Fluorescent Peptide Substrate Research Articles

Interleukin-1 receptor-dependent and -independent caspase-1 activity in retinal cells mediated by receptor interacting protein 2.

Inflammation and cell death play an important role in the pathogenesis of diabetic retinopathy. Previously we observed sustained activation of pro-inflammatory caspase-1 in retinas of diabetic animals and patients. In this study, we aimed to look at mechanisms underlying chronic caspase-1 activation in vitro and in vivo. Non-diabetic and diabetic wild type and IL-1 receptor (IL-1R1) knockout mice were used for in vivo experiments. Diabetes was induced using STZ (streptozotocin). Human Müller cells were used for in vitro studies. Cells were treated with either 5mM or 25mM glucose or interleukin-1beta (IL-1β) in the presence or absence of IL-1 receptor antagonist (IL-1ra) or siRNA against RIP2 (receptor interacting protein-2) for up to 96h. Outcome measurements to assess Müller cell functions included measurements of caspase-1 activity using a fluorescence peptide substrate, production of IL-1β by Elisa, and cell death using trypan blue exclusion assays. Our in vivo results demonstrate that caspase-1 activation progresses from an IL-1R1 independent mechanism at 10weeks of diabetes to an IL-1R1 dependent mechanism at 20weeks indicating that feedback through IL-1R1 is crucial for sustained caspase-1 activity in retinas of mice. A similar hyperglycemia-mediated caspase-1/IL-1β/IL-1R1 feedback signaling was detected in vitro in human Müller cells which was prevented by treatment with IL-1ra. Our data also indicate that hyperglycemia induces caspase-1 activation initially but IL-1β sustains caspase-1 activation via caspase-1/IL-1β/IL-1R1 feedback and we identified RIP2 as mediator for both hyperglycemia- and IL-1β-induced caspase-1 activation. Activation of caspase-1/IL-1β/IL-1R1 feedback signaling caused Müller cell death which was prevented by RIP2 knockdown. We conclude that any intervention in caspase-1/IL-1β/IL-1R1 feedback signaling presents novel therapeutic options for the treatment of diabetic retinopathy.

Abstract B054: Protease activity as a biomarker and potential target among pancreatic cancer patients

Abstract Background: The pancreas produces digestive proteases, which are often elevated in diseased states and capable of indiscriminate cell surface receptor cleavage. High levels of digestive protease activity have been implicated in the pathophysiology of pancreatic ductal adenocarcinoma (PDAC). We hypothesize that elevated digestive protease activity might downregulate or cleave immune cell receptors from the surface of cancer cells and contribute to the resistance of PDAC to immunotherapy. We have focused initially on major histocompatibility complex (MHC)-1, a ubiquitous receptor essential for antigen presentation, and CD155, an adhesion molecule over-expressed on cancer cells that regulates immune surveillance through binding to the TIGIT and CD226 receptors on leukocytes. Methods: The Rapid Assay for Protease Detection (RAPD) assay is a novel assay developed at our institution that utilizes fluorescent charge-changing peptide substrates to produce a positively charged fluorescent product fragment upon cleavage by the target protease. The RAPD assay was used to analyze protease activity levels in PDAC patients (N=50) and healthy (N=25) plasma samples. We also analyzed plasma samples obtained from an orthotopic KPC-derived mouse model for PDAC. We investigated the effects of specific proteases as well as plasma samples from PDAC and healthy subjects on MHC-1 and CD-155 receptor cleavage in cultured cells in vitro. Utilizing flow cytometry and immunofluorescent imaging, receptor loss was quantified for different proteases and plasma samples. Results: We observed elevated activity levels of digestive protease chymotrypsin as well as cathepsin-S and MMP-2 in the plasma of PDAC patients compared to healthy subjects. Similar patterns of protease activity were seen in plasma from tumor-bearing mice compared to non-tumor-bearing mice. Additionally, our results suggest loss of CD155 and MHC-1 from pancreatic cancer cells when incubated with trypsin and cathepsin-S. Incubation of cells with PDAC plasma also decreases the number of cells expressing MHC-1 and CD155 compared to control plasma, with greater differences observed in plasma with higher protease levels. Higher levels of cleaved CD155 are also detectable by ELISA in human and mouse PDAC plasma than in control plasma. Also, the protease activity (MMP2 and trypsin) correlates with plasma levels of CD155 and PD-L1. Conclusions: These studies begin to elucidate a possible role of digestive proteases in the ability of PDAC to evade the immune system. Ongoing experiments are exploring other immune receptors (including checkpoints) and whether specific protease inhibitors can block immune receptor cleavage. These experiments will inform subsequent experiments combining protease inhibitors with immunotherapy in mouse models. Citation Format: Utsav Joshi, Heather Farris, Jorge De La Torre De La Torre, kim Nguyen-Ta1, Michael Heller, Geert Schmid-Schonbein, Rebekah White. Protease activity as a biomarker and potential target among pancreatic cancer patients [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pancreatic Cancer Research; 2024 Sep 15-18; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(17 Suppl_2):Abstract nr B054.

Establishment of a stable THP-1 cell line expressing PSMB9-eGFP-His protein and detection of immunoproteasome activity

The ubiquitin/proteasome system (UPS) plays a crucial role in maintaining cellular protein homeostasis. The catalytic activity of proteasome in the UPS is regulated by β1 (PSMB6), β2 (PSMB7), and β5 (PSMB5) subunits. Interferon (IFN)-γ, tumor necrosis factor (TNF)-α, inflammation, and oxidative stress can induce the replacement of β1, β2, and β5 with their respective immuno-subunits β1i (PSMB9), β2i (PSMB10), and β5i (PSMB8), which can be assembled into the immunoproteasome. Compared with the standard proteasome, the immunoproteasome exerts enhanced regulatory effects on immune responses, such as processing and presenting MHC class Ⅰ antigens, production of pro-inflammatory cytokines, and T cell differentiation and proliferation. Abnormal aggregation of immunoproteasomes can cause neurodegenerative diseases like Parkinson's disease, Alzheimer's disease, and amyotrophic lateral sclerosis. To explore the function of PSMB9 after bacterial infection, we constructed a lentivirus plasmid overexpressing PSMB9-eGFP-His and transfected the plasmid into HEK293T cells for packaging by using a triple-plasmid system in this study. After screening with puromycin, we obtained a stable human leukemia monocytic THP-1 cell line expressing the fusion protein of PSMB9. Western blotting (WB) and fluorescence microscopy verified the expression of the fusion protein in the stable THP-1 cells. Quantitative PCR (qPCR) was employed to measure the copies of PSMB9-eGFP in THP-1 cells. Immunofluorescence results found that eGFP-His did not affect the subcellular localization of PSMB9. The purification with nickel affinity chromatography confirmed that the fusion protein could be assembled into the 20S immunoproteasome and exhibited cleaving activity for fluorescent peptide substrates. These results indicated that the PSMB9-eGFP fusion gene was integrated into the chromosome, and could be stably expressed in the constructed THP-1 cell line. This cell line can be utilized for the research on subcellular localization, dynamic expression, and activity of PSMB9 in live cells at different infection conditions and disease stages. It also provides a model for the stable cell lines construction of other immunoproteasome subunits PSMB8 and PSMB10.

Optimization of quenched fluorescent peptide substrates of SARS-CoV-2 3CLpro main protease (Mpro) from proteomic identification of P6-P6' active site specificity.

SARS-CoV-2 3C-like main protease (3CLpro) is essential for protein excision from the viral polyprotein. 3CLpro inhibitor drug development to block SARS-CoV-2 replication focuses on the catalytic non-prime (P) side for specificity and potency, but the importance of the prime (P') side in substrate specificity and for drug development remains underappreciated. We determined the P6-P6' specificity for 3CLpro from >800 cleavage sites that we identified using Proteomic Identification of Cleavage site Specificity (PICS). Cleavage occurred after the canonical P1-Gln and non-canonical P1-His and P1-Met residues. Moreover, P3 showed a preference for Arg/Lys and P3' for His. Essential H-bonds between the N-terminal Ser1 of protomer-B in 3CLpro dimers form with P1-His, but not with P1-Met. Nonetheless, cleavage occurs at P1-Met456 in native MAP4K5. Elevated reactive oxygen species in SARS-CoV-2 infection oxidize methionines. Molecular simulations revealed P1-MetOX forms an H-bond with Ser1 and notably, strong positive cooperativity between P1-Met with P3'-His was revealed, which enhanced peptide-cleavage rates. The highly plastic S3' subsite accommodates P3'-His that displays stabilizing backbone H-bonds with Thr25 lying central in a "'threonine trio" (Thr24-Thr25-Thr26) in the P'-binding domain I. Molecular docking simulations unveiled structure-activity relationships impacting 3CLpro-substrate interactions, and the role of these structural determinants was confirmed by MALDI-TOF-MS cleavage assays of P1'- and P3'-positional scanning peptide libraries carrying a 2nd optimal cut-site as an internal positive control. These data informed the design of two new and highly soluble 3CLproquenched-fluorescent peptide substrates for improved FRET monitoring of 3CLpro activity with 15× improved sensitivity over current assays.IMPORTANCEFrom global proteomics identification of >800 cleavage sites, we characterized the P6-P6' active site specificity of SARS-CoV-2 3CLpro using proteome-derived peptide library screens, molecular modeling simulations, and focussed positional peptide libraries. In P1', we show that alanine and serine are cleaved 3× faster than glycine and the hydrophobic small amino acids Leu, Ile, or Val prevent cleavage of otherwise optimal non-prime sequences. In characterizing non-canonical non-prime P1 specificity, we explored the unusual P1-Met specificity, discovering enhanced cleavage when in the oxidized state (P1-MetOX). We unveiled unexpected amino acid cooperativity at P1-Met with P3'-His and noncanonical P1-His with P2-Phe, and the importance of the threonine trio (Thr24-Thr25-Thr26) in the prime side binding domain I in defining prime side binding in SARS-CoV-2 3CLpro. From these analyses, we rationally designed quenched-fluorescence natural amino acid peptide substrates with >15× improved sensitivity and high peptide solubility, facilitating handling and application for screening of new antiviral drugs.

Steady State and Time-Dependent Fluorescent Peptide Assays for Protein Kinases.

Protein kinases catalyze the phosphorylation of proteins most commonly on Ser, Thr, and Tyr residues and regulate many cellular events in eukaryotic cells, such as cell cycle progression, transcription, metabolism, and apoptosis. Protein kinases each have a conserved ATP-binding site and one or more substrate-binding site(s) that exhibit recognition features for different protein substrates. By bringing ATP and a substrate into proximity, each protein kinase can transfer the γ phosphate of the ATP molecule to a hydroxyl group of the target residue on the substrate. In such a way, signaling pathways downstream from the substrate can be regulated based on the phosphorylated versus dephosphorylated status of the substrate. Although there are a number of ways to assay the activity of protein kinases, most of them are technically cumbersome and/or are indirect or based on quenched reactions. This protocol describes an assay employing a fluorescent peptide substrate to detect phosphorylation by protein kinases in real time. The assay is based on the principle that the phosphorylation of the peptide substrate leads to an increase in the fluorescence emission intensity of an appended fluorophore. We extend the application of this assay to an example of how to assess time-dependent covalent inhibition of kinases as well. © 2024 Wiley Periodicals LLC. Basic Protocol 1: Measuring protein kinase activity using fluorescent peptides Alternate Protocol: Measuring protein kinase activity using a fluorescence plate reader Support Protocol: Labeling peptides with sox fluorophore Basic Protocol 2: Measuring time-dependent ATP-competitive inhibition of protein kinases using fluorescent peptides.

Salivary macrophage activation-related chemokines and mitogen-activated kinase kinase-degrading proteolytic activity in type 1 diabetes mellitus.

This cross-sectional study aimed to evaluate salivary concentrations of macrophage activation-related chemokines and mitogen-activated kinase kinase (MAPKK)-degrading proteolytic activity in children and adolescents with and without type 1 diabetes mellitus (T1DM). A total of 122 children and adolescents (65 T1DM patients, 50.8% female, mean age:10.9 years; 57 systemically healthy controls, 36.8% female, mean age: 9.5 years) were included in the study. Salivary concentrations of interferon gamma inducible protein-10 (IP-10), monocyte chemoattractant protein (MCP)-1, MCP-2, MCP-3, MCP-4, macrophage-derived chemokine (MDC), macrophage migration inhibitory factor (MIF), monokine induced by interferon gamma (MIG), and macrophage inflammatory protein-1 alpha (MIP-1α) were quantified using a bead-based technique. MAPKK-degrading proteolytic activity was detected using fluorescent peptide substrates. The T1DM group had higher plaque index (PI%, p=0.032) and bleeding on probing (BOP%, p=0.045) scores, and lower decayed, missing, filled teeth (dmft/DMFT, p=0.002) index scores compared to the healthy controls. Compared to the controls, salivary MCP-1 (p=0.007), MCP-3 (p<0.001), MIG (p=0.007), and MIP-1α (p=0.033) concentrations were elevated whereas MCP-4 concentrations decreased (p<0.001) in the T1DM group. After adjusting for age, PI%, BOP%, and dmft/DMFT scores, significant differences in salivary concentrations of MIG (p=0.033) and MIP-1α (p=0.017) were observed between the groups. Moreover, protease activities directed to the cleavage sites of MEK23-18 (p=0.001), MKK6b7-22 (p=0.007), MKK451-66 (p=0.005), MKK7b37-52 (p=0.034), and MKK7b69-84 (p=0.009) were elevated in the T1DM group. T1DM disrupts the salivary macrophage activation-related chemokine profile and dysregulates proteolytic MAPKK cleavage. These findings can be an outcome of the impaired systemic immune response in T1DM.

Luteolin Attenuates Diabetic Myocardial Hypertrophy by Inhibiting Proteasome Activity

Introduction: Luteolin is a flavonoid polyphenolic compound exerting broad pharmacological and medicinal properties. Diabetes-related obesity increases the total blood volume and cardiac output and may increase the myocardial hypertrophy progression. However, the mechanism of luteolin in diabetic myocardial hypertrophy remains uncertain. Therefore, this study aimed to evaluate whether luteolin improved diabetic cardiomyopathy (DCM) by inhibiting the proteasome activity. Methods: Cardiomyopathy was induced in streptozotocin-treated diabetes mellitus (DM) and db/db mice. Luteolin (20 mg kg−1·day−1) was administrated via gavage for 12 weeks. In vitro, high glucose and high insulin (HGI, glucose at 25.5 mM and insulin at 0.1 µM) inducing primary neonatal rat cardiomyocytes (NRCMs) were treated with or without luteolin for 48 h. Echocardiography, reverse transcription quantitative polymerase chain reaction, histology, immunofluorescence, and Western blotting were conducted. Proteasome activities were also detected using a fluorescent peptide substrate. Results: Luteolin administration significantly prevented the onset of cardiac hypertrophy, fibrosis, and dysfunction in type 1 DM (T1DM) and type 2 DM (T2DM). Compared with DCM mice, luteolin groups showed lower serum triglyceride and total cholesterol levels. Furthermore, luteolin attenuated HGI-induced myocardial hypertrophy and reduced atrial natriuretic factor mRNA level in NRCMs. Proteasome activities were inhibited by luteolin in vitro. Luteolin also reduces the proteasome subunit levels (PSMB) 1, PSMB2, and PSMB5 of the 20S proteasome, as well as proteasome-regulated particles (Rpt) 1 and Rpt4 levels of 19S proteasome. Furthermore, luteolin treatment increased protein kinase B (AKT) and GSK-3α/β (inactivation of GSK-3) phosphorylation. The phosphorylation level of AMPK activity was also reversed after the treatment with luteolin in comparison with the HGI-treated group. Conclusion: This study indicates that luteolin protected against DCM in mice, including T1DM and T2DM, by upregulating phosphorylated protein AMPK and AKT/GSK-3 pathways while decreasing the proteasome activity. These findings suggest that luteolin may be a potential therapeutic agent for DCM.

The role of polymeric chains as a protective environment for improving the stability and efficiency of fluorogenic peptide substrates

We have faced the preparation of fully water-soluble fluorescent peptide substrate with long-term environmental stability (in solution more than 35 weeks) and, accordingly, with stable results in the use of this probe in determining the activity of enzymes. We have achieved this goal by preparing a co-polymer of the commercial N-vinyl-2-pyrrolidone (99.5% mol) and a fluorescent substrate for trypsin activity determination having a vinylic group (0.5%). The activity of trypsin has been measured in water solutions of this polymer over time, contrasted against the activity of both the commercial substrate Z-L-Arg-7-amido-4-methylcoumarin hydrochloride and its monomeric derivative, prepared ad-hoc. Initially, the activity of the sensory polymer was 74.53 ± 1.72 nmol/min/mg of enzyme, while that of the commercial substrate was 20.44 ± 0.65 nmol/min/mg of enzyme, the former maintained stable along weeks and the latter with a deep decay to zero in three weeks. The ‘protection’ effect exerted by the polymer chain has been studied by solvation studies by UV–Vis spectroscopy, steady-state & time resolved fluorescence, thermogravimetry and isothermal titration calorimetry.

Assay for Protealysin-like Protease Inhibitor Activity.

Here, we present the first quantitative method for the activity analysis of protealysin-like protease (PLP) inhibitors. This approach is based on a previously developed method for protealysin activity determination by hydrolysis of internally quenched fluorescent peptide substrate 2-aminobenzoyl-L-arginyl-L-seryl-L-valyl-L-isoleucyl-L-(ε-2,4-dinitrophenyl)lysine. In this protocol, we significantly reduced enzyme concentration and introduced some minor modifications to decrease variation between replicates. The protocol was validated using emfourin, a novel proteinaceous metalloprotease inhibitor. Data obtained demonstrates that the developed assay method is an affordable approach for characterizing and screening various PLP inhibitors. Graphical abstract.

Reynoutria Rhizomes as a Natural Source of SARS-CoV-2 Mpro Inhibitors-Molecular Docking and In Vitro Study.

More than a year has passed since the world began to fight the novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) responsible for the Coronavirus disease 2019 (COVID-19) pandemic, and still it spreads around the world, mutating at the same time. One of the sources of compounds with potential antiviral activity is Traditional Chinese Medicinal (TCM) plants used in China in the supportive treatment of COVID-19. Reynoutria japonica is important part of the Shu Feng Jie Du Granule/Capsule-TCM herbal formula, recommended by China Food and Drug Administration (CFDA) for treatment of patients with H1N1- and H5N9-induced acute lung injury and is also used in China to treat COVID-19, mainly combined with other remedies. In our study, 25 compounds from rhizomes of R. japonica and Reynoutria sachalinensis (related species), were docked into the binding site of SARS-CoV-2 main protease. Next, 11 of them (vanicoside A, vanicoside B, resveratrol, piceid, emodin, epicatechin, epicatechin gallate, epigallocatechin gallate, procyanidin B2, procyanidin C1, procyanidin B2 3,3’-di-O-gallate) as well as extracts and fractions from rhizomes of R. japonica and R. sachalinensis were tested in vitro using a fluorescent peptide substrate. Among the tested phytochemicals the best results were achieved for vanicoside A and vanicoside B with moderate inhibition of SARS-CoV-2 Mpro, IC50 = 23.10 µM and 43.59 µM, respectively. The butanol fractions of plants showed the strongest inhibition of SARS-CoV-2 Mpro (IC50 = 4.031 µg/mL for R. sachalinensis and IC50 = 7.877 µg/mL for R. japonica). As the main constituents of butanol fractions, besides the phenylpropanoid disaccharide esters (e.g., vanicosides), are highly polymerized procyanidins, we suppose that they could be responsible for their strong inhibitory properties. As inhibition of SARS-CoV-2 main protease could prevent the replication of the virus our research provides data that may explain the beneficial effects of R. japonica on COVID-19 and identify the most active compounds worthy of more extensive research.

Repurposing existing drugs: identification of SARS-CoV-2 3C-like protease inhibitors

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is responsible for coronavirus disease 2019 (COVID-19). Since its emergence, the COVID-19 pandemic has not only distressed medical services but also caused economic upheavals, marking urgent the need for effective therapeutics. The experience of combating SARS-CoV and MERS-CoV has shown that inhibiting the 3-chymotrypsin-like protease (3CLpro) blocks the replication of the virus. Given the well-studied properties of FDA-approved drugs, identification of SARS-CoV-2 3CLpro inhibitors in an FDA-approved drug library would be of great therapeutic value. Here, we screened a library consisting of 774 FDA-approved drugs for potent SARS-CoV-2 3CLpro inhibitors, using an intramolecularly quenched fluorescence (IQF) peptide substrate. Ethacrynic acid, naproxen, allopurinol, butenafine hydrochloride, raloxifene hydrochloride, tranylcypromine hydrochloride, and saquinavir mesylate have been found to block the proteolytic activity of SARS-CoV-2 3CLpro. The inhibitory activity of these repurposing drugs against SARS-CoV-2 3CLpro highlights their therapeutic potential for treating COVID-19 and other Betacoronavirus infections.

A quaternary ammonium silane antimicrobial triggers bacterial membrane and biofilm destruction

To study the antimicrobial effects of quaternary ammonium silane (QAS) exposure on Streptococcus mutans and Lactobacillus acidophilus bacterial biofilms at different concentrations. Streptococcus mutans and Lactobacillus acidophilus biofilms were cultured on dentine disks, and incubated for bacterial adhesion for 3-days. Disks were treated with disinfectant (experimental QAS or control) and returned to culture for four days. Small-molecule drug discovery-suite was used to analyze QAS/Sortase-A active site. Cleavage of a synthetic fluorescent peptide substrate, was used to analyze inhibition of Sortase-A. Raman spectroscopy was performed and biofilms stained for confocal laser scanning microscopy (CLSM). Dentine disks that contained treated dual-species biofilms were examined using scanning electron microscopy (SEM). Analysis of DAPI within biofilms was performed using CLSM. Fatty acids in bacterial membranes were assessed with succinic-dehydrogenase assay along with time-kill assay. Sortase-A protein underwent conformational change due to QAS molecule during simulation, showing fluctuating alpha and beta strands. Spectroscopy revealed low carbohydrate intensities in 1% and 2% QAS. SEM images demonstrated absence of bacterial colonies after treatment. DAPI staining decreased with 1% QAS (p < 0.05). Fatty acid compositions of dual specie biofilm increased in both 1% and 2% QAS specimens (p < 0.05). Quaternary ammonium silane demonstrated to be a potent antibacterial cavity disinfectant and a plaque inhibitor and can be of potential significance in eliminating caries-forming bacteria.

An Internally Quenched Fluorescent Peptide Substrate for Protealysin

Protealysin, a metalloprotease of Serratia proteamaculans, is the prototype of a subgroup of the M4 peptidase family. Protealysin-like proteases (PLPs) are widely spread in bacteria but also occur in fungi and certain archaea. The interest in PLPs is primarily due to their putative involvement in the bacterial pathogenesis in animals and plants. Studying PLPs requires an efficient quantitative assay for their activity; however, no such assay has been reported so far. Here, we used the autoprocessing site sequence of the protealysin precursor to construct an internally quenched fluorescent peptide substrate 2-aminobenzoyl-L-arginyl-L-seryl-L-valyl-L-isoleucyl-L-(ε-2,4-dinitrophenyl)lysine. Protealysin and thermolysin, the prototype of the M4 family, proved to hydrolyze only the Ser-Val bond of the substrate. The substrate exhibited a KM = 35 ± 4 μM and kcat = 21 ± 1 s−1 for protealysin as well as a KM = 33 ± 8 μM and kcat = 7 ± 1 s−1 for thermolysin at 37 °C. Comparison of the effect of different enzymes (thermolysin, trypsin, chymotrypsin, savinase, and pronase E) on the substrate has demonstrated that it is not strictly specific for protealysin; however, this enzyme has higher molar activity even compared to the closely related thermolysin. Thus, the proposed substrate can be advantageous for quantitative studies of protealysin as well as for activity assays of other M4 peptidases.

Relationship between blastocoel cell-free DNA and day-5 blastocyst morphology.

Cell-free DNA (cfDNA) which is present in the blastocoel cavity of embryos is believed to result from physiological apoptosis during development. This study assessed cfDNA content and caspase-3 protease activity in day-5 IVF blastocysts to determine if there was a correlation with embryo morphology. Day-5 IVF blastocysts were scored according to the Gardner and Schoolcraft system (modified to generate a numerical value) and cfDNA was collected following laser-induced blastocoel collapsing prior to cryopreservation in 25μL of media. cfDNA was quantified via fluorospectrometry and apoptotic activity was assessed via a caspase-3 protease assay using a fluorescent peptide substrate. Data were compared by linear regression. A total of 32 embryos were evaluated. There was a significant (p < 0.01) and positive correlation (cfDNA = 104.753 + (11.281 × score); R2 = 0.200) between embryo score and cfDNA content. A significant (p < 0.05) and positive correlation (cfDNA = 115.9 + (0.05 × caspase-3); R2= 0.128) was observed between caspase-3 activity and cfDNA levels. There was no significant relationship between caspase-3 activity and embryo morphology score. This study provides further evidence that cfDNA is present in blastocoel fluid, can be quantified, and positively correlates with embryonic morphology. There is also evidence that at least a portion of the cfDNA present is from intracellular contents of embryonic cells that underwent apoptosis. Additional studies are warranted to determine other physiological sources of the cfDNA in blastocyst fluid and to determine the relationship with cfDNA content, embryo morphology, and chromosomal ploidy status plus implantation potential.

Quercetin Prevents In Vivo and In Vitro Myocardial Hypertrophy Through the Proteasome-GSK-3 Pathway.

Quercetin, a flavonoid, has been reported to ameliorate cardiovascular diseases, such as cardiac hypertrophy. However, the mechanism is not completely understood. In this study, a mechanism related to proteasome-glycogen synthesis kinase 3 (GSK-3) was elucidated in rats and primary neonatal cardiomyocytes. Rats were subjected to sham or constriction of abdominal aorta surgery groups and treated with or without quercetin for 8weeks. Angiotensin II (Ang II)-induced primary cardiomyocytes were cultured with quercetin treatment or not for 48h. Echocardiography, real-time RT-PCR, histology, immunofluorescence, and Western blotting were conducted. Proteasome activities were also detected using a fluorescent peptide substrate. Echocardiography showed that quercetin prevented constriction of abdominal aorta-induced cardiac hypertrophy and improved the cardiac diastolic function. In addition, quercetin also significantly reduced the Ang II-induced hypertrophic surface area and atrial natriuretic factor (ANF) mRNA level in primary cardiomyocytes. Proteasome activities were obviously inhibited in the quercetin-treated group both in vivo and in vitro. Quercetin also decreased the levels of proteasome subunit beta type (PSMB) 1, PSMB2, and PSMB5 of the 20S proteasome as well as the levels of proteasome regulatory particle (Rpt) 1 and Rpt4 of the 19S proteasome. In particular, the PSMB5 level in the nucleus was reduced after quercetin treatment. Furthermore, phosphorylated GSK-3α/β (inactivation of GSK-3) was decreased, which means that GSK-3 activity was increased. The phosphorylation levels of upstream AKT (PKB (protein kinase B)) and liver kinase B1/AMP activated protein kinase (LKB1/AMPKα) and those of downstream extracellular signal-regulated kinase (ERK), histone H3, β-catenin, and GATA binding protein 4 (GATA4) were reduced after quercetin treatment, while hypertrophy was reversed after treatment with the GSK-3 inhibitor. In summary, quercetin prevents cardiac hypertrophy, which is related to proteasome inhibition and activation of GSK-3α/β. Upstream (AKT, LKB1/AMPKα) and downstream hypertrophic factors, such as ERK, histone H3, β-catenin, and GATA4, may also be involved.

A microfluidics-based mobility shift assay to identify new inhibitors of β-secretase for Alzheimer's disease.

The β-secretase (BACE1) initiates the generation of toxic amyloid-β peptide (Aβ) from amyloid-β precursor protein (APP), which was widely considered to play a key role in the pathogenesis of Alzheimer's disease (AD). Here, a novel microfluidics-based mobility shift assay (MMSA) was developed, validated, and applied for the screening of BACE1 inhibitors for AD. First, the BACE1 activity assay was established with a new fluorescent peptide substrate (FAM-EVNLDAEF) derived from the Swedish mutant APP, and high-quality ratiometric data were generated in both endpoint and kinetic modes by electrophoretic separation of peptide substrate from the BACE1 cleaved product (FAM-EVNL) before fluorescence quantification. To validate the assay, the inhibition and kinetic parameter values of two known inhibitors (AZD3839 and AZD3293) were evaluated, and the results were in good agreement with those reported by other methods. Finally, the assay was applied to screen for new inhibitors from a 900-compound library in a 384-well format, and one novel hit (IC50 = 26.5 ± 1.5μM) was identified. Compared with the common fluorescence-based assays, the primary advantage of the direct MMSA was to discover novel BACE1 inhibitors with lower auto-fluorescence interference, and its superb capability for kinetic study. Graphical abstract Microfluidics-based mobility shift assay for BACE1.

Highly sensitive and adaptable fluorescence-quenched pair discloses the substrate specificity profiles in diverse protease families

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.

Novel fluorescent substrates for detection of trypsin activity and inhibitor screening by self-quenching

A group of self-quenching-based substrates with two fluorescent peptides for detection of trypsin activity was designed and synthesized. The substrates could be easily synthesized using simple solid-phase peptide synthesis techniques. Two fluorescent peptide substrates for trypsin were conjugated to the amino groups of lysine as a branched unit. The fluorescence of these substrates was self-quenched owing to the highly assembled fluorophores on the substrates. The release of these concentrated fluorophores by proteases allows for fluorescence recovery. Self-quenching reduced the fluorescence of the substrates by 64.1%, and the fluorescence intensity was recovered by the release of the fluorophores from the substrate peptides via tryptic cleavage. The kinetic assay revealed that the kcat/Km values of the substrates were almost comparable to those of the standard fluorescent probe, peptide-MCA. The detection limit for trypsin was 111pM, and the calculation of IC50 and Ki values for the Bowman–Birk inhibitor was achieved using these substrates. These easily synthesizable self-quenching-based substrates have the potential to be useful for the detection of other disease-related protease activities.

Thrombin generation assay in untreated whole human blood.

Present coagulation assays fail to detect mild coagulation disorders, while thrombin-generation (TG) assays solve this problem. However, most of them only work with threated blood samples, which makes them labor intensive, time consuming, unreliable, and expensive. We have developed a TG electrophoretic assay that uses a thrombin specific charge-changing fluorescent peptide substrate, electrophoretic separation, and requires a drop of blood. The limit of detection of the assay was 1.97 nM in phosphate buffer saline and 6.82 nM in citrated whole blood. The assay was used to determine the TG in whole blood from healthy volunteers (n = 6, one aspirin user), over 30 min, after the blood was drawn; the TG increased from a baseline level of 2 × 10(6) RFU to 1.2 × 10(13) RFU. The lag time between the blood draw and initial burst of TG was 6 min for the volunteers (n = 5) and 15 min for the aspirin user. Specificity of the assay was evaluated by reacting our substrate with the heparinized blood samples and other proteases. The TG electrophoretic assay was designed and tested in the whole human blood, requiring no sample preparation, 5 μL of blood, 45 min, and it detected differences in coagulation patterns between a volunteer taking aspirin and non-aspirin users.

Fluorous-assisted metal chelate affinity extraction technique for analysis of protein kinase activity

We have developed a fluorous affinity-based extraction method for measurement of protein kinase activity. In this method, a fluorescent peptide substrate was phosphorylated by a protein kinase, and the obtained phosphopeptide was selectively captured with Fe(III)-immobilized perfluoroalkyliminodiacetic acid reagent via a metal chelate affinity technique. Next, the captured phosphopeptide was selectively extracted into a fluorous solvent mixture, tetradecafluorohexane and 1H,1H,2H,2H-tridecafluoro-1-n-octanol (3:1, v/v), using the specificity of fluorous affinity (fluorophilicity). In contrast, the remained substrate peptide in the aqueous (non-fluorous) phase was easily measured fluorimetrically. Finally, the enzyme activity could be assayed by measuring the decrease in fluorescence. The feasibility of this method was demonstrated by applying the method for measurement of the activity of cAMP-dependent protein kinase (PKA) using its substrate peptide (kemptide) pre-labeled with carboxytetramethylrhodamine (TAMRA).