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Journal articles on the topic 'Cleavage assay'

Author: Grafiati

Published: 4 June 2021

Last updated: 1 February 2022

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1

Huitema, Carly, and Lindsay D. Eltis. "A Fluorescent Protein-Based Biological Screen of Proteinase Activity." Journal of Biomolecular Screening 15, no. 2 (2010): 224–29. http://dx.doi.org/10.1177/1087057109357790.

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A cell-based fluorescent protein reporter assay for proteinase activity amenable to high-throughput applications was developed. This assay is based on Förster resonance energy transfer (FRET) between 2 variants of the green fluorescent protein connected by a short cleavable linker and expressed in Escherichia coli as tagged proteins. A library to assay proteinase specificity was generated by randomizing a portion of the linker using PCR. The library could be grown in microplates, allowing cells to be lysed in situ and substrate cleavage to be monitored through loss of FRET signal using a plate

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2

Han, Yue, and X. Long Zheng. "A Sensitive and Specific Shear-Based Assay for Plasma ADAMTS13 Activity and Inhibitors-the Diagnostic Utility for Thrombotic Microangiopathies." Blood 114, no. 22 (2009): 2114. http://dx.doi.org/10.1182/blood.v114.22.2114.2114.

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Abstract Abstract 2114 Poster Board II-91 Severe deficiency of plasma ADAMTS13 activity causes a potentially fatal thrombotic thrombocytopenic purpura (TTP). To date, functional assays for plasma ADAMTS13 activity and inhibitors rely on cleavage of von Willebrand factor (VWF) in the presence of 1.5 M urea or guanidine-HCl or small peptides derived from the central VWF-A2 domain. To develop a more physiological assay under fluid shear stress, we assessed the proteolytic cleavage of plasma-derived VWF by plasma ADAMTS13 under constant vortexing (2,500 rpm) in a PCR tube mini-mixer that mimics ar

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3

Pope, Mark R., Leonard L. Saari, and Paul W. Ludden. "Fluorometric assay for ADP-ribosylarginine cleavage enzymes." Analytical Biochemistry 160, no. 1 (1987): 68–77. http://dx.doi.org/10.1016/0003-2697(87)90615-4.

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4

Belliot, Gaël, Stanislav V. Sosnovtsev, Tanaji Mitra, Carl Hammer, Mark Garfield, and Kim Y. Green. "In Vitro Proteolytic Processing of the MD145 Norovirus ORF1 Nonstructural Polyprotein Yields Stable Precursors and Products Similar to Those Detected in Calicivirus-Infected Cells." Journal of Virology 77, no. 20 (2003): 10957–74. http://dx.doi.org/10.1128/jvi.77.20.10957-10974.2003.

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ABSTRACT The MD145-12 strain (GII/4) is a member of the genus Norovirus in the Caliciviridae and was detected in a patient with acute gastroenteritis in a Maryland nursing home. The open reading frame 1 (ORF1) (encoding the nonstructural polyprotein) was cloned as a consensus sequence into various expression vectors, and a proteolytic cleavage map was determined. The virus-encoded cysteine proteinase mediated at least five cleavages (Q330/G331, Q696/G697, E875/G876, E1008/A1009, and E1189/G1190) in the ORF1 polyprotein in the following order: N-terminal protein; nucleoside triphosphatase; 20-k

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5

Ferracci, Géraldine, Raymond Miquelis, Shunji Kozaki, Michael Seagar, and Christian Lévêque. "Synaptic vesicle chips to assay botulinum neurotoxins." Biochemical Journal 391, no. 3 (2005): 659–66. http://dx.doi.org/10.1042/bj20050855.

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BoNTs (botulinum neurotoxins), considered to be the most toxic of all biological substances, inhibit neurotransmission through proteolytic cleavage of SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) proteins [VAMP (vesicle-associated membrane protein, or synaptobrevin), SNAP-25 (25 kDa synaptosome-associated protein) or syntaxin]. Expansion in the use of BoNTs as therapeutic and cosmetic agents, and the potential threat they constitute as biological weapons, underlines the need for rapid and sensitive in vitro assays. Here, we present new automatized bioassays to

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6

Amberg, Sean M., and Charles M. Rice. "Mutagenesis of the NS2B-NS3-Mediated Cleavage Site in the Flavivirus Capsid Protein Demonstrates a Requirement for Coordinated Processing." Journal of Virology 73, no. 10 (1999): 8083–94. http://dx.doi.org/10.1128/jvi.73.10.8083-8094.1999.

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ABSTRACT Analysis of flavivirus polyprotein processing has revealed the presence of a substrate for the virus-encoded NS2B-NS3 protease at the carboxy-terminal end of the C (capsid or core) protein. Cleavage at this site has been implicated in the efficient generation of the amino terminus of prM via signal peptidase cleavage. Yellow fever virus has four basic residues (Arg-Lys-Arg-Arg) in the P1 through P4 positions of this cleavage site. Multiple alanine substitutions were made for these residues in order to investigate the substrate specificity and biological significance of this cleavage.

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7

Russell, Anthony G., Holger Ebhardt, and Patrick P. Dennis. "Substrate Requirements for a Novel Archaeal Endonuclease That Cleaves Within the 5′ External Transcribed Spacer of Sulfolobus acidocaldarius Precursor rRNA." Genetics 152, no. 4 (1999): 1373–85. http://dx.doi.org/10.1093/genetics/152.4.1373.

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Abstract During ribosome biogenesis in the hyperthermophilic archaeon Sulfolobus acidocaldarius, at least three separate precursor endonucleolytic cleavages occur within the 144-nucleotide-long 5′ external transcribed spacer (5′ ETS) region of the rRNA operon primary transcript. The 5′ ETS sequence contains three regions of very stable helical structure. One cleavage (5′ to position -98) is in the single-stranded region between the 5′ and the central helical domains; a second cleavage (5′ to position -31) is in the single-stranded region between the central and the 3′ helical domains; and a th

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8

Damodaran, Suresh, Sajag Adhikari, Marie Turner, and Senthil Subramanian. "Quantitative Amplification of Cleaved Ends (qACE) to assay miRNA-directed target cleavage." F1000Research 3 (July 1, 2015): 240. http://dx.doi.org/10.12688/f1000research.5266.2.

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microRNA (miRNA) regulation is crucial to achieve precise spatio-temporal expression patterns of their target genes. This makes it crucial to determine the levels of cleavage of a particular target mRNA in different tissues and under different conditions. We developed a quantitative PCR method “quantitative Amplification of Cleaved Ends (qACE)” to assay levels of specific cleavage products in order to determine the extent of miRNA-directed target cleavage of a specific target gene. qACE uses cDNA generated from adapter-ligated RNA molecules and relies on a carefully designed fusion primer that

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9

Nakajima, Yuto, Keiji Nogami, Hiroaki Minami, Kana Sasai, and Midori Shima. "Residues 1680-1684 in the A3 Domain of Factor VIII Contain a Novel Thrombin-Interactive Site Responsible for Proteolytic Cleavage at Arg1689." Blood 134, Supplement_1 (2019): 1107. http://dx.doi.org/10.1182/blood-2019-129313.

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Thrombin-catalyzed activation of factor (F)VIII by proteolytic cleavages at Arg372, Arg740, and Arg1689 is essential for the propagation phase of blood coagulation cascade. Activated FVIII (FVIIIa) forms the tenase complex and markedly amplifies FX activation as a cofactor of FIXa. We previously reported that thrombin interacts with FVIII through the A2 domain (residues 392-394 and 484-509) and C2 domain, and these interactions governed the cleavages at Arg740, Arg372 and Arg1689, respectively (Nogami, JBC 2000, 2005, BJH 2008). A previous report suggested, however, that FVIII lacking the C2 d

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10

Damodaran, Suresh, Sajag Adhikari, Marie Turner, and Senthil Subramanian. "Quantitative Amplification of Cleaved Ends (qACE) to assay miRNA-directed target cleavage." F1000Research 3 (October 9, 2014): 240. http://dx.doi.org/10.12688/f1000research.5266.1.

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microRNA (miRNA) regulation is crucial to achieve precise spatio-temporal expression patterns of their target genes. This makes it crucial to determine the levels of cleavage of a particular target mRNA in different tissues and under different conditions. We developed a quantitative PCR method “quantitative Amplification of Cleaved Ends (qACE)” to assay levels of specific cleavage products in order to determine the extent of miRNA regulation for a specific target gene. qACE uses cDNA generated from adapter-ligated RNA molecules and relies on a carefully designed fusion primer that spans the ad

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11

Pruss, Cynthia M., Carol A. Hegadorn, Colleen R. P. Notley, Rouzbeh Chegeni, Aly S. Dhala, and David Lillicrap. "Human von Willebrand Disease Mutations in Mouse von Willebrand Factor Alter Its Cleavage by ADAMTS13." Blood 110, no. 11 (2007): 2708. http://dx.doi.org/10.1182/blood.v110.11.2708.2708.

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Abstract Von Willebrand Factor (VWF) is a large multimeric glycoprotein that mediates platelet adhesion to the damaged blood vessel wall and subsequent platelet aggregation at the site of vascular injury. The size of VWF multimers in plasma is regulated by the specific VWF cleaving protease, ADAMTS13, that cleaves VWF at the Y1605-M1606 bond in the VWF A2 domain. The adhesive properties of VWF is directly related to the multimer size, with loss of high molecular weight VWF leading to the bleeding phenotype in Type 2A von Willebrand disease (VWD) and ultra high molecular weight VWF multimers ob

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12

Leichnitz, S., J. Heinrich, and N. Kulak. "A fluorescence assay for the detection of hydrogen peroxide and hydroxyl radicals generated by metallonucleases." Chemical Communications 54, no. 95 (2018): 13411–14. http://dx.doi.org/10.1039/c8cc06996d.

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ROS quench assays for metal-based DNA cleavage show low selectivity and reliability – a fluorogenic assay was thus developed to reliably, selectively and sensitively detect H<sub>2</sub>O<sub>2</sub> and HO˙.

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13

Rawson, Jonathan M. O., Alice Duchon, Olga A. Nikolaitchik, Vinay K. Pathak, and Wei-Shau Hu. "Development of a Cell-Based Luciferase Complementation Assay for Identification of SARS-CoV-2 3CLpro Inhibitors." Viruses 13, no. 2 (2021): 173. http://dx.doi.org/10.3390/v13020173.

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The 3C-like protease (3CLpro) of SARS-CoV-2 is considered an excellent target for COVID-19 antiviral drug development because it is essential for viral replication and has a cleavage specificity distinct from human proteases. However, drug development for 3CLpro has been hindered by a lack of cell-based reporter assays that can be performed in a BSL-2 setting. Current efforts to identify 3CLpro inhibitors largely rely upon in vitro screening, which fails to account for cell permeability and cytotoxicity of compounds, or assays involving replication-competent virus, which must be performed in a

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14

De Araujo Herculano, Bruno, Zhe Wang та Weihong Song. "A Novel Cell-based β-secretase Enzymatic Assay for Alzheimer’s Disease". Current Alzheimer Research 16, № 2 (2019): 128–34. http://dx.doi.org/10.2174/1567205016666181212151540.

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Background: Deposition of the amyloid β protein (Aβ) into neuritic plaques is the neuropathological hallmark of Alzheimer’s Disease (AD). Aβ is generated through the cleavage of the Amyloid Precursor Protein (APP) by β-secretase and γ-secretase. Currently, the evaluation of APP cleavage by β-secretase in experimental settings has largely depended on models that do not replicate the physiological conditions of this process. Objective: To establish a novel live cell-based β-secretase enzymatic assay utilizing a novel chimeric protein that incorporates the natural sequence of APP and more closely

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15

Calvo-Garrido, Javier, Sergio Carilla-Latorre, Ana Mesquita, and Ricardo Escalante. "A proteolytic cleavage assay to monitor autophagy inDictyostelium discoideum." Autophagy 7, no. 9 (2011): 1063–68. http://dx.doi.org/10.4161/auto.7.9.16629.

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16

Corcoran, Robert, Marc Labelle, Anthony W. Czarnik, and Ronald Breslow. "An assay to determine the kinetics of RNA cleavage." Analytical Biochemistry 144, no. 2 (1985): 563–68. http://dx.doi.org/10.1016/0003-2697(85)90154-x.

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17

Nuss, Jonathan E., Gordon Ruthel, Lyal E. Tressler, et al. "Development of Cell-Based Assays to Measure Botulinum Neurotoxin Serotype A Activity Using Cleavage-Sensitive Antibodies." Journal of Biomolecular Screening 15, no. 1 (2009): 42–51. http://dx.doi.org/10.1177/1087057109354779.

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Botulinum neurotoxins (BoNTs) are zinc-metalloproteases that cleave components of the SNARE (soluble N-ethylmaleimidesensitive factor attachment protein receptor) protein complex, inhibiting acetylcholine release into neuromuscular junctions, resulting in flaccid paralysis and eventual death. The potential for the malicious misuse of these toxins as bioweapons has created an urgent need to develop effective therapeutic countermeasures. Robust cell-based assays will be essential for lead identification and the optimization of therapeutic candidates. In this study, the authors developed novel Bo

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18

Jung, Soo Bin, Chae young Lee, Kwang-Ho Lee, Kyu Heo, and Si Ho Choi. "A cleavage-based surrogate reporter for the evaluation of CRISPR–Cas9 cleavage efficiency." Nucleic Acids Research 49, no. 15 (2021): e85-e85. http://dx.doi.org/10.1093/nar/gkab467.

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Abstract CRISPR–Cas9 is a powerful tool for genome engineering, but its efficiency largely depends on guide RNA (gRNA). There are multiple methods available to evaluate the efficiency of gRNAs, including the T7E1 assay, surveyor nuclease assay, deep sequencing, and surrogate reporter systems. In the present study, we developed a cleavage-based surrogate that we have named the LacI-reporter to evaluate gRNA cleavage efficiency. The LacI repressor, under the control of the EF-1α promoter, represses luciferase or EGFP reporter expression by binding to the lac operator. Upon CRISPR–Cas9 cleavage a

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19

Zhang, Kaixiang, Ruijie Deng, Yue Li, Ling Zhang, and Jinghong Li. "Cas9 cleavage assay for pre-screening of sgRNAs using nicking triggered isothermal amplification." Chemical Science 7, no. 8 (2016): 4951–57. http://dx.doi.org/10.1039/c6sc01355d.

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A novel Cas9 cleavage assay was developed for quantitative evaluation of Cas9 cleavage efficiency and pre-screening of sgRNA to achieve highly specific and highly efficient CRISPR/Cas9 genome editing.

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20

Barhoover, Melissa A., and Michael Kalafatis. "Clevage at Both Arg306 and Arg506 Are Required for Timely and Efficient Inactivation of Factor Va by Activated Protein C: Influence of the Assay." Blood 114, no. 22 (2009): 2121. http://dx.doi.org/10.1182/blood.v114.22.2121.2121.

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Abstract Abstract 2121 Poster Board II-98 The coagulation cascade culminates in the production of thrombin from prothrombin by the prothrombinase complex (factor Xa (fXa), factor Va (fVa), Ca2+, on a membrane surface). Once enough thrombin is produced to stop bleeding when there is an injury to the vasculature, down-regulation of the coagulation cascade begins. Thrombin is a procoagulant (promotes clotting by aiding in the formation of the fibrin plug) and also an anti-coagulant (initiates the down-regulation of the coagulation cascade). The anti-coagulant function of thrombin is to activate p

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21

Børglum Jensen, Tine, Linda Hilsted, and Jens F. Rehfeld. "Library of Sequence-specific Radioimmunoassays for Human Chromogranin A." Clinical Chemistry 45, no. 4 (1999): 549–60. http://dx.doi.org/10.1093/clinchem/45.4.549.

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Abstract Background: Human chromogranin A (CgA) is an acidic protein widely expressed in neuroendocrine tissue and tumors. The extensive tissue- and tumor-specific cleavages of CgA at basic cleavage sites produce multiple peptides. Methods: We have developed a library of RIAs specific for different epitopes, including the NH2 and COOH termini and three sequences adjacent to dibasic sites in the remaining part of CgA. Results: The antisera raised against CgA(210–222) and CgA(340–348) required a free NH2 terminus for binding. All antisera displayed high titers, high indexes of heterogeneity (∼1.

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22

Kalafatis, M., PE Haley, D. Lu, RM Bertina, GL Long, and KG Mann. "Proteolytic events that regulate factor V activity in whole plasma from normal and activated protein C (APC)-resistant individuals during clotting: an insight into the APC-resistance assay." Blood 87, no. 11 (1996): 4695–707. http://dx.doi.org/10.1182/blood.v87.11.4695.bloodjournal87114695.

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Human factor V is activated to factor Va by alpha-thrombin after cleavages at Arg709, Arg1018, and Arg1545. Factor Va is inactivated by activated protein C (APC) in the presence of a membrane surface after three sequential cleavages of the heavy chain. Cleavage at Arg506 provides for efficient exposure of the inactivating cleavages at Arg306 and Arg679. Membrane-bound factor V is also inactivated by APC after cleavage at Arg306. Resistance to APC is associated with a single nucleotide change in the factor V gene (G1691-->A) corresponding to a single amino acid substitution in the factor V m

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23

Anantacharya, Rajpurohit, Nayak D. Satyanarayan, Bhuvanesh Sukhlal Kalal, and Vinitha Ramanath Pai. "Cytotoxic, DNA Cleavage and Pharmacokinetic Parameter Study of Substituted Novel Furan C-2 Quinoline Coupled 1, 2, 4-Triazole and Its Analogs." Open Medicinal Chemistry Journal 12, no. 1 (2018): 60–72. http://dx.doi.org/10.2174/1874104501812010060.

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Background: Furan, quinoline and triazoles are known for their wide spectrum biologically active molecules. A series of novel furan C-2 quinoline and 1, 2, 4-triazole (FQT) coupled hybrids were designed and synthesized to evaluate for their DNA cleavage and cytotoxic studies. Objectives: In this work we describe the synthesis and biological evaluation of furan C-2 quinoline coupled triazoles exposed for cytotoxic and DNA cleavage study. Methods: The electrophoretic DNA cleavage studies on λ-DNA (Eco-RI/Hinda-III double digest) using agarose gelelectrophoresis and the cytotoxic activity were ca

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24

Kanjanahaluethai, Amornrat, Dalia Jukneliene, and Susan C. Baker. "Identification of the Murine Coronavirus MP1 Cleavage Site Recognized by Papain-Like Proteinase 2." Journal of Virology 77, no. 13 (2003): 7376–82. http://dx.doi.org/10.1128/jvi.77.13.7376-7382.2003.

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ABSTRACT The replicase polyprotein of murine coronavirus is extensively processed by three proteinases, two papain-like proteinases (PLPs), termed PLP1 and PLP2, and a picornavirus 3C-like proteinase (3CLpro). Previously, we established a trans-cleavage assay and showed that PLP2 cleaves the replicase polyprotein between p210 and membrane protein 1 (MP1) (A. Kanjanahaluethai and S. C. Baker, J. Virol. 74:7911-7921, 2000). Here, we report the results of our studies identifying and characterizing this cleavage site. To determine the approximate position of the cleavage site, we expressed constru

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25

Herold, Jens, Alexander E. Gorbalenya, Volker Thiel, Barbara Schelle, and Stuart G. Siddell. "Proteolytic Processing at the Amino Terminus of Human Coronavirus 229E Gene 1-Encoded Polyproteins: Identification of a Papain-Like Proteinase and Its Substrate." Journal of Virology 72, no. 2 (1998): 910–18. http://dx.doi.org/10.1128/jvi.72.2.910-918.1998.

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ABSTRACT Expression of the coronavirus gene 1-encoded polyproteins, pp1a and pp1ab, is linked to a series of proteolytic events involving virus-encoded proteinases. In this study, we used transfection and immunoprecipitation assays to show that the human coronavirus 229E-encoded papain-like cysteine proteinase, PCP1, is responsible for the release of an amino-terminal protein, p9, from the gene 1-encoded polyproteins. The same protein, p9, has also been identified in virus-infected cells. Furthermore, using an in vitrotrans-cleavage assay, we defined the proteolytic cleavage site at the carbox

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26

Karvinen, Jarkko, Pertti Hurskainen, Sujatha Gopalakrishnan, David Burns, Usha Warrior, and Ilkka Hemmilä. "Homogeneous Time-Resolved Fluorescence Quenching Assay (LANCE) for Caspase-3." Journal of Biomolecular Screening 7, no. 3 (2002): 223–31. http://dx.doi.org/10.1177/108705710200700306.

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In addition to kinases and G protein—coupled receptors, proteases are one of the main targets in modern drug discovery. Caspases and viral proteases, for instance, are potential targets for new drugs. To satisfy the current need for fast and sensitive high-throughput screening for inhibitors, new homogeneous protease assays are needed. We used a caspase-3 assay as a model to develop a homogeneous time-resolved fluorescence quenching assay technology. The assay utilizes a peptide labeled with both a luminescent europium chelate and a quencher. Cleavage of the peptide by caspase-3 separates the

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27

Takeyama, Masahiro, Hironao Wakabayashi, and Philip Fay. "Contribution of factor VIII light-chain residues 2007–2016 to an activated protein C-interactive site." Thrombosis and Haemostasis 109, no. 02 (2013): 187–98. http://dx.doi.org/10.1160/th12-08-0561.

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SummaryAlthough factor (F) VIIIa is inactivated by activated protein C (APC) through cleavages in the FVIII heavy chain-derived A1 (Arg336) and A2 subunits (Arg562), the FVIII light chain (LC) contributes to catalysis by binding the enzyme. ELISA-based binding assays showed that FVIII and FVIII LC bound to immobilised active site-modified APC (DEGRAPC) (apparent K d ~270 nM and 1.0 μM, respectively). Fluid-phase binding studies using fluorescence indicated an estimated K d of ~590 nM for acrylodan-labelled LC binding to DEGR-APC. Furthermore, FVIII LC effectively competed with FVIIIa in blocki

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28

Naqvi, Tabassum, Anice Lim, Riaz Rouhani, Raj Singh, and Richard M. Eglen. "Galactosidase Enzyme Fragment Complementation as a High-Throughput Screening Protease Technology." Journal of Biomolecular Screening 9, no. 5 (2004): 398–408. http://dx.doi.org/10.1177/1087057104264040.

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The authors describe a homogeneous, high-throughput screening (HTS) assay for measuring protease activity and detection of inhibitors. The assay comprises a cyclic β-galactosidase (β-gal) enzyme donor peptide (ED) containing a protease-selective cleavage sequence. Alone, the cyclic peptide is inactive, but when linearized following protease cleavage, ED complements with β-gal enzyme acceptor forming active β-gal enzyme. This then catalyzes the formation of either fluorescent or chemiluminescent products, with β-gal turnover providing a highly amplified signal, and thus an assay technology of h

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29

Rolli, Veronique, Nathalie Enjolras, Cecile Ducasse, et al. "Generation of Factor VIII Molecules Partially Resistant to Activated Protein C." Blood 104, no. 11 (2004): 1729. http://dx.doi.org/10.1182/blood.v104.11.1729.1729.

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Abstract Following a vascular injury, factor VIII (FVIII) is rapidly activated by thrombin cleavage at arginine (Arg) 372, 740 and 1689. Activated FVIII, an heterotrimer composed by the association of A1, A2 and A3-C1-C2 domains, is rapidly degraded to limit thrombosis risk. Two main phenomenons that account for the disappearence of FVIIIa consist of an intrinsic dissociation of the trimer due to the loss of A2 domain, and the cleavage of the molecule by activated protein C (APC). APC cleaves FVIIIa at Arg 336 and 562. Mutant FVIII molecules were already generated, with one or two arginines su

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30

Miceli, Matteo, Silvana Casati, Pietro Allevi, et al. "A New Ultrasensitive Bioluminescence-Based Method for Assaying Monoacylglycerol Lipase." International Journal of Molecular Sciences 22, no. 11 (2021): 6148. http://dx.doi.org/10.3390/ijms22116148.

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A novel bioluminescent Monoacylglycerol lipase (MAGL) substrate 6-O-arachidonoylluciferin, a D-luciferin derivative, was synthesized, physico-chemically characterized, and used as highly sensitive substrate for MAGL in an assay developed for this purpose. We present here a new method based on the enzymatic cleavage of arachidonic acid with luciferin release using human Monoacylglycerol lipase (hMAGL) followed by its reaction with a chimeric luciferase, PLG2, to produce bioluminescence. Enzymatic cleavage of the new substrate by MAGL was demonstrated, and kinetic constants Km and Vmax were dete

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31

Amano, Kagehiro, Donna Michnick, Micheline Moussalli, and Randal Kaufman. "Mutation at either Arg336 or Arg562 in Factor VIII Is Insufficient for Complete Resistance to Activated Protein C (APC)-mediated Inactivation: Implications for the APC Resistance Test." Thrombosis and Haemostasis 79, no. 03 (1998): 557–63. http://dx.doi.org/10.1055/s-0037-1614944.

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SummaryActivated protein C (APC)-mediated inactivation of factor VIII (FVIII) correlates with cleavage at either Arg336 and/or Arg562. To elucidate the APC cleavage requirements for inactivation of FVIII, APC cleavage site mutants in FVIII (R336I, R562K and R336I/R562K) were made by site-directed mutagenesis. Analysis of these FVIII mutants expressed in COS-1 monkey cells demonstrated the thrombin-cleaved mutant R562K was resistant to APC cleavage at residue 562 but not at Arg336 and the thrombin cleaved mutant R336I was mostly resistant to APC cleavage at residue 336, but was sensitive to APC

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32

Smith, Maria, Kenneth Smith, Alan Olstein, Andrew Oleinikov, and Andrey Ghindilis. "Restriction Endonuclease-Based Assays for DNA Detection and Isothermal Exponential Signal Amplification." Sensors 20, no. 14 (2020): 3873. http://dx.doi.org/10.3390/s20143873.

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Application of restriction endonuclease (REase) enzymes for specific detection of nucleic acids provides for high assay specificity, convenience and low cost. A direct restriction assay format is based on the specific enzymatic cleavage of a target–probe hybrid that is accompanied with the release of a molecular marker into the solution, enabling target quantification. This format has the detection limit in nanomolar range. The assay sensitivity is improved drastically to the attomolar level by implementation of exponential signal amplification that is based on a cascade of self-perpetuating r

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33

Podolska, Katerina, David Sedlak, Petr Bartunek, and Petr Svoboda. "Fluorescence-Based High-Throughput Screening of Dicer Cleavage Activity." Journal of Biomolecular Screening 19, no. 3 (2013): 417–26. http://dx.doi.org/10.1177/1087057113497400.

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Production of small RNAs by ribonuclease III Dicer is a key step in microRNA and RNA interference pathways, which employ Dicer-produced small RNAs as sequence-specific silencing guides. Further studies and manipulations of microRNA and RNA interference pathways would benefit from identification of small-molecule modulators. Here, we report a study of a fluorescence-based in vitro Dicer cleavage assay, which was adapted for high-throughput screening. The kinetic assay can be performed under single-turnover conditions (35 nM substrate and 70 nM Dicer) in a small volume (5 µL), which makes it sui

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Eleraky, Nasser Z., Stephen A. Kania, James F. Evermann, and Leon N. D. Potgieter. "Comparison of Targeting F and G Protein Genes to Detect Bovine and Ovine Respiratory Syncytial Viruses." Journal of Veterinary Diagnostic Investigation 15, no. 3 (2003): 277–80. http://dx.doi.org/10.1177/104063870301500310.

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In this study, 2 reverse transcriptase–polymerase chain reaction (RT-PCR) assays were developed and compared for simultaneous detection of bovine and ovine respiratory syncytial viruses (RSVs). One assay was based on a set of primers, which amplified a 426-bp fragment of either bovine or ovine RSV F gene (RT-PCR F). The F products could be distinguished by EcoRI or BstYI restriction endonuclease cleavage. In the other assay, a set of primers amplified a 542-bp fragment of either ovine or bovine RSV G gene (RT-PCR G). EcoO1091 and RsaI restriction enzymes were used to differentiate between the

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Ason, B. "A high-throughput assay for Tn5 Tnp-induced DNA cleavage." Nucleic Acids Research 32, no. 10 (2004): e83-e83. http://dx.doi.org/10.1093/nar/gnh080.

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Baroso, Rémi, Pauline Sellier, Federica Defendi, et al. "Kininogen Cleavage Assay: Diagnostic Assistance for Kinin-Mediated Angioedema Conditions." PLOS ONE 11, no. 9 (2016): e0163958. http://dx.doi.org/10.1371/journal.pone.0163958.

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Eis, Peggy S., Marilyn C. Olson, Tsetska Takova, et al. "An invasive cleavage assay for direct quantitation of specific RNAs." Nature Biotechnology 19, no. 7 (2001): 673–76. http://dx.doi.org/10.1038/90290.

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Lu, Manchun, Michael R. Shortreed, Jeff G. Hall, et al. "A surface invasive cleavage assay for highly parallel SNP analysis." Human Mutation 19, no. 4 (2002): 416–22. http://dx.doi.org/10.1002/humu.10071.

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Ausländer, Simon, David Fuchs, Samuel Hürlemann, David Ausländer, and Martin Fussenegger. "Engineering a ribozyme cleavage-induced split fluorescent aptamer complementation assay." Nucleic Acids Research 44, no. 10 (2016): e94-e94. http://dx.doi.org/10.1093/nar/gkw117.

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Dexheimer, Thomas S., and Yves Pommier. "DNA cleavage assay for the identification of topoisomerase I inhibitors." Nature Protocols 3, no. 11 (2008): 1736–50. http://dx.doi.org/10.1038/nprot.2008.174.

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Gambino, Stefano, Briana Mousley, Lindsay Cathcart, Janelle Winship, Joseph J. Loparo, and Allen C. Price. "A single molecule assay for measuring site-specific DNA cleavage." Analytical Biochemistry 495 (February 2016): 3–5. http://dx.doi.org/10.1016/j.ab.2015.11.013.

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Meng, Xiang Xian, Xiao Hai Yang, Ke Min Wang, Wei Hong Tan, and Qiu Ping Guo. "Real-time monitoring of DNAzyme cleavage process using fluorescent assay." Chinese Chemical Letters 20, no. 8 (2009): 990–94. http://dx.doi.org/10.1016/j.cclet.2009.03.028.

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Bao, Yu, Simone Marini, Takeyuki Tamura, et al. "Toward more accurate prediction of caspase cleavage sites: a comprehensive review of current methods, tools and features." Briefings in Bioinformatics 20, no. 5 (2018): 1669–84. http://dx.doi.org/10.1093/bib/bby041.

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AbstractAs one of the few irreversible protein posttranslational modifications, proteolytic cleavage is involved in nearly all aspects of cellular activities, ranging from gene regulation to cell life-cycle regulation. Among the various protease-specific types of proteolytic cleavage, cleavages by casapses/granzyme B are considered as essential in the initiation and execution of programmed cell death and inflammation processes. Although a number of substrates for both types of proteolytic cleavage have been experimentally identified, the complete repertoire of caspases and granzyme B substrate

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Laajala, Mira, Minna M. Hankaniemi, Juha A. E. Määttä, Vesa P. Hytönen, Olli H. Laitinen, and Varpu Marjomäki. "Host Cell Calpains Can Cleave Structural Proteins from the Enterovirus Polyprotein." Viruses 11, no. 12 (2019): 1106. http://dx.doi.org/10.3390/v11121106.

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Enteroviruses are small RNA viruses that cause diseases with various symptoms ranging from mild to severe. Enterovirus proteins are translated as a single polyprotein, which is cleaved by viral proteases to release capsid and nonstructural proteins. Here, we show that also cellular calpains have a potential role in the processing of the enteroviral polyprotein. Using purified calpains 1 and 2 in an in vitro assay, we show that addition of calpains leads to an increase in the release of VP1 and VP3 capsid proteins from P1 of enterovirus B species, detected by western blotting. This was prevente

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Eriksson, Anna, and Mari Norgren. "Cleavage of Antigen-Bound Immunoglobulin G by SpeB Contributes to Streptococcal Persistence in Opsonizing Blood." Infection and Immunity 71, no. 1 (2003): 211–17. http://dx.doi.org/10.1128/iai.71.1.211-217.2003.

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ABSTRACT Group A streptococci (GAS) express a superantigen, SpeB, having cysteine protease activity. SpeB exhibits several properties that might contribute to virulence, the most recently discovered being the ability to cleave immunoglobulin G (IgG) in a manner similar to that of papain. In the present study, we confirmed this latter finding and found that the irreversible inhibition of SpeB protease activity completely abolishes IgG cleavage. SpeB cleavage of IgG was not species restricted since SpeB cleaved both human, rabbit, and mouse IgG. In order to investigate the nature of the SpeB cle

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Gutierrez, Pedro A., Kirk Baughman, Yadong Sun, and Liang Tong. "A real-time fluorescence assay for CPSF73, the nuclease for pre-mRNA 3′-end processing." RNA 27, no. 10 (2021): 1148–54. http://dx.doi.org/10.1261/rna.078764.121.

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CPSF73 is the endonuclease that catalyzes the cleavage reaction for 3′-end processing of mRNA precursors (pre-mRNAs) in two distinct machineries, a canonical machinery for the majority of pre-mRNAs and a U7 snRNP (U7 machinery) for replication-dependent histone pre-mRNAs in animal cells. CPSF73 also possesses 5′–3′ exonuclease activity in the U7 machinery, degrading the downstream cleavage product after the endonucleolytic cleavage. Recent studies show that CPSF73 is a potential target for developing anticancer, antimalarial, and antiprotozoal drugs, spurring interest in identifying new small-

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Gale, Andrew J., Diana Rozenshteyn, and Justin Riceberg. "The Neutrophil Proteases, Elastase and Cathepsin G, May Modulate the Activity of Factor VIII." Blood 108, no. 11 (2006): 1716. http://dx.doi.org/10.1182/blood.v108.11.1716.1716.

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Abstract Neutrophils and monocytes express cathepsin G and elastase and also can bind to activated platelets, thus they can be localized to the site of active coagulation. Early studies suggested that cathepsin G and elastase inactivated factor VIII (FVIII) and were thus anticoagulant. But other studies have suggested procoagulant functions for cathepsin G and elastase in activation of factor V or activation of platelets among other possible mechanisms. Therefore, we investigated the effects of human neutrophil elastase and human neutrophil cathepsin G on FVIII/VIIIa. Elastase does inactivate

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Shan, Yu-Fei, and Gen-Jun Xu. "Study on Substrate Specificity at Subsites for Severe Acute Respiratory Syndrome Coronavirus 3CL Protease." Acta Biochimica et Biophysica Sinica 37, no. 12 (2005): 807–13. http://dx.doi.org/10.1111/j.1745-7270.2005.00114.x.

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AbstractAutocleavage assay and peptide-based cleavage assay were used to study the substrate specificity of 3CL protease from the severe acute respiratory syndrome coronavirus. It was found that the recognition between the enzyme and its substrates involved many positions in the substrate, at least including residues from P4 to P2′. The deletion of either P4 or P2′ residue in the substrate would decrease its cleavage efficiency dramatically. In contrast to the previous suggestion that only small residues in substrate could be accommodated to the S1′ subsite, we have found that bulky residues s

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Giuliani, Isabelle, François Rieunier, Catherine Larue, et al. "Assay for Measurement of Intact B-Type Natriuretic Peptide Prohormone in Blood." Clinical Chemistry 52, no. 6 (2006): 1054–61. http://dx.doi.org/10.1373/clinchem.2005.061770.

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Abstract Background: B-Type natriuretic peptide (BNP1–32) as well as the N-terminal fragment of the prohormone containing residues 1–76 (NT-proBNP1–76), both cleavage products of the precursor proBNP1–108, are reported to be powerful markers for prognosis and risk stratification of heart failure. However, the intact precursor also circulates in the bloodstream. Assays for the detection of these cleavage products have been developed, but most of these assays may overestimate the concentrations of the cleavage products because they also measure the precursor form. It is therefore important to de

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He, Chuan, Jinli Zhang, Wei Li, and Yan Fu. "Engineering oligonucleotide-based peroxidase mimetics for the colorimetric assay of S1 nuclease." Analytical Methods 10, no. 12 (2018): 1405–12. http://dx.doi.org/10.1039/c7ay02830j.

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