To investigate the in vitro effect of caffeic acid phenethyl ester (CAPE), a NF-κB inhibitor, on the apoptosis of osteoarthritic (OA) chondrocytes and on the regulation of the gelatinases matrix metalloproteinase 2 (MMP-2) and matrix metalloproteinase 9 (MMP-9).
Our ability of screening broad communities for clinically asymptomatic diseases critically drives population health. Sensory chewing gums are presented targeting the tongue as 24/7 detector allowing diagnosis by “anyone, anywhere, anytime”. The chewing gum contains peptide sensors consisting of a protease cleavable linker in between a bitter substance and a microparticle. Matrix metalloproteinases in the oral cavity, as upregulated in peri-implant disease, specifically target the protease cleavable linker while chewing the gum, thereby generating bitterness for detection by the tongue. The peptide sensors prove significant success in discriminating saliva collected from patients with peri-implant disease versus clinically asymptomatic volunteers. Superior outcome is demonstrated over commercially available protease-based tests in saliva. “Anyone, anywhere, anytime” diagnostics are within reach for oral inflammation. Expanding this platform technology to other diseases in the future features this diagnostic as a massive screening tool potentially maximizing impact on population health.Early detection of gum inflammation caused by dental implants helps prevent tissue damage. Here, the authors present a peptide sensor that generates a bitter taste when cleaved by proteases present in peri-implant disease, embed it in a chewing gum, and compare the probe to existing sensors using patient saliva.
Periodontitis is a group of inflammatory diseases affecting the tissues supporting the teeth that will progressively cause the loss of alveolar bone and periodontal ligaments and eventually the dentition. Activation of osteoclast activity by RANKL and released enzymes such as matrix metalloproteinases (MMPs) are among the factors involved in the destruction of the periodontium. However, the mechanisms regulating their production in periodontitis are poorly understood. Endothelin signaling via the activation of the endothelin-A receptor (EDNRA) by endothelin-1 may play a role in the disease since the expression of the receptor and ligand is elevated in the periodontal tissues of patients with periodontitis.
Acute lung injury (ALI) is one of the most common extra-pancreatic complications of acute pancreatitis. In this study, we examined the protective effect of protease inhibitor aprotinin and a matrix metalloproteinase inhibitor (MMPi) on pulmonary inflammation in rats with severe pancreatitis-associated ALI.
θ-defensins constitute a family of macrocyclic peptides expressed exclusively in Old World monkeys. The peptides are pleiotropic effectors of innate immunity, possessing broad spectrum antimicrobial activities and immunoregulatory properties. Here we report that rhesus θ-defensin 1 (RTD-1) is highly effective in arresting and reversing joint disease in a rodent model of rheumatoid arthritis (RA). Parenteral RTD-1 treatment of DA/OlaHsd rats with established pristane-induced arthritis (PIA) rapidly suppressed joint disease progression, restored limb mobility, and preserved normal joint architecture. RTD-1 significantly reduced joint IL-1β levels compared with controls. RTD-1 dose-dependently inhibited fibroblast-like synoviocyte (FLS) invasiveness and FLS IL-6 production. Consistent with the inhibition of FLS invasiveness, RTD-1 was a potent inhibitor of arthritogenic proteases including ADAMs 17 and 10 which activate TNFα, and inhibited matrix metalloproteases, and cathepsin K. RTD-1 was non-toxic, non-immunogenic, and effective when administered as infrequently as once every five days. Thus θ-defensins, which are absent in humans, have potential as retroevolutionary biologics for the treatment of RA.
We have characterised the proteolytic cleavage events responsible for the shedding of triggering receptor expressed on myeloid cells 2 (TREM2) from primary cultures of human macrophages, murine microglia and TREM2-expressing human embryonic kidney (HEK293) cells. In all cell types, a soluble 17 kDa N-terminal cleavage fragment was shed into the conditioned media in a constitutive process that is inhibited by G1254023X and metalloprotease inhibitors and siRNA targeting ADAM10. Inhibitors of serine proteases and matrix metalloproteinases 2/9, and ADAM17 siRNA did not block TREM2 shedding. Peptidomimetic protease inhibitors highlighted a possible cleavage site, and mass spectrometry confirmed that shedding occurred predominantly at the H157-S158 peptide bond for both wild-type and H157Y human TREM2 and for the wild-type murine orthologue. Crucially, we also show that the Alzheimer’s disease-associated H157Y TREM2 variant was shed more rapidly than wild type from HEK293 cells, possibly by a novel, batimastat- and ADAM10-siRNA-independent, sheddase activity. These insights offer new therapeutic targets for modulating the innate immune response in Alzheimer’s and other neurological diseases.
Internally quenched fluorescent (IQF) peptide substrates originating from FRET (Förster Resonance Energy Transfer) are powerful tool for examining the activity and specificity of proteases, and a variety of donor/acceptor pairs are extensively used to design individual substrates and combinatorial libraries. We developed a highly sensitive and adaptable donor/acceptor pair that can be used to investigate the substrate specificity of cysteine proteases, serine proteases and metalloproteinases. This novel pair comprises 7-amino-4-carbamoylmethylcoumarin (ACC) as the fluorophore and 2,4-dinitrophenyl-lysine (Lys(DNP)) as the quencher. Using caspase-3, caspase-7, caspase-8, neutrophil elastase, legumain, and two matrix metalloproteinases (MMP2 and MMP9), we demonstrated that substrates containing ACC/Lys(DNP) exhibit 7 to 10 times higher sensitivity than conventional 7-methoxy-coumarin-4-yl acetic acid (MCA)/Lys(DNP) substrates; thus, substantially lower amounts of substrate and enzyme can be used for each assay. We therefore propose that the ACC/Lys(DNP) pair can be considered a novel and sensitive scaffold for designing substrates for any group of endopeptidases. We further demonstrate that IQF substrates containing unnatural amino acids can be used to investigate protease activities/specificities for peptides containing post-translationally modified amino acids. Finally, we used IQF substrates to re-investigate the P1-Asp characteristic of caspases, thus demonstrating that some human caspases can also hydrolyze substrates after glutamic acid.
- Proceedings of the National Academy of Sciences of the United States of America
- Published over 3 years ago
Fibrillar collagen, an essential structural component of the extracellular matrix, is remarkably resistant to proteolysis, requiring specialized matrix metalloproteinases (MMPs) to initiate its remodeling. In the context of native fibrils, remodeling is poorly understood; MMPs have limited access to cleavage sites and are inhibited by tension on the fibril. Here, single-molecule recordings of fluorescently labeled MMPs reveal cleavage-vulnerable binding regions arrayed periodically at ∼1-µm intervals along collagen fibrils. Binding regions remain periodic even as they migrate on the fibril, indicating a collective process of thermally activated and self-healing defect formation. An internal strain relief model involving reversible structural rearrangements quantitatively reproduces the observed spatial patterning and fluctuations of defects and provides a mechanism for tension-dependent stabilization of fibrillar collagen. This work identifies internal-strain-driven defects that may have general and widespread regulatory functions in self-assembled biological filaments.
Targeted cancer therapies require a precise determination of the underlying biological processes driving tumorigenesis within the complex tumor microenvironment. Therefore, new diagnostic tools that capture the molecular activity at the disease site in vivo are needed to better understand tumor behavior and ultimately maximize therapeutic responses. Matrix metalloproteinases (MMPs) drive multiple aspects of tumorigenesis, and their activity can be monitored using engineered peptide substrates as protease-specific probes. To identify tumor specific activity profiles, local sampling of the tumor microenvironment is necessary, such as through remote control of probes, which are only activated at the tumor site. Alternating magnetic fields (AMFs) provide an attractive option to remotely apply local triggering signals because they penetrate deep into the body and are not likely to interfere with biological processes due to the weak magnetic properties of tissue. Here, we report the design and evaluation of a protease-activity nanosensor that can be remotely activated at the site of disease via an AMF at 515 kHz and 15 kA/m. Our nanosensor was composed of thermosensitive liposomes containing functionalized protease substrates that were unveiled at the target site by remotely triggered heat dissipation of coencapsulated magnetic nanoparticles (MNPs). This nanosensor was combined with a unique detection assay to quantify the amount of cleaved substrates in the urine. We applied this spatiotemporally controlled system to determine tumor protease activity in vivo and identified differences in substrate cleavage profiles between two mouse models of human colorectal cancer.
The practical realization of disease modulation by catalytic degradation of a therapeutic target protein suffers from the difficulty to identify candidate proteases, or to engineer their specificity. We identified 23 measurable, specific, and new protease activities using combinatorial screening of 27 human proteases against 24 therapeutic protein targets. We investigate the cleavage of monocyte chemoattractant protein 1, interleukin-6 (IL-6), and IL-13 by matrix metalloproteinases (MMPs) and serine proteases, and demonstrate that cleavage of IL-13 leads to potent inhibition of its biological activity in vitro. MMP-8 degraded human IL-13 most efficiently in vitro and ex vivo in human IL-13 transgenic mouse bronchoalveolar lavage. Hence, MMP-8 is a therapeutic protease lead against IL-13 for inflammatory conditions whereby reported genetic and genomics data suggest an involvement of MMP-8. This work describes the first exploitation of human enzyme promiscuity for therapeutic applications, and reveals both starting points for protease-based therapies and potential new regulatory networks in inflammatory disease.