prodomain  (R&D Systems)

Journal: Biochemical Journal

Article Title: Activin E is a transforming growth factor β ligand that signals specifically through activin receptor-like kinase 7

doi: 10.1042/BCJ20230404

Figure Lengend Snippet: ( A–C ) Luciferase reporter assay response to recombinant mature ActA, ActC, or GDF11 (5 nM) along with conditioned media of ActE Construct I, Construct II, or empty vector (EV) in (CAGA 12 )-luciferase HEK293T293 cells transfected with SB-431542 resistant ALK4-ST ( A ), ALK5-ST ( B ), or ALK7-ST ( C ) type I receptors. Media was concentrated 20× and diluted two-fold for the titration (2.5×, 5×, 10×, 20×). Data normalized to transfected cells with no ligand treatment. ( D ) Effects of the ALK7 neutralizing antibody (nAb) on recombinant ActA or ActE conditioned media samples on (CAGA 12 )-luciferase promoter activity in cells expressing the designated SB-431542 resistant type I receptor. (ns = not significant) (**** P < 0.0001) (**** P < 0.0001) ( E ). Western blot analysis under reducing and non-reducing conditions of elution samples from the purification of flag-tagged mature ActE from construct 2 using flag affinity resin. ( F ) Luciferase reporter activity of pooled fractions from ( E ) in (CAGA 12 )-luciferase HEK293T cells transfected with the ALK7-ST. Data normalized to untreated control cells. In ( A–D, and F ) each point represents a technical replicate within triplicate experiments with bars displaying the mean ± SD. In ( A–D, and F ), cells were treated with 10 μM SB-431542 to inhibit signaling activity of endogenous receptors.

Article Snippet: Conditioned media produced with a construct containing GDF8 prodomain and GDF11 mature (8Pro/11Mature) was provided by Elevian.

Techniques: Luciferase, Reporter Assay, Recombinant, Construct, Plasmid Preparation, Transfection, Titration, Activity Assay, Expressing, Western Blot, Purification, Control

Journal: Biochemical Journal

Article Title: Activin E is a transforming growth factor β ligand that signals specifically through activin receptor-like kinase 7

doi: 10.1042/BCJ20230404

Figure Lengend Snippet: ( A ) (CAGA 12 )-luciferase HEK293T cells transfected with ALK4-ST or ALK7-ST receptor constructs and treated with recombinant GDF11 (0.62 nM), ActE conditioned media (20×), EV conditioned media (20×), and ActC (0.62 nM) in the presence or absence of increasing amounts of FS288 (12.5 and 25 nM) (**** P < 0.0001) (* P = 0.0105) (*** P = 0.0004) ( P = 0.2746) ( P = 0.8552) (ns = not significant). ( B ) (CAGA 12 )-luciferase HEK293T cells transfected with ALK4-ST or ALK7-ST receptor constructs and treated with recombinant GDF11 (0.62 nM), ActE conditioned media (20×), EV conditioned media (20×), and ActC (0.62 nM) in the presence or absence of increasing amounts of WFIKKN2 (12.5 and 25 nM) (**** P < 0.0001) ( P = 0.3687) ( P = 0.1983) (ns = not significant). Each point represents a technical replicate within triplicate experiments with bars displaying the mean ± SD. Data are normalized to untreated control cells. In ( A and B ), cells were treated with 10 μM SB-431542 to inhibit signaling activity of endogenous receptors.

Article Snippet: Conditioned media produced with a construct containing GDF8 prodomain and GDF11 mature (8Pro/11Mature) was provided by Elevian.

Techniques: Luciferase, Transfection, Construct, Recombinant, Control, Activity Assay

Journal: The Plant Cell

Article Title: Arabidopsis metacaspase MC1 localizes in stress granules, clears protein aggregates, and delays senescence

doi: 10.1093/plcell/koad172

Figure Lengend Snippet: MC1 contains intrinsically disordered and aggregation-prone regions. A) Prediction of IDRs of MC1. Top, scheme of MC1 protein structure. Zf: LSD1-Zinc finger domain within the prodomain (amino acids 1-77); H164 and C220 correspond to the amino acids of the catalytic dyad within the large p20 catalytic subunit; 360 loop (amino acids 318-346): hydrophobic loop within the small p10 catalytic subunit. Middle and bottom, prediction of the disordered regions by D 2 P 2 ( https://d2p2.pro ) and DISOPRED3 ( http://bioinf.cs.ucl.ac.UK/psipred ), respectively. Precision above the dotted line show disordered scores higher than 0.5 and precision dots below the dotted line show disordered scores lower than 0.5. B) A3D structure of MC1. The prodomain and the 360 loop are highlighted. The amino acid sequences of the prodomain and 360 loop are shown and the amino acids with high A3D scores (aggregation-prone) are highlighted.

Article Snippet: For purification of rMC1, the MC1 coding sequences lacking the 360 loop and with or without the prodomain (MC1Δ360 or ΔNMC1Δ360/rMC1) ( ) were synthesized (Twist Bioscience) with codon optimization for expression in E. coli .

Techniques:

Journal: The Plant Cell

Article Title: Arabidopsis metacaspase MC1 localizes in stress granules, clears protein aggregates, and delays senescence

doi: 10.1093/plcell/koad172

Figure Lengend Snippet: The prodomain and the 360 loop confer insolubility to MC1. A) Schematic representation of MC1 full length, MC1Δ360 (without 360 loop) and ΔNMC1Δ360 (without prodomain and 360 loop) domain architecture. B) SDS–PAGE Coomassie-stained gels (upper panels) and western blot analysis (lower panels) of lysates from total, soluble, or insoluble fractions of E. coli cells expressing either MC1Δ360 or ΔNMC1Δ360 (rMC1) carrying an N-terminal 6xHis tag. Arrow denotes expected molecular weight of each of the 2 MC1 variants. C) Size-exclusion chromatography of concentrated eluates obtained by nickel-affinity chromatography . The inlet shows an SDS–PAGE Coomassie-stained gel of fractions 14 to 16 ml of the eluted volume from a Superdex 75 column.

Article Snippet: For purification of rMC1, the MC1 coding sequences lacking the 360 loop and with or without the prodomain (MC1Δ360 or ΔNMC1Δ360/rMC1) ( ) were synthesized (Twist Bioscience) with codon optimization for expression in E. coli .

Techniques: SDS Page, Staining, Western Blot, Expressing, Molecular Weight, Size-exclusion Chromatography, Affinity Chromatography

Journal: The Plant Cell

Article Title: Arabidopsis metacaspase MC1 localizes in stress granules, clears protein aggregates, and delays senescence

doi: 10.1093/plcell/koad172

Figure Lengend Snippet: The 360 loop and the prodomain of MC1 are involved, respectively, in recruitment to and clearance from SGs. A) Five-day-old mc1 seedlings expressing Pro35S:MC1-GFP , Pro35S:MC1C220A-GFP , Pro35S:ΔNMC1-GFP , or Pro35S:MC1Δ360loop-GFP were heat stressed at 39 °C for 40 min (HS), followed by incubation at 22 °C for up to 120 min (recovery). Images of root tips were taken at indicated time points. Bars = 5 μ m. B) Quantification of the number of condensates in the experiment shown in A) . C) Quantification of the area ( µ m 2 ) of condensates in the experiment shown in B) . D) Selected time frames (prebleach and 0, 3, and 15 s after bleaching) from FRAP analysis of MC1-GFP, MC1C220A-GFP, ΔNMC1-GFP, and MC1Δ360loop-GFP foci formed upon heat stress (40 min at 39 °C) in root tip cells of mc1 seedlings expressing Pro35S:MC1-GFP , Pro35S:MC1C220A-GFP , Pro35S:ΔNMC1-GFP , or Pro35S:MC1Δ360loop-GFP , respectively. Bars = 2 μ m. E) Initial signal recovery (%) of the experiment shown in C) . In B) , C) , and E) , upper and lower box boundaries represent the first and third quantiles, respectively; horizontal lines mark the median and whiskers mark the highest and lowest values. In B) and C) , 3 independent experiments, each including 3 to 5 seedlings. For each seedling, ∼30 cells from the root meristem were analyzed. In E) , 3 independent experiments, each containing at least 8 measurements of different SGs, were performed. Means with different letters are significantly different at P < 0.05 (1-way ANOVA).

Article Snippet: For purification of rMC1, the MC1 coding sequences lacking the 360 loop and with or without the prodomain (MC1Δ360 or ΔNMC1Δ360/rMC1) ( ) were synthesized (Twist Bioscience) with codon optimization for expression in E. coli .

Techniques: Expressing, Incubation

Journal: The Plant Cell

Article Title: Arabidopsis metacaspase MC1 localizes in stress granules, clears protein aggregates, and delays senescence

doi: 10.1093/plcell/koad172

Figure Lengend Snippet: Working model on the role of MC1 in SGs. A) Upper panel (WT plants): Under basal conditions, no SGs are detectable and MC1 presents a diffuse nucleo-cytoplasmic localization pattern. Upon perception of an acute, reversible stress, MC1 is recruited to SGs where it hypothetically clears misfolded/aggregated proteins. Under chronic or irreversible stress, the proteostatic capacity of the cell is surpassed and toxic protein aggregates that cannot be cleared start accumulating in the cytoplasm. Lower panel ( mc1 mutant plants): Plants devoid of MC1 cannot cope as WT with proteotoxic stress. Any stress may result in accumulation of protein aggregates that over time manifest as the observed accelerated senescence phenotype. B) MC1 is recruited to SGs via its 360 loop. Once in SGs, it clears aggregated proteins via its disaggregase activity. Release of MC1 from SGs is dependent on the prodomain. Nuclear schemes in A) and B) were obtained from Bioicons/Servier/license CC BY 3.0. C) MC1 is recruited to SGs via its 360 loop. Once in SGs, it clears aggregated proteins via its disaggregase activity. Release of MC1 from SGs is dependent on the prodomain.

Article Snippet: For purification of rMC1, the MC1 coding sequences lacking the 360 loop and with or without the prodomain (MC1Δ360 or ΔNMC1Δ360/rMC1) ( ) were synthesized (Twist Bioscience) with codon optimization for expression in E. coli .

Techniques: Mutagenesis, Activity Assay