All posts in Raw-Journal Peptide Science

The non-random chromosomal translocations t(10;11)(p13;q23) and t(10;11)(p13;q14–21) result in leukemogenic fusion proteins comprising the coiled coil domain of the transcription factor AF10 and the proteins MLL or CALM, respectively, and subsequently cause certain types of acute leukemia. The AF10 coiled-coil domain, which is crucial for the leukemogenic effect, has been shown to interact with GAS41, a protein previously identified as the product of an amplified gene in glioblastoma. Using sequential synthetic peptides, we mapped the potential AF10/GAS41 interaction site, which was subsequently be used as scaffold for a library targeting the AF10 coiled-coil domain. Using phage display, we selected a peptide that binds the AF10 coiled-coil domain with higher affinity than the respective coiled-coil region of wild-type GAS41, as demonstrated by phage ELISA, CD, and PCAs. Furthermore, we were able to successfully deploy the inhibitory peptide in a mammalian cell line to lower the expression of Hoxa genes that have been described to be overexpressed in these leukemias. This work dissects molecular determinants mediating AF10-directed interactions in leukemic fusions comprising the N-terminal parts of the proteins MLL or CALM and the C-terminal coiled-coil domain of AF10. Furthermore, it outlines the first steps in recognizing and blocking the leukemia-associated AF10 interaction in histiocytic lymphoma cells and therefore, may have significant implications in future diagnostics and therapeutics. Copyright © 2014 European Peptide Society and John Wiley & Sons, Ltd.

This article describes the design and selection of an interfering peptide that binds and blocks the AF10 coiled-coil domain of the leukemia-associated CALM–AF10 fusion protein. The anti-AF10 peptide binds tighter than AF10 wild-type interaction partners and is able to lower hDOT1L-mediated overexpression of Hoxa genes that have been linked to leukemogenesis.

Members of the Chordopoxvirinae subfamily possess an unusual 11 protein entry–fusion complex (EFC) that is highly conserved and present in all species. The mode of action of this EFC is unknown, and the interactions of the constituent proteins are uncharacterised. Here, we present the chemical synthesis of membrane domain truncated linear constructs of two EFC proteins in orf virus, ORFV036 and 049. By using Boc solid phase peptide synthesis and native chemical ligation methods, these truncated proteins have been readily prepared in milligram quantities. These robust synthetic protocols allow ready access to these polypeptides to facilitate biological studies. Copyright © 2014 European Peptide Society and John Wiley & Sons, Ltd.

Orf virus requires an unusually large ≥11-protein entry fusion complex to enter host cells. Through chemical synthesis, we have prepared truncated constructs of two of these entry fusion proteins (ORFV036 and 049), which were previously found to be intractable to bacterial expression. The robust synthetic protocols used allow access to milligram quantities in high purity for further biological studies.

Two glycosylated peptides have been studied using NMR spectroscopy supported by molecular modeling. Peptide I is an oxytocin (OT) analogue in which glutamine 4 was replaced by serine with attached α-d-mannose through the oxygen β atom, whereas peptide II is a lysine-vasopressin (LVP) analogue with lysine 8 side chain modified by the attachment of glucuronic acid through an amide bond. Both peptides exhibit very weak uterotonic effect and are less susceptible to proteolytic degradation than the mother hormones. Additionally, peptide II reveals very weak pressor and antidiuretic activities. Our results have shown that the conformational preferences of glycosylated analogues are highly similar to those of their respective mother hormones. OT glycosylated analogue (I) exhibits a 3,4 β-turn characteristic of OT-like peptides, and vasopressin-glycosylated analogue (II) exhibits β-turns typical of vasopressin-like peptides. Therefore, the lack of binding of the glycosylated analogues to the receptors can be attributed to a steric interference between the carbohydrate moieties and the receptors. We also consider this to be the reason of the very low activity of the analyzed glycopeptides. We expect that results from these studies will be helpful in designing new OT-like and vasopressin-like drugs. Copyright © 2014 European Peptide Society and John Wiley & Sons, Ltd.

The introduction of the sterically restricted bulky glycoconjugates significantly reduces the activity of the neurohypophyseal hormones analogues. We consider that the reason of this can be steric repulsion between the carbohydrate moiety and the receptors. Therefore, despite that oxytocin-glycosylated analogue I exhibits 3,4 β-turn characteristic of oxytocin-like peptides and vasopressin-glycosylated analogue II exhibits β-turns typical of vasopressin-like peptides, these analogues have very low activity.

Vicinal disulfide rings (VDRs) occur when a disulfide bond forms between adjacent cysteine residues in a protein and results in a rare eight-membered ring structure. This eight-membered ring has been found to exist in four major conformations in solution, divided between cis and trans conformers. Some selenoenzymes use a special type of VDR in which selenium replaces sulfur, generating a vicinal selenosulfide ring (VSeSR). Here, we provide evidence that this substitution reduces ring strain, resulting in a strong preference for the trans conformation relative to cis in a VSeSR (cis:trans – 9:91). This was determined by using the ‘γ-gauche effect’, which makes use of both ¹H-NMR and two-dimensional (2D) NMR techniques for determining the amide bond conformeric ratio. The presence of selenium in a VSeSR also lowers the dihedral strain energy (DSE) of the selenosulfide bond relative to the disulfide bond of VDRs. While cis amide geometry decreases strain on the amide bond, it increases strain on the scissile disulfide bond of the VDR found in thioredoxin reductase from Drosophila melanogaster (DmTR). We hypothesize that the cis conformation of the VDR is the catalytically competent conformer for thiol/disulfide exchange. This hypothesis was investigated by computing the DSE of VDR and VSeSR conformers, the structure of which was determined by 2D NMR spectroscopy and energy minimization. The computed values of the VDR from DmTR are 16.5 kJ/mol DSE and 14.3 kJ/mol for the C+ and T− conformers, respectively, supporting the hypothesis that the enzyme uses the C+ conformer for thiol/disulfide exchange. Copyright © 2014 European Peptide Society and John Wiley & Sons, Ltd.

Some enzymes use a rare vicinal disulfide ring for catalysis. These disulfide rings are strained and may fluctuate in the enzyme between cis and trans amide conformations. We show that the cis conformer may be important for thiol/disulfide exchange. Mammalian thioredoxin reductase uses such a motif, except that one sulfur atom of the ring is replaced with selenium. The consequence of this substitution is that the trans form of the ring is stabilized and is the predominant conformer in solution.

While genome-wide association studies as well as candidate gene studies have revealed a great deal of insight into the contribution of genetics to obesity development and susceptibility, advances in adipose tissue research have substantially changed the understanding of adipose tissue function. Its perception has changed from passive lipid storage tissue to active endocrine organ regulating and modulating whole-body energy homeostasis and metabolism and inflammatory and immune responses by secreting a multitude of bioactive molecules, termed adipokines.

The expression of human vaspin (serpinA12) is positively correlated to body mass index and insulin sensitivity and increases glucose tolerance in vivo, suggesting a compensatory role in response to diminished insulin signaling in obesity. Recently, considerable insight has been gained into vaspin structure, function, and specific target tissue-dependent effects, and several lines of evidence suggest vaspin as a promising candidate for drug development for the treatment of obesity-related insulin resistance and inflammation. These will be summarized in this review with a focus on molecular mechanisms and pathways. Copyright © 2014 European Peptide Society and John Wiley & Sons, Ltd.

Considerable insight has been gained into vaspin structure, function, and specific target tissue-dependent effects, and several lines of evidence suggest vaspin as a promising candidate for drug development for the treatment of obesity-related insulin resistance and inflammation. This review will summarize recent findings with a focus on molecular mechanisms and pathways.

O-Acyl isopeptides, in which the N-acyl linkage on the hydroxyamino acid residue (e.g. Ser and Thr) is replaced by an O-acyl linkage, generally suppress unfavorable aggregation properties derived from the corresponding parent peptides. Here, we report the synthesis of an O-acyl isopeptide of 34-mer pyroGlu-ADan (2), a component of amyloid deposits in hereditary familial Danish dementia, by using native chemical ligation. Native chemical ligation of pyroGlu¹-ADan(1-21)-SCH₂CH₂SO₃⁻Na⁺ (3) and Cys²²-O-acyl isopeptide (4), in which the amino group of the Ser²⁹ residue at the isopeptide moiety was protected by an allyloxycarbonyl group, proceeded well in an aqueous solvent to yield a ligated O-acyl isopeptide (5). Subsequent disulfide bond formation and deprotection of the allyloxycarbonyl group followed by HPLC purification gave 2 with a reasonable overall yield. 2 was converted to the parent peptide 1 via an O-to-N acyl migration reaction. The sequential method, namely (i) native chemical ligation of the O-acyl isopeptide, (ii) HPLC purification as the O-acyl isopeptide form, and (iii) O-to-N acyl migration into the desired polypeptide, would be helpful to solve problems with HPLC purification of hydrophobic polypeptides in the process of chemical protein synthesis. Copyright © 2014 European Peptide Society and John Wiley & Sons, Ltd.

We here reported the synthesis of an O-acyl isopeptide by using native chemical ligation in an aqueous solvent, in combination with orthogonal allyloxycarbonyl protecting group at the amino group of the isopeptide moiety. The O-acyl isopeptide was quantitatively converted to the parent native peptide via an O-to-N acyl migration reaction. The present method would be helpful to solve the problem of HPLC purification of hydrophobic polypeptides in the process of chemical protein synthesis by using ligation chemistry.

We studied the interaction of the SH3 domain of Bin1 with a 15-mer peptide of HCV NS5A and show its potency to competitively displace a 15-mer human c-Myc fragment, which is a physiological ligand of Bin1, using NMR spectroscopy. Fluorescence spectroscopy and ITC were employed to determine the affinity of Bin1 SH3 to NS5A(347–361), yielding a submicromolar affinity to NS5A. Our study compares the binding dynamics and affinities of the relevant regions for binding of c-Myc and NS5A to Bin1 SH3. The result gives further insights into the potential role of NS5A in Bin1-mediated apoptosis. Copyright © 2014 European Peptide Society and John Wiley & Sons, Ltd.

Studying the interactions of Bin1 SH3 with peptides derived from the HCV protein NS5A and c-Myc revealed that NS5A can displace c-Myc.