Complexity of Signaling Networks Old Protein, New Tricks Biplab Bose.

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Presentation transcript:

Complexity of Signaling Networks Old Protein, New Tricks Biplab Bose

Exotoxin of Corynebacterium diphtheriae. Diphtheria Toxin

Diphtheria Toxin in Therapeutics

Diphtheria Toxin in Cancer Therapeutics

HB-EGF: DT Receptor Normal Function Cell proliferation Developmental process Wound-healing In Oncogenesis: Increases proliferation Induction of migration and invasion Promotion of angiogenesis Overexpressed in tumors: Pancreatic, Liver, Gastric and Glioma Heparin-binding epidermal growth factor (EGF)-like growth factor Soluble & Membrane bound

HB-EGF Signaling

Receptor Binding Domain of DT (RDT) Diphtheria Toxin R-domain of Diphtheria Toxin PDB ID: 1F0L

Cloning of RDT SOURCE: Full Length DT cloned in pET-22b 8

Expression & Purification of RDT E. coli BL21(DE3) Induction at 28 0 C,1 mM IPTG. Purification by His-Trap Column. Western Blot using anti-His Ab SDS-PAGE of purified RDT

RDT binds to HB-EGF HB-EGF Coated on 96-well ELISA plate. Detection: anti-His Ab followed by anti-mouse Ab Solid Phase ELISA:

RDT Binds to Cell Surface HB-EGF Cell line: U-87 MG. Detection: anti-His Ab followed by FITC-Conjugated anti-mouse Ab Immunofluorescence: 11

Binding Affinity of RDT Single cycle Kinetics. CM5 Chip, HB-EGF immobilized SPR, Biacore X-100 k on (1/M.1/s) k off (1/s) K D (M) RDT 4.4 x x x DT 3.9 x x x 10 -8

Can a Drug Bind to RDT ? blue = hydrophilic and orange red = hydrophobic.

Curcumin: a Potential Therapeutic Agent

Curcumin: a Good Probe Fluorescence of Curcumin depends upon environment  Water quenches curcumin fluorescence.

Curcumin binds to some proteins Protein binding increases fluorescence of Curcumin 16 Curcumin: a Good Probe

Docking of Curcumin on RDT Docking server: SwissDock Docking Criteria: Blind, no flexibility for RDT. RDT Structure of Curcumin (from ZINC) Source: R-domain of B-chain of 1FOL 17

Blue = Hydrophilic Orange red = Hydrophobic. Docking of Curcumin on RDT Ribbon Diagram Surface Diagram A potential binding pose

Docking of Curcumin on RDT Important interactions between RDT and curcumin:

Curcumin Binds to RDT Fluorescence spectroscopy: Curcumin (10 µM) in PBS, Molar ratio of Curcumin:Protein (10:1) Incubation at 4 0 C, 2 hr Excitation at 430 nm.

Curcumin Binds to RDT Fluorescence spectroscopy: Curcumin (10 µM) in PBS, with RDT varied (0 to 2 µM) Incubation at 4 0 C, 2 hr Excitation at 430 nm.

Curcumin Binds to RDT Average Life time of Fluorescence decay (ns) Curcumin0.86 Curcumin- RDT 1.04 Time-resolved fluorescence spectroscopy: Excitation at 405 nm Curcumin (10 µM) in PBS, Molar ratio of Curcumin:Protein(10:1) Decay measured in ns/channel. Exponential component analysis.

20X Curcumin-RDT enhances accumulation of curcumin Cell Line: U-87 MG Curcumin (2 µM); molar ratio curcumin:protein (10:1) 23

24 * No significant difference between these two (p = 0.143); **significantly different from others (p < 0.001). One-way ANOVA with pairwise comparison Curcumin-RDT increases cellular uptake of curcumin Cell Line : U-87 MG Curcumin:1 µM; Curcumin-protein (molar ratio:10:1) Incubation at 37 0 C, 2 hr, Serum. C18 column; methanolic cell extract HPLC : * * **

Curcumin-RDT potentiate curcumin Significant difference between Curcumin and Curcumin-RDT: 2-way ANOVA, p<0.001 Cell line: U-87 MG RDT: 0.1 µM Incubation at 37 0 C, 72 hr, Serum-Free MTT assay:

Effect of Curcumin-RDT is not synergistic Cell line: U-87 MG RDT: 0.1µM; Curcumin:20 µM; Curcumin-RDT: 20 µM:0.1 µM; MTT Assay ** Significantly different from other treatment groups (p < 0.001)

Effect of Curcumin-RDT on cell cycle Flowcytometry Cell line: U-87 MG RDT: 0.1 µM; Curcumin:20 µM; Curcumin-RDT: 20 µM:0.1 µM; 48 hr.

Curcumin-RDT enhances apoptosis PI Annexin V Flowcytometry Cell line: U-87 MG RDT: 0.1 µM; Curcumin:20 µM; Curcumin-RDT: 20 µM:0.1 µM; Incubation at 37 0 C, 48 hr.

Using RDT to enhance drug delivery