OMA1 clears traffic jam in TOM tunnel in mammals.

Using an engineered mitochondrial clogger, Krakowczyk et al. (https://doi.org/10.1083/jcb.202306051) identified the OMA1 protease as a critical component that eliminates import failure at the TOM translocase in mammalian cells, providing a novel quality control mechanism that is distinct from those described in yeast.

Engineering metalloproteinase inhibitors: tissue inhibitors of metalloproteinases or antibodies, that is the question.

Targeting metalloproteinases (MPs) has been the center of attention for developing therapeutics due to their contribution to a wide range of diseases, including cancer, cardiovascular, neurodegenerative disease, and preterm labor. Protein-based MP inhibitors offer higher stability and selectivity, which is critical for developing efficient therapeutics with low off-target effects. Tissue inhibitors of metalloproteinases (TIMPs), natural inhibitors of MPs, and antibodies provide excellent protein scaffolds for engineering selective or multispecific MP inhibitors. Advances in protein engineering and design techniques, such as rational design and directed evolution using yeast display to develop potent MP inhibitors, are discussed, including but not limited to loop grafting, swapping, and counterselective selection.

An up-to-date review of biomedical applications of serratiopeptidase and its biobetter derivatives as a multi-potential metalloprotease.

Serratiopeptidase is a bacterial metalloprotease used in a variety of medical applications. The multidimensional properties of serratiopeptidase make it noticeable as a miraculous enzyme. Anti-coagulant, anti-inflammatory and anti-biofilm activity of serratiopeptidase making it useful in reducing pain and swelling associated with various conditions including arthritis, diabetes, cancer, swelling, pain and also thrombolytic disorders. It breaks down fibrin, thins the fluids formed during inflammation and due to its anti-biofilm activity, can be used in the combination of antibiotics to reduce development of antibiotic resistance. However, some drawbacks like sensitivity to environmental conditions and low penetration into cells due to its large size have limited its usage as a potent pharmaceutical agent. To overcome such limitations, improved versions of the enzyme were introduced using protein engineering in our previous studies. Novel functional serratiopeptidases with shorter length and higher stability have seemingly created a hope for using this enzyme as a more effective therapeutic enzyme. This review explains the structural properties and functional aspects of serratiopeptidase, its main characteristics and properties, pre-clinical and clinical applications of the enzyme, improved qualities of the modified forms, different formulations of the enzyme, and the potential future developments.

Miao medicine Gu Yan Xiao tincture inhibits mTOR to stimulate chondrocyte autophagy in a rabbit model of osteoarthritis.

ETHNOPHARMACOLOGICAL RELEVANCE: The Gu Yan Xiao tincture, a blend of traditional Chinese herbs, is traditionally used for osteoarthritis and related pain. This study investigated its mechanism of action in order to rationalize and validate its therapeutic use. AIM OF THE STUDY: This study analyzed, in a rabbit model of knee osteoarthritis, whether and how Gu Yan Xiao tincture exerts therapeutic benefits by modulating chondrocyte autophagy. MATERIALS AND METHODS: The active constituents within the GYX tincture were identified using liquid chromatography-mass spectrometry. The rabbit model was established by injecting animals with type II collagenase intra-articularly, and the effects of topically applied tincture were examined on osteoarthritis lesions of the knee using histopathology, micro-computed tomography and x-ray imaging. Effects of the tincture were also evaluated on levels of inflammatory cytokines, matrix metalloproteases, and autophagy in chondrocytes. As a positive control, animals were treated with sodium diclofenac. RESULTS: The tincture mitigated the reduction in joint space, hyperplasia of the synovium and matrix metalloproteases in serum that occurred after injection of type II collagenase in rabbits. These therapeutic effects were associated with inhibition of mTOR and activation of autophagy in articular chondrocytes. Inhibiting mTOR with rapamycin potentiated the therapeutic effects of the tincture, while inhibiting autophagy with 3-methyladenine antagonized them. CONCLUSIONS: Gu Yan Xiao tincture mitigates tissue injury in a rabbit model of osteoarthritis, at least in part by inhibiting mTOR and thereby promoting autophagy in chondrocytes. These results rationalize the use of the tincture not only against osteoarthritis but also potentially other diseases involving inhibition of autophagy in bones and joints.

Antiangiogenic properties of BthMP, a P-I metalloproteinase from Bothrops moojeni snake venom by VEGF pathway in endothelial cells.

Angiogenesis is a process that is controlled by a delicate combination of proangiogenic and antiangiogenic molecules and can be disrupted in various illnesses, including cancer. Non-cancerous diseases can also have an abnormal or insufficient vascular growth, inflammation and hypoxia, which exacerbate angiogenesis. These conditions include atherosclerosis, psoriasis, endometriosis, asthma, obesity and AIDS. Based on that, the present work assessed the in vitro and ex vivo antiangiogenic properties stemming from BthMP, a P-I metalloproteinase from Bothrops moojeni snake venom, via the VEGF pathway. BthMP at a concentration of 5 and 40 µg/mL showed no toxicity to endothelial cells (HUVEC) in the MTT assay and was not able to induce necrosis and colony proliferation. Interestingly, BthMP inhibited adhesion, migration and invasion of HUVECs in Matrigel and arrested in vitro angiogenesis by reducing the average number of nodules in toxin-treated cells by 9.6 and 17.32 at 5 and 40 µg/mL, respectively, and the number of tubules by 15.9 at 5 µg/mL and 21.6 at 40 µg/mL in a VEGF-dependent way, an essential proangiogenic property. Furthermore, BthMP inhibited the occurrence of the angiogenic process in an ex vivo aortic ring test by decreasing new vessel formation by 52% at 5 µg/mL and by 66% at 40 µg/mL and by increasing the expression of an antiangiogenic gene, SFLT-1, and decreasing the expression of the proangiogenic genes VEGFA and ANGPT-1. Finally, this toxin reduces the production of nitric oxide, a marker that promotes angiogenesis and VEGF modulation, and decreases the protein expression of VEGFA in the supernatant of the HUVEC culture by about 30 %. These results suggest that BthMP has a promising antiangiogenic property and proves to be a biotechnological mechanism for understanding the antiangiogenic responses induced by snake venom metalloproteinases, which could be applied to a variety of diseases that exhibit an imbalance of angiogenesis mechanisms.

Neurotensin contributes to cholestatic liver disease potentially modulating matrix metalloprotease-7.

The diagnosis and treatment of biliary atresia pose challenges due to the absence of reliable biomarkers and limited understanding of its etiology. The plasma and liver of patients with biliary atresia exhibit elevated levels of neurotensin. To investigate the specific role of neurotensin in the progression of biliary atresia, the patient's liver pathological section was employed. Biliary organoids, cultured biliary cells, and a mouse model were employed to elucidate both the potential diagnostic significance of neurotensin and its underlying mechanistic pathway. In patients' blood, the levels of neurotensin were positively correlated with matrix metalloprotease-7, interleukin-8, and liver function enzymes. Neurotensin and neurotensin receptors were mainly expressed in the intrahepatic biliary cells and were stimulated by bile acids. Neurotensin suppressed the growth and increased expression of matrix metalloprotease-7 in biliary organoids. Neurotensin inhibited mitochondrial respiration, oxidative phosphorylation, and attenuated the activation of calmodulin-dependent kinase kinase 2-adenosine monophosphate-activated protein kinase (CaMKK2-AMPK) signaling in cultured biliary cells. The stimulation of neurotensin in mice and cultured cholangiocytes resulted in the upregulation of matrix metalloprotease-7 expression through binding to its receptors, namely neurotensin receptors 1/3, thereby attenuating the activation of the CaMKK2-AMPK pathway. In conclusion, these findings revealed the changes of neurotensin in patients with cholestatic liver disease and its mechanism in the progression of the disease, providing a new understanding of the complex mechanism of hepatobiliary injury in children with biliary atresia.

Comparative analysis of Deinagkistrodon acutus venom from Taiwan and China utilizing chromatographic, electrophoretic, and bioinformatic approaches, along with ELISA employing a monospecific antivenom.

Deinagkistrodon acutus is a medically important pitviper inhabiting mainly South China and Taiwan. The hemorrhagic effects of its envenoming are compatible to its venom, which is abundant in metalloproteases (svMPs) and C-type lectin-like proteins. In this study, we investigated geographic variations in the venom of D. acutus collected from Taiwan and four Mainland Chinese provinces: Fujian, Jiangxi, Anhui, and Hunan. The variations were assessed through high-performance liquid chromatography, non-metric multidimensional scaling analysis, gel electrophoresis, and enzyme-linked immunosorbent assay (ELISA) with a monospecific antivenom (DaMAV) generated against the Taiwanese D. acutus venom, and discussed based on venom-protein sequences in databases and literature related to D. acutus venom. Additionally, the cross-reactivity of DaMAV against Crotalus horridus and Calloselasma rhodostoma venoms was investigated. We noted differential abundances of D. acutus venom metalloproteases, C-type lectin-like proteins, and phospholipase A2, along with point mutations and selective expression of serine protease isoforms. The ELISA results revealed that the venom from Taiwan was more reactive toward Taiwanese DaMAV than the four Mainland Chinese venoms, consistent with chromatographic profile differences, whereas C. horridus venom presented moderate cross-reactivity with DaMAV. The observed immunoreactivities of these venom with DaMAV can be attributed to the high prevalence of their PIII-svMPs, which are the dominant antigens, and the conservation of PIII-svMP epitopes.

Induction of human-fetal-membrane remodeling in-vitro by the alpha hemolysin of Escherichia coli.

INTRODUCTION: Almost 80% of urinary tract infections during pregnancy are caused by uropathogenic strains of Escherichia coli. Alpha-hemolysin, toxin secreted by them, has a fundamental role in this pathology development. Considering that urinary tract infections are related with premature rupture of fetal membranes, we proposed to evaluate the effects that alpha-hemolysin induces on human-fetal-membranes. METHODS: Thirteen fetal membranes obtained from elective cesarean sections (>37 weeks) were mounted in a transwell-device generating two independent chambers. To mimic an ascendant-urinary-tract infection, membranes were incubated with different concentrations of pure alpha-hemolysin from the choriodecidual side during 24h. Extensive histological analyses were performed and transepithelial electrical-resistance were determined. Cell viability, metalloproteinase activity and cyclooxygenase-2- gene expression was estimated by lactate-dehydrogenase-release assay, zymography and RT-qPCR, respectively. Finally, four fetal membranes were treated with hemolysin preincubated with polyclonal anti-hemolysin antibodies. Cell viability and metalloproteinase activity were monitored. RESULTS: After 24 h of treatment, fetal membranes evidenced a structural damage and a decrease in membrane resistance that progressed as the concentration of alpha hemolysin increased. While the amniotic-epithelial-layer remained practically unaffected, the chorion cells manifested an increase in vacuolization and necrosis. In addition, the extracellular matrix exhibited collagen-fiber disorganization, a marked decrease in fiber content, and became thicker in presence of the toxin. Cyclooxigenase-2 expression and metalloproteinase activity were also higher in the treated groups than in untreated ones. Finally, a preincubation of hemolysin with specific antibodies prevented the cytotoxicity on the chorion cells and the increase in metalloproteinase activity. DISCUSSION: Hemolysin induces structural and molecular changes associated with the remodeling of human-fetal-membranes in-vitro.

The Deinococcus protease PprI senses DNA damage by directly interacting with single-stranded DNA.

Bacteria have evolved various response systems to adapt to environmental stress. A protease-based derepression mechanism in response to DNA damage was characterized in Deinococcus, which is controlled by the specific cleavage of repressor DdrO by metallopeptidase PprI (also called IrrE). Despite the efforts to document the biochemical, physiological, and downstream regulation of PprI-DdrO, the upstream regulatory signal activating this system remains unclear. Here, we show that single-stranded DNA physically interacts with PprI protease, which enhances the PprI-DdrO interactions as well as the DdrO cleavage in a length-dependent manner both in vivo and in vitro. Structures of PprI, in its apo and complexed forms with single-stranded DNA, reveal two DNA-binding interfaces shaping the cleavage site. Moreover, we show that the dynamic monomer-dimer equilibrium of PprI is also important for its cleavage activity. Our data provide evidence that single-stranded DNA could serve as the signal for DNA damage sensing in the metalloprotease/repressor system in bacteria. These results also shed light on the survival and acquired drug resistance of certain bacteria under antimicrobial stress through a SOS-independent pathway.

ADAM19 cleaves the PTH receptor and associates with brachydactyly type E.

Brachydactyly type E (BDE), shortened metacarpals, metatarsals, cone-shaped epiphyses, and short stature commonly occurs as a sole phenotype. Parathyroid hormone-like protein (PTHrP) has been shown to be responsible in all forms to date, either directly or indirectly. We used linkage and then whole genome sequencing in a small pedigree, to elucidate BDE and identified a truncated disintegrin-and-metalloproteinase-19 (ADAM19) allele in all affected family members, but not in nonaffected persons. Since we had shown earlier that the extracellular domain of the parathyroid hormone receptor (PTHR1) is subject to an unidentified metalloproteinase cleavage, we tested the hypothesis that ADAM19 is a sheddase for PTHR1. WT ADAM19 cleaved PTHR1, while mutated ADAM-19 did not. We mapped the cleavage site that we verified with mass spectrometry between amino acids 64-65. ADAM-19 cleavage increased Gq and decreased Gs activation. Moreover, perturbed PTHR1 cleavage by ADAM19 increased ß-arrestin2 recruitment, while cAMP accumulation was not altered. We suggest that ADAM19 serves as a regulatory element for PTHR1 and could be responsible for BDE. This sheddase may affect other PTHrP or PTH-related functions.

The Role of Rosavin in the Pathophysiology of Bone Metabolism.

Rosavin, a phenylpropanoid in Rhodiola rosea's rhizome, and an adaptogen, is known for enhancing the body's response to environmental stress. It significantly affects cellular metabolism in health and many diseases, particularly influencing bone tissue metabolism. In vitro, rosavin inhibits osteoclastogenesis, disrupts F-actin ring formation, and reduces the expression of osteoclastogenesis-related genes such as cathepsin K, calcitonin receptor (CTR), tumor necrosis factor receptor-associated factor 6 (TRAF6), tartrate-resistant acid phosphatase (TRAP), and matrix metallopeptidase 9 (MMP-9). It also impedes the nuclear factor of activated T-cell cytoplasmic 1 (NFATc1), c-Fos, the nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB), and mitogen-activated protein kinase (MAPK) signaling pathways and blocks phosphorylation processes crucial for bone resorption. Moreover, rosavin promotes osteogenesis and osteoblast differentiation and increases mouse runt-related transcription factor 2 (Runx2) and osteocalcin (OCN) expression. In vivo studies show its effectiveness in enhancing bone mineral density (BMD) in postmenopausal osteoporosis (PMOP) mice, restraining osteoclast maturation, and increasing the active osteoblast percentage in bone tissue. It modulates mRNA expressions by increasing eukaryotic translation elongation factor 2 (EEF2) and decreasing histone deacetylase 1 (HDAC1), thereby activating osteoprotective epigenetic mechanisms, and alters many serum markers, including decreasing cross-linked C-telopeptide of type I collagen (CTX-1), tartrate-resistant acid phosphatase 5b (TRACP5b), receptor activator for nuclear factor κ B ligand (RANKL), macrophage-colony-stimulating factor (M-CSF), and TRAP, while increasing alkaline phosphatase (ALP) and OCN. Additionally, when combined with zinc and probiotics, it reduces pro-osteoporotic matrix metallopeptidase 3 (MMP-3), interleukin 6 (IL-6), and tumor necrosis factor α (TNF-α), and enhances anti-osteoporotic interleukin 10 (IL-10) and tissue inhibitor of metalloproteinase 3 (TIMP3) expressions. This paper aims to systematically review rosavin's impact on bone tissue metabolism, exploring its potential in osteoporosis prevention and treatment, and suggesting future research directions.

Ligand and structure-based discovery of phosphorus-containing compounds as potential metalloproteinase inhibitors.

In this study, a methodology is proposed, combining ligand- and structure-based virtual screening tools, for the identification of phosphorus-containing compounds as inhibitors of zinc metalloproteases. First, we use Dragon molecular descriptors to develop a Linear Discriminant Analysis classification model, which is widely validated according to the OECD principles. This model is simple, robust, stable and has good discriminating power. Furthermore, it has a defined applicability domain and it is used for virtual screening of the DrugBank database. Second, docking experiments are carried out on the identified compounds that showed good binding energies to the enzyme thermolysin. Considering the potential toxicity of phosphorus-containing compounds, their toxicological profile is evaluated according to Protox II. Of the five molecules evaluated, two show carcinogenic and mutagenic potential at small LD50, not recommended as drugs, while three of them are classified as non-toxic, and could constitute a starting point for the development of new vasoactive metalloprotease inhibitor drugs. According to molecular dynamics simulation, two of them show stable interactions with the active site maintaining coordination with the metal. A high agreement is evident between QSAR, docking and molecular dynamics results, demonstrating the potentialities of the combination of these tools.

Determination of the role of specific amino acids in the binding of Zn(II), Ni(II), and Cu(II) to the active site of the M10 family metallopeptidase.

Metallopeptidases are a group of metal-dependent enzymes that hydrolyze peptide bonds. These enzymes found in Streptococcus pneumoniae assist the pathogen in infecting the host by breaking down host tissues and extracellular matrix proteins. Considering metallopeptidases' significant role in bacterial virulence, inhibiting this enzyme represents a promising avenue for research. These enzymes are characterized by the presence of Zn(II) in the active site, proper coordination of which is essential for their catalytic function. This work aims to determine the significance of the specific amino acids in the metal binding domain of metallopeptidase from S. pneumoniae. For this purpose, we investigated the coordination chemistry of Zn(II), Ni(II), and Cu(II) ions with point-mutated peptide models of the metal-binding domain. Mutations were introduced at His-2 (L1) and Glu-1. Studies have shown that at pH 7.14 (pH of infected lungs by S. pneumoniae), point mutation on glutamic acid caused only minor effects on the binding of Zn(II) and Ni(II), while significantly improving Cu(II) binding. The stability of copper complexes is greater with the mutant Glu-1 â†’ Gln-1 than with the original domain due to a hydrogen bonding network created by the Gln backbone with its side chain. Substituting histidine resulted in a significant reduction in metal binding for all metal ions, highlighting the crucial role of His-2 in metal coordination. Introduced mutations at neutral pH did not significantly affect the secondary structure of metal complexes. However, at alkaline pH, the peptides showed a higher percentage of antiparallel ß-sheet structures upon the addition of Cu(II), Ni(II) and Zn(II).

The knockdown of stathmin with si-RNA inhibits invasion of mesothelioma.

BACKGROUND: To investigate the mechanism of action of stathmin1 (STMN1) in mesothelioma (MSM) and whether it has any role in its treatment. METHODS: STMN1 expression was examined using immunohistochemistry in biopsy tissues taken from MSM patients. The relationships between the levels of STMN1 expression in the pathology preparations of MSM patients, and the clinicopathological characteristics of these patients, and their survival times were investigated. Transfection of STMN1-specific siRNA into SPC212 cells was compared to negative control siRNAs. The mRNA levels of genes that may play a role in invasion, apoptosis, and autophagy were evaluated by RT-PCR. RESULTS: The expression of STMN1 was shown to be high in MSM tissues (p < 0.05). It was found that the only independent predictor factor affecting the survival time of MSM patients was the disease stage (p < 0.05). STMN1 was significantly reduced after siRNA intervention (81.5%). STMN1 with specific siRNA has been shown to suppress invasion by reducing the mRNA levels of cadherin-6 (CDH6), fibroblast growth factor-8 (FGF8), hypoxia-inducible factor 1 (HIF1A), matrix metallopeptidase 1-2 (gelatinase A) (MMP1-2), and TIMP metallopeptidase inhibitor 2 (TIMP2), which are important markers for invasion. Although the expression of apoptosis and autophagy-related genes, caspase-2 (Casp2) and LC-3, was reduced by silencing STMN1 with specific siRNA in western blot analysis, this effect was not observed in PCR results. CONCLUSIONS: Immunohistochemical analysis of STMN1 may contribute to the differential diagnosis of MSM, and STMN1 may also be considered as a potential therapeutic target in the early invasive stage of MSM therapy.

Identification, Biochemical Characterization, and In Vivo Detection of a Zn-Metalloprotease with Collagenase Activity from Mannheimia haemolytica A2.

Respiratory diseases in ruminants are a main cause of economic losses to farmers worldwide. Approximately 25% of ruminants experience at least one episode of respiratory disease during the first year of life. Mannheimia haemolytica is the main etiological bacterial agent in the ruminant respiratory disease complex. M. haemolytica can secrete several virulence factors, such as leukotoxin, lipopolysaccharide, and proteases, that can be targeted to treat infections. At present, little information has been reported on the secretion of M. haemolytica A2 proteases and their host protein targets. Here, we obtained evidence that M. haemolytica A2 proteases promote the degradation of hemoglobin, holo-lactoferrin, albumin, and fibrinogen. Additionally, we performed biochemical characterization for a specific 110 kDa Zn-dependent metalloprotease (110-Mh metalloprotease). This metalloprotease was purified through ion exchange chromatography and characterized using denaturing and chaotropic agents and through zymography assays. Furthermore, mass spectrometry identification and 3D modeling were performed. Then, antibodies against the 110 kDa-Mh metalloprotease were produced, which achieved great inhibition of proteolytic activity. Finally, the antibodies were used to perform immunohistochemical tests on postmortem lung samples from sheep with suggestive histology data of pneumonic mannheimiosis. Taken together, our results strongly suggest that the 110-Mh metalloprotease participates as a virulence mechanism that promotes damage to host tissues.

The mitochondrial protease PARL is required for spermatogenesis.

Mitochondrial function plays an important role in the maintenance of male fertility. However, the mechanisms underlying mitochondrial defect-related infertility remain mostly unclear. Here we show that a deficiency of PARL (Parl-/-), a mitochondrial protease, causes complete arrest of spermatogenesis during meiosis I. PARL deficiency led to severe downregulation of proteins of respiratory chain complex IV in testes that did not occur in other tested organs, causing a deficit in complex IV activity and ATP production. Furthermore, Parl-/- testes showed an almost complete loss of HSD17B3, a protein of the sER responsible for the last step in testosterone synthesis. While testosterone production appeared to be restored by overexpression of HSD17B12, loss of the canonical testosterone synthesis led to an upregulation of luteinizing hormone (LH) and of LH-regulated responses. These results suggest an important impact of the downstream regulation of mitochondrial defects that manifest in a cell-type-specific manner and extend beyond mitochondria.

Recombinant protein production and functional analysis of a M60-like-2 metallopeptidase enzyme from the carcinogenic liver fluke Opisthorchis viverrini.

Mucin plays a crucial role in safeguarding mucosal tissues by obstructing the translocation of microorganisms. Mucosal tissue-dwelling parasites must devise a strategy to surmount this mucin barrier in order to establish colonization. In a recent discovery, it was observed that the liver fluke Opisthorchis viverrini secretes two mucinases, namely Ov-M60-like-1 and Ov-M60-like-2. Ov-M60-like-1 was previously characterized. Here, we study the Ov-M60-like-2 by utilizing the wheat germ expression system to produce recombinant proteins and conducted a functional analysis of its enzymatic activity on bovine submaxillary mucin (BSM). Subsequently, we delved deeper into understanding the role of this enzyme in host-parasite interactions by evaluating its mucinase activity on mucins from the bile duct of O. viverrini-infected hamsters. Through successful production of recombinant proteins using the wheat germ expression system, we observed that this enzyme displayed mucinase activity over a wide pH range (pH 2 to pH 10) against BSM. Our investigations revealed it ability to digest mucin from the bile duct. These findings suggest that Ov-M60-like-2 possess a mucinase activity, together with Ov-M60-like-1, enabling the liver fluke to successful colonization of the host's bile duct.

Effects of toxin metalloproteinases from jellyfish Nemopilema nomurai nematocyst on the dermal toxicity and potential treatment of jellyfish dermatitis.

Jellyfish dermatitis is a common medical problem in many countries due to the jellyfish envenomation. However, there are no specific and targeted medications for their treatment. Here we investigated the possible therapeutic effects of metalloproteinase inhibitors on the dermal toxicity of Nemopilema nomurai nematocyst venom (NnNV), a giant venomous jellyfish from China, using the jellyfish dermatitis model, focusing on inflammatory effector molecules during jellyfish envenomation. Metalloproteinase may further stimulate inflammation by promoting oxidative stress in the organism and play key roles by activating MAPK and NF-κB, in the pathogenesis of jellyfish dermatitis. And the metalloproteinase inhibitors batimastat and EDTA disodium salt may treat the Jellyfish dermatitis by inhibiting the metalloproteinase activity in NnNV. These observations suggest that the metalloproteinase components of NnNV make a considerable contribution to dermal toxicity as the inflammation effect molecular, and metalloproteinase inhibitors can be regarded as novel therapeutic medicines in jellyfish envenomation. This study contributes to understanding the mechanism of jellyfish dermatitis and suggests new targets and ideas for the treatment of jellyfish envenomation.

Substrate profiling of the metalloproteinase ovastacin uncovers specific enzyme-substrate interactions and discloses fertilization-relevant substrates.

The metalloproteinase ovastacin is released by the mammalian egg upon fertilization and cleaves a distinct peptide bond in zona pellucida protein 2 (ZP2), a component of the enveloping extracellular matrix. This limited proteolysis causes zona pellucida hardening, abolishes sperm binding, and thereby regulates fertility. Accordingly, this process is tightly controlled by the plasma protein fetuin-B, an endogenous competitive inhibitor. At present, little is known about how the cleavage characteristics of ovastacin differ from closely related proteases. Physiological implications of ovastacin beyond ZP2 cleavage are still obscure. In this study, we employed N-terminal amine isotopic labeling of substrates (N-TAILS) contained in the secretome of mouse embryonic fibroblasts to elucidate the substrate specificity and the precise cleavage site specificity. Furthermore, we were able to unravel the physicochemical properties governing ovastacin-substrate interactions as well as the individual characteristics that distinguish ovastacin from similar proteases, such as meprins and tolloid. Eventually, we identified several substrates whose cleavage could affect mammalian fertilization. Consequently, these substrates indicate newly identified functions of ovastacin in mammalian fertilization beyond zona pellucida hardening.

PSD-95 inhibitor Tat-NR2B9c (NA-1) protects the integrity of the blood-brain barrier after transient middle artery occlusion in rats by downregulating matrix metalloprotease-9 and upregulating endothelial nitric oxide synthase.

BACKGROUND: Protection against ischemic stroke may be most effective when multiple components of the neurovascular unit are protected, yet current treatments target mainly neurons. Here we explored whether the PSD-95 inhibitor Tat-NR2B9c (NA-1) can protect not only neurons but also the blood-brain barrier. METHODS: Adult male Sprague-Dawley rats were randomly divided into three groups, which were subjected to either sham surgery or transient cerebral ischemia-reperfusion, after which some animals were treated with Tat-NR2B9c. The therapeutic efficacy of Tat-NR2B9c was assessed in terms of the degree of neurological deficit and cerebral infarction, integrity of the blood-brain barrier, cerebral water content, as well as expression of PSD-95, nitric oxide synthase, and matrix metalloprotease-9. RESULTS: Tat-NR2B9c (NA-1) ameliorated neurofunctional deficit, reduced cerebral infarction, mitigated blood-brain barrier injury and improved its integrity following ischemia-reperfusion, leading to less cerebral edema. These improvements were associated with upregulation of tight junction proteins in the blood-brain barrier. At the same time, Tat-NR2B9c (NA-1) downregulated neuronal nitric oxide synthase and matrix metalloprotease-9, while reversing the ischemia-induced downregulation of endothelial nitric oxide synthase in brain. We report here the first evidence that PSD-95 is expressed in vascular endothelial cells in the brain. CONCLUSION: Our experiments in a rat model of transient occlusion of the middle cerebral artery suggest that Tat-NR2B9c (NA-1) can mitigate ischemic injury to the blood-brain barrier, and that it may do so by downregulating matrix metalloprotease-9 and upregulating endothelial nitric oxide synthase.