BIO-PROCESS ENGINEERING AND

PROTEIN SCIENCE FOR DRUG

DEVELOPMENT

Masafumi YOHDA
1

Tokyo University of
Agriculture and Technology

MYSTERIES IN PCR
Authors

of the first paper.

Why Dr. K. Mullis purified a thermostable DNA

polymerase by himself?

Why Dr. K. Mullis retired from Cetus?

Why the patent was transferred from Cetus to

Roche?
2

THE FIRST PAPER ON PCR

Saiki RK, Scharf S, Faloona F, Mullis KB, Horn GT,
Erlich HA, Arnheim N.
Enzymatic amplification of beta-globin genomic
sequences and restriction site analysis for diagnosis
of sickle cell anemia.
Science. 1985 Dec 20;230(4732):13503

KARY MULLIS
1962 Georgia Tech, Department of Chemical Engineering
Interest in Physics (including cosmology)
1966 UC Berkeley, Doctoral program in biochemistry
1968 Submit an article The Cosmological Significance of Time Reversal to Nature.
It was finally accepted after two rejections.
Ph.D. Structure and Organic Synthesis of Microbial Iron Transport Agents
1972-1975 Worked for a pediatric cardiology laboratory
1975-1977 Manager of a local restaurant and coffee shop.
1977 Started to work with Tom White of UCSF, DNA synthesis
1979 Tom White joined Cetus.
Tom White was appointed as a head of the Recombinant Molecular Research department.
He appointed Mullis as head a head of the DNA synthesis lab.
Development of DNA synthesizer. Mullis was active in suggesting improvement
1983 First presentation on the concept of PCR at the regular Cetus seminar
1984 Cetus scientific meeting.
Mullis presented a poster showing amplification of a beta globin gene.
However, the poster was generally ignored.
He scraped with colleagues, and was excluded from DNA synthesis group.
He could concentrate on development of PCR.
1993 Novel Prize in Chemistry
4

From Making PCR

BACKGROUNDS OF PCR (1)

of Bio-ventures in the 1st generations are
cloning and development of novel biopharmaceuticals
from human proteins.
A mission of Dr. Mullis is to synthesize DNA probes for
cloning DNAs.
As he was tried of Southern hybridization or colony
hybridization, he tried to devise a new method.
Most of researchers of Cetus thought that it is more
important to clone genes than developing new
technology, PCR. It seems reasonable from the
success stories of other companies (Amgen or
Genentech)
Targets

Backgrounds of PCR (2)

Although

many biochemists worked for Cetus, they

did not support Dr. Mullis to purify DNA polymerase
from thermophilic bacteria. They are too busy to purify
human proteins that will be candidates of biopharmaceuticals.
As the patent is owned by the company in USA. Dr.
Mullis got small money for the patent on PCR, and
finally left Cetus.
Cetus
was
interested
in
developing
biopharmaceuticals from Interleukin-2. The patent on PCR
was sold to Roche to obtain the license to use the
patent on Interleukin-2. It was good deal for Roche.
6

SALES OF BIOPHARMACEUTICALS IN JAPAN

IN THE 1ST STAGE

INCREASE OF BIO-PHARMACEUTICALS
2010

Ratio

2011

Ratio

2012

Ratio

Bio

70,832

31.6%

78,301

34.0%

84.327

39.3%

Small
molecule

153.307

68.4%

151.961

66.0%

130,394

60.7%

Total

224,139

230,262

214.721

Top 50s, in million dollars

Top 10 Losers (All small molecules)
Name

2012

Decrease

Lipidor

Statin/ Cholesterol Drug

5,028

-5,832

Plavix

Anti Platelet drug

5,277

-4,452

Seroquel

Anti psychotic drug

3,135

-3,052

Zyprexa

Anti depressant drug

1,734

-2,962

Lecsapro

Anti depressant drug

1,380

-2,593

Actos

Type 3 diabate drug

1,521

-2,486

18,075

-21,377

Total

STATIN HMG-COA REDUCTASE INHIBITOR

MEVALONIC ACID PATHWAY

10

DR. AKIRA ENDO

- DISCOVERY OF STATIN Akira Endo is the medical research scientist
who discovered the first statin drug-compactin.
The drugs used before this discovery to lower
cholesterol did so by increasing the removal of
cholesterol from the body or by inhibiting its
absorption from food. These methods were not
very effective and produced severe side effects.
Statins are a class of drugs with remarkable
cholesterol-lowering properties. They lower the
part of cholesterol known as bad cholesterol,
technically known as low density lipoprotein or
LDL cholesterol.
They work by limiting
cholesterol synthesis within the liver and have
proved to be a much safer and effective
alternative. In fact, these drugs have created a
revolution in the prevention and treatment of
coronary heart disease within over the past
couple of decades.
11

2010 PROBLEM OF PHARMACEUTICAL

COMPANIES
Patents on block basters have expired around 2010.

2008
Norvacs (Pfizer)
2010
Lipidor (Phizer)
2009
Takepron (Takeda)
2011
Actos (Takeda)
2012
Blopres (Takeda)
2010
Aricept (Eisai)
2010
Pariet Eisai)
2008 -2011 Harnal, Prograf (Astellas)
12

DIFFICULTIES IN DEVELOPING NEW DRUGS

Failure of Pfizer - Torcetrapib

Expected to decrease LDL (bad cholesterole) and increase LDL

(good cholesterol
Prevent cholesterosis and decrease cardiovacsular disturbance.
Expected maximum sales: 20 billion dollars.
Research and development expenditure800 million dollars
Unexpectedly, it increased the number of heat attacks in clinical
study.
10% of employees were fired, and the research laboratory in
Japan was closed.

It is difficult to develop new drugs nowadays.

1998 FDA approved 53 new drugs.
After 2000 There has been no year, in which more than 30 new
drugs were approved.
13
20007 Only 18 new drugs.

DRUGS IN 21ST CENTURY

Bio-Pharmaceuticals
It is difficult to produce Bio-Pharmaceuticals
with the same quality by Generic Maker.
Molecular Target Drugs
Small Molecule: Glivec
Chronic Myelogenous Leukemia (CML)
Bio-Pharmacetuticals: Antibody
Taylormade Drugs
Based on the genomic information,
appropriate drugs will be selected to each
patient.

14

EFFICACY AND SIDE EFFECTS OF MEDICINES VARY

WITH THE GENETIC POLYMORPHISMS IN DRUGMETABOLIZING ENZYMES, TRANSPORTERS,
RECEPTORS, AND OTHER DRUG TARGETS

Drug-metabolizing
enzymes

Blood

target cell

Drug

Receptor

metabolize

signal
transduction
Response

excretion

f. e. Effect of Genetic polymorphisms of Drug-metabolizing enzymes

Blood

Blood

Drug

excretion
EM

Drug

excretion
PM

extensive metabolizer
intermediate metabolizer
poor metabolizer

CYTOCHROME P450 2C19 (CYP2C19)

The CYP2C19 gene is located on chromosome 10q24. So far, 21 SNPs
have been found in CYP2C19. But in the Japanese have variants only *2
and *3. (*1 is wild type)
*3

exon 4
*1Wild type

*2

The *2 (m1) alleles (subtypes A and B) have a

defining mutation of a G681 to A substitution
that results in a splicing defect. Subtypes are
not differentiated.

exon 5

The *3 (m2) allele has a defining mutation of a

G636 to A substitution that results in a Trp212
to stop codon change.

1. Most proton pump inhibitor drugs are metabolized by

CYP2C19.
2. Number of poor metabolozer in asian people are
relatively abundant compared with other ethnics.
- Relatively high risk of side effects.

SUCCESS OF AMGEN
- EPO & G-CSF -

EPO

G-CSF

17

INCREASE OF DIALYSIS PATIENTS IN JAPAN

18


EPO

EPO is produced in Kidney

19

EFFECTS OF EPO (ERYTHROPOETIN)

Dialysis

Dialysis 2 or 3 times a week

Removal uremix toxins
Decrease of EPO
Decrease of erythrocytes
being in anemia

Renal Failure

EPO

Increase of erythrocyte
Improvement of QOL

20

RECOMBINANT HUMAN ERYTHROPOETIN

1
Ala Pro Pro Arg Leu

Ile

ss

Leu Leu Glu Ala Lys Glu Ala Glu Asn

160
Thr
Tyr

150

Leu

Asn
Phe

Val

Lys Thr Asp Pro Val Thr

Ile Asn Glu Asn Leu Ser Cys

130
Thr Asp Ala Thr

Ala Trp Lys Arg Met Glu Val Gly Gln Gln Ala Val Glu

120
Gly
Leu
Ala

Ala Gln Lys Glu Ala

110

70

80

Ile Thr Arg Leu Pro Ala Ala Ser Ala Ala

Ile Ser

Asp
Pro
Pro

Leu

30
Glu
Ala
His

60

Lys
Phe

Thr Thr Gly Cys

s
s

50

Tyr

Leu Phe Arg Val Tyr Ser Asn Phe Leu Arg Gly Lys Leu Lys

Arg

Ile

40

Asp Gly Thr Arg Cys Ala Glu Gly

140

20

10
Cys Asp Ser Arg Val Leu Glu Arg Tyr

Val
Trp
Gln

Ala Gln Gly Arg Leu Val Ala Glu Ser Leu Leu Ala Leu Gly

Leu
Val

Asn Ser Ser Gln Pro Trp Glu

90
Pro
Leu

100

Arg Leu Leu Thr Thr Leu Ser Arg Leu Gly Ser Val Ala Lys Asp Val His Leu

Gln

:N-Glycoside carbohydrate chain

: O-Glycoside carbohydrate chain

: Disulfide bond

21

PRODUCTION OF EPO USING ANIMAL CELL

22

PRODUCTION OF EPO
Culture

Purification

Bulk Drug

Preparation
Filling
23

Packaging

Product

EPO PRODUCTION SYSTEM DEVELOPED

BY KIRIN

24

Roller Bottle

25

LARGE SCALE CULTURE SYSTEM

26

PURIFICATION SYSTEM

27

COMMERCIAL HUMAN ERYTHROPOETIN

28

EFFECT OF G-CSF (GRANULOCYTE

COLONY STIMULATION FACTOR)
Anti-cancer drugs (e.g. Cisplatin, Adryamicin)
Death of malignant cells
Death of normal cells
Weaken immune system
Treatment in sterile room

Cancer
Patient

G-CSF

Increase of
white blood cells
Recovery of Immune system
Increase of survival ratio

29

RECOMBINANT G-CSF

30

PRODUCTION OF RECOMBINANT G-CSF

BY E. COLI

31

FERMENTER FOR RECOMBINANT E. COLI

32

CELL DISRUPTOR FOR E. COLI

33

REFOLDING FROM INCLUSION BODY

34

COMMERCIAL HUMAN G-CSF

35

ADRENERGIC RECEPTORS

36

ALPHA BLOCKER AND BETA BLOCKER

37

DEVELOPMENT OF DRUG BASED ON

STRUCTURAL INFORMATION
- SUCCESS OF HIV PROTEASE INHIBITORS HIV protease inhibitors were first invented between 1989
and 1994 by researchers working for the pharmaceutical
companies of Hoffmann- La Roche Inc. (of Nutley, New
Jersey), Abbott Laboratories and Merck & Co., Inc. HIV
protease inhibitors are used in the treatment of patients
with AIDS and were considered the first breakthrough in
over a decade of AIDS research. HIV protease inhibitors
can
lower
the
viral
load
carried
by
AIDS
patents. Currently, there are five HIV protease inhibitors
approved by FDA for the treatment of HIV infection. These
medications work at the final stage of viral replication and
attempt to prevent HIV from making new copies of itself
by interfering with the HIV protease enzyme. As a result,
the new copies of HIV are not able to infect new cells.
38

ANTI HIV DRUGS

Reverse transcriptase inhibitor

HIV belongs to the family of retrovirus, and
depends on reverse transcriptase. As we
dont have reverse transcriptase, it was first
target of anti-HIV drug
- Selected from nucleotide analogues.

HIV protease inhibitor

Proteins of HIV are firstly translated and then it
is cleaved and released to functional proteins.
The protease was ideal drug target as it
showed unique characters.
39
- Designed based on the structure

SURVIVAL OF CD4+ CELLS BY REVERSE

TRANSCRIPTASE INHIBITORS

By Prof. Hiroaki Mitsuya

He did not apply for the patent
on AZT. Afterward, he applied
patent ddC, another inhibitor.
Why?

40

In 1985 Dallas, homophobic, drug addicted

party boy Ron Woodroof is diagnosed
with HIV and is given 30 days to live. He
starts taking the Food and Drug
Administration (FDA)-approved AZT, the
only drug legally available in the U.S,
which brings him to the brink of death. To
survive,
he
smuggles
anti-viral
medications from all over the world, which
were still unapproved and unavailable in
the U.S. Other AIDS patients seek out his
medications forgoing hospitals, doctors,
and AZT. With the help of his doctor, Eve
Saks (Garner) and a fellow patient, Rayon,
Ron creates the Dallas Buyers Club, one
of the dozens which form around the
country, providing its paying members
with these alternative treatments. The
clubs, growing in numbers and clientele,
are brought to the attention of the FDA
and pharmaceutical companies, which
wage an all out war on Ron.
41

STRUCTURE OF HIV PROTEASE

42

STRUCTURAL GENOME PROJECT

Although almost all protein coding genes were

identified by Human Genome Project, most of their
structural and functional information of them were
still unveiled.
Structural Genome Project has started to determine
3D structures of proteins encoded on the human
genome.
Based on the assumption that basic structures of
proteins should be at most 10,000, the tentative
purpose was 10,000 structures.
Japan undertook 30%. Protein 30000 project.
43

SUCCESS AND FAILURE OF STRUCTURAL

GENOME PROJECT

Achievement of Protein 3000 project

More than 3000 structures were determined

Infrastructures of structural biology
Human resources for structural biology

Failure of Protein 3000 Project and its cause

Limited number of basic structures

Especially membrane proteins

Limited number of drug targets

Difference between HIV Protease and adrenergic G

protein coupled receptor

44

45

DR. KOBILKA RECEIVED NOBEL PRIZE JUST AFTER

5 YEARS FROM STRUCTURE DETERMINATION OF GPCR

46

Mechanism of Breast Cancer

47

HER2 and Molecular Targeting Drug

48

ANOTHER APPROACH
- ANTIBODY DRUG

Herceptin (trastuzumab) is cancer medication that interferes with

the growth and spread of cancer cells in the body. It is a
humanized antibody against HER2, the product of human
oncogene HER2/neu(c-erbB-2). cancer medication that interferes
with the growth and spread of cancer cells in the body.

Tocilizumab(INN, or atlizumab, developed by HoffmannLa

Roche and Chugai and sold under the trade names Actemra and
RoActemra) is an immunosuppressive drug, mainly for the
treatment of rheumatoid arthritis (RA) and systemic juvenile
idiopathic arthritis, a severe form of RA in children. It is a
humanized monoclonal antibody against the interleukin-6
receptor (IL-6R). Interleukin 6 (IL-6) is a cytokine that plays an
important role in immune response and is implicated in the
pathogenesis of many diseases, such as autoimmune diseases,
multiple myeloma and prostate cancer.
49

PRESCRIPTION OF HERCEPTIN

For use in the treatment of metastatic breast cancer:

Administer trastuzumab, alone or in combination with paclitaxel.
Initial dose: 4 mg/kg IV infusion over 90 minutes
Subsequent therapy: 2 mg/kg IV infusion over 30 minutes once
weekly until disease progression
Extremely large amount is required. Thus, too expensive.

50

STRUCTURE OF ANTIBODY

51

HUMANIZED ANTIBODY

52

ADVANCEMENT OF ANTIBODY
PRODUCTION

53

IMPORTANCE OF PRODUCTION TECHNOLOGY

To help people in disease, drugs must be cheap and be

supplied sufficiently.

54

STUDIES ON NITRILE HYDRATASE

AND MOLECULAR CHAPERONES

Masafumi YOHDA

Department of Biotechnology and Life Science

Tokyo University of Agriculture and Technology

ACRYLAMIDE
Industrially important material
Used for production of polymer coagulant, waste
water treatment reagent, soil modifier, paper
strengthening agent, paint, resin et al.
Produced from acrylnitrile

H
H

C C

H
CN

Acrylnitrile

H2O

H
H

C C

H
CONH2

Acrylamide

COMPARISON OF ACRYLAMIDE PRODUCTION

PROCESS

Cupper Catalyst
Method

Acrylnitrile
H2 O

Hydrolysis

Catalyst
Preparatio
n
Removal
of catalyst

Concentration

Removal
of Cu ion
Acrylamide

Unreacted AN

Bio-Catalyst
Method

Acrylnitrile
H2O

Removal
of catalyst

Hydrolysis

Acrylamide

Acrylnitrile
H2 O

Acrylamide

CO2 Production [kg-CO2/ kg-AAm]

COMPARISON OF CO2 PRODUCTION

5
4
3

2
1
0

Raw
Materials
Steam
Electric
Power
Catalytic
Process

Enzymatic
Old-Process

Enzymatic
New-Process

Environmental Information Science (1996) 25(3) 61

SCREENING MICROBES TO
PRODUCE ACRYLAMINDE
Nitrilase
R-CO2H

R-CN

Nitrile hydratase

R-CONH2

NH3

Amidase

Activity
Measuremen
t

Inoculatio
n
Soil Sample
Subcultur
e

Subculture

Plate

HISTORY OF MICROBES USED FOR

ACRYLAMIDE PRODUCTION
CH2=CHCN + H2O CH2=CHCONH2
Nitrile Hydratase

3rd Generation
Rhodococcus rhodochrous J1
2nd Generation
Pseudomonas chlororaphis B23

1st Generation

Rhodococcus sp.N-774

HISTORY OF INDUSTRIAL ACRYLAMIDE PRODUCTION

Copper catalyst
1969
Sulfuric acid
catalyst
1954

AA production by
Sulfuric acid catalyst
Nitto Chem. Co.
1957

1974

Discovery of acrylamide producing microorganisms

Rhodococcus sp. N7741978 Nitto Chem. Co.
P. chlororaphis B23 1981 Kyoto Univ.
R. rhodochrous J1 1986 Kyoto Univ.

A.A. production by
Bio-plants
Nitto Chem. Co.
1985
N774
4,000/year
1985

B23
6,000/year
1988

SNF Co. France

was licensed to use
Bio-plants.
1999
J1
15,000/year20,000/year
19911995

These microorganisms possess nitrile hydratase which catalyzes

the hydration of acrylonitrile to acrylamide.

IMPROVEMENT OF NITRILE HYDRATASE FOR

THE PRODUCTION OF ACRYLAMIDE
Microorganism used
Rhodococcus
sp. N-774

Pseudomonas
chlororaphis
B23

Rhodococcus
rhodochrous
J1

Enzyme type

Fe

Fe

Co

Tolerance to acrylamide (%)

27

40

50

Acrylic acid formation

vl*

bd*

bd*

Acrylamide productivity(g/g-cells)

500

850

>7000

Final concentration of acrylamide

(%)

20

27

40

*Abbriviations: vl, very little; bd, barely detected.

TIBTECH, 10, 402-408, 1992

APPLICATION OF NITRILE HYDRATASE

Rhodococcus
rhodochrous J1
3-Cyanopyridine

Adiponitrile

Nicotineamide

Pseudomonas
chlororaphis B23

5-cyanovaleramide

PRODUCTION OF NICTINEAMIDE
BY NITRILE HYDRATASE

0 :
J1
1 :
6 :
18:

Material (3-Cyanopyridine) with NHase from Rhodococcus rhodochrous

Reaction intermediates
Product (Acrylamide)
Product (acrylamide)

NHASE IS USED FOR INDUSTRIAL

ACRYLAMIDE PRODUCTION
Acrylamide Production (kilo-ton)

50
45
40

BioNHase
process
Chemical catalyst
Copper
catalyst

35
30
25
20
15
10
5
0

80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 00 01 02 03 04 05

Year

PRODUCTION PLANT FOR ACRYLAMIDE

YOKOHAMA Factory of Mitsubishi Rayon CC.

300,000 t of acrylamide is produced annually.

DISCOVERY OF PHOTOACTIVATION
NHase activity of Rhodococcus sp.N771 and N774
varies with cultivation conditions.

Small Scale
High Activity
Large Scale
Low Activity

NHase Activity [units/mg-dry-cell]

PHOTOACTIVATION OF NHASE
50

Light Activation
40
30
20
10

0
0

Dark Inactivation
10

20

30

Time [h]

40

50

Photoreactivity of NHase is
Regulated by NO
hu

NO

b NO a
Fe

RCN
H2 O

RCONH2

a
Fe

Inactive NHase
(Nitrosylated)

dark

NO

Active NHase

The reaction of NHases in a crystal can be simultaneously

started by light activation

DIFFERENCE FTIR SPECTRA OF NHASE

BETWEEN BEFORE AND AFTER
PHOTOACTIVATION

A: Natural

B: N15 Label

Noguchi et al., FEBS. Lett. (1995) 358,9-12

PHOTOREACTIVITY OF NHASE IS REGULATED BY NO

1853
1865

Activity [U/mg]

1844
0.002

A. Inactive form

6.5

h
B. Active form

831

NO
NO

D. Active NO
Light

718

C. Active
h

1900

1880

1860

1840

Wavenumber

1820 1800
(cm-1)

(Odaka et al., J. Am. Chem. Soc. 1997)

REGULATION OF NHASE BY NO
hu
hu

NO

b NO a
Fe

RCN
H2 O

RCONH2

a
Fe

Dark
Inactive
NO Bound Form

NO

Active
Without NO

SPECTRAL CHANGE INDUCED BY

PHOTOACTIVATION

PHOTOREACTIVITY IS INDEPENDENT
OF 3D STRUCTURE

ISOLATION OF PHOTOREACTIVE
SUBUNIT

PHOTOREACTIVE PROTEASE
DIGESTED FRAGMENT

Tsujimura J Biol Chem. 1997) 272,29454

MASS SPECTROMETRY OF
PHOTOREACTIVE PEPTIDE

Tsujimura et al. J Biol Chem. 1997) 272,29454

MODIFICATION OF CYS TO
CYSTEINE SULFINIC ACID

Tsujimura J Biol Chem. 1997) 272,29454

NITRILE HYDRATASE (NHASE)

Catalytic center
Fe (III) or Co(III)
Hetero-dimer ,
(MW=23kDa)

OH2

R-CN
Nitriles

R-CO-NH2
Amides

NHase is used for the industrial production of

acrylamide and nicotinamide.

CRYSTAL STRUCTURE OF FE-TYPE NHASE

(NO-BOUND FORM) AND ITS METALLOCENTER
1,4-dioxane
Cys114
subunit

Arg56

NO

Active Center

Arg141
Cys112

subunit
Nitrosylated form
(inactive)

Fe

STRUCTURE OF FE-CENTER OF NHASE

NO
Ser113

Cysteine sulfenic
acid
Cys114-SOH 2 main chain amide

notrogen as
coordinated.

oxidation state of 3 Cys

sulfures are different.

FeIII
Amido
nitoroge
n

NHase is the first

example having posttranslationally modified
cysteine ligands.

Cys109-SH
Cysteine sulfinic
acid

Oxygen

Carbon Cys112-SO2H

Nitrogen

Sulfur (Nagashima et al., Nat. Struct, Biol. (1998) 5, 34

METHOD FOR TIME RESOLVED

X-RAY CRYSTALLOGRAPHY
1. Crystals of NO bound NHase was soaked
with tBuNC.
2. NHasetBuNC complexes were photoactivated.
3. At each time, the crystals were flash cooled
with Nh
2 gas at 95K (Reaction stop).

293K

293K

293K

293K

95K

293K
Flash-Cooling

0 min

0 min

X min

TIME RESOLVED CRYSTALLOGRAPHY

0 min

18
min

NO

tert-butylisonitrile
(tBuNC)

120 min

440 min

> 440
min

120 MIN
FeBrown
N Blue
S Green
C Yellow
O Red

t-BuNC directly binds Fe ion at the catalytic site

440 MIN
FeBrown
N Blue
S Green
C Yellow
O Red

Hydrolysis intermediate of
tBuNH2

PREDICTED REACTION MECHANISM

K. Hashimoto et al., J. Biol. Chem., 283, 36617 (2008)

DOGMA OF ANFINSEN
Christian B. Anfinsen

1972 Nobel Prize in Chemistry

Anfinsens Experiment

BOILED EGG PROBLEM

Heat

Raw Egg

Cool, but
irreversible

Boiled Egg Fried Egg

DISEASE OF PROTEIN
-IF IT IS ISOLATED, OK. BUT ---

Denature

Aggregation

Renature

Boiled Egg

CROWDED CONDITION IN CELL

There exist 30 ribosomes, 340 tRNAs,

2 GroEL, 500 other proteins in
100nm3 of E. coli cytosol.

PROTEIN AGGREGATION DISEASES

Alzheimers Disease
Parkinsons Disease
Poly-Glutamine Disease

Huntingtons Disease

Prion Disease

Creutzfeldt-Jakob disease: CJD

Mud Cow Disease

ALZHEIMERS DISEASE

AMYLOID PLAQUES

FORMATION OF AMYLOID

POLYGLUTAMINE DISEASE

POLYGLUTAMINE DISEASES

Huntingtin

PRION DISEASES

TRANSMISSION OF PRION DISEASES

DISEASE OF PROTEIN
-RESCUE BY CHAPERONEDenature

Isolation

Cure

By Chaperone
Chaperones

CHAPERONIN

GroES Heptamer

GroEL-ES Coplex

Chaperonin is the representative of molecular chaperones. It captures an

unfolded protein in its cavity and refold it in ATP dependent manner.

2011 LASKER AWARDS

Chaperones

LIFE OF PROTEINS

HEAT SHOCK PROTEINS

Heat shock proteins (HSP) are a family of proteins that are
produced by cells in response to exposure to stressful conditions.
They were first described in relation to heat shock, but are now
known to also be expressed during other stresses including
exposure to cold, UV light, and during wound healing or tissue
remodeling.[4] Many members of this group perform chaperone
function by stabilizing new proteins to ensure correct folding or by
helping to refold proteins that were damaged by the cell stress. This
increase in expression is transcriptionally regulated. The dramatic
upregulation of the heat shock proteins is a key part of the heat
shock response and is induced primarily by heat shock factor
(HSF). HSPs are found in virtually all living organisms, from bacteria
to humans.

HISTORY OF HEAT SHOCK PROTEINS

It is known that rapid heat hardening can be elicited by a brief
exposure of cells to sub-lethal high temperature, which in turn
provides protection from subsequent and more severe temperature.
In 1962, Italian geneticist Ferruccio Ritossa reported that heat and
the metabolic uncoupler 2,4-dinitrophenol induced a characteristic
pattern of puffing in the chromosomes of Drosophila. This
discovery eventually led to the identification of the heat-shock
proteins (HSP) or stress proteins whose expression these puffs
represented. Increased synthesis of selected proteins in Drosophila
cells following stresses such as heat shock was first reported in
1974.
Beginning in the mid-1960s, investigators recognized that many
HSPs function as molecular chaperones and thus play a critical role
in protein folding, intracellular trafficking of proteins, and coping
with proteins denatured by heat and other stresses. Therefore, the
study of stress proteins has undergone explosive growth.

DROSOPHILA - HEAT SHOCK

PROTEINS, CHROMOSOMAL
PUFFS

CLASSIFICATION OF HEAT SHOCK

PROTEINS
Molecular Weight
(kDa)

Prokaryotic
Proteins

Eukaryotic
Proteins

Function

10kDa

GroES

Hsp10

Co-factor of
GroEL/Hsp60

20 kDa 30kDa

sHsps

HspB group
proteins, Hsp27

40kDa

DanJ

Hsp40

Co-factor of Hsp40

60kDa

GroEL

Hsp60

Protein folding

DanK

HspA group proteins,

Hsp70, Grp 78

Protein folding or
preventing protein
folding

70 kDa

90kDa

HtpG

Hsp90, Grp94

Maintenance steroid
receptors, protein
Kinases

100kDa

ClpA, B, X

Hsp104, Hsp110

Protein degradation,
disaggregation

STRUCTURE OF GROELS

FUNCTION CYCLE OF CHAPERONIN

QUANTUM DOT (Q-DOT)

CdS or CdSe

Several nm

1. Wavelength of the fluorescence varies with its size.

2. The size of Q-dot is about several nm.
3. The fluorescence is very stable.
4. Q-dots coagulate and lose fluorescence in aqueous solution due to
its hydrophobicity.
5. In medical or biological applications, Q-dots coated by hydrophilic
materials are used.
Drs Aida and Kinbara have realized that Q-dot has similar characteristics as
unfolded proteins.

CHAPERONIN-MEDIATED STABILIZATION AND

ATP-TRIGGERED RELEASE OF Q-DOT

Ishii et al. (2003) Nature

FORMATION OF T.TH CPNCDS Q-DOT COMPLEX

AND ITS SPECIFIC RESPONSE TO ATP

Ishii et al. (2003) Nature

TRANSMISSION ELECTRON MICROGRAPHS

OF T.TH CPNCDS Q-DOT

Ishii et al. (2003) Nature

Features of group I and group II

chaperonins
Group I chaperonins

Group II chaperonins

Are found in eubacteria and in

Exist in eukaryotic cytosol (CCT)

and in archaea (thermosome)

endosymbiotic organelles (mitochondria

and chloroplasts)

Are independent of GroES - Built-in

lid

Have a detachable lid (GroES)

GroES

ATP
binding

ATP
hydrolysis

GroEL

ATP
binding

?
ATP
hydrolysis
How does it work without co-chaperonin ?

Bukau & Horwich (1998) Cell 92, 351-366

Chaperonin from a hyperthermophilic

archaeum, Thermococcus strain KS-1
Exhibits high protein folding
activity
Is composed of two highly
homologous subunits (a, b)
Each subunit forms homooligomer and functions as a
chaperonin.
Crystal structure of aG65C/I125T mutant
Shomura et al. (2004) J Mol Biol

Helical protrusion of group II

chaperonin
The protrusion is thought to
seal off the central cavity
(Built-in lid).
The region is also assumed

Helical
protrusion

to be involved in the binding

of substrate proteins.

However, the exact role

is quite unclear.

Subunit structure of group II

chaperonin

Model for functional cycle of

T. KS-1 chaperonin
Native
polypeptide

Non-native
polypeptide

ATP
H
A
I
E

Nucleotidefree form

ATP-bound
form

Pi

ADP-bound
form

A, I, and E refer to the apical, intermediate, and equatorial domains, respectively.

H represents the helical protrusion.

Iizuka et al (2004) J Biol Cem

CONFORMATIONAL CHANGE MODEL

Booth CB, et al. (2008) Nat Struct Mol Biol, 15(7):746-53

ATP-INDUCED STRUCTURAL CHANGE OF

GROUP II CHAPERONIN

Kanzaki et al. (2008) J Biol Chem

Cryo-EM, single particle

reconstruction
lid-closure with twisting
motion
C. R. Booth et al. (2008)
Nature Struct. Mol. Biol

TO INVESTIGATE PROTEINS DYNAMICS

Specialized Method for Protein Dynamics
labeling

Labeling is also valid for

synchrotron radiation analysis

Tracer: nanocrystal
Diffracted X-ray Tracking

nanocrystal

Observed Rotational motion of F1-ATPase

Nature 386:299(1997)

protein
120

DIFFRACTED X-RAY TRACKING (DXT)

Laue spot

X-rays

Laue spot
moves

gold nanocrystal
(20-50 nm)

protein

Structural
Change

Features:
1. High accuracy(m rad, pico meter level)
2. Time-resolved information (ms to s)
3. Independent from chemical conditions
4. Applied to in vivo measurement

THE METHOD FOR MONITORING TWISTING MOTION:

DIFFRACTED X-RAY TRACKING (DXT)

Au

DIFFRACTED X-RAY TARCKING: DXT

Twisting

Tilting

White
X-ray

White
X-ray

Resolution of DXT
direction = 1 mrad (0.057)

direction = 6 mrad (0.34)

INSTRUMENTATION OF DXT
D263C
Image
Intensifier

Chaperonin
KS-1
(D263C/C366S)

Sample

V7739P (Hamamatsu
Photonics)

Thermococcus chaperonin

polyimide film

polyimide film
aqueous
gold
solution

White Xray
heater

X-rays
gold nano
crystal
protein

50 m

CCD:
C4880-80(Hamamatsu
photonics)

Sample
PF-AR NW14A
(35-60mA)

TWISTING MOTION WAS DETECTED

2=
0.2rad

Sekiguchi et al. (2013) PLoS One

TRACES OF DIFFRACTION SPOTS

60, 0mM ATP

RT, 2mM ATP

60 , 2mM ATP

50mM MOPS, 10mM MgCl2, 100mM KCl, pH=7.5

Chaperonin (KS-1 0.5mg/ml) on Au-surface
PF-AR NW14@KEK
Sekiguchi et al. (2013) PLoS One

MEAN-SQUARE DISPLACEMENT
25

20

15

140

60, 2mM ATP

25, 2mM ATP
60, 0mM ATP

120

MSD
(mrad2)

100

MSD
(mrad2)

80

60

10

40
5
2
0
0

100

200

time (ms)

300

400

100

200

time (ms)

300

400

THE RING TWISTS CW OR CCW ?

UV
irradiation

caged ATP
(inactive ATP)

ATP

transparent teflon
film
gold
X-rays

UV-triggered DXT :
To confirm the twisting direction of
chaperonins ring when ATP binds to
the ring

Au-crystal
protein

Laser
355nm

70 m thick

Sekiguchi et al. (2013) PLoS One

128

Angular displacement

CAGED ATP: TWISTS CCW AFTER UV FLASH

129

THANK YOU VERY MUCH