YME1L-mediated mitophagy protects renal tubular cells against cellular senescence under diabetic conditions.

BACKGROUND: The senescence of renal tubular epithelial cells (RTECs) is crucial in the progression of diabetic kidney disease (DKD). Accumulating evidence suggests a close association between insufficient mitophagy and RTEC senescence. Yeast mitochondrial escape 1-like 1 (YME1L), an inner mitochondrial membrane metalloprotease, maintains mitochondrial integrity. Its functions in DKD remain unclear. Here, we investigated whether YME1L can prevent the progression of DKD by regulating mitophagy and cellular senescence. METHODS: We analyzed YME1L expression in renal tubules of DKD patients and mice, explored transcriptomic changes associated with YME1L overexpression in RTECs, and assessed its impact on RTEC senescence and renal dysfunction using an HFD/STZ-induced DKD mouse model. Tubule-specific overexpression of YME1L was achieved through the use of recombinant adeno-associated virus 2/9 (rAAV 2/9). We conducted both in vivo and in vitro experiments to evaluate the effects of YME1L overexpression on mitophagy and mitochondrial function. Furthermore, we performed LC-MS/MS analysis to identify potential protein interactions involving YME1L and elucidate the underlying mechanisms. RESULTS: Our findings revealed a significant decrease in YME1L expression in the renal tubules of DKD patients and mice. However, tubule-specific overexpression of YME1L significantly alleviated RTEC senescence and renal dysfunction in the HFD/STZ-induced DKD mouse model. Moreover, YME1L overexpression exhibited positive effects on enhancing mitophagy and improving mitochondrial function both in vivo and in vitro. Mechanistically, our LC-MS/MS analysis uncovered a crucial mitophagy receptor, BCL2-like 13 (BCL2L13), as an interacting partner of YME1L. Furthermore, YME1L was found to promote the phosphorylation of BCL2L13, highlighting its role in regulating mitophagy. CONCLUSIONS: This study provides compelling evidence that YME1L plays a critical role in protecting RTECs from cellular senescence and impeding the progression of DKD. Overexpression of YME1L demonstrated significant therapeutic potential by ameliorating both RTEC senescence and renal dysfunction in the DKD mice. Moreover, our findings indicate that YME1L enhances mitophagy and improves mitochondrial function, potentially through its interaction with BCL2L13 and subsequent phosphorylation. These novel insights into the protective mechanisms of YME1L offer a promising strategy for developing therapies targeting DKD.

Identification and Characterization of Two Structurally Related Dipeptides that Enhance Catalytic Efficiency of Neurolysin.

Neurolysin (Nln) is a recently recognized endogenous mechanism functioning to preserve the brain from ischemic injury. To further understand the pathophysiological function of this peptidase in stroke and other neurologic disorders, the present study was designed to identify small molecule activators of Nln. Using a computational approach, the structure of Nln was explored, which was followed by docking and in silico screening of ∼140,000 molecules from the National Cancer Institute Developmental Therapeutics Program database. Top ranking compounds were evaluated in an Nln enzymatic assay, and two hit histidine-dipeptides were further studied in detail. The identified dipeptides enhanced the rate of synthetic substrate hydrolysis by recombinant (human and rat) and mouse brain-purified Nln in a concentration-dependent manner (micromolar A50 and Amax ≥ 300%) but had negligible effect on activity of closely related peptidases. Both dipeptides also enhanced hydrolysis of Nln endogenous substrates neurotensin, angiotensin I, and bradykinin and increased efficiency of the synthetic substrate hydrolysis (Vmax/Km ratio) in a concentration-dependent manner. The dipeptides and competitive inhibitor dynorphin A (1-13) did not affect each other's affinity for Nln, suggesting differing nature of their respective binding sites. Lastly, drug affinity responsive target stability (DARTS) and differential scanning fluorimetry (DSF) assays confirmed concentration-dependent interaction of Nln with the activator molecule. This is the first study demonstrating that Nln activity can be enhanced by small molecules, although the peptidic nature and low potency of the activators limit their application. The identified dipeptides provide a chemical scaffold to develop high-potency, drug-like molecules as research tools and potential drug leads. SIGNIFICANCE STATEMENT: This study describes discovery of two molecules that selectively enhance activity of peptidase Nln-a newly recognized cerebroprotective mechanism in the poststroke brain. The identified molecules will serve as a chemical scaffold for development of drug-like molecules to further study Nln and may become lead structures for a new class of drugs. In addition, our conceptual and methodological framework and research findings might be used for other peptidases and enzymes, the activation of which bears therapeutic potential.

Heterologous production of active form of beta-lytic protease by Bacillus subtilis and improvement of staphylolytic activity by protein engineering.

BACKGROUND: Most of the proteases classified into the M23 family in the MEROPS database exhibit staphylolytic activity and have potential as antibacterial agents. The M23 family is further classified into two subfamilies, M23A and M23B. Proteases of the M23A subfamily are thought to lack the capacity for self-maturation by auto-processing of a propeptide, which has been a challenge in heterologous production and application research. In this study, we investigated the heterologous expression, in Bacillus subtilis, of the Lysobacter enzymogenes beta-lytic protease (BLP), a member of the M23A subfamily. RESULTS: We found that B. subtilis can produce BLP in its active form. Two points were shown to be important for the production of BLP in B. subtilis. The first was that the extracellular proteases produced by the B. subtilis host are essential for BLP maturation. When the host strain was deficient in nine extracellular proteases, pro-BLP accumulated in the supernatant. This observation suggested that BLP lacks the capacity for self-maturation and that some protease from B. subtilis contributes to the cleavage of the propeptide of BLP. The second point was that the thiol-disulfide oxidoreductases BdbDC of the B. subtilis host are required for efficient secretory production of BLP. We infer that intramolecular disulfide bonds play an important role in the formation of the correct BLP conformation during secretion. We also achieved efficient protein engineering of BLP by utilizing the secretory expression system in B. subtilis. Saturation mutagenesis of Gln116 resulted in a Q116H mutant with enhanced staphylolytic activity. The minimum bactericidal concentration (MBC) of the wild-type BLP and the Q116H mutant against Staphylococcus aureus NCTC8325 was 0.75 µg/mL and 0.375 µg/mL, respectively, and the MBC against Staphylococcus aureus ATCC43300 was 6 µg/mL and 3 µg/mL, respectively. CONCLUSIONS: In this study, we succeeded in the secretory production of BLP in B. subtilis. To our knowledge, this work is the first report of the successful heterologous production of BLP in its active form, which opens up the possibility of industrial use of BLP. In addition, this study proposes a new strategy of using the extracellular proteases of B. subtilis for the maturation of heterologous proteins.

Integration of VEK-30 peptide enhances fibrinolytic properties of staphylokinase.

Staphylokinase (SAK), a 136 amino acid bacterial protein with profibrinolytic properties, has emerged as an important thrombolytic agent because of its fibrin specificity and reduced inhibition by α-2 antiplasmin. In an attempt to enhance the clot dissolution ability of SAK, a 30 amino acid peptide (VEK-30) derived from a plasminogen (Pg) binding protein (PAM), was fused at the C-terminal end of SAK with a RGD (Arg-Gly-Asp) linker. The chimeric protein, SAKVEK, was expressed in E. coli and purified as a soluble protein. Pg activation by equimolar complexes of SAKVEK and SAK with plasmin revealed that the fusion of VEK-30 peptide has significantly enhanced the catalytic activity of SAK. The kinetic constant, kcat /Km , of SAKVEK for the substrate Pg appeared 2.7 times higher than that of SAK and the time required for the fibrin and platelet rich clot lysis was shortened by 30% and 50%, respectively. The binary activator complex of SAKVEK with plasmin gets inhibited by α2- antiplasmin but remains protected in the presence of fibrin, very similar to SAK. Thus, the present study suggests that SAKVEK is more potent and effective as a thrombolytic agent due to its higher catalytic activity for Pg activation in a fibrin-specific manner and its ability to clear platelet-rich plasma clot faster than SAK.

Interaction between a Novel Oligopeptide Fragment of the Human Neurotrophin Receptor TrkB Ectodomain D5 and the C-Terminal Fragment of Tetanus Neurotoxin.

This article presents experimental evidence and computed molecular models of a potential interaction between receptor domain D5 of TrkB with the carboxyl-terminal domain of tetanus neurotoxin (Hc-TeNT). Computational simulations of a novel small cyclic oligopeptide are designed, synthesized, and tested for possible tetanus neurotoxin-D5 interaction. A hot spot of this protein-protein interaction is identified in analogy to the hitherto known crystal structures of the complex between neurotrophin and D5. Hc-TeNT activates the neurotrophin receptors, as well as its downstream signaling pathways, inducing neuroprotection in different stress cellular models. Based on these premises, we propose the Trk receptor family as potential proteic affinity receptors for TeNT. In vitro, Hc-TeNT binds to a synthetic TrkB-derived peptide and acts similar to an agonist ligand for TrkB, resulting in phosphorylation of the receptor. These properties are weakened by the mutagenesis of three residues of the predicted interaction region in Hc-TeNT. It also competes with Brain-derived neurotrophic factor, a native binder to human TrkB, for the binding to neural membranes, and for uptake in TrkB-positive vesicles. In addition, both molecules are located together In Vivo at neuromuscular junctions and in motor neurons.

Cloning, Expression and Inhibitory Effects on Lewis Lung Carcinoma Cells of rAj-Tspin from Sea Cucumber (Apostichopus japonicus).

In recent years, sea cucumber has become a favorite healthcare food due to its characteristic prevention of cardiovascular diseases, suppression of tumors, as well as enhancement of immunity. In order to screen the anti-tumoral proteins or peptides from sea cucumber (Apostichopus japonicus), its cDNA library was analyzed, and a disintegrin-like and metalloproteinase with thrombospondin type 1 motif, member 13 (ADAMTS13)-like was found. ADAMTS13-like contains 10 thrombospondin 1 (TSP1) domains. Based on analysis of bioinformatics, the third TSP1 domain of this protein, which is further named Aj-Tspin, contains an arginine-glycine-aspartate (RGD) motif. Since our previous studies showed that the recombinant RGD-containing peptide from lampreys showed anti-tumoral activity, the third TSP1 domain of ADAMTS13-like was chosen to evaluate it's effect on tumor proliferation and metastasis, despite the fact it shares almost no homologue with disintegrins from other species. After artificial synthesis, its cDNA sequence, Aj-Tspin, which is composed of 56 amino acids, was subcloned into a pET23b vector and expressed as a recombinant Aj-Tspin (rAj-Tspin) in a soluble form with a molecular weight of 6.976 kDa. Through affinity chromatography, rAj-Tspin was purified as a single protein. Both anti-proliferation and immunofluorescence assays showed that rAj-Tspin suppressed the proliferation of Lewis lung carcinoma (LLC) cells through apoptosis. Adhesion assay also displayed that rAj-Tspin inhibited the adhesion of LLC cells to ECM proteins, including fibronectin, laminin, vitronectin and collagen. Lastly, rAj-Tspin also suppressed the migration and invasion of LLC cells across the filter in transwells. Thus, the above indicates that rAj-Tspin might act as a potential anti-tumoral drug in the future and could also provide information on the nutritional value of sea cucumber.

Antiviral activity of hexapeptides derived from conserved regions of bacterial proteases against HCV NS3 protease.

The main cause of hepatitis C is hepatitis C virus or HCV and for the cure of hepatitis C, NS3/4A protease has been found an important and emerging target. A number of HCV NS3/4A protease inhibitors have been discovered which have shown subsequent reduction in reducing the viral load leading to this infection however they are still undergoing clinical trials for improvement. Bacterial proteases are of great pharmaceutical importance and have a key role in various biological processes and in life cycle of several pathogens. The current study was planned to explore hexapeptides derived from conserved regions of bacterial proteases for their potential in blocking the NS3 protease activity of HCV which would finally inhibit HCV multiplication. For this, a novel protease gene nprB was isolated from a thermophilic bacterium Streptomyces thermovulgaris and bioinformatics analyses were performed. PCR amplification and sequencing of nprB gene indicated an open reading frame of 178 aa (20191.18 Dalton).The peptide GGVHIN was the top ranked with minimum S-score of -17.21) followed by hexapeptides VDAHAN, GVGREA, GALNES and VHINSS with their S-scores of -14.73, -13.78, -10.72 and -10.70, respectively. A phylogram was also reconstructed to reveal evolutionary relationships of nprB with its various homologs. The provided data will serve as a background to further reveal pharmaceutical and biotechnological importance of this novel protease gene from S. thermovulgaris in future.

Pseudomonas aeruginosa virulence proteins pseudolysin and protease IV impede cutaneous wound healing.

The intricate biological process of cutaneous wound healing is achieved through precise and highly programmed events. Dermal fibroblasts and keratinocytes play a significant role in the process of reepithelialization during wound healing. Pathogenic bacteria such as Pseudomonas aeruginosa (P. aeruginosa) may delay the proliferative phase of wound repair by secreting their proteins leading to delayed or impaired wound healing. We have analyzed three virulent strains of P. aeruginosa isolated from the wound environment which also differed in their ability to produce biofilms. Mass spectrometric analysis of differentially expressed secreted proteins by three virulent strains of P. aeruginosa revealed peptides from pseudolysin and protease IV expressed from lasB and prpL genes. Pseudolysin and protease IV recombinant proteins were tested for their ability to modulate wound healing in several cell types of wound microenvironment in in vitro and in vivo models. Both pseudolysin and protease IV inhibited migration and survival of fibroblasts, keratinocytes, and endothelial cells. In three dimensional spheroid endothelial models and matrigel assays these proteins impeded sprouting and tube formation. In a mouse model of excision wound, pseudolysin and protease IV treatment showed reduced collagen content, inhibited neovascularization and epithelialization, and delayed wound contraction. Furthermore, pseudolysin and protease IV treatment resulted in a significant increase in plasma IL-6 levels when compared to vehicle control and control, suggesting the induction of a state of prolonged inflammation. Taken together, our data indicate pseudolysin and protease IV secreted from biofilm producing and antibiotic resistant P. aeruginosa in wound microenvironment produce both local and systemic effects that is detrimental to the maintenance of tissue homeostasis. Hence, these proteins may serve as potential therapeutic targets toward better clinical management of wounds.

Purification and in vivo stability and half-life of recombinant lipid modified staphylokinase.

Staphylokinase (SAK), the thrombolytic protein holds a significant position in treating cardiovascular diseases. However, the rapid clearance of this protein from blood circulation reduces its effective usage and as a strategy to increase the half-life of SAK, initial work focussed on lipid modification of SAK (LMSAK) in E. coli GJ1158. Effective purification of the modified protein achieved using the two step method of hydrophobic interaction chromatography in succession with size exclusion chromatography, indicated a better yield. The thrombolytic activity of purified LMSAK analysed in heated plasma agar plate assay confirmed an enhanced activity. In vivo pharmacokinetic studies carried out for determining the half-life of LMSAK in blood circulation of mice presented that it has a half-life of 43.3 ± 3.4 min which is much higher than 21.6 ± 2.1 min that of the unmodified version of SAK. The studies confirmed the role of lipid modification as a crucial factor in confirming in vivo stability of LMSAK and proves to be beneficial in therapeutic usage.

Intestinal Epithelial Cells Exposed to Bacteroides fragilis Enterotoxin Regulate NF-κB Activation and Inflammatory Responses through ß-Catenin Expression.

The Bacteroides fragilis enterotoxin (BFT), a virulence factor of enterotoxigenic B. fragilis (ETBF), interacts with intestinal epithelial cells and can provoke signals that induce mucosal inflammation. Although ß-catenin signaling is reported to be associated with inflammatory responses and BFT is known to cleave E-cadherin linked with ß-catenin, little is known about the ß-catenin-mediated regulation of inflammation in ETBF infection. This study was conducted to investigate the role of ß-catenin as a cellular signaling intermediate in the induction of proinflammatory responses to stimulation of intestinal epithelial cells with BFT. Expression of ß-catenin in intestinal epithelial cells was reduced relatively early after stimulation with BFT and then recovered to normal levels relatively late after stimulation. In contrast, phosphorylation of ß-catenin in BFT-exposed cells occurred at high levels early in stimulation and decreased as time passed. Concurrently, late after stimulation the nuclear levels of ß-catenin were relatively higher than those early after stimulation. Suppression of ß-catenin resulted in increased NF-κB activity and interleukin-8 (IL-8) expression in BFT-stimulated cells. However, suppression or enhancement of ß-catenin expression neither altered the phosphorylated IκB kinase α/ß complex nor activated activator protein 1 signals. Furthermore, inhibition of glycogen synthase kinase 3ß was associated with increased ß-catenin expression and attenuated NF-κB activity and IL-8 expression in BFT-exposed cells. These findings suggest the negative regulation of NF-κB-mediated inflammatory responses by ß-catenin in intestinal epithelial cells stimulated with BFT, resulting in attenuation of acute inflammation in ETBF infection.

Recombinant N-acyl homoserine lactone-Lactonase AiiAQSI-1 Attenuates Aeromonas hydrophila Virulence Factors, Biofilm Formation and Reduces Mortality in Crucian Carp.

Quorum quenching (QQ) is a promising alternative infection-control strategy to antibiotics that controls quorum-regulated virulence without killing the pathogens. Aeromonas hydrophila is an opportunistic gram-negative pathogen living in freshwater and marine environments. A. hydrophila possesses an N-acyl homoserine lactone (AHL)-based quorum-sensing (QS) system that regulates virulence, so quorum signal-inactivation (i.e., QQ) may represent a new way to combat A. hydrophila infection. In this study, an AHL lactonase gene, aiiA was cloned from Bacillus sp. strain QSI-1 and expressed in Escherichia coli strain BL21(DE3). The A. hydrophila hexanoyl homoserine lactone (C6-HSL) QS signal molecule was degraded by AiiAQSI-1, which resulted in a decrease of bacterial swimming motility, reduction of extracellular protease and hemolysin virulence factors, and inhibited the biofilm formation of A. hydrophila YJ-1 in a microtiter assay. In cell culture studies, AiiAQSI-1 decreased the ability of A. hydrophila adherence to and internalization by Epithelioma papulosum cyprini (EPC) cells. During in vivo studies, oral administration of AiiAQSI-1 via feed supplementation attenuated A. hydrophila infection in Crucian Carp. Results from this work indicate that feed supplementation with AiiAQSI-1 protein has potential to control A. hydrophila aquaculture disease via QQ.

Preparation and Identification of Antioxidative Peptides from Pacific Herring (Clupea pallasii) Protein.

The aim of this study was to isolate and purify antioxidative peptides from Pacific herring (Clupea pallasii) protein. Five enzymes (pepsin, trypsin, papain, flavourzyme, and neutrase) were used for protein hydrolysis, and Pacific herring protein hydrolysates (PHPH) were separated by ultrafiltration. The fraction with the molecular weight below 3500 Da exhibited the highest in vitro antioxidant activities and cellular antioxidant activity. The PHPH was isolated and purified by consecutive chromatographic methods including gel filtration chromatography and reverse high-performance liquid chromatography (RP-HPLC). The purified antioxidant peptides were identified as Leu-His-Asp-Glu-Leu-Thr (MW = 726.35 Da) and Lys-Glu-Glu-Lys-Phe-Glu (MW = 808.40 Da), and the IC50 values of cellular antioxidant activity were 1.19 ± 0.05 mg/mL and 1.04 ± 0.06 mg/mL. The results demonstrate that is possible to produce natural antioxidative peptides from Pacific herring protein via enzymatic hydrolysis and purification.

Conjugation with Eight-Arm PEG Markedly Improves the In Vitro Activity and Prolongs the Blood Circulation of Staphylokinase.

Staphylokinase (SAK) is a profibrinolytic protein and can be used for therapy of acute myocardial infarction and coronary thrombosis. However, SAK suffers from a short serum half-life time (∼6 min) that limits its clinical application. PEGylation prolongs the half-life time of SAK, whereas it significantly decreases the bioactivity of SAK for the steric shielding effect of PEG. To improve the bioactivity and prolong the half-life time of SAK, 8-arm PEG maleimide (8-arm PEG) was used for conjugation of multiple SAK molecules in one entity. C terminus of SAK was engineered with cysteine residue, followed by reaction with the maleimide moieties of 8-arm PEG to obtain the conjugate (SAKp-PEG). Conjugation with 8-arm PEG retained the secondary structure of SAK, slightly perturbed the tertiary structure of SAK, and essentially maintained its in vitro bioactivity by the multivalence of SAK. Conjugation with 8-arm PEG increased the hydrodynamic volume and thus significantly prolonged the half-life time of SAK. SAKp-PEG elicited a 1.4-fold increase in the SAK-specific IgG titers as compared with SAK, and rendered no apparent toxicity to the cardiac, liver and renal functions of mice. Thus, multiple conjugation of a protein with 8-arm PEG was an effective strategy to develop a long-acting protein drug with improved bioactivity and prolonged blood circulation.

The role of LPD-nanoparticles containing recombinant major surface glycoprotein of Leishmania (rgp63) in protection against leishmaniasis in murine model.

CONTEXT: Leishmaniasis is a major public health problem. Despite numerous attempts, yet there is no effective vaccine against human leishmaniasis, mainly due to a lack of an effective vaccine delivery system as well as adjuvant. OBJECTIVE(S): The aim of this study was to evaluate the ability of recombinant glycoprotein 63 (rgp63) as a model of Leishmania antigen, entrapped in liposome-polycation-DNA (LPD) complexes nanoparticles in inducing cell mediated immune (CMI) response and protecting against L. major in BALB/c mice. MATERIALS AND METHODS: To this end, the abundant leishmania promastigote cell surface glycoprotein, gp63, was entrapped in nano-sized LPD (CpG) particles, (LPD (CpG)-rgp63), and BALB/c mice were immunized three times with either (LPD (CpG)-rgp63) or rgp63-CpG DNA or LPD (CpG) or free rgp63 and dextrose 5%. Various parameters including footpad thickness, splenic load of L. major parasites, rgp63-binding IgGs and also cytokine levels of rgp63-reactive T lymphocytes were then compared among different vaccinated animals. RESULTS: The lowest number of parasites in spleen, the higher levels of IgG2a after challenge infection, the minimal footpad swelling and high level of IFN-γ secretion, all indicated that adjuvants and antigen-delivery systems are essential in modifying immune responses; as mice received LPD (CpG)-rgp63 induced immune response stronger than the other groups. CONCLUSIONS: This study demonstrates that LPD nanoparticle is a promising and adaptable delivery system which could be modified towards specific vaccine targets to induce a more potent immune response in combination with rgp63.

IL-8 and IP-10 expression from human bronchial epithelial cells BEAS-2B are promoted by Streptococcus pneumoniae endopeptidase O (PepO).

BACKGROUND: The bronchial epithelium serves as the first defendant line of host against respiratory inhaled pathogens, mainly through releasing chemokines (e.g. interleukin-8 (IL-8), interferon-induced protein 10 (IP-10) etc.) responsible for neutrophil or lymphocyte recruitment to promote the clearance of inhaled pathogens including Streptococcus pneumoniae (S. pneumoniae). Previous studies have shown that IL-8 expression is induced by pneumococcal virulence factors (e.g. pneumolysin, peptidoglycan-polysaccharides, pneumococcal surface protein A (PspA) etc.), which contributes to the pathogenesis of pneumonia. Whether other pneumococcal virulence factors are involved in inducing chemokines expression in epithelium is still unknown. RESULTS: We studied the effect of PepO, a widely expressed and newly discovered pneumococcal virulence protein, on the release of proinflammatory cytokines, IL-8 and IP-10, from human bronchial epithelial cell line BEAS-2B and identified the relevant signaling pathways. Incubation of BEAS-2B with PepO resulted in increased synthesis and release of IL-8 and IP-10 in a dose and time independent manner. We also detected the increased and sustained expression of TLR2 and TLR4 transcripts in BEAS-2B stimulated by PepO. PepO activation leaded to the phosphorylation of MAPKs, Akt and p65. Pharmacologic inhibitors of MAPKs, PI3K and IκB-α phosphorylation attenuated IL-8 release, while IP-10 production was just suppressed by inhibitors of IκB-α phosphorylation, PI3K and P38 MAPK. CONCLUSION: These results suggest that PepO enhances IL-8 and IP-10 production in BEAS-2B in a MAPKs-PI3K/Akt-p65 dependent manner, which may play critical roles in the pathogenesis of pneumonia.

Staphylokinase Control of Staphylococcus aureus Biofilm Formation and Detachment Through Host Plasminogen Activation.

Staphylococcus aureus biofilms, a leading cause of persistent infections, are highly resistant to immune defenses and antimicrobial therapies. In the present study, we investigated the contribution of fibrin and staphylokinase (Sak) to biofilm formation. In both clinical S. aureus isolates and laboratory strains, high Sak-producing strains formed less biofilm than strains that lacked Sak, suggesting that Sak prevents biofilm formation. In addition, Sak induced detachment of mature biofilms. This effect depended on plasminogen activation by Sak. Host-derived fibrin, the main substrate cleaved by Sak-activated plasminogen, was a major component of biofilm matrix, and dissolution of this fibrin scaffold greatly increased susceptibility of biofilms to antibiotics and neutrophil phagocytosis. Sak also attenuated biofilm-associated catheter infections in mouse models. In conclusion, our results reveal a novel role for Sak-induced plasminogen activation that prevents S. aureus biofilm formation and induces detachment of existing biofilms through proteolytic cleavage of biofilm matrix components.

Hemagglutinin protease secreted by V. cholerae induced apoptosis in breast cancer cells by ROS mediated intrinsic pathway and regresses tumor growth in mice model.

Conventional anticancer therapies are effective but have side effects, so alternative targets are being developed. Bacterial toxins that can kill cells or alter the cellular processes like proliferation, apoptosis and differentiation have been reported for cancer treatment. In this study we have shown antitumor activity of hemagglutinin protease (HAP) secreted by Vibrio cholerae. One µg of HAP showed potent antitumor activity when injected into Ehrlich ascites carcinoma (EAC) tumors in Swiss albino mice. Weekly administration of this dose is able to significantly diminish a large tumor volume within 3 weeks and increases the survival rates of cancerous mice. HAP showed apoptotic activity on EAC and other malignant cells. Increased level of pro-apoptotic p53 with increased ratio of pro-apoptotic Bax to anti-apoptotic Bcl-2 signify that HAP induced apoptogenic signals lead to death of the tumor cells. In vivo and ex vivo studies suggest that mitochondrial dependent intrinsic pathway is responsible for this apoptosis. The level of ROS in malignant cells is reported to be higher than the normal healthy cells. HAP induces oxidative stress and increases the level of ROS in malignant cells which is significantly higher than the normal healthy cells. As a result the malignant cells cross the threshold level of ROS for cell survival faster than normal healthy cells. This mechanism causes HAP mediated apoptosis in malignant cells, but normal cells remain unaltered in the same environment. Our study suggests that HAP may be used as a new candidate drug for cancer therapy.

Moojenactivase, a novel pro-coagulant PIIId metalloprotease isolated from Bothrops moojeni snake venom, activates coagulation factors II and X and induces tissue factor up-regulation in leukocytes.

Coagulopathies following snakebite are triggered by pro-coagulant venom toxins, in which metalloproteases play a major role in envenomation-induced coagulation disorders by acting on coagulation cascade, platelet function and fibrinolysis. Considering this relevance, here we describe the isolation and biochemical characterization of moojenactivase (MooA), a metalloprotease from Bothrops moojeni snake venom, and investigate its involvement in hemostasis in vitro. MooA is a glycoprotein of 85,746.22 Da, member of the PIIId group of snake venom metalloproteases, composed of three linked disulfide-bonded chains: an N-glycosylated heavy chain, and two light chains. The venom protease induced human plasma clotting in vitro by activating on both blood coagulation factors II (prothrombin) and X, which in turn generated α-thrombin and factor Xa, respectively. Additionally, MooA induced expression of tissue factor (TF) on the membrane surface of peripheral blood mononuclear cells (PBMC), which led these cells to adopt pro-coagulant characteristics. MooA was also shown to be involved with production of the inflammatory mediators TNF-α, IL-8 and MCP-1, suggesting an association between MooA pro-inflammatory stimulation of PBMC and TF up-regulation. We also observed aggregation of washed platelets when in presence of MooA; however, the protease had no effect on fibrinolysis. Our findings show that MooA is a novel hemostatically active metalloprotease, which may lead to the development of coagulopathies during B. moojeni envenomation. Moreover, the metalloprotease may contribute to the development of new diagnostic tools and pharmacological approaches applied to hemostatic disorders.

Recombinant ADAMTS 13 Attenuates Brain Injury After Intracerebral Hemorrhage.

BACKGROUND AND PURPOSE: Inflammatory responses and blood-brain barrier (BBB) dysfunction play important roles in brain injury after intracerebral hemorrhage (ICH). The metalloprotease ADAMTS 13 (a disintegrin and metalloprotease with thrombospondin type I motif, member 13) was shown to limit inflammatory responses through its proteolytic effects on von Willebrand factor. In the present study, we addressed the role of ADAMTS 13 after experimental ICH. METHODS: ICH was induced in mice by intracerebral infusion of autologous blood. The peri-hematomal inflammatory responses, levels of matrix metalloproteinase-9 and intercellular adhesion molecule-1, pericyte coverage on brain capillaries, and BBB permeability were quantified at 24 hours. Functional outcomes, cerebral edema, and hemorrhagic lesion volume were quantified at day 3. RESULTS: Treatment with recombinant ADAMTS 13 (rADAMTS 13) reduced the levels of chemokines and cytokines, myeloperoxidase activity, and microglia activation and neutrophil recruitment after ICH. rADAMTS 13 also decreased interleukin-6 expression in brain endothelial cells stimulated by lipopolysaccharide, whereas recombinant von Willebrand factor reversed this effect. The anti-inflammatory effect of rADAMTS 13 was accompanied by reduced expression of intercellular adhesion molecule-1 and less activation of matrix metalloproteinase, enhanced pericyte coverage of brain microvessels, and attenuated BBB disruption. Furthermore, neutrophil depletion protected against BBB damage, and rADAMTS 13 treatment had no further beneficial effect. Finally, treatment of mice with rADAMTS 13 reduced cerebral edema and hemorrhagic lesion volume and improved neurological functions. CONCLUSIONS: Our findings reveal the importance of rADAMTS 13 in regulating pathological inflammation and BBB function and suggest that rADAMTS 13 may provide a new therapeutic strategy for ICH.

Enhanced Peptide Stability Against Protease Digestion Induced by Intrinsic Factor Binding of a Vitamin B12 Conjugate of Exendin-4.

Peptide digestion from proteases is a significant limitation in peptide therapeutic development. It has been hypothesized that the dietary pathway of vitamin B12 (B12) may be exploited in this area, but an open question is whether B12-peptide conjugates bound to the B12 gastric uptake protein intrinsic factor (IF) can provide any stability against proteases. Herein, we describe a new conjugate of B12 with the incretin peptide exendin 4 that demonstrates picomolar agonism of the glugacon-like peptide-1 receptor (GLP1-R). Stability studies reveal that Ex-4 is digested by pancreatic proteases trypsin and chymotrypsin and by the kidney endopeptidase meprin ß. Prebinding the B12 conjugate to IF, however, resulted in up to a 4-fold greater activity of the B12-Ex-4 conjugate relative to Ex-4, when the IF-B12-Ex-4 complex was exposed to 22 µg/mL of trypsin, 2.3-fold greater activity when exposed to 1.25 µg/mL of chymotrypsin, and there was no decrease in function at up to 5 µg/mL of meprin ß.