biam  (MedChemExpress)

Journal: The EMBO Journal

Article Title: Redox regulation of G protein oligomerization and signaling by the glutaredoxin WG1 controls grain size in rice

doi: 10.1038/s44318-025-00462-9

Figure Lengend Snippet: ( A ) Flow diagram of BIAM-labeling assay detecting the presence of active thiol groups in GS3. MBP-GS3 proteins were expressed and purified, then treated with different concentrations of H 2 O 2 and followed by incubation with the same amount of BIAM, which could react with active thiol groups (①). S-BIAM represents the thiol labeled with BIAM. BIAM-labeled MBP-GS3 was detected by an anti-Biotin antibody (②). ( B ) The BIAM-labeling assay confirmed the presence of active thiol groups in GS3. According to the flow diagram outlined in ( A ), with an increase in the concentration of H 2 O 2 , the level of BIAM-labeled MBP-GS3 proteins progressively decreased. In contrast, the purified MBP-FLAG protein (a negative control) did not exhibit any BIAM-labeled bands. To ensure uniformity of protein levels in each reaction system, both MBP-GS3 and MBP-FLAG in the reaction mixtures were detected using an anti-MBP antibody. All the proteins were separated on 10% reducing SDS–PAGE gels. ( C , D ) GS3 can interact with itself in yeast cells. The protein structure of GS3 is shown. The N-terminus of GS3 (aa:1–94) including GGL/OSR domain and the C-terminus (aa:95–232) including Cys-rich domain are shown ( D ). The indicated construct pairs were co-transformed into yeast strain AH109. Interactions between bait and prey were examined on the control media SD-2 (SD/-Leu/-Trp) and selective media SD-4 (SD/-Leu/-Trp/-His/-Ade). ( E ) The relative β-galactosidase activity was quantified for each pair of bait and prey proteins as indicated in ( D ). The values are means ± SD ( n = 3). The average value of GS3-BD/GS3 1-94 -AD pair was set at 1. Different lowercase letters denote significant differences among the various pairs, as determined by one-way ANOVA with Tukey’s multiple comparisons test. p = 0.0189 (GS3-BD/AD vs. BD/GS3 1-94 -AD), 0.3062 (GS3-BD/AD vs. BD/GS3 95-232 -AD), 0.3951 (BD/GS3 1-94 -AD vs. BD/GS3 95-232 -AD), <0.0001 for other comparisons. ( F ) GS3 can interact with itself in LCI assay. N.benthamiana leaves were transformed by injection of Agrobacterium GV3101 cells harboring GS3-nLUC and cLUC-GS3 plasmids. Co-transformation of GS3-nLUC with cLUC-EOG1 and EOG1-nLUC with cLUC-GS3 served as negative controls. Strong luciferase complementation signal was observed for GS3-nLUC and cLUC-GS3 combination, while no obvious signal was observed for the negative controls. ( G ) GS3 interacts with itself in BiFC assays in N.benthamiana leaves. cYFP-GS3/nYFP-GS3, nYFP-GS3/cYFP and cYFP-GS3/nYFP were co-expressed with the plasma membrane marker (PIP2-mCherry), nuclear marker (H2B-mCherry) or endosome marker (VPS23A-mCherry) in leaves of N.benthamiana , respectively. Strong YFP fluorescence was observed in plasma membrane and endosome for cYFP-GS3/nYFP-GS3 combination, while no obvious signal was observed for the negative controls. Scale bars represent 50 μm. ( H ) GS3 associates with itself through intermolecular disulfide bonds in vitro. Purified MBP-GS3 and MBP-FLAG proteins, treated with or without NEM (N-ethylmaleimide), were separated on a 10% non-reducing SDS-PAGE gel, and subsequently immunoblotted using an anti-MBP antibody. Bands representing monomers, dimers and oligomers of MBP-GS3 were clearly observed in the absence of NEM. The oligomerization capability of MBP-GS3 decreased with the addition of NEM. In contrast, the negative control MBP-FLAG exclusively exhibited monomeric forms regardless of NEM treatment. ( I ) GS3 can form oligomers in rice plants. GFP-GS3 proteins extracted from 10–15 cm young panicles of pro35S:GFP-GS3 transgenic rice plants were separated on a 10% non-reducing SDS-PAGE gel. The presence of GFP-GS3 oligomers in rice plants was evident, and these oligomers could be disrupted by 30% β-Mercaptoethanol (β-ME) treatment. ACTIN protein was separated on a 10% reducing SDS-PAGE gel and detected with an anti-ACTIN antibody as a loading control. ( J ) The conformation of GS3 is subject to redox regulation by DTT and H 2 O 2 . Purified MBP-GS3 proteins, treated with varying concentrations of H 2 O 2 and DTT, were separated on an 8% non-reducing SDS-PAGE gel. With increasing DTT concentration, there was a gradual increase in the levels of monomers and dimers, accompanied by a decrease in the levels of oligomers. The addition of H 2 O 2 led to a decrease in monomers and dimers, while the levels of oligomers increased proportionally with the concentration of H 2 O 2 . M, D, and O in ( H – J ) denote monomers, dimers, and oligomers, respectively. .

Article Snippet: The pellets were washed for three times with cold acetone (no more than 80%) and dissolved in a BIAM labeling buffer contained 500 μM BIAM (Anaspec, AS-60644), then reacted at a room temperature with gentle shaking in darkness for 1 h. The reaction was stopped by the adding final concentration of 20 mM β-mercaptoethanol.

Techniques: Labeling, Purification, Incubation, Concentration Assay, Negative Control, SDS Page, Construct, Transformation Assay, Control, Activity Assay, Injection, Luciferase, Clinical Proteomics, Membrane, Marker, Fluorescence, In Vitro, Transgenic Assay

Journal: The EMBO Journal

Article Title: Redox regulation of G protein oligomerization and signaling by the glutaredoxin WG1 controls grain size in rice

doi: 10.1038/s44318-025-00462-9

Figure Lengend Snippet: ( A ) The catalytically active site CCMC of WG1 is crucial to its interaction with GS3. The indicated construct pairs were co-transformed into yeast strain AH109. Interactions between bait and prey were examined on the control media SD-2 (SD/-Leu/-Trp) and selective media SD-4 (SD/-Leu/-Trp/-His/-Ade). AD-WG1 and BD pair, BD-GS3 and AD pair as well as AD-WG1 C46,49S and BD pair served as negative controls. The notation “C46, 49S” indicates that the 46th and 49th cysteine residues of WG1 protein have been mutated to serine, resulting in a catalytically inactive version of WG1. ( B ) The relative β-galactosidase activity was quantified for each pair of bait and prey proteins as indicated in ( A ). The values are means ± SD ( n = 3). The average value of GS3-BD/WG1 C46,49S -AD pair was set at 1. Different lowercase letters denote significant differences among the various pairs, as determined by one-way ANOVA with Tukey’s multiple comparisons test. p = 0.0242 (GS3-BD/AD vs. BD/WG1-AD), 0.1794 (GS3-BD/AD vs. BD/WG1 C46,49S -AD), 0.6906 (BD/WG1-AD vs. BD/WG1 C46,49S -AD), 0.0028 (BD/WG1-AD vs. GS3-BD/WG1 C46,49S -AD), 0.0005 (BD/WG1 C46,49S -AD vs. GS3-BD/WG1 C46,49S -AD), <0.0001 for other comparisons. ( C ) Flow diagram of biotin-switch assay for detecting whether WG1 could reduce oxidized thiol of GS3. Purified MBP-GS3 were treated with or without H 2 O 2 (①), then incubated with or without FLAG-WG1/FLAG-WG1 C46,49S (②) and followed by reacting with NEM (③), DTT (④), and BIAM (⑤). The BIAM labeled MBP-GS3 were detected using an anti-Biotin antibody (⑥). ( D ) Oxidized thiol of GS3 can be reduced by WG1 in biotin-switch assay. Light BIAM-labeling bands for MBP-GS3 without H 2 O 2 treatment indicated that most active thiols of MBP-GS3 were blocked by NEM. Strong BIAM-labeling bands for MBP-GS3 with H 2 O 2 treatment indicated that H 2 O 2 can protect the active thiol of MBP-GS3 from being blocked by NEM. This protective effect was significantly decreased with FLAG-WG1 treatment, whereas it was not decreased by the treatment with the catalytically active site mutation version of FLAG-WG1 (FLAG-WG1 C46,49S ), indicating the reduction of MBP-GS3 oxidized thiol by FLAG-WG1. The inputs of MBP-GS3 and FLAG-WG1 were detected using an anti-MBP and anti-FLAG antibodies, respectively. All proteins were separated on 10% reducing SDS–PAGE gels for analysis. ( E ) WG1 can reduce the intermolecular disulfide bonds of GS3 in vitro. Purified MBP-GS3 proteins, incubated with or without varying concentrations of FLAG-WG1 in the presence of 50 mM GSH, were separated on a 6% non-reducing SDS-PAGE gel and immunoblotted using an anti-MBP antibody. The inputs of MBP-GS3 and FLAG-WG1 were separated on a 10% reducing SDS–PAGE gel and immunoblotted using an anti-MBP and anti-FLAG antibodies, respectively. With an increase in the concentration of FLAG-WG1, the monomers of MBP-GS3 showed a gradual increase obviously. The protein level of MBP-GS3 monomers/inputs MBP-GS3 ratio, representing the monomer level of MBP-GS3 from three independent repeats ( E , Fig. ), was illustrated in the bar chart below, with the treatment lacking FLAG-WG1 set at 1. The protein level was quantified by ImageJ. The values are means ± SD ( n = 3). Different lowercase letters denote significant differences among the various groups, as determined by one-way ANOVA with Tukey’s multiple comparisons test. p = 0.0026 (CK vs. 1 X FLAG-WG1), 0.0001 (CK vs. 2 X FLAG-WG1), 0.0094 (1 X FLAG-WG1 vs. 2 X FLAG-WG1). ( F ) FLAG-WG1 C46,49S loses its ability to reduce the intermolecular disulfide bonds of GS3 in vitro. Similar experimental procedures were conducted as presented in ( E ), with FLAG-WG1 replaced with FLAG-WG1 C46,49S . After mutating the catalytically active sites CCMC of WG1 to an inactive SCMS form, the oligomerization level of MBP-GS3 could no longer be reduced. The protein level of MBP-GS3 monomers/inputs MBP-GS3 ratio, representing the monomer level of MBP-GS3 from three independent repeats ( F , Fig. ), was illustrated in the bar chart below, with the treatment lacking FLAG-WG1 C46,49S set at 1. The protein level was quantified by ImageJ. The values are means ± SD ( n = 3). The p value was determined using an unpaired two-tailed t test with Welch’s correction compared with the absence of FLAG-WG1 C46,49S treatment. ns represents no significant difference. p = 0.7346 (CK vs. 1 X FLAG-WG1 C46,49S ), 0.9887 (CK vs. 2 X FLAG-WG1 C46,49S ). ( G ) MYC-WG1 diminishes the oligomerization level of GFP-GS3 in rice. Total proteins extracted from 10 to 15 cm young panicles of pro35S:GFP-GS3 (homozygous, 1) and pro35S:GFP-GS3/pro35S:MYC-WG1 F1 (heterozygous, 2) plants were separated on a 10% non-reducing SDS-PAGE gel and immunoblotted using an anti-GFP antibody. The inputs of MYC-WG1 and ACTIN were separated on 10% reducing SDS–PAGE gels and immunoblotted using anti-MYC and anti-ACTIN antibodies, respectively. ( H ) FLAG-GS3 exhibited a stronger oligomerization ability in the wg1-2 mutant compared to the wild type in rice protoplasts. The pro35S:FLAG-GS3 vector was transiently transformed into both wg1-2 and wild-type protoplasts. FLAG-GS3 proteins were expressed and separated on a 10% partial-reducing (4% β-ME treatment) SDS-PAGE gel and immunoblotted with an anti-FLAG antibody. M, D, and O in ( E – H ) denote monomers, dimers, and oligomers, respectively. .

Article Snippet: The pellets were washed for three times with cold acetone (no more than 80%) and dissolved in a BIAM labeling buffer contained 500 μM BIAM (Anaspec, AS-60644), then reacted at a room temperature with gentle shaking in darkness for 1 h. The reaction was stopped by the adding final concentration of 20 mM β-mercaptoethanol.

Techniques: Construct, Transformation Assay, Control, Activity Assay, Biotin Switch Assay, Purification, Incubation, Labeling, Mutagenesis, SDS Page, In Vitro, Concentration Assay, Two Tailed Test, Plasmid Preparation