Fluorogenic Protease Research Articles

Spying on SARS-CoV-2 with Fluorescent Tags and Protease Reporters.

The SARS-CoV-2 coronavirus has caused worldwide disruption through the COVID-19 pandemic, providing a sobering reminder of the profound impact viruses can have on human well-being. Understanding virus life cycles and interactions with host cells lays the groundwork for exploring therapeutic strategies against virus-related diseases. Fluorescence microscopy plays a vital role in virus imaging, offering high spatiotemporal resolution, sensitivity, and spectroscopic versatility. In this opinion piece, we first highlight two recent techniques, SunTag and StayGold, for the in situ imaging of viral RNA translation and viral assembly. Next, we discuss a new class of genetically encoded fluorogenic protease reporters, such as FlipGFP, which can be customized to monitor SARS-CoV-2's main (Mpro) or papain-like (PLpro) protease activity. These assays have proven effective in identifying potential antivirals through high-throughput screening, making fluorogenic viral protease reporters a promising platform for viral disease diagnostics and therapeutics.

Direct monitoring of protease activity using an integrated microchip coated with multilayered fluorogenic nanofilms.

Proteases play an essential role in the four sequential but overlapping phases of wound healing: hemostasis, inflammation, proliferation, and remodeling. In chronic wounds, excessive protease secretion damages the newly formed extracellular matrix, thereby delaying or preventing the normal healing process. Peptide-based fluorogenic sensors provide a visual platform to sense and analyze protease activity through changes in the fluorescence intensity. Here, we have developed an integrated microfluidic chip coated with multilayered fluorogenic nanofilms that can directly monitor protease activity. Fluorogenic protease sensors were chemically conjugated to polymer films coated on the surface of parallel microfluidic channels. Capillary flow layer-by-layer (CF-LbL) was used for film assembly and combined with subsequent sensor modification to establish a novel platform sensing technology. The benefits of our platform include facile fabrication and processing, controllable film nanostructure, small sample volume, and high sensitivity. We observed increased fluorescence of the LbL nanofilms when they were exposed to model recombinant proteases, confirming their responsiveness to protease activity. Increases in the nanofilms' fluorescence intensity were also observed during incubation with liquid extracted from murine infected wounds, demonstrating the potential of these films to provide real-time, in situ information about protease activity levels.

Designing a Green Fluorogenic Protease Reporter by Flipping a Beta Strand of GFP for Imaging Apoptosis in Animals.

A family of proteases called caspases mediate apoptosis signaling in animals. We report a GFP-based fluorogenic protease reporter, dubbed "FlipGFP", by flipping a beta strand of the GFP. Upon protease activation and cleavage, the beta strand is restored, leading to reconstitution of the GFP and fluorescence. FlipGFP-based TEV protease reporter achieves 100-fold fluorescence change. A FlipGFP-based executioner caspase reporter visualized apoptosis in live zebrafish embryos with spatiotemporal resolution. FlipGFP also visualized apoptotic cells in the midgut of Drosophila. Thus, the FlipGFP-based caspase reporter will be useful for monitoring apoptosis during animal development and for designing reporters of proteases beyond caspases. The design strategy can be further applied to a red fluorescent protein for engineering a red fluorogenic protease reporter.

Quest for novel fluorogenic xanthene dyes: Synthesis, spectral properties and stability of 3-imino-3H-xanthen-6-amine (pyronin) and its silicon analog

To expand the range of primary aniline fluorophores available and suitable for the design of fluorogenic protease probes, the synthesis of 3-imino-3H-xanthen-6-amine (known as pyronin) and its silicon analog (Si-pyronin) was explored and presented here. A comprehensive photophysical study of these two fluorescent anilines, confirms the effectiveness of the heteroatom-substitution approach (O → SiMe2) to yield dramatic red-shifts in absorption and fluorescence maxima of the xanthene scaffold (+85 nm). However, it also revealed its adverse effect on the hydrolytic stability of the Si-pyronin, especially at physiological pH. The pro-fluorescent character and utility of these two fluorogenic (hetero)xanthene dyes are also proved by the preparation and in vitro validation of activatable fluorescence “turn-on” probes for penicillin G acylase (PGA).

Hydrodabcyl: A Superior Hydrophilic Alternative to the Dark Fluorescence Quencher Dabcyl.

Dark fluorescence quenchers are nonfluorescent dyes that can modulate the fluorescence signal of an appropriate fluorophore donor in a distance-dependent manner. Dark quenchers are extensively used in many biomolecular analytical applications, such as studies with fluorogenic protease substrates or nucleic acids probes. A very popular dark fluorescence quencher is dabcyl, which is a hydrophobic azobenzene derivative. However, its insolubility in water may constitute a major drawback, especially during the investigation of biochemical systems whose natural solvent is water. We designed and synthesized a new azobenzene-based dark quencher with excellent solubility in aqueous media, which represents a superior alternative to the much-used dabcyl. The advantage of hydrodabcyl over dabcyl is exemplarily demonstrated for the cleavage of the fluorogenic substrate hydrodabcyl-Ser-Phe-EDANS by the proteases thermolysin and papain.

Incorporation of a Valine-Leucine-Lysine-Containing Substrate in the Bacterial Cell Wall.

The emergence of antibiotic-resistant bacteria is a major public health threat, and therefore novel antimicrobial targets and strategies are urgently needed. In this regard, cell-wall-associated proteases are envisaged as interesting antimicrobial targets due to their role in cell wall remodeling. Here, we describe the discovery and characteristics of a protease substrate that is processed by a bacterial cell-wall-associated protease. Stationary-phase grown Gram-positive bacteria were incubated with fluorogenic protease substrates, and their cleavage and covalent incorporation into the cell wall was analyzed. Of all of the substrates used, only one substrate, containing a valine-leucine-lysine (VLK) motif, was covalently incorporated into the bacterial cell wall. Linkage of the VLK-peptide substrate appeared unrelated to sortase A and B activity, as both wild-type and sortase A and B knock out Staphylococcus aureus strains incorporated this substrate into their cell wall with comparable efficiency. Additionally, the VLK-peptide substrate showed significantly higher incorporation in the cell wall of VanA-positive Enterococcus faecium strains than in VanB- and vancomycin-susceptible isolates. In conclusion, the VLK-peptide substrate identified in this study shows promise as a vehicle for targeting antimicrobial compounds and diagnostic contrast agents to the bacterial cell wall.

Rational Design of a GFP-Based Fluorogenic Caspase Reporter for Imaging Apoptosis In Vivo

Fluorescence resonance energy transfer-based executioner caspase reporters using GFP are important tools for imaging apoptosis. While these reporters are useful for imaging apoptosis in cultured cells, their invivo application has been handicapped by poor signal to noise. Here, we report the design and characterization of a GFP-based fluorogenic protease reporter, dubbed ZipGFP. ZipGFP-based TEV protease reporter increased fluorescence 10-fold after activation by protease. A ZipGFP-based executioner caspase reporter visualized apoptosis in live zebrafish embryos with spatiotemporal resolution. Thus, the ZipGFP-based caspase reporter may be useful for monitoring apoptosis during animal development and for designing reporters of proteases beyond the executioner caspases.

PAI-2/SerpinB2 inhibits proteolytic activity in a P. gingivalis-dominated multispecies bacterial consortium

ObjectiveThe aim of this study was to investigate the ability of the serine protease inhibitor plasminogen activator inhibitor type 2 (PAI-2/Serpin B2) to inhibit proteases produced by a multispecies bacterial consortium in vitro. BackgroundGingival and periodontal inflammation is associated with an increased flow of protein-rich gingival fluid. This nutritional change in the microenvironment favors bacteria with a proteolytic phenotype, triggering inflammation and associated tissue breakdown. PAI-2 is produced by macrophages and keratinocytes and is present in very high concentrations in gingival crevicular fluid; the highest level in the body. DesignA multispecies bacterial consortium comprising nine bacterial strains, resembling the conditions in a periodontal pocket, was grown planktonically and as a biofilm. After seven days PAI-2 was added to the consortium and the proteolytic activity was assayed with fluorogenic protease substrates; FITC-labeled casein to detect global protease activity, fluorescent H-Gly-Pro-AMC for serine protease activity and fluorescent BIKKAM-10 for Porphyromonas gingivalis-associated protease activity. Protease activity associated with biofilm cells was examined by confocal scanning laser microscopy. ResultsPAI-2 inhibited proteolytic activity of the bacterial consortium, as seen by decreased fluorescence of all substrates. PAI-2 specifically inhibited P. gingivalis proteolytic activity. ConclusionTo our knowledge, this is the first time that PAI-2 has been shown to inhibit bacterial proteases. Given the high concentration of PAI-2 in the gingival region, our results indicate that PAI-2 might play a role for the integrity of the epithelial barrier.

Short communication: Protease activity measurement in milk as a diagnostic test for clinical mastitis in dairy cows

Due to the increasing use of automated milking systems, automated detection of clinical mastitis is becoming more important. Various in- or on-line diagnostic tests are in use, but generally suffer from false mastitis alerts. In this study, we explored a new diagnostic approach based on measurement of protease activity using fluorogenic protease substrates, which can be performed on site, at high speed, and at low costs. Samples from cows with clinical mastitis submitted for bacteriological culture at the University Farm Animal Practice were collected during several months and kept at −20°C until protease activity measurement. A reference set of milk samples from clinically healthy cows were collected on 9 different farms and were tested for protease activity directly and after freezing at −20°C to allow for comparison with the samples from clinical cases. The protease activity in mastitic milk samples was significantly higher than in samples from healthy animals. Based on 71 clinical mastitis samples and 180 milk samples from clinically healthy quarters, the area under the receiver-operating characteristic curve was estimated to be between 0.88 and 0.90, and at a threshold of 38 fluorescence per minute the test had a specificity of 0.99 at a sensitivity of 0.58. Protease activity measured in fresh milk from clinically healthy cows was significantly associated with somatic cell count and parity, but not with electrical conductivity, whereas protease activity in milk that had been frozen was statistically significantly associated with all 3 parameters. This study indicates that protease activity measurement as a stand-alone test can be used for detecting mastitis samples, using milk samples that have been frozen. Because protease activity acts in part on a different biological mechanism than somatic cell count or electrical conductivity, this test may increase the accuracy of mastitis diagnosis in combination with currently available in- or on-line tests in automated milking systems.

Rationally designed fluorogenic protease reporter visualizes spatiotemporal dynamics of apoptosis in vivo

Fluorescence resonance energy transfer-based reporters have been widely used in imaging cell signaling; however, their in vivo application has been handicapped because of poor signal. Although fluorogenic reporters overcome this problem, no such reporter of proteases has been demonstrated for in vivo imaging. Now we have redesigned an infrared fluorescent protein so that its chromophore incorporation is regulated by protease activity. Upon protease activation, the infrared fluorogenic protease reporter becomes fluorescent with no requirement of exogenous cofactor. To demonstrate biological applications, we have designed an infrared fluorogenic executioner-caspase reporter, which reveals spatiotemporal coordination between cell apoptosis and embryonic morphogenesis, as well as dynamics of apoptosis during tumorigenesis in Drosophila. The designed scaffold may be used to engineer reporters of other proteases with specific cleavage sequence.

Genetically Encoded Protease Substrate Based on Lanthanide-Binding Peptide for Time-Gated Fluorescence Detection

The study of biomolecular interactions is at the heart of biomedical research. Fluorescence and Förster resonance energy transfer (FRET) are potent and versatile tools in studying these interactions. Fluorescent proteins enable genetic encoding which facilitates their use in recombinant protein and in vivo applications. To eliminate the autofluorescence background encountered in applications based on fluorescent proteins, lanthanide labels can be used as donor fluorophores. Their long emission lifetime enables the use of time-gating that significantly improves assay sensitivity. In this work, we have combined the favorable characteristics of a terbium-ion-containing lanthanide-binding peptide (Tb(3+)-LBP) and green fluorescent protein (GFP) in a FRET-based homogeneous protease activity assay. The used genetically engineered construct had LBP and GFP sequences at adjacent ends of a linker that encoded the recognition sequence for caspase-3. Caspase proteases are central mediators in apoptosis and, consequently, are of great interest in the pharmaceutical industry. The designed fluorogenic protease substrate was applied for the detection of caspase-3 activity. We were able to demonstrate, for the first time, the applicability of a Tb(3+)-LBP-GFP energy-transfer pair in a protease activity assay. The intrinsically fluorescent and genetically encodable components enable easy expression of the construct without the need of cumbersome chemical labeling. By varying the fluorescent protein and the protease specificity of the internal linker sequence, the method can be applied for the detection of a wide variety of proteases.

Angiotensin II type I receptor agonistic autoantibody-induced apoptosis in neonatal rat cardiomyocytes is dependent on the generation of tumor necrosis factor-α

Angiotensin II type I receptor agonistic autoantibodies (AT1-AA) are related to pre-eclampsia and hypertension and have a direct effect of stimulating the production of tumor necrosis factor-alpha (TNF-α) in the placenta. TNF-α is a known mediator of apoptosis. However, few studies have reported the role of TNF-α and its relationship within AT1-AA-induced apoptosis of cardiomyocytes. In this study, neonatal rat cardiomyocytes were treated with various concentrations of AT1-AA. The apoptosis of neonatal rat cardiomyocytes was determined using TUNEL assay and flow cytometry. The level of secreted TNF-α was measured by enzyme-linked immunosorbent assay, and caspase-3 activity was measured by a fluorogenic protease assay kit. AT1 receptor blockade and TNF inhibitor were added to determine whether they could inhibit the apoptotic effect of AT1-AA. Results showed that AT1-AA induced the apoptosis of neonatal rat cardiomyocytes in a dose-dependent and time-dependent manner. AT1-AA increased TNF secretion and caspase-3 activities. AT1 receptor blockade completely abrogated AT1-AA-induced TNF-α secretion, caspase-3 activation, and cardiomyocyte apoptosis. TNF-α receptor inhibitor significantly attenuated AT1-AA-induced neonatal rat cardiomyocyte apoptosis. AT1-AA in the plasma of pre-eclamptic patients promoted neonatal rat cardiomyocyte apoptosis through a TNF-caspase signaling pathway.

Enzymatic Analysis of Recombinant Japanese Encephalitis Virus NS2B(H)-NS3pro Protease with Fluorogenic Model Peptide Substrates

BackgroundJapanese encephalitis virus (JEV), a member of the Flaviviridae family, causes around 68,000 encephalitis cases annually, of which 20–30% are fatal, while 30–50% of the recovered cases develop severe neurological sequelae. Specific antivirals for JEV would be of great importance, particularly in those cases where the infection has become persistent. Being indispensable for flaviviral replication, the NS2B-NS3 protease is a promising target for design of anti-flaviviral inhibitors. Contrary to related flaviviral proteases, the JEV NS2B-NS3 protease is structurally and mechanistically much less characterized. Here we aimed at establishing a straightforward procedure for cloning, expression, purification and biochemical characterization of JEV NS2B(H)-NS3pro protease.Methodology/Principal FindingsThe full-length sequence of JEV NS2B-NS3 genotype III strain JaOArS 982 was obtained as a synthetic gene. The sequence of NS2B(H)-NS3pro was generated by splicing by overlap extension PCR (SOE-PCR) and cloned into the pTrcHisA vector. Hexahistidine-tagged NS2B(H)-NS3pro, expressed in E. coli as soluble protein, was purified to >95% purity by a single-step immobilized metal affinity chromatography. SDS-PAGE and immunoblotting of the purified enzyme demonstrated NS2B(H)-NS3pro precursor and its autocleavage products, NS3pro and NS2B(H), as 36, 21, and 10 kDa bands, respectively. Kinetic parameters, K m and k cat, for fluorogenic protease model substrates, Boc-GRR-amc, Boc-LRR-amc, Ac-nKRR-amc, Bz-nKRR-amc, Pyr-RTKR-amc and Abz-(R)4SAG-nY-amide, were obtained using inner filter effect correction. The highest catalytic efficiency k cat /K m was found for Pyr-RTKR-amc (k cat/K m: 1962.96±85.0 M−1 s−1) and the lowest for Boc-LRR-amc (k cat/K m: 3.74±0.3 M−1 s−1). JEV NS3pro is inhibited by aprotinin but to a lesser extent than DEN and WNV NS3pro.Conclusions/SignificanceA simplified procedure for the cloning, overexpression and purification of the NS2B(H)-NS3pro was established which is generally applicable to other flaviviral proteases. Kinetic parameters obtained for a number of model substrates and inhibitors, are useful for the characterization of substrate specificity and eventually for the design of high-throughput assays aimed at antiviral inhibitor discovery.

Targeting Proteases in Cardiovascular Diseases by Mass Spectrometry-Based Proteomics

Proteases hydrolyze peptide bonds, thereby controlling the function of proteins and peptides on the posttranslational level. In the cardiovascular system, proteases play pivotal roles in the regulation of blood pressure, coagulation and other essential physiological processes. Accordingly, proteases are prime targets for therapeutic interventions and diagnostics. Proteases are part of complex proteolytic networks comprised of enzymes, inhibitors, activators, substrates and cleavage products. Analyzing these networks on a system-wide level is essential to understanding cardiovascular function and how dysregulation can lead to pathological conditions. Mass spectrometry-based quantitative and dynamic proteomics approaches are leading the way to enhance our knowledge of proteolytic networks such as the renin-angiotensin-system. Here, we critically review proteomics tools utilized in protease biology and provide an overview on how these methods can be used to characterize and validate protease function.

Morpholinecarbonyl-Rhodamine 110 Based Substrates for the Determination of Protease Activity with Accurate Kinetic Parameters

Commonly used fluorogenic substrate analogues for the detection of protease activity contain two enzyme-cleavable bonds conjugated to the fluorophore. Enzymatic cleavage follows a two-step reaction with a monoamide intermediate. This intermediate shows fluorescence at the same wavelength as the final product complicating the kinetic analysis of fluorescence-based assays. Fluorogenic substrate analogues for α-chymotrypsin with one cleavable peptide bond have been prepared from morpholinecarbonyl-Rhodamine 110 (MC-Rh110). A comparison of their kinetic properties with the corresponding (peptide)(2)-Rh110 derivatives revealed that these frequently used double-substituted substrate analogues yield only apparent K(m) and k(cat) values that are quite different from the kinetic parameters obtained from the monosubstituted MC-Rh110 based substrate analogues. Although both the monoamide intermediate and MC-Rh110 are monosubstituted Rhodamine 110 derivatives, they show different spectroscopic properties. The data from the spectroscopic analysis clearly show that these properties are directly related to the electron structure of the fluorophore and not to the previously proposed equilibrium between the lactone form and the open ionic form of the fluorophore. This knowledge about the determinants of the spectroscopic properties of monosubstituted Rhodamine 110 introduces a way for a more systematic development of new fluorogenic protease substrate analogues.

A comparative evaluation of microarray slides as substrates for the development of protease assay biosensors

The application of commercially available microarray slides as substrates for fluorogenic protease assays has been explored in terms of binding efficiency, stability, and activity. A fluorescent, biotinylated substrate for botulinum neurotoxin A (BoNTA) was attached via self-assembled monolayer of Streptavidin to amine-reactive aldehyde, epoxy, hydrogel, and polymer slides. Nexterion Slide P® was found to have optimal protein binding efficiency and stability of the slides examined. Addition of glycerol to the printing buffer improved spot morphology significantly and polyvinylpyrrolidone provided long-term stability, allowing chips to be stored for up to 1 month with good viability. Detection of a recombinant BoNTA light chain was then carried out at 37 °C and a sub-lethal dose was detected in 2 hours.

Localization and Purification of Phagosome‐Associated Proteases in Tetrahymena thermophila

Phagosome maturation involves sequential introduction of oxidases, hydrolases and proteases that degrade encapsulated materials. In the ciliate Tetrahymena thermophila, we found that protease activity is associated with small vesicle‐like structures at the phagosome surface and occasionally in the lumen. Our current focus is to localize and purify phagosome associated cysteine proteases from T. thermophila using fluorescent and affinity‐tagged conjugates of an irreversible inhibitor DCG‐04 (lys(Boc)‐6‐aminohexanoic acid‐tyr‐leu‐ethyl (2S, 3S)‐oxirane‐2, 3‐dicarboxylate after Greenbaum et al, 2000). Two irreversible active‐site directed fluorescent cysteine protease inhibitors (6 FAM SE DCG‐04 and BODIPY 588/616‐DCG‐04) were synthesized using a combination of solution phase and standard F‐moc solid phase peptide chemistry. The products were confirmed by MALDI‐TOF mass spectrometry and purified by reverse phase HPLC. The binding of 6 FAM SE DCG‐04 to formaldehyde fixed, detergent permeabilized cells was assessed by fluorescence microscopy in the presence and absence of a 100‐fold excess of E‐64, a competing irreversible cysteine protease inhibitor. There was extensive punctate binding of the fluorescent inhibitor only in the absence of E‐64. 6 FAM SE DCG‐04 was taken up by live cells and following fixation, the fluor was observed to co‐localize with a fluorogenic protease substrate. Biotinylated DCG‐04 was used to isolate inhibited cysteine proteases by affinity chromatography. One‐dimensional electrophoresis revealed at least 21 distinct proteins ranging in size from about 18 ‐ 200 kDa. Future efforts will focus on proteomic analysis of these isolated proteins.

Tetra-chloro-(bis-(3,5-dimethylpyrazolyl)methane)gold(III) chloride: An HIV-1 reverse transcriptase and protease inhibitor

The title compound ([3,5-Me(2)bpzaH(2)][AuCl(4)]Cl, 1) (Me(2)bpza=bis(3,5-dimethylpyrazolyl)acetic acid), was prepared by reacting H[AuCl(4)] with 3,5-Me(2)bpza; and spectroscopically and structurally characterized. In the solid state structure of 1, the pyrazolyl ligand is doubly protonated to form two strong charge assisted hydrogen bonds of the type N(+)Hcdots, three dots, centeredCl(-) with the single chloride anion whilst the [AuCl(4)](-) anion remains discrete. The anti-HIV-1 activity of 1 was determined by a colorimetric direct enzyme reverse transcriptase (RT) assay and a fluorogenic protease (PR) assay. Compound 1 significantly (p<0.05) inhibited RT over a concentration range of 5-250muM and inhibited HIV-1 protease at 100muM. Compound 1 inhibited two very important HIV-1 enzymes (RT and PR) in direct enzyme assays and therefore warrants further evaluation.

Intracellular protease activation in apoptosis and cell-mediated cytotoxicity characterized by cell-permeable fluorogenic protease substrates

Over the past decade the importance of signaling from reporter molecules inside live cells and tissues has been clearly established. Biochemical events related to inflammation, tumor metastasis and proliferation, and viral infectivity and replication are examples of processes being further defined as more molecular tools for live cell measurements become available. Moreover, in addition to quantitating parameters related to physiologic processes, real-time imaging of molecular interactions that compose basic cellular activities are providing insights into understanding disease mechanisms as well as extending clinical efficacy of therapeutic regimens. In this review the use of highly cell-permeable fluorogenic substrates that report protease activities inside live cells is described; applications to defining the molecular events of two cellular processes, i.e., apoptosis and cell-mediated cytotoxicity, are then illustrated.

Assembly and selective “in synthesis” labeling of quenched fluorogenic protease substrates

Because impaired cellular protease activities are linked to many diseases, such as cancer, inflammation, neurodegeneration, and infection, internally quenched fluorescent peptides have recently been developed as tools for analyzing the specificities of these enzymes. Here we report convenient and cost-effective approaches for the selective “in synthesis” assembly of such substrate peptides for protease assays. Fluorescein and Dabcyl groups were covalently and selectively attached during synthesis to ε-amino groups of internal lysines. Functionality was then tested by digestion with leucine aminopeptidase, chymotrypsin, and microsomal vesicles. All peptides proved to be appropriate substrates of the enzymes tested and of the endogenous peptidases in the microsomal vesicles. In summary, we describe an innovative and cheap method to develop completely functional quenched fluorescent peptides that are usable in specific detection of individual proteases, in particular aminopeptidases, in both in vitro and in vivo systems.