Investigation of acyl transfer auxiliary-assisted glycoconjugation for glycoprotein semi-synthesis.

Homogeneous glycoprotein syntheses have become possible in the last decade due to advances in chemical ligation strategies, particularly Native Chemical Ligation (NCL). For native glycoproteins this still requires laborious and technically challenging syntheses of glycopeptide components, combined with multi-segment ligation reactions. Here we explore new reactions between sugar-linked acyl transfer auxiliaries and peptide thioesters. We show that native glycoproteins are difficult to produce using this approach but various related analogues are accessible. The results show that site-specific neoglycoconjugation is a viable route to simply glycosylated proteins, which may be extended using well-documented enzymatic processes.

Novel electrochemically-mediated peptide dethiylation in processes relevant to native chemical ligation.

Here we explore electrochemical dethiylation in processes relevant to Native Chemical Ligation (NCL). NCL's reliance on the redox active amino acid cysteine and ß-mercaptamine derivatives suggests a potential role for electrochemistry. We show that the application of a 1 V potential to platinum electrodes in 0.15 M TCEP solution is sufficient to convert Cys to Ala in cyclic peptides, and to cleave the 2-mercapto-2-phenethyl class of acyl transfer auxiliary.

Persistent kallikrein 5 activation induces atopic dermatitis-like skin architecture independent of PAR2 activity.

BACKGROUND: Upregulation of kallikreins (KLKs) including KLK5 has been reported in atopic dermatitis (AD). KLK5 has biological functions that include degrading desmosomal proteins and inducing proinflammatory cytokine secretion through protease-activated receptor 2 (PAR2). However, due to the complex interactions between various cells in AD inflamed skin, it is difficult to dissect the precise and multiple roles of upregulated KLK5 in AD skin. OBJECTIVE: We investigated the effect of upregulated KLK5 on the expression of epidermal-related proteins and cytokines in keratinocytes and on skin architecture. METHODS: Lesional and nonlesional AD skin biopsies were collected for analysis of morphology and protein expression. The relationship between KLK5 and barrier-related molecules was investigated using an ex vivo dermatitis skin model with transient KLK5 expression and a cell model with persistent KLK5 expression. The influence of upregulated KLK5 on epidermal morphology was investigated using an in vivo skin graft model. RESULTS: Upregulation of KLK5 and abnormal expression of desmoglein 1 (DSG1) and filaggrin, but not PAR2 were identified in AD skin. PAR2 was increased in response to transient upregulation of KLK5, whereas persistently upregulated KLK5 did not show this effect. Persistently upregulated KLK5 degraded DSG1 and stimulated secretion of IL-8, IL-10, and thymic stromal lymphopoietin independent of PAR2 activity. With control of higher KLK5 activity by the inhibitor sunflower trypsin inhibitor G, restoration of DSG1 expression and a reduction in AD-related cytokine IL-8, thymic stromal lymphopoietin, and IL-10 secretion were observed. Furthermore, persistently elevated KLK5 could induce AD-like skin architecture in an in vivo skin graft model. CONCLUSIONS: Persistently upregulated KLK5 resulted in AD-like skin architecture and secretion of AD-related cytokines from keratinocytes in a PAR2 independent manner. Inhibition of KLK5-mediated effects may offer potential as a therapeutic approach in AD.

Expanding the scope of N → S acyl transfer in native peptide sequences.

Understanding the factors that influence N → S acyl transfer in native peptide sequences, and discovery of new reagents that facilitate it, will be key to expanding its scope and applicability. Here, through a study of short model peptides in thioester formation and cyclisation reactions, we demonstrate that a wider variety of Xaa-Cys motifs than originally envisaged are capable of undergoing efficient N → S acyl transfer. We present data for the relative rates of thioester formation and cyclisation for a representative set of amino acids, and show how this expanded scope can be applied to the production of the natural protease inhibitor Sunflower Trypsin Inhibitor-1 (SFTI-1).

The molecular basis of ATM-dependent dimerization of the Mdc1 DNA damage checkpoint mediator.

Mdc1 is a large modular phosphoprotein scaffold that maintains signaling and repair complexes at double-stranded DNA break sites. Mdc1 is anchored to damaged chromatin through interaction of its C-terminal BRCT-repeat domain with the tail of γH2AX following DNA damage, but the role of the N-terminal forkhead-associated (FHA) domain remains unclear. We show that a major binding target of the Mdc1 FHA domain is a previously unidentified DNA damage and ATM-dependent phosphorylation site near the N-terminus of Mdc1 itself. Binding to this motif stabilizes a weak self-association of the FHA domain to form a tight dimer. X-ray structures of free and complexed Mdc1 FHA domain reveal a 'head-to-tail' dimerization mechanism that is closely related to that seen in pre-activated forms of the Chk2 DNA damage kinase, and which both positively and negatively influences Mdc1 FHA domain-mediated interactions in human cells prior to and following DNA damage.

Making ends meet: chemically mediated circularization of recombinant proteins.

A selective N→S acyl transfer reaction facilitates semi-synthesis of the plant cyclotide kalata B1 from a linear precursor peptide of bacterial origin, through simple appendage of N-terminal cysteine and a thiol-labile C-terminal Gly-Cys motif. This constitutes the first synthesis of a ribosomally derived circular miniprotein, without recourse to protein splicing elements.

Investigation of peptide thioester formation via N→Se acyl transfer.

Native chemical ligation is widely used for the convergent synthesis of proteins. The peptide thioesters required for this process can be challenging to produce, particularly when using Fmoc-based solid-phase peptide synthesis. We have previously reported a route to peptide thioesters, following Fmoc solid-phase peptide synthesis, via an N→S acyl shift that is initiated by the presence of a C-terminal cysteine residue, under mildly acidic conditions. Under typical reaction conditions, we occasionally observed significant thioester hydrolysis as a consequence of long reaction times (~48 h) and sought to accelerate the reaction. Here, we present a faster route to peptide thioesters, by replacing the C-terminal cysteine residue with selenocysteine and initiating thioester formation via an N→Se acyl shift. This modification allows thioester formation to take place at lower temperatures and on shorter time scales. We also demonstrate how application of this strategy also accelerates peptide cyclization, when a linear precursor is furnished with an N-terminal cysteine and C-terminal selenocysteine.

Cysteine promoted C-terminal hydrazinolysis of native peptides and proteins.

Tagging the terminus: N→S acyl transfer in native peptides and proteins can be reliably intercepted with hydrazine. The method allows selective labeling and ligation, without recourse to the use of protein-splicing elements. NCL=native chemical ligation.

Small-molecule inhibition of c-MYC:MAX leucine zipper formation is revealed by ion mobility mass spectrometry.

The leucine zipper interaction between MAX and c-MYC has been studied using mass spectrometry and drift time ion mobility mass spectrometry (DT IM-MS) in addition to circular dichroism spectroscopy. Peptides comprising the leucine zipper sequence with (c-MYC-Zip residues 402-434) and without a postulated small-molecule binding region (c-MYC-ZipΔDT residues 406-434) have been synthesized, along with the corresponding MAX leucine zipper (MAX-Zip residues 74-102). c-MYC-Zip:MAX-Zip complexes are observed both in the absence and in the presence of the reported small-molecule inhibitor 10058-F4 for both forms of c-MYC-Zip. DT IM-MS, in combination with molecular dynamics (MD), shows that the c-MYC-Zip:MAX-Zip complex [M+5H](5+) exists in two conformations, one extended with a collision cross section (CCS) of 1164 ± 9.3 Å(2) and one compact with a CCS of 982 ± 6.6 Å(2); similar values are observed for the two forms of c-MYC-ZipΔDT:MAX-Zip. Candidate geometries for the complexes have been evaluated with MD simulations. The helical leucine zipper structure previously determined from NMR measurements (Lavigne, P.; et al. J. Mol. Biol. 1998, 281, 165), altered to include the DT region and subjected to a gas-phase minimization, yields a CCS of 1247 Å(2), which agrees with the extended conformation we observe experimentally. More extensive MD simulations provide compact complexes which are found to be highly disordered, with CCSs that correspond to the compact form from experiment. In the presence of the ligand, the leucine zipper conformation is completely inhibited and only the more disordered species is observed, providing a novel method to study the effect of interactions of disordered systems and subsequent inhibition of the formation of an ordered helical complex.

Native N-glycopeptide thioester synthesis through N→S acyl transfer.

Peptide thioesters are important tools for the total synthesis of proteins using native chemical ligation (NCL). Preparation of glycopeptide thioesters, that enable the assembly of homogeneously glycosylated proteins, is complicated by the perceived fragile nature of the sugar moiety. Herein, we demonstrate the compatibility of thioester formation via N→S acyl transfer with native N-glycopeptides and report observations that will aid in their preparation.

Shifting Native Chemical Ligation into Reverse through N→S Acyl Transfer.

Peptide thioester synthesis by N→S acyl transfer is being intensively explored by many research groups the world over. Reasons for this likely include the often straightforward method of precursor assembly using Fmoc-based chemistry and the fundamentally interesting acyl migration process. In this review we introduce recent advances in this exciting area and discuss, in more detail, our own efforts towards the synthesis of peptide thioesters through N→S acyl transfer in native peptide sequences. We have found that several peptide thioesters can be readily prepared and, what's more, there appears to be ample opportunity for further development and discovery.

Rapid access to Asp/Glu sidechain hydrazides as thioester precursors for peptide cyclization and glycosylation.

Head-to-sidechain macrocylic peptides, and neoglycopeptides, were readily prepared by site-specific amidation of aspartic and glutamic acid sidechain hydrazides. Hydrazides, serving as latent thioesters, were introduced through regioselective opening of the corresponding Nα-Fmoc protected anhydride precursors.

Peptide fragments of a beta-defensin derivative with potent bactericidal activity.

Beta-defensins are known to be both antimicrobial and able to chemoattract various immune cells. Although the sequences of paralogous genes are not highly conserved, the core defensin structure is retained. Defb14-1C(V) has bactericidal activity similar to that of its parent peptide (murine beta-defensin Defb14) despite all but one of the canonical six cysteines being replaced with alanines. The 23-amino-acid N-terminal half of Defb14-1C(V) is a potent antimicrobial while the C-terminal half is not. Here, we use a library of peptide derivatives to demonstrate that the antimicrobial activity can be localized to a particular region. Overlapping fragments of the N-terminal region were tested for their ability to kill Gram-positive and Gram-negative bacteria. We demonstrate that the most N-terminal fragments (amino acids 1 to 10 and 6 to 17) are potent antimicrobials against Gram-negative bacteria whereas fragments based on sequence more C terminal than amino acid 13 have very poor activity against both Gram-positive and -negative types. We further test a series of N-terminal deletion peptides in both their monomeric and dimeric forms. We find that bactericidal activity is lost against both Gram types as the deletion region increases, with the point at which this occurs varying between bacterial strains. The dimeric form of the peptides is more resistant to the peptide deletions, but this is not due just to increased charge. Our results indicate that the primary sequence, together with structure, is essential in the bactericidal action of this beta-defensin derivative peptide and importantly identifies a short fragment from the peptide that is a potent bactericide.

Defensin-related peptide 1 (Defr1) is allelic to Defb8 and chemoattracts immature DC and CD4+ T cells independently of CCR6.

Beta-defensins comprise a family of cationic, antimicrobial and chemoattractant peptides. The six cysteine canonical motif is retained throughout evolution and the disulphide connectivities stabilise the conserved monomer structure. A murine beta-defensin gene (Defr1) present in the main defensin cluster of C57B1/6 mice, encodes a peptide with only five of the canonical six cysteine residues. In other inbred strains of mice, the allele encodes Defb8, which has the six cysteine motif. We show here that in common with six cysteine beta-defensins, defensin-related peptide 1 (Defr1) displays chemoattractant activity for CD4(+) T cells and immature DC (iDC), but not mature DC cells or neutrophils. Murine Defb2 replicates this pattern of attraction. Defb8 is also able to attract iDC but not mature DC. Synthetic analogues of Defr1 with the six cysteines restored (Defr1 Y5C) or with only a single cysteine (Defr1-1c(V)) chemoattract CD4(+) T cells with reduced activity, but do not chemoattract DC. Beta-defensins have previously been shown to attract iDC through CC receptor 6 (CCR6) but neither Defr1 or its related peptides nor Defb8, chemoattract cells overexpressing CCR6. Thus, we demonstrate that the canonical six cysteines of beta-defensins are not required for the chemoattractant activity of Defr1 and that neither Defr1 nor the six cysteine polymorphic variant allele Defb8, act through CCR6.

Peptide and protein thioester synthesis via N-->S acyl transfer.

Peptide and protein thioesters are playing an increasingly prominent role in the chemical toolbox for protein assembly and modification through Native Chemical Ligation (NCL). In this Emerging Area we highlight recent developments in a somewhat surprising route to thioesters: selective disruption of amides, the more stable carboxylic acid derivatives.

Access to phosphoproteins and glycoproteins through semi-synthesis, Native Chemical Ligation and N→S acyl transfer.

Peptide thioesters are important tools for protein synthesis and semi-synthesis through their use in Native Chemical Ligation (NCL). NCL can be employed to assemble site-specifically modified proteins that can help elucidate the mechanisms of biomolecular processes. In this article we explore the compatibility of phosphopeptide synthesis and glycopeptide synthesis with thioester production through N→S acyl transfer.

Exploring neoglycoprotein assembly through native chemical ligation using neoglycopeptide thioesters prepared via N-->S acyl transfer.

Sugars and simplified oligosaccharide "mimics" can be joined with protein fragments at pre-defined sites using reliable chemical reactions such as thiol alkylation and Cu(I) catalysed azide/acetylene ligation (click chemistry). These fragments have the potential to be assembled into neoglycoprotein therapeutics using native chemical ligation.

Binding a heparin derived disaccharide to defensin inspired peptides: insights to antimicrobial inhibition from gas-phase measurements.

Due to the ubiquitous presence of polysaccharide moieties on bacterial surfaces, it is hypothesised that a peptide-saccharide interaction plays a key role during the recognition of invading microorganisms by beta-defensins. We have employed different gas-phase methods to investigate these interactions. This manuscript describes: an MS-based titration assay measuring the gas-phase binding of ten beta-defensin related peptides to a sulfated disaccharide derived from heparin (HDD); ion mobility-mass spectrometry-determined collision cross sections of 3 peptides (both free and binding HDD); and results from molecular modelling with the aim of reconciling some of our experimental observations. We observe a clear qualitative correlation between the antimicrobial activity of several beta-defensins and related peptides and their gas-phase binding to a heparin-derived disaccharide (HDD). Four of the ten peptides show >100 micromolar K(d) values with HDD, and no bacteriocidal activity, illustrating that HDD binding correlates with peptide antimicrobial activity. For five of the remaining six peptides, bacteriocidal activity was re-measured with HDD present. For the peptides containing intramolecular disulfide bonds in two out of five, bacteriocidal activity was reduced approximately 10-fold; for the remaining three peptides, which lack intramolecular disulfide bonds, HDD addition had little effect on bacteriocidal activity. The latter results are suggested to arise from the greater degree of flexibility imparted by the removal of disulfide bonds giving the peptides the ability to envelope HDD and assume a "defensin-like" fold. Thus gas-phase analysis is put forward as a powerful tool for assessing the properties of antimicrobial peptides providing valuable insights in the mechanism of antimicrobial inhibition.

3-Mercaptopropionic acid-mediated synthesis of peptide and protein thioesters.

Peptides and proteins fragment sequence-specifically in the presence of 3-mercaptopropionic acid to afford thioesters which can be used in native chemical ligation reactions.

Peptide thioester synthesis through N-->S acyl-transfer: application to the synthesis of a beta-defensin.

Peptide thioesters readily prepared through N-->S acyl transfer of a specific C-terminal motif provide access to biologically active mini-proteins using native chemical ligation.