Peptides are fragments of proteins that carry out biological functions. They act as signaling entities via all domains of life and interfere with protein-protein interactions, which are indispensable in bio-processes. Short peptides include fundamental molecular information for a prelude to the symphony of life. They have aroused considerable interest due to their unique features and great promise in innovative bio-therapies. This work focusing on the current state-of-the-art short peptide-based therapeutical developments is the first global review written by researchers from all continents, as a celebration of 100 years of peptide therapeutics since the commencement of insulin therapy in the 1920s. Peptide “drugs” initially played only the role of hormone analogs to balance disorders. Nowadays, they achieve numerous biomedical tasks, can cross membranes, or reach intracellular targets. The role of peptides in bio-processes can hardly be mimicked by other chemical substances. The article is divided into independent sections, which are related to either the progress in short peptide-based theranostics or the problems posing challenge to bio-medicine. In particular, the SWOT analysis of short peptides, their relevance in therapies of diverse diseases, improvements in (bio)synthesis platforms, advanced nano-supramolecular technologies, aptamers, altered peptide ligands and in silico methodologies to overcome peptide limitations, modern smart bio-functional materials, vaccines, and drug/gene-targeted delivery systems are discussed.
Angiotensin-converting enzyme 2 (ACE2) is critically involved in cardiovascular physiology and pathology, and is currently clinically evaluated to treat acute lung failure. Here we show that the B38-CAP, a carboxypeptidase derived from Paenibacillus sp. B38, is an ACE2-like enzyme to decrease angiotensin II levels in mice. In protein 3D structure analysis, B38-CAP homolog shares structural similarity to mammalian ACE2 with low sequence identity. In vitro, recombinant B38-CAP protein catalyzed the conversion of angiotensin II to angiotensin 1-7, as well as other known ACE2 target peptides. Treatment with B38-CAP suppressed angiotensin II-induced hypertension, cardiac hypertrophy, and fibrosis in mice. Moreover, B38-CAP inhibited pressure overload-induced pathological hypertrophy, myocardial fibrosis, and cardiac dysfunction in mice. Our data identify the bacterial B38-CAP as an ACE2-like carboxypeptidase, indicating that evolution has shaped a bacterial carboxypeptidase to a human ACE2-like enzyme. Bacterial engineering could be utilized to design improved protein drugs for hypertension and heart failure.
Angiotensin-converting enzyme 2 (ACE2) is a carboxypeptidase which is highly homologous to angiotensin-converting enzyme (ACE). ACE2 produces vasodilator peptides angiotensin 1-7 from angiotensin II. In the present study, we synthesized various internally quenched fluorogenic (IQF) substrates (fluorophore-Xaa-Pro-quencher) based on the cleavage site of angiotensin II introducing N-terminal fluorophore N-methylanthranilic acid (Nma) and C-terminal quencher N ε -2,4-dinitrophenyl-lysine [Lys(Dnp)]. The synthesized mixed substrates "Nma-Xaa-Pro-Lys(Dnp)" were hydrolyzed by recombinant human (rh) ACE2. The amount of each product was determined by liquid chromatography mass spectrometry (LC-MS) with fluorescence detection and it was found that Nma-His-Pro-Lys(Dnp) is the most suitable substrate for rhACE2. The K m , k cat , and k cat /K m values of Nma-His-Pro-Lys(Dnp) on rhACE2 were determined to be 23.3 μM, 167 s −1 , and 7.17 μM −1 s −1 , respectively. Using the rhACE2 and the newly developed IQF substrate, we found rhACE2 inhibitory activity in soybean and isolated the active compound soybean ACE2 inhibitor (ACE2iSB). The physicochemical data on the isolated ACE2iSB were identical to those of nicotianamine. ACE2iSB strongly inhibited rhACE2 activity with an IC 50 value of 84 nM. This is the first demonstration of an ACE2 inhibitor from foodstuffs.
During over a decade of study on aspartic protease inhibitors and water‐soluble prodrugs, in 2003, we discovered that the presence of an O‐acyl instead of N‐acyl residue within the peptide backbone significantly changed the secondary structure of the native peptide. In addition, the target peptide was subsequently generated by an O‐N intramolecular acyl migration reaction. These findings led to the development of a novel method, called “O‐acyl isopeptide method,” for the synthesis of peptides containing difficult sequence. Further application of the method to Alzheimer's Aβ1‐42 revealed that the O‐acyl isopeptide of Aβ1‐42 could be effectively synthesized and stored without spontaneous self‐assembly. Intact monomer Aβ1‐42 could then be obtained from the isopeptide under physiological experimental conditions. We named the O‐acyl isopeptide as “Click Peptide,” because of its “quick and easy one‐way conversion” to the parent Aβ1‐ 42. Application of the click peptide has provided a new basis for the investigation of the biological functions of Aβ1‐42 by inducible activation of its self‐assembly. The O‐acyl isopeptide method has further evolved as a general method for peptides synthesis with our recent developments of “O‐acyl isodipeptide units” and “racemization‐free segment condensation methodology.” Isodipeptide units have enabled routine use of the O‐acyl isopeptide method by avoiding the often difficult esterification reaction on resin. “Racemizationfree segment condensation methodology” has been achieved by employing N‐segments possessing a C‐terminal urethaneprotected O‐acyl Ser/Thr residues. The synthesis of long peptides/proteins by racemization‐free segment condensation has thus become possible at Ser/Thr residues instead of Cterminal Gly/Pro residues. As the O‐acyl isopeptide method becomes more widely utilized, we have composed this review to facilitate its application for the production of peptides and proteins. © 2007 Wiley Periodicals, Inc. Biopolymers (Pept Sci) 88: 253–262, 2007. This article was originally published online as an accepted preprint. The ‘Published Online’ date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com
A solubilizing Trt-K tag was developed for the effective chemical preparation of peptides/proteins with low solubility. The Trt-K tag comprises a hydrophilic oligo-Lys sequence and a trityl anchor, and can be selectively introduced to a side chain thiol of Cys of deprotected peptides/proteins with a trityl alcohol-type introducing reagent Trt(OH)-K under acidic conditions. Significantly, the ligation product in the reaction mixture of a thiol-additive-free native chemical ligation can be modified directly in a one-pot manner to facilitate the isolation of the product by high-performance liquid chromatography. Finally, the Trt-K tag can be readily removed with a standard trifluoroacetic acid cocktail. Using this easy-to-attach/detach tag-aided method, a hepatitis B virus capsid protein that is usually difficult to handle was synthesized successfully.
Native chemical ligation (NCL) performed without resorting to the use of thiol additives was demonstrated to be an efficient and effective procedure for synthesizing Cys-rich peptides. This method using tris(2-carboxyethyl)phosphine (TCEP) as a reducing agent facilitates the ligation reaction even at the Thr-Cys or Ile-Cys site and enables one-pot synthesis of Cys-rich peptides throughout NCL and oxidative folding.
Inducing newly synthesized proteins to appropriate locations is an indispensable biological function in every organism. Integration of proteins into biomembranes in Escherichia coli is mediated by proteinaceous factors, such as Sec translocons and an insertase YidC. Additionally, a glycolipid named MPIase (membrane protein integrase), composed of a long sugar chain and pyrophospholipid, was proven essential for membrane protein integration. We reported that a synthesized minimal unit of MPIase possessing only one trisaccharide, mini-MPIase-3, involves an essential structure for the integration activity. Here, to elucidate integration mechanisms using MPIase, we analyzed intermolecular interactions of MPIase or its synthetic analogs with a model substrate, the Pf3 coat protein, using physicochemical methods. Surface plasmon resonance (SPR) analyses revealed the importance of a pyrophosphate for affinity to the Pf3 coat protein. Compared with mini-MPIase-3, natural MPIase showed faster association and dissociation due to its long sugar chain despite the slight difference in affinity. To focus on more detailed MPIase substructures, we performed docking simulations and saturation transfer difference-nuclear magnetic resonance. These experiments yielded that the 6-O-acetyl group on glucosamine and the phosphate of MPIase play important roles leading to interactions with the Pf3 coat protein. The high affinity of MPIase to the hydrophobic region and the basic amino acid residues of the protein was suggested by docking simulations and proven experimentally by SPR using protein mutants devoid of target regions. These results demonstrated the direct interactions of MPIase with a substrate protein and revealed detailed mechanisms of membrane protein integration.
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