Neuroinflammation is an important factor in the pathogenesis of neurodegenerative diseases. Microglia-derived lysosomal cathepsins have been increasingly recognized as important inflammatory ...mediators that trigger signaling pathways that aggravate neuroinflammation.
, a contribution to neuroinflammation processes has been shown for cathepsin X: however, the expression patterns and functional role of cathepsin X in neuroinflammatory brain pathology remain elusive. In this study we analyzed the expression, activity, regional distribution and cellular localization of cathepsin X in the rat brain with neuroinflammation-induced neurodegeneration. The unilateral injection of lipopolysaccharide (LPS) induced a strong upregulation of cathepsin X expression and its activity in the ipsilateral striatum. In addition to the striatum, cathepsin X overexpression was detected in other brain areas such as the cerebral cortex, corpus callosum, subventricular zone and external globus pallidus, whereas the upregulation was mainly restricted to activated microglia and reactive astrocytes. Continuous administration of the cathepsin X inhibitor AMS36 indicated protective effects against LPS-induced striatal degeneration, as seen by the attenuated LPS-mediated dilation of the lateral ventricles and partial decreased extent of striatal lesion. Taken together, our results indicate that cathepsin X plays a role as a pathogenic factor in neuroinflammation-induced neurodegeneration and represents a potential therapeutic target for neurodegenerative diseases associated with neuroinflammation.
The cysteine protease inhibitors, clitocypin and macrocypins, from higher fungi (mycocypins), together with the serine protease inhibitors highly specific for trypsin cospin and cnispin from higher ...fungi (mycospins), display several characteristics that distinguish them from protease inhibitors from other sources. Their high genetic heterogeneity affects their functionality and/or stability and results in numerous protein variants with slightly different inhibitory profiles that influence the type of protease inhibited and/or the strength of inhibition. They possess the β-trefoil fold that shows high plasticity in their utilization of the 11 diverse loops for the inhibition of various families of proteases through different mechanisms of inhibition. Their high versatility is also seen in their regulatory and defence functions and in their potential applications in biotechnology, crop protection and medicine.
Natural killer (NK) cells are the key immune effectors with the ability to mediate selection and differentiation of a number of different cancer stem cells/undifferentiated tumors via lysis, and ...secreted or membrane-bound interferon (IFN)-γ and tumor necrosis factor (TNF)-α, respectively, leading to curtailment of tumor growth and metastasis. In this review, we present an overview of our recent findings on the biology and significance of NK cells in selection and differentiation of stem-like tumors using in vitro and in vivo studies conducted in humanized-BLT mice and in cancer patients. In addition, we present current advances in NK cell expansion and therapeutic delivery, and discuss the utility of allogeneic supercharged NK cells in the treatment of cancer patients. Moreover, we discuss the potential loss of NK cell numbers and function at the neoplastic and pre-neoplastic stages of tumorigenesis in induction and progression of pancreatic cancer. Therefore, because of their indispensable role in targeting cancer stem-like/undifferentiated tumors, NK cells should be placed high in the armamentarium of tumor immunotherapy. A combination of allogeneic supercharged NK cells with other immunotherapeutic strategies such as oncolytic viruses, antibody-dependent cellular cytotoxicity (ADCC)-inducing antibodies, checkpoint inhibitors, chimeric antigen receptor (CAR) T cells, CAR NK cells, and chemotherapeutic and radiotherapeutic strategies can be used for the ultimate goal of tumor eradication.
Summary
Development of targeted treatment for colorectal cancer is crucial to avoid side effects. To harness the possibilities offered by microbiome engineering, we prepared safe multifunctional ...cancer cell‐targeting bacteria Lactococcus lactis. They displayed, on their surface, binding proteins for cancer‐associated transmembrane receptors epithelial cell adhesion molecule (EpCAM) and human epidermal growth factor receptor 2 (HER2) and co‐expressed an infrared fluorescent protein for imaging. Binding of engineered L. lactis to tumour antigens EpCAM and HER2 was confirmed and characterised in vitro using soluble receptors. The proof‐of‐principle of targeting was demonstrated on human cell lines HEK293, HT‐29 and Caco‐2 with fluorescent microscopy and flow cytometry. The highest L. lactis adhesion was seen for the HEK293 cells with the overexpressed tumour antigens, where colocalisation with their tumour antigens was seen for 39% and 67% of EpCAM‐targeting and HER2‐targeting bacteria, respectively. On the other hand, no binding was observed to HEK293 cells without tumour antigens, confirming the selectivity of the engineered L. lactis. Apart from cell targeting in static conditions, targeting ability of engineered L. lactis was also shown in conditions of constant flow of bacterial suspension over the HEK293 cells. Successful targeting by engineered L. lactis support the future use of these bacteria in biopharmaceutical delivery for the treatment of colorectal cancer.
Multifunctional cancer cell‐targeting bacteria Lactococcus lactis with surface displayed binding proteins for cancer‐associated transmembrane receptors EpCAM and HER2 were tested for specific binding to corresponding receptors. Concomitantly, an infrared fluorescent protein was co‐expressed for imaging. We confirmed binding of engineered L. lactis to tumor antigens EpCAM and HER2 in vitro using soluble receptors and on human cell lines HEK293, HT‐29 and Caco‐2.
Cystatin F is a cysteine peptidase inhibitor which, unlike other cystatin family members, is targeted to endosomal/lysosomal compartments. It is synthesized as an inactive disulfide-linked dimer ...which is then converted to an active monomer by proteolytic cleavage of 15 N-terminal residues. Cystatin F has been suggested to regulate the cytotoxicity of natural killer (NK) cells by inhibiting the major granzyme convertases, cathepsins C and H. To test this hypothesis, we prepared variants of cystatin F and analyzed their uptake, subcellular trafficking, and peptidase inhibition, as well as their impact on the cytotoxicity of NK-92 cells and primary NK cells. The N-glycosylation pattern is responsible for the secretion, uptake, and subcellular sorting of cystatin F in HeLa and Hek293 cells, whereas the legumain binding site had no effect on these processes. Active, N-terminally truncated, monomeric cystatin F can also be internalized by recipient cells and targeted to endo/lysosomes, affecting also cells lacking the activating peptidase. Cystatin F mutants capable of cell internalization and trafficking through the endo/lysosomal pathway significantly decreased cathepsin C and H activities, both
, following transfection and in trans, using conditioned media. Further, incubation of IL-2 stimulated NK-92 and primary NK cells with full-length and N-terminally truncated cystatin F mutants led to suppression of their granule-mediated cytotoxicity. This effect was most significant with the N-terminally truncated mutants. These results suggest that cystatin F can be an important mediator within tumor microenvironment affecting the cytotoxicity of NK cells and consequently antitumor immune response.
Abstract
Patients infected with SARS-CoV-2 risk co-infection with Gram-positive bacteria, which severely affects their prognosis. Antimicrobial drugs with dual antiviral and antibacterial activity ...would be very useful in this setting. Although glycopeptide antibiotics are well-known as strong antibacterial drugs, some of them are also active against RNA viruses like SARS-CoV-2. It has been shown that the antiviral and antibacterial efficacy can be enhanced by synthetic modifications. We here report the synthesis and biological evaluation of seven derivatives of teicoplanin bearing hydrophobic or superbasic side chain. All but one teicoplanin derivatives were effective in inhibiting SARS-CoV-2 replication in VeroE6 cells. One lipophilic and three perfluoroalkyl conjugates showed activity against SARS-CoV-2 in human Calu-3 cells and against HCoV-229E, an endemic human coronavirus, in HEL cells. Pseudovirus entry and enzyme inhibition assays established that the teicoplanin derivatives efficiently prevent the cathepsin-mediated endosomal entry of SARS-CoV-2, with some compounds inhibiting also the TMPRSS2-mediated surface entry route. The teicoplanin derivatives showed good to excellent activity against Gram-positive bacteria resistant to all approved glycopeptide antibiotics, due to their ability to dually bind to the bacterial membrane and cell-wall. To conclude, we identified three perfluoralkyl and one monoguanidine analog of teicoplanin as dual inhibitors of Gram-positive bacteria and SARS-CoV-2.
The concept of multitarget-directed ligands (MTDLs) has attracted considerable interest in the development of agents against Alzheimer’s disease. Cathepsin B, a cysteine protease, and ...butyrylcholinesterase, a serine hydrolase, play important function in the development and progression of Alzheimer’s disease. We present herein the evaluation of 8-hydroxyquinoline-based analogues with activities against butyrylcholinesterase and β-secretase activity of cathepsin B. Furthermore, inhibition of amyloid β aggregation, chelation of copper and zinc ions coupled with antioxidative properties and safe cytotoxicity profile of (
1R
,
2S
)-
N
-ethyl-2-(8-hydroxy-5-nitroquinolin-7-yl)methylaminocyclohexane-1-carboxamide suggest that this derivative is a good starting point for optimization towards an effective multifunctional ligand.
Graphic abstract
Ralstonia solanaceraum is the quarantine plant pathogenic bacterium that causes bacterial wilt in over 200 host plants, which include economically important crops such as potato, tomato, tobacco, ...banana, and ginger. Alternative biological methods of disease control that can be used in integrated pest management are extensively studied. In search of new proteins with antibacterial activity against R. solanacearum, we identified L-amino acid oxidases (LAOs) from fruiting bodies of Amanita phalloides (ApLAO) and Infundibulicybe geotropa (CgLAO). We describe an optimized isolation procedure for their biochemical characterization, and show that they are dimeric proteins with estimated monomer molecular masses of 72 and 66 kDa, respectively, with isoelectric point of pH 6.5. They have broad substrate specificities for hydrophobic and charged amino acids, with highest Km for L-Leu, and broad pH optima at pH 5 and pH 6, respectively. An enzyme with similar properties is also characterized from the mycelia of I. geotropa (CgmycLAO). Fractionated aqueous extracts of 15 species of mushrooms show that LAO activity against L-Leu correlates with antibacterial activity. We confirm that the LAO activities mediate the antibacterial actions of ApLAO, CgLAO, and CgmycLAO. Their antibacterial activities are greater against Gram-negative versus Gram-positive bacteria, with inhibition of growth rate, prolongation of lag-phase, and decreased endpoint biomass. In Gram-positive bacteria, they mainly prolong the lag phase. These in vitro antibacterial activities of CgLAO and CgmycLAO are confirmed in vivo in tomato plants, while ApLAO has no effect on disease progression in planta. Transmission electron microscopy shows morphological changes of R. solanacearum upon LAO treatments. Finally, broad specificity of the antibacterial activities of these purified LAOs were seen for in vitro screening against 14 phytopathogenic bacteria. Therefore, these fungal LAOs show great potential as new biological phytoprotective agents and show the fruiting bodies of higher fungi to be a valuable source of antimicrobials with unique features.
Glioblastoma (GBM) is the most common and deadly primary brain tumor in adults. Understanding GBM pathobiology and discovering novel therapeutic targets are critical to finding efficient treatments. ...Upregulation of the lysosomal cysteine carboxypeptidase cathepsin X has been linked to immune dysfunction and neurodegenerative diseases, but its role in cancer and particularly in GBM progression in patients is unknown. In this study, cathepsin X expression and activity were found to be upregulated in human GBM tissues compared to low-grade gliomas and nontumor brain tissues. Cathepsin X was localized in GBM cells as well as in tumor-associated macrophages and microglia. Subsequently, potent irreversible (AMS36) and reversible (Z7) selective cathepsin X inhibitors were tested in vitro. Selective cathepsin X inhibitors decreased the viability of patient-derived GBM cells as well as macrophages and microglia that were cultured in conditioned media of GBM cells. We next examined the expression pattern of neuron-specific enzyme γ-enolase, which is the target of cathepsin X. We found that there was a correlation between high proteolytic activity of cathepsin X and
-terminal cleavage of γ-enolase and that cathepsin X and γ-enolase were colocalized in GBM tissues, preferentially in GBM-associated macrophages and microglia. Taken together, our results on patient-derived material suggest that cathepsin X is involved in GBM progression and is a potential target for therapeutic approaches against GBM.
Fungal ricin B-like lectins and protease inhibitors, mycocypins and mycospins, are important mediators in fungal defence against antagonists and all possess the β-trefoil fold. We demonstrate here ...that fungal β-trefoil proteins interact with each other, in addition to their apparent targets, and that these interactions modulate their biological activity. Such regulation of carbohydrate binding or inhibitory activity is observed for the first time in β-trefoil proteins and could constitute a mechanism for regulating their physiological functions. It could also have implications in molecular recognition of other combinations of β-trefoil proteins in other species.
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