carrying a yellow fluorescent protein, we showed that the tethered membranes comprised fully functional receptors in terms of ligand and G protein binding. The interactions between the different signaling partners during the formation and subsequent dissociation of the ternary signaling complex on single beads could be observed in real time using multicolor fluorescence microscopy. This approach of tethering inside-out native membranes accessible from both sides is straightforward and readily applied to other transmembrane proteins. It represents a generic platform suitable for ensemble as well as single-molecule measurements to investigate signaling processes at plasma membranes.     

Interaction between the mouse homologue of CD99 and its ligand PILR as a mechanism of T cell receptor-independent thymocyte apoptosis

Here, we show that the interaction between two membrane proteins, the mouse homologue of CD99 (designated D4) and its ligand, paired immunoglobulin-like type 2 receptor (PILR), is one of the major mechanisms of thymocyte apoptosis. Using the polymeric fusion protein of PILR and IgG1 (PILR-Ig), we demonstrated that D4 ligation in the absence of T cell receptor (TCR) engagement leads to the induction of apoptosis, mainly at the double-positive stage of thymocytes. This was further confirmed by a blocking study in which blocking the interaction between D4 and PILR by soluble D4 protein led to reduced apoptosis in the fetal thymic organ culture with wild type and TCRα^-/- mice. Furthermore, the dissection of intracellular signaling pathway demonstrated that D4 cross-linking led to caspase activation without any change in mitochondrial membrane potential. Based on these data, we propose a mechanism for thymocyte depletion in which the interaction between D4 and PILR delivers an active signal.     

A diverse set of oligomeric class II MHC-peptide complexes for probing T-cell receptor interactions

BACKGROUND: T-cells are activated by engagement of their clonotypic cell surface receptors with peptide complexes of major histocompatibility complex (MHC) proteins, in a poorly understood process that involves receptor clustering on the membrane surface. Few tools are available to study the molecular mechanisms responsible for initiation of activation processes in T-cells. RESULTS: A topologically diverse set of oligomers of the human MHC protein HLA-DR1, varying in size from dimers to tetramers, was produced by varying the location of an introduced cysteine residue and the number and spacing of sulfhydryl-reactive groups carried on novel and commercially available cross-linking reagents. Fluorescent probes incorporated into the cross-linking reagents facilitated measurement of oligomer binding to the T-cell surface. Oligomeric MHC-peptide complexes, including a variety of MHC dimers, trimers and tetramers, bound to T-cells and initiated T-cell activation processes in an antigen-specific manner. CONCLUSION: T-cell receptor dimerization on the cell surface is sufficient to initiate intracellular signaling processes, as a variety of MHC-peptide dimers differing in intramolecular spacing and orientation were each able to trigger early T-cell activation events. The relative binding affinities within a homologous series of MHC-peptide oligomers suggest that T-cell receptors may rearrange in the plane of the membrane concurrent with oligomer binding.     

The effect of magnetic nanoparticles containing hyaluronic acid and methotrexate on the expression of genes involved in apoptosis and metastasis in A549 lung cancer cell lines

Lung cancer has been shown to be resistant to treatment with some chemotherapy drugs due to epithelial-mesenchymal transmission (EMT). Because the rate of cytotoxicity and induction of apoptosis by methotrexate (MTX) is negligible in A549 lung cancer cells, a CD44 positive cell line, we decided to synthesize magnetic nanoparticles (MNPs) containing hyaluronic acid (HA) and MTX to evaluate the effect of CD44 receptor targeting on the expression of genes involved in apoptosis. The TNF genes can modulate the expression of CD44 and implicate carcinogenesis and metastases. Therefore, inhibition of the TNF gene and study of its interaction with the CD44 receptor can determine the success of a treatment method. The results of the MTT assay confirmed that the MNPs-HA-MTX offered better cellular cytotoxic effects on cell viability than free MTX. The real-time PCR test also showed that the Bak1/Bclx ratio was 52.5 times higher than the control. On the other hand, the expression of the TNF gene was severely reduced, which could be due to the binding of HA-moiety of the MNPs-HA-MTX to the receptor and endocytosis. All the results gave us hope that we could increase the effectiveness of methotrexate in lung cancer by targeting the CD44 receptor.     

Synergistic induction of cancer cell migration regulated by Gβγ and phosphatidylinositol 3-kinase

Phosphatidylinositol 3-kinase (PI3K) is essential for both G protein-coupled receptor (GPCR)- and receptor tyrosine kinase (RTK)-mediated cancer cell migration. Here, we have shown that maximum migration is achieved by full activation of phosphatidylinositol 3,4,5-trisphosphate-dependent Rac exchanger 1 (P-Rex1) in the presence of Gβγ and PI3K signaling pathways. Lysophosphatidic acid (LPA)- induced migration was higher than that of epidermal growth factor (EGF)-induced migration; however, LPA-induced activation of Akt was lower than that stimulated by EGF. LPA-induced migration was partially blocked by either Gβγ or RTK inhibitor and completely blocked by both inhibitors. LPA-induced migration was synergistically increased in the presence of EGF and vice versa. In correlation with these results, sphingosine-1-phosphate (S1P)-induced migration was also synergistically induced in the presence of insulin-like growth factor-1 (IGF-1). Finally, silencing of P-Rex1 abolished the synergism in migration as well as in Rac activation. Moreover, synergistic activation of MMP-2 and cancer cell invasion was attenuated by silencing of P-Rex1. Given these results, we suggest that P-Rex1 requires both Gβγ and PI3K signaling pathways for synergistic activation of Rac, thereby inducing maximum cancer cell migration and invasion.     

Analysis of leukocyte membrane protein interactions using protein microarrays

Background  

Protein microarrays represent an emerging class of proteomic tools to investigate multiple protein-protein interactions in parallel. A sufficient proportion of immobilized proteins must maintain an active conformation and an orientation that allows for the sensitive and specific detection of antibody and ligand binding. In order to establish protein array technology for the characterization of the weak interactions between leukocyte membrane proteins, we selected the human leukocyte membrane protein CD200 (OX2) and its cell surface receptor (hCD200R) as a model system. As antibody-antigen reactions are generally of higher affinity than receptor-ligand binding, we first analyzed the reactivity of monoclonal antibodies (mAb) to normal and mutant forms of immobilized CD200R.