Development and Implementation of a 384-Well Homogeneous Fluorescence Intensity High-Throughput Screening Assay to Identify Mitogen-Activated Protein Kinase Phosphatase-1 Dual-Specificity Protein Phosphatase Inhibitors

2007 ◽  
Vol 5 (3) ◽  
pp. 319-332 ◽  
Author(s):  
Paul A. Johnston ◽  
Caleb A. Foster ◽  
Tong Ying Shun ◽  
John J. Skoko ◽  
Sunita Shinde ◽  
...  
Keyword(s):  
High Throughput Screening ◽  
Mitogen Activated Protein Kinase ◽  
Screening Assay ◽  
Phosphatase Inhibitors ◽  
Dual Specificity ◽  
High Throughput Screening Assay ◽  
Protein Phosphatase Inhibitors ◽  
Mitogen Activated Protein ◽  
Dual Specificity Protein Phosphatase ◽  
Specificity Protein

scholarly journals Structure ofMycobacterium smegmatisEis in complex with paromomycin

2014 ◽  
Vol 70 (9) ◽  
pp. 1173-1179 ◽  
Author(s):  
Kyoung Hoon Kim ◽  
Doo Ri An ◽  
Hye Jin Yoon ◽  
Jin Kuk Yang ◽  
Se Won Suh
Keyword(s):  
Crystal Structure ◽  
Aminoglycoside Antibiotic ◽  
Intracellular Survival ◽  
Mitogen Activated Protein Kinase ◽  
Host Immune Responses ◽  
Dual Specificity ◽  
Mitogen Activated Protein ◽  
Dual Specificity Protein Phosphatase ◽  
High Level ◽  
Specificity Protein

TheRv2416cgene ofMycobacterium tuberculosis(Mtb) encodes the enhanced intracellular survival (Eis) protein that enhances intracellular survival of the pathogen in host macrophages during infection. TheMtbEis protein is released into the cytoplasm of the phagocyte during intracellular infection and modulates the host immune response. It also contributes to drug resistance by acetylating multiple amine groups of aminoglycosides. Interestingly, the nonpathogenicM. smegmatis(Msm) contains a homologouseisgene (MSMEG_3513). The overall structures ofMtbEis andMsmEis are highly similar to each other, reflecting the high level (58%) of amino-acid sequence identity between them. BothMtbEis andMsmEis are active as aminoglycoside acetyltransferases, while onlyMtbEis functions as anN∊-acetyltransferase to acetylate Lys55 of dual-specificity protein phosphatase 16 (DUSP16)/mitogen-activated protein kinase phosphatase 7 (MKP-7), leading to the suppression of host immune responses. Here, the crystal structure ofMsmEis in the paromomycin-bound form is reported, revealing detailed interactions between an aminoglycoside antibiotic andMsmEis. The crystal structure ofMsmEis in the paromomycin-bound form has been determined at 3.3 Å resolution. This work provides potentially useful information for structure-guided discovery of Eis inhibitors as a novel antituberculosis drug against drug-resistantMtb.

scholarly journals Expression, purification and characterization of recombinant mitogen-activated protein kinase kinases

1994 ◽  
Vol 303 (1) ◽  
pp. 105-112 ◽  
Author(s):  
P Dent ◽  
Y H Chow ◽  
J Wu ◽  
D K Morrison ◽  
R Jove ◽  
...  
Keyword(s):  
Peptide Mapping ◽  
Mitogen Activated Protein Kinase ◽  
Recombinant Viruses ◽  
Dual Specificity ◽  
Protein Map ◽  
Mitogen Activated Protein ◽  
Michaelis Constants ◽  
High Level ◽  
Specificity Protein

Mitogen-activated protein (MAP) kinase kinases (MKKs) are dual-specificity protein kinases which activate p42mapk and p44mapk by phosphorylation of regulatory tyrosine and threonine residues. cDNAs for two isotypes of MKK, MKK1 and MKK2, have been isolated from several species. Here we describe construction of recombinant baculoviruses for high-level expression of histidine-tagged rat MKK1 and MKK2, and procedures for production of nearly homogeneous MKK1 and MKK2 fusion proteins, in both inactive and active forms. Co-infection of Sf9 cells with either MKK1 or MKK2 virus together with recombinant viruses for Raf-1, pp60src (Y527F) and c-Ha-Ras resulted in activations of 250-fold and 150-fold for MKK1 and MKK2 respectively. Specific activities towards kinase-defective p42mapk were of the order of several hundred nanomoles of phosphate transferred/min per mg of MKK protein. The Michaelis constants for both enzymes were approx. 1 microM. Preparations of activated MKK were apparently free of Raf-1 as assessed by Western blotting. Raf-1 phosphorylated MKK1 on one major tryptic phosphopeptide, the phosphorylation of which increased with time. This phosphopeptide contained only phosphoserine and possessed neutral overall charge at pH 1.9 on two-dimensional peptide mapping. Phosphorylation of MKK1 by Raf-1 correlated with activation and reached a plateau of approximately 2 mol/mol.

SIRT2 is involved in cisplatin-induced acute kidney injury through regulation of mitogen-activated protein kinase phosphatase-1

2020 ◽  
Vol 35 (7) ◽  
pp. 1145-1156 ◽  
Author(s):  
Yu Jin Jung ◽  
Woong Park ◽  
Kyung Pyo Kang ◽  
Won Kim
Keyword(s):  
Protein Kinase ◽  
Epithelial Cells ◽  
Renal Injury ◽  
Kidney Injury ◽  
Mitogen Activated Protein Kinase ◽  
Tubular Epithelial Cells ◽  
Dual Specificity ◽  
Mitogen Activated Protein ◽  
Proximal Tubular ◽  
Dual Specificity Protein Phosphatase

Abstract Background Activation of mitogen-activated protein kinase phosphatase-1 (MKP-1), a dual-specificity protein phosphatase, regulates mitogen-activated protein kinase signaling. C-Jun N-terminal kinase (JNK) and p38 are activated in cisplatin-induced renal injury. However, the change of MKP-1 expression in cisplatin-induced renal injury and the regulatory effect of sirtuin 2 (SIRT2), a nicotinamide adenine dinucleotide–dependent deacetylase, on MKP-1 remains unknown. Methods To address these issues, we used constitutional Sirt2 knockout (KO) mice, transgenic (TG) mice with increased expression of SIRT2 specifically in proximal tubular epithelial cellsand wild-type (WT) mice. Cisplatin nephrotoxicity was induced by intraperitoneal injection of cisplatin. Results MKP-1 expression in the kidney was decreased after cisplatin treatment. Cisplatin-induced downregulation of MKP-1 was reversed in Sirt2 KO mice kidney and further decreased in Sirt2 TG mice kidney. We observed similar phenomenon with SIRT2-knockdown or SIRT2-overexpressed tubular epithelial cells. Phosphorylation of p38 and JNK, a downstream signal pathway of MKP-1, increased in WT mice kidney following treatment with cisplatin. A decrease in SIRT2 suppressed cisplatin-induced phosphorylation of p38 and JNK in kidney and tubular epithelial cells. Overexpression of SIRT2 further increased phosphorylation of p38 and JNK in kidney and tubular epithelial cells. Acetylation of MKP-1 was significantly increased in SIRT2-knockdown cells and decreased in SIRT2-overexpressed cells after cisplatin stimulation. Sirt2 KO mice and Sirt2 TG mice showed amelioration and aggravation of renal injury, apoptosis, necroptosis and inflammation induced by cisplatin. Conclusion Our data show that SIRT2 is associated with cisplatin-induced renal injury through regulation of MKP-1 expression.

scholarly journals Dual-Specificity Phosphatase Regulation in Neurons and Glial Cells

2019 ◽  
Vol 20 (8) ◽  
pp. 1999 ◽  
Author(s):  
Pérez-Sen ◽  
Queipo ◽  
Gil-Redondo ◽  
Ortega ◽  
Gómez-Villafuertes ◽  
...  
Keyword(s):  
Map Kinases ◽  
Protein Phosphatases ◽  
Mapk Signaling ◽  
Protein Stabilization ◽  
Mitogen Activated Protein Kinase ◽  
Brain Diseases ◽  
General Terms ◽  
Dual Specificity ◽  
Mitogen Activated Protein ◽  
Specificity Protein

Dual-specificity protein phosphatases comprise a protein phosphatase subfamily with selectivity towards mitogen-activated protein (MAP) kinases, also named MKPs, or mitogen-activated protein kinase (MAPK) phosphatases. As powerful regulators of the intensity and duration of MAPK signaling, a relevant role is envisioned for dual-specificity protein phosphatases (DUSPs) in the regulation of biological processes in the nervous system, such as differentiation, synaptic plasticity, and survival. Important neural mediators include nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF) that contribute to DUSP transcriptional induction and post-translational mechanisms of DUSP protein stabilization to maintain neuronal survival and differentiation. Potent DUSP gene inducers also include cannabinoids, which preserve DUSP activity in inflammatory conditions. Additionally, nucleotides activating P2X7 and P2Y13 nucleotide receptors behave as novel players in the regulation of DUSP function. They increase cell survival in stressful conditions, regulating DUSP protein turnover and inducing DUSP gene expression. In general terms, in the context of neural cells exposed to damaging conditions, the recovery of DUSP activity is neuroprotective and counteracts pro-apoptotic over-activation of p38 and JNK. In addition, remarkable changes in DUSP function take place during the onset of neuropathologies. The restoration of proper DUSP levels and recovery of MAPK homeostasis underlie the therapeutic effect, indicating that DUSPs can be relevant targets for brain diseases.

scholarly journals Recurrent Somatic MAP2K1 Mutations in Papillary Thyroid Cancer and Colorectal Cancer

2021 ◽  
Vol 11 ◽  
Author(s):  
Rong Bu ◽  
Abdul K. Siraj ◽  
Tariq Masoodi ◽  
Sandeep Kumar Parvathareddy ◽  
Kaleem Iqbal ◽  
...  
Keyword(s):  
Colorectal Cancer ◽  
Thyroid Cancer ◽  
Protein Kinase ◽  
Papillary Thyroid Cancer ◽  
Middle Eastern ◽  
Mitogen Activated Protein Kinase ◽  
Papillary Thyroid ◽  
Dual Specificity ◽  
Mitogen Activated Protein ◽  
Specificity Protein

Mitogen-activated protein kinase kinase 1 (MAP2K1) is a dual specificity protein kinase that phosphorylates both threonine and tyrosine residues in ERK. MAP2K1 mutations have been identified in several cancers. However, their role in Middle Eastern papillary thyroid cancer (PTC) and colorectal cancer (CRC) is lacking. In this study, we evaluated the prevalence of MAP2K1 mutations in a large cohort of Middle Eastern PTC and CRC using whole-exome and Sanger sequencing technology. In the discovery cohort of 100 PTC and 100 CRC cases (comprising 50 MAPK mutant and 50 MAPK wildtype cases each), we found one MAP2K1 mutation each in PTC and CRC, both of which were MAPK wildtype. We further analyzed 286 PTC and 289 CRC MAPK wildtype cases and found three MAP2K1 mutant PTC cases and two MAP2K1 mutant CRC cases. Thus, the overall prevalence of MAP2K1 mutation in MAPK wildtype cases was 1.1% (4/336) in PTC and 0.9% (3/339) in CRC. Histopathologically, three of the four MAP2K1 mutant PTC cases were follicular variant and all four tumors were unifocal with absence of extra-thyroidal extension. All the three CRC cases harboring MAP2K1 mutation were of older age (> 50 years) and had moderately differentiated stage II/III tumors located in the left colon. In conclusion, this is the first comprehensive report of MAP2K1 somatic mutations prevalence in PTC and CRC from this ethnicity. The mutually exclusive nature of MAP2K1 and MAPK mutations suggests that each of these mutation may function as an initiating mutation driving tumorigenesis through MAPK signaling pathway.

High Throughput Screening Assay to Identify Modulators of IL-17 Expression

2018 ◽  
Vol 20 (9) ◽  
pp. 804-819 ◽  
Author(s):  
Mohamed Boudjelal ◽  
Ana Maria Ruiz-Avendano ◽  
Gonzalo Colmenarejo ◽  
Sergio A. Senar-Sancho ◽  
Ashley Barnes ◽  
...  
Keyword(s):  
High Throughput ◽  
High Throughput Screening ◽  
Screening Assay ◽  
High Throughput Screening Assay

scholarly journals A high-throughput screening assay based on automated microscopy for monitoring antibiotic susceptibility of Mycobacterium tuberculosis phenotypes

2021 ◽  
Vol 21 (1) ◽  
Author(s):  
Sadaf Kalsum ◽  
Blanka Andersson ◽  
Jyotirmoy Das ◽  
Thomas Schön ◽  
Maria Lerm
Keyword(s):  
Mycobacterium Tuberculosis ◽  
High Throughput ◽  
High Throughput Screening ◽  
Live Cell Imaging ◽  
Cell Imaging ◽  
Imaging System ◽  
Aggregate Size ◽  
Live Cell ◽  
Screening Assay ◽  
High Throughput Screening Assay

Abstract Background Efficient high-throughput drug screening assays are necessary to enable the discovery of new anti-mycobacterial drugs. The purpose of our work was to develop and validate an assay based on live-cell imaging which can monitor the growth of two distinct phenotypes of Mycobacterium tuberculosis and to test their susceptibility to commonly used TB drugs. Results Both planktonic and cording phenotypes were successfully monitored as fluorescent objects using the live-cell imaging system IncuCyte S3, allowing collection of data describing distinct characteristics of aggregate size and growth. The quantification of changes in total area of aggregates was used to define IC50 and MIC values of selected TB drugs which revealed that the cording phenotype grew more rapidly and displayed a higher susceptibility to rifampicin. In checkerboard approach, testing pair-wise combinations of sub-inhibitory concentrations of drugs, rifampicin, linezolid and pretomanid demonstrated superior growth inhibition of cording phenotype. Conclusions Our results emphasize the efficiency of using automated live-cell imaging and its potential in high-throughput whole-cell screening to evaluate existing and search for novel antimycobacterial drugs.

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