A miniaturized peptidyl-prolyl isomerase enzyme assay

Vivoli, Mirella; Renou, Julien; Chevalier, Arnaud; Norville, Isobel H.; Diaz, Suraya; Juli, Christina; Atkins, Helen S.; Holzgrabe, Ulrike; Renard, Pierre; Sarkar‐Tyson, Mitali; Harmer, Nicholas J.

doi:10.1016/j.ab.2017.08.004

Cited by 11 publications

(10 citation statements)

References 63 publications

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“…This assay was first described by Fischer et al, but it is not appropriate for large scale screening, due to some drawbacks like high consumption of peptide and enzyme and its high susceptibility to temperature variation. In order to develop a medium throughput assay with improved robustness and higher reproducibility, Vivoli et al optimized the assay conditions and created an improved version of PPIase assay . This involved a temperature controlled plate reader which enabled semiautomated medium throughput assays increasing the number of samples to 84, a potentially 4-fold increase in the number of assays that can be run in a single day.…”

Section: Development Of Inhibitors To the Mip Protein In Gram-negativ...mentioning

confidence: 96%

“…In order to develop a medium throughput assay with improved robustness and higher reproducibility, Vivoli et al optimized the assay conditions and created an improved version of PPIase assay. 81 This involved a temperature controlled plate reader which enabled semiautomated medium throughput assays increasing the number of samples to 84, a potentially 4-fold increase in the number of assays that can be run in a single day.…”

Section: ■ Fk506 Binding Proteins In Bacteriamentioning

confidence: 99%

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Targeting Protein Folding: A Novel Approach for the Treatment of Pathogenic Bacteria

et al. 2020

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Infectious diseases are a major cause of morbidity and mortality worldwide, exacerbated by increasing antibiotic resistance in many bacterial species. The development of drugs with new modes of action is essential. A leading strategy is antivirulence, with the aim to target bacterial proteins that are important in disease causation and progression but do not affect growth, resulting in reduced selective pressure for resistance. Immunophilins, a superfamily of peptidyl-prolyl cis–trans isomerase (PPIase) enzymes have been shown to be important for virulence in a broad-spectrum of pathogenic bacteria. This Perspective will provide an overview of the recent advances made in understanding the role of each immunophilin family, cyclophilins, FK506 binding proteins (FKBPs), and parvulins in bacteria. Inhibitor design and medicinal chemistry strategies for development of novel drugs against bacterial FKBPs will be discussed. Furthermore, drugs against human cyclophilins and parvulins will be reviewed in their current indication as antiviral and anticancer therapies.

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Section: Development Of Inhibitors To the Mip Protein In Gram-negativ...mentioning

confidence: 96%

Section: ■ Fk506 Binding Proteins In Bacteriamentioning

confidence: 99%

Targeting Protein Folding: A Novel Approach for the Treatment of Pathogenic Bacteria

et al. 2020

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“…To verify the interaction of CTS with the potential target protein FKBP1A, a coupled activity assay of this peptidyl-prolyl-cistrans-isomerase was performed [28]. FKBP1A isomerizes the cisconfigurated substrate N-succinyl-Ala-Ala-Pro-Phe p-nitroanilide (Suc-AAPF-pNA) to the trans-configurated proline peptide.…”

Section: Resultsmentioning

confidence: 99%

“…Enzymatic activity of FKBP1A [28] All reagents were stored on ice for the assay. Suc-AAPF-pNA (3 mM) in LiCl-TFE solution (20 mg/mL) were prepared freshly prior to all tests.…”

Section: Ms-based Protein Expression Analysismentioning

confidence: 99%

Cryptotanshinone from Salvia miltiorrhiza Roots Reduces Cytokeratin CK1/10 Expression in Keratinocytes by Activation of Peptidyl-prolyl-cis-trans-isomerase FKBP1A

et al. 2018

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Cryptotanshinone (CTS) (1 µM) from the roots of exerts a strong influence on the terminal differentiation of human keratinocytes (HaCaT cell line, primary natural human keratinocytes) and downregulates the expression of differentiation-specific cytokeratins CK1 and CK10 on protein and gene level. Other differentiation specific proteins as involucrin, filaggrin, loricrin, and transglutaminase were not affected to a higher extent. CTS (1 µM) did not influence the cell viability and the proliferation of keratinocytes. Using a combination of drug affinity response target stability assay in combination with a proteomic approach and multivariate statistics for target elucidation, peptidyl-prolyl-cis-trans-isomerase FKBP1A (known target of inhibitors such as tacrolimus or rapamycin) was addressed as potential molecular target of CTS. The interaction of CTS with FKBP1A was additionally shown by thermal shift and enzymatic activity assays. Interestingly, CTS served as an activator of FKBP1A, which led to a reduced activity of the TGF receptor pathway and therefore to a diminished CK1 and CK10 expression. The combination of the FKBP1A activator CTS with the inhibitor tacrolimus neutralized the effects of both compounds. From these data, a potential dermatological use of CTS and CTS-containing plant extracts (e.g., hydroalcoholic extract from the roots ) for keratinopathic ichthyosis, a disease characterized by overexpression of CK1 and CK10, is discussed. This study displays an experimental strategy for combining phytochemical aspects on active natural products with systematic identification of molecular targets on gene, protein, and cell level.

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“…Reported efforts to introduce novel peptide substrates − with better properties for such an assay have yet to yield significant success. Moreover, the physiological function of CypD is likely not the interconversion of cis and trans conformers of short peptides but that of proteins in the process of protein folding, as inferred from the functions of other cyclophilins .…”

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confidence: 99%

RNase T1 Refolding Assay for Determining Mitochondrial Cyclophilin D Activity: A Novel In Vitro Method Applicable in Drug Research and Discovery

et al. 2020

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Human cyclophilin D is a mitochondrial peptidyl-prolyl isomerase that plays a role in regulating the opening of the mitochondrial permeability transition pore. It is considered a viable and promising molecular target for the treatment of diseases for which disease development is associated with pore opening, e.g., Alzheimer’s disease or ischemia/reperfusion injury. Currently available and widely used in vitro methods based on Kofron’s assay for determining cyclophilin D activity suffer from serious drawbacks and limitations. In this study, a completely novel approach for an in vitro assay of cyclophilin D activity using RNase T1 refolding is introduced. The method is simple and is more in line with the presumed physiological role of cyclophilin D in protein folding than Kofron’s assay, which relies on a peptide substrate. The method is applicable for identifying novel inhibitors of cyclophilin D as potential drugs for the treatment of the diseases mentioned above. Moreover, the description of CypD activity in the in vitro RNase T1 refolding assay reveals new possibilities for investigating the role of cyclophilin D in protein folding in cells and may lead to a better understanding of its pathological and physiological roles.

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A miniaturized peptidyl-prolyl isomerase enzyme assay

Cited by 11 publications

References 63 publications

Targeting Protein Folding: A Novel Approach for the Treatment of Pathogenic Bacteria

Targeting Protein Folding: A Novel Approach for the Treatment of Pathogenic Bacteria

Cryptotanshinone from Salvia miltiorrhiza Roots Reduces Cytokeratin CK1/10 Expression in Keratinocytes by Activation of Peptidyl-prolyl-cis-trans-isomerase FKBP1A

RNase T1 Refolding Assay for Determining Mitochondrial Cyclophilin D Activity: A Novel In Vitro Method Applicable in Drug Research and Discovery

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