Apo-Lactoferrin Inhibits the Proteolytic Activity of the 110 kDa Zn Metalloprotease Produced by Mannheimia haemolytica A2.

Mannheimia haemolytica is the main etiological bacterial agent in ruminant respiratory disease. M. haemolytica secretes leukotoxin, lipopolysaccharides, and proteases, which may be targeted to treat infections. We recently reported the purification and in vivo detection of a 110 kDa Zn metalloprotease with collagenase activity (110-Mh metalloprotease) in a sheep with mannheimiosis, and this protease may be an important virulence factor. Due to the increase in the number of multidrug-resistant strains of M. haemolytica, new alternatives to antibiotics are being explored; one option is lactoferrin (Lf), which is a multifunctional iron-binding glycoprotein from the innate immune system of mammals. Bovine apo-lactoferrin (apo-bLf) possesses many properties, and its bactericidal and bacteriostatic effects have been highlighted. The present study was conducted to investigate whether apo-bLf inhibits the secretion and proteolytic activity of the 110-Mh metalloprotease. This enzyme was purified and sublethal doses of apo-bLf were added to cultures of M. haemolytica or co-incubated with the 110-Mh metalloprotease. The collagenase activity was evaluated using zymography and azocoll assays. Our results showed that apo-bLf inhibited the secretion and activity of the 110-Mh metalloprotease. Molecular docking and overlay assays showed that apo-bLf bound near the active site of the 110-Mh metalloprotease, which affected its enzymatic activity.

Importance of the Cysteine-Rich Domain of Snake Venom Prothrombin Activators: Insights Gained from Synthetic Neutralizing Antibodies.

Snake venoms are cocktails of biologically active molecules that have evolved to immobilize prey, but can also induce a severe pathology in humans that are bitten. While animal-derived polyclonal antivenoms are the primary treatment for snakebites, they often have limitations in efficacy and can cause severe adverse side effects. Building on recent efforts to develop improved antivenoms, notably through monoclonal antibodies, requires a comprehensive understanding of venom toxins. Among these toxins, snake venom metalloproteinases (SVMPs) play a pivotal role, particularly in viper envenomation, causing tissue damage, hemorrhage and coagulation disruption. One of the current challenges in the development of neutralizing monoclonal antibodies against SVMPs is the large size of the protein and the lack of existing knowledge of neutralizing epitopes. Here, we screened a synthetic human antibody library to isolate monoclonal antibodies against an SVMP from saw-scaled viper (genus Echis) venom. Upon characterization, several antibodies were identified that effectively blocked SVMP-mediated prothrombin activation. Cryo-electron microscopy revealed the structural basis of antibody-mediated neutralization, pinpointing the non-catalytic cysteine-rich domain of SVMPs as a crucial target. These findings emphasize the importance of understanding the molecular mechanisms of SVMPs to counter their toxic effects, thus advancing the development of more effective antivenoms.

Potential of Marine Bacterial Metalloprotease A69 in the Preparation of Peanut Peptides with Angiotensin-Converting Enzyme (ACE)-Inhibitory and Antioxidant Properties.

Marine bacterial proteases have rarely been used to produce bioactive peptides, although many have been reported. This study aims to evaluate the potential of the marine bacterial metalloprotease A69 from recombinant Bacillus subtilis in the preparation of peanut peptides (PPs) with antioxidant activity and angiotensin-converting enzyme (ACE)-inhibitory activity. Based on the optimization of the hydrolysis parameters of protease A69, a process for PPs preparation was set up in which the peanut protein was hydrolyzed by A69 at 3000 U g-1 and 60 °C, pH 7.0 for 4 h. The prepared PPs exhibited a high content of peptides with molecular weights lower than 1000 Da (>80%) and 3000 Da (>95%) and contained 17 kinds of amino acids. Moreover, the PPs displayed elevated scavenging of hydroxyl radical and 1,1-diphenyl-2-picryl-hydrazyl radical, with IC50 values of 1.50 mg mL-1 and 1.66 mg mL-1, respectively, indicating the good antioxidant activity of the PPs. The PPs also showed remarkable ACE-inhibitory activity, with an IC50 value of 0.71 mg mL-1. By liquid chromatography mass spectrometry analysis, the sequences of 19 ACE inhibitory peptides and 15 antioxidant peptides were identified from the PPs. These results indicate that the prepared PPs have a good nutritional value, as well as good antioxidant and antihypertensive effects, and that the marine bacterial metalloprotease A69 has promising potential in relation to the preparation of bioactive peptides from peanut protein.

The Balance between Protealysin and Its Substrate, the Outer Membrane Protein OmpX, Regulates Serratia proteamaculans Invasion.

Serratia are opportunistic bacteria, causing infections in plants, insects, animals and humans under certain conditions. The development of bacterial infection in the human body involves several stages of host-pathogen interaction, including entry into non-phagocytic cells to evade host immune cells. The facultative pathogen Serratia proteamaculans is capable of penetrating eukaryotic cells. These bacteria synthesize an actin-specific metalloprotease named protealysin. After transformation with a plasmid carrying the protealysin gene, noninvasive E. coli penetrate eukaryotic cells. This suggests that protealysin may play a key role in S. proteamaculans invasion. This review addresses the mechanisms underlying protealysin's involvement in bacterial invasion, highlighting the main findings as follows. Protealysin can be delivered into the eukaryotic cell by the type VI secretion system and/or by bacterial outer membrane vesicles. By cleaving actin in the host cell, protealysin can mediate the reversible actin rearrangements required for bacterial invasion. However, inactivation of the protealysin gene leads to an increase, rather than decrease, in the intensity of S. proteamaculans invasion. This indicates the presence of virulence factors among bacterial protealysin substrates. Indeed, protealysin cleaves the virulence factors, including the bacterial surface protein OmpX. OmpX increases the expression of the EGFR and ß1 integrin, which are involved in S. proteamaculans invasion. It has been shown that an increase in the invasion of genetically modified S. proteamaculans may be the result of the accumulation of full-length OmpX on the bacterial surface, which is not cleaved by protealysin. Thus, the intensity of the S. proteamaculans invasion is determined by the balance between the active protealysin and its substrate OmpX.

Deregulation of Metalloproteinase Expression in Gray Horse Melanoma Ex Vivo and In Vitro.

The ability of human melanoma cells to switch from an epithelial to a mesenchymal phenotype contributes to the metastatic potential of disease. Metalloproteinases (MPs) are crucially involved in this process by promoting the detachment of tumor cells from the primary lesion and their migration to the vasculature. In gray horse melanoma, epithelial-mesenchymal transition (EMT) is poorly understood, prompting us to address MP expression in lesions versus intact skin by transcriptome analyses and the immunofluorescence staining (IF) of gray horse tumor tissue and primary melanoma cells. RNAseq revealed the deregulation of several MPs in gray horse melanoma and, notably, a 125-fold upregulation of matrix metalloproteinase 1 (MMP1) that was further confirmed by RT-qPCR from additional tumor material. The IF staining of melanoma tissue versus intact skin for MMP1 and tumor marker S100 revealed MMP1 expression in all lesions. The co-expression of S100 was observed at different extents, with some tumors scoring S100-negative. The IF staining of primary tumor cells explanted from the tumors for MMP1 showed that the metalloproteinase is uniformly expressed in the cytoplasm of 100% of tumor cells. Overall, the presented data point to MP expression being deregulated in gray horse melanoma, and suggest that MMP1 has an active role in gray horse melanoma by driving EMT-mediated tumor cell dissemination via the degradation of the extracellular matrix. Whilst S100 is considered a reliable tumor marker in human MM, gray horse melanomas do not seem to regularly express this protein.

Galectin-13 and Laeverin Levels Interfere with Human Fetoplacental Growth.

Galectin-13 (Gal-13) is predominantly produced by the syncytiotrophoblast, while laeverin is expressed on the outgrowing extravillous trophoblast, and both are thought to be biomarkers of preeclampsia. The aim of this study was to assess the correlation between concentrations of Gal-13 and laeverin measured in maternal serum and amniotic fluid at 16-22 weeks of gestation and the sonographic assessment of the fetoplacental measurements. Fetal biometric data and placental volume and perfusion indices were measured in 62 singleton pregnancies. Serum and amniotic levels of Gal-13 and laeverin levels were measured using a sandwich ELISA. Both amniotic fluid and serum Gal-13 levels expressed a negative correlation to the plasma laeverin level in mid-pregnancy. Serum laeverin level correlated positively with the gestational length at delivery (ß = 0.39, p < 0.05), while the amniotic laeverin level correlated well with the abdominal circumference of the fetus (ß = 0.44, p < 0.05). Furthermore, laeverin level in the amnion correlated positively with the estimated fetal weight (ß = 0.48, p < 0.05) and with the placental volume (ß = 0.32, p < 0.05). Logistic regression analyses revealed that a higher circulating Gal-13 level represents a slightly significant risk factor (OR: 1.01) for hypertension-related diseases during pregnancy. It is a novelty that laeverin can be detected in the amniotic fluid, and amnion laeverin concentration represents a potential biomarker of fetoplacental growth.

Cloning and expression of recombinant arazyme with anti-inflammatory and anti-breast cancer potential.

Arazyme is an extracellular metalloprotease which is secreted by a Gram-negative symbiotic bacterium called Serratia proteomaculans. There are limited studies on various biological activities of arazyme. This preliminary study was designed to investigate the anti-cancer and anti-inflammatory capacities of recombinant arazyme (rAra) in vitro and in vivo. Arazyme gene, araA was cloned and expressed in E. coli BL21 (DE3) using pET-28a as a vector. Nickel column purification was used to obtain pure rAra. SDS-PAGE and protein assay were used to identify the product and to measure protein content, respectively. Skimmed milk test and casein assay were carried out to assess protease activity. MCF7 cells as a breast cancer cell model were exposed to different concentrations of rAra to study anti-breast cancer potentials using MTT assay. The anti-inflammatory property of rAra was investigated using a murine air-pouch model. PCR and SDS-PAGE data showed that cloning and expression of rAra was successful and the enzyme of interest was observed at 52 KDa. Protein assay indicated that 1 mg/ml of rAra was obtained through purification. A clear zone around the enzyme on skimmed milk agar confirmed the proteolytic activity of rAra and the enzymatic activity was 320 U/mg protein in the casein assay. Cytotoxic effects of rAra reported as IC50 were 16.2 µg/ml and 13.2 mg/ml after 24 h and 48 h, respectively. In the air-pouch model, both the neutrophil count and myeloperoxidase activity, which are measures of inflammation, were significantly reduced. The results showed that rAra can be used in future mechanistic studies and R&D activities in the pharmaceutical industry to investigate the safety and efficacy of the recombinant arazyme.

Dual therapy with phospholipase and metalloproteinase inhibitors from Sinonatrix annularis alleviated acute kidney and liver injury caused by multiple snake venoms.

Snakebite envenomation often induces acute kidney injury (AKI) and acute liver injury (ALI), leading to augmented injuries and poor rehabilitation. Phospholipase A2 (PLA2) and metalloproteinase (SVMP) present in venom are responsible for the envenomation-associated events. In this study, mice envenomed with Deinagkistrodon acutus, Naja atra, or Agkistrodon halys pallas venom exhibited typical AKI and ALI symptoms, including significantly increased plasma levels of myoglobin, free hemoglobin, uric acid, aspartate aminotransferase, and alanine aminotransferase and upregulated expression of kidney NGAL and KIM-1. These effects were significantly inhibited when the mice were pretreated with natural inhibitors of PLA2 and SVMP isolated from Sinonatrix annularis (SaPLIγ and SaMPI). The inhibitors protected the physiological structural integrity of the renal tubules and glomeruli, alleviating inflammatory infiltration and diffuse hemorrhage in the liver. Furthermore, the dual therapy alleviated oxidative stress and apoptosis in the kidneys and liver by mitigating mitochondrial damage, thereby effectively reducing the lethal effect of snake venom in the inhibitor-treated mouse model. This study showed that dual therapy with inhibitors of metalloproteinase and phospholipase can effectively prevent ALI and AKI caused by snake bites. Our findings suggest that intrinsic inhibitors present in snakes are prospective therapeutic agents for multi-organ injuries caused by snake envenoming.

Highly conserved and extremely variable: The paradoxical pattern of toxin expression revealed by comparative venom-gland transcriptomics of Phalotris (Serpentes: Dipsadidae).

Although non-front fanged snakes account for almost two-thirds of snake diversity, most studies on venom composition and evolution focus exclusively on front-fanged species, which comprise most of the clinically relevant accidents. Comprehensive reports on venom composition of non-front fanged snakes are still scarce for several groups. In this study, we address such shortage of knowledge by providing new insights about the venom composition among species of Phalotris, a poorly studied Neotropical dipsadid genus. Phalotris are known for their specialized venom delivery system and toxic venoms, which can cause life-threatening accidents in humans. We evaluate the venom-gland transcriptome of Phalotris, comparing the following three South American species: P. reticulatus for the Araucaria Pine forests, P. lemniscatus for the Pampa grasslands, and P. mertensi for the Brazilian Cerrado. Our results indicate similar venom profiles, in which they share a high expression level of Kunitz-type inhibitors (KUNZ). On the other hand, comparative analyses revealed substantial differences in the expression levels of C-type lectins (CTL) and snake venom metalloproteinases (SVMP). The diverse set of SVMP and CTL isoforms shows signals of positive selection, and we also identified truncated forms of type III SVMPs, which resemble type II and type I SVMPs of viperids. Additionally, we identified a CNP precursor hosting a proline-rich region containing a BPP motif resembling those commonly detected in viperid venoms with hypotensive activity. Altogether, our results suggest an evolutionary history favoring high expression levels of few KUNZ isoforms in Phalotris venoms, contrasting with a highly diverse set of SVMP and CTL isoforms. Such diversity can be comparable with the venom variability observed in some viperids. Our findings highlight the extreme phenotypic diversity of non-front fanged snakes and the importance to allocate greater effort to study neglected groups of Colubroidea.

Insights into the discovery and intervention of metalloproteinase in marine hazardous jellyfish.

The proliferation of toxic organisms caused by changes in the marine environment, coupled with the rising human activities along the coastal lines, has resulted in an increasing number of stinging incidents, posing a serious threat to public health. Here, we evaluated the systemic toxicity of the venom in jellyfish Chrysaora quinquecirrha at both cellular and animal levels, and found that jellyfish tentacle extract (TE) has strong lethality accompanied by abnormal elevation of blood biochemical indicators and pathological changes. Joint analysis of transcriptome and proteome indicated that metalloproteinases are the predominant toxins in jellyfish. Specially, two key metalloproteinases DN6695_c0_g3 and DN8184_c0_g7 were identified by mass spectrometry of the red blood cell membrane and tetracycline hydrochloride (Tch) inhibition models. Structurally, molecular docking and kinetic analysis are employed and observed that Tch could inhibit the enzyme activity by binding to the hydrophobic pocket of the catalytic center. In this study, we demonstrated that Tch impedes the metalloproteinase activity thereby reducing the lethal effect of jellyfish, which suggests a potential strategy for combating the health threat of marine toxic jellyfish.

Anti-arthritic and immunomodulatory efficacy of Micromeria biflora Benth extract and its fractions in rats by restoring oxidative stress, metalloproteinases, pro-inflammatory and anti-inflammatory cytokines network.

Micromeria biflora (M.B) Benth has proven anti-inflammatory efficacy, thereby, the goal of the current investigation was to assess the anti-arthritic potential of M.B ethanolic extract and fractions as well as to investigate the likely mechanism of action. The effectiveness of M.B against acute arthritic manifestations was assessed using an arthritic model prompted by formaldehyde, whereas a chronic model was developed using an adjuvant called Complete Freund's in Sprague-Dawley rats. Weekly evaluations were conducted for parameters involving paw volume, body weight, and arthritic score; at the completion of the CFA model, hematological, biochemical and oxidative stress parameters as well as the level of various mediators (PGE2, IL-1ß, TNFα, IL6, MMP2, 3, 9, VEGF, NF-ĸB, IL-10, and IL-4) were evaluated. The results demonstrated the plant's ability to treat arthritis by showing a significant decrease in paw volume, arthritic score, and histological characteristics. The levels of NF-ĸB, MMP2, 3, 9, IL6, IL1ß, TNFα, and VEGF were all significantly reduced after treatment with plant extract and fractions. Plant extract and its fractions substantially preserved body weight loss, oxidative stress markers and levels of IL-4 and 1L-10. PGE2 levels were also shown to be reduced in the treatment groups, supporting the M.B immunomodulatory ability. Hematological and biochemical indicators were also normalized after M.B administration. Outcomes of the study validated the anti-arthritic and immunomodulatory attributes of M.B probably through modulating oxidative stress, inflammatory, pro-inflammatory and anti-inflammatory biomarkers.

BjussuMP-II, a venom metalloproteinase, induces the release and cleavage of pro-inflammatory cytokines and disrupts human umbilical vein endothelial cells.

Snake venom metalloproteases (SVMPs) are hydrolytic enzymes dependent on metal binding, primarily zinc (Zn2+), at their catalytic site. They are classified into three classes (P-I to P-III). BjussuMP-II, a P-I SVMP isolated from Bothrops jararacussu snake venom, has a molecular mass of 24 kDa. It exhibits inhibitory activity on platelet aggregation and hydrolyzes fibrinogen. TNF-α upregulates the expression of adhesion molecules on endothelial cell surfaces, promoting leukocyte adhesion and migration during inflammation. Literature indicates that SVMPs may cleave the TNF-α precursor, possibly due to significant homology between metalloproteases from mammalian extracellular matrix and SVMPs. This study aimed to investigate BjussuMP-II's effects on human umbilical vein endothelial cells (HUVEC), focusing on viability, detachment, adhesion, release, and cleavage of TNF-α, IL-1ß, IL-6, IL-8, and IL-10. HUVEC were incubated with BjussuMP-II (1.5-50 µg/mL) for 3-24 h. Viability was determined using LDH release, MTT metabolization, and 7AAD for membrane integrity. Adhesion and detachment were assessed by incubating cells with BjussuMP-II and staining with Giemsa. Cytokines were quantified in HUVEC supernatants using EIA. TNF-α cleavage was evaluated using supernatants from PMA-stimulated cells or recombinant TNF-α. Results demonstrated BjussuMP-II's proteolytic activity on casein. It was not toxic to HUVEC at any concentration or duration studied but interfered with adhesion and promoted detachment. PMA induced TNF-α release by HUVEC, but this effect was not observed with BjussuMP-II, which cleaved TNF-α. Additionally, BjussuMP-II cleaved IL-1ß, IL-6, and IL-10. These findings suggest that the zinc metalloprotease BjussuMP-II could be a valuable biotechnological tool for treating inflammatory disorders involving cytokine deregulation.

Assessing Target Specificity of the Small Molecule Inhibitor MARIMASTAT to Snake Venom Toxins: A Novel Application of Thermal Proteome Profiling.

New treatments that circumvent the pitfalls of traditional antivenom therapies are critical to address the problem of snakebite globally. Numerous snake venom toxin inhibitors have shown promising cross-species neutralization of medically significant venom toxins in vivo and in vitro. The development of high-throughput approaches for the screening of such inhibitors could accelerate their identification, testing, and implementation and thus holds exciting potential for improving the treatments and outcomes of snakebite envenomation worldwide. Energetics-based proteomic approaches, including thermal proteome profiling and proteome integral solubility alteration (PISA) assays, represent "deep proteomics" methods for high throughput, proteome-wide identification of drug targets and ligands. In the following study, we apply thermal proteome profiling and PISA methods to characterize the interactions between venom toxin proteoforms in Crotalus atrox (Western Diamondback Rattlesnake) and the snake venom metalloprotease (SVMP) inhibitor marimastat. We investigate its venom proteome-wide effects and characterize its interactions with specific SVMP proteoforms, as well as its potential targeting of non-SVMP venom toxin families. We also compare the performance of PISA thermal window and soluble supernatant with insoluble precipitate using two inhibitor concentrations, providing the first demonstration of the utility of a sensitive high-throughput PISA-based approach to assess the direct targets of small molecule inhibitors for snake venom.

Inhibition of Verticillium Wilt in Cotton through the Application of Pseudomonas aeruginosa ZL6 Derived from Fermentation Residue of Kitchen Waste.

To isolate and analyze bacteria with Verticillium wilt-resistant properties from the fermentation residue of kitchen wastes, as well as explore their potential for new applications of the residue. A total of six bacterial strains exhibiting Verticillium wilt-resistant capabilities were isolated from the biogas residue of kitchen waste fermentation. Using a polyphasic approach, strain ZL6, which displayed the highest antagonistic activity against cotton Verticillium wilt, was identified as belonging to the Pseudomonas aeruginosa. Bioassay results demonstrated that this strain possessed robust antagonistic abilities, effectively inhibiting V. dahliae spore germination and mycelial growth. Furthermore, P. aeruginosa ZL6 exhibited high temperature resistance (42°C), nitrogen fixation, and phosphorus removal activities. Pot experiments revealed that P. aeruginosa ZL6 fermentation broth treatment achieved a 47.72% biological control effect compared to the control group. Through activity tracking and protein mass spectrometry identification, a neutral metalloproteinase (Nml) was hypothesized as the main virulence factor. The mutant strain ZL6ΔNml exhibited a significant reduction in its ability to inhibit cotton Verticillium wilt compared to the strain P. aeruginosa ZL6. While the inhibitory activities could be partially restored by a complementation of nml gene in the mutant strain ZL6CMΔNml. This research provides a theoretical foundation for the future development and application of biogas residue as biocontrol agents against Verticillium wilt and as biological preservatives for agricultural products. Additionally, this study presents a novel approach for mitigating the substantial amount of biogas residue generated from kitchen waste fermentation.

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.

Plasmodium falciparum proteases as new drug targets with special focus on metalloproteases.

Malaria poses a significant global health threat particularly due to the prevalence of Plasmodium falciparum infection. With the emergence of parasite resistance to existing drugs including the recently discovered artemisinin, ongoing research seeks novel therapeutic avenues within the malaria parasite. Proteases are promising drug targets due to their essential roles in parasite biology, including hemoglobin digestion, merozoite invasion, and egress. While exploring the genomic landscape of Plasmodium falciparum, it has been revealed that there are 92 predicted proteases, with only approximately 14 of them having been characterized. These proteases are further distributed among 26 families grouped into five clans: aspartic proteases, cysteine proteases, metalloproteases, serine proteases, and threonine proteases. Focus on metalloprotease class shows further role in organelle processing for mitochondria and apicoplasts suggesting the potential of metalloproteases as viable drug targets. Holistic understanding of the parasite intricate life cycle and identification of potential drug targets are essential for developing effective therapeutic strategies against malaria and mitigating its devastating global impact.

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.

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.

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.

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.