complexity of signaling networks old protein, new tricks biplab bose

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 ToxinR-domain of

Diphtheria Toxin

PDB ID: 1F0L

Cloning of RDT

SOURCE: Full Length DT cloned in pET-22b

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Expression & Purification of RDT

• E. coli BL21(DE3)

• Induction at 28 0C ,1 mM IPTG.

• Purification by His-Trap Column.

Western Blot using anti-His AbSDS-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:

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Binding Affinity of RDT

• Single cycle Kinetics.

• CM5 Chip, HB-EGF immobilized

SPR, Biacore X-100

kon

(1/M.1/s) koff (1/s)

KD

(M)

 RDT 4.4 x 104 3.1 x 10-3 7.4 x 10-8

 DT 3.9 x 103 1.5 x 10-4 3.9 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

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Curcumin: a Good Probe

Docking of Curcumin on RDT

Docking server: SwissDockDocking Criteria: Blind, no flexibility for RDT.

RDT Structure of Curcumin (from ZINC)

Source: R-domain of B-chain of 1FOL17

• 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 0C, 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 0C, 2 hr• Excitation at 430 nm.

Curcumin Binds to RDT

Average Life time of

Fluorescence decay (ns)

Curcumin 0.86

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 MGCurcumin (2 µM); molar ratio curcumin:protein (10:1)

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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 0C, 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 0C, 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 0C, 48 hr.

Using RDT to enhance drug delivery