complexity of signaling networks old protein, new tricks biplab bose
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 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
