Biochemical Characterisation of Sis: A Distinct Thermophilic PETase with Enhanced NanoPET Substrate Hydrolysis and Thermal Stability.

Polyethylene terephthalate (PET) degradation by enzymatic hydrolysis is significant for addressing plastic pollution and fostering sustainable waste management practices. Identifying thermophilic and thermostable PET hydrolases is particularly crucial for industrial bioprocesses, where elevated temperatures may enhance enzymatic efficiency and process kinetics. In this study, we present the discovery of a novel thermophilic and thermostable PETase enzyme named Sis, obtained through metagenomic sequence-based analysis. Sis exhibits robust activity on nanoPET substrates, demonstrating effectiveness at temperatures up to 70 °C and displaying exceptional thermal stability with a melting temperature (Tm) of 82 °C. Phylogenetically distinct from previously characterised PET hydrolases, Sis represents a valuable addition to the repertoire of enzymes suitable for PET degradation.

The Antimicrobial, Antibiofilm and Anti-Inflammatory Activities of P13#1, a Cathelicidin-like Achiral Peptoid.

Cationic antimicrobial peptides (CAMPs) are powerful molecules with antimicrobial, antibiofilm and endotoxin-scavenging activities. These properties make CAMPs very attractive drugs in the face of the rapid increase in multidrug-resistant (MDR) pathogens, but they are limited by their susceptibility to proteolytic degradation. An intriguing solution to this issue could be the development of functional mimics of CAMPs with structures that enable the evasion of proteases. Peptoids (N-substituted glycine oligomers) are an important class of peptidomimetics with interesting benefits: easy synthetic access, intrinsic proteolytic stability and promising bioactivities. Here, we report the characterization of P13#1, a 13-residue peptoid specifically designed to mimic cathelicidins, the best-known and most widespread family of CAMPs. P13#1 showed all the biological activities typically associated with cathelicidins: bactericidal activity over a wide spectrum of strains, including several ESKAPE pathogens; the ability to act in combination with different classes of conventional antibiotics; antibiofilm activity against preformed biofilms of Pseudomonas aeruginosa, comparable to that of human cathelicidin LL-37; limited toxicity; and an ability to inhibit LPS-induced proinflammatory effects which is comparable to that of "the last resource" antibiotic colistin. We further studied the interaction of P13#1 with SDS, LPSs and bacterial cells by using a fluorescent version of P13#1. Finally, in a subcutaneous infection mouse model, it showed antimicrobial and anti-inflammatory activities comparable to ampicillin and gentamicin without apparent toxicity. The collected data indicate that P13#1 is an excellent candidate for the formulation of new antimicrobial therapies.

Simultaneous Irradiation with UV-A, -B, and -C Lights Promotes Effective Decontamination of Planktonic and Sessile Bacteria: A Pilot Study.

Surfaces in highly anthropized environments are frequently contaminated by both harmless and pathogenic bacteria. Accidental contact between these contaminated surfaces and people could contribute to uncontrolled or even dangerous microbial diffusion. Among all possible solutions useful to achieve effective disinfection, ultraviolet irradiations (UV) emerge as one of the most "Green" technologies since they can inactivate microorganisms via the formation of DNA/RNA dimers, avoiding the environmental pollution associated with the use of chemical sanitizers. To date, mainly UV-C irradiation has been used for decontamination purposes, but in this study, we investigated the cytotoxic potential on contaminated surfaces of combined UV radiations spanning the UV-A, UV-B, and UV-C spectrums, obtained with an innovative UV lamp never conceived so far by analyzing its effect on a large panel of collection and environmental strains, further examining any possible adverse effects on eukaryotic cells. We found that this novel device shows a significant efficacy on different planktonic and sessile bacteria, and, in addition, it is compatible with eukaryotic skin cells for short exposure times. The collected data strongly suggest this new lamp as a useful device for fast and routine decontamination of different environments to ensure appropriate sterilization procedures.

Bioconversion of 4-hydroxyestradiol by extradiol ring-cleavage dioxygenases from Novosphingobium sp. PP1Y.

Livestock breeding activities and pharmaceutical wastes lead to considerable accumulation of steroid hormones and estrogens in wastewaters. Here estrogens act as pro-cancerogenic agents and endocrine disruptors interfering with the sexual development of aquatic animals and having toxic effects in humans. Environmental bacteria play a vital role in estrogens degradation. Their wide reservoir of enzymes, such as ring cleavage dioxygenases (RCDs), can degrade the steroid nucleus, catalyzing the meta-cleavage of A, B or D steroid rings. In this work, 4 extra-diol ring cleavage dioxygenases (ERCDs), PP28735, PP26077, PP00124 and PP00193, were isolated from the marine sphingomonad Novosphingobium sp. PP1Y and characterized. Enzymes kinetic parameters were determined on different synthetic catecholic substrates. Then, the bioconversion of catechol estrogens was evaluated. PP00124 showed to be an efficient catalyst for the degradation of 4-hydroxyestradiol (4-OHE2), a carcinogenic hydroxylated derivate of E2. 4-OHE2 complete cleavage was obtained using PP00124 both in soluble form and in whole recombinant E. coli cells. LC-MS/MS analyses confirmed the generation of a semialdehyde product, through A-ring meta cleavage. To the best of our knowledge, PP00124 is the first characterized enzyme able to directly degrade 4-OHE2 via meta cleavage. Moreover, the complete 4-OHE2 biodegradation using recombinant whole cells highlighted advantages for bioremediation purposes.

Protective Effects of Recombinant Human Angiogenin in Keratinocytes: New Insights on Oxidative Stress Response Mediated by RNases.

Human angiogenin (ANG) is a 14-kDa ribonuclease involved in different pathophysiological processes including tumorigenesis, neuroprotection, inflammation, innate immunity, reproduction, the regeneration of damaged tissues and stress cell response, depending on its intracellular localization. Under physiological conditions, ANG moves to the cell nucleus where it enhances rRNA transcription; conversely, recent reports indicate that under stress conditions, ANG accumulates in the cytoplasmic compartment and modulates the production of tiRNAs, a novel class of small RNAs that contribute to the translational inhibition and recruitment of stress granules (SGs). To date, there is still limited and controversial experimental evidence relating to a hypothetical role of ANG in the epidermis, the outermost layer of human skin, which is continually exposed to external stressors. The present study collects compelling evidence that endogenous ANG is able to modify its subcellular localization on HaCaT cells, depending on different cellular stresses. Furthermore, the use of recombinant ANG allowed to determine as this special enzyme is effectively able to counter at various levels the alterations of cellular homeostasis in HaCaT cells, actually opening a new vision on the possible functions that this special enzyme can support also in the stress response of human skin.

Human Cryptic Host Defence Peptide GVF27 Exhibits Anti-Infective Properties against Biofilm Forming Members of the Burkholderia cepacia Complex.

Therapeutic solutions to counter Burkholderia cepacia complex (Bcc) bacteria are challenging due to their intrinsically high level of antibiotic resistance. Bcc organisms display a variety of potential virulence factors, have a distinct lipopolysaccharide naturally implicated in antimicrobial resistance. and are able to form biofilms, which may further protect them from both host defence peptides (HDPs) and antibiotics. Here, we report the promising anti-biofilm and immunomodulatory activities of human HDP GVF27 on two of the most clinically relevant Bcc members, Burkholderia multivorans and Burkholderia cenocepacia. The effects of synthetic and labelled GVF27 were tested on B. cenocepacia and B. multivorans biofilms, at three different stages of formation, by confocal laser scanning microscopy (CLSM). Assays on bacterial cultures and on human monocytes challenged with B. cenocepacia LPS were also performed. GVF27 exerts, at different stages of formation, anti-biofilm effects towards both Bcc strains, a significant propensity to function in combination with ciprofloxacin, a relevant affinity for LPSs isolated from B. cenocepacia as well as a good propensity to mitigate the release of pro-inflammatory cytokines in human cells pre-treated with the same endotoxin. Overall, all these findings contribute to the elucidation of the main features that a good therapeutic agent directed against these extremely leathery biofilm-forming bacteria should possess.

Environment-Sensitive Fluorescent Labelling of Peptides by Luciferin Analogues.

Environment-sensitive fluorophores are very valuable tools in the study of molecular and cellular processes. When used to label proteins and peptides, they allow for the monitoring of even small variations in the local microenvironment, thus acting as reporters of conformational variations and binding events. Luciferin and aminoluciferin, well known substrates of firefly luciferase, are environment-sensitive fluorophores with unusual and still-unexploited properties. Both fluorophores show strong solvatochromism. Moreover, luciferin fluorescence is influenced by pH and water abundance. These features allow to detect local variations of pH, solvent polarity and local water concentration, even when they occur simultaneously, by analyzing excitation and emission spectra. Here, we describe the characterization of (amino)luciferin-labeled derivatives of four bioactive peptides: the antimicrobial peptides GKY20 and ApoBL, the antitumor peptide p53pAnt and the integrin-binding peptide RGD. The two probes allowed for the study of the interaction of the peptides with model membranes, SDS micelles, lipopolysaccharide micelles and Escherichia coli cells. Kd values and binding stoichiometries for lipopolysaccharide were also determined. Aminoluciferin also proved to be very well-suited to confocal laser scanning microscopy. Overall, the characterization of the labeled peptides demonstrates that luciferin and aminoluciferin are previously neglected environment-sensitive labels with widespread potential applications in the study of proteins and peptides.

Host defence peptides identified in human apolipoprotein B as promising antifungal agents.

Therapeutic options to treat invasive fungal infections are still limited. This makes the development of novel antifungal agents highly desirable. Naturally occurring antifungal peptides represent valid candidates, since they are not harmful for human cells and are endowed with a wide range of activities and their mechanism of action is different from that of conventional antifungal drugs. Here, we characterized for the first time the antifungal properties of novel peptides identified in human apolipoprotein B. ApoB-derived peptides, here named r(P)ApoBLPro, r(P)ApoBLAla and r(P)ApoBSPro, were found to have significant fungicidal activity towards Candida albicans (C. albicans) cells. Peptides were also found to be able to slow down metabolic activity of Aspergillus niger (A. niger) spores. In addition, experiments were carried out to clarify the mechanism of fungicidal activity of ApoB-derived peptides. Peptides immediately interacted with C. albicans cell surfaces, as indicated by fluorescence live cell imaging analyses, and induced severe membrane damage, as indicated by propidium iodide uptake induced upon treatment of C. albicans cells with ApoB-derived peptides. ApoB-derived peptides were also tested on A. niger swollen spores, initial hyphae and branched mycelium. The effects of peptides were found to be more severe on swollen spores and initial hyphae compared to mycelium. Fluorescence live cell imaging analyses confirmed peptide internalization into swollen spores with a consequent accumulation into hyphae. Altogether, these findings open interesting perspectives to the application of ApoB-derived peptides as effective antifungal agents. KEY POINTS: Human cryptides identified in ApoB are effective antifungal agents. ApoB-derived cryptides exert fungicidal effects towards C. albicans cells. ApoB-derived cryptides affect different stages of growth of A. niger. Graphical abstract.

Antimicrobial D-amino acid oxidase-derived peptides specify gut microbiota.

The flavoenzyme D-amino acid oxidase (DAAO) is deputed to the degradation of D-enantiomers of amino acids. DAAO plays various relevant physiological roles in different organisms and tissues. Thus, it has been recently suggested that the goblet cells of the mucosal epithelia secrete into the lumen of intestine, a processed and active form of DAAO that uses the intestinal D-amino acids to generate hydrogen peroxide (H2O2), an immune messenger that helps fighting gut pathogens, and by doing so controls the homeostasis of gut microbiota. Here, we show that the DAAO form lacking the 1-16 amino acid residues (the putative secretion signal) is unstable and inactive, and that DAAO is present in the epithelial layer and the mucosa of mouse gut, where it is largely proteolyzed. In silico predicted DAAO-derived antimicrobial peptides show activity against various Gram-positive and Gram-negative bacteria but not on Lactobacilli species, which represent the commensal microbiota. Peptidomic analysis reveals the presence of such peptides in the mucosal fraction. Collectively, we identify a novel mechanism for gut microbiota selection implying DAAO-derived antimicrobial peptides which are generated by intestinal proteases and that are secreted in the gut lumen. In conclusion, we herein report an additional, ancillary role for mammalian DAAO, unrelated to its enzymatic activity.

Transglutaminase-mediated crosslinking of a host defence peptide derived from human apolipoprotein B and its effect on the peptide antimicrobial activity.

Background Microbial transglutaminase (mTG) has been successfully used to produce site-specific protein conjugates derivatized at the level of Gln and/or Lys residues for different biotechnological applications. Here, a recombinant peptide identified in human apolipoprotein B sequence, named r(P)ApoBL and endowed with antimicrobial activity, was studied as a possible acyl acceptor substrate of mTG with at least one of the six Lys residues present in its sequence. Methods The enzymatic crosslinking reaction was performed in vitro using N,N-dimethylcasein, substance P and bitter vetch (Vicia ervilia) seed proteins, well known acyl donor substrates in mTG-catalyzed reactions. Mass spectrometry analyses were performed for identifying the Lys residue(s) involved in the crosslinking reaction. Finally, bitter vetch protein-based antimicrobial films grafted with r(P)ApoBL were prepared and, their biological activity evaluated. Results r(P)ApoBL was able to be enzymatically modified by mTG. In particular, it was demonstrated the highly selective crosslinking of the peptide under study by mTG at level of Lys-18. Interestingly, the biological activity of the peptide when grafted into protein-based films was found to be lost following mTG-catalyzed crosslinking. Conclusions r(P)ApoBL was shown to be an effective acyl acceptor substrate of mTG. The involvement of Lys-18 in the enzymatic reaction was demonstrated. In addition, films grafted with r(P)ApoBL in the presence of mTG lost antimicrobial property. General significance A possible role of mTG as biotechnological tool to modulate the r(P)ApoBL antimicrobial activity was hypothesized, and a potential use in food packaging of protein-based films grafted with r(P)ApoBL was suggested.

Antimicrobial peptide Temporin-L complexed with anionic cyclodextrins results in a potent and safe agent against sessile bacteria.

Concern over antibiotic resistance is growing, and new classes of antibiotics, particularly against Gram-negative bacteria, are needed. Antimicrobial peptides (AMPs) have been proposed as a new class of clinically useful antimicrobials. Special attention has been devoted to frog-skin temporins. In particular, temporin L (TL) is strongly active against Gram-positive, Gram-negative bacteria and yeast strains. With the aim of overcoming some of the main drawbacks preventing the widespread clinical use of this peptide, i.e. toxicity and unfavorable pharmacokinetics profile, we designed new formulations combining TL with different types of cyclodextrins (CDs). TL was associated to a panel of neutral or negatively charged, monomeric and polymeric CDs. The impact of CDs association on TL solubility, as well as the transport through bacterial alginates was assessed. The biocompatibility on human cells together with the antimicrobial and antibiofilm properties of TL/CD systems was explored.

Enzymes as a Reservoir of Host Defence Peptides.

Host defence peptides (HDPs) are powerful modulators of cellular responses to various types of insults caused by pathogen agents. To date, a wide range of HDPs, from species of different kingdoms including bacteria, plant and animal with extreme diversity in structure and biological activity, have been described. Apart from a limited number of peptides ribosomally synthesized, a large number of promising and multifunctional HDPs have been identified within protein precursors, with properties not necessarily related to innate immunity, consolidating the fascinating hypothesis that proteins have a second or even multiple biological mission in the form of one or more bio-active peptides. Among these precursors, enzymes constitute certainly an interesting group, because most of them are mainly globular and characterized by a fine specific internal structure closely related to their catalytic properties and also because they are yet little considered as potential HDP releasing proteins. In this regard, the main aim of the present review is to describe a panel of HDPs, identified in all canonical classes of enzymes, and to provide a detailed description on hydrolases and their corresponding HDPs, as there seems to exist a striking link between these structurally sophisticated catalysts and their high content in cationic and amphipathic cryptic peptides.

Novosphingobium sp. PP1Y as a novel source of outer membrane vesicles.

Outer membrane vesicles (OMVs) are nanostructures of 20-200 nm diameter deriving from the surface of several Gram-negative bacteria. OMVs are emerging as shuttles involved in several mechanisms of communication and environmental adaptation. In this work, OMVs were isolated and characterized from Novosphingobium sp. PP1Y, a Gram-negative non-pathogenic microorganism lacking LPS on the outer membrane surface and whose genome was sequenced and annotated. Scanning electron microscopy performed on samples obtained from a culture in minimal medium highlighted the presence of PP1Y cells embedded in an extracellular matrix rich in vesicular structures. OMVs were collected from the exhausted growth medium during the mid-exponential phase, and purified by ultracentrifugation on a sucrose gradient. Atomic force microscopy, dynamic light scattering and nanoparticle tracking analysis showed that purified PP1Y OMVs had a spherical morphology with a diameter of ca. 150 nm and were homogenous in size and shape. Moreover, proteomic and fatty acid analysis of purified OMVs revealed a specific biochemical "fingerprint", suggesting interesting details concerning their biogenesis and physiological role. Moreover, these extracellular nanostructures do not appear to be cytotoxic on HaCaT cell line, thus paving the way to their future use as novel drug delivery systems.

Membrane disintegration by the antimicrobial peptide (P)GKY20: lipid segregation and domain formation.

Antimicrobial peptides (AMPs) are membrane-active peptides with a broad spectrum of activity against different pathogenic organisms and they represent promising new drugs to overcome the emergence of resistance to antibiotics in bacteria. (P)GKY20 is an antimicrobial peptide with a low hemolytic effect on eukaryotic cells and a strong antimicrobial activity especially against Gram-negative bacteria. However, its mechanism of action is still unknown. Here, we use fluorescence spectroscopy and differential scanning calorimetry combined with atomic force microscopy to characterise the binding of (P)GKY20 with model biomembranes and its effect on the membrane's microstructure and thermotropic properties. We found that (P)GKY20 selectively perturbs the bacterial-like membrane via a carpet-like mechanism employing peptide conformational changes, lipid segregation and domain formation as key steps in promoting membrane disruption. These results shed a first light on the action mechanism of (P)GKY20 and could represent an important contribution to the development of new peptides serving as antimicrobial agents.

Cost-effective production of recombinant peptides in Escherichia coli.

Among bioactive peptides, cationic antimicrobial peptides (AMPs), also referred to as host defence peptides (HDPs), are valuable tools to treat infections, being able to kill a wide variety of microbes directly and/or modulate host immunity. HDPs have great therapeutic potential against antibiotic-resistant bacteria, viruses and even parasites. However, high manufacturing costs have greatly limited their development as drugs, thus highlighting the need to develop novel and competitive production strategies. Here, a cost-effective procedure was established to produce the high amounts of peptides required for basic and clinical research. Firstly, a novel culture medium was designed, which was found to support significantly higher cell densities and recombinant expression levels of peptides under test compared to conventional media. The procedure has been also efficiently scaled up by using a 5 L fermenter, while the costs have been lowered significantly by developing a successful auto-induction strategy, which has been found to support higher yields of target constructs and cell biomass compared to conventional strategies based on expression induction by IPTG. Interestingly, it was estimated that by increasing production scale from 100 to 1000 mg/batch, unit costs decreased strongly from 253 to 42 €/mg. These costs appear highly competitive when compared to chemical synthesis strategies. Altogether, the data indicate that the strategy represents an important starting point for the future development of large-scale manufacture of HDPs.

Rapid Affinity Maturation of Novel Anti-PD-L1 Antibodies by a Fast Drop of the Antigen Concentration and FACS Selection of Yeast Libraries.

The affinity engineering is a key step to increase the efficacy of therapeutic monoclonal antibodies and yeast surface display is the most widely used and powerful affinity maturation approach, achieving picomolar binding affinities. In this study, we provide an optimization of the yeast surface display methodology, applied to the generation of potentially therapeutic high affinity antibodies targeting the immune checkpoint PD-L1. In this approach, we coupled a 10-cycle error-prone mutagenesis of heavy chain complementarity determining region 3 of an anti-PD-L1 scFv, previously identified by phage display, with high-throughput sequencing, to generate scFv-yeast libraries with high mutant frequency and diversity. In addition, we set up a novel, faster and effective selection scheme by fluorescence-activated cell sorting, based on a fast drop of the antigen concentration between the first and the last selection cycles, unlike the gradual decrease typical of current selection protocols. In this way we isolated 6 enriched mutated scFv-yeast clones overall, showing an affinity improvement for soluble PD-L1 protein compared to the parental scFv. As a proof of the potency of the novel approach, we confirmed that the antibodies converted from all the mutated scFvs retained the affinity improvement. Remarkably, the best PD-L1 binder among them also bound with a higher affinity to PD-L1 expressed in its native conformation on human-activated lymphocytes, and it was able to stimulate lymphocyte proliferation in vitro more efficiently than its parental antibody. This optimized technology, besides the identification of a new potential checkpoint inhibitor, provides a tool for the quick isolation of high affinity binders.

Identification of Novel Cryptic Multifunctional Antimicrobial Peptides from the Human Stomach Enabled by a Computational-Experimental Platform.

Novel approaches are needed to combat antibiotic resistance. Here, we describe a computational-experimental framework for the discovery of novel cryptic antimicrobial peptides (AMPs). The computational platform, based on previously validated antimicrobial scoring functions, indicated the activation peptide of pepsin A, the main human stomach protease, and its N- and C-terminal halves as antimicrobial peptides. The three peptides from pepsinogen A3 isoform were prepared in a recombinant form using a fusion carrier specifically developed to express toxic peptides in Escherichia coli. Recombinant pepsinogen A3-derived peptides proved to be wide-spectrum antimicrobial agents with MIC values in the range 1.56-50 µM (1.56-12.5 µM for the whole activation peptide). Moreover, the activation peptide was bactericidal at pH 3.5 for relevant foodborne pathogens, suggesting that this new class of previously unexplored AMPs may contribute to microbial surveillance within the human stomach. The peptides showed no toxicity toward human cells and exhibited anti-infective activity in vivo, reducing by up to 4 orders of magnitude the bacterial load in a mouse skin infection model. These peptides thus represent a promising new class of antibiotics. We envision that computationally guided data mining approaches such as the one described here will lead to the discovery of antibiotics from previously unexplored sources.

Structural and functional insights into RHA-P, a bacterial GH106 α-L-rhamnosidase from Novosphingobium sp. PP1Y.

α-L-Rhamnosidases (α-RHAs, EC 3.2.1.40) are glycosyl hydrolases (GHs) hydrolyzing terminal α-l-rhamnose residues from different substrates such as heteropolysaccharides, glycosylated proteins and natural flavonoids. Although the possibility to hydrolyze rhamnose from natural flavonoids has boosted the use of these enzymes in several biotechnological applications over the past decades, to date only few bacterial rhamnosidases have been fully characterized and only one crystal structure of a rhamnosidase of the GH106 family has been described. In our previous work, an α-l-rhamnosidase belonging to this family, named RHA-P, was isolated from the marine microorganism Novosphingobium sp. PP1Y. The initial biochemical characterization highlighted the biotechnological potential of RHA-P for bioconversion applications. In this work, further functional and structural characterization of the enzyme is provided. The recombinant protein was obtained fused to a C-terminal His-tag and, starting from the periplasmic fractions of induced recombinant cells of E. coli strain BL21(DE3), was purified through a single step purification protocol. Homology modeling of RHA-P in combination with a site directed mutagenesis analysis confirmed the function of residues D503, E506, E644, likely located at the catalytic site of RHA-P. In addition, a kinetic characterization of the enzyme on natural flavonoids such as naringin, rutin, hesperidin and quercitrin was performed. RHA-P showed activity on all flavonoids tested, with a catalytic efficiency comparable or even higher than other bacterial α-RHAs described in literature. The results confirm that RHA-P is able to hydrolyze both α-1,2 and α-1,6 glycosidic linkages, and suggest that the enzyme may locate different polyphenolic aromatic moities in the active site.

Cryptic Antimicrobial Peptides: Identification Methods and Current Knowledge of their Immunomodulatory Properties.

Cationic antimicrobial peptides (CAMPs), also known as host defence peptides (HDPs), are essential evolutionarily conserved components of innate immunity, constitutively or inducibly expressed in response to invasion by pathogens. In addition to a direct antimicrobial action, they are able to synergistically operate with other defence molecules to combat infection by neutralization of endotoxins, chemokine-like activities, induction of angiogenesis and wound repair. The importance of CAMPs has been highlighted in animal models and supported by observations in patient studies. CAMPs are attractive alternative candidates to antibiotic treatment, because they offer several advantages over the currently used drugs, moreover, knowledge on these peptides, especially regarding the intertwinement between their structure, function and mechanism of action, could be applied in the rational design of antimicrobial/anti-inflammatory/wound healing enhancing drugs. CAMPs combat pathogens by targeting bacterial membranes and essential membrane-related functions, and, in some cases, also target intracellular components. Despite differences in their size and sequence, many of them share a net positive charge and fold into amphipathic structures after contact with bacterial surfaces or endotoxins like lipopolysaccharides and lipoteichoic acid. Due to their peculiar mechanism, acquisition of resistance towards these peptides would be difficult for the bacteria. Very interestingly it has been demonstrated that several proteins, including proteins apparently not involved in immunity, can behave as sources of CAMPs hidden in their primary structures and released by the action of host and/or bacterial proteases. The existence of these "cryptic CAMPs" suggests that the panel of antimicrobial peptides present in higher eukaryotes and the variety of functions they perform could be much wider and more complex than previously suspected. This review focuses on source, structure and mechanism of action of cryptic CAMPs, with special attention to their immunomodulatory functions.

Chemical Cleavage of an Asp-Cys Sequence Allows Efficient Production of Recombinant Peptides with an N-Terminal Cysteine Residue.

Peptides with an N-terminal cysteine residue allow site-specific modification of proteins and peptides and chemical synthesis of proteins. They have been widely used to develop new strategies for imaging, drug discovery, diagnostics, and chip technologies. Here we present a method to produce recombinant peptides with an N-terminal cysteine residue as a convenient alternative to chemical synthesis. The method is based on the release of the desired peptide from a recombinant fusion protein by mild acid hydrolysis of an Asp-Cys sequence. To test the general validity of the method we prepared four fusion proteins bearing three different peptides (20-37 amino acid long) at the C-terminus of a ketosteroid isomerase-derived and two Onconase-derived carriers for the production of toxic peptides in E. coli. The chosen peptides were (C)GKY20, an antimicrobial peptide from the C-terminus of human thrombin, (C)ApoBL, an antimicrobial peptide from an inner region of human Apolipoprotein B, and (C)p53pAnt, an anticancer peptide containing the C-terminal region of the p53 protein fused to the cell penetrating peptide Penetratin. Cleavage efficiency of Asp-Cys bonds in the four fusion proteins was studied as a function of pH, temperature, and incubation time. In spite of the differences in the amino acid sequence (GTGDCGKY, GTGDCHVA, GSGTDCGSR, SQGSDCGSR) we obtained for all the proteins a cleavage efficiency of about 70-80% after 24 h incubation at 60 °C and pH 2. All the peptides were produced with very good yield (5-16 mg/L of LB cultures), high purity (>96%), and the expected content of free thiol groups (1 mol per mole of peptide). Furthermore, (C)GKY20 was modified with PyMPO-maleimide, a commercially available fluorophore bearing a thiol reactive group, and with 6-hydroxy-2-cyanobenzothiazole, a reagent specific for N-terminal cysteines, with yields of 100% thus demonstrating that our method is very well suited for the production of fully reactive peptides with an N-terminal cysteine residue.