A Thioethylalkylamido (TEA) Thioester Surrogate in the Synthesis of a Cyclic Peptide via a Tandem Acyl Shift

The cyclic cystine-knot peptide, kalata B1, was synthesized by employing a novel Fmoc-compatible thioethylalkylamido (TEA) thioester surrogate via an N–S acyl shift followed by a thiol-thioester exchange reaction. TEA thioester surrogate is cost-effective, conveniently prepared in one-step with starting materials, readily available from commercial sources, and highly efficient in preparing peptide thioesters

Schematic representation of the peptide N- and C-terminal dimerization strategies.

<p>N-terminal dimerization employed a linker molecule consisted of two serine residues branching from a lysine, to which two peptides were attached at their N-termini via a thiazolidine linkage. C-terminal dimerization employed a MBHA resin.</p

Membrane-Active Sequences within gp41 Membrane Proximal External Region (MPER) Modulate MPER-Containing Peptidyl Fusion Inhibitor Activity and the Biosynthesis of HIV-1 Structural Proteins

<div><p>The membrane proximal external region (MPER) is a highly conserved membrane-active region located at the juxtamembrane positions within class I viral fusion glycoproteins and essential for membrane fusion events during viral entry. The MPER in the human immunodeficiency virus type I (HIV-1) envelope protein (Env) interacts with the lipid bilayers through a cluster of tryptophan (Trp) residues and a C-terminal cholesterol-interacting motif. The inclusion of the MPER N-terminal sequence contributes to the membrane reactivity and anti-viral efficacy of the first two anti-HIV peptidyl fusion inhibitors T20 and T1249. As a type I transmembrane protein, Env also interacts with the cellular membranes during its biosynthesis and trafficking. Here we investigated the roles of MPER membrane-active sequences during both viral entry and assembly, specifically, their roles in the design of peptidyl fusion inhibitors and the biosynthesis of viral structural proteins. We found that elimination of the membrane-active elements in MPER peptides, namely, penta Trp→alanine (Ala) substitutions and the disruption of the C-terminal cholesterol-interacting motif through deletion inhibited the anti-viral effect against the pseudotyped HIV-1. Furthermore, as compared to C-terminal dimerization, N-terminal dimerization of MPER peptides and N-terminal extension with five helix-forming residues enhanced their anti-viral efficacy substantially. The secondary structure study revealed that the penta-Trp→Ala substitutions also increased the helical content in the MPER sequence, which prompted us to study the biological relevance of such mutations in pre-fusion Env. We observed that Ala mutations of Trp664, Trp668 and Trp670 in MPER moderately lowered the intracellular and intraviral contents of Env while significantly elevating the content of another viral structural protein, p55/Gag and its derivative p24/capsid. The data suggest a role of the gp41 MPER in the membrane-reactive events during both viral entry and budding, and provide insights into the future development of anti-viral therapeutics.</p></div

Expression and viral incorporation of viral structural proteins in the context of pseudotyped HIV-1.

<p><b>A.</b> p55/Gag-derived p24 levels in pseudovirus-producing HLtat cells. HIV(WT), HIV(W5A), HIV(W3A) and HIV(W2A) were produced by co-transfecting HLtat cells with pNLHIVxΔu∆ss and pNL1.5EU+, pNL1.5EU+W5A, pNL1.5EU+W3A, or pNL1.5EU+W2A, respectively. The p24 levels (ng) in cell lysates were quantified by the automated system Architect (Abbott). ***P < 0.001 as compared to WT by the unpaired Student’s t test. <b>B.</b> Steady-state intracellular levels of viral proteins in pseudovirus-producing HLtat cells. HLtat cells from A were harvested 48 h post-transfection and the lysates were resolved by SDS-PAGE and immunoblotted with antibodies against gp41, p24, Vif, Nef and β-actin. Vif and Nef expression served as the transfection control. Un-transfected HLtat cells served as negative control. <b>C.</b> Densitometric analysis of protein bands in blots from two independent experiments as described in in B was performed in ImageJ and presented as means ± SD, with gp160, p55/Gag, and p24 levels in HIV(WT) standardized to 100%. *P < 0.05; **P < 0.01 as compared to WT by the unpaired Student’s t test. <b>D.</b> p24 levels (ng) in the culture supernatants of pseudovirus-producing HLtat cells. p24 levels in the culture supernatant of HLtat cells in A was quantified by the automated system Architect (Abbott). ****P < 0.0001 as compared to WT by the unpaired Student’s t test. <b>E.</b> Env gp41, p55/Gag and p24 levels in precipitated HIV(WT), HIV(W5A), HIV(W3A) and HIV(W2A). Viral particles from the cell culture supernatant from A were precipitated, lysed, separated by SDS-PAGE and immunoblotted with antibodies against gp41 and p24. <b>F</b> Densitometric analysis of the blot in E was performed in ImageJ and presented as means, with gp41, p55/Gag and p24 levels in HIV(WT) standardized to 100%. <b>G.</b> Entry of the cell-free pseudotyped HIV-1 into TZM-bl cells. Cell culture supernatant from A containing HIV(WT), HIV(W5A), HIV(W3A) or HIV(W2A) was clarified through centrifugation and 0.45μm filtration, and applied to 10⁴ TZM-bl cells. Seventy-two h post-infection, tat-activated luciferase activities in the TZM-bl cells were measured and plotted, with the luciferase activity in HIV(WT)-infected TZM-bl cells standardized to 100%.</p

The influence of N- and C-terminal dimerization on the anti-viral effects of the MPER-derived peptides.

<p><b>A.</b> Test for inhibition of pseudo HIV-1 (NL4-3) infection of TZM-bl cells by dimerized peptide QK26. <b>B.</b> Test for inhibition of pseudo HIV-1 (NL4-3) infection of TZM-bl cells by dimerized peptide EK30. <b>C.</b> Test for inhibition of pseudo HIV-1 (NL4-3) infection of TZM-bl cells by dimerized peptide EK37. <b>D.</b> Summary of the concentrations of peptides yielding a 50% and 80% reduction in tat-activated luciferase activity, as tested in B, C and D. The concentrations were estimated with GraphPad Prism. <b>E.</b> No cytotoxicity effect was observed for the MPER-derived dimeric peptides at 100 μM in TZM-bl cells. Monomeric and dimeric peptides (100 μM) were incubated with 10,000 Vero cells for 24 h. PrestoBlue cell viability reagent was subsequently added to the cells, and cytotoxicity effects were monitored as absorbance values (OD) at 570 nm and 600 nm (baseline).</p

Schematic representation of HIV-1 gp41 and partial sequence alignment of the gp41 from different groups.

<p>Partial HR2, the MPER and the TMD sequences of HIV-1 group M subtypes A, B, C and D; group O and the experimental strain of this study, HIV(NL4-3) were aligned[<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0134851#pone.0134851.ref015" target="_blank">15</a>]. Sequences of the anti-HIV-1 first and second generation fusion inhibitor, T20 and T1498, respectively, are shown together with the MPER-containing peptides tested in this study, EK37, EL30, QK26, QT19, LK21 and LK21-5W5A, and all are aligned with the MPER sequence. The MPER sequence is highlighted in bold with its conserved residues shaded. Peptide LK21-5W5A have all five tryptophan residues in MPER sequence substituted by Ala.</p

Total Synthesis of Circular Bacteriocins by Butelase 1

Circular bacteriocins, ranging from 35 to 70 amino acids, are the largest cyclic peptides produced by lactic acid bacteria to suppress growth of other bacteria. Their end-to-end cyclized backbone that enhances molecular stability is an advantage to survive in pasteurization and cooking processes in food preservation, but becomes a disadvantage and challenge in chemical synthesis. They also contain unusually long and highly hydrophobic segments which pose an additional synthetic challenge. Here we report the total synthesis of the three largest circular bacteriocins, AS-48, uberolysin, and garvicin ML, by an efficient chemoenzymatic strategy. A key feature of our synthetic scheme is the use of an Asn-specific butelase-mediated cyclization of their linear precursors, prepared by microwave stepwise synthesis. Antimicrobial assays showed that the AS-48 linear precursor is inactive at concentrations up to 100 μM, whereas the macrocyclic AS-48 is potently active against pathogenic and drug-resistant bacteria, with minimal inhibitory concentrations in a sub-micromolar range

Biophysical Properties and Supramolecular Structure of Self-Assembled Liposome/ε-Peptide/DNA Nanoparticles: Correlation with Gene Delivery

Using solid-phase synthesis, lysine can be oligomerized by a reaction of the peptide carboxylate with the ε-amino group to produce nontoxic, biodegradable cationic peptides, ε-oligo­(l-lysines). Here α-substituted derivatives of such ε-oligo­(l-lysines) containing arginine and histidine in the side chain were tested as vectors for in vitro gene delivery. Combination of ε-oligolysines with the cationic lipid DOTAP and plasmid DNA resulted in transfection efficiency exceeding that of DOTAP alone, without significant increase in cytotoxicity. Synchrotron small-angle X-ray scattering studies revealed self-assembly of the DOTAP, ε-oligolysines, and DNA to ordered lamellar complexes. High transfection efficiency of the nanoparticles correlates with increase in zeta potential above +20 mV and requires particle size to be below 500 nm. The synergistic effect of branched ε-oligolysines and DOTAP in gene delivery can be explained by the increase in surface charge and by the supramolecular structure of the DOTAP/ε-oligolysine/DNA nanoparticles

Regulation of Mcl-1 expression by MEK-1, PI3K and GADD153.

<p>(A) Induction of Mcl-1 in IBV-infected cells in the presence or absence of either 20 mM of MEK-1 inhibitor U0126 or 40 mM of PI3K inhibitor LY294002. Vero, and H1299 cells were incubated with normal medium (DMSO-), LY294002 in DMSO, U0129 in DMSO and DMSO only (DMSO+) for 1 hour, and then infected with IBV at a multiplicity of infection of approximately 2 in the presence or absence of the inhibitors. Cells were harvested at 16 hours post-infection, and total RNA extracted. The relative amounts of Mcl-1 transcripts were determined by quantitative RT-PCR and normalized against GAPDH. The relative fold of Mcl-1 induction in IBV-infected cells was determined by comparing with mock-infected cells. (B) Induction of Mcl-1 in IBV-infected cells by the pro-apoptotic transcription factor GADD153. H1299 cells were transfected with either siGADD153 or a non-targeting control for 72 hours and subsequently infected with IBV. Cells were harvested at 16, 18 and 20 hours post infection for western blot analysis using specific antibodies against the indicated proteins, with anti-actin as a loading control. M, mock infection.</p

Evaluation of the Effect of Trypsin Digestion Buffers on Artificial Deamidation

Nonenzymatic deamidation occurs readily under the condition of trypsin digestion, resulting in the identification of many artificial deamidation sites. To evaluate the effect of trypsin digestion buffers on artificial deamidation, we compared the three commonly used buffers Tris-HCl (pH 8), ammonium bicarbonate (ABC), and triethylammonium bicarbonate (TEAB), and ammonium acetate (pH 6), which was reported to reduce Asn deamidation. iTRAQ quantification on rat kidney tissue digested in these four buffers indicates that artificial Asn deamidation is produced in the order of ammonium acetate < Tris-HCl < ABC < TEAB, and Gln deamidation has no significant differences in all tested buffers. Label-free experiments show the same trend, while protein and unique peptide identification are comparable using these four buffers. To explain the differences of these four buffers in producing artificial Asn deamidation, we determined the half-life of Asn deamidation in these buffers using synthetic peptides containing -Asn-Gly- sequences. It is 51.4 ± 6.0 days in 50 mM of ammonium acetate (pH 6) at 37 °C, which is about 23, 104, and 137 times that in Tris-HCl, ABC, and TEAB buffers, respectively. In conclusion, ammonium acetate (pH 6) is more suitable than other tested buffers for characterizing endogenous deamidation and N-glycosylation