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One of the most desirable properties that biomaterials designed for tissue engineering or drug delivery applications should fulfill is biodegradation and resorption without toxicity. Therefore, there is an increasing interest in the development of biomaterials able to be enzymatically degraded once implanted at the injury site or once delivered to the target organ. In this paper, we demonstrate the protease sensitivity of self-assembling amphiphilic peptides, in particular, RAD16-I (AcN-RADARADARADARADA-CONH2), which contains four potential cleavage sites for trypsin. We detected that when subjected to thermal denaturation, the peptide secondary structure suffers a transition from β-sheet to random coil. We also used Matrix-Assisted Laser Desorption/Ionization-Time-Of-Flight (MALDI-TOF) to detect the proteolytic breakdown products of samples subjected to incubation with trypsin as well as atomic force microscopy (AFM) to visualize the effect of the degradation on the nanofiber scaffold. Interestingly, thermally treated samples had a higher extent of degradation than non-denatured samples, suggesting that the transition from β-sheet to random coil leaves the cleavage sites accessible and susceptible to protease degradation. These results indicate that the self-assembling peptide can be reduced to short peptide sequences and, subsequently, degraded to single amino acids, constituting a group of naturally biodegradable materials optimal for their application in tissue engineering and regenerative medicine. [ABSTRACT FROM AUTHOR]

Milk proteins are recognized as the main source of biologically active peptides. Casein's primary structure contains several bioactive amino acid sequences on its latent inactive form. These potential active sequences can be released during cheese manufacture and ripening, giving rise to peptides with biological activity such as antihypertensive, antidiabetic, antioxidant, immunomodulatory, and mineral‐binding properties. However, the presence of biopeptides in cheese does not imply actual biological activity in vivo because these peptides can be further hydrolyzed during gastrointestinal transit. This paper reviews the recent advances in biopeptide formation in ripened cheeses production, focusing on the influence of technological parameters affecting proteolysis and the consequent release of peptides. The main discoveries in the field of cheese peptide digestion through recent in vivo and in vitro model studies are also reviewed. © 2021 Society of Chemical Industry [ABSTRACT FROM AUTHOR]

Incubation of calf tens cortex homogenate with [14C]putrescine or dansylcadaverine, followed by two-dimensional gel electrophoresis and fluorography, enabled the identification of three different β-crystallin chains as the endogenous substrates of Ca2+-dependent lens transglutaminase (R-glutaminyl-peptide:amine-γ-glutamylyltransferase, EC 2.3.2.13). One of these is βBp, the predominant subunit of β-crystallin, of which the amino acid sequence is known. The site of amine-labeling in βBp could be located, by limited proteolysis, in the N-terminal domain of this chain. Tryptic digestion of the N-terminal domain and subdigestion with elastase of the N-terminal tryptic peptide identified glutamine-7 as the single residue to which the amines are bound. This is the first example of an endogenous substrate of intracellular transglutaminase in which the site of the acyl-donor glutamine residue has been established. Tryptic digestion of the putrescine-labeled β-crystallin aggregate, followed by high-voltage paper electrophoresis, provided a preliminary characterization of the labeled peptides originating from the other two labeled β subunits. [ABSTRACT FROM AUTHOR]

Many useful properties are assigned to camel ( Camelus dromedarius ) milk, which is traditionally used for the treatment of tuberculosis, gastroenteritis, and allergy in many countries. Some amino acid sequences, which are encrypted in the camel proteins, may play a beneficial role in human health once they are released from milk either in vivo during normal digestion or by proteolysis with purified enzymes or during bacterial fermentation. Similar to the bovine milk counterparts, camel milk bioactive peptides may display a variety of potential activities that were almost always unveiled from in vitro analyses: anti-microbial, anti-oxidative, anti-hypertensive, anti-inflammatory, and immunomodulatory activities. Today, there is a growing interest for bioactive peptides generated from camel milk. This paper reviews available data on the potential biological activities of the camel milk proteins and their peptides liberated either during milk fermentation with proteolytic bacterial strains or by enzyme hydrolysis with specific proteases or simulated gastro-intestinal digestion. [ABSTRACT FROM AUTHOR]

Oxidative stress is an automatic mechanism responsible for the commencement and continuance of liver injury. In this study, an antioxidative peptide Val-Thr-Ala-Leu (VTAL) was purified from simulated gastrointestinal digestion of protein hydrolysates of the triploid oyster Magallana gigas. Significant antioxidant activity was identified, as well as a protective effect against acetaminophen (APAP)-induced human liver cancer (HepG2) cells. The results suggested that the antioxidant activity improved in a dose-dependent manner. The highest cell viability (88.105 ± 3.62%) was observed in 15 mM APAP-induced cells when treated with 25 μg/mL M. gigas peptide [M.g (pep)]. The peptide sequences include hydrophobic amino acids, which could be responsible for its chemoprotective and antioxidant activities. Treatment with M.g (pep) significantly promoted the proliferation of HepG2 cells, thus protecting them against APAP and imbuing them with significant antioxidant capacity. M.g (pep) could be beneficial for treating drug-induced oxidative stress and liver damage. Additionally, M.g (pep) could serve as an alternative to synthetic antioxidant drugs. [ABSTRACT FROM AUTHOR]

The amino acid sequence of the first domain (positions 1·175) of Panulirus interruptus hemocyanin subunit a has been determined. The sequence of residues 1·158 (18-kDa fragment obtained by limited proteolysis) was derived from peptides obtained by digestion of this fragment with CNBr and trypsin and by subdigestion of these peptides with other enzymes. The peptides were sequenced automatically or manually. The amino acid sequence has been fitted into the electron-density map at 0.32-nm resolution. The residues of domain 1 are folded into a larger, mainly helical, globular part, containing one disulfide bridge, and a smaller part near the molecular twofold axis. The latter part consists of an α helix and a β strand which contains a covalently attached carbohydrate moiety. The sites susceptible to limited proteolytic cleavage of the subunit are discussed. Comparison of the N-terminal sequence with those of other arthropod hemocyanins revealed, besides an N- terminal extension of five residues, the presence of a 21-residue loop (positions 22–42) in the crustacean sequences. This loop contains helix 1.2, a less defined region in the electron-density map. It is absent in chelicerate sequences. Strong evidence is presented that: (a) the structure of the first 21 residues (including helix 1.1) is the same in all arthropod hemocyanins with known amino acid sequence; (b) a stretch containing about 15 residues (including part of helix 1.3) following the 21-residue loop has a different structure in crustaceans and chelicerates; (c) the rest of domain 1 has the same structure again. It is shown that all conserved residues are in the contact region with the other two domains. [ABSTRACT FROM AUTHOR]

Performic-acid-oxidized isoinhibitor K of snails (Helix pomatia) was subjected to arginine-directed tryptic proteolysis. Six peptide fragments including one overlap peptide from limited cleavage of the Arg-3-Pro-4 bond were purified to homogeneity. Four arginine peptides and the C-terminal peptide were sequenced by automatic Edman degradation using a special peptide program. The phenylthiohydantoins were all identified by chemical ionization mass spectrometry, except four cysteic acid residues that were identified on an amino acid analyzer after acid hydrolysis. Quantitative evaluation of the phenylthiohydantoins by chemical ionization mass spectrometry using total molecular-ion beam integration greatly facilitated sequencing. The mass spectrum of the dipeptide < GluGly revealed that the N-terminus was blocked by pyroglutamic acid. The complete amino acid sequence of isoinhibitor K was determined, An almost 50% homology between the sequences of the snail inhibitor and the bovine trypsin-kallikrein® inhibitor (Kunitz) became obvious. A comparison of all homologous sequences of this particular class of proteins known to date is presented. [ABSTRACT FROM AUTHOR]

Simple Summary: Recently, dairy proteins, in addition to their basic nutritional role in the diet, were recognized as a source of bioactive peptides. Such peptides are encoded within the primary structure of the protein and can be released by enzymatic hydrolysis. The growing interest in the development of functional foods for the benefit of consumer health led to a recent increase in research on the production of bioactive peptides from different matrices and production methods. The use of aspartic proteases from stigmas of mature artichoke (Cynara scolymus L.) flowers to obtain hydrolytic enzymes (cinarases) in the production of bioactive peptides would involve the utilization of an agricultural residue of a plant species of great socio-economic importance. In the present study, the characterization of the optimal hydrolysis conditions of artichoke flower extracts was carried out for the production of peptides from bovine casein. Furthermore, the angiotensin-converting enzyme-I inhibitory activity and the antioxidant capacity against 1,1-diphenyl-2-picrylhydrazyl (DPPH) and 2,2'-azino-bis (3-ethylbenzothiazoline-6-sulphonic acid (ABTS) free radicals in vitro were determined for the obtained hydrolysates. The results revealed that the water-soluble extract of artichoke flower could be suitable for the production of bioactive peptides from whole bovine casein. The aim of this work is to establish the most suitable proteolysis conditions to obtain bovine casein hydrolysates containing peptides with antioxidant and antihypertensive capacity. To this end, the proteolytic activity of Cynara scolymus L. flower extracts was characterized on whole bovine casein, evaluating the effect of several factors (pH, temperature, substrate concentration, enzyme concentration, and hydrolysis time). The optimal conditions to carry out the hydrolysis with the C. scolymus L. extract were as follows: pH 6.2, 50 °C, and 0.023 mg·mL−1 of extract-protein concentration. A Michaelis constant (Km) value of 5.66 mg·mL−1 and a maximum rate of reaction (Vmax) of 8.47 mUAbs∙min−1 were observed. The optimal hydrolysis time was 17 h. The casein hydrolysates obtained with these conditions contained peptides with antioxidant activity (1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging capacity: 30.89%; Trolox equivalent antioxidant capacity (TEAC) against 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) free radical (ABTS●+): 4.43 mM Trolox equivalent·mg−1 peptide) and antihypertensive activity, showing 55.05% angiotensin-converting enzyme-I inhibition in vitro. [ABSTRACT FROM AUTHOR]

The BIOPEP-UWM™ database of bioactive peptides (formerly BIOPEP) has recently become a popular tool in the research on bioactive peptides, especially on these derived from foods and being constituents of diets that prevent development of chronic diseases. The database is continuously updated and modified. The addition of new peptides and the introduction of new information about the existing ones (e.g., chemical codes and references to other databases) is in progress. New opportunities include the possibility of annotating peptides containing D-enantiomers of amino acids, batch processing option, converting amino acid sequences into SMILES code, new quantitative parameters characterizing the presence of bioactive fragments in protein sequences, and finding proteinases that release particular peptides. [ABSTRACT FROM AUTHOR]

(Arginine-alanine-aspartic acid-alanine)4 ((RADA)4) nanoscaffolds are excellent candidates for use as peptide delivery vehicles: they are relatively easy to synthesize with custom bio-functionality, and assemble in situ to allow a focal point of release. This enables (RADA)4 to be utilized in multiple release strategies by embedding a variety of bioactive molecules in an all-in-one "construct". One novel strategy focuses on the local, on-demand release of peptides triggered via proteolysis of tethered peptide sequences. However, the spatial-temporal morphology of self-assembling nanoscaffolds may greatly influence the ability of enzymes to both diffuse into as well as actively cleave substrates. Fine structure and its impact on the overall effect on peptide release is poorly understood. In addition, fractal networks observed in nanoscaffolds are linked to the fractal nature of diffusion in these systems. Therefore, matrix morphology and fractal dimension of virgin (RADA)4 and mixtures of (RADA)4 and matrix metalloproteinase 2 (MMP-2) cleavable substrate modified (RADA)4 were characterized over time. Sites of high (glycine-proline-glutamine-glycine+isoleucine-alanine-serine-glutamine (GPQG+IASQ), CP1) and low (glycine-proline-glutamine-glycine+proline-alanine-glycine-glutamine (GPQG+PAGQ), CP2) cleavage activity were chosen. Fine structure was visualized using transmission electron microscopy. After 2 h of incubation, nanofiber networks showed an established fractal nature; however, nanofibers continued to bundle in all cases as incubation times increased. It was observed that despite extensive nanofiber bundling after 24 h of incubation time, the CP1 and CP2 nanoscaffolds were susceptible to MMP-2 cleavage. The properties of these engineered nanoscaffolds characterized herein illustrate that they are an excellent candidate as an enzymatically initiated peptide delivery platform. [ABSTRACT FROM AUTHOR]