Apolipoprotein E derived HDL mimetic peptide ATI-5261 promotes nascent HDL formation and reverse cholesterol transport in vitro

Modulation of the reverse cholesterol transport (RCT) pathway may provide a therapeutic target for the prevention and treatment of atherosclerotic cardiovascular disease (CVD). In the present study, we evaluated a novel 26-amino acid apolipoprotein mimetic peptide (ATI-5261) designed from the carboxyl terminal of apoE, in its ability to mimic apoA-I functionality in RCT in vitro. Our data shows that nascent HDL-like (nHDL) particles generated by incubating cells over-expressing ABCA1 with ATI-5261 increase the rate of specific ABCA1 dependent lipid efflux, with high affinity interactions with ABCA1. We also show that these nHDL particles interact with membrane micro-domains in a manner similar to nHDL apoA-I. These nHDL particles then interact with the ABCG1 transporter and are remodeled by plasma HDL-modulating enzymes. Finally, we show that these mature HDL-like particles are taken up by SR-BI for cholesterol delivery to liver cells. This ATI-5621-mediated process mimics apoA-I and may provide a means to prevent cholesterol accumulation in the artery wall. In this study, we propose an integrative physiology approach of HDL biogenesis with the synthetic peptide ATI-5261. These experiments provide new insights for potential therapeutic use of apolipoprotein mimetic peptides.     

An apoA-I mimetic peptide increases LCAT activity in mice through increasing HDL concentration

Lecithin cholesterol acyltransferase (LCAT) plays a key role in the reverse cholesterol transport (RCT) process by converting cholesterol to cholesteryl ester to form mature HDL particles, which in turn deliver cholesterol back to the liver for excretion and catabolism. HDL levels in human plasma are negatively correlated with cardiovascular risk and HDL functions are believed to be more important in atheroprotection. This study investigates whether and how D-4F, an apolipoprotein A-I (apoA-I) mimetic peptide, influences LCAT activity in the completion of the RCT process. We demonstrated that the apparent rate constant value of the LCAT enzyme reaction gives a measure of LCAT activity and determined the effects of free metals and a reducing agent on LCAT activity, showing an inhibition hierarchy of Zn²⁺>Mg²⁺>Ca²⁺ and no inhibition with β-mercaptoethanol up to 10 mM. We reconstituted nano-disc particles using apoA-I or D-4F with phospholipids. These particles elicited good activity in vitro in the stimulation of cholesterol efflux from macrophages through the ATP-binding cassette transporter A1 (ABCA1). With these particles we studied the LCAT activity and demonstrated that D-4F did not activate LCAT in vitro. Furthermore, we have done in vivo experiments with apoE-null mice and demonstrated that D-4F (20 mg/kg body weight, once daily subcutaneously) increased LCAT activity and HDL level as well as apoA-I concentration at 72 hours post initial dosing. Finally, we have established a correlation between HDL concentration and LCAT activity in the D-4F treated mice.     

An apoA-I mimetic peptide facilitates off-loading cholesterol from HDL to liver cells through scavenger receptor BI

Apolipoprotein A-I (apoA-I) mimetic peptides have been pursued as new therapeutic agents for the treatment of atherosclerosis, yet their precise mechanism responsible for atheroprotection remains unclear. Like apoA-I itself, most of these peptides are capable of stimulating cholesterol efflux from macrophages or foam cells, and some of them stimulate lecithin cholesterol acyltransferase (LCAT) activity in the reverse cholesterol transport (RCT) pathway. However, the ability of mimetic peptides to deliver cholesterol into hepatocytes (off-loading), the last step of the RCT pathway, has not been demonstrated. In this study, we compared a mimetic peptide D-4F to purified apoA-I, to address the role that mimetics play during the off-loading process. Both D-4F and apoA-I formed spherical nano-particles when reconstituted with cholesteryl ester and phospholipids. Compared to apoA-I, D-4F particles were 20 times more efficient in off-loading cholesterol to HepG2 hepatocytes with an apparent K_t (transport) of 0.74 μg/mL. Furthermore, D-4F also facilitated cholesteryl ester offloading from HDL particles into HepG2 cells when it was pre-incubated with these HDL particles. Using an inducible HEK293 cell line, we demonstrated that these nano-particles were able to be taken up through SR-BI, a HDL selective receptor. Cholesterol uptake by HepG2 cells was completely blocked by a neutralizing monoclonal antibody against SR-BI, demonstrating that D-4F particles, similar to HDL, specifically off-loaded cholesterol through SR-BI. Overall our data provides evidence that D-4F is capable of mimicking apoA-I to form HDL-like particles, and off-loads cholesterol for catabolism and excretion, thus completing RCT.     

Distinct structural elements that direct solution aggregation and membrane assembly in the channel-forming peptide M2GlyR

Restoration of chloride conductance via the introduction of an anion selective pore, formed by a channel-forming peptide, has been hypothesized as a novel treatment modality for patients with cystic fibrosis (CF). Delivery of these peptide sequences to airway cells from an aqueous environment in the absence of organic solvents is paramount. New highly soluble COOH- and NH(2)-terminal truncated peptides, derived from the second transmembrane segment of the glycine receptor alpha-subunit (M2GlyR), were generated, with decreasing numbers of amino acid residues. NH(2)-terminal lysyl-adducted truncated peptides with lengths of 22, 25, and 27 amino acid residues are equally able to stimulate short circuit current (I(SC)). Peptides with as few as 16 amino acid residues are able to stimulate I(SC), although to a lesser degree. In contrast, COOH-terminal truncated peptides show greatly reduced induced I(SC) values for all peptides fewer than 27 residues in length and show no measurable activity for peptides fewer than 21 residues in length. CD spectra for both the NH(2)- and COOH-truncated peptides have random structure in aqueous solution, and those sequences that stimulated the highest maximal I(SC) are predominantly helical in 40% trifluoroethanol. Peptides with a decreased propensity to form helical structures in TFE also failed to stimulate I(SC). Palindromic peptide sequences based on both the NH(2)- and COOH-terminal halves of M2GlyR were synthesized to test roles of the COOH- and NH(2)-terminal halves of the molecule in solution aggregation and channel forming ability. On the basis of the study presented here, there are distinct, nonoverlapping regions of the M2GlyR sequence that define solution aggregation and membrane channel assembly. Peptides that eliminate solution aggregation with complete retention of channel forming activity were generated.     

The C-terminal t peptide of acetylcholinesterase forms an alpha helix that supports homomeric and heteromeric interactions.

Acetylcholinesterase subunits of type T (AChET) possess an alternatively spliced C-terminal peptide (t peptide) which endows them with amphiphilic properties, the capacity to form various homo-oligomers and to associate, as a tetramer, with anchoring proteins containing a proline rich attachment domain (PRAD). The t peptide contains seven conserved aromatic residues. By spectroscopic analyses of the synthetic peptides covering part or all of the t peptide of Torpedo AChET, we show that the region containing the aromatic residues adopts an alpha helical structure, which is favored in the presence of lipids and detergent micelles: these residues therefore form a hydrophobic cluster in a sector of the helix. We also analyzed the formation of disulfide bonds between two different AChET subunits, and between AChET subunits and a PRAD-containing protein [the N-terminal fragment of the ColQ protein (QN)] possessing two cysteines upstream or downstream of the PRAD. This shows that, in the complex formed by four T subunits with QN (T4-QN), the t peptides are not folded on themselves as hairpins but instead are all oriented in the same direction, antiparallel to that of the PRAD. The formation of disulfide bonds between various pairs of cysteines, introduced by mutagenesis at various positions in the t peptides, indicates that this complex possesses a surprising flexibility.     

Targeting and assembly of an oligomeric bacterial enterotoxoid in the endoplasmic reticulum of Saccharomyces cerevisiae

A hybrid protein consisting of the Escherichia coli lipoprotein signal sequence attached to the mature sequence of the B subunit of heat-labile enterotoxin (Lipo-EtxB) was expressed in yeast and E. coli. Analyses of cell lysates from Saccharomyces cerevisiae and E. coli expressing the protein revealed that both organisms were able to assemble Lipo-EtxB into oligomers that were (i) stable in the presence of sodium dodecyl sulphate, (ii) resistant to proteinase K degradation, and (iii) able to bind to GM1-ganglioside receptors. Each of these properties are characteristic of the wild-type B subunit pentamer produced in E. coli. Assembly of Lipo-EtxB was found to be unaffected in a sec18 mutant of S. cerevisiae, which possesses a temperature-sensitive defect in protein transport from the endoplasmic reticulum (ER) to the Golgi apparatus, but was found not to assemble in a sec53 mutant, which causes the misfolding of proteins targeted to the ER. A kar2-1 mutation with a defect in the yeast homologue of BiP caused an 18-fold reduction in Lipo-EtxB assembly at the non-permissive temperature in S. cerevisiae. However, introduction of the wild-type KAR2 gene on a plasmid into the kar2-1 mutant completely suppressed the inhibition of Lipo-EtxB assembly. This provides the first evidence that KAR2 facilitates the assembly of an oligomeric protein in yeast and thus implicates KAR2 as a 'molecular chaperone'. The possible mechanisms of enterotoxoid assembly in E. coli and S. cerevisiae are discussed.     

BSD2 is a Rubisco‐specific assembly chaperone,forms intermediary hetero‐oligomeric complexes,and is nonlimiting to growth in tobacco

The folding and assembly of Rubisco large and small subunits into L₈S₈ holoenzyme in chloroplasts involves many auxiliary factors, including the chaperone BSD2. Here we identify apparent intermediary Rubisco‐BSD2 assembly complexes in the model C₃ plant tobacco. We show BSD2 and Rubisco content decrease in tandem with leaf age with approximately half of the BSD2 in young leaves (~70 nmol BSD2 protomer.m²) stably integrated in putative intermediary Rubisco complexes that account for <0.2% of the L₈S₈ pool. RNAi‐silencing BSD2 production in transplastomic tobacco producing bacterial L₂ Rubisco had no effect on leaf photosynthesis, cell ultrastructure, or plant growth. Genetic crossing the same RNAi‐bsd2 alleles into wild‐type tobacco however impaired L₈S₈ Rubisco production and plant growth, indicating the only critical function of BSD2 is in Rubisco biogenesis. Agrobacterium mediated transient expression of tobacco, Arabidopsis, or maize BSD2 reinstated Rubisco biogenesis in BSD2‐silenced tobacco. Overexpressing BSD2 in tobacco chloroplasts however did not alter Rubisco content, activation status, leaf photosynthesis rate, or plant growth in the field or in the glasshouse at 20°C or 35°C. Our findings indicate BSD2 functions exclusively in Rubisco biogenesis, can efficiently facilitate heterologous plant Rubisco assembly, and is produced in amounts nonlimiting to tobacco growth.     

Molecular dynamics study on an adipokinetic hormone peptide in aqueous solution

Lom-AKH-I is a member of the adipokinetic hormone/red pigment concentrating hormone (AKH/RPCH) family of peptides found in flying insects. A molecular dynamics simulation at room temperature (293 K) in water has been performed to survey the folding path of the Lom-AKH-I peptide in water and to establish the secondary structure of Lom-AKH-I. The obtained results indicate the presence of an undefined extended conformation.     

HIV-1 and Ebola virus encode small peptide motifs that recruit Tsg101 to sites of particle assembly to facilitate egress.

Retroviral Gag proteins encode sequences, termed late domains, which facilitate the final stages of particle budding from the plasma membrane. We report here that interactions between Tsg101, a factor involved in endosomal protein sorting, and short peptide motifs in the HIV-1 Gag late domain and Ebola virus matrix (EbVp40) proteins are essential for efficient egress of HIV-1 virions and Ebola virus-like particles. EbVp40 recruits Tsg101 to sites of particle assembly and a short, EbVp40-derived Tsg101-binding peptide sequence can functionally substitute for the HIV-1 Gag late domain. Notably, recruitment of Tsg101 to assembling virions restores budding competence to a late-domain-defective HIV-1 in the complete absence of viral late domain. These studies define an essential virus-host interaction that is conserved in two unrelated viruses. Because the Tsg101 is recruited by small, conserved viral sequence motifs, agents that mimic these structures are potential inhibitors of the replication of these lethal human pathogens.     

A monomeric membrane peptide that lives in three worlds: in solution, attached to, and inserted across lipid bilayers

The membrane peptide pH (low) insertion peptide (pHLIP) lives in three worlds, being soluble in aqueous solution at pH 7.4, binding to the surface of lipid bilayers, and inserting as a transbilayer helix at low pH. With low pH driving the process, pHLIP can translocate cargo molecules attached to its C-terminus via a disulfide and release them in the cytoplasm of a cell. Here we examine a key aspect of the mechanism, showing that pHLIP is monomeric in each of its three major states: soluble in water near neutral pH (state I), bound to the surface of a membrane near neutral pH (state II), and inserted across the membrane as an alpha-helix at low pH (state III). The peptide does not induce fusion or membrane leakage. The unique properties of pHLIP made it attractive for the biophysical investigation of membrane protein folding in vitro and for the development of a novel class of delivery peptides for the transport of therapeutic and diagnostic agents to acidic tissue sites associated with various pathological processes in vivo.     

4Ca2+.troponin C forms dimers in solution at neutral pH that dissociate upon binding various peptides: small-angle X-ray scattering studies of peptide-induced structural changes.

Small-angle X-ray scattering data have been measured for rabbit skeletal muscle troponin C and its complexes with the venom peptides melittin and mastoparan as well as synthetic peptides based on regions of the troponin I sequence implicated in troponin C binding. At the neutral pH used in this study (pH 6.8), troponin C shows a tendency to form dimers in the presence of 4 mol equiv of Ca2+, but is monomeric in solution when 2 or less mol equiv of Ca2+ is present. The 4Ca2+.troponin C dimers dissociate upon binding melittin, mastoparan, and peptides based on residues 96-115, 1-30, and 1-40 in the troponin I sequence. This result suggests that the peptide-binding sites overlap with the regions of contact between troponin C molecules forming a dimer. Like the structurally homologous calcium-binding protein calmodulin, troponin C shows conformational flexibility upon binding different peptides. Upon binding melittin, troponin C contracts in a similar manner to calmodulin when it binds peptides known to form amphiphilic helices (e.g., melittin, mastoparan, or MLCK-I). In contrast, mastoparan binding to troponin C does not result in a contracted structure. The scattering data indicate troponin C also remains in an extended structure upon binding the inhibitory peptides having the same sequence as residues 96-115 in troponin I.     

Proteins binding to site C2 (muE3) in the immunoglobulin heavy-chain enhancer exist in multiple oligomeric forms.

We describe the purification to near homogeneity of proteins binding to site C2 (muE3) in the immunoglobulin heavy-chain enhancer. Proteins binding to this site produce four protein-DNA complexes which are distinguished by their mobility in gel retardation assays and their elution properties in an anion exchange column. DNA affinity-purified preparations of three chromatographically separated pools, containing different subsets of the four complexes, each contained three polypeptides of 42.5, 44, and 45 kilodaltons (kDa). UV crosslinking of protein to enhancer DNA demonstrated that site C2-binding activities in the three different pools bound DNA through proteins of similar sizes (about 45 kDa), even though the protein-DNA complexes formed by these binding activities were quite distinct. Gel exclusion chromatography and equilibrium binding analyses indicated that the distinct protein-DNA complexes were due to different oligomeric forms of the individual subunits and that a larger multimeric form bound with high affinity to the heavy-chain enhancer site C2, while a smaller species had a much lower affinity for heavy-chain enhancer sequences. Purified protein has been used to map high-affinity binding sites for site C2-binding proteins within an immunoglobulin heavy-chain promoter and at site KE3 in the kappa light-chain enhancer.     

Crystal structures of MKK4 kinase domain reveal that substrate peptide binds to an allosteric site and induces an auto-inhibition state

MKK4 activates both JNKs and p38s. We determined the crystal structures of human non-phosphorylated MKK4 kinase domain (npMKK4) complexed with AMP-PNP (npMKK4/AMP) and a ternary complex of npMKK4, AMP-PNP and p38α peptide (npMKK4/AMP/p38). These crystal structures revealed that the p38α peptide-bound npMKK4 at the allosteric site rather than at the putative substrate binding site and induced an auto-inhibition state. While the activation loop of the npMKK4/AMP complex was disordered, in the npMKK4/AMP/p38 complex it configured a long α-helix, which prevented substrate access to the active site and αC-helix movement to the active configuration of MKK4.     

An in vitro fluorescence screen to identify antivirals that disrupt hepatitis B virus capsid assembly

Virus assembly has not been routinely targeted in the development of antiviral drugs, in part because of the lack of tractable methods for screening in vitro. We have developed an in vitro assay of hepatitis B virus (HBV) capsid assembly, based on fluorescence quenching of dye-labeled capsid protein, for testing potential inhibitors. This assay is adaptable to high-throughput screening and can identify small-molecule inhibitors of virus assembly that prevent, inappropriately accelerate and/or misdirect capsid formation to yield aberrant particles. An in vitro primary screen has the advantage of identifying promising lead compounds affecting assembly without the requirement that they be taken up by cells in culture and be nontoxic. Our approach may facilitate the identification of antivirals targeting viruses other than HBV, such as avian influenza and HIV.     

Theoretical study of volume changes associated with the helix-coil transition of an alanine-rich peptide in aqueous solution

The changes in the partial molar volume (PMV) associated with the conformational transition of an alanine-rich peptide AK16 from the alpha-helix structure to various random coil structures are calculated by the three-dimensional interaction site model (3D-RISM) theory coupled with the Kirkwood-Buff theory. The volume change is analyzed by decomposing it into contributions from geometry and hydration: the changes in the van der Waals, void, thermal, and interaction volume. The total change in the PMV is positive. This is primarily due to the growth of void space within the peptide, which is canceled in part by the volume reduction resulting from the increase in the electrostatic interaction between the peptide and water molecules. The changes in the void and thermal volume of the coil structures are widely distributed and tend to compensate each other. Additionally, the relations between the hydration volume components and the surface properties are investigated. We categorize coil structures into extended coils with the PMV smaller than helix and general coils with the PMV larger than helix. The pressure therefore can both stabilize and destabilize the coil structures. The latter seems to be a more proper model of random coil structures of the peptide.     

Epstein-barr virus-induced resistance to drugs that activate the mitotic spindle assembly checkpoint in Burkitt's lymphoma cells

Epstein-Barr virus (EBV) is associated with a number of human cancers, and latent EBV gene expression has been reported to interfere with cell cycle checkpoints and cell death pathways. Here we show that latent EBV can compromise the mitotic spindle assembly checkpoint and rescue Burkitt's lymphoma (BL)-derived cells from caspase-dependent cell death initiated in aberrant mitosis. This leads to unscheduled mitotic progression, resulting in polyploidy and multi- and/or micronucleation. The EBV latent genes responsible for this phenotype are expressed from the P3HR1 strain of virus and several viruses with similar genomic deletions that remove the EBNA2 gene. Although EBNA2 and the latent membrane proteins are not expressed, the EBNA3 proteins are present in these BL cells. Survival of the EBV-positive cells is not consistently associated with EBV lytic gene expression or with the genes that are expressed in EBV latency I BL cells (i.e., EBNA1, EBERs, and BARTs) but correlates with reduced expression of the cellular proapoptotic BH3-only protein Bim. These data suggest that a subset of latent EBV gene products may increase the likelihood of damaged DNA being inherited because of the impaired checkpoint and enhanced survival capacity. This could lead to greater genetic diversity in progeny cells and contribute to tumorigenesis. Furthermore, since it appears that this restricted latent EBV expression interferes with the responses of Burkitt's lymphoma-derived cells to cytotoxic drugs, the results of this study may have important therapeutic implications in the treatment of some BL.     

NMR analysis of the binding of a rhodanese peptide to a minichaperone in solution.

A detailed structural analysis of interactions between denatured proteins and GroEL is essential for an understanding of its mechanism. Minichaperones constitute an excellent paradigm for obtaining high-resolution structural information about the binding site and conformation of substrates bound to GroEL, and are particularly suitable for NMR studies. Here, we used transferred nuclear Overhauser effects to study the interaction in solution between minichaperone GroEL(193-335) and a synthetic peptide (Rho), corresponding to the N-terminal alpha-helix (residues 11 to 23) of the mitochondrial rhodanese, a protein whose in vitro refolding is mediated by minichaperones. Using a 60 kDa maltose-binding protein (MBP)-GroEL(193-335) fusion protein to increase the sensitivity of the transferred NOEs, we observed characteristic sequential and mid-range transferred nuclear Overhauser effects. The peptide adopts an alpha-helical conformation upon binding to the minichaperone. Thus the binding site of GroEL is compatible with binding of alpha-helices as well as extended beta-strands. To locate the peptide-binding site on GroEL(193-335), we analysed changes in its chemical shifts on adding an excess of Rho peptide. All residues with significant chemical shift differences are localised in helices H8 and H9. Non-specific interactions were not observed. This indicates that the peptide Rho binds specifically to minichaperone GroEL(193-335). The binding region identified by NMR in solution agrees with crystallographic studies with small peptides and with fluorescence quenching studies with denatured proteins.