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Hydrolytic enzymes play crucial roles in cellular processes, and dysregulation of their activities is implicated in various physiological and pathological conditions. These enzymes cleave substrates such as peptide bonds, phosphodiester bonds, glycosidic bonds, and other esters. Detecting aberrant hydrolase activity is vital for understanding disease mechanisms and developing targeted therapeutic interventions. This study introduces a novel approach to measuring hydrolase activity using giant magnetoresistive (GMR) spin valve sensors. These sensors change resistance in response to magnetic fields, and here, they are functionalized with specific substrates for hydrolases conjugated to magnetic nanoparticles (MNPs). When a hydrolase cleaves its substrate, the tethered magnetic nanoparticle detaches, causing a measurable shift in the sensor’s resistance. This design translates hydrolase activity into a real-time, activity-dependent signal. The assay is simple, rapid, and requires no washing steps, making it ideal for point-of-care settings. Unlike fluorescent methods, it avoids issues like autofluorescence and photobleaching, broadening its applicability to diverse biofluids. Furthermore, the sensor array contains 80 individually addressable sensors, allowing for the simultaneous measurement of multiple hydrolases in a single reaction. The versatility of this method is demonstrated with substrates for nucleases, Bcu I and DNase I, and the peptidase, human neutrophil elastase. To demonstrate a clinical application, we show that neutrophil elastase in sputum from cystic fibrosis patients hydrolyze the peptide-GMR substrate, and the cleavage rate strongly correlates with a traditional fluorogenic substrate. This innovative assay addresses challenges associated with traditional enzyme measurement techniques, providing a promising tool for real-time quantification of hydrolase activities in diverse biological contexts.

It is now evident that the M1 family of aminopeptidases play important roles in many pathophysiological processes. Among them, the enzymatic properties of arginyl aminopeptidase-like 1 (RNPEPL1) are characterized only by its truncated form. No peptide substrate has been identified. To characterize the enzymatic properties of RNPEPL1 in more detail, the full-length protein was expressed in Escherichia coli and purified to homogeneity. The full-length RNPEPL1 showed rather restricted substrate specificity and basic amino acid preference towards synthetic substrates, which was different from the previously reported specificity characterized by the truncated form. Searching for peptide substrates, we found that several peptides, such as Met-enkephalin and kallidin, were cleaved. RNPEPL1 cleaved bradykinin to de-[Arg]-bradykinin despite the presence of proline at the P2'-position. The enzyme cleaved Met-enkephalin but not dynorphin A1–17. Similar to aminopeptidase B, the full-length RNPEPL1 showed basic amino acid preference towards both synthetic and peptide substrates. In addition to the unusual cleavage of bradykinin, this enzyme shows chain length-dependent cleavage of peptide substrates sharing N-terminal amino acid sequence. This is the first study to report the enzymatic properties of the full-length human RNPEPL1 as an aminopeptidase enzyme. [ABSTRACT FROM AUTHOR]

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Several attempts have been made over the past decade to explore the concept of prodrug strategies that exploit PSA as a molecular target for the release of anticancer drugs in prostate tumors using various prostate specific antigen (PSA)-cleavable peptide linkers, but the desired antitumor and antimetastatic efficacy has not yet been fully achieved. We set out to look for new PSA-cleavable peptide substrates that could be cleaved more rapidly and efficiently than the previously used peptides. To look for the most susceptible PSA-cleavable peptide substrates, we used the so-called spot technology. With the following general formula, we designed 25 different fluorogenic heptapeptides; Cellulose-P5-P4-P3-P2-P1-P1′-P2’ (Fluorophore). The increase of the fluorescence in the supernatant of the reaction mixture was monitored using a 96-well fluorometric plate reader with excitation of λex 485 nm and λem 535 nm. Three sequences showed a high fluorogenic liberation after incubation with PSA, i.e., Arg-Arg-Leu-His-Tyr-Ser-Leu (7), Arg-Arg-Leu-Asn-Tyr-Ser-Leu (8) and Arg-Ser-Ser-Tyr-Arg-Ser-Leu (23). Future incorporation of these optimized substrates in the PSA-cleavable prodrug formulations could further optimize the cleavage pattern and so the release characteristics of these prodrugs to rapidly and efficiently liberate the free cytotoxic agents inside the tumor tissues.

The G protein-coupled receptor kinase (GRK) family consists of seven cytosolic serine/threonine (Ser/Thr) protein kinases, and among them, GRK2 is involved in the regulation of an enormous range of both G protein-coupled receptors (GPCRs) and non-GPCR substrates that participate in or regulate many critical cellular processes. GRK2 dysfunction is associated with multiple diseases, including cancers, brain diseases, cardiovascular and metabolic diseases, and therefore GRK2-specific substrates/inhibitors are needed not only for studies of GRK2-mediated cellular functions but also for GRK2-targeted drug development. Here, we first review the structure, regulation and functions of GRK2, and its synthetic substrates and inhibitors. We then highlight recent work on synthetic peptide substrates/inhibitors as promising tools for fundamental studies of the physiological functions of GRK2, and as candidates for applications in clinical diagnostics. [ABSTRACT FROM AUTHOR]

Efficient deposition of biomolecules on the surface, maintaining their full activity and stability, is a most significant factor in biosensor construction. For this reason, more and more research is focused on the development of electrochemical biosensors that have the ability to electrically detect adsorbed molecules on electrode surface with high selectivity and sensitivity. The presented research aims to develop an efficient methodology that allows quantification of processes related to the evaluation of enzyme activity (proprotein convertase) using electrochemical methods. In this study we used impedance spectroscopy to investigate the immobilization of peptide substrate (Arg-Val-Arg-Arg) modified with 11-mercaptoundecanoic acid on the surface of gold electrode. Both the synthesis of the peptide substrate as well as the full electrochemical characteristics of the obtained electrode materials have been described. Experimental conditions, including concentration of peptide substrate immobilization, modification time, linker, and the presence of additional blocking groups have been optimized. The main advantages of the described method is that it makes it possible to observe the peptide substrate–enzyme interaction without the need to use fluorescent labels. This also allows observation of this interaction at a very low concentration. Both of these factors make this new technique competitive with the standard spectrofluorimetric method.

Clostridium botulinum produces botulinum neurotoxins (BoNTs) that are one of the most poisonous substances. In order to respond to public health emergencies, there is a need to develop sensitive and specific methods for detecting botulinum toxin in various clinical matrices. Our laboratory has developed a mass spectrometry-based Endopep-MS assay that is able to rapidly detect and differentiate BoNT serotypes A–G by immunoaffinity capture of toxins and detection of unique cleavage products of peptide substrates. To improve the sensitivity of the Endopep-MS assay for the detection of BoNT serotype G, we report here the optimization of synthetic peptide substrates through systematic substitution, deletion, and incorporation of unnatural amino acids. Our data show that the resulting optimized peptides produced a significant improvement (two orders of magnitude) in assay sensitivity and allowed the detection of 0.01 mouseLD50 toxin present in buffer solution. [ABSTRACT FROM AUTHOR]

BACKGROUND: Since the common protease substrates did not give satisfactory results for the determination of Sunn pest protease activity in damaged wheat, different peptide substrates derived from the repeated sequences of high molecular weight glutenin subunits were synthesized. RESULTS: Hydrolysis of peptides by pest protease was determined by high‐performance liquid chromatography. Among three peptides having the same consensus motifs, peptide1 (PGQGQQGYYPTSPQQ) showed the best catalytic efficiency. A novel assay was described for monitoring the enzymatic activity of protease extracted from damaged wheat flour. The selected peptide was labeled with a fluorophore (EDANS) and quencher (Dabcyl) to display fluorescence resonance energy transfer. The proteolytic activity was measured by the change in fluorescence intensity that occurred when the protease cleaved the peptide substrate. Furthermore, the assay developed was modified for rapid and easy detection of bug damage in flour. Flour samples were suspended in water and mixed with fluorescence peptide substrate. After centrifugation, the fluorescence intensities of the supernatants, which are proportional to the protease content of the flour, were determined. CONCLUSION: The total analysis time for the assay developed is estimated as 15 min. The assay developed permits a significant decrease in time and labor, offering sensitive detection of Sunn pest damage in wheat flour. © 2018 Society of Chemical Industry [ABSTRACT FROM AUTHOR]

Amyloid-β (Aβ) is generated by proteolytic processing of amyloid precursor protein (APP) by beta-secretase (BACE-1) and gamma-secretase. Amyloid-β is responsible for the formation of senile plaques in Alzheimer's disease (AD). Consequently, inhibition of β-secretase (BACE-1), a rate-limiting enzyme in the production of Aβ, constitutes an attractive therapeutic approach to the treatment of AD. This paper reports an inkjet printing-based fluorescence assay for high throughput screening of β-secretase inhibitors achieved by employing a BACE-1 FRET substrate (Rh-Glu-Val-Asn-Leu-Asp-Ala-Glu-Phe-Lys-Quencher). This peptide substrate is known to be a readily available and suitable substrate for proteolytic activity, and it has high affinity to BACE-1. The BACE-1 peptide substrate printed on parchment paper was effectively cleaved by BACE-1, which was printed on the same spot. The amount of enzyme and substrate required for this inkjet printing-based BACE-1 assay can be less than 1.4ⅹ10 3 , permitting the evaluation of inhibitor activity with femtomolar potency. The inkjet-printing-based BACE-1 inhibitory assay revealed inhibitory effects of inhibitor IV and STA on BACE-1 with an IM 50 of 1.00 × 10 −15  mol and 1.01 × 10 −14  mol, respectively. These data confirm that both BACE-1 inhibitors (inhibitor IV and STA) actively inhibited the BACE-1 proteolysis of BACE-1 substrate on parchment paper. It important to note that the number of mole of BACE-1-substrate and enzyme utilized in the printing-based enzymatic assay are 1.4ⅹ10 3 smaller than the amount used in the conventional well-plate assay. The inkjet printing-based inhibitory assay constitutes a versatile high throughput technique and the IM 50 values of the inhibitors were obtained with satisfactory reproducibility, suggesting that this inkjet-printing BACE-1 inhibitory assay could be quite suitable for the screening of new potential BACE-1 inhibitors for AD. [ABSTRACT FROM AUTHOR]

A peptide probe-based MMP-9 inhibitory assay was developed to assess anticancer activity of compounds on parchment paper using FRET-based inkjet printing. The fluorescent peptide containing 5-FAM (donor) and QXL520 (acceptor) was used as a substrate with the sequence of Pro-Leu-Gly-Cys-His-Ala-Arg-Lys specific to MMP-9. The suitability of the peptide as a substrate of MMP-9 was confirmed by HPLC. When both MMP-9 and the peptide were printed on the identical reaction spot of parchment paper, the peptide was cleaved and fragmented by MMP-9. The fluorescence was then recovered by 5-FAM and detected at Ex/Em = 490 ± 20 nm/520 ± 20 nm. Based on the successful performance of FRET on the parchment paper without any surface treatment, the assay platform was applied to test compounds, and a successful anticancer assay could be accomplished. This methodology holds notable strengths in versatile applications to the MMP enzyme family, as well as control of ejection volume, reduction of consumed enzyme amount, and rapid reaction time. Hence, we suggest the FRET peptide-based assay using inkjet printing as a viable replacement for sensitive and high-throughput evaluation of anticancer compounds. [ABSTRACT FROM AUTHOR]

Rapid and sensitive detection of botulinum neurotoxins (BoNTs), which cause botulism, is essential in a public health emergency or bioterrorism event. We have previously developed a mass spectrometry (MS)-based functional method, Endopep-MS assay, for the fast detection and differentiation of all BoNT serotypes by affinity enriching the toxin and detecting the serotype-specific cleavage products of peptide substrates derived from the in vivo targets. To improve the performance of the Endopep-MS assay, we report here the further optimization of the peptide substrate for the detection of serotype A botulinum neurotoxins. An increased substrate cleavage was achieved by extending the original peptide N-terminus with optimized amino acid sequence, increasing the detection sensitivity of the method. In addition, the resistance of the substrate to nonspecific hydrolysis was dramatically improved by selectively substituting amino acids at the scissile bond and various other positions of the extended peptide. Moreover, incorporating the N-terminal hydrophobic residues dramatically improved the relative intensity of the cleavage products in the mass spectra. This allowed easy detection of the cleavage products, further enhancing the performance of the assay. The limit of detection for spiked serum sample was enhanced from 0.5 to 0.1 mouseLD and from 0.5 to 0.2 mouseLD for spiked stool. [ABSTRACT FROM AUTHOR]

Botulinum neurotoxins (BoNTs) are the most toxic proteins in nature. Endopeptidase-mass-spectrometry (Endopep-MS) is used as a specific and rapid in - vitro assay to detect BoNTs. In this assay, immunocaptured toxin cleaves a serotype-specific-peptide-substrate, and the cleavage products are then detected by MS. Here we describe the design of a new peptide substrate for improved detection of BoNT type A (BoNT/A). Our strategy was based on reported BoNT/A-SNAP-25 interactions integrated with analysis method efficiency considerations. Integration of the newly designed substrate led to a 10-fold increase in the assay sensitivity both in buffer and in clinically relevant samples. [ABSTRACT FROM AUTHOR]

This manuscript reports on a novel substrate for the amperometric detection of cellular apoptosis, Asp-Glu-Val-Asp- p-methoxyaniline (DEVD- pMA). This substrate showed electrochemical inertia from −0.2 to 0.7 V versus Ag/AgCl in phosphate-buffered saline (PBS), but was cleaved by caspase-3, releasing pMA, which was detected with cyclic voltammetry and chronoamperometry. Compared with p-aminophenol ( pAP), pMA showed lower toxicity to the human hepatocellular carcinoma cell line (HepG2) and higher stability in PBS. By using this substrate, we successfully detected caspase-3 in the range of 0.31-2.5×10−2 units/mL and cellular apoptosis in HepG2 cells by using simple amperometry without cell lysis. This work will help to develop a simple method for the electrochemical monitoring of apoptosis, which can be applied in biological research and drug development. [ABSTRACT FROM AUTHOR]

Matrix metalloproteinase-9 (MMP-9) is a core protease of cancer invasion and metastasis. In this paper, we report an electrochemical biosensor integrated with a redox reporter labeled peptide for the detection of MMP-9. The peptide substrate conjugated with methylene blue (MB) specific to MMP-9 was immobilized on Au electrodes using a thiol group in cysteine. The peptide cleavage by MMP-9 caused the release of electroactive MB from the electrodes and resulted in the decrease of the electrical tunneling current. The concentric electrode device consisted of an island electrode and an enclosing electrode which is ca. 1000 folds larger than the island electrode. The self-gating effect from the concentric electrode device was able to provide a reliable electrical measurement without a reference electrode. From the electrochemical biosensor, a linear correlation between the concentration of MMP-9 and the electrical signal reduction was found in a range from 1 pM to 1 nM and the limit of detection was determined at 7 pM. [ABSTRACT FROM AUTHOR]

In this work we report the detection of plasmin protease by means of the thickness shear mode (TSM) acoustic method. The biorecognition element consists of a peptide substrate (PS) specific to plasmin immobilized on a piezoelectric quartz crystal electrode. After enzymatic reaction with plasmin, it cleaves a short fragment of the peptide causing increase in the resonance frequency of the piezo crystal. Plasmin was detected in the range of concentrations 1–20 nM, a target interval in which its presence presumably affects the quality of milk. The PS exhibited negligible response against to similar protease trypsin. This has been confirmed also by electrochemical detection method. Limit of detection of this acoustic transducer was found to be 0.65 nM. Formation of the sensing surface and kinetic effect of plasmin on the peptide substrate was studied by atomic force microscopy (AFM). The PS response was also validated in pretreated milk samples spiked by known concentrations of plasmin achieving an average recovery of 63 ± 0.6%. [ABSTRACT FROM AUTHOR]

We presented a strategy for fabricating graphene oxide (GO)-based fluorescent biosensors to monitor the change of phosphorylation state and detect phosphatase activity. By regulating the interaction between the negatively charged phosphate group and the positively charged amino residue, we found that GO showed different quenching efficiency toward the phosphorylated and dephosphorylated dye-labeled peptides. To demonstrate the application of our method, alkaline phosphatase (ALP) was tested as a model enzyme with phosphorylated fluorescein isothiocyanate (FITC)-labeled short peptide FITC-Gly-Gly-Gly-Tyr(PO32-)-Arg as the probe. When the negatively charged phosphate group in the Tyr residue was removed from the peptide substrate by enzymatic hydrolysis, the resulting FITC-Gly-Gly-Gly-Tyr-Arg was readily adsorbed onto the GO surface through electrostatic interaction. As a result, fluorescence quenching was observed. Furthermore, the method was applied for the screening of phosphatase inhibitors. [ABSTRACT FROM AUTHOR]

Botulinum neurotoxins (BoNTs) are the most toxic proteins in nature. Rapid and sensitive detection of BoNTs is achieved by the endopeptidase–mass spectrometry (Endopep–MS) assay. In this assay, BoNT cleaves a specific peptide substrate and the cleaved products are analyzed by MS. Here we describe the design of a new peptide substrate for improved detection of BoNT type B (BoNT/B) in the Endopep–MS assay. Our strategy was based on reported BoNT/B–substrate interactions integrated with analysis method efficiency considerations. Incorporation of the new peptide led to a 5-fold increased sensitivity of the assay both in buffer and in a clinically relevant human spiked serum. [ABSTRACT FROM AUTHOR]

We report a comparative analysis of plasmin detection by means of electrochemical biosensor and photometric assays. The biosensor has been based on ferrocene-modified peptide (Fc-P) specific to plasmin. Fc-P substrate is enzymatically cleaved by plasmin and the short fragment of peptide contained Fc is released causing decrease of redox current measured by cyclic voltammetry. The biosensor was tested in the range 1-20 nM of freshly prepared plasmin and it was highly selective ( LOD=0.56±0.03 nM) demonstrating lower reaction to other proteins such as thrombin and β-casein. The concentration of plasmin has been determined also by a photometric analysis based on UV-vis spectroscopy ( LOD=3.68±0.04 nM). The assay has been validated in milk samples. [ABSTRACT FROM AUTHOR]

Botulinum neurotoxins (BoNTs) produced by Clostridium botulinum are the most poisonous substances known to humankind. It is essential to have a simple, quick, and sensitive method for the detection and quantification of botulinum toxin in various media, including complex biological matrices. Our laboratory has developed a mass spectrometry-based Endopep–MS assay that is able to rapidly detect and differentiate all types of BoNTs by extracting the toxin with specific antibodies and detecting the unique cleavage products of peptide substrates. Botulinum neurotoxin type E (BoNT/E) is a member of a family of seven distinctive BoNT serotypes (A–G) and is the causative agent of botulism in both humans and animals. To improve the sensitivity of the Endopep–MS assay, we report here the development of novel peptide substrates for the detection of BoNT/E activity through systematic and comprehensive approaches. Our data demonstrate that several optimal peptides could accomplish 500-fold improvement in sensitivity compared with the current substrate for the detection of both not-trypsin-activated and trypsin-activated BoNT/E toxin complexes. A limit of detection of 0.1 mouse LD 50 /ml was achieved using the novel peptide substrate in the assay to detect not-trypsin-activated BoNT/E complex spiked in serum, stool, and food samples. [ABSTRACT FROM AUTHOR]

Highlights: [•] GRK2 appears to require an acidic amino acid at the −2, −3, or −4 positions. [•] The consensus phosphorylation site motifs for GRK2 are (D/E)X1–3(S/T), (D/E)X1–3(S/T)(D/E), or (D/E)X0–2(D/E)(S/T). [•] Among 17 peptide substrates, a peptide fragment of β-tubulin (DEMEFTEAESNMN) shows the highest affinity for GRK2. [Copyright &y& Elsevier]

Highlights: [•] Myristoylated peptides derived from amino acid residues 2–19 of the preS1 domain selectively recognized human hepatocellular carcinoma cells. [•] Non-myristoylated peptides showed much lower affinity for human hepatocellular carcinoma cells than myristoylated peptides. [•] A modified peptide exhibited the highest affinity for human hepatocellular carcinoma cells. [ABSTRACT FROM AUTHOR]

Abstract: Matriptase, a type II transmembrane serine protease, is responsible for the early stage proliferation of a wide range of human cancers. It is highly expressed on the surface of tumor cells, allowing the protease to serve as a biomarker of cancer detection. Hence, in this study, we designed two molecular beacons consisting of a fluorescent dye–peptide conjugate attached to gold nanoparticles (Au NPs) for in vitro and in vivo detection of the matriptase expression on tumor cells. The peptide substrate (GRQSRAGC) for matriptase served as the linker between dye (donor) and Au NP (acceptor). In this configuration, the dye fluorescence was quenched by Au NP under physiological conditions and recovered after selective cleavage of the GRQSRAGC by matriptase. To maximize spectral overlap between dye fluorescence and Au NP absorption and optimize the quenching efficiency mediated by fluorescence resonance energy transfer (FRET), the visible dye (fluorescein) and near-infrared (NIR) dye peptide conjugates were each attached to spherical and rod-shaped Au NP for in vitro and in vivo detection, respectively. Both in vitro cell and in vivo animal studies indicate that these two molecular beacons are sensitive and specific for the detection of matriptase expression in the tumor, thereby paving the way to image the activities of this diagnostic protease in living organisms. [Copyright &y& Elsevier]

BACKGROUND: Several serum biomarkers such as antigens, soluble proteins, metabolites, and genes have been identified for the diagnosis of cancers and for monitoring the recurrence after cancer treatment. METHODS: In the present study, a protein kinase C (PKC) α-specific peptide substrate was phosphorylated with serum samples collected from cancer-bearing mice (U87, A431, HepG2, and A549) and the phosphorylation ratio was detected by matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry (MALDI-TOF MS). RESULTS: The phosphorylation ratio for peptide substrates significantly increased in the serum of cancer-bearing mice compared with the ratio found in control mice. The addition of a PKCα inhibitor induced a concentration-dependent decrease in phosphorylation ratios, but the non-PKCα inhibitors, rottlerin and H-89, did not significantly effect phosphorylation ratios. CONCLUSIONS: These results suggest that serum activated PKCα is a good biomarker applicable to cancer diagnosis. [ABSTRACT FROM AUTHOR]

Abstract: We identified nine small-molecule hit compounds of Heat shock 70kDa protein 5 (HSPA5) from cascade in silico screening based on the binding modes of the tetrapeptides derived from the peptide substrate or inhibitors of Escherichia coli HSP70. Two compounds exhibit promising inhibition activities from cancer cell viability and tumor inhibition assays. The binding modes of the hit compounds provide a platform for development of selective small molecule inhibitors of HSPA5. [Copyright &y& Elsevier]

Abstract: Botulinum neurotoxins (BoNTs) are a family of seven toxin serotypes that are the most toxic substances known to humans. Intoxication with BoNT causes flaccid paralysis and can lead to death if untreated with serotype-specific antibodies. Supportive care, including ventilation, may be necessary. Rapid and sensitive detection of BoNT is necessary for timely clinical confirmation of clinical botulism. Previously, our laboratory developed a fast and sensitive mass spectrometry (MS) method termed the Endopep–MS assay. The BoNT serotypes are rapidly detected and differentiated by extracting the toxin with serotype-specific antibodies and detecting the unique and serotype-specific cleavage products of peptide substrates that mimic the sequence of the BoNT native targets. To further improve the sensitivity of the Endopep–MS assay, we report here the optimization of the substrate peptide for the detection of BoNT/A. Modifications on the terminal groups of the original peptide substrate with acetylation and amidation significantly improved the detection of BoNT/A cleavage products. The replacement of some internal amino acid residues with single or multiple substitutions led to further improvement. An optimized peptide increased assay sensitivity 5-fold with toxin spiked into buffer solution or different biological matrices. [Copyright &y& Elsevier]

Abstract: Protein kinase C (PKC), a phospholipid-dependent serine/threonine kinase, appears to be involved in the signal transduction response to many hormones and growth factors; there are 11 different PKC isozymes. Because PKC isozymes directly and/or indirectly participate in signal transduction pathways of normal and transformed cells through phosphorylation of target proteins, it is critical to understand the diversity of the intracellular signaling pathways regulated by each PKC isozyme. Thus, PKC isozyme-specific substrates are useful to understand the characterization of the intracellular signaling pathways for each PKC isozyme. Consensus sequences and sequence information obtained from PKC target proteins are very important to design PKC isozyme-specific peptide substrates. Moreover, computational prediction programs of phosphorylation sites using a library of peptide substrates aid in the fast design of PKC isozyme-specific peptide substrates. Although a large number of target proteins and synthetic peptides for PKCs are known, only two peptide substrates (peptide 422–426 of murine elongation factor-1α and Alphatomega peptide) have been reported as PKC isozyme-specific peptide substrates. This discussion will review the literature concerning these native and synthetic PKC isozyme-specific peptide substrates and their design. [Copyright &y& Elsevier]

Many cellular signaling events are regulated by tyrosine phosphorylation and mediated by the opposing actions of protein tyrosine kinases and phosphatases. Protein tyrosine phosphatases are emerging as drug targets, but poor cell permeability of inhibitors has limited the development of drugs targeting these enzymes [Tautz L, et al. (2006) Expert Opin Ther Targets 10:157-177]. Here we developed a method to monitor tyrosine phosphatase activity at the single-cell level and applied it to the identification of cell-permeable inhibitors. The method takes advantage of the fluorogenic properties of phosphorylated coumaryl amino propionic acid (pCAP), an analog of phosphotyrosine, which can be incorporated into peptides. Once delivered into cells, pCAP peptides were dephosphorylated by protein tyrosine phosphatases, and the resulting cell fluorescence could be monitored by flow cytometry and high-content imaging. The robustness and sensitivity of the assay was validated using peptides preferentially dephosphorylated by CD45 and T-cell tyrosine phosphatase and available inhibitors of these two enzymes. The assay was applied to high-throughput screening for inhibitors of CD45, an important target for autoimmunity and infectious diseases [Hermiston ML, et al. (2003) Annu Rev Immunol 21:107-137]. We identified four CD45 inhibitors that showed activity in T cells and macrophages. These results indicate that our assay can be applied to primary screening for inhibitors of CD45 and of other protein tyrosine phosphatases to increase the yield of biologically active inhibitors. [ABSTRACT FROM AUTHOR]

Abstract: The unique and physiologically vital bacterial enzyme, prolipoprotein diacylglyceryl transferase (Lgt), which catalyzes the committed first step in the posttranslational transfer of diacylglyceryl group from phosphatidylglycerol to the prospective N-terminal cysteine of prolipoproteins, remains to be characterized for want of a simpler but equally sensitive nonradioactive assay. We, for the first time, report a coupled enzymatic fluorescence assay for Lgt using the de novo synthetic peptide substrate MKATKSAVGSTLAGCSSHHHHHH. The assay is based on the conversion of the by-product, glycerol-1-phosphate, to dihydroxyacetone using an alkaline phosphatase–glycerol dehydrogenase combination and estimating the fluorescence of the coupled reduction of resazurin to resorufin. The minimum amount of glycerol-1-phosphate, and hence the modified peptide, detected by this method is approximately 20pmol, thereby making this assay a promising alternative to the radioactive assays. The assay is rapid, more convenient, less laborious, and suitable for purification and characterization of Lgt. [Copyright &y& Elsevier]

Background: The Luminescent Oxygen Channeling Immunoassay (LOCI®) technology is a well-established homogeneous assay format that allows for fast, accurate, and highly sensitive quantitation of analytes. We set out to develop and prove a novel concept to establish a LOCI format that should principally allow for the determination of the activity of coagulation factors and anticoagulants of clinical relevance. Methods: The concept is based on the linkage of LOCI nano-beads by a peptide that can be cleaved by a coagulation factor. To prove the principle, we used a peptide that can be cleaved by thrombin. Results: We were able to show that coagulation activation of plasma or whole blood samples that were combined with the LOCI components degraded the thrombin-sensitive peptide and consequently, led to a reduction of the LOCI signal. Signal reduction was proportional to the amount of active thrombin generated. The research prototype assay allowed for the detection of factor deficiencies in both the extrinsic and intrinsic coagulation pathways, and for the quantification of hirudin, a direct thrombin inhibitor. Conclusions: Taken together, we conclude that the LOCI technology has the potential for extension to functional blood coagulation assays. [ABSTRACT FROM AUTHOR]

Abstract: Gene therapy is a new medical approach for the treatment of tumors. For safe and efficient gene therapy, therapeutic genes need to be delivered efficiently into the target tumor cells. Development of gene delivery systems to specifically recognize and target tumor cells and to distinguish them from normal cells, especially in the same tissue or organ, is one of the most important issues regarding the present gene delivery methodologies. The enhanced permeability and retention (EPR) effect using the characteristics of angiogenic tumor blood vessels, as well as gene delivery systems recognizing hyperactivated receptors or intracellular signals, is broadly applied to tumor-targeted gene therapy. In addition, bacterial vectors can be a useful means for targeting hypoxic or anoxic regions of a tumor. [ABSTRACT FROM AUTHOR]

Abstract: Recently, there is a growing interest in the intracellular signal-targeting gene therapy or diagnosis, mainly by using the reaction of targeting enzymes with peptide substrates. In the present study, we proved the importance of target intracellular signal-specificity peptide substrate for intracellular signals-targeting gene therapy or diagnosis. Protein kinase C (PKC) was used as a trigger to activate the transgene expression. Two peptides, a positive peptide showing phosphorylation levels on several PKC isozymes (PKCα, βII, γ, ε, η, ζ, and ι/λ) and a negative peptide in which the phosphorylation site was destroyed by changing from serine to alanine, were designed. Moreover, two polymers possessing each peptide as a pendant chain, a PKC-responsive conjugate [PPC(S)] and a negative control conjugate [PPC(A)], were synthesized. After the introduction of complexes into cells or tissues, gene expression for PPC(S)/DNA complexes was higher that for PPC(A)/DNA complexes. However, no difference in gene expression between B16 melanoma tumors and normal skin tissues was identified. These results suggest that a peptide substrate specific to a target intracellular signal is very important for intracellular signals-targeting gene therapy or diagnosis. [Copyright &y& Elsevier]

Abstract: Calpains are intracellular proteases that selectively cleave proteins in response to calcium signals. Although calpains cut many different sequences, residue preferences within peptide substrates were recently determined and incorporated into a superior FRET (fluorescence resonance energy transfer)-based substrate (PLFAER). Here we show PLFAER is cleaved by calpain at the intended F-A scissile bond. Sequential replacement of individual residues by alanine reduced activity except with PLFAAR, which is cleaved 2.3 times faster than PLFAER. The rates of hydrolysis of the alanine-substituted substrates were used to compare substrate preferences of calpain, papain and cathepsins B and L. The preferences of the two major isoforms, calpains 1 and 2, were virtually indistinguishable and were very similar to those of the calpain 1 protease core and papain. However, the activity profiles with the FRET substrate series were significantly different for the cathepsins, particularly cathepsin B. [Copyright &y& Elsevier]

Abstract: Sirtuins are the class III histone deacetylases that catalyze the deacetylation of acetyl-lysine residues of histones and other proteins using nicotinamide adenine dinucleotide (NAD+) as the cofactor. The reaction yields the deacetylated protein, nicotinamide, and 2’-O-acetyl-ADP-ribose. Three 9-fluorenylmethoxycarbonyl (Fmoc)-labeled peptides derived from the amino acid sequence of p53, Fmoc–KK(Ac)-NH2, Fmoc–KK(Ac)L-NH2, and Fmoc–RHKK(Ac)-NH2, were characterized as substrates for two of the human sirtuins: SIRT1 and SIRT2. The deacetylation was monitored by a validated capillary electrophoresis assay. Efficient deacetylation by SIRT1 and SIRT2 was demonstrated for all three peptide substrates. The kinetics of the enzymatic reaction was determined with the Michaelis constants (K m) varying between 16.7 and 34.6μM for SIRT1 and between 34.7 and 58.6μM for SIRT2. Resveratrol did not function as an activator for SIRT1 using the Fmoc-labeled peptides as SIRT substrates. The IC50 values of sirtinol using the three peptide substrates were determined. Further sirtuin inhibitors were also evaluated. [Copyright &y& Elsevier]

• Enzymatic hydrolysis of a specific substrate for plasmin activity is studied. • The effect of peptide sequence on enzyme activity was observed. • APPPPAKAPPPPC was the superior peptide sequence with optimal alignment and conformation. • A new SERS-based sensitive method was developed for the detection of plasmin activity. • The method was successfully used for the detection of plasmin activity in real milk samples. In this study, a new surface-enhanced Raman scattering (SERS)-based method has been developed for the detection of plasmin activity. Firstly, different peptide sequences, which are specific to plasmin, were examined. Then, SERS substrates were prepared by chosen peptide substrate. Enzyme activity was determined by pursuing the reduction of DTNB band at 1331 cm−1 with Raman spectroscopy. The reduction in SERS intensity was related to the plasmin activity, and changes in SERS intensity vs. plasmin concentration graph was obtained. Limit of detection (LOD) and limit of quantification (LOQ) values were calculated as 2.14 U/mL and 6.42 U/mL, respectively. Intra- and inter-day repeatability results were determined as 1.45% and 1.47% relative standard deviation (RSD). Also, recovery of the method was determined for the plasmin spiked milk samples. The results demonstrated that the proposed method could be successfully used to detect the plasmin activity in milk samples. [ABSTRACT FROM AUTHOR]

Abstract: Bacteria produce a range of proteolytic enzymes. In an attempt to detect and identify bacteria on the basis of their protease activity, a panel of protease substrates was investigated. Peptides conjugated to the fluorophore 7-amino-4-methylcoumarin (AMC) are well-established substrates for measuring protease activity. Although peptide–AMC substrates are generally not specific for a single protease, a unique pattern can be achieved for both highly specific enzymes and those with a broader substrate range by comparing different peptide substrates. The panel of 7 peptide–AMC substrates chosen exhibited a unique pattern for nine microbial proteases. The selected peptides were used to determine protease activity in cultured strains of Pseudomonas aeruginosa and Staphylococcus aureus. A signal pattern obtained with peptides with arginine, lysine, and tyrosine in the P1 position characterized the bacterial protease activities in these samples. The kinetic parameters for the three best substrates for the P. aeruginosa sample were calculated. Further information about substrate specificity was gained by the selective use of protease inhibitors. The results presented show that peptide–AMC substrates provide a simple and sensitive tool to characterize protease activity in microbiological samples and that they have the potential to identify and distinguish different bacterial species. [Copyright &y& Elsevier]

FLT3 (fms-like tyrosine kinase 3) is frequently activated by mutation in acute myeloid leukemia, and is therefore under study as a drug target. Testing and characterization of tyrosine kinase inhibitors is facilitated by the availability of efficient peptide substrates. Searching for FLT3 peptide substrates using phosphorylation experiments on peptide arrays and in solution revealed that the peptide F-T-D-R-L-Q-Q-Y8-I-S-T-R-G-L-G is efficiently phosphorylated (apparent Km 10 μmol/l), with Y8 as the phosphorylated site. This peptide presents a novel tool for identifying and characterizing FLT3 kinase inhibitors. [ABSTRACT FROM AUTHOR]

Abstract: Lipid modification of proteins is an essential post-translational event that can be targeted for protein engineering and pharmaceutical applications. In this regard, the unique and ubiquitous bacterial N-terminal lipid modification (N-acyl S-diacylglyceryl modification of N-terminal cysteine) is particularly attractive. It is initiated by phosphatidylglycerol:prolipoprotein diacylglyceryl transferase (Lgt) and therefore its properties, which remain uninvestigated, largely define the specifics of the modification. A synthetic peptide-substrate (MKATKSAVGSTLAGCSSHHHHHH) with a short hydrophilic h-region, unlike that of the prototypical substrate used so far, demonstrated lack of enzyme''s substrate preference based on hydrophobicity, perhaps accounting for a significant number of lipoproteins possessing hydrophilic signal peptides. Solubilization experiments revealed a peripheral and possibly reversible hydrophobic association of Lgt with the inner-membrane on the cytosolic side contradicting its deduced transmembrane topology. Except for heat stability, the soluble enzyme was indistinguishable from the membrane-bound enzyme in kinetic behaviour, indicating that the committed first step of bacterial lipid modification may be aqueous compatible. The direct, more accurate, precise and easier paper electrophoretic assay, designed anew, and Lgt''s ready extraction with water or low ionic strength solutions from inverted vesicles could aid better understanding and exploitation of the enzyme and bacterial lipid modification. [Copyright &y& Elsevier]

Abstract: ClpP and its ATPase compartment, ClpX or ClpA, remove misfolded proteins in cells and are of utmost importance in protein quality control. The ring hexamers of ClpA or ClpX recognize, unfold, and translocate target substrates into the degradation chamber of the double-ring tetradecamer of ClpP. The overall reaction scheme catalyzed by ClpXP or ClpAP has been proposed; however, the molecular mechanisms associated with substrate recognition and degradation have not yet been clarified in detail. To investigate these mechanisms, we determined the crystal structures of ClpP from Helicobacter pylori in complex with product peptides bound to the active site as well as in the apo state. In the complex structure, the peptides are zipped with two antiparallel strands of ClpP and point to the adjacent active site, thus providing structural explanations for the broad substrate specificity, the product inhibition and the processive degradation of substrates in the chamber. The structures also suggest that substrate binding causes local conformational changes around the active site that ultimately induce the active conformation of ClpP. [Copyright &y& Elsevier]

Abstract: Regulated phosphorylation by protein tyrosine kinases (PTKs), such as c-Abl, is critical to cellular homeostasis. In turn, once deregulated as in the chronic myeloid leukemia (CML) fusion protein Bcr-Abl, PTKs can promote cancer onset and progression. The dramatic success of the Bcr-Abl inhibitor imatinib as therapy for CML has inspired interest in other PTKs as targets for cancer drug discovery. Here we report a novel PTK activity and inhibition screening method using hydrogel-immobilized peptide substrates. Using acrylate crosslinkers, we tether peptides via terminal cysteines to thiol-presenting hydrogels in 96-well plates. These surfaces display low background and high reproducibility, allowing semiquantitative detection of peptide phosphorylation by recombinant c-Abl or by Bcr-Abl activity in cell extracts using traditional anti-phosphotyrosine immunodetection and chemifluorescence. The capabilities of this assay are demonstrated by performing model screens for inhibition with several commercially available PTK inhibitors and a collection of pyridopyrimidine Src/Abl dual inhibitors. This assay provides a practical method to measure the activity of a single kinase present in a whole cell lysate with high sensitivity and specificity as a valuable means for efficient small molecule screening. [Copyright &y& Elsevier]

Abstract: The present study was initiated to characterize angiotensin-converting enzyme (ACE) in Crustaceans. Using degenerate DNA primers deduced from consensus sequences located upward and downward from the active site of ACEs from different arthropod species, several tissues from the crayfish Astacus leptodactylus were screened by RT-PCR. Amplicons were obtained from hepatopancreas, testis and hemocytes. Analysis of the predicted protein sequences after cloning and Northern blot experiments revealed an original and complex polymorphism of the ACE-like active site. Two variants were obtained in the hepatopancreas, one displaying a 6.4kb size transcript, probably corresponding to a double domain ACE, with an unusual active site structure while the other had a transcript size of 2.5kb, close to the size of the transcript obtained in testis and hemocytes (2 and 3kb, respectively), likely representing single domain enzymes. Functional assays using a synthetic substrate were performed from the different tissues and showed a maximal ACE-like activity associated to membrane fraction from testis and hepatopancreas. [Copyright &y& Elsevier]

Abstract: The study of substrate specificity of protein phosphatases (PPs) is very challenging since it is difficult to prepare a suitable phosphorylated substrate. Phosphoproteins, phosphorylated by a protein kinase, or chemically synthesized phosphopeptides are commonly used substrates for PPs. Both types of these substrates have their advantages and limitations. Phosphoproteins mimic more closely the physiologically relevant PP substrates, but their preparation is technically demanding. Synthetic phosphopeptides present advantages over proteins because they can be easily produced in large quantity and their amino acid sequence can be designed to contain potential determinants of substrate specificity. However, short peptides are less optimal compared to in vivo PP substrates and often display poor and variable binding to different matrices, resulting in low sensitivity in analysis of PP activity on solid support. In this work we utilize the intein-mediated protein ligation (IPL) technique to generate substrates for PPs, combining the advantages of proteins and synthetic peptides in one molecule. The ligation of a synthetic phosphopeptide to an intein-generated carrier protein (CP) with a one-to-one stoichiometry results in the formation of a ligated phosphoprotein (LPP). Three widely used assays, dot blot array, Western blot and ELISA were employed to study the PP activity on LPP substrates. Dephosphorylation was measured by detection of the remaining phosphorylation, or lack of it, with a phospho-specific antibody. The data show the advantage of LPPs over free peptides in assays on solid supports. LPPs exhibited enhanced binding to the matrices used in the study, which significantly improved sensitivity and consistency of the assays. In addition, saturation of the signal was circumvented by serial dilution of the assay samples. This report describes detailed experimental procedures for preparation of LPP substrates and their use in PP assays based on immobilization on solid supports. [Copyright &y& Elsevier]

The Pim family of Ser/Thr kinases has been implicated in the process of lymphomagenesis and cell survival. Known substrates of Pim kinases are few and poorly characterized. In this study we set out to identify novel Pim-2 substrates using the Kinase Substrate Tracking and Elucidation (KESTREL) approach. Two potential substrates, eukaryotic initiation factor 4B (eIF4B) and apoptosis inhibitor 5 (API-5), were identified from rat thymus extracts. Sequence comparison of the Pim-2 kinase phosphorylation sites of eIF4B and mouse BAD, the only other known Pim-2 substrate, revealed conserved amino acids preceding the phosphorylated serine residue. Stepwise replacement of the conserved residues produced a consensus sequence for Pim kinase recognition: RXRHXS. Pim-1 and Pim-2 catalyzed the phosphorylation of this recognition sequence 20-fold more efficiently than the original (K/R-K/R-R-K/R-L-S/T-a; a = small chain amino acid) Pim-1 phosphorylation site. The identification of the novel Pim kinase consensus sequence provides a more sensitive and versatile peptide based assay for screening modulators of Pim kinase activity. [ABSTRACT FROM PUBLISHER]

Protein tyrosine phosphatases (PTPs) play key roles in regulating tyrosine phosphorylation levels in cells. Since the discovery of PTP1B as a major drug target for diabetes and obesity, PTPs have emerged as a new and promising class of signaling targets for drug development in a variety of therapeutic areas. The routine use of generic substrate 6,8-difluoro-4-methylumbelliferyl phosphate (DiFMUP) in our hands led to the discovery of very similar and often not very selective molecules. Therefore, to increase the chances to discover novel chemical scaffolds, a side-by-side comparison between the DiFMUP assay and a chip-based mobility shift assay with a specific phosphopeptide was performed, on 1 PTP, using a focused set of compounds. Assay robustness and sensitivity were comparable for both the DiFMUP and mobility shift assays. The off-chip mobility shift assay required a longer development time because of identification, synthesis, and characterization of a specific peptide, and its cost per point was higher. However, although most potent scaffolds found with the DiFMUP assay were confirmed in the mobility shift format, the off-chip mobility shift assay led to the identification of previously unidentified chemical scaffolds with improved druglike properties. [ABSTRACT FROM PUBLISHER]