No abstract

The release of hormones is subject to a complex and finely tuned regulation system. The biosynthesis plays a key role by specifically converting the prohormone precursor into its biological active product(s). A family of mammalian proteases could be identified to be responsible for the endoproteolytic processing. These subtilisin/kexin-like prohormone convertases (PC) recognize their substrates at single or pairs of basic residues with a high substrate specificity. The so far known seven members include PC1/3, PC2, furin/PACE, PACE4, PC4, PC5/6 and PC7/SPC7/LPC/PC8. PC1/3 and PC2 are the most important enzymes for the processing of prohormones, whereas furin is the only one that causes lethality in knock-out models. Tissue-specific co-localization of the prohormone and the PC as well as distinct characteristics of both, like the secondary structures, determine the possible conversion processes. Identification of such determinants implies a great potential for the development of novel drug targets. To obtain sufficient amounts for the in vitro characterization of prohormones, chemical and recombinant synthesis methods have been developed. Application of expressed protein ligation lead to the semisynthesis of the first chemically modified analogs of a full-length proneurohormone (pro-neuropeptide Y). Structural analyses mainly on peptides of the pro-oxytocin/neurophysin system and on prosomatostatin highlighted the importance of flexible turn or loop structures adjacent to the cleavage site for the specific substrate-enzyme active site interaction. Prohormones and their processing show multiple functions. Therapeutic application including PC inhibitors is very promising for the treatment of disorders like cancer.

Chemical synthesis is the most powerful way to assemble selectively modified proteins and is a tool for engineering proteins that are inaccessible by standard site-directed mutagenesis. A number of methods for the chemical synthesis of proteins have been developed, of which native chemical ligation (NCL) [1] is currently the most widely applied. This ligation approach is based on the chemoselective reaction of a C-terminal peptide thioester with a peptide that has a cysteine residue at its N terminus and results in the formation of a native peptide bond. Various attempts have been made to overcome the requirement for the cysteine residue at the ligation site. However, although some of these were successful, for example, with glycine [2] or selenocysteine [3] in place of cysteine, this feature still limits the flexibility of NCL. Expressed protein ligation (EPL) [4] has opened up a whole new dimension in terms of accessible protein targets, because it applies recombinant techniques. However, this method suffers from restrictions similar to those of native ligation. Proteases that are used as ligation catalysts tolerate greater structural diversity at the coupling site. However, the limited primary substrate specificity of these enzymes reduces the scope of their use in synthesis. In the subtiligase-catalyzed fragment-condensation strategy (SCFC), which is one of the most successful enzymatic ligation methods to date, a genetically optimized enzyme is used for the synthesis of native and modified ribonuclease A.[5] Although this method increased the size of synthetic targets to entire proteins, it still suffers from the typical requirement for the careful selection of suitable coupling sites, because of limited enzyme specificity. Thus, despite significant progress in protein synthesis, alternative ligation strategies with broader applicability are still needed.Herein we report a further step toward this ambitious aim-expressed enzymatic ligation (EEL), which combines the advantages of EPL with those of the substrate-mimeticmediated ligation strategy. The latter eliminates the specificity problem of classical protease-catalyzed synthesis through the attachment of an active-site-specific ester leaving group at the C-terminus of a nonspecific peptide moiety.[6] This combined approach also takes advantage of the recombinant expression of long-chain peptide thioesters, which can then act as substrate mimetics. Through this technique, peptide coupling can be carried out by an ordinary protease, for example, the Glu/Asp-specific serine protease V8 from Staphylococcus aureus, at nonspecific ligation sites, regardless of the specificity of the enzyme.The feasibility of EEL has been demonstrated through the synthesis of a cysteine-free carboxyfluorescein (CF)-labeled analogue of the 69-mer prohormone neuropeptide Y (proNPY; Scheme 1). ProNPY processing leads to one of the most abundant neuropeptides in the brain, neuropeptide Y (NPY), which is involved in numerous biological events, such as the regulation of food intake and...

Enzymatische Synthese und exprimierte Proteinligation werden hier zur Proteinsynthese kombiniert, wobei Proteinthioester mit spezifischen Abgangsgruppen von der Protease V8 aus Staphylococcus aureus zur Bildung von Peptidbindungen genutzt werden. Auf diesem Weg wurden Fluoreszenz‐markierte Analoga des Pro‐Neuropeptids Y synthetisiert.

Enzymatic cleavage of prohormone neuropeptide Y (proNPY) leads to mature neuropeptide Y (NPY), a widely distributed neuropeptide with multiple functions both peripherally and centrally. A single dibasic pair of amino acids, Lys38-Arg39, represents the recognition motif for a class of hormone-processing enzymes known as prohormone convertases (PCs). Two members of this PC family, PC1/3 and PC2, are involved in proNPY cleavage. The aim of this work was to establish an effective method for the generation of full-length 69-amino acid proNPY analogues for further studies of prohormone convertase interaction. We have chosen two ligation sites in order to perform the semisynthesis of proNPY analogues by expressed protein ligation (EPL). By using the intein-mediated purification system (IMPACT) with improved conditions for intein splicing, we were able to isolate proNPY 1-40 and proNPY 1-54 fragments as Cterminal thioesters. Peptides bearing Nterminal cysteine instead of the naturally occurring Ser41 and Thr55 residues, respectively, were generated by solid-phase peptide synthesis. Moreover, labels (carboxyfluorescein and biotin) were inserted into the peptide sequences. The synthesis of the [C41]proNPY 41-69 fragment, which proved to be a difficult peptide sequence, could be achieved by the incorporation of two pseudo-proline derivatives. Western blot analysis revealed that all five proNPY analogues are recognized by monoclonal antibodies directed against NPY as well as against the Cflanking peptide of NPY (CPON).