Structure and lipid interactions of an anti-inflammatory and anti-atherogenic 10-residue class G(*) apolipoprotein J peptide using solution NMR

The surprising observation that a 10-residue class G(?) peptide from apolipoprotein J, [113-122]apoJ, possesses anti-inflammatory and anti-atherogenic properties prompted us to delineate its structural characteristics in the presence of normal and oxidized lipid. Towards this, we have determined high-resolution structure of [113-122]apoJ in solution using nuclear magnetic resonance (NMR) spectroscopy and studied its interaction with lipids, including oxidized lipids, using a number of biophysical methods. Circular dichroism and NMR studies established that in the presence of dodecylphosphocholine (DPC) micelle, this peptide adopts amphipathic α-helical structure. The observed Nuclear Overhauser effects indicate that the amphipathic helical structure of the peptide is stabilized by the N-terminal acetyl and C-terminal amide blocking groups. We used isothermal titration calorimetry to measure binding enthalpy of the peptide with DPC micelle, an oxidized lipid, 1-(palmitoyl)-2-(5-keto-6-octene-dioyl) phosphatidylcholine (KOdiA-PC), and the mixture of these two lipids (5mol% KOdiA-PC in DPC micelle). We find that the peptide binding with DPC micelle is associated with an enthalpy change (-16.75±0.16 Kcal/mol) much larger than that resulting from the binding with KodiA-PC (-3.67±0.13 Kcal/mol). Incorporation of a small amount of KOdiA-PC (5mol%) in DPC micelle also results in the lowering of peptide binding enthalpy (-13.43±0.18 Kcal/mol). These results are consistent with overall negative charge and altered conformational properties of oxidized sn-2 chain of KOdiA-PC. Our results have unambiguously established the amphipathic α-helical structure of [113-122]apoJ peptide in the presence of DPC micelle as well as its ability to bind oxidized lipid. These in vitro results help explain the previously observed anti-inflammatory and anti-atherosclerotic properties of this peptide.     

Sequence conservation of apolipoprotein A-I affords novel insights into HDL structure-function

We performed alignment of apolipoprotein A-I (apoA-I) sequences from 31 species of animals. We found there is specific conservation of salt bridge-forming residues in the first 30 residues of apoA-I and general conservation of a variety of residue types in the central domain, helix 2/3 to helix 7/8. In the lipid-associating domain, helix 7 and helix 10 are the most and least conserved helixes, respectively. Furthermore, eight residues are completely conserved: P66, R83, P121, E191, and P220, and three of seven Tyr residues in human apoA-I, Y18, Y115, and Y192, are conserved. Residue Y18 appears to be important for assembly of HDL. E191-Y192 represents the only completely conserved pair of adjacent residues in apoA-I; Y192 is a preferred target for site-specific oxidative modification within atheroma, and molecular dynamic simulations suggest that the conserved pair E191-Y192 is in a solvent-exposed loop-helix-loop. Molecular dynamics testing of human apoA-I showed that M112 and M148 interact with Y115, a microenvironment unique to human apoA-I. Finally, conservation of Arg residues in the α11/3 helical wheel position 7 supports several possibilities: interactions with adjacent phospholipid molecules and/or oxidized lipids and/or binding of antioxidant enzymes through cation-π orbital interactions. We conclude that sequence alignment of apoA-I provides unique insights into apoA-I structure-function relationship.     

Structural requirements for antioxidative and anti-inflammatory properties of apolipoprotein A-I mimetic peptides

Recently, attention has been focused on pharmacological treatments that increase HDL cholesterol to prevent coronary artery disease. Despite three decades of extensive research of human apolipoprotein A-I (apoA-I), the major protein component of HDL, the molecular basis for its antiatherogenic and anti-inflammatory functions remain elusive. Another protein component of HDL, apoA-II, has structural features similar to those of apoA-I but does not possess atheroprotective properties. To understand the molecular basis for the effectiveness of apoA-I, we used model synthetic peptides. We designed analogs of the class A amphipathic helical motif in apoA-I that is responsible for solubilizing phospholipids. None of these analogs has sequence homology to apoA-I, but all are similar in their lipid-associating structural motifs. Although all of these peptide analogs interact with phospholipids to form peptide:lipid complexes, the biological properties of these analogs are different. Physical-chemical and NMR studies of these peptides have enabled the delineation of structural requirements for atheroprotective and anti-inflammatory properties in these peptides. It has been shown that peptides that interact strongly with lipid acyl chains do not have antiatherogenic and anti-inflammatory properties. In contrast, peptides that associate close to the lipid head group (and hence do not interact strongly with the lipid acyl chain) are antiatherogenic and anti-inflammatory. Understanding the structure and function of apoA-I and HDL through studies of the amphipathic helix motif may lead to peptide-based therapies for inhibiting atherosclerosis and other related inflammatory lipid disorders.     

ApoA-I mimetic peptides with differing ability to inhibit atherosclerosis also exhibit differences in their interactions with membrane bilayers

Two homologous apoA-I mimetic peptides, 3F-2 and 3F(14), differ in their in vitro antiatherogenic properties (Epand, R. M., Epand, R. F., Sayer, B. G., Datta, G., Chaddha, M., and Anantharamaiah, G. M. (2004) J. Biol. Chem. 279, 51404-51414). In the present work, we demonstrate that the peptide 3F-2, which has more potent anti-inflammatory activity in vitro when administered intraperitoneally to female apoE null mice (20 microg/mouse/day) for 6 weeks, inhibits atherosclerosis (lesion area 15,800 +/- 1000 microm(2), n = 29), whereas 3F(14) does not (lesion area 20,400 +/- 1000 microm(2), n = 26) compared with control saline administered (19,900 +/- 1400 microm(2), n = 22). Plasma distribution of the peptides differs in that 3F-2 preferentially associates with high density lipoprotein, whereas 3F(14) preferentially associates with apoB-containing particles. After intraperitoneal injection of (14)C-labeled peptides, 3F(14) reaches a higher maximal concentration and has a longer half-time of elimination than 3F-2. A study of the effect of these peptides on the motional and organizational properties of phospholipid bilayers, using several NMR methods, demonstrates that the two peptides insert to different extents into membranes. 3F-2 with aromatic residues at the center of the nonpolar face partitions closer to the phospholipid head group compared with 3F(14). In contrast, only 3F(14) affects the terminal methyl group of the acyl chain, decreasing the (2)H order parameter and at the same time also decreasing the molecular motion of this methyl group. This dual effect of 3F(14) can be explained in terms of the cross-sectional shape of the amphipathic helix. These results support the proposal that the molecular basis for the difference in the biological activities of the two peptides lies with their different interactions with membranes.     

GM2 gangliosidoses in Spain: Analysis of the HEXA and HEXB genes in 34 Tay-Sachs and 14 Sandhoff patients

Acid α-glucosidase (GAA) is a lysosomal enzyme that hydrolyzes glycogen to glucose. Deficiency of GAA causes Pompe disease. Mammalian GAA is synthesized as a precursor of ~ 110,000 Da that is N-glycosylated and targeted to the lysosome via the M6P receptors. In the lysosome, human GAA is sequentially processed by proteases to polypeptides of 76-, 19.4-, and 3.9-kDa that remain associated. Further cleavage between R²⁰⁰ and A²⁰⁴ inefficiently converts the 76-kDa polypeptide to the mature 70-kDa form with an additional 10.4-kDa polypeptide. GAA maturation increases its affinity for glycogen by 7-10 fold. In contrast to human GAA, processing of bovine and hamster GAA to the 70-kDa form is more rapid. A comparison of sequences surrounding the cleavage site revealed human GAA contains histidine at 201 while other species contain hydrophobic amino acids at position 201 in the otherwise conserved sequence. Recombinant human GAA (rhGAA) containing the H201L substitution was expressed in 293 T cells by transfection. Pulse chase experiments in 293 T cells expressing rhGAA with or without the H201L substitution revealed rapid processing of rhGAA^H201L but not rhGAA^WT to the 70-kDa form. Similarly, when GAA precursor was endocytosed by human Pompe fibroblasts rhGAA^H201L but not rhGAA^WT was rapidly converted to the 70-kDa mature GAA. These studies indicate that the amino acid at position 201 influences the rate of conversion of 76-kDa GAA to 70-kDa GAA. The GAA sequence rather than the lysosomal protease environment explains the predominance of the 76-kDa form in human tissues.     

The effect of three years of TNF alpha blocking therapy on markers of bone turnover and their predictive value for treatment discontinuation in patients with ankylosing spondylitis: a prospective longitudinal observational cohort study

Introduction

Statins (hydroxymethylglutaryl coenzyme A reductase inhibitors) are effective in reducing the risk of cardiovascular morbidity and mortality in patients with hyperlipidemia, hypertension, or type II diabetes. Next to their cholesterol-lowering activity, statins have immunomodulatory properties. Based on these properties, we hypothesized that statin use may eventually lead to dysregulation of immune responses, possibly resulting in autoimmunity. We have recently shown in an observational study that statin use was associated with an increased risk of developing rheumatoid arthritis. Our objective was to investigate whether a causal relationship could be established for this finding.

Methods

The mouse collagen type II (CII)-induced arthritis (CIA) model was used, with immunization, challenge, and euthanasia at days 0, 21, and 42, respectively. Statins were given orally before (day -28 until day 21) or after (day 21 until day 42) CIA induction. Atorvastatin (0.2 mg/day) or pravastatin (0.8 mg/day) was administered. Arthritis was recorded three times a week. Serum anti-CII autoantibodies and cytokines in supernatants from Concanavalin-A-stimulated lymph node cells and CII-stimulated spleen cells were measured.

Results

Statin administration accelerated arthritis onset and resulted in 100% arthritic animals, whereas only seven out of 12 nonstatin control animals developed arthritis. Atorvastatin administration after CIA induction resulted in earlier onset than atorvastatin administration before induction, or than pravastatin administration before or after induction. The arthritic score of animals given pravastatin before CIA induction was similar to that of the nonstatin controls, whereas the other groups that received statins showed higher arthritic scores. Atorvastatin administration, especially before CIA induction, increased anti-CII autoantibody production. IL-2 and IL-17 production by lymph node and spleen cells was higher in CIA animals than in PBS controls, but was not affected by statin administration. While IFN?? production was not affected by CIA induction, atorvastatin administration before CIA induction increased the production of this cytokine.

Conclusion

These data support previous results from our observational studies, indicating a role for statins in the induction of autoimmunity.     

Anti-inflammatory apoA-I-mimetic peptides bind oxidized lipids with much higher affinity than human apoA-I

4F is an anti-inflammatory, apolipoprotein A-I (apoA-I)-mimetic peptide that is active in vivo at nanomolar concentrations in the presence of a large molar excess of apoA-I. Physiologic concentrations ( approximately 35 microM) of human apoA-I did not inhibit the production of LDL-induced monocyte chemotactic activity by human aortic endothelial cell cultures, but adding nanomolar concentrations of 4F in the presence of approximately 35 microM apoA-I significantly reduced this inflammatory response. We analyzed lipid binding by surface plasmon resonance. The anti-inflammatory 4F peptide bound oxidized lipids with much higher affinity than did apoA-I. Initially, we examined the binding of PAPC (1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphatidylcholine) and observed that its oxidized products bound 4F with an affinity that was approximately 4-6 orders of magnitude higher than that of apoA-I. This high binding affinity was confirmed in studies with defined lipids and phospholipids. 3F-2 and 3F(14) are also amphipathic alpha-helical octadecapeptides, but 3F-2 inhibits atherosclerosis in mice and 3F(14) does not. Like 4F, 3F-2 also bound oxidized phospholipids with very high affinity, whereas 3F(14) resembled apoA-I. The extraordinary ability of 4F to bind pro-inflammatory oxidized lipids probably accounts for its remarkable anti-inflammatory properties.     

Nisin inducible production of listeriolysin O in <Emphasis Type="Italic">Lactococcus lactis</Emphasis> NZ9000

Background  

Listeria monocytogenesis a well-characterized food-borne pathogen that infects pregnant women and immunocompromised individuals. Listeriolysin O (LLO) is the major virulence factor of the pathogen and is often used as a diagnostic marker for detection of L. monocytogenes. In addition, LLO represents a potent antigen driving T cell-mediated immunity during infection. In the present work, Lactococcus lactisNZ9000 was used as an expression host to hyper-produce LLO under inducible conditions using the NICE (NIsin Controlled Expression) system. We created a modified pNZ8048 vector encoding a six-His-tagged LLO downstream of the strong inducible PnisA promoter.