Journal: Molecular oral microbiology

Article Title: Characterization of a novel family of fibronectin-binding proteins with M23 peptidase domains from Treponema denticola

doi: 10.1111/j.2041-1014.2010.00584.x

Figure Lengend Snippet: (A) Predicted identifiable peptide motifs (www.ebi.ac.uk/InterProScan/) of Treponema pallidum fibronectin-binding protein Tp0155 and potential orthologues identified from the Treponema denticola genome using BLAST similarity searches. LysM domains are shown with vertical bars (A) or dots (B). Treponema pallidum and T. denticola M23 peptidase domains (diagonal bars) retain 37% identity. Potential signal sequences (solid black) were identified using http://bioinformatics.leeds.ac.uk/prot_analysis/signal.html. (B) Alignments of LysM(A) and LysM(B) domain sequences from T. pallidum Tp0155 and potential T. denticola orthologues showing high degree of consensus across the different peptide sequences. Degree of identity is shown from highest (e.g. ) to lowest (e.g. H). The LysM motif YxxxxGxx(Hyd) (Turner et al., 2004) is boxed. Asterisk indicates residues that do not fit the specified motif.

Article Snippet: The E. coli was cultured on Luria Bertani (LB) agar ( Sambrook et al ., 1989 ) or in LB broth, containing appropriate antibiotics (ampicillin, 100μg ml −1 ; kanamycin 25μg ml −1 ). table ft1 table-wrap mode="anchored" t5 caption a7 Strain/plasmid Relevant characteristics Source Treponema denticola ATCC 35405 Type strain ( Chan et al ., 1993 ) Escherichia coli XL-1 Blue recA1 endA1 gyrA96 thi hsdR17 supE44 relA1 lac [ F ‘proAB lacl q Z Δ M15 Tn10 (Tet R )] Stratagene M15 (pREP-4 Kn R ) nal s str s rif s thi − lac − ara + gal + mtl − F − recA + uvr + lon + Qiagen Plasmids pQE30 bla ColE1 ori (Ap R ) Qiagen pQE30-TDE2318 bla ColE1 ori This study pQE30-TDE2753 bla ColE1 ori This study pQE30-TDE1738 bla ColE1 ori This study pQE30-TDE0110 bla ColE1 ori This study pQE30-TDE2189 bla ColE1 ori This study pQE30-TDE1136 bla ColE1 ori This study pQE30-TDE1297 bla ColE1 ori This study Open in a separate window Bacterial strains and plasmids used in this study

Techniques: Binding Assay

Journal: Molecular oral microbiology

Article Title: Characterization of a novel family of fibronectin-binding proteins with M23 peptidase domains from Treponema denticola

doi: 10.1111/j.2041-1014.2010.00584.x

Figure Lengend Snippet: Detection of proteins on the surface of Treponema denticola ATCC 35405 using antibodies to the Treponema pallidum protein Tp0155 which had first been pre-adsorbed against Treponema phagedenis (Kazan). (A, C, E, G, I, K and M) Phase-contrast images; (B, D, F, H, J, L and N) corresponding immunofluorescence. (A, B) Preimmune serum for Tp0155-specific antibodies; (C, D), Tp0155-specific antibodies, non-detergent-treated T. denticola; (E, F), Tp0155-specific antibodies, Triton X-114-treated T. denticola; (G, H), preimmune serum for Tp0249-specific antibodies; (I, J), Tp0249-specific antibodies, non-detergent-treated T. denticola (negative control); (K, L), Tp0249-specific antibodies, Triton X-114-treated T. denticola; (M, N), anti-T. denticola antibodies (control) (Edwards et al., 2005).

Article Snippet: The E. coli was cultured on Luria Bertani (LB) agar ( Sambrook et al ., 1989 ) or in LB broth, containing appropriate antibiotics (ampicillin, 100μg ml −1 ; kanamycin 25μg ml −1 ). table ft1 table-wrap mode="anchored" t5 caption a7 Strain/plasmid Relevant characteristics Source Treponema denticola ATCC 35405 Type strain ( Chan et al ., 1993 ) Escherichia coli XL-1 Blue recA1 endA1 gyrA96 thi hsdR17 supE44 relA1 lac [ F ‘proAB lacl q Z Δ M15 Tn10 (Tet R )] Stratagene M15 (pREP-4 Kn R ) nal s str s rif s thi − lac − ara + gal + mtl − F − recA + uvr + lon + Qiagen Plasmids pQE30 bla ColE1 ori (Ap R ) Qiagen pQE30-TDE2318 bla ColE1 ori This study pQE30-TDE2753 bla ColE1 ori This study pQE30-TDE1738 bla ColE1 ori This study pQE30-TDE0110 bla ColE1 ori This study pQE30-TDE2189 bla ColE1 ori This study pQE30-TDE1136 bla ColE1 ori This study pQE30-TDE1297 bla ColE1 ori This study Open in a separate window Bacterial strains and plasmids used in this study

Techniques: Immunofluorescence, Negative Control, Control

Journal: Molecular oral microbiology

Article Title: Characterization of a novel family of fibronectin-binding proteins with M23 peptidase domains from Treponema denticola

doi: 10.1111/j.2041-1014.2010.00584.x

Figure Lengend Snippet: (A) Alignments of M23 peptidase domains from TDE1738 of Treponema denticola and homologous regions of peptidoglycan-degrading enzymes lysostaphin from Staphylococcus simulans biovar staphylolyticus, zoocin A from Streptococcus equi subsp zooepidemicus and ALE-1 from Staphylococcus capitis. The M23 peptidase domain of TDE1738 retains 43% identity to lysostaphin, 43% identity to zoocin A and 40% identity to ALE-1 with conservation across the HxGxD and HLH active sites. (B) Alignments of a 92 amino acid (aa) residue portion regions of TDE1739 of T. denticola and lysostaphin immunity factor from S. simulans biovar staphylolyticus, demonstrating homology (29% identity) between these peptides. Sequence consensus is shown as is degree of identity from highest (e.g. ) to lowest (e.g. A). (C) Diagram depicting genes encoding potential and known peptidoglycan degradation enzymes and adjacent immunity factors which provide protection from autolysis by the peptidoglycanolytic enzyme. (i) The chromosomal gene TDE1738 from T. denticola encodes for a potential peptidoglycan-degrading enzyme, and TDE1739 for a potential immunity factor. (ii) Lysostaphin and adjacent lysostaphin immunity factor (Lif/Epr) are encoded on the plasmid pACK1 of S. simulans biovar staphylolyticus. (iii) The gene encoding zoocin A and corresponding zoocin A immunity factor (Zif) is on the chromosome of Streptococcus equi subsp zooepidemicus, while (iv) ale-1 and adjacent endopeptidase resistance (epr) genes are located on a plasmid in Staphylococcus capitis. (v) Tp0706 from the Treponema pallidum chromosome, with homology to TDE1738 may also be an enzyme with peptidoglycan-degrading activity and the adjacent gene Tp0705 is annotated as a pencillin-binding protein in the same manner as zoocin A immunity factor (Zif).

Article Snippet: The E. coli was cultured on Luria Bertani (LB) agar ( Sambrook et al ., 1989 ) or in LB broth, containing appropriate antibiotics (ampicillin, 100μg ml −1 ; kanamycin 25μg ml −1 ). table ft1 table-wrap mode="anchored" t5 caption a7 Strain/plasmid Relevant characteristics Source Treponema denticola ATCC 35405 Type strain ( Chan et al ., 1993 ) Escherichia coli XL-1 Blue recA1 endA1 gyrA96 thi hsdR17 supE44 relA1 lac [ F ‘proAB lacl q Z Δ M15 Tn10 (Tet R )] Stratagene M15 (pREP-4 Kn R ) nal s str s rif s thi − lac − ara + gal + mtl − F − recA + uvr + lon + Qiagen Plasmids pQE30 bla ColE1 ori (Ap R ) Qiagen pQE30-TDE2318 bla ColE1 ori This study pQE30-TDE2753 bla ColE1 ori This study pQE30-TDE1738 bla ColE1 ori This study pQE30-TDE0110 bla ColE1 ori This study pQE30-TDE2189 bla ColE1 ori This study pQE30-TDE1136 bla ColE1 ori This study pQE30-TDE1297 bla ColE1 ori This study Open in a separate window Bacterial strains and plasmids used in this study

Techniques: Residue, Sequencing, Plasmid Preparation, Activity Assay, Binding Assay

Journal: bioRxiv

Article Title: Targeting the PI5P4K lipid kinase family in cancer using novel covalent inhibitors

doi: 10.1101/819961

Figure Lengend Snippet: (A) Chemical structures of compound JNK-IN-7 as previously reported in and novel compound THZ-P1-2 with its reversible and desthiobiotinylated counterparts. ( B) THZ-P1-2 potently inhibits PI5P4Kα kinase activity in the ADP-Glo luminescence assay. The curve obtained is representative of two independent experiments and the IC50 value is an average of values obtained from both experiments with three technical replicates each. (C) THZ-P1-2 shows potent inhibition of kinase activity of all three PI5P4K isoforms in a radiometric TLC assay measuring radiolabeled PI-4,5-P 2 . TLC image shown is representative of two independent experiments. ( D) Quantification of (C). Radiolabeled PI-4,5-P 2 spots were imaged by autoradiography and quantified by densitometry.

Article Snippet: A construct of human PI5P4Kα covering residues 35-405 in the pNIC28Bsa4 vector (Addgene #42494) was overexpressed in E. coli BL21 (DE3) in TB medium in the presence of 50 μg/ml of kanamycin.

Techniques: Activity Assay, Luminescence Assay, Inhibition, Autoradiography

Journal: bioRxiv

Article Title: Targeting the PI5P4K lipid kinase family in cancer using novel covalent inhibitors

doi: 10.1101/819961

Figure Lengend Snippet: (A) list of selected targets, including PI5P4Kγ (PIP4K2C), from KiNativ profiling of JNK-IN-7 at 1 μM for 3 hours in A375 cells. A higher percent inhibition of kinase labeling by ATP-biotin is indicates stronger binding and inhibition of the target kinase. Full list is available in . (B) JNK-IN-7 inhibits kinase activity of all three PI5P4K isoforms in a radiometric TLC assay measuring radiolabeled PI-4,5-P 2 . JNK-IN-7 was incubated with purified protein for 30min, followed by a 10min kinase reaction, quenching, lipid extraction and TLC development. (C) Quantification of (B). Radiolabeled PI-4,5-P 2 spots were imaged by autoradiography and quantified by densitometry. (D) Zero charge masses of recombinant PI5P4Kα and γ incubated with JNK-IN-7 for 2 hours at 37°C demonstrates covalent labeling of PI5P4K isoforms as determined by intact mass spectrometry. See Supplemental Intact MS Spectra for representative raw MS spectra used for charge deconvolution. (E) Subsequent protease digestion and tandem mass spectrometry confirms that THZ-P1-2 covalently labels cysteine residues on all three PI5P4K isoforms. The peptide for PI5P4Kβ was exclusively observed to be singly labeled at either Cys-307 or Cys-318. See Supplemental Intact MS Spectra for representative raw MS spectra used for charge deconvolution.

Article Snippet: A construct of human PI5P4Kα covering residues 35-405 in the pNIC28Bsa4 vector (Addgene #42494) was overexpressed in E. coli BL21 (DE3) in TB medium in the presence of 50 μg/ml of kanamycin.

Techniques: Inhibition, Labeling, Binding Assay, Activity Assay, Incubation, Purification, Autoradiography, Recombinant, Mass Spectrometry

Journal: bioRxiv

Article Title: Targeting the PI5P4K lipid kinase family in cancer using novel covalent inhibitors

doi: 10.1101/819961

Figure Lengend Snippet: (A) Electrospray mass spectrometry of recombinant PI5P4Kα/β/γ incubated with THZ-P1-2 demonstrates covalent labeling of PI5P4K isoforms. (B) Subsequent protease digestion and tandem mass spectrometry confirms that THZ-P1-2 covalently labels cysteine residues. (C) Crystal structure of PI5P4Kα in complex with THZ-P1-2, colored according to B factor and shown with covalent warhead extended out towards the covalently-targeted cysteine, C293 (labeled; not resolved in crystal structure). (D) Ligand interaction map of THZ-P1-2 with residues in the ATP-binding pocket of PI5P4Kα. (E) Modeled THZ-P1-2 binding showed for all three isoforms based on alignment of published PI5P4Kβ and γ structures with obtained PI5P4Kα structure. PI5P4Kα (magenta), PI5P4Kβ (blue), PI5P4Kγ (orange).

Article Snippet: A construct of human PI5P4Kα covering residues 35-405 in the pNIC28Bsa4 vector (Addgene #42494) was overexpressed in E. coli BL21 (DE3) in TB medium in the presence of 50 μg/ml of kanamycin.

Techniques: Mass Spectrometry, Recombinant, Incubation, Labeling, Binding Assay

Journal: bioRxiv

Article Title: Targeting the PI5P4K lipid kinase family in cancer using novel covalent inhibitors

doi: 10.1101/819961

Figure Lengend Snippet: (A) Protease digestion and tandem mass spectrometry following intact mass labeling confirms that THZ-P1-2 covalently labels cysteine residues on all three PI5P4K isoforms. The peptide for PI5P4Kβ was exclusively observed to be singly labeled at either Cys-307 or Cys-318. Related to . See Supplemental Intact MS Spectra for representative raw MS spectra used for charge deconvolution. (B) THZ-P1-2-R was found not to covalently label recombinant PI5P4Kα and γ when incubated with purified protein for 2 hours at 37°C. Mass labeling plot shown here is compared to mass shift observed with THZ-P1-2 from . (C) Surface representation of co-crystal structure of PI5P4Kα in complex with THZ-P1-2. Related to . (D) Zoom-in of ligand electron density within the active site of PI5P4Kα. See for diffraction data collection and refinement statistics.

Article Snippet: A construct of human PI5P4Kα covering residues 35-405 in the pNIC28Bsa4 vector (Addgene #42494) was overexpressed in E. coli BL21 (DE3) in TB medium in the presence of 50 μg/ml of kanamycin.

Techniques: Mass Spectrometry, Labeling, Recombinant, Incubation, Purification

Journal: bioRxiv

Article Title: Targeting the PI5P4K lipid kinase family in cancer using novel covalent inhibitors

doi: 10.1101/819961

Figure Lengend Snippet: (A) THZ-P1-2-R and dtb-THZ-P1-2 were found to inhibit PI5P4Kα in an ADP-Glo assay, with a slight loss in potency with THZ-P1-2-R. Both compounds were incubated with PI5P4Ka for 15min before proceeding with a 1 hour reaction with ATP and development with ADP-Glo reagents. Plots are shown in comparison with THZ-P1-2 from . (B) THZ-P1-2 engages PI5P4K isoforms at 2h and 4h timepoints, exhibiting prolonged engagement when a washout is performed. HEK293T cells were treated with DMSO or THZ-P1-2 at time, t=0 and cells were washed 2X with PBS at either 2 or 4h and harvested at the end of 6h. A streptavidin pulldown was then conducted in lysates after normalization of protein content with a BCA assay. (C) THZ-P1-2 inhibits the Type 1 PI4P5K kinases at a lower extent but shows potent inhibition on PIKfyve by ADP-Glo. In vitro kinase assays were performed by Carna Biosciences. (D) THZ-P1-2 causes vacuolar enlargement, characteristic of PIKfyve inhibition, at a concentration of 10 μ M but not 1 μ M, compared to PIKfyve inhibitor apilimod at 10 nM treatment. Vero cells were treated with DMSO or compound for 6h and imaged. (E) Incubation of Vero cells with 1 μM THZ-P1-2 was unable to impair the PIKfyve inhibitory phenotype of apilimod. Preincubation: Vero cells were incubated with DMSO/THZ-P1-2 for 1 or 6h, washed with PBS to completely remove compound, treated with DMSO/apilimod and imaged after 6h. Coincubation: Vero cells were incubated with DMSO/THZ-P1-2 for 1 or 6h, and then co-treated with DMSO/apilimod and imaged after 6h.

Article Snippet: A construct of human PI5P4Kα covering residues 35-405 in the pNIC28Bsa4 vector (Addgene #42494) was overexpressed in E. coli BL21 (DE3) in TB medium in the presence of 50 μg/ml of kanamycin.

Techniques: Glo Assay, Incubation, BIA-KA, Inhibition, In Vitro, Concentration Assay

Journal: bioRxiv

Article Title: Targeting the PI5P4K lipid kinase family in cancer using novel covalent inhibitors

doi: 10.1101/819961

Figure Lengend Snippet: (A) Similar to genetic loss of PIP4K2A/B , inhibition of PI5P4K activity with THZ-P1-2 increases LAMP1-positive lysosomal size, number and contact with LC3B-positive autophagosomes. HeLa cells were cultured overnight with either DMSO, 0.25 μM, 0.5 μM or 1.0 μM THZ-P1-2 and stained for LC3B (magenta) or LAMP1 (green) with nuclei in blue. Scale bars, 10 μM. (B) Inhibition of PI5P4K activity with THZ-P1-2 increases TFEB nuclear localization. HeLa cells were cultured overnight with either DMSO, 0.25 μM, 0.5 μM or 1.0 μM THZ-P1-2 and stained for TFEB (green) with nuclei in blue. Scale bars, 10 μM. (C) Quantification of results in (B). The intensity of TFEB immunofluorescence was quantified in the nucleus and the cytoplasm, and used to calculate the ratio. Statistical significance determined by ANOVA (***p < 0.0005) with Dunnett multiple comparison post-test. Each group was compared to control control HeLa cells treated with DMSO, (n ≥ 30). (D) Expression of PI5P4K cysteine to serine mutants alleviates lysosomal dysfunction induced by THZ-P1-2 treatment. HeLa cells were infected with viruses expressing GFP, GFP-PI5P4Kα, GFP-PI5P4Kα C293S, GFP-PI5P4Kβ, or GFP-PI5P4Kβ C307S C318S and treated with either DMSO or 250 nM THZ-P1-2 overnight. Expression of both the PI5P4Kα and PI5P4Kβ cysteine to serine mutants alleviated dysfunctional lysosomes (green) with nuclei in blue. Scale bars, 10 μM. (E) Quantification of results in (D). The number of lysosomes was quantified per cell. Statistical significance determined by ANOVA (***p < 0.0005) with Dunnett multiple comparison post-test. Each group was compared to control HeLa cells expressing GFP and treated with DMSO, (n ≥ 30). (F) HeLa cells expressing GFP, GFP-PI5P4Kα, GFP-PI5P4Kα C293S, GFP-PI5P4Kβ, or GFP-PI5P4Kβ C307S C318S were treated with either DMSO or 250 nM THZ-P1-2 overnight for 16 hours and subsequently harvested for qPCR of TFEB targets. Fold change is calculated by comparison to HeLa cells expressing GFP treated with DMSO. *p < 0.05, Student’s t-test, (n ≥ 8).

Article Snippet: A construct of human PI5P4Kα covering residues 35-405 in the pNIC28Bsa4 vector (Addgene #42494) was overexpressed in E. coli BL21 (DE3) in TB medium in the presence of 50 μg/ml of kanamycin.

Techniques: Inhibition, Activity Assay, Cell Culture, Staining, Immunofluorescence, Expressing, Infection

Journal:

Article Title: Design and Evaluation of Useful Bacterium-Specific PCR Primers That Amplify Genes Coding for Bacterial 16S rRNA

doi:

Figure Lengend Snippet: Species of bacteria and archaea tested in PCRs using amplimer 63f and 1387f to amplify 16S rRNA genes

Article Snippet: Treponema denticola ATCC 35405, ATCC 33520 , Spirochaeta-Treponema-Borrelia subdivision , +.

Techniques: Amplification

Journal:

Article Title: Design and Evaluation of Useful Bacterium-Specific PCR Primers That Amplify Genes Coding for Bacterial 16S rRNA

doi:

Figure Lengend Snippet: Alignment of sequences of amplimers 63f-1387r and 27f-1392r with the corresponding regions of 16S rDNA genes of representative species used in PCR

Article Snippet: Treponema denticola ATCC 35405, ATCC 33520 , Spirochaeta-Treponema-Borrelia subdivision , +.

Techniques: Sequencing