Bactericidal Permeability-Increasing Protein (BPI) Inhibits Mycobacterium tuberculosis Growth.

Bactericidal permeability-increasing protein (BPI) is a multifunctional cationic protein produced by neutrophils, eosinophils, fibroblasts, and macrophages with antibacterial anti-inflammatory properties. In the context of Gram-negative infection, BPI kills bacteria, neutralizes the endotoxic activity of lipopolysaccharides (LPSs), and, thus, avoids immune hyperactivation. Interestingly, BPI increases in patients with Gram-positive meningitis, interacts with lipopeptides and lipoteichoic acids of Gram-positive bacteria, and significantly enhances the immune response in peripheral blood mononuclear cells. We evaluated the antimycobacterial and immunoregulatory properties of BPI in human macrophages infected with Mycobacterium tuberculosis. Our results showed that recombinant BPI entered macrophages, significantly reduced the intracellular growth of M. tuberculosis, and inhibited the production of the proinflammatory cytokine tumor necrosis factor-alpha (TNF-α). Furthermore, BPI decreased bacterial growth directly in vitro. These data suggest that BPI has direct and indirect bactericidal effects inhibiting bacterial growth and potentiating the immune response in human macrophages and support that this new protein's broad-spectrum antibacterial activity has the potential for fighting tuberculosis.

Ubiquitin ligase MARCH5 localizes to peroxisomes to regulate pexophagy.

Mitochondria and peroxisomes are independent but functionally closely related organelles. A few proteins have been characterized as dual-organelle locating proteins with distinct or similar roles on mitochondria and peroxisomes. MARCH5 is a mitochondria-associated ubiquitin ligase best known for its regulatory role in mitochondria quality control, fission, and fusion. Here, we used a proximity tagging system, PUP-IT, and identified new interacting proteins of MARCH5. Our data uncover that MARCH5 is a dual-organelle locating protein that interacts with several peroxisomal proteins. PEX19 binds the transmembrane region on MARCH5 and targets it to peroxisomes. On peroxisomes, MARCH5 binds and mediates the ubiquitination of PMP70. Furthermore, we find PMP70 ubiquitination and pexophagy induced by mTOR inhibition are blocked in the absence of MARCH5. Our study suggests novel roles of MARCH5 on peroxisomes.

An Ultrapotent and Selective Cyclic Peptide Inhibitor of Human ß-Factor XIIa in a Cyclotide Scaffold.

Cyclotides are plant-derived peptides with complex structures shaped by their head-to-tail cyclic backbone and cystine knot core. These structural features underpin the native bioactivities of cyclotides, as well as their beneficial properties as pharmaceutical leads, including high proteolytic stability and cell permeability. However, their inherent structural complexity presents a challenge for cyclotide engineering, particularly for accessing libraries of sufficient chemical diversity to design potent and selective cyclotide variants. Here, we report a strategy using mRNA display enabling us to select potent cyclotide-based FXIIa inhibitors from a library comprising more than 1012 members based on the cyclotide scaffold of Momordica cochinchinensis trypsin inhibitor-II (MCoTI-II). The most potent and selective inhibitor, cMCoFx1, has a pM inhibitory constant toward FXIIa with greater than three orders of magnitude selectivity over related serine proteases, realizing specific inhibition of the intrinsic coagulation pathway. The cocrystal structure of cMCoFx1 and FXIIa revealed interactions at several positions across the contact interface that conveyed high affinity binding, highlighting that such cyclotides are attractive cystine knot scaffolds for therapeutic development.

The SZT2 Interactome Unravels New Functions of the KICSTOR Complex.

Seizure threshold 2 (SZT2) is a component of the KICSTOR complex which, under catabolic conditions, functions as a negative regulator in the amino acid-sensing branch of mTORC1. Mutations in this gene cause a severe neurodevelopmental and epileptic encephalopathy whose main symptoms include epilepsy, intellectual disability, and macrocephaly. As SZT2 remains one of the least characterized regulators of mTORC1, in this work we performed a systematic interactome analysis under catabolic and anabolic conditions. Besides numerous mTORC1 and AMPK signaling components, we identified clusters of proteins related to autophagy, ciliogenesis regulation, neurogenesis, and neurodegenerative processes. Moreover, analysis of SZT2 ablated cells revealed increased mTORC1 signaling activation that could be reversed by Rapamycin or Torin treatments. Strikingly, SZT2 KO cells also exhibited higher levels of autophagic components, independent of the physiological conditions tested. These results are consistent with our interactome data, in which we detected an enriched pool of selective autophagy receptors/regulators. Moreover, preliminary analyses indicated that SZT2 alters ciliogenesis. Overall, the data presented form the basis to comprehensively investigate the physiological functions of SZT2 that could explain major molecular events in the pathophysiology of developmental and epileptic encephalopathy in patients with SZT2 mutations.

LEAP-2: An Emerging Endogenous Ghrelin Receptor Antagonist in the Pathophysiology of Obesity.

Liver-expressed antimicrobial peptide 2 (LEAP-2), originally described as an antimicrobial peptide, has recently been recognized as an endogenous blocker of growth hormone secretagogue receptor 1a (GHS-R1a). GHS-R1a, also known as ghrelin receptor, is a G protein-coupled receptor (GPCR) widely distributed on the hypothalamus and pituitary gland where it exerts its major functions of regulating appetite and growth hormone (GH) secretion. The activity of GHS-R1a is controlled by two counter-regulatory endogenous ligands: Ghrelin (activation) and LEAP-2 (inhibition). Ghrelin activates GHS-R1a on the neuropeptide Y/Agouti-related protein (NPY/AgRP) neurons at the arcuate nucleus (ARC) to promote appetite, and on the pituitary somatotrophs to stimulate GH release. On the flip side, LEAP-2, acts both as an endogenous competitive antagonist of ghrelin and an inverse agonist of constitutive GHS-R1a activity. Such a biological property of LEAP-2 vigorously blocks ghrelin's effects on food intake and hormonal secretion. In circulation, LEAP-2 displays an inverse pattern as to ghrelin; it increases with food intake and obesity (positive energy balance), whereas decreases upon fasting and weight loss (negative energy balance). Thus, the LEAP-2/ghrelin molar ratio fluctuates in response to energy status and modulation of this ratio conversely influences energy intake. Inhibiting ghrelin's activity has shown beneficial effects on obesity in preclinical experiments, which sheds light on LEAP-2's anti-obesity potential. In this review, we will analyze LEAP-2's effects from a metabolic point of view with a focus on metabolic hormones (e.g., ghrelin, GH, and insulin), and discuss LEAP-2's potential as a promising therapeutic target for obesity.

Chemical "Butterfly Effect" Explaining the Coordination Chemistry and Antimicrobial Properties of Clavanin Complexes.

Can a minor difference in the nonmetal binding sequence of antimicrobial clavanins explain the drastic change in the coordination environment and antimicrobial efficiency? This study answers the question with a definite "yes", showing the details of the bioinorganic chemistry of Zn(II) and Cu(II) complexes with clavanins, histidine-rich, antimicrobial peptides from hemocytes of the tunicate Styela clava.

mTOR inhibitors potentially reduce TGF-ß2-induced fibrogenic changes in trabecular meshwork cells.

We examined the effects of mTOR inhibitors on the fibrotic response induced by transforming growth factor-beta2 (TGF-ß2) in cultured human trabecular meshwork (hTM) cells. TGF-ß2-induced expression of fibronectin, collagen type I, alpha 1 chain (COL1A1), and alpha-smooth muscle actin (αSMA) in hTM cells was examined in the presence or absence of mTOR inhibitors using quantitative real-time polymerase chain reaction, Western blotting, and immunohistochemistry. The migration rates of hTM cells were examined in the presence of TGF-ß2 with or without mTOR inhibitors. An in vitro study showed that the expression of fibronectin, COL1A1, and αSMA was upregulated by TGF-ß2 treatment of hTM cells; such upregulation was significantly suppressed by mTOR inhibitors. The inhibitors significantly reduced the migration rate of TGF-ß2-stimulated hTM cells. mTOR inhibitors may usefully reduce the fibrotic response of hTM cells and we may have to explore if it is also effective in in vivo model.

The proline-rich myticalins from Mytilus galloprovincialis display a membrane-permeabilizing antimicrobial mode of action.

Bivalve mollusks are continuously exposed to potentially pathogenic microorganisms living in the marine environment. Not surprisingly, these filter-feeders developed a robust innate immunity to protect themselves, which includes a broad panel of antimicrobial peptides. Among these, myticalins represent a recently discovered family of linear cationic peptides expressed in the gills of Mytilus galloprovincialis. Even though myticalins and insect and mammalian proline-rich antimicrobial peptides (PrAMPs) share a similar amino acid composition, we here show that none of the tested mussel peptides use a non-lytic mode of action relying on the bacterial transporter SbmA. On the other hand, all the tested myticalins perturbed and permeabilized the membranes of E. coli BW25113, as shown by flow-cytometry and atomic force microscopy. Circular dichroism spectra revealed that most myticalins did not adopt recognizable secondary structures in the presence of amphipathic environments, such as biological membranes. To explore possible uses of myticalins for biotech, we assessed their biocompatibility with a human cell line. Non-negligible cytotoxic effects displayed by myticalins indicate that their optimization would be required before their further use as lead compounds in the development of new antibiotics.

Anti-diabetic Role of Adropin in Streptozotocin Induced Diabetic Rats via Alteration of PI3K/Akt and Insulin Signaling Pathway.

Diabetes mellitus (DM) is a hyperglycemia-related multifactorial condition with an elevated risk of microvascular and microvascular complications associated with this disease. The current experimental study was to examine the antidiabetic activity of streptozotocin (STZ)-induced adropin against diabetic rats by altering the PI3K/Akt and insulin signaling pathways. STZ (60 mg/kg) was used for the induction of DM and rats were divided into different groups and received the adropin (20, 40 and 80 mg/kg) and glibenclamide (10 mg/kg) till 28 days. Body weight, plasma insulin, blood glucose and food intake were estimated, respectively. Biochemical enzymes, carbohydrate enzymes, lipid parameters, AMPK and insulin signalling pathway parameters were estimated. GLUT4 and PPARγ expression were also estimated. Oral administration of adropin significantly (p < 0.001) increased the glycogen, glucose-6-phosphatase dehydrogenase, insulin, hexokinase and belittled the blood glucose level, fructose 1-6-biphosphatase, glucose-6-phosphatase at dose dependent manner. Adropin significantly (p < 0.001) reduced the level of triglyceride, cholesterol, low density lipoprotein, very low density lipoprotein and increased the level of high density lipoprotein at dose dependent manner. Adropin significantly (p < 0.001) activated the Akt, IRS-2, IRS-1, IR, p-AKT and PI3k, which are the key modulator molecules of PI3K/Akt, AMPK and insulin signalling pathway in DM rats. The current experimental study confirms the anti-diabetic effect of adropin on DM rats induced by AMPK and insulin signalling pathway against STZ.

Adropin-based dual treatment enhances the therapeutic potential of mesenchymal stem cells in rat myocardial infarction.

Both weak survival ability of stem cells and hostile microenvironment are dual dilemma for cell therapy. Adropin, a bioactive substance, has been demonstrated to be cytoprotective. We therefore hypothesized that adropin may produce dual protective effects on the therapeutic potential of stem cells in myocardial infarction by employing an adropin-based dual treatment of promoting stem cell survival in vitro and modifying microenvironment in vivo. In the current study, adropin (25 ng/ml) in vitro reduced hydrogen peroxide-induced apoptosis in rat bone marrow mesenchymal stem cells (MSCs) and improved MSCs survival with increased phosphorylation of Akt and extracellular regulated protein kinases (ERK) l/2. Adropin-induced cytoprotection was blocked by the inhibitors of Akt and ERK1/2. The left main coronary artery of rats was ligated for 3 or 28 days to induce myocardial infarction. Bromodeoxyuridine (BrdU)-labeled MSCs, which were in vitro pretreated with adropin, were in vivo intramyocardially injected after ischemia, following an intravenous injection of 0.2 mg/kg adropin (dual treatment). Compared with MSCs transplantation alone, the dual treatment with adropin reported a higher level of interleukin-10, a lower level of tumor necrosis factor-α and interleukin-1ß in plasma at day 3, and higher left ventricular ejection fraction and expression of paracrine factors at day 28, with less myocardial fibrosis and higher capillary density, and produced more surviving BrdU-positive cells at day 3 and 28. In conclusion, our data evidence that adropin-based dual treatment may enhance the therapeutic potential of MSCs to repair myocardium through paracrine mechanism via the pro-survival pathways.

Characterization of blood protein adsorption on PM2.5 and its implications on cellular uptake and cytotoxicity of PM2.5.

In biological fluids, micro- or nano-size particles are prone to adsorb proteins and form a layer. The ambient air fine particulate matter (PM2.5) is inhaled via the lung, penetrates biological barriers and eventually reaches systemic blood circulation. However, there are very few data available regarding the adsorption of proteins on PM2.5. Here, we compared protein corona formed in plasma after bronchoalveolar lavage fluid (BALF) exposure with those formed in plasma alone. Using purified coronal proteins, we explored their adsorption behaviors on PM2.5 and their influence on biological reactivity of PM2.5. Liquid-chromatography tandem mass-spectrometry (LC-MS/MS) analysis revealed that exposure to BALF significantly changed the blood protein profile on PM2.5. Regardless of the presence of BALF, the protein corona on PM2.5 contained an abundance of serum albumin, hemoglobin (Hb) and fibrinogen (Fg) proteins. Using Fg as a corona surrogate, we found that van der Waals interactions, hydrophobic interactions, π-π stacking and electrostatic attractions contributed to the Fg adsorption and led to the conformational changes of Fg. In addition, Fg decoration decreased cellular internalization of PM2.5 and corresponding subsequent oxidative stress responses in a murine RAW264.7 macrophage. These results support the view that the formation of PM2.5 corona should be considered for toxicity assessment of PM2.5.

Pronociceptive Roles of Schwann Cell-Derived Galectin-3 in Taxane-Induced Peripheral Neuropathy.

Chemotherapy-induced peripheral neuropathy (CIPN) is a severe dose-limiting side effect of taxanes such as paclitaxel and docetaxel. Despite the high medical needs, insufficient understanding of the complex mechanism underlying CIPN pathogenesis precludes any endorsed causal therapy to prevent or relieve CIPN. In this study, we report that elevation of plasma galectin-3 level is a pathologic change common to both patients with taxane-treated breast cancer with CIPN and a mouse model of taxane-related CIPN. Following multiple intraperitoneal injections of paclitaxel in mice, galectin-3 levels were elevated in Schwann cells within the sciatic nerve but not in other peripheral organs or cells expressing galectin-3. Consistent with this, paclitaxel treatment of primary cultures of rat Schwann cells induced upregulation and secretion of galectin-3. In vitro migration assays revealed that recombinant galectin-3 induced a chemotactic response of the murine macrophage cell line RAW 264.7. In addition, perineural administration of galectin-3 to the sciatic nerve of naive mice mimicked paclitaxel-induced macrophage infiltration and mechanical hypersensitivity. By contrast, chemical depletion of macrophages by clodronate liposomes suppressed paclitaxel-induced mechanical hypersensitivity despite the higher level of plasma galectin-3. Deficiency (Galectin-3 -/- mice) or pharmacologic inhibition of galectin-3 inhibited paclitaxel-induced macrophage infiltration and mechanical hypersensitivity. In conclusion, we propose that Schwann cell-derived galectin-3 plays a pronociceptive role via macrophage infiltration in the pathogenesis of taxane-induced peripheral neuropathy. Therapies targeting this phenomenon, which is common to patients with CIPN and mouse models, represent a novel approach to suppress taxane-related CIPN. SIGNIFICANCE: These findings demonstrate that the elevation of plasma galectin-3 is a CIPN-related pathologic change common to humans and mice, and that targeting galectin-3 is a therapeutic option to delay CIPN progression.

Production and characterization of chicken blood hydrolysate with antihypertensive properties.

Chicken blood has limited utilization despite its high protein content. Production of a blood hydrolysate exhibiting angiotensin I-converting enzyme (ACE)-inhibitory activity would be means of valorizing chicken blood. The optimized conditions used to produce chicken blood corpuscle hydrolysate (BCH) by Alcalase were 51.1°C, 4% enzyme, and pH 9.6 for 6 h, resulting in a 35.8% degree of hydrolysis and 37.7% ACE inhibition at a peptide concentration of 0.2 mg/mL. The permeate of a 1-kDa membrane, BCH-III, showed a 2.5-fold increase in ACE inhibition compared with that of BCH. BCH-III was resistant to in vitro gastrointestinal digestion, whereas the BCH digesta exhibited an increased ACE-inhibitory activity after digestion. Both BCH and BCH-III were rich in hydrophobic amino acids. A single administration of BCH and BCH-III to spontaneously hypertensive rats at concentrations of 600 and 100 mg/kg, respectively, lowered the systolic blood pressure by -57.7 and -70.9 mmHg, respectively, 6 h after oral administration compared with the control group. The blood pressure-lowering effect of the 600 mg/kg BCH dose was comparable with that of the 100 mg/kg BCH-III dose after 4 wk of oral administration. Both BCH and BCH-III could be developed for use as nutraceutical products with antihypertensive effects.

Dietary Supplementation with Spray-Dried Porcine Plasma Attenuates Colon Inflammation in a Genetic Mouse Model of Inflammatory Bowel Disease.

Dietary supplementation with spray-dried porcine plasma (SDP) can modulate the immune response of gut-associated lymphoid tissue. SDP supplementation reduces acute mucosal inflammation, as well as chronic inflammation associated with aging. The aim of this study was to analyze if SDP supplementation could ameliorate colitis in a genetic mouse model of inflammatory bowel disease (IBD). Wild-type mice and Mdr1a knockout (KO) mice were administered a control diet or an SDP-supplemented diet from day 21 (weaning) until day 56. The histopathological index, epithelial barrier, and intestinal immune system were analyzed in the colonic mucosa. KO mice had higher epithelial permeability, increased Muc1 and Muc4 expression, and lower abundance of E-cadherin and Muc2 (all p < 0.001). SDP prevented these effects (all p < 0.05) and decreased the colonic inflammation observed in KO mice, reducing neutrophil and monocyte infiltration and activation and the percentage of activated T helper lymphocytes in the colonic mucosa (all p < 0.05). SDP also diminished proinflammatory cytokine expression and increased the anti-inflammatory IL-10 concentration in the colonic mucosa (all p < 0.05). In conclusion, dietary supplementation with SDP enhances colon barrier function and reduces mucosal inflammation in a mouse model of IBD.

Adropin suppresses insulin expression and secretion in INS-1E cells and rat pancreatic islets.

Adropin is a peptide hormone which is produced in brain and peripheral tissues such as liver. It was found that adropin modulates lipid and glucose homeostasis by interacting with hepatocytes and myocytes. Adropin enhances insulin sensitivity and alleviates hyperinsulinemia in animal models with high-fat diet-induced insulin resistance. However, it is unknown whether adropin regulates insulin secretion and proliferation of beta cells. Therefore, we studied the effects of adropin on insulin secretion in INS-1E cells as well as isolated pancreatic islets. Furthermore, we assessed the influence of adropin on insulin mRNA expression, cell viability and proliferation in INS-1E cells. Pancreatic islets were isolated from male Wistar rats. mRNA expression was evaluated using real-time PCR and cell viability by MTT assay. Cell replication was measured by BrdU incorporation and insulin secretion by RIA. We found that adropin suppresses insulin mRNA expression in INS-1E cells. Moreover, adropin attenuates glucose-induced insulin secretion in INS-1E cells as well as in isolated pancreatic islets. In addition, using INS-1E cells we found that adropin suppresses glucose-induced cAMP production. However, adropin fails to modulate INS-1E cell viability and proliferation. In summary, we found adropin suppresses insulin mRNA expression and secretion, without affecting beta cell viability or proliferation.

Purification and Assays of Tachylectin-5.

Tachylectin-5, a 41-kDa protein with a common fold of the C-terminal globular domain of the γ-chain of fibrinogen, is purified from horseshoe crab hemolymph plasma by affinity column chromatography, using acetyl-group-immobilized resin. Two types of isolectins, tachylectin-5A and tachylectin-5B, are obtained by stepwise elution with GlcNAc at 25 and 250 mM, respectively. Tachylectins-5A and -5B exhibit extraordinarily strong hemagglutinating activity against all types of human erythrocytes (the minimum agglutinating concentration of 0.004-0.008 µg/mL for tachylectin-5A and 0.077-0.27 µg/mL for tachylectin-5B). Their hemagglutinating activities are inhibited by acetyl group-containing sugars and noncarbohydrates such as sodium acetate, acetylcholine, and acetyl CoA (the minimum inhibitory concentrations of 1.3-1.6 mM), indicating that the acetyl group is required and sufficient for recognition by tachylectins-5A and -5B. EDTA inhibits their hemagglutinating activity, whereas the inhibition is overcome by adding an excess amount of Ca2+. Tachylectins-5A and -5B also exhibit bacterial agglutinating activity against both Gram-negative bacteria (the minimum agglutinating concentrations of 0.04-0.08 µg/mL for tachylectin-5A and 0.05-0.11 µg/mL for tachylectin-5B) and Gram-positive bacteria (the minimum agglutinating concentrations of 0.3-2.4 µg/mL for tachylectin-5A and 15.1-26.8 µg/mL for tachylectin-5B). Interestingly, tachylectins-5A and -5B enhance the antimicrobial activity of a hemocyte-derived peptide, big defensin.

Purification and Assays of Tachycitin.

An antimicrobial peptide tachycitin (73 amino acids) is purified by steps of chromatography, including Sephadex G-50 and S Sepharose FF, from the acid extract of hemocyte debris of horseshoe crabs. Tachycitin is present in monomer form in solution, revealed by ultracentrifugation analysis. Tachycitin exhibits bacterial agglutination activity and inhibits the growth of both Gram-negative bacteria, Gram-positive bacteria, and fungus Candida albicans. Interestingly, tachycitin shows synergistic antimicrobial activity in corporation with another antimicrobial peptide, big defensin. Tachycitin shows a specific binding activity to chitin but not to cellulose, mannan, xylan, and laminarin. Tachycitin is composed of the N-terminal three-stranded ß-sheet and the C-terminal two-stranded ß-sheet following a short helical turn, and the C-terminal structural motif shares a significant structural similarity with the chitin-binding domain derived from a plant chitin-binding protein, hevein.

Francisella novicida Two-Component System Response Regulator BfpR Modulates iglC Gene Expression, Antimicrobial Peptide Resistance, and Biofilm Production.

Response regulators are a critical part of the two-component system of gene expression regulation in bacteria, transferring a signal from a sensor kinase into DNA binding activity resulting in alteration of gene expression. In this study, we investigated a previously uncharacterized response regulator in Francisella novicida, FTN_1452 that we have named BfpR (Biofilm-regulating Francisella protein Regulator, FTN_1452). In contrast to another Francisella response regulator, QseB/PmrA, BfpR appears to be a negative regulator of biofilm production, and also a positive regulator of antimicrobial peptide resistance in this bacterium. The protein was crystallized and X-ray crystallography studies produced a 1.8 Å structure of the BfpR N-terminal receiver domain revealing interesting insight into its potential interaction with the sensor kinase. Structural analysis of BfpR places it in the OmpR/PhoP family of bacterial response regulators along with WalR and ResD. Proteomic and transcriptomic analyses suggest that BfpR overexpression affects expression of the critical Francisella virulence factor iglC, as well as other proteins in the bacterium. We demonstrate that mutation of bfpR is associated with an antimicrobial peptide resistance phenotype, a phenotype also associated with other response regulators, for the human cathelicidin peptide LL-37 and a sheep antimicrobial peptide SMAP-29. F. novicida with mutated bfpR replicated better than WT in intracellular infection assays in human-derived macrophages suggesting that the down-regulation of iglC expression in bfpR mutant may enable this intracellular replication to occur. Response regulators have been shown to play important roles in the regulation of bacterial biofilm production. We demonstrate that F. novicida biofilm formation was highly increased in the bfpR mutant, corresponding to altered glycogen synthesis. Waxworm infection experiments suggest a role of BfpR as a negative modulator of iglC expression with de-repression by Mg2+. In this study, we find that the response regulator BfpR may be a negative regulator of biofilm formation, and a positive regulator of antimicrobial peptide resistance in F. novicida.

A Potent Host Defense Peptide Triggers DNA Damage and Is Active against Multidrug-Resistant Gram-Negative Pathogens.

Gram-negative bacteria are some of the biggest threats to public health due to a large prevalence of antibiotic resistance. The difficulty in treating bacterial infections, stemming from their double membrane structure combined with efflux pumps in the outer membrane, has resulted in a much greater need for antimicrobials with activity against these pathogens. Tunicate host defense peptide (HDP), Clavanin A, is capable of not only inhibiting Gram-negative growth but also potentiating activity in the presence of Zn(II). Here, we provide evidence that the improvements of Clavanin A activity in the presence of Zn(II) are due to its novel mechanism of action. We employed E. coli TD172 (ΔrecA::kan) and the terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay to show in cellulae that DNA damage occurs upon treatment with Clavanin A. In vitro assays demonstrated that Zn(II) ions are required for the nuclease activity of the peptide. The quantum mechanics/molecular mechanics (QM/MM) calculations were used to investigate the mechanism of DNA damage. In the rate-determining step of the proposed mechanism, due to its Lewis acidity, the Zn(II) ion activates the scissile P-O bond of DNA and creates a hydroxyl nucleophile from a water molecule. A subsequent attack by this group to the electrophilic phosphorus cleaves the scissile phosphoester bond. Additionally, we utilized bacterial cytological profiling (BCP), circular dichroism (CD) spectroscopy in the presence of lipid vesicles, and surface plasmon resonance combined with electrical impedance spectroscopy in order to address the apparent discrepancies between our results and the previous studies regarding the mechanism of action of Clavanin A. Finally, our approach may lead to the identification of additional Clavanin A like HDPs and promote the development of antimicrobial peptide based therapeutics.

Transcriptomic Analysis Reveals the Wound Healing Activity of Mussel Myticin C.

Myticin C is the most studied antimicrobial peptide in the marine mussel Mytilusgalloprovincialis. Although it is constitutively expressed in mussel hemocytes and displays antibacterial, antiviral, and chemotactic functions, recent work has suggested that this molecule is mainly activated after tissue injury. Therefore, the main objective of this work was to characterize the hemocytes' transcriptomic response after a myticin C treatment, in order to understand the molecular changes induced by this cytokine-like molecule. The transcriptome analysis revealed the modulation of genes related to cellular movement, such as myosin, transgelin, and calponin-like proteins, in agreement with results of functional assays, where an implication of myticin C in the in vitro activation of hemocytes and migration was evidenced. This was also observed in vivo after a tissue injury, when hemocytes, with high concentrations of myticin C, migrated to the damaged area to heal the wound. All these properties allowed us to think about the biotechnological application of these molecules as wound healers. Human keratinocytes and larvae zebrafish models were used to confirm this hypothesis. Accelerated regeneration after a wound or tail fin amputation was observed after treatment with the myticin C peptide, supporting the chemotactic and healing activity of myticin C.