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Based on the in vitro trypanocidal efficacy of previously synthesized N -aryl-1,10-phenanthroline-2-amines ( Phen1-20 ) (aryl = R-phenyl, 1- or 2-naphthyl), we explored the potential interactions of these derivatives as ligands of our comparative model of T. cruzi GP63 ( Tc GP63). This surface metalloprotease plays a crucial role in parasite adhesion to host cells and aids in cell invasion during T. cruzi infection in Chagas disease. Ligand-protein consensus docking simulations using four GOLD scoring functions revealed that N -(R-phenyl) derivatives (R = CH 3 , OCH 3 , CF 3 , CN, NO 2 , F, Cl, and Br) presented poses with higher fitness scores than the N -naphthyl ones, with the six para -substituted derivatives ( Phen4 , p -CH 3 ; Phen7 , p -OCH 3 ; Phen10 , p -CN; Phen14 , p -F; Phen17 , p -Cl; and Phen18 , p -Br) being more favorable than the ortho or meta ones. Subsequent aqueous molecular dynamics simulation (GROMACS package, CHARMM36 force field, and TIP3P water model) of the ligand-protein complexes for these six top-ranking compounds showed persistent interactions within the Tc GP63 active site, primarily through coordination with Zn(II)-cofactor, and H-bonding with catalytic Glu221 and zinc-binding His224. RMSD and RMSF analyses confirmed the stability of these interactions, particularly for compounds with electron-withdrawing groups by inductive effect as R-substituents, such as p -OCH 3 ( Phen7 ) and p -CN ( Phen10 ). Binding free energy calculations by the linear interaction energy (LIE) approach corroborated the favorable interactions observed in simulations, highlighting Phen7 and Phen10 as the most promising candidates. This study underscores the potential of N -phenyl-1,10-phenanthroline-2-amines as putative inhibitors targeting the T. cruzi GP63 enzyme.

Keratinases are exciting proteolytic enzymes that display the capability to degrade the insoluble protein keratin. These enzymes are produced by diverse microorganisms belonging to the Eucarya, Bacteria, and Archea domains. Keratinases display a great diversity in their biochemical and biophysical properties. Most keratinases are optimally active at neutral to alkaline pH and 40-60°C, but examples of microbial keratinolysis at alkalophilic and thermophilic conditions have been well documented. Several keratinases have been associated to the subtilisin family of serine-type proteases by analysis of their protein sequences. Studies with specific substrates and inhibitors indicated that keratinases are often serine or metalloproteases with preference for hydrophobic and aromatic residues at the P1 position. Keratinolytic enzymes have several current and potential applications in agroindustrial, pharmaceutical, and biomedical fields. Their use in biomass conversion into biofuels may address the increasing concern on energy conservation and recycling.

Predator-prey interactions play important roles in the cycling of marine organic matter. Here we show that a Gram-negative bacterium isolated from marine sediments ( Pseudoalteromonas sp. strain CF6-2) can kill Gram-positive bacteria of diverse peptidoglycan (PG) chemotypes by secreting the metalloprotease pseudoalterin. Secretion of the enzyme requires a Type II secretion system. Pseudoalterin binds to the glycan strands of Gram positive bacterial PG and degrades the PG peptide chains, leading to cell death. The released nutrients, including PG-derived D-amino acids, can then be utilized by strain CF6-2 for growth. Pseudoalterin synthesis is induced by PG degradation products such as glycine and glycine-rich oligopeptides. Genes encoding putative pseudoalterin-like proteins are found in many other marine bacteria. This study reveals a new microbial interaction in the ocean. Predator-prey interactions play important roles in the cycling of marine organic matter. Here the authors show that a Gram-negative bacterium isolated from marine sediments can kill and feed on Gram-positive bacteria by secreting a peptidoglycan-degrading enzyme.

Key Points ADAMs (a disintegrin and metalloproteinase) are membrane-associated metalloproteinases with a complex multi-domain structure. About half of the family have proteolytic potential, as well as domains with adhesive properties and a cytoplasmic domain involved in cell signalling. The upregulation of proteolytic ADAMs has been documented for a number of cancers, with some correlation to parameters of disease progression. The 'shedding' activities of ADAMs — cleaving transmembrane proteins and solubilizing the complete ectodomain of cytokines, growth factors, receptors and adhesion molecules — places them in pivotal positions in the extracellular regulation of cellular signalling. They can potentially regulate many activities within the tumour microenvironment, including inflammatory responses, immune regulation, angiogenesis, and cell migration and proliferation. ADAM proteolysis has been implicated in key signalling pathways identified in many tumour cells. The generation of soluble ligands for the family of Erbb receptors, including in response to the activation of G-protein-coupled receptors and leading to epidermal growth factor receptor transactivation, have been described. ADAM sheddase activity appears to modulate the process of regulated intramembrane proteolysis (RIPping) by γ-secretase: the intracellular portions of transmembrane proteins are released either into proteasomal degradation pathways or into the cytosol prior to nuclear trafficking where they function as transcription factors. ADAM activity is regulated in part by cellular trafficking and changes in the interaction with the membrane-bound substrate. Numerous protein adaptors and modulators that alter either enzyme activity or substrate availability have been documented and these may build specificity into ADAM sheddase activities in vivo . The data from cell and animal models of cancer indicate that ADAM proteolytic activities could drive aspects of tumorigenesis. Before their consideration as therapeutic targets it will be necessary to further define the correlation of their expression in relation to different aspects of tumour development, and the mechanism by which they do this. Furthermore, the design of specific inhibitors, possibly targeting extracatalytic sites or adaptor proteins, will be essential. The disintegrin metalloproteinases of the Adam (a disintegrin and metalloproteinase) family mediate proteolytic 'shedding' of membrane-associated proteins and hence rapidly modulate key cell signalling pathways in the tumour microenvironment. What is the biological and clinical relevance of the ADAMs? Over the last few years disintegrin metalloproteinases of the Adam (a disintegrin and metalloproteinase) family have been associated with the process of proteolytic 'shedding' of membrane-associated proteins and hence the rapid modulation of key cell signalling pathways in the tumour microenvironment. Furthermore, numerous members of the Adam family have been associated with tumorigenesis and tumour progression. The question now arises of whether pharmacological manipulation of their functions would be a useful adjunct to therapies targeting intercellular communications. To learn from the lessons of matrix metalloproteinase inhibitors as anticancer agents, there are many facets of the biological and clinical relevance of the ADAMs that need to be understood before embarking with confidence on such an approach.

Following fertilization, cortical granules exocytose ovastacin, a metalloendopeptidase that cleaves ZP2 in the zona pellucida surrounding mouse eggs to prevent additional sperm binding. Using high- and super-resolution imaging with ovastacin mCherry as a fluorescent marker, we characterize cortical granule dynamics at single granule resolution in transgenic mouse eggs. Newly-developed imaging protocols provide an unprecedented view of vesicular dynamics near the plasma membrane in mouse eggs. We discover that cortical granule anchoring in the cortex is dependent on maternal MATER and document that myosin IIA is required for biphasic trafficking to the plasma membrane. We observe local clearance of cortical actin during exocytosis and determine that pharmacologic or genetic disruption of trafficking to the plasma membrane impairs secretion of cortical granules and results in polyspermy. Thus, the regulation of cortical granule dynamics at the cortex-plasma membrane interface is critical for exocytosis and the post-fertilization block to sperm binding that ensures monospermic fertilization. Mammalian eggs require a single sperm for viable fertilization, and cortical granule exocytosis prevents additional sperm binding. Vogt et al. image at single granule resolution to document that cortical granule anchoring in the cortex ensures proper trafficking, exocytosis and polyspermy block.

Snake venom contains mixture of bioactive proteins and polypeptides. Most of these proteins and polypeptides exist as monomers, but some of them form complexes in the venom. These complexes exhibit much higher levels of pharmacological activity compared to individual components and play an important role in pathophysiological effects during envenomation. They are formed through covalent and/or non-covalent interactions. The subunits of the complexes are either identical (homodimers) or dissimilar (heterodimers; in some cases subunits belong to different families of proteins). The formation of complexes, at times, eliminates the non-specific binding and enhances the binding to the target molecule. On several occasions, it also leads to recognition of new targets as protein-protein interaction in complexes exposes the critical amino acid residues buried in the monomers. Here, we describe the structure and function of various protein complexes of snake venoms and their role in snake venom toxicity.

Human immunoglobulin A (IgA), comprising the isotypes IgA1 and IgA2, protects ~400 m 2 of mucosal surfaces against microbial infections but can also lead to aberrant IgA deposits that cause disease. Certain bacteria have evolved peptidases that cleave the hinge between the F ab and F c fragments of IgA, undermining its immune function. These peptidases specifically target IgA1, but not IgA2, which predominates in the gut and possesses a structurally distinct hinge region. The only known IgA2-specific peptidase is IgAse from the gut microbiome member Thomasclavelia ramosa , which also targets IgA1 but no other proteins. IgAse is a ~ 140-kDa, seven-domain, membrane-bound metallopeptidase (MP). Differential scanning fluorimetry, small-angle X-ray scattering, AI-based structural predictions, mass spectrometry, and high-resolution crystallography and cryo-electron microscopy of multidomain fragments of IgAse revealed a novel 313-residue catalytic domain (CD) from the igalysin family within the metzincin MP clan. The CD is flanked by an N-terminal globular C-type lectin-like domain and a wrapping domain (WD), followed by four all-β domains. Functional studies involving a comprehensive set of constructs (wild-type and mutant), authentic and recombinant IgA fragments, and inhibitors demonstrated that the minimal functional assembly requires the CD and WD, along with the F ab and hinge region (F ab ’). Modelling studies suggested that the F ab heavy-chain constant domain interacts with the N-terminal subdomain of the CD, positioning the hinge peptide for cleavage–a mechanism confirmed by mutational analysis. These findings open avenues for therapeutic strategies to inhibit the only known IgA1/IgA2 peptidase and to develop it for dissolving pathologic IgA deposits.

Overcoming the blood-brain barrier to deliver therapeutics is a major hurdle in treating diseases of the central nervous system. We engineered 4-arm carboxyl terminated poly(D,L-lactide-co-glycolide) nanoparticles with the fungal metalloprotease Mpr1, an enzyme utilized by the neurotropic pathogen Cryptococcus neoformans ( Cn ) to cross the blood-brain barrier. Nanoparticles were prepared using a modified single emulsion solvent evaporation technique, and characterized in terms of shape, size, zeta potential, encapsulation efficiency, and toxicity to brain microvascular endothelial cells. Mpr1-functionalized nanoparticles had increased penetration over non-functionalized nanoparticles in an in vitro model of the blood-brain barrier. When encapsulating amphotericin B, a potent antifungal drug, Mpr1-functionalized nanoparticles reduced fungal burden in an in vitro model of neural cryptococcosis. Loaded nanoparticles also had an 8-fold lower minimum inhibitory concentration against Cn and Candida albicans ( Ca ) compared to unencapsulated amphotericin B. Results indicate that Mpr1-coating of polymeric nanoparticles is a promising strategy to enhance drug delivery to the brain.

The genome of Weissella oryzae SG25T was recently sequenced and a botulinum neurotoxin (BoNT) like gene was identified by bioinformatics methods. The typical three-domains organization of BoNTs with a N-terminal metalloprotease domain, a translocation and a cell binding domains could be identified. The BoNT family of neurotoxins is rapidly growing, but this was the first indication of the possible expression of a BoNT toxin outside the Clostridium genus. We performed molecular modeling and dynamics simulations showing that the 50 kDa N-terminal domain folds very similarly to the metalloprotease domain of BoNT/B, whilst the binding part is different. However, neither the recombinant metalloprotease nor the binding domains showed cross-reactivity with the standard antisera that define the seven serotypes of BoNTs. We found that the purified Weissella metalloprotease cleaves VAMP at a single site untouched by the other VAMP-specific BoNTs. This site is a unique Trp-Trp peptide bond located within the juxtamembrane segment of VAMP which is essential for neurotransmitter release. Therefore, the present study identifies the first non-Clostridial BoNT-like metalloprotease that cleaves VAMP at a novel and relevant site and we propose to label it BoNT/Wo.

Monospermic fertilization is mediated by the extracellular zona pellucida composed of ZP1, ZP2 and ZP3. Sperm bind to the N-terminus of ZP2 which is cleaved after fertilization by ovastacin (encoded by Astl) exocytosed from egg cortical granules to prevent sperm binding. AstlNull mice lack the post-fertilization block to sperm binding and the ability to rescue this phenotype with AstlmCherry transgenic mice confirms the role of ovastacin in providing a definitive block to polyspermy. During oogenesis, endogenous ovastacin traffics through the endomembrane system prior to storage in peripherally located cortical granules. Deletion mutants of ovastacinmCherry expressed in growing oocytes define a unique 7 amino acid motif near its N-terminus that is necessary and sufficient for cortical granule localization. Deletion of the 7 amino acids by CRISPR/Cas9 at the endogenous locus (AstlΔ) prevents cortical granule localization of ovastacin. The misdirected enzyme is present within the endomembrane system and ZP2 is prematurely cleaved. Sperm bind poorly to the zona pellucida of AstlΔ/Δ mice with partially cleaved ZP2 and female mice are sub-fertile.