Journal: ACS infectious diseases

Article Title: Clostridium difficile ClpP Homologues are Capable of Uncoupled Activity and Exhibit Different Levels of Susceptibility to Acyldepsipeptide Modulation

doi: 10.1021/acsinfecdis.8b00199

Figure Lengend Snippet: Composition and in vitro behavior of reported multi-ClpP systems. “Mixing” denotes that this result was obtained by mixing ClpP1 and ClpP2 homotetradecamers.

Article Snippet: clpP1 ( {"type":"entrez-protein","attrs":{"text":"YP_001089821.1","term_id":"126700924"}} YP_001089821.1 ) and clpp2 ( {"type":"entrez-protein","attrs":{"text":"YP_001089868.1","term_id":"126700971"}} YP_001089868.1 ) were synthesized with a Poly-His(6x) C-terminal tag, codon optimized, and cloned into pET28a by Genscript USA (Piscataway, NJ). clpX ( {"type":"entrez-protein","attrs":{"text":"YP_001089820.1","term_id":"126700923"}} YP_001089820.1 ) with a TEV-cleavable Poly-His(6x) N-terminal tag was also synthesized by Genscript USA, codon optimized, and cloned into pET21a.

Techniques: In Vitro

Journal: ACS infectious diseases

Article Title: Clostridium difficile ClpP Homologues are Capable of Uncoupled Activity and Exhibit Different Levels of Susceptibility to Acyldepsipeptide Modulation

doi: 10.1021/acsinfecdis.8b00199

Figure Lengend Snippet: Relative quantification of clpP1 and clpP2 mRNA expression during exponential and stationary growth phases in BHIS broth. Transcript levels were normalized to the reference gene rpoB, with clpP2 transcript level reported relative to clpP1. The resulting data were analyzed via multiple comparison two-way ANOVA and corrected by the Sidak method. ****, P ≤ 0.0001.

Article Snippet: clpP1 ( {"type":"entrez-protein","attrs":{"text":"YP_001089821.1","term_id":"126700924"}} YP_001089821.1 ) and clpp2 ( {"type":"entrez-protein","attrs":{"text":"YP_001089868.1","term_id":"126700971"}} YP_001089868.1 ) were synthesized with a Poly-His(6x) C-terminal tag, codon optimized, and cloned into pET28a by Genscript USA (Piscataway, NJ). clpX ( {"type":"entrez-protein","attrs":{"text":"YP_001089820.1","term_id":"126700923"}} YP_001089820.1 ) with a TEV-cleavable Poly-His(6x) N-terminal tag was also synthesized by Genscript USA, codon optimized, and cloned into pET21a.

Techniques: Expressing

Journal: ACS infectious diseases

Article Title: Clostridium difficile ClpP Homologues are Capable of Uncoupled Activity and Exhibit Different Levels of Susceptibility to Acyldepsipeptide Modulation

doi: 10.1021/acsinfecdis.8b00199

Figure Lengend Snippet: Tetradecameric assembly and thermal stability of ClpP1 and ClpP2 homomeric complexes. (A) size exclusion chromatograph of purified recombinant ClpP1 and ClpP2 following Ni-affinity purification. B. subtilis ClpP (BsClpP) is included for comparison. (B) time-dependent oligomerization of ClpP2. An aliquot of ClpP2 was injected onto a S300 SEC (blue, solid) and again 14 days later (blue, dashed). (C and D) SYPRO Orange TSA of tetradecameric ClpP1 (C) and ClpP2 (D) in the presence and absence of varying concentrations of an ADEP activator.

Article Snippet: clpP1 ( {"type":"entrez-protein","attrs":{"text":"YP_001089821.1","term_id":"126700924"}} YP_001089821.1 ) and clpp2 ( {"type":"entrez-protein","attrs":{"text":"YP_001089868.1","term_id":"126700971"}} YP_001089868.1 ) were synthesized with a Poly-His(6x) C-terminal tag, codon optimized, and cloned into pET28a by Genscript USA (Piscataway, NJ). clpX ( {"type":"entrez-protein","attrs":{"text":"YP_001089820.1","term_id":"126700923"}} YP_001089820.1 ) with a TEV-cleavable Poly-His(6x) N-terminal tag was also synthesized by Genscript USA, codon optimized, and cloned into pET21a.

Techniques: Purification, Recombinant, Affinity Purification, Injection

Journal: ACS infectious diseases

Article Title: Clostridium difficile ClpP Homologues are Capable of Uncoupled Activity and Exhibit Different Levels of Susceptibility to Acyldepsipeptide Modulation

doi: 10.1021/acsinfecdis.8b00199

Figure Lengend Snippet: Susceptibility of ClpP1 and ClpP2 to ADEP activation. (A) Synthetic ADEP utilized in these experiments. (B) Dose-dependent ADEP-induced degradation of the decapeptide Abz-DFAPKMALVPYNO2. (C) Time-dependent ADEP (100 μM) induced degradation of FITC β-casein; ClpP1 (black, triangles) and ClpP2 (blue, squares).

Article Snippet: clpP1 ( {"type":"entrez-protein","attrs":{"text":"YP_001089821.1","term_id":"126700924"}} YP_001089821.1 ) and clpp2 ( {"type":"entrez-protein","attrs":{"text":"YP_001089868.1","term_id":"126700971"}} YP_001089868.1 ) were synthesized with a Poly-His(6x) C-terminal tag, codon optimized, and cloned into pET28a by Genscript USA (Piscataway, NJ). clpX ( {"type":"entrez-protein","attrs":{"text":"YP_001089820.1","term_id":"126700923"}} YP_001089820.1 ) with a TEV-cleavable Poly-His(6x) N-terminal tag was also synthesized by Genscript USA, codon optimized, and cloned into pET21a.

Techniques: Activation Assay

Journal: ACS infectious diseases

Article Title: Clostridium difficile ClpP Homologues are Capable of Uncoupled Activity and Exhibit Different Levels of Susceptibility to Acyldepsipeptide Modulation

doi: 10.1021/acsinfecdis.8b00199

Figure Lengend Snippet: Peptidolytic activity of ClpP1 and ClpP2. (A) Time-dependent degradation of SLY-AMC by ClpP1 expressed from ΔEcClpP cells (black, solid, solid triangle), ClpP1 from Rosetta DE3 (black, dashed, hollow triangle), ClpP2 from ΔEcClpP (blue, solid, solid square), and ClpP2 from Rosetta DE3 (blue, dashed, hollow square). Experiments conducted with 1 μM ClpP tetradecamer and 0.5 mM SLY-AMC. (B) Michaelis-Menten analysis of ClpP1 and ClpP2 degradation of SLY-AMC. ClpP1 (DE3) Vmax = 1213 ± 38.0; ClpP1 (ΔEcClpP) Vmax = 1357 ± 39.9. ClpP2 Vmax values were not determined due to relatively negligible cleavage of the substrate. (C) Time-dependent (0–30 min) degradation of Z-GGL-AMC by ClpP isoforms. Experiments conducted with 1 μM ClpP tetradecamer and 0.1 mM Z-GGL-AMC. (D) Degradation of Z-GGL-AMC by ClpP isoforms after extended periods (30 and 46 h) of incubation. Means of each data set were compared to the control (Z-GGL-AMC alone) at each time point for statistical significance within a 95% confidence interval by two-way ANOVA analysis with Dunnett’s multiple comparison test. (**, P < 0.05). (E) Serine-protease active site labeling with ActivX TAMRA-FP visualized in an SDS-PAGE gel with chymotrypsin (CHY) as a positive control.

Article Snippet: clpP1 ( {"type":"entrez-protein","attrs":{"text":"YP_001089821.1","term_id":"126700924"}} YP_001089821.1 ) and clpp2 ( {"type":"entrez-protein","attrs":{"text":"YP_001089868.1","term_id":"126700971"}} YP_001089868.1 ) were synthesized with a Poly-His(6x) C-terminal tag, codon optimized, and cloned into pET28a by Genscript USA (Piscataway, NJ). clpX ( {"type":"entrez-protein","attrs":{"text":"YP_001089820.1","term_id":"126700923"}} YP_001089820.1 ) with a TEV-cleavable Poly-His(6x) N-terminal tag was also synthesized by Genscript USA, codon optimized, and cloned into pET21a.

Techniques: Activity Assay, Incubation, Labeling, SDS Page, Positive Control

Journal: ACS infectious diseases

Article Title: Clostridium difficile ClpP Homologues are Capable of Uncoupled Activity and Exhibit Different Levels of Susceptibility to Acyldepsipeptide Modulation

doi: 10.1021/acsinfecdis.8b00199

Figure Lengend Snippet: ClpXP mediated degradation of ssrA-GFP and ClpP induced reduction in ATP hydrolysis. (A) ClpXP1 and ClpXP2 mediated degradation of ssrA-GFP. Experiment was performed in triplicate, and SDS-PAGE results were quantified with ImageJ (NIH) to provide the quantified results (graph). (B) Fraction of ssrA-GFP remaining when incubated with ClpP variants in the absence of ClpX. (C) Rate of C. difficile ClpX ATP hydrolysis in the presence or absence of C. difficile ClpP1 or ClpP2. C. difficile ClpX hydrolyzed ATP at a rate of 149.2 ± 6.0 min−1·ClpX6−1. In the presence of ClpP1, ClpX hydrolyzed ATP at a rate of 122.5 ± 0.3 min−1·ClpX6−1. In the presence of ClpP2, ClpX hydrolyzed ATP at a rate of 138.7 ± 0.4 min−1·ClpX6−1. Km values for ClpX, ClpXP1, ClpXP2 are 375.2, 318.9, and 402.9 μM, respectively.

Article Snippet: clpP1 ( {"type":"entrez-protein","attrs":{"text":"YP_001089821.1","term_id":"126700924"}} YP_001089821.1 ) and clpp2 ( {"type":"entrez-protein","attrs":{"text":"YP_001089868.1","term_id":"126700971"}} YP_001089868.1 ) were synthesized with a Poly-His(6x) C-terminal tag, codon optimized, and cloned into pET28a by Genscript USA (Piscataway, NJ). clpX ( {"type":"entrez-protein","attrs":{"text":"YP_001089820.1","term_id":"126700923"}} YP_001089820.1 ) with a TEV-cleavable Poly-His(6x) N-terminal tag was also synthesized by Genscript USA, codon optimized, and cloned into pET21a.

Techniques: SDS Page, Incubation

Journal: ACS infectious diseases

Article Title: Clostridium difficile ClpP Homologues are Capable of Uncoupled Activity and Exhibit Different Levels of Susceptibility to Acyldepsipeptide Modulation

doi: 10.1021/acsinfecdis.8b00199

Figure Lengend Snippet: Depiction of the (A) ClpP1 and (B) ClpP2 ADEP binding pockets based on homology modeling with B. subtilis ClpP. Prime numbers indicate one ClpP subunit and nonprime numbers another. Gray indicates the culled surface of ClpP with a solvent radius set to 2.0 Å.

Article Snippet: clpP1 ( {"type":"entrez-protein","attrs":{"text":"YP_001089821.1","term_id":"126700924"}} YP_001089821.1 ) and clpp2 ( {"type":"entrez-protein","attrs":{"text":"YP_001089868.1","term_id":"126700971"}} YP_001089868.1 ) were synthesized with a Poly-His(6x) C-terminal tag, codon optimized, and cloned into pET28a by Genscript USA (Piscataway, NJ). clpX ( {"type":"entrez-protein","attrs":{"text":"YP_001089820.1","term_id":"126700923"}} YP_001089820.1 ) with a TEV-cleavable Poly-His(6x) N-terminal tag was also synthesized by Genscript USA, codon optimized, and cloned into pET21a.

Techniques: Binding Assay

Journal: Molecular cell

Article Title: Native Chromatin Proteomics Reveals Role for Specific Nucleoporins in Heterochromatin Organization and Maintenance

doi: 10.1016/j.molcel.2019.10.018

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Q800 Sonicator , Qsonica Sonicators , Cat# {"type":"entrez-protein","attrs":{"text":"Q800R3","term_id":"82241717","term_text":"Q800R3"}} Q800R3 –110.

Techniques: Purification, Recombinant, Silver Staining, Protease Inhibitor, Hybridization, Western Blot, Staining, Microscopy, Mass Spectrometry, Software, Real-time Polymerase Chain Reaction