Protein Nanotube based Probes for Cancer Cell Imaging

Protein Nanotube based Probes

for Cancer Cell Imaging

Hanudatta S. Atreya

Indian Institute of Science, Bangalore

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

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

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

Carbon Nanotubes: An OverviewCarbon Nanotubes: An Overview

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

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

Visualizing nanotubes

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

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

Protein Nanotubes as antibacterial drugs Bacterial

Cell Wall

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

Insulin-like growth factor (IGF) system

Growth Hormone

Insulin-like

growth factor

The Human Endocrine System

growth factor

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

Drugs targeting the IGF system in Cancer

16

Block this pathway

Insulin-like growth factor system

CANCER

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)

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

Cysteine: A peculiar amino acid

DTT AIR

Even number of

Cysteines

INSULIN

S

S

S

Odd number of Cysteines

S

S

S

S

S

S

S

S

SS S S S

Dimer

Odd number of Cysteines

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

IGFBP-2 (249-289)

TEM

IGFBP-2 Nanotubes

Oxidized Reduced (+DTT)

Reversible

Fluorescence spectroscopy

IGFBP-2 (249-289)

Mutation of the extra cysteine

R

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)

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

IGFBP-2 Nanotubes

3D Structure of Monomer

By NMR

Basic unit of Nanotube

8-monomer units

~25Å~25Å

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

IGFBP-2 Nanotubes in Diabetes?

Applications of IGFBP-2249-289 nanotubes

Cancer Cell Imaging

34

Cancer Cell Imaging

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

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

RGD peptides for Tumor imaging

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 ?

Application of IGFBP-2 Nanotubes

Cell Adhesion: HT1080 Cancer line

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

IGFBP-2 (249-289) Nanotube

FUTURE STUDIES