Mechanisms involved in the antiplatelet activity of magnesium in human platelets

In this study, magnesium sulphate dose-dependently (0.6-3.0 mmol/l) inhibited platelet aggregation in human platelets stimulated by agonists. Furthermore, magnesium sulphate (3.0 mmol/l) markedly interfered with the binding of fluorescein isothiocanate-triflavin to the glycoprotein (GP)IIb/IIIa complex in platelets stimulated by collagen. Magnesium sulphate (1.5 and 3.0 mmol/l) also inhibited phosphoinositide breakdown and intracellular Ca+2 mobilization in human platelets stimulated by collagen. Magnesium sulphate (3.0 mmol/l) significantly inhibited thromboxane A2 formation stimulated by collagen in platelets. Moreover, magnesium sulphate (1.5 and 3.0 mmol/l) obviously increased the fluorescence of platelet membranes tagged with diphenylhexatriene. In addition, magnesium sulphate (1.5 and 3.0 mmol/l) increased the formation of cyclic adenosine monophosphate (AMP) in platelets. Phosphorylation of a protein of Mr 47 000 (P47) was markedly inhibited by magnesium sulphate (1.5 mmol/l). In conclusion, the antiplatelet activity of magnesium sulphate may involve the following two pathways. (1) Magnesium sulphate may initially induce membrane fluidity changes with resulting interference of fibrinogen binding to the GPIIb/IIIa complex, followed by inhibition of phosphoinositide breakdown and thromboxane A2 formation, thereby leading to inhibition of both intracellular Ca2+ mobilization and phosphorylation of P47. (2) Magnesium sulphate might also trigger the formation of cyclic AM, ultimately resulting in inhibition of the phosphorylation of P47 and intracellular Ca+2 mobilization.     

The antiplatelet activity of Escherichia coli lipopolysaccharide is mediated through a nitric oxide/cyclic GMP pathway.

In this study, Escherichia coli LPS dose-dependently (100-500 microg/ml) and time-dependently (10-60 min) inhibited platelet aggregation in human and rabbit platelets stimulated by agonists. LPS also dose-dependently inhibited the intracellular Ca2+ mobilization in human platelets stimulated by collagen. In addition, LPS (200 and 500 microg/ml) significantly increased the formation of cyclic GMP but not cyclic AMP in platelets. LPS (200 microg/ml) significantly increased the production of nitrate within a 10-min incubation period. Furthermore, LPS also dose-dependently inhibited platelet aggregation induced by PDBu (30 nmol/l), a protein kinase C activator. These results indicate that the antiplatelet activity of E. coli LPS may be involved in the activation of a nitric oxide/cyclic GMP pathway in platelets, resulting in inhibition of platelet aggregation. Therefore, LPS-mediated alteration of platelet function may contribute to bleeding diathesis in septicemic and endotoxemic patients.     

Peroxynitrite causes phosphorylation of vasodilator-stimulated phosphoprotein through a PKC dependent mechanism

Peroxynitrite is a potent nitrating and oxidizing agent that exerts differential effects on platelets. In the present study we investigated the influence of peroxynitrite on vasodilator-stimulated phosphoprotein (VASP), a protein that plays a key role in inhibition of platelet adhesion and spreading. In platelets, VASP is a substrate for protein kinase A (PKA), PKC and PKG and phosphorylation by these kinases is thought to block VASP-mediated actin cytoskeletal rearrangement. In the present study, we demonstrate that peroxynitrite phosphorylates VASP by a PKC-dependent mechanism. Peroxynitrite (0–100 µM) induced a concentration and time-dependent increase in phosphorylation of VASP at serine¹⁵⁷ (Ser¹⁵⁷) and Ser²³⁹. Inhibition of soluble guanylyl cyclase (sGC) did not significantly reduce peroxynitrite-mediated phosphorylation, indicating a cGMP-independent pathway for VASP phosphorylation. In contrast nitric oxide-mediated VASP phosphorylation was abolished under conditions of sGC inhibition. Further exploration of the mechanisms underlying VASP phosphorylation indicated a requirement for Ca²⁺ mobilization, but was independent of protein kinase A, Src kinases and protein nitration. Consistent with previous reports phorbol 12-myristate 13-acetate (PMA; 300 nM) induced phosphorylation of VASP at Ser¹⁵⁷, but not Ser²³⁹, which was blocked by general protein kinase C (PKC) inhibitors, Ro31-8220 and Bisindolylmaleimide I (BIM-1), and Gö6976, an inhibitor of conventional PKC isoforms. Interestingly, treatment of platelets with these PKC inhibitors significantly reduced peroxynitrite-mediated phosphorylation of both sites, indicating that phosphorylation occurred through PKC-dependent mechanism. Consistent with these findings peroxynitrite caused a small increase in PKC activity as evidenced by increased phosphorylation of PKC substrates. Together these data indicate that peroxynitrite may inhibit platelet function by inducing the phosphorylation of VASP through a mechanism that requires the activation of PKC.     

Effects of Escherichia coli lipopolysaccharide on the phosphoinositide metabolism and serotonin secretion in thrombin-activated platelets

Lipopolysaccharide treatment of platelets in basal conditions does not produce any effect on serotonin secretion or on phosphatidic acid synthesis or arachidonic acid release. However, a brief exposure of platelets to endotoxin enhances the thrombin-stimulated activation of these parameters, whereas a more prolonged treatment with lipopolysaccharide impairs thrombin action. The results presented here also suggest that very short-term treatment of platelets with lipopolysaccharide activates the inositol 1,4,5-trisphosphate 3-kinase. The long-term effects of endotoxin could be mediated by protein kinase C activation.     

Structural basis for substrate specificity in the Escherichia coli maltose transport system

ATP-binding cassette (ABC) transporters are molecular pumps that harness the chemical energy of ATP hydrolysis to translocate solutes across the membrane. The substrates transported by different ABC transporters are diverse, ranging from small ions to large proteins. Although crystal structures of several ABC transporters are available, a structural basis for substrate recognition is still lacking. For the Escherichia coli maltose transport system, the selectivity of sugar binding to maltose-binding protein (MBP), the periplasmic binding protein, does not fully account for the selectivity of sugar transport. To obtain a molecular understanding of this observation, we determined the crystal structures of the transporter complex MBP-MalFGK₂ bound with large malto-oligosaccharide in two different conformational states. In the pretranslocation structure, we found that the transmembrane subunit MalG forms two hydrogen bonds with malto-oligosaccharide at the reducing end. In the outward-facing conformation, the transmembrane subunit MalF binds three glucosyl units from the nonreducing end of the sugar. These structural features explain why modified malto-oligosaccharides are not transported by MalFGK₂ despite their high binding affinity to MBP. They also show that in the transport cycle, substrate is channeled from MBP into the transmembrane pathway with a polarity such that both MBP and MalFGK₂ contribute to the overall substrate selectivity of the system.The ATP-binding cassette (ABC) transporter family contains more than 2,000 members sharing a common architecture of two transmembrane domains (TMDs) that form the translocation pathway and two cytoplasmic nucleotide-binding domains (NBDs) that hydrolyze ATP (1). Importers found in prokaryotes require additional soluble proteins that bind substrates with high affinity and deliver them to the TMDs. Some ABC transporters recognize only a single substrate, whereas others are more promiscuous. For example, ABC transporters that secrete toxins, hydrolytic enzymes, and antibiotic peptides are dedicated to one specific substrate (2), but in contrast, the multidrug transporter P-glycoprotein interacts with more than 200 chemically diverse compounds (3). MRP1, ABCG2, and TAP also have broad substrate spectra (2).Regardless of substrate specificity, the ATPase activity of ABC transporters is regulated by the presence of substrates. Thus, substrate binding must generate a signal that enables ATP hydrolysis. Understanding how ABC transporters interact with their substrates has been a major challenge in the field.A controversial issue in the ABC transporter field is whether the transmembrane components contain a well-defined substrate-binding site. It has been suggested that for binding protein-dependent ABC transporters, substrate specificity is defined exclusively by the binding protein, which interacts with the substrate with high affinity. The transmembrane components act as a nonspecific pore for substrate to diffuse through the membrane (4). However, for the Escherichia coli maltose transporter, it has been well established that the selectivity of sugar binding to the maltose-binding protein (MBP) does not fully account for the selectivity of sugar transport. For example, cyclic maltodextrins, maltodextrins containing more than seven glucosyl units, and maltose analogs with a modified reducing end are not transported despite their high-affinity binding to MBP (5, 6). Further evidence for selectivity through the ABC transporter MalFGK₂ itself comes from mutant transporters that function independently of MBP. In the absence of MBP, these mutants constitutively hydrolyze ATP and specifically transport maltodextrins (7, 8).In this study, we determined the crystal structures of the maltose transport complex MBP-MalFGK₂ bound with large maltodextrin in two conformational states. The determination of these structures, along with previous studies of maltoporin and MBP, allow us to define how overall substrate specificity is achieved for the maltose transport system.     

Influence of vitamin E on the antiplatelet effect of acetylsalicylic acid in human blood

We analysed the in vitro interaction between acetylsalicylic acid and vitamin E on the principal antiplatelet sites of action of acetylsalicylic acid, i.e., platelet aggregation, prostanoid production in platelets and leukocytes, and nitric oxide synthesis. Aggregation was measured in whole blood and in platelet-rich plasma (PRP) with ADP, collagen or arachidonic acid as platelet inducers, and we measured the production of thromboxane B₂, prostacyclin and nitric oxide. Vitamin E potentiated the antiplatelet effect of acetylsalicylic acid in both whole blood and PRP. In PRP induced with collagen the IC₅₀ for acetylsalicylic acid alone was 339?±?11.26, and that of acetylsalicylic acid?+?vitamin E was 0.89?±?0.09 (P?<?0.05). Vitamin E did not enhance inhibition of platelet thromboxane production by acetylsalicylic acid. Vitamin E spared or even increased prostacyclin levels, and acetylsalicylic acid?+?vitamin E diminished the inhibition of prostacyclin synthesis by acetylsalicylic acid (IC₅₀ acetylsalicylic acid alone?=?1.81?±?0.15?µM; IC₅₀ acetylsalicylic acid?+?vitamin E?= 12.92?±?1.10?µM, P?<?0.05). Vitamin E increased the effect of acetylsalicylic acid on neutrophil nitric oxide production 42-fold (P?<?0.05). We conclude that vitamin E potentiates the antiplatelet effect of acetylsalicylic acid in vitro, and thus merits further research in ex vivo studies.     

Crosstalk between the lipopolysaccharide and phospholipid pathways during outer membrane biogenesis in Escherichia coli

The outer membrane of gram-negative bacteria is composed of phospholipids in the inner leaflet and lipopolysaccharides (LPS) in the outer leaflet. LPS is an endotoxin that elicits a strong immune response from humans, and its biosynthesis is in part regulated via degradation of LpxC (EC 3.5.1.108) and WaaA (EC 2.4.99.12/13) enzymes by the protease FtsH (EC 3.4.24.-). Because the synthetic pathways for both molecules are complex, in addition to being produced in strict ratios, we developed a computational model to interrogate the regulatory mechanisms involved. Our model findings indicate that the catalytic activity of LpxK (EC 2.7.1.130) appears to be dependent on the concentration of unsaturated fatty acids. This is biologically important because it assists in maintaining LPS/phospholipids homeostasis. Further crosstalk between the phospholipid and LPS biosynthetic pathways was revealed by experimental observations that LpxC is additionally regulated by an unidentified protease whose activity is independent of lipid A disaccharide concentration (the feedback source for FtsH-mediated LpxC regulation) but could be induced in vitro by palmitic acid. Further experimental analysis provided evidence on the rationale for WaaA regulation. Overexpression of waaA resulted in increased levels of 3-deoxy-d-manno-oct-2-ulosonic acid (Kdo) sugar in membrane extracts, whereas Kdo and heptose levels were not elevated in LPS. This implies that uncontrolled production of WaaA does not increase the LPS production rate but rather reglycosylates lipid A precursors. Overall, the findings of this work provide previously unidentified insights into the complex biogenesis of the Escherichia coli outer membrane.The outer membrane of gram-negative bacteria is decorated with a potent endotoxin (called lipid A), which plays a significant role in bacterial pathogenicity and immune evasion (1). It also acts as a physical barrier protecting the cell from chemical attack and represents a significant obstacle for the effective delivery of numerous antimicrobial agents (2, 3). The outer membrane is composed of phospholipids in the inner leaflet and lipopolysaccharides (LPS) in the outer leaflet (4). Phospholipids consist of a glycerol molecule, a phosphate group, and two fatty acid moieties (except for cardiolipins) (5) (see reviews (5, 6) and SI Appendix for the biosynthesis and regulation of phospholipids). LPS, on the other hand, contains three distinct components: lipid A, core oligosaccharides, and O-antigen (7, 8). Lipid A is the sole essential component of LPS, and its biosynthesis involves nine enzyme-catalyzed reactions (8). The lipid A pathway has been widely investigated, and we recently produced a pathway model that incorporates all of the known regulatory mechanisms (9). Briefly, the first reaction step catalyzed by LpxA is highly unfavorable, which makes the proceeding enzyme, LpxC, the first committed enzyme (10). LpxC is regulated by the protease FtsH (11, 12), and we recently postulated that the negative feedback signal arises from lipid A disaccharide, the substrate for LpxK (9). Furthermore, FtsH regulates WaaA (formerly called KdtA), an enzyme downstream of LpxC (13). The exact rationale for WaaA regulation remains unknown.A wealth of research exists for either LPS or phospholipids biosynthesis; however, our current understanding on the crosstalk between both pathways is limited at the moment. Because both pathways are synchronized to ensure a proper balance of membrane components (11, 14), studies underpinning the underlying mechanisms would appear valuable. There are a number of experimental findings that indicate the existence of strong links between both biosynthetic pathways (11, 15, 16). Thus, in the context of outer membrane biogenesis, the role involving phospholipids cannot be ignored in the study of LPS regulation. Furthermore, during membrane synthesis, ∼20 million molecules of fatty acids are synthesized in Escherichia coli (8). Yu et al. (17) reconstituted an in vitro steady-state kinetic system of fatty acid biosynthesis using purified enzymes and observed that the maximum fatty acid production rate obtainable was 100 µM/min. This production rate falls far below the amount of fatty acids required by a cell in vivo [if one assumes a cell volume of 6.7 × 10⁻¹⁶ L (18) and a generation time of 30 min (19)]. Therefore, to test the consistency of reported in vitro parameters and investigate the role of the biosynthetic enzymes on fatty acids turnover rate, a “systems” approach is necessary. Similarly, ever since the regulation of WaaA by FtsH was first reported (13), no study has investigated the underlying regulatory mechanism to date. This would also appear important because under wild-type conditions, WaaA catalyzes a step that is required for the endotoxic activity of lipid A (20).In this work, we present a detailed picture of the crosstalk between the LPS and phospholipids biosynthetic machinery. Our work involves a computational kinetic model spanning 81 chemical reactions and involving 90 chemical species. Additionally, we used a series of E. coli fatty acid biosynthesis mutants to investigate the effect of substrate flux into the saturated and unsaturated fatty acid pathway on LpxC stability. Our complete model agrees qualitatively with published datasets and with our own experiments. Our results imply that the catalytic activation of LpxK is dependent on unsaturated fatty acids. Furthermore, our experimental investigations have implicated a secondary protease involved in LpxC regulation. Finally, we have provided experimental evidence to explain the rationale for WaaA regulation.     

Alterations of phosphoinositide-specific phospholipase C and protein kinase C in the myocardium of spontaneously hypertensive rats

Summary In order to determine whether phosphoinositide metabolism is altered in hypertensive cardiac hypertrophy, phospholipase C (PLC) and protein kinase C activities were measured in hearts from 4- and 20-week-old spontaneously hypertensive rats (SHR) and age-matched, normotensive Wister-Kyoto rats (WKY). PLC activities were assayed using phosphatidylinositol (PI) and phosphatidylinositol-4,5-bisphosphate (PIP₂) as substrates to assess the substrate specificity. PI-hydrolyzing PLC activity (PI-PLC) was predominantly located in the cytosol, and its activity was similar in both strains. Membrane-bound PIP₂-hydrolyzing PLC activity (PIP₂-PLC) was significantly lower in 20-week-old SHR than in WKY, but there was no significant difference in soluble PIP₂-PLC. Protein kinase C activity was significantly elevated in 20-week-old SHR and Ca²⁺-phospholipid-dependent phosphorylation was observed in the proteins of molecular weight 26, 32, 43, and 95 KDa. In 4-week-old prehypertensive SHR, there were no significant differences in PI-PLC, PIP₂-PLC, or protein kinase C activities as compared with age-matched WKY. These data demonstrated that protein kinase C and membrane-bound PIP₂-PLC are altered during the period of hypertension development. These alterations may have important roles in the development or maintenance of hypertensive cardiac hypertrophy in SHR.Until April 31, 1990 N. Makita, Department of Cardiovascular Medicine, Hokkaido University School of Medicine, Kita 15, Nishi 7, Kita-Ku Sapporo 060, Japan After May 1, 1990 N. Makita, Division of Nephrology, S-3223, Medical Center North, Vanderbilt University, Nashville. Tennessee 37232, USA     

一氧化氮参与了缺血预处理后5′-核苷酸酶的延迟升高

目的 :观察在缺血预处理期间抑制一氧化氮合酶对 2 4h后 5′-核苷酸酶活性的影响和对缺血预处理第二保护窗的影响。方法 :阻断兔冠脉 5 m in,再灌注 10 min,重复 4次 ,造成缺血预处理。在缺血预处理期间静脉注射一氧化氮合酶抑制剂 NG-硝基 - L-精氨酸甲酯 （L- NAME） ,缺血预处理后 2 4h,提取心肌标本测量 5′-核苷酸酶活性 ,或阻断冠脉 30 m in再灌注 12 0 min,测量心肌梗死面积。结果 :缺血预处理 2 4h后 ,心肌细胞膜和胞浆 5′-核苷酸酶活性均较假手术对照组明显升高。静脉注射 L- NAME阻断了缺血预处理所致的细胞膜和胞浆 5′-核苷酸酶活性的升高。缺血预处理组心梗面积明显小于对照组 ,而 L- NAME阻断了这种保护作用。结论 :一氧化氮参与了缺血预处理所致的 5′-核苷酸酶活性的延迟升高。延迟升高的 5′-核苷酸酶活性可能参与了缺血预处理的第二保护窗。     

Signal Transduction Pathways in Enhanced Microvascular Permeability

We have been investigating the molecular mechanisms underlying pathophysiological regulation of microvascular permeability on isolated venules and cultured venular endothelial monolayers. Physiological approaches have been employed in combination with molecular analyses to probe the signal transduction pathways leading to enhanced microvascular permeability. A newly developed technique of protein transfection into cells and intact microvessels enables the correlation of functional reactions and signaling events at the molecular level in a direct and specific fashion. The results indicate that inflammatory mediators increase microvascular permeability via intracellular signaling pathways involving the activation of phospholipase C, cytosolic calcium, protein kinase C, nitric oxide synthase, guanylate cyclase, and protein kinase G. In response to the signaling stimulation, complex biochemical and conformational reactions occur at the endothelial structural proteins. Specifically, myosin light‐chain activation‐mediated myosin light‐chain phosphorylation can result in cell contraction. VE‐cadherin and β‐catenin phosphorylation may induce dissociation of the junctional proteins and their connection to the cytoskeleton, leading to a loose or opened intercellular junction. Focal adhesion phosphorylation and redistribution further provide an anchorage support for the conformational changes in the cells and at the cell junction. The three processes may act in concert to facilitate the flux of fluid and macromolecules across the microvascular endothelium.     

冠心病患者血小板PKC、PKCI及红细胞PKC活性变化

目的 研究蛋白激酶C(PKC)在冠心病的发生发展中的作用。方法 测定87例心绞痛(AP)患者、91例急性心肌梗死(AMl)患者、90例健康对照(HC)者的血小板胞膜、胞浆PKC、胞浆蛋白激酶C抑制剂(PKCl)活性和红细胞胞膜、胞浆PKC活性。结果 AP组和AMI组血小板胞膜中PKC活性明显高于HC组,而胞浆中PKC活性明显低于HC组;AP组和AMI组血小板胞浆中PKCI活性明显低于HC组;AP组和AMI组红细胞胞膜中PKC活性明显高于HC组,而胞浆中PKC活性低于HC组。结论 PKC可能参与冠心病的发病。     

反义MAPK寡核苷酸可抑制血管升压素诱导的心肌成纤维细胞一氧化氮合酶活性

目的探讨反义丝裂素活化蛋白激酶（MAPK）寡核苷酸对血管升压素（VP）诱导的心肌成纤维细胞（CFs）一氧化氮合酶（NOS）活性的抑制作用。方法分离培养SD仔鼠CFs,采用分光光度法和RT-PCR测定CFs NOS活性和诱导型一氧化氮合酶（iNOS）mRNA表达。结果VP显著提高CFs NOS活性和iNOS mRNA表达;反义MAPK寡核苷酸剂量依赖性地抑制VP诱导下CFs NOS活性增高和iNOS mRNA表达增强,其中0.2μmol/L和0.3μmol/L反义MAPK寡核苷酸可将VP诱导下CFs NOS活性和iNOS mRNA表达抑制到与对照组近似水平。结论MAPK参与了VP诱导下CFs NOS活性增高和iNOS mRNA表达增强。     

Vitamin E potentiates the antiplatelet activity of aspirin in collagen-stimulated platelets

BACKGROUND AND OBJECTIVES: The combination of vitamin E with aspirin is becoming an attractive therapeutic approach to prevent thrombotic vascular accidents. In this study we investigated the capacity of vitamin E (50 and 100 M) to enhance the antiplatelet effect of aspirin. DESIGN AND METHODS: The dose-response curves of platelet aggregation, dense body secretion, phospholipase C activation and calcium mobilization were measured in aspirin-treated platelets with and without added vitamin E (50 and 100 M). The role of vitamin E in reducing platelet adhesion to collagen was also studied. RESULTS: We demonstrated that, in platelets incubated with 100 M vitamin E, collagen-concentration ( g/mL) able to induce 50% of the maximal platelet aggregation and of the calcium mobilization was higher than in controls (11.6 versus 3.8 and 21.3 versus 9.8, respectively). Furthermore, 50 M vitamin E reduced platelet adhesion to collagen by about 80%. INTERPRETATION AND CONCLUSIONS: These data demonstrate that vitamin E can potentiate the antiplatelet activity of aspirin by inhibiting the early events of platelet activation pathways induced by collagen. This finding provides a rationale for combining aspirin and vitamin E to prevent thrombotic complications in atherosclerotic patients.     

Single-cell FRET imaging of phosphatase activity in the Escherichia coli chemotaxis system

Two-component signaling systems, in which a receptor-coupled kinase is used to control the phosphorylation level of a response regulator, are commonly used in bacteria to sense their environment. In the chemotaxis system of Escherichia coli, the receptors, and thus the kinase, are clustered on the inner cell membrane. The phosphatase of this system also is recruited to receptor clusters, but the reason for this association is not clear. By using FRET imaging of single cells, we show that in vivo the phosphatase activity is substantially larger at the cluster, indicating that the signaling source (the kinase) and the signaling sink (the phosphatase) tend to be located at the same place in the cell. When this association is disrupted, a gradient in the concentration of the phosphorylated response regulator appears, and the chemotactic response is degraded. Such colocalization is inevitable in systems in which the activity of the kinase and the phosphatase are produced by the same enzyme. Evidently, this design enables a more rapid and spatially uniform response.     

Role of ERK/MAPK signalling pathway in anti-inflammatory effects of Ecklonia cava in activated human mast cell line-1 cells

Objective:The anti-inflammatory effects of Ecklonia cava(EC)and its mechanism of action were examined in phorbol-12 myristate 13-acetate(30 nmol/L)and A23187(1μmol/L)(PMACI)stimulated human mast cell line-1 cells.Methods:Nitric oxide content,inducible nitric oxide synthase and cyclooxygenase-2 protein expression,pro-inflammatory cytokines including IL-1β,TNF-α,and IL-6 mRNA and protein expressions were determined.In addition,extracellular regulated protein kinases/mitogen-activated protein kinase(ERK/MAPK)activation was examined.Results:EC dose-dependently suppressed inducible nitric oxide synthase and cyclooxygenase-2 protein expression and subsequently it reduces nitric oxide content in PMACI stimulated human mast cell line-1 cells.EC dose-dependently inhibited the mRNA as well as protein expression of TNF-α,IL—1β,and TL-6 in the PMACI stimulated human mast cell line-1 cells without any cytotoxic effect.Furthermore,EC significantly inhibited PMACI induced phosphorylation of ERK1/2 in a dose-dependent manner without affecting the total protein levels.Conclusions:EC exert its anti-inflammatory actions via inhibition of ERK/MAPK signalling pathway,suggesting that EC is a potent and efficacious anti-inflammatory agent for mast cellmediated inflammatory diseases.     

Structure of signaling-competent neurotensin receptor 1 obtained by directed evolution in Escherichia coli

Crystallography has advanced our understanding of G protein–coupled receptors, but low expression levels and instability in solution have limited structural insights to very few selected members of this large protein family. Using neurotensin receptor 1 (NTR1) as a proof of principle, we show that two directed evolution technologies that we recently developed have the potential to overcome these problems. We purified three neurotensin-bound NTR1 variants from Escherichia coli and determined their X-ray structures at up to 2.75 Å resolution using vapor diffusion crystallization experiments. A crystallized construct was pharmacologically characterized and exhibited ligand-dependent signaling, internalization, and wild-type–like agonist and antagonist affinities. Our structures are fully consistent with all biochemically defined ligand-contacting residues, and they represent an inactive NTR1 state at the cytosolic side. They exhibit significant differences to a previously determined NTR1 structure (Protein Data Bank ID code 4GRV) in the ligand-binding pocket and by the presence of the amphipathic helix 8. A comparison of helix 8 stability determinants between NTR1 and other crystallized G protein–coupled receptors suggests that the occupancy of the canonical position of the amphipathic helix is reduced to various extents in many receptors, and we have elucidated the sequence determinants for a stable helix 8. Our analysis also provides a structural rationale for the long-known effects of C-terminal palmitoylation reactions on G protein–coupled receptor signaling, receptor maturation, and desensitization.Neurotensin is a 13-amino-acid peptide, which plays important roles in the pathogenesis of Parkinson’s disease, schizophrenia, antinociception, and hypothermia and in lung cancer progression (1–4). It is expressed throughout the central nervous system and in the gut, where it binds to at least three different neurotensin receptors (NTRs). NTR1 and NTR2 are class A G protein–coupled receptors (GPCRs) (5, 6), whereas NTR3 belongs to the sortilin family. Most of the effects of neurotensin are mediated through NTR1, where the peptide acts as an agonist, leading to GDP/GTP exchange within heterotrimeric G proteins and subsequently to the activation of phospholipase C and adenylyl cyclase, which produce second messengers in the cytosol (5, 7). Activated NTR1 is rapidly phosphorylated and internalizes by a β-arrestin– and clathrin-mediated process (8), which is crucial for desensitizing the receptor (9). Several lines of evidence suggest that internalization is also linked to G protein–independent NTR1 signaling (10, 11). To improve our mechanistic understanding of NTR1 and to gain additional insight into GPCR features such as helix 8 (H8), we were interested in obtaining a structure of this receptor in a physiologically relevant state.To date, by far the most successful strategy for GPCR structure determination requires the replacement of the intracellular loop 3 by a fusion protein, as the intracellular domain is otherwise too small to provide crystal contacts. The fusion protein approach has provided a wealth of valuable structural data on GPCRs, but as it renders the crystallized constructs signaling-inactive, the most important functionality—the activation of G proteins—cannot be confirmed for these structures. This leads inevitably to a degree of uncertainty regarding the physiological relevance of intracellular structural aspects, and it also impedes the elucidation of signaling mechanisms, as functional assays and structure determination cannot be performed with the same GPCR constructs.Crystallization in the absence of fusion proteins was so far mainly possible for rhodopsin (12), the A_2A adenosine receptor (A2AR) (13), and the β₁-adrenergic receptor (14). Together, they share a high stability, which is either given naturally (rhodopsin) or it is due to stabilizing mutations. High stability appeared to be crucial for crystallographic success, as it allowed the application of harsh short-chain detergents. These tend to form small micelles, which may explain why crystal contact formation can occur under these conditions despite the small extra- and intracellular domains of class A GPCRs.Besides the stability requirement and/or the necessity of fusion proteins, structural studies of GPCRs have also been complicated by the need of eukaryotic expression systems [e.g., Spodoptera frugiperda (Sf9) insect cells], as prokaryotes exhibit generally low functional expression levels of wild-type GPCRs. However, prokaryotes such as Escherichia coli offer several advantages compared with insect cells, including quick genetic modification strategies, growth to high cell densities, fast doubling times, inexpensive media, absence of glycosylation, and robust handling. Furthermore, E. coli is well suited for producing fully isotope-labeled proteins—a crucial requirement for many NMR studies, which are limited to date.To exploit these advantages, we recently developed a directed evolution method for high functional GPCR expression levels in E. coli (15). In contrast to screening a few hundred mutants one by one, this strategy allows the simultaneous, competitive testing of >10⁸ different protein variants for highest prokaryotic expression and functionality. Briefly, diverse libraries of NTR1 variants were either obtained synthetically (16, 17) or by error-prone PCR on the wild-type sequence (15). The libraries were ligated to a plasmid encoding an inducible promoter, which was subsequently used to transform E. coli. Selection pressure for high functional expression levels was applied by incubating the induced cells with fluorescently labeled neurotensin, which allowed enrichment of the best expressing cells by fluorescence-activated cell sorting (FACS). The outlined procedure was performed in cycles, leading to a gradual adaptation of the NTR1 population toward high functional expression levels, and additionally, it gave rise to an increase in thermostability for certain variants.In a second technology, termed CHESS (cellular high-throughput encapsulation, solubilization and screening), we adapted this concept to directly evolve NTR1 variants for high thermostability in short-chain detergent micelles—a property that is not only beneficial for structural studies but also for in vitro drug screening (18). The crucial development of CHESS was to surround, simultaneously, every E. coli cell by a semipermeable polysaccharide capsule. This allows us to solubilize the receptor mutants with harsh short-chain detergents, each mutant inside its own encapsulated cell, all at once and in the same test tube. Both the solubilized receptors and their encoding plasmids are maintained within the same capsules. Long-term incubation under these conditions followed by labeling of the encapsulated solubilized receptors with fluorescent neurotensin and rounds of FACS enrichment ensured a strong selection pressure and a gradual adaption of the NTR1 population toward high stability in harsh short-chain detergents (18).In this work, we present the crystal structures of three evolved NTR1 variants, which were either obtained by evolving high functional expression levels in E. coli or by directed evolution for stability in detergent micelles. In contrast to the majority of crystallized GPCRs, our NTR1 variants are devoid of bulky modifications at the cytoplasmic face and can thus remain signaling-active, which allows us to gain unique insights into the structure–function relationship of NTR1.     

Retinal blood flow in diabetes

OBJECTIVE: This is a review of work focused on characterizing retinal blood flow in diabetes. The review describes results on validation of the methodology for retinal blood flow measurements, the mechanisms of action of various factors that contribute to abnormalities in retinal blood flow in diabetic rodent models, and the translation of these results to clinical studies demonstrating the effectiveness of different therapeutic agents in normalizing retinal blood flow abnormalities in patients with diabetes. METHODS: Retinal blood flow measurements were performed using video fluorescein angiography, a methodology that is based on the measurement of fluorescein dye circulation times through the retinal circulation. RESULTS: The results of a number of experiments are summarized, detailing the effects of hyperglycemia and the roles of factors such as protein kinase C activation, endothelin-1 and endothelin-3, angiotensin-II, and nitric oxide in the development of retinal blood flow abnormalities in diabetes. CONCLUSION: The measurement of retinal blood flow both in animals and in clinical trials using the same retinal blood flow measurement methodology can provide a valuable method of quantitation allowing characterization of physiological effects and their association with metabolic alterations in diabetes and their effects on the development and incidence of microvascular complications.     

Identification of two inner-membrane proteins required for the transport of lipopolysaccharide to the outer membrane of Escherichia coli

The outer membrane (OM) of most Gram-negative bacteria contains lipopolysaccharide (LPS) in the outer leaflet. LPS, or endotoxin, is a molecule of important biological activities. In the host, LPS elicits a potent immune response, while in the bacterium, it plays a crucial role by establishing a barrier to limit entry of hydrophobic molecules. Before LPS is assembled at the OM, it must be synthesized at the inner membrane (IM) and transported across the aqueous periplasmic compartment. Much is known about the biosynthesis of LPS but, until recently, little was known about its transport and assembly. We applied a reductionist bioinformatic approach that takes advantage of the small size of the proteome of the Gram-negative endosymbiont Blochmannia floridanus to search for novel factors involved in OM biogenesis. This led to the discovery of two essential Escherichia coli IM proteins of unknown function, YjgP and YjgQ, which are required for the transport of LPS to the cell surface. We propose that these two proteins, which we have renamed LptF and LptG, respectively, are the missing transmembrane components of the ABC transporter that, together with LptB, functions to extract LPS from the IM en route to the OM.