protein nanotube based probes for cancer cell imaging
TRANSCRIPT
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Protein Nanotube based Probes
for Cancer Cell Imaging
Hanudatta S. Atreya
Indian Institute of Science, Bangalore
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NMR Research Centre, IISc
• Started in 1977 with the first superconducting magnet based high field NMR• Started in 1977 with the first superconducting magnet based high field NMR
spectrometer in the country
•The Centre at present has seven NMR spectrometers under one roof, a unique
national distinction.
•The Centre has Six Faculty members and ~30 Students
•It also serves as a National NMR Facility for users from academia/Industry
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500 MHz Solids
NMR Research Centre, IISc
500 MHz Liquids
700 MHz Liquids
(cryo-probe) 500 MHz Liquids
400 MHz Liquids 300 MHz solids
800 MHz
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Fast NMR Methods
Isotope Labeling
Methods Development
Protein nanotubes
Structural Studies of
IGF-binding proteins
Malaria Vaccine
Research
Projects
Biology/Application
Automated
Analysis
Malaria VaccineProjects
@IISc
Metabolomics
Protein folding
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Carbon Nanotubes: An OverviewCarbon Nanotubes: An Overview
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The Discovery……The Discovery……
Monthioux, Marc; Kuznetsov, Vladimir L. (2006). "Who should be given the credit for the discovery
of carbon nanotubes?". Carbon 44: 1621.
Nature 354: 56–58 , 1991
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Carbon Nanotubes
• A single-walled carbon nanotube (SWNT) can be imagined as a rolled-up rectangular strip of
• Now recognized as an allotrope of carbon
(belongs to Fullerene family)
be imagined as a rolled-up rectangular strip of a graphene layer.
MWCNT
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Visualizing nanotubes
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Tools for studying Microstructures
• Microscopes based on optical image formation
The Optical Microscope
The Transmission Electron Microscope
• Microscopes based on projection image formation• Microscopes based on projection image formation
The Field ion Microscope
• Microscopes based on scanning image formation
Scanning Electron Microscope
Scanning Tunneling Microscope
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Application of bio-material based nanotubes
• New materials/Instrumentation (Molecular electronics)
• Drug Delivery
• Chemical and Bio-sensors
• Healthcare: therapeutics and diagonostics
• Antibacterial drugs
• Polymeric nanostructures
• Robotics
• Molecular Motors
• Environmental Improvement
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Protein Nanotubes as antibacterial drugs Bacterial
Cell Wall
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Insulin-like growth factor binding proteins
• 3D structure of full-length protein is not known for any
of the IGFBPs.
(i) difficult to over-express in E. coli due to large number of cysteines
(ii) Difficult to crystallize due to the flexible central domain
• 3D structure of individual domains of IGFBPs have been well studied.
N-terminal; IGFBP-4 C-terminal; IGFBP-2
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Insulin-like growth factor (IGF) system
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Growth Hormone
Insulin-like
growth factor
The Human Endocrine System
growth factor
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IGFBP
Proteases
IGF1/IGF2
IGF
System
IGFBP1-6
IGF
Receptors
The Insulin like growth Factor (IGF) System
Cancer
15
L
N C
IGF
IGFBP Proteases
IGFBPsIGF-R
Intracellular
signaling
N
C
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Drugs targeting the IGF system in Cancer
16
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Block this pathway
Insulin-like growth factor system
CANCER
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Insulin-like growth factor binding proteins (IGFBP)
• There are Six IGFBPs in our body (IGFBP-1 to IGFBP-6). IGFBP-3 is the most
abundant followed by IGFBP-2. They bind IGF-1 and IGF-2 strongly (KD ~ nM-pM)
N-terminal domain Central domain C-terminal domain
~100 aa ~100 aa ~100 aa
Flexible
• 3D structure of full length protein and/or its complex with IGFs
is not known for any of the IGFBPs.
(i) difficult to over-express in E. coli due to large number of cysteines
(ii) Difficult to crystallize due to the flexible central domain
• 3D structure of individual domains of IGFBPs have been studied.
Rosenzweig and Atreya, Biochem. Pharmacol. (2010)
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Insulin-like Growth Factor Binding Protein-2
IGFBP-2 1-96
IGFBP-2
6HIS.IGFBP-2
IGFBP-2 1-248
IGFBP-2 1-190
IGFBP-2 249-289
IGFBP-2 191-289
IGFBP-2 96-190
52 amino acids
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Cysteine: A peculiar amino acid
DTT AIR
Even number of
Cysteines
INSULIN
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S
S
S
Odd number of Cysteines
S
S
S
S
S
S
S
S
SS S S S
Dimer
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Odd number of Cysteines
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IGFBP-2 (249-289)
23
91
1.1
59
1SLin, Sm oothed
1000
1250
1500
Inte
ns. [a
.u.]
Oxidized
29
89
1.7
90
21
88
2.2
26
27
79
1.8
93
35
63
3.3
20
33
77
2.4
71
0
250
500
750
20000 22000 24000 26000 28000 30000 32000 34000 36000 38000
m /z
20,000 Da 30,000 Da
No peak < 10 kDaReduced IGFBP-2249-289
Oxidized
IGFBP-2249-289
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IGFBP-2 (249-289)
TEM
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IGFBP-2 Nanotubes
Oxidized Reduced (+DTT)
Reversible
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Fluorescence spectroscopy
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IGFBP-2 (249-289)
Mutation of the extra cysteine
R
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S
S
S
IGFBP-2 (249-289) Nanotube
Model for formation
S
S
S
S
S
S
S
S
SS S S S
Dimer
(Not a Nanotube)
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IGFBP-2 (249-289) Nanotube
Model for formation
S S S
S S
S
SS
S
S
S
S
SS
SS S S
SSSSS
Snucleation
Nanotube formationIGFBP-2249-289 (monomers)
Nanotube formation
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IGFBP-2 Nanotubes
3D Structure of Monomer
By NMR
Basic unit of Nanotube
8-monomer units
~25Å~25Å
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Reducing conditions
Oxidizing conditions
IGFBP-2 monomers ~ 5.6 kDa
SH
HSSH
SH
HS
SH
SH
HS
SH
SH H
S
SH
SH
HS
SH
SH H
S
SH
SH
HS
SH
SH
HS
SH
ss
s
~ 35 nm
Mechanism of nanotube formation
IGFBP-2249-289 monomers ~ 5.6 kDa
s
s
s
s
s
s
s
s
s
s
s
s
s
s
s
ss
s
ss
s
s
s
s
s
s
s s
s
s
s
ss
s
ss
s
s s
s
s s
s
s
s
s s
s
s s
s
ss
s
ss
s
s
s
Monalisa Swain et. al. Chem. Commun., 2010, 46, 216–218
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IGFBP-2 Nanotubes in Diabetes?
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Applications of IGFBP-2249-289 nanotubes
Cancer Cell Imaging
34
Cancer Cell Imaging
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INTEGRINS
Integrins are receptors that mediate attachment between a cell and
the tissues surrounding it.
They also play a role in cell signaling and thereby regulate cellular
shape, motility, and the cell cycle.
Integrin
35
Extracellular Matrix
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RGD
In Cancer cells integrins are present in large amount, and they
are known to bind to proteins containing an
Arg-Gly-Asp (RGD) motif
INTEGRINS
RGD
36
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RGD peptides for Tumor imaging
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CVNPNTGKLIQGAPTIRGDPECHLFYNEQQEACGVHTQRMQ
Can IGFBP-2 nanotube having a RGD motif
(IGFBP-2249-289) nanotubes
38
Can IGFBP-2 nanotube having a RGD motif
be used for
Cancer cell imaging ?
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Application of IGFBP-2 Nanotubes
Cell Adhesion: HT1080 Cancer line
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IGFBP-2 (249-289) Nanotube
FUTURE STUDIES
1. Analytical Ultra-centrifuge
(to find the size distribution of nanotubes)
2. Kinetics of formation of nanotubes (Can we trap the intermediate?)
using NMR Or Tyrosine fluoroscence
3 Diffusion Studies by NMR3 Diffusion Studies by NMR
4. Rigidity estimation of nanotubes by AFM
5. Formation of nantubes under: Different concentration,
different pH and temperature dependence
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IGFBP-2 (249-289) Nanotube
FUTURE STUDIES