Palmitoylation of Hedgehog proteins by Hedgehog acyltransferase: roles in signalling and disease

Hedgehog acyltransferase (Hhat), a member of the membrane-bound O-acyltransferase (MBOAT) family, catalyses the covalent attachment of palmitate to the N-terminus of Hedgehog proteins. Palmitoylation is a post-translational modification essential for Hedgehog signalling. This review explores the mechanisms involved in Hhat acyltransferase enzymatic activity, similarities and differences between Hhat and other MBOAT enzymes, and the role of palmitoylation in Hedgehog signalling. In vitro and cell-based assays for Hhat activity have been developed, and residues within Hhat and Hedgehog essential for palmitoylation have been identified. In cells, Hhat promotes the transfer of palmitoyl-CoA from the cytoplasmic to the luminal side of the endoplasmic reticulum membrane, where Shh palmitoylation occurs. Palmitoylation is required for efficient delivery of secreted Hedgehog to its receptor Patched1, as well as for the deactivation of Patched1, which initiates the downstream Hedgehog signalling pathway. While Hhat loss is lethal during embryogenesis, mutations in Hhat have been linked to disease states or abnormalities in mice and humans. In adults, aberrant re-expression of Hedgehog ligands promotes tumorigenesis in an Hhat-dependent manner in a variety of different cancers, including pancreatic, breast and lung. Targeting hedgehog palmitoylation by inhibition of Hhat is thus a promising, potential intervention in human disease.     

Membrane Topology of Hedgehog Acyltransferase

Hedgehog acyltransferase (Hhat) is a multipass transmembrane enzyme that mediates the covalent attachment of the 16-carbon fatty acid palmitate to the N-terminal cysteine of Sonic Hedgehog (Shh). Palmitoylation of Shh by Hhat is critical for short and long range signaling. Knowledge of the topological organization of Hhat transmembrane helices would enhance our understanding of Hhat-mediated Shh palmitoylation. Bioinformatics analysis of transmembrane domains within human Hhat using 10 different algorithms resulted in highly consistent predictions in the C-terminal, but not in the N-terminal, region of Hhat. To empirically determine the topology of Hhat, we designed and exploited Hhat constructs containing either terminal or 12 different internal epitope tags. We used selective permeabilization coupled with immunofluorescence as well as a protease protection assay to demonstrate that Hhat contains 10 transmembrane domains and 2 re-entrant loops. The invariant His and highly conserved Asp residues within the membrane-bound O-acyltransferase (MBOAT) homology domain are segregated on opposite sides of the endoplasmic reticulum membrane. The localization of His-379 on the lumenal membrane surface is consistent with a role for this invariant residue in catalysis. Analysis of the activity and stability of the Hhat constructs revealed that the C-terminal MBOAT domain is especially sensitive to manipulation. Moreover, there was remarkable similarity in the overall topological organization of Hhat and ghrelin O-acyltransferase, another MBOAT family member. Knowledge of the topological organization of Hhat could serve as an important tool for further design of selective Hhat inhibitors.     

Identification of N-terminal Residues of Sonic Hedgehog Important for Palmitoylation by Hedgehog Acyltransferase

Sonic Hedgehog (Shh) is a secreted morphogen that regulates embryonic development. After removal of the signal peptide, Shh is processed to the mature, active form through autocleavage and a series of lipid modifications, including the attachment of palmitate. Covalent attachment of palmitate to the N-terminal cysteine of Shh is catalyzed by Hedgehog acyltransferase (Hhat) and is critical for proper signaling. The sequences within Shh that are responsible for palmitoylation by Hhat are not known. Here we show that the first six amino acids of mature Shh (CGPGRG) are sufficient for Hhat-mediated palmitoylation. Alanine scanning mutagenesis was used to determine the role of each amino acid and the positional sequence requirement in a cell-based Shh palmitoylation assay. Mutation of residues in the GPGR sequence to Ala had no effect on palmitoylation, provided that a positively charged residue was present within the first seven residues. The N-terminal position exhibited a strong but not exclusive requirement for Cys. Constructs with an N-terminal Ala were not palmitoylated. However, an N-terminal Ser served as a substrate for Hhat, but not the Drosophila melanogaster ortholog Rasp, highlighting a critical difference between the mammalian and fly enzymes. These findings define residues and regions within Shh that are necessary for its recognition as a substrate for Hhat-mediated palmitoylation. Finally, we report the results of a bioinformatics screen to identify other potential Hhat substrates encoded in the human genome.     

Hhat is a palmitoylacyltransferase with specificity for N-palmitoylation of Sonic Hedgehog

Palmitoylation of Sonic Hedgehog (Shh) is critical for effective long- and short-range signaling. Genetic screens uncovered a potential palmitoylacyltransferase (PAT) for Shh, Hhat, but the molecular mechanism of Shh palmitoylation remains unclear. Here, we have developed and exploited an in vitro Shh palmitoylation assay to purify Hhat to homogeneity. We provide direct biochemical evidence that Hhat is a PAT with specificity for attaching palmitate via amide linkage to the N-terminal cysteine of Shh. Other palmitoylated proteins (e.g. PSD95 and Wnt) are not substrates for Hhat, and Porcupine, a putative Wnt PAT, does not palmitoylate Shh. Neither autocleavage nor cholesterol modification is required for Shh palmitoylation. Both the Shh precursor and mature protein are N-palmitoylated by Hhat, and the reaction occurs during passage through the secretory pathway. This study establishes Hhat as a bona fide Shh PAT and serves as a model for understanding how secreted morphogens are modified by distinct PATs.     

Click chemistry armed enzyme-linked immunosorbent assay to measure palmitoylation by hedgehog acyltransferase

Hedgehog signaling is critical for correct embryogenesis and tissue development. However, on maturation, signaling is also found to be aberrantly activated in many cancers. Palmitoylation of the secreted signaling protein sonic hedgehog (Shh) by the enzyme hedgehog acyltransferase (Hhat) is required for functional signaling. To quantify this important posttranslational modification, many in vitro Shh palmitoylation assays employ radiolabeled fatty acids, which have limitations in terms of cost and safety. Here we present a click chemistry armed enzyme-linked immunosorbent assay (click–ELISA) for assessment of Hhat activity through acylation of biotinylated Shh peptide with an alkyne-tagged palmitoyl-CoA (coenzyme A) analogue. Click chemistry functionalization of the alkyne tag with azido-FLAG peptide allows analysis through an ELISA protocol and colorimetric readout. This assay format identified the detergent n-dodecyl β-d-maltopyranoside as an improved solubilizing agent for Hhat activity. Quantification of the potency of RU-SKI small molecule Hhat inhibitors by click–ELISA indicated IC₅₀ values in the low- or sub-micromolar range. A stopped assay format was also employed that allows measurement of Hhat kinetic parameters where saturating substrate concentrations exceed the binding capacity of the streptavidin-coated plate. Therefore, click–ELISA represents a nonradioactive method for assessing protein palmitoylation in vitro that is readily expandable to other classes of protein lipidation.     

SARS-CoV envelope protein palmitoylation or nucleocapid association is not required for promoting virus-like particle production

Background

Coronavirus membrane (M) proteins are capable of interacting with nucleocapsid (N) and envelope (E) proteins. Severe acute respiratory syndrome coronavirus (SARS-CoV) M co-expression with either N or E is sufficient for producing virus-like particles (VLPs), although at a lower level compared to M, N and E co-expression. Whether E can release from cells or E/N interaction exists so as to contribute to enhanced VLP production is unknown. It also remains to be determined whether E palmitoylation or disulfide bond formation plays a role in SARS-CoV virus assembly.

Results

SARS-CoV N is released from cells through an association with E protein-containing vesicles. Further analysis suggests that domains involved in E/N interaction are largely located in both carboxyl-terminal regions. Changing all three E cysteine residues to alanines did not exert negative effects on E release, E association with N, or E enhancement of VLP production, suggesting that E palmitoylation modification or disulfide bond formation is not required for SARS-CoV virus assembly. We found that removal of the last E carboxyl-terminal residue markedly affected E release, N association, and VLP incorporation, but did not significantly compromise the contribution of E to efficient VLP production.

Conclusions

The independence of the SARS-CoV E enhancement effect on VLP production from its viral packaging capacity suggests a distinct SARS-CoV E role in virus assembly.     

Primary Cilia Are Not Required for Normal Canonical Wnt Signaling in the Mouse Embryo

Background

Sonic hedgehog (Shh) signaling in the mouse requires the microtubule-based organelle, the primary cilium. The primary cilium is assembled and maintained through the process of intraflagellar transport (IFT) and the response to Shh is blocked in mouse mutants that lack proteins required for IFT. Although the phenotypes of mouse IFT mutants do not overlap with phenotypes of known Wnt pathway mutants, recent studies report data suggesting that the primary cilium modulates responses to Wnt signals.

Methodology/Principal Findings

We therefore carried out a systematic analysis of canonical Wnt signaling in mutant embryos and cells that lack primary cilia because of loss of the anterograde IFT kinesin-II motor (Kif3a) or IFT complex B proteins (Ift172 or Ift88). We also analyzed mutant embryos with abnormal primary cilia due to defects in retrograde IFT (Dync2h1). The mouse IFT mutants express the canonical Wnt target Axin2 and activate a transgenic canonical Wnt reporter, BAT-gal, in the normal spatial pattern and to the same quantitative level as wild type littermates. Similarly, mouse embryonic fibroblasts (MEFs) derived from IFT mutants respond normally to added Wnt3a. The switch from canonical to non-canonical Wnt also appears normal in IFT mutant MEFs, as both wild-type and mutant cells do not activate the canonical Wnt reporter in the presence of both Wnt3a and Wnt5a.

Conclusions

We conclude that loss of primary cilia or defects in retrograde IFT do not affect the response of the midgestation embryo or embryo-derived fibroblasts to Wnt ligands.     

A systematic analysis of protein palmitoylation in Caenorhabditis elegans

Background

Palmitoylation is a reversible post-translational protein modification which involves the addition of palmitate to cysteine residues. Palmitoylation is catalysed by the DHHC family of palmitoyl-acyl transferases (PATs) and reversibility is conferred by palmitoyl-protein thioesterases (PPTs). Mutations in genes encoding both classes of enzymes are associated with human diseases, notably neurological disorders, underlining their importance. Despite the pivotal role of yeast studies in discovering PATs, palmitoylation has not been studied in the key animal model Caenorhabditis elegans.

Results

Analysis of the C. elegans genome identified fifteen PATs, using the DHHC cysteine-rich domain, and two PPTs, by homology. The twelve uncategorised PATs were officially named using a dhhc-x system. Genomic data on these palmitoylation enzymes and those in yeast, Drosophila and humans was collated and analysed to predict properties and relationships in C. elegans. All available C. elegans strains containing a mutation in a palmitoylation enzyme were analysed and a complete library of RNA interference (RNAi) feeding plasmids against PAT or PPT genes was generated. To test for possible redundancy, double RNAi was performed against selected closely related PATs and both PPTs. Animals were screened for phenotypes including size, longevity and sensory and motor neuronal functions. Although some significant differences were observed with individual mutants or RNAi treatment, in general there was little impact on these phenotypes, suggesting that genetic buffering exists within the palmitoylation network in worms.

Conclusions

This study reports the first characterisation of palmitoylation in C. elegans using both in silico and in vivo approaches, and opens up this key model organism for further detailed study of palmitoylation in future.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-841) contains supplementary material, which is available to authorized users.     

Lipid modifications of Sonic hedgehog ligand dictate cellular reception and signal response

Background

Sonic hedgehog (Shh) signaling regulates cell growth during embryonic development, tissue homeostasis and tumorigenesis. Concentration-dependent cellular responses to secreted Shh protein are essential for tissue patterning. Shh ligand is covalently modified by two lipid moieties, cholesterol and palmitate, and their hydrophobic properties are known to govern the cellular release and formation of soluble multimeric Shh complexes. However, the influences of the lipid moieties on cellular reception and signal response are not well understood.

Methodology/Principal Findings

We analyzed fully lipidated Shh and mutant forms to eliminate one or both adducts in NIH3T3 mouse embryonic fibroblasts. Quantitative measurements of recombinant Shh protein concentration, cellular localization, and signaling potency were integrated to determine the contributions of each lipid adduct on ligand cellular localization and signaling potency. We demonstrate that lipid modification is required for cell reception, that either adduct is sufficient to confer cellular association, that the cholesterol adduct anchors ligand to the plasma membrane and that the palmitate adduct augments ligand internalization. We further show that signaling potency correlates directly with cellular concentration of Shh ligand.

Conclusions/Significance

The findings of this study demonstrate that lipid modification of Shh determines cell concentration and potency, revealing complementary functions of hydrophobic modification in morphogen signaling by attenuating cellular release and augmenting reception of Shh protein in target tissues.     

In the absence of Sonic hedgehog, p53 induces apoptosis and inhibits retinal cell proliferation, cell-cycle exit and differentiation in zebrafish

Background

Sonic hedgehog (Shh) signaling regulates cell proliferation during vertebrate development via induction of cell-cycle regulator gene expression or activation of other signalling pathways, prevents cell death by an as yet unclear mechanism and is required for differentiation of retinal cell types. Thus, an unsolved question is how the same signalling molecule can regulate such distinct cell processes as proliferation, cell survival and differentiation.

Methodology/Principal Findings

Analysis of the zebrafish shh ^−/− mutant revealed that in this context p53 mediates elevated apoptosis during nervous system and retina development and interferes with retinal proliferation and differentiation. While in shh ^−/− mutants there is activation of p53 target genes and p53-mediated apoptosis, an increase in Hedgehog (Hh) signalling by over-expression of dominant-negative Protein Kinase A strongly decreased p53 target gene expression and apoptosis levels in shh ^−/− mutants. Using a novel p53 reporter transgene, I confirm that p53 is active in tissues that require Shh for cell survival. Proliferation assays revealed that loss of p53 can rescue normal cell-cycle exit and the mitotic indices in the shh ^−/− mutant retina at 24, 36 and 48 hpf. Moreover, generation of amacrine cells and photoreceptors was strongly enhanced in the double p53 ^−/− shh ^−/− mutant retina suggesting the effect of p53 on retinal differentiation.

Conclusions

Loss of Shh signalling leads to the p53-dependent apoptosis in the developing nervous system and retina. Moreover, Shh-mediated control of p53 activity is required for proliferation and cell cycle exit of retinal cells as well as differentiation of amacrine cells and photoreceptors.     

Direct Activation of RhoA by Reactive Oxygen Species Requires a Redox-Sensitive Motif

Background

Rho family GTPases are critical regulators of the cytoskeleton and affect cell migration, cell-cell adhesion, and cell-matrix adhesion. As with all GTPases, their activity is determined by their guanine nucleotide-bound state. Understanding how Rho proteins are activated and inactivated has largely focused on regulatory proteins such as guanine nucleotide exchange factors (GEFs) and GTPase activating proteins (GAPs). However, recent in vitro studies have indicated that GTPases may also be directly regulated by redox agents. We hypothesized that this redox-based mechanism occurs in cells and affects cytoskeletal dynamics, and in this report we conclude this is indeed a novel mechanism of regulating the GTPase RhoA.

Methodology/Principal Findings

In this report, we show that RhoA can be directly activated by reactive oxygen species (ROS) in cells, and that this requires two critical cysteine residues located in a unique redox-sensitive motif within the phosphoryl binding loop. First, we show that ROS can reversibly activate RhoA and induce stress fiber formation, a well characterized readout of RhoA activity. To determine the role of cysteine residues in this mechanism of regulation, we generated cysteine to alanine RhoA mutants. Mutation of these cysteines abolishes ROS-mediated activation and stress fiber formation, indicating that these residues are critical for redox-regulation of RhoA. Importantly, these mutants maintain the ability to be activated by GEFs.

Conclusions/Significance

Our findings identify a novel mechanism for the regulation of RhoA in cells by ROS, which is independent of classical regulatory proteins. This mechanism of regulation may be particularly relevant in pathological conditions where ROS are generated and the cellular redox-balance altered, such as in asthma and ischemia-reperfusion injury.     

Importance of post-translational modifications for functionality of a chloroplast-localized carbonic anhydrase (CAH1) in Arabidopsis thaliana

Background

The Arabidopsis CAH1 alpha-type carbonic anhydrase is one of the few plant proteins known to be targeted to the chloroplast through the secretory pathway. CAH1 is post-translationally modified at several residues by the attachment of N-glycans, resulting in a mature protein harbouring complex-type glycans. The reason of why trafficking through this non-canonical pathway is beneficial for certain chloroplast resident proteins is not yet known. Therefore, to elucidate the significance of glycosylation in trafficking and the effect of glycosylation on the stability and function of the protein, epitope-labelled wild type and mutated versions of CAH1 were expressed in plant cells.

Methodology/Principal Findings

Transient expression of mutant CAH1 with disrupted glycosylation sites showed that the protein harbours four, or in certain cases five, N-glycans. While the wild type protein trafficked through the secretory pathway to the chloroplast, the non-glycosylated protein formed aggregates and associated with the ER chaperone BiP, indicating that glycosylation of CAH1 facilitates folding and ER-export. Using cysteine mutants we also assessed the role of disulphide bridge formation in the folding and stability of CAH1. We found that a disulphide bridge between cysteines at positions 27 and 191 in the mature protein was required for correct folding of the protein. Using a mass spectrometric approach we were able to measure the enzymatic activity of CAH1 protein. Under circumstances where protein N-glycosylation is blocked in vivo, the activity of CAH1 is completely inhibited.

Conclusions/Significance

We show for the first time the importance of post-translational modifications such as N-glycosylation and intramolecular disulphide bridge formation in folding and trafficking of a protein from the secretory pathway to the chloroplast in higher plants. Requirements for these post-translational modifications for a fully functional native protein explain the need for an alternative route to the chloroplast.     

Mutational Analysis of Highly Conserved Residues in the Phage PhiC31 Integrase Reveals Key Amino Acids Necessary for the DNA Recombination

Background

Amino acid sequence alignment of phage phiC31 integrase with the serine recombinases family revealed highly conserved regions outside the catalytic domain. Until now, no system mutational or biochemical studies have been carried out to assess the roles of these conserved residues in the recombinaton of phiC31 integrase.

Methodology/Principal Findings

To determine the functional roles of these conserved residues, a series of conserved residues were targeted by site-directed mutagenesis. Out of the 17 mutants, 11 mutants showed impaired or no recombination ability, as analyzed by recombination assay both in vivo and in vitro. Results of DNA binding activity assays showed that mutants (R18A, I141A, L143A,E153A, I432A and V571A) exhibited a great decrease in DNA binding affinity, and mutants (G182A/F183A, C374A, C376A/G377A, Y393A and V566A) had completely lost their ability to bind to the specific target DNA attB as compared with wild-type protein. Further analysis of mutants (R18A, I141A, L143A and E153A) synapse and cleavage showed that these mutants were blocked in recombination at the stage of strand cleavage.

Conclusions/Significance

This data reveals that some of the highly conserved residues both in the N-terminus and C-terminus region of phiC31 integrase, play vital roles in the substrate binding and cleavage. The cysteine-rich motif and the C-tail val-rich region of phiC31 integrase may represent the major DNA binding domains of phiC31 integrase.     

Crucial Residue Involved in L-Lactate Recognition by Human Monocarboxylate Transporter 4 (hMCT4)

Background

Monocarboxylate transporters (MCTs) transport monocarboxylates such as lactate, pyruvate and ketone bodies. These transporters are very attractive therapeutic targets in cancer. Elucidations of the functions and structures of MCTs is necessary for the development of effective medicine which targeting these proteins. However, in comparison with MCT1, there is little information on location of the function moiety of MCT4 and which constituent amino acids govern the transport function of MCT4. The aim of the present work was to determine the molecular mechanism of L-lactate transport via hMCT4.

Experimental approach

Transport of L-lactate via hMCT4 was determined by using hMCT4 cRNA-injected Xenopus laevis oocytes. hMCT4 mediated L-lactate uptake in oocytes was measured in the absence and presence of chemical modification agents and 4,4′-diisothiocyanostilbene-2,2′-disulphonate (DIDS). In addition, L-lactate uptake was measured by hMCT4 arginine mutants. Immunohistochemistry studies revealed the localization of hMCT4.

Results

In hMCT4-expressing oocytes, treatment with phenylglyoxal (PGO), a compound specific for arginine residues, completely abolished the transport activity of hMCT4, although this abolishment was prevented by the presence of L-lactate. On the other hand, chemical modifications except for PGO treatment had no effect on the transport activity of hMCT4. The transporter has six conserved arginine residues, two in the transmembrane-spanning domains (TMDs) and four in the intracellular loops. In hMCT4-R278 mutants, the uptake of L-lactate is void of any transport activity without the alteration of hMCT4 localization.

Conclusions

Our results suggest that Arg-278 in TMD8 is a critical residue involved in substrate, L-lactate recognition by hMCT4.     

Sonic Hedgehog Gene Delivery to the Rodent Heart Promotes Angiogenesis via iNOS/Netrin-1/PKC Pathway

Background

We hypothesized that genetic modification of mesenchymal stem cells (MSCs) with Sonic Hedgehog (Shh) transgene, a morphogen during embryonic development and embryonic and adult stem cell growth, improved their survival and angiogenic potential in the ischemic heart via iNOS/netrin/PKC pathway.

Methods/Principal Findings

MSCs from young Fisher-344 rat bone marrow were purified and transfected with pCMV Shh plasmid (^ShhMSCs). Immunofluorescence, RT-PCR and Western blotting showed higher expression of Shh in ^ShhMSCs which also led to increased expression of angiogenic and pro-survival growth factors in ^ShhMSCs. Significantly improved migration and tube formation was seen in ^ShhMSCs as compared to empty vector transfected MSCs (^EmpMSCs). Significant upregulation of netrin-1 and iNOS was observed in ^ShhMSCs in PI3K independent but PKC dependent manner. For in vivo studies, acute myocardial infarction model was developed in Fisher-344 rats. The animals were grouped to receive 70 µl basal DMEM without cells (group-1) or containing 1×10^{6 Emp}MSCs (group-2) and ^ShhMSCs (group-3). Group-4 received recombinant netrin-1 protein injection into the infarcted heart. FISH and sry-quantification revealed improved survival of ^ShhMSCs post engraftment. Histological studies combined with fluorescent microspheres showed increased density of functionally competent blood vessels in group-3 and group-4. Echocardiography showed significantly preserved heart function indices post engraftment with ^ShhMSCs in group-3 animals.

Conclusions/Significance

Reprogramming of stem cells with Shh maximizes their survival and angiogenic potential in the heart via iNOS/netrin-1/PKC signaling.     

The hedgehog receptor patched is involved in cholesterol transport

Background

Sonic hedgehog (Shh) signaling plays a crucial role in growth and patterning during embryonic development, and also in stem cell maintenance and tissue regeneration in adults. Aberrant Shh pathway activation is involved in the development of many tumors, and one of the most affected Shh signaling steps found in these tumors is the regulation of the signaling receptor Smoothened by the Shh receptor Patched. In the present work, we investigated Patched activity and the mechanism by which Patched inhibits Smoothened.

Methodology/Principal Findings

Using the well-known Shh-responding cell line of mouse fibroblasts NIH 3T3, we first observed that enhancement of the intracellular cholesterol concentration induces Smoothened enrichment in the plasma membrane, which is a crucial step for the signaling activation. We found that binding of Shh protein to its receptor Patched, which involves Patched internalization, increases the intracellular concentration of cholesterol and decreases the efflux of a fluorescent cholesterol derivative (BODIPY-cholesterol) from these cells. Treatment of fibroblasts with cyclopamine, an antagonist of Shh signaling, inhibits Patched expression and reduces BODIPY-cholesterol efflux, while treatment with the Shh pathway agonist SAG enhances Patched protein expression and BODIPY-cholesterol efflux. We also show that over-expression of human Patched in the yeast S. cerevisiae results in a significant boost of BODIPY-cholesterol efflux. Furthermore, we demonstrate that purified Patched binds to cholesterol, and that the interaction of Shh with Patched inhibits the binding of Patched to cholesterol.

Conclusion/Significance

Our results suggest that Patched may contribute to cholesterol efflux from cells, and to modulation of the intracellular cholesterol concentration. This activity is likely responsible for the inhibition of the enrichment of Smoothened in the plasma membrane, which is an important step in Shh pathway activation.     

The accessibility in the external part of the TM5 of the glutamate transporter EAAT1 is conformationally sensitive during the transport cycle

Background

Excitatory amino acid transporter 1 (EAAT1) is a glutamate transporter which is a key element in the termination of the synaptic actions of glutamate. It serves to keep the extracellular glutamate concentration below neurotoxic level. However the functional significance and the change of accessibility of residues in transmembrane domain (TM) 5 of the EAAT1 are not clear yet.

Methodology/Principal Findings

We used cysteine mutagenesis with treatments with membrane-impermeable sulfhydryl reagent MTSET [(2-trimethylammonium) methanethiosulfonate] to investigate the change of accessibility of TM5. Cysteine mutants were introduced from position 291 to 300 of the cysteine-less version of EAAT1. We checked the activity and kinetic parameters of the mutants before and after treatments with MTSET, furthermore we analyzed the effect of the substrate and blocker on the inhibition of the cysteine mutants by MTSET. Inhibition of transport by MTSET was observed in the mutants L296C, I297C and G299C, while the activity of K300C got higher after exposure to MTSET. V_max of L296C and G299C got lower while that of K300C got higher after treated by MTSET. The L296C, G299C, K300C single cysteine mutants showed a conformationally sensitive reactivity pattern. The sensitivity of L296C to MTSET was potentiated by glutamate and TBOA,but the sensitivity of G299C to MTSET was potentiated only by TBOA.

Conclusions/Significance

All these facts suggest that the accessibility of some positions of the external part of the TM5 is conformationally sensitive during the transport cycle. Our results indicate that some residues of TM5 take part in the transport pathway during the transport cycle.     

Microparticles Carrying Sonic Hedgehog Favor Neovascularization through the Activation of Nitric Oxide Pathway in Mice

Background

Microparticles (MPs) are vesicles released from plasma membrane upon cell activation and during apoptosis. Human T lymphocytes undergoing activation and apoptosis generate MPs bearing morphogen Shh (MPs^Shh+) that are able to regulate in vitro angiogenesis.

Methodology/Principal Findings

Here, we investigated the ability of MPs^Shh+ to modulate neovascularization in a model of mouse hind limb ischemia. Mice were treated in vivo for 21 days with vehicle, MPs^Shh+, MPs^Shh+ plus cyclopamine or cyclopamine alone, an inhibitor of Shh signalling. Laser doppler analysis revealed that the recovery of the blood flow was 1.4 fold higher in MPs^Shh+-treated mice than in controls, and this was associated with an activation of Shh pathway in muscles and an increase in NO production in both aorta and muscles. MPs^Shh+-mediated effects on flow recovery and NO production were completely prevented when Shh signalling was inhibited by cyclopamine. In aorta, MPs^Shh+ increased activation of eNOS/Akt pathway, and VEGF expression, being inhibited by cyclopamine. By contrast, in muscles, MPs^Shh+ enhanced eNOS expression and phosphorylation and decreased caveolin-1 expression, but cyclopamine prevented only the effects of MPs^Shh+ on eNOS pathway. Quantitative RT-PCR revealed that MPs^Shh+ treatment increased FGF5, FGF2, VEGF A and C mRNA levels and decreased those of α5-integrin, FLT-4, HGF, IGF-1, KDR, MCP-1, MT1-MMP, MMP-2, TGFβ1, TGFβ2, TSP-1 and VCAM-1, in ischemic muscles.

Conclusions/Significance

These findings suggest that MPs^Shh+ may contribute to reparative neovascularization after ischemic injury by regulating NO pathway and genes involved in angiogenesis.