Inhibits Membrane Binding Research Articles

Dynamics of membrane tubulation coupled with fission by a two-component module

Membrane tubulation coupled with fission (MTCF) is a widespread phenomenon but mechanisms for their coordination remain unclear, partly because of the lack of assays to monitor dynamics of membrane tubulation and subsequent fission. Using polymer cushioned bilayer islands, we analyze the membrane tubulator Bridging Integrator 1 (BIN1) mixed with the fission catalyst dynamin2 (Dyn2). Our results reveal this mixture to constitute a minimal two-component module that demonstrates MTCF. MTCF is an emergent property and arises because BIN1 facilitates recruitment but inhibits membrane binding of Dyn2 in a dose-dependent manner. MTCF is therefore apparent only at high Dyn2 to BIN1 ratios. Because of their mutual involvement in T-tubules biogenesis, mutations in BIN1 and Dyn2 are associated with centronuclear myopathies and our analysis links the pathology with aberrant MTCF. Together, our results establish cushioned bilayer islands as a facile template for the analysis of membrane tubulation and inform of mechanisms that coordinate MTCF.

A highly conserved glutamic acid in ALFY inhibits membrane binding to aid in aggregate clearance.

Autophagy-linked FYVE protein (ALFY) is a large, multidomain protein involved in the degradation of protein aggregates by selective autophagy. The C-terminal FYVE domain of ALFY has been shown to bind phosphatidylinositol 3-phosphate (PI(3)P); however, ALFY only partially colocalizes with other FYVE domains in cells. Thus, we asked if the FYVE domain of ALFY has distinct membrane binding properties compared to other FYVE domains and whether these properties might affect its function in vivo. We found that the FYVE domain of ALFY binds weakly to PI(3)P containing membranes in vitro. This weak binding is the result of a highly conserved glutamic acid within the membrane insertion loop in the FYVE domain of ALFY that is not present in any other human FYVE domain. In addition, not only does this glutamic acid reduce binding to membranes in vitro and inhibits its targeting to membranes in vivo, but it is also important for the ability of ALFY to clear protein aggregates.

Structure of Membrane-Bound Huntingtin Exon 1 Reveals Membrane Interaction and Aggregation Mechanisms

Huntington's disease is caused by a polyQ expansion in the first exon of huntingtin (Httex1). Membrane interaction of huntingtin is of physiological and pathological relevance. Using electron paramagnetic resonance and Overhauser dynamic nuclear polarization, we find that the N-terminal residues 3-13 of wild-type Httex1(Q25) form a membrane-bound, amphipathic α helix. This helix is positioned in the interfacial region, where it is sensitive to membrane curvature and electrostatic interactions with head-group charges. Residues 14-22, which contain the first five residues of the polyQ region, are in a transition region that remains in the interfacial region without taking up a stable, α-helical structure. The remaining C-terminal portion is solvent exposed. The phosphomimetic S13D/S16D mutations, which are known to protect from toxicity, inhibit membrane binding and attenuate membrane-mediated aggregation of mutant Httex1(Q46) due to electrostatic repulsion. Targeting the N-terminal membrane anchor using post-translational modifications or specific binders could be a potential means to reduce aggregation and toxicity invivo.

Toward Understanding the Mechanism of Calcium-Inhibited Membrane Binding of the SLP-2 C2A Domain

Synaptotagmin-like proteins (Slps) are membrane-binding proteins that play key roles in trafficking of large dense-core vesicles for exocytosis. In particular, Slp-2 is responsible for docking hormone secretory vesicles to sites of exocytosis in pancreatic alpha cells and other cell types. Like all Slp family members, Slp-2 contains an N-terminal Slp-homology domain, a linker region of undefined structure, and two C-terminal membrane-binding C2 domains (C2A and C2B). The C2A domain of Slp-2 has been shown to have a unique response to Ca2+ ions, in that it binds anionic liposome membranes in the absence of Ca2+ but dissociates upon addition of sufficient Ca2+. This Ca2+ dependence contrasts with most other C2 domains, which bind lipid membranes in either a Ca2+-dependent or Ca2+-independent fashion. To investigate the mechanism of this unusual Ca2+ inhibition, a series of biophysical and spectroscopic measurements has been completed. First, the affinity of the well-purified C2A domain for liposomes containing different compositions of key anionic lipids was measured using steady-state and stopped-flow fluorescence spectroscopy. Next, the effect of Ca2+ on C2A domain binding to each of the liposome compositions was measured. It was found that Ca2+ does inhibit membrane binding, albeit at significantly higher concentrations than previously reported. Although the protein-liposome affinity varies by >10-fold among the different lipid compositions tested, the Ca2+ concentration required for inhibition varies relatively little, suggesting a nonspecific mechanism. Current efforts are directed toward quantifying Ca2+ and other metal binding affinities using a luminescence-based Tb3+ displacement assay, and measuring effects of Ca2+ and lipids on protein structure using EPR and NMR.

Cellular uptake of proMMP-2:TIMP-2 complexes by the endocytic receptor megalin/LRP-2

Matrix metalloproteinases (MMPs) are regulated at multiple transcriptional and post-transcriptional levels, among which receptor-mediated endocytic clearance. We previously showed that low-density lipoprotein receptor-related protein-1 (LRP-1) mediates the clearance of a complex between the zymogen form of MMP-2 (proMMP-2) and tissue inhibitor of metalloproteinases, TIMP-2, in HT1080 human fibrosarcoma cells. Here we show that, in BN16 rat yolk sac cells, proMMP-2:TIMP-2 complex is endocytosed through a distinct LRP member, megalin/LRP-2. Addition of receptor-associated protein (RAP), a natural LRP antagonist, caused accumulation of endogenous proMMP-2 and TIMP-2 in conditioned media. Incubation with RAP also inhibited membrane binding and cellular uptake of exogenous iodinated proMMP-2:TIMP-2. Moreover, antibodies against megalin/LRP-2, but not against LRP-1, inhibited binding of proMMP-2:TIMP-2 to BN16 cell surface. BIAcore analysis confirmed direct interaction between the complex and megalin/LRP-2. Conditional renal invalidation of megalin/LRP-2 in mice resulted in accumulation of proMMP-2 and TIMP-2 in their urine, highlighting the physiological relevance of the binding. We conclude that megalin/LRP-2 can efficiently mediate cell-surface binding and endocytosis of proMMP-2:TIMP-2 complex. Therefore megalin/LRP-2 can be considered as a new actor in regulation of MMP-2 activity, an enzyme crucially involved in many pathological processes.

Phosphorylation Weakens but Does Not Inhibit Membrane Binding and Clustering of K-Ras4B.

K-Ras4B is one of the most frequently mutated Ras isoforms in cancer. The signaling activity of K-Ras4B depends on its localization to the plasma membrane (PM), which is mainly mediated by its polybasic farnesylated C-terminus. On top of the constitutive cycles that maintain the PM enrichment of K-Ras4B, conditional phosphorylation at Ser181 located within this motif has been found to be involved in regulating K-Ras4B's cell distribution and signaling activity. However, discordant observations have undermined our understanding of the role this phosphorylation plays. Here, we report an efficient strategy for producing K-Ras4B simultaneously bearing phosphate, farnesyl, and methyl modifications on a preparative scale, a very useful in vitro system when used in concert with model biomembranes. By using this system, we determined that phosphorylation at Ser181 does not fully inhibit membrane binding and clustering of K-Ras4B but reduces its membrane binding affinity, depending on membrane fluidity. In addition, phosphorylated K-Ras4B maintains tight association with its cytosolic shuttle protein PDEδ. After delivering K-Ras4B containing nonhydrolyzable phosphoserine mimetic into cells, the protein displayed a decreasing PM distribution compared with nonphosphorylable K-Ras4B, implying that phosphorylation might facilitate the dissociation of K-Ras4B from the PM. In addition, phosphorylation does not alter the localization of K-Ras4B in the liquid-disordered lipid subdomains of the membrane but slightly alters the thermotropic properties of K-Ras4B-incorporated membranes probably due to minor differences in membrane partitioning and dynamics. These results provide novel mechanistic insights into the role that phosphorylation at Ser181 plays in regulating K-Ras4B's distribution and activity.

A novel cholesterol-insensitive mode of membrane binding promotes cytolysin-mediated translocation by Streptolysin O.

Cytolysin-mediated translocation (CMT), performed by Streptococcus pyogenes, utilizes the cholesterol-dependent cytolysin Streptolysin O (SLO) to translocate the NAD(+) -glycohydrolase (SPN) into the host cell during infection. SLO is required for CMT and can accomplish this activity without pore formation, but the details of SLO's interaction with the membrane preceding SPN translocation are unknown. Analysis of binding domain mutants of SLO and binding domain swaps between SLO and homologous cholesterol-dependent cytolysins revealed that membrane binding by SLO is necessary but not sufficient for CMT, demonstrating a specific requirement for SLO in this process. Despite being the only known receptor for SLO, this membrane interaction does not require cholesterol. Depletion of cholesterol from host membranes and mutation of SLO's cholesterol recognition motif abolished pore formation but did not inhibit membrane binding or CMT. Surprisingly, SLO requires the coexpression and membrane localization of SPN to achieve cholesterol-insensitive membrane binding; in the absence of SPN, SLO's binding is characteristically cholesterol-dependent. SPN's membrane localization also requires SLO, suggesting a co-dependent, cholesterol-insensitive mechanism of membrane binding occurs, resulting in SPN translocation.

Replacing the Factor VIII C1 Domain with a Second C2 Domain Reduces Factor VIII Stability and Affinity for Factor IXa

Factor VIII (FVIII) consists of a heavy chain (A1(a1)A2(a2)B domains) and light chain ((a3)A3C1C2 domains). To gain insights into a role of the FVIII C domains, we eliminated the C1 domain by replacing it with the homologous C2 domain. FVIII stability of the mutant (FVIIIC2C2) as measured by thermal decay at 55 °C of FVIII activity was markedly reduced (~11-fold), whereas the decay rate of FVIIIa due to A2 subunit dissociation was similar to WT FVIIIa. The binding affinity of FVIIIC2C2 for phospholipid membranes as measured by fluorescence resonance energy transfer was modestly lower (~2.8-fold) than that for WT FVIII. Among several anti-FVIII antibodies tested (anti-C1 (GMA8011), anti-C2 (ESH4 and ESH8), and anti-A3 (2D2) antibody), only ESH4 inhibited membrane binding of both WT FVIII and FVIIIC2C2. FVIIIa cofactor activity measured in the presence of each of the above antibodies was examined by FXa generation assays. The activity of WT FVIIIa was inhibited by both GMA8011 and ESH4, whereas the activity of FVIIIC2C2 was inhibited by both the anti-C2 antibodies, ESH4 and ESH8. Interestingly, factor IXa (FIXa) binding affinity for WT FVIIIa was significantly reduced in the presence of GMA8011 (~10-fold), whereas the anti-C2 antibodies reduced FIXa binding affinity of FVIIIC2C2 variant (~4-fold). Together, the reduced stability plus impaired FIXa interaction of FVIIIC2C2 suggest that the C1 domain resides in close proximity to FIXa in the FXase complex and contributes a critical role to FVIII structure and function.

Membrane Penetrating Ability of Ebola Matrix Protein, VP40

Ebola from the filoviridae family of viruses causes severe and mostly fatal hemorrhagic fevers in primates and has been listed as a category IV pathogen by the NIH. Viral Protein 40 (VP40), the major matrix protein of Ebola virus, regulates the assembly and budding of the virus and alone harbors the ability to form virus-like particles (VLPs) from human cells. We hypothesize that VP40 is a high affinity lipid binding and membrane curvature-inducing protein with specificity for plasma membrane (PM) lipids. This specificity leads to localization of VP40 to the PS-rich inner leaflet of the PM and formation of VLPs. Using fluorescence spectroscopy to investigate VP40 binding and insertion within lipid vesicles (POPC:POPS) containing brominated lipids. Because the fluorescence of the tryptophan is variably quenched depending on its distance from the bromine atoms on the lipid acyl chain, a tryptophan introduced into the membrane binding interface was utilized as a probe to detect the depth of membrane penetration of VP40. Results were indicative of VP40's high affinity and specificity for PS in a PS-concentration dependent manner demonstrating the robust ability of VP40 to penetrate membranes. Further analysis of VP40 membrane insertion revealed a depth of penetration more than halfway into one monolayer of the membrane. Data also confirmed that VP40 binds with nanomolar affinity to vesicles that recapitulated the PM in comparison to the nuclear membrane. In addition VP40-mutants, which inhibit membrane binding and penetration, obstruct VLP formation and release. Cellular assays confirmed the lipid-binding specificity of VP40 in the PM of different cell lines and also demonstrated that deep membrane penetration is essential for VLP. We predict that these results will elucidate the molecular basis of VP40 induced membrane curvature changes, a prerequisite to the PM deformation required for VLP production.

A Large Extension to HIV-1 Gag, Like Pol, Has Negative Impacts on Virion Assembly

The GagPol protein of HIV-1 harbors viral enzymes, such as protease (PR), reverse transcriptase, and integrase, that are all crucial for virion infectivity. Previous studies have suggested that expression of GagPol alone does not produce viral particles and that the budding defect is caused by the presence of the Pol region. However, it has remained unknown why GagPol fails to produce viral particles. We show here that HIV-1 GagPol is incapable of membrane binding and subsequent particle assembly. Our confocal data indicated that, despite full N-myristoylation, GagPol protein failed to target plasma membrane with diffuse distribution in the cytoplasm. Membrane flotation analysis confirmed these findings. Progressive C-terminal truncation of GagPol to give GagPR allowed for plasma membrane targeting but still not for particle production. Conversely, the C-terminal addition of a noncognate protein, such as ß-galactosidase or 4 tandem GFP, to Gag impaired the membrane affinity, indicating that the Pol region, a large extension to Gag, inhibits membrane binding in the context of GagPol. The addition of the 10 N-terminal amino acids of Fyn kinase [Fyn(10)], a tight membrane-binding signal, conferred plasma membrane targeting on GagPol, but the Fyn(10)GagPol did not produce viral particles. The defect in particle budding was not rescued by the introduction of the PTAP motif, which is responsible for a late stage of viral particle budding. Rather, electron microscopy suggested that the budding defect of GagPR occurred at an early stage of particle morphogenesis. Our data, which were consistent with previous observations, demonstrate the defects of GagPol in membrane binding and particle assembly.

Hypothermia translocates nitric oxide synthase from cytosol to membrane in snail neurons

Neuronal nitric oxide (NO) levels are modulated through the control of catalytic activity of NO synthase (NOS). Although signals limiting excess NO synthesis are being extensively studied in the vertebrate nervous system, our knowledge is rather limited on the control of NOS in neurons of invertebrates. We have previously reported a transient inactivation of NOS in hibernating snails. In the present study, we aimed to understand the mechanism leading to blocked NO production during hypothermic periods of Helix pomatia. We have found that hypothermic challenge translocated NOS from the cytosol to the perinuclear endoplasmic reticulum, and that this cytosol to membrane trafficking was essential for inhibition of NO synthesis. Cold stress also downregulated NOS mRNA levels in snail neurons, although the amount of NOS protein remained unaffected in response to hypothermia. Our studies with cultured neurons and glia cells revealed that glia-neuron signaling may inhibit membrane binding and inactivation of NOS. We provide evidence that hypothermia keeps NO synthesis "hibernated" through subcellular redistribution of NOS.

Ubiquitination of PTEN (Phosphatase and Tensin Homolog) Inhibits Phosphatase Activity and Is Enhanced by Membrane Targeting and Hyperosmotic Stress

The PTEN (phosphatase and tensin homolog) tumor suppressor is a phosphatase that inhibits phosphoinositide 3-kinase-dependent signaling by metabolizing the phosphoinositide lipid phosphatidylinositol 3,4,5-trisphosphate (PtdInsP(3)) at the plasma membrane. PTEN can be mono- or polyubiquitinated, and this appears to control its nuclear localization and stability, respectively. Although PTEN phosphorylation at a cluster of C-terminal serine and threonine residues has been shown to stabilize the protein and inhibit polyubiquitination and plasma membrane localization, details of the regulation of ubiquitination are unclear. Here, we show that plasma membrane targeting of PTEN greatly enhances PTEN ubiquitination and that phosphorylation of PTEN in vitro does not affect subsequent ubiquitination. These data suggest that C-terminal phosphorylation indirectly regulates ubiquitination by controlling membrane localization. We also show that either mono- or polyubiquitination in vitro greatly reduces PTEN phosphatase activity. Finally, we show that hyperosmotic stress increases both PTEN ubiquitination and cellular PtdInsP(3) levels well before a reduction in PTEN protein levels is observed. Both PTEN ubiquitination and elevated PtdInsP(3) levels were reduced within 10 min after removal of the hyperosmotic stress. Our data indicate that ubiquitination may represent a regulated mechanism of direct reversible control over the PTEN enzyme.

The Hydrophobic Cysteine-rich Domain of SNAP25 Couples with Downstream Residues to Mediate Membrane Interactions and Recognition by DHHC Palmitoyl Transferases

SNAP25 is synthesized as a soluble protein but must associate with the plasma membrane to function in exocytosis; however, this membrane-targeting pathway is poorly defined. SNAP25 contains a palmitoylated cysteine-rich domain with four cysteines, and we show that coexpression of specific DHHC palmitoyl transferases is sufficient to promote SNAP25 membrane association in HEK293 cells. siRNA-mediated knockdown of its SNARE partner, syntaxin 1A, does not affect membrane interaction of SNAP25 in PC12 cells, whereas specific cysteine-to-alanine mutations perturb membrane binding, which is restored by leucine substitutions. These results suggest a role for cysteine hydrophobicity in initial membrane interactions of SNAP25, and indeed other hydrophobic residues in the cysteine-rich domain are also important for membrane binding. In addition to the cysteine-rich domain, proline-117 is also essential for SNAP25 membrane binding, and experiments in HEK293 cells revealed that mutation of this residue inhibits membrane binding induced by coexpression with DHHC17, but not DHHC3 or DHHC7. These results suggest a model whereby SNAP25 interacts autonomously with membranes via its hydrophobic cysteine-rich domain, requiring only sufficient expression of partner DHHC proteins for stable membrane binding. The role of proline-117 in SNAP25 palmitoylation is one of the first descriptions of elements within substrate proteins that modulate DHHC specificity.

Point Mutations in the HIV-1 Matrix Protein Turn Off the Myristyl Switch

During the late phase of human immunodeficiency virus type-1 (HIV-1) replication, newly synthesized retroviral Gag proteins are targeted to lipid raft regions of specific cellular membranes, where they assemble and bud to form new virus particles. Gag binds preferentially to the plasma membrane (PM) of most hematopoietic cell types, a process mediated by interactions between the cellular PM marker phosphatidylinositol-(4,5)-bisphosphate (PI(4,5)P 2) and Gag's N-terminally myristoylated matrix (MA) domain. We recently demonstrated that PI(4,5)P 2 binds to a conserved cleft on MA and promotes myristate exposure, suggesting a role as both a direct membrane anchor and myristyl switch trigger. Here we show that PI(4,5)P 2 is also capable of binding to MA proteins containing point mutations that inhibit membrane binding in vitro, and in vivo, including V7R, L8A and L8I. However, these mutants do not exhibit PI(4,5)P 2 or concentration-dependent myristate exposure. NMR studies of V7R and L8A MA reveal minor structural changes that appear to be responsible for stabilizing the myristate-sequestered (myr(s)) species and inhibiting exposure. Unexpectedly, the myristyl group of a revertant mutant with normal PM targeting properties (V7R,L21K) is also tightly sequestered and insensitive to PI(4,5)P 2 binding. This mutant binds PI(4,5)P 2 with twofold higher affinity compared with the native protein, suggesting a potential compensatory mechanism for membrane binding.

Depletion of cellular cholesterol inhibits membrane binding and higher-order multimerization of human immunodeficiency virus type 1 Gag

Recent studies have suggested that the plasma membrane contains cholesterol-enriched microdomains known as lipid rafts. HIV-1 Gag binds raft-rich regions of the plasma membrane, and cholesterol depletion impairs HIV-1 particle production. In this study, we sought to define the block imposed by cholesterol depletion. We observed that membrane binding and higher-order multimerization of Gag were markedly reduced upon cholesterol depletion. Fusing to Gag a highly efficient, heterologous membrane-binding sequence reversed the defects in Gag–membrane binding and multimerization caused by cholesterol depletion, indicating that the impact of reducing the membrane cholesterol content on Gag–membrane binding and multimerization can be circumvented by increasing the affinity of Gag for membrane. Virus release efficiency of this Gag derivative was minimally affected by cholesterol depletion. Altogether, these results are consistent with the hypothesis that cholesterol-enriched membrane microdomains promote HIV-1 particle production by facilitating both Gag–membrane binding and Gag multimerization.

Matrix-Induced Inhibition of Membrane Binding Contributes to Human Immunodeficiency Virus Type 1 Particle Assembly Defects in Murine Cells

Defective human immunodeficiency virus type 1 (HIV-1) assembly in murine cells is accompanied by poor plasma membrane binding and proteolytic processing of the HIV-1 Gag precursor. Here, we show that such defects are induced by the propensity of the HIV-1 MA globular head to inhibit membrane binding and particle assembly, particularly at the low expression levels observed in murine cells. Simple additions to or deletion of the MA globular head can improve the yield of infectious virions from murine cells by >50-fold. Expression level and autoinhibition can be important confounding variables in studies of HIV-1 assembly and contribute to defects encountered in murine cells.

Investigation of the pore-forming mechanism of a cytolytic delta-endotoxin from Bacillus thuringiensis.

Cyt2Aa1 is a cytolytic protein produced by Bacillus thuringiensis subsp. kyushuensis. Penetration of the toxin into membranes has been studied to learn more about membrane-insertion mechanisms and transmembrane-pore formation. The haemolysis assay of Cyt2Aa1 showed a steep and sigmoidal dose-response curve, indicating that toxin aggregation or oligomerization is required for pore formation. Studies of the effect of temperature on pore formation and fluorimetric studies of acrylodan-labelled toxin suggest that toxin inserts into the membrane before oligomerizing to form a pore. Low temperature neither inhibited membrane binding nor closed pores that have been formed, but markedly inhibited oligomerization of the toxin molecules. When toxin-treated red blood cells at 4 degrees C were transferred to a toxin-free solution at 37 degrees C, no significant increase in haemolysis was observed. This result suggests that membrane-bound toxin could not diffuse laterally and interact with other molecules to form a pore. From these results, we propose that Cyt2Aa1 binds and inserts into the membrane as a monomer. Oligomerization occurs when toxin molecules have bound in close proximity to each other and pores are formed from large oligomers.

Antisense oligodeoxynucleotide inhibits expression of recombinant porcine follicle-stimulating hormone receptor.

Follicle-stimulating hormone (FSH) regulates folliculogenesis in the ovary and spermatogenesis in the testis via specific, high affinity membrane-bound receptors (FSHR). To assess the role of FSHR gene expression in regulating expression of FSHR protein in the plasma membrane, the effects of a porcine FSHR cDNA antisense oligodeoxynucleotide (ODN) on FSHR mRNA levels and (125)I-FSH binding were determined in Chinese hamster ovary cells stably expressing recombinant porcine FSHR (pFSHR-CHO cells). An 18-mer phosphorothioated antisense ODN corresponding to the region surrounding the translation initiation codon of the porcine FSHR cDNA was synthesized. An 18-mer phosphorothioated nonsense sequence of identical nucleotide composition was synthesized for use as a control. pFSHR-CHO cells were cultured in the absence or presence of 1-20 microM antisense or nonsense ODN for 24 hr and then assayed for porcine FSHR mRNA, using quantitative reverse transcription and competitive polymerase chain reaction, and for (125)I-FSH binding activity. Treatment with 10 microM antisense ODN caused a paradoxical increase in porcine FSHR mRNA. Nonsense ODN had no effect on porcine FSHR mRNA. Antisense, but not nonsense, ODN (10 microM ) inhibited membrane binding of (125)I-FSH by 13.6 +/- 0.8% (mean +/- SEM, n = 3, P < 0.05) in 24 hr. Treatment of cells with antisense ODN (10 microM) for 48 hr resulted in a 76 +/- 1.5% (P < 0.05) inhibition of (125)I-FSH binding. These results indicate that an FSHR antisense ODN effectively inhibits porcine FSHR synthesis and inhibition of receptor synthesis causes a decrease in functional membrane-bound FSHR.

Inhibition of Protein Palmitoylation, Raft Localization, and T Cell Signaling by 2-Bromopalmitate and Polyunsaturated Fatty Acids

The ability of the Src family kinases Fyn and Lck to participate in signaling through the T cell receptor is critically dependent on their dual fatty acylation with myristate and palmitate. Here we identify a palmitate analog, 2-bromopalmitate, that effectively blocks Fyn fatty acylation in general and palmitoylation in particular. Treatment of COS-1 cells with 2-bromopalmitate blocked myristoylation and palmitoylation of Fyn and inhibited membrane binding and localization of Fyn to detergent-resistant membranes (DRMs). In Jurkat T cells, 2-bromopalmitate blocked localization of the endogenous palmitoylated proteins Fyn, Lck, and LAT to DRMs. This resulted in impaired signaling through the T cell receptor as evidenced by reductions in tyrosine phosphorylation, calcium release, and activation of mitogen-activated protein kinase. We also examined the ability of long chain polyunsaturated fatty acids (PUFAs) to inhibit protein fatty acylation. PUFAs have been reported to inhibit T cell signaling by excluding Src family kinases from DRMs. Here we show that the PUFAs arachidonic acid and eicosapentaenoic acid inhibit Fyn palmitoylation and consequently block Fyn localization to DRMs. We propose that inhibition of protein palmitoylation represents a novel mechanism by which PUFAs exert their immunosuppressive effects.

Antisense oligodeoxynucleotide inhibits expression of recombinant porcine follicle-stimulating hormone receptor.

To assess the role of follicle-stimulating hormone receptor (FSHR) gene expression in regulating expression of FSHR protein in the plasma membrane, the effects of a porcine FSHR cDNA antisense oligodeoxynucleotide (ODN) on FSHR mRNA levels and 125I-FSH binding were determined in Chinese hamster ovary cells expression recombinant porcine FSHR (pFSHR-CHO cells). An 18-mer phosphorothioate-endcapped antisense ODN that corresponded to the region surrounding the translation initiation codon of the porcine FSHR cDNA was synthesized. An 18-mer nonsense sequence of identical nucleotide composition, which had little homology to known DNA sequences, was synthesized for use as a control. pFSHR-CHO cells were cultured in 24-well plates (10(5) cells/well) in the absence or presence of 1-20 mumol/L antisense or nonsense ODN for 24 h and then assayed for porcine FSHR mRNA, using quantitative reverse transcription and competitive polymerase chain reaction, and for 125I-FSH binding activity. Treatment with 10 mumol/L antisense ODN caused a paradoxical increase in porcine FSHR mRNA from 0.89 +/- 0.06 to 1.64 +/- 0.08 ng/mg total RNA (P < 0.05). Transfection with lipofectamine and 0.33 mumol/L antisense ODN caused an increase in porcine mRNA from 0.95 +/- 0.08 to 1.53 +/- 0.07 ng/mg total RAN. This was probably due to upregulation of mRNA synthesis resulting from inhibition of porcine FSHR protein translation. The nonsense ODN had no effect on porcine FSHR mRNA. Antisense, but not nonsense, ODN (10 mumol/L) inhibited membrane binding of 125I-FSH by 13.6 +/- 0.8% (P < 0.05) in 24 h. Treatment of cells with antisense ODN (10 mumol/L) for 48 h resulted in a 76 +/- 1.5% (P < 0.05) inhibition of 125I-FSH binding. In contrast, transfection with lipofectamine and 0.33 mumol/L antisense ODN at 0 h caused a 76.1 +/- 1.3% (P < 0.05) reduction in binding within 24 h. Binding had returned to 52.3 +/- 2.3% (P < 0.05) of normal by 48 h. These results indicate that an antisense ODN corresponding to the region of the translation start site of the porcine FSHR cDNA is an effective specific inhibitor of porcine FSHR synthesis and that inhibition of receptor synthesis causes a decrease in functional membrane-bound FSHR.