DNA-maleimide: An improved maleimide compound for electrophoresis-based titration of reactive thiols in a specific protein

Hara, Satoshi; Nojima, Tatsuya; Seio, Kohji; Yoshida, Masasuke; Hisabori, Toru

doi:10.1016/j.bbagen.2013.01.012

Cited by 15 publications

(10 citation statements)

References 31 publications

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“…After incubation for 30 min at 25°C, proteins were TCA-precipitated, labeled with AMS, and subjected to nonreducing SDS-PAGE as described earlier. Labeling of free thiols using DNA-maleimide (DNA-Mal) was performed as described previously (28).…”

Section: Methodsmentioning

confidence: 99%

Thioredoxin Selectivity for Thiol-based Redox Regulation of Target Proteins in Chloroplasts

Yoshida

Hara

Hisabori

2015

Journal of Biological Chemistry

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101

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Background: Thioredoxin (Trx) plays a pivotal role in the redox regulation of target proteins. Results: Functional diversity of chloroplast Trxs was determined by observing Trx-dependent redox shifts of several thiolmodulated enzymes in vitro and in vivo. Conclusion: Novel insights into the chloroplast redox network were provided. Significance: Our results shed light on the molecular basis of the light-responsive adjustment of chloroplast functions.

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Section: Methodsmentioning

confidence: 99%

Thioredoxin Selectivity for Thiol-based Redox Regulation of Target Proteins in Chloroplasts

Yoshida

Hara

Hisabori

2015

Journal of Biological Chemistry

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101

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“…5c). This discordance is likely due to the absence of a charge on PEG, so that PEG-labeling at multiple sites has the potential to affect the charge to mass ratio, and thus its mobility in SDS-PAGE 38 . SA or SA+TNX treatment did not affect protein band migration patterns, indicating that the redox state of cysteines in NPR1 are not affected by SA or SA+TNX treatment.…”

Section: Tnx Inhibits Sa-induced Cellular Redox Shiftmentioning

confidence: 99%

Oxicam-type nonsteroidal anti-inflammatory drugs inhibit NPR1-mediated salicylic acid pathway

Ishihama

Choi

Noutoshi

et al. 2020

Preprint

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Salicylic acid (SA) and its structural analogs are nonsteroidal anti-inflammatory drugs (NSAIDs) that target mammalian cyclooxygenases. In plants, SA acts as a defense hormone that regulates NON-EXPRESSOR OF PATHOGENESIS RELATED GENES 1 (NPR1), the master transcriptional regulator of immunity-related genes. We identified a number of NSAIDs that enhance bacterial effector-induced cell death. Among them, the oxicam-type NSAIDs tenoxicam (TNX), meloxicam, and piroxicam, but not other types of NSAIDs, exhibit an inhibitory effect on immunity to bacteria and SA-dependent immune responses in plants. TNX treatment reduces NPR1 levels, independently from the proposed SA receptors NPR3 and NPR4. Instead, TNX induces oxidation of cytosolic redox status, which is also affected by SA and regulates NPR1 homeostasis. Surprisingly, however, cysteine modification associated with NPR1 oligomerization via intermolecular disulfide bonds is not affected by either SA or TNX. Therefore, oxicam-type NSAIDs highlight importance of SA effects on the cytosolic redox status, but not on cysteine modification or oligomerization of NPR1.

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“…The expression and purification of Trxb, NTR [12], and roGFP [14] were performed as described. A gene encoding Arabidopsis thaliana methionine synthase (MetE) (AT5G17920) with his-tag appended to its N-terminus was cloned into the expression vector pET23a.…”

Section: Proteinsmentioning

confidence: 99%

“…The introduction of the maleimide moiety into 5 0 -aminohexyl PC ssDNA and the subsequent purification were performed as previously described [12], with slight modifications. After the anion exchange chromatography, the purified DNA-PCMal was desalted by ultrafiltration and then lyophilized.…”

Section: Synthesis Of Dna-pcmalmentioning

confidence: 99%

“…However, determination of the number of reduced thiols is sometimes very difficult owing to the chemical properties of these modifiers. To overcome the difficulties of these maleimide compounds, we have developed the single-stranded DNA (ssDNA)-based maleimide compound (DNA-Mal) [12]. When a protein is labeled with DNA-Mal and analyzed on SDS-PAGE, DNA-Mal shifts its mobility by an identical 9.3 kDa per molecule of DNA-Mal.…”

Section: Introductionmentioning

confidence: 99%

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Direct determination of the redox status of cysteine residues in proteins in vivo

Hara

Tatenaka

Ohuchi

et al. 2015

Biochemical and Biophysical Research Communications

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The redox states of proteins in cells are key factors in many cellular processes. To determine the redox status of cysteinyl thiol groups in proteins in vivo, we developed a new maleimide reagent, a photocleavable maleimide-conjugated single stranded DNA (DNA-PCMal). The DNA moiety of DNA-PCMal is easily removed by UV-irradiation, allowing DNA-PCMal to be used in Western blotting applications. Thereby the state of thiol groups in intracellular proteins can be directly evaluated. This new maleimide compound can provide information concerning redox proteins in vivo, which is important for our understanding of redox networks in the cell.

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DNA-maleimide: An improved maleimide compound for electrophoresis-based titration of reactive thiols in a specific protein

Cited by 15 publications

References 31 publications

Thioredoxin Selectivity for Thiol-based Redox Regulation of Target Proteins in Chloroplasts

Thioredoxin Selectivity for Thiol-based Redox Regulation of Target Proteins in Chloroplasts

Oxicam-type nonsteroidal anti-inflammatory drugs inhibit NPR1-mediated salicylic acid pathway

Direct determination of the redox status of cysteine residues in proteins in vivo

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