BIO-PROCESS ENGINEERING AND

PROTEIN SCIENCE FOR DRUG

DEVELOPMENT

Masafumi YOHDA

Tokyo University of

Agriculture and Technology

1

2

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?

MYSTERIES IN PCR

3

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):1350-

4

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

KARY MULLIS

From Making PCR

5

Targets 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)

BACKGROUNDS OF PCR (1)

6

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 bio-

pharmaceuticals.

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 bio-

pharmaceuticals 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.

SALES OF BIOPHARMACEUTICALS IN JAPAN

IN THE 1ST STAGE

7

INCREASE OF BIO-PHARMACEUTICALS

8

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

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

Total 18,075 -21,377

Top 10 Losers (All small molecules)

STATIN HMG-COA REDUCTASE INHIBITOR

9

MEVALONIC ACID PATHWAY

10

11

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.

DR. AKIRA ENDO

- DISCOVERY OF STATIN -

2010 PROBLEM OF PHARMACEUTICAL

COMPANIES

12

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)

13

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.

20007 Only 18 new drugs.

DIFFICULTIES IN DEVELOPING NEW DRUGS

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

DRUGS IN 21ST CENTURY

Drug-metabolizing

enzymes

Blood

Drug Receptor

signal

transduction

Response

target cell

metabolize

excretion

extensive metabolizer

intermediate metabolizer

poor metabolizer Blood

Drug

excretion

Blood

Drug

excretion

EM PM

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

EFFICACY AND SIDE EFFECTS OF MEDICINES VARY

WITH THE GENETIC POLYMORPHISMS IN DRUG-

METABOLIZING ENZYMES, TRANSPORTERS,

RECEPTORS, AND OTHER DRUG TARGETS

CYTOCHROME P450 2C19 (CYP2C19)

*3 *2

exon 4 exon 5

*1Wild type

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.

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

G636 to A substitution that results in a Trp212

to stop codon change.

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)

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.

17

SUCCESS OF AMGEN - EPO & G-CSF -

EPO

G-CSF

INCREASE OF DIALYSIS PATIENTS IN JAPAN

18

EPO

19

EPO is produced in Kidney

EPO

Dialysis

Renal Failure

Dialysis 2 or 3 times a week Removal uremix toxins Decrease of EPO Decrease of erythrocytes being in anemia

Increase of erythrocyte Improvement of QOL

EFFECTS OF EPO (ERYTHROPOETIN)

20

:N-Glycoside carbohydrate chain

: O-Glycoside carbohydrate chain

: Disulfide bond

Glu Ala

Ala Ala Ala Arg Ile Leu Arg Pro Pro Arg Leu Ile Asp Ser Val Leu Leu Glu Glu Glu Glu Tyr Lys Asn Thr Thr Gly Cys Cys

Pro

Leu

Leu

Leu

Ile

Asp

Val

Lys

Leu Leu

Tyr Tyr

Asn

Lys

Lys

His

His Cys

Asn

Asn

Ile

Thr

Asp Asp

Pro Ser Val Leu Asn Asn Thr Glu Ile Thr Asp

Phe

Phe

Phe

Ala

Ala

Trp

Trp

Trp

Arg Arg

Arg

Val

Val

Gly

Ala Arg Val Lys Gly Glu Met Val Gln Gln

Gln

Gln

Gln

Glu

Glu

Glu

Leu Leu

Ser Ser

Ser

Ala Ala

Ala Ala

Val Val

Val Gly Gln

Leu Leu

Leu

Ser

Ser Ser

Pro

Pro Pro Glu Gln Lys

Val Gly

Gly

Gly Leu Leu Leu

Thr Thr

Thr

Leu

Arg

Arg Ala

Ala

Ser

Ser Pro Ala Ala Ala Ala Thr

Lys Lys Phe

Tyr

Leu Leu Arg

Cys Glu Ala Asp Arg Gly Gly Thr Thr

s s

Leu

30

40

60 50

70 80

90

100 110

120

130

10 20

1

150

140

160 s s

Ala

Leu

Arg

RECOMBINANT HUMAN ERYTHROPOETIN

21

22

PRODUCTION OF EPO USING ANIMAL CELL

Culture

Purification

Preparation

Filling

Packaging

Bulk Drug

Product

PRODUCTION OF EPO

23

EPO PRODUCTION SYSTEM DEVELOPED

BY KIRIN

24

Roller Bottle

25

LARGE SCALE CULTURE SYSTEM

26

PURIFICATION SYSTEM

27

COMMERCIAL HUMAN ERYTHROPOETIN

28

Increase of

white blood cells Recovery of Immune system

Increase of survival ratio

Death of malignant cells

Death of normal cells

Weaken immune system

Treatment in sterile room

G-CSF

Anti-cancer drugs (e.g. Cisplatin, Adryamicin)

Cancer

Patient

EFFECT OF G-CSF (GRANULOCYTE

COLONY STIMULATION FACTOR)

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

37

ALPHA BLOCKER AND BETA BLOCKER

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

DEVELOPMENT OF DRUG BASED ON

STRUCTURAL INFORMATION

- SUCCESS OF HIV PROTEASE INHIBITORS -

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.

- Designed based on the structure 39

ANTI HIV DRUGS

SURVIVAL OF CD4+ CELLS BY REVERSE

TRANSCRIPTASE INHIBITORS

40

By Prof. Hiroaki Mitsuya

He did not apply for the patent

on AZT. Afterward, he applied

patent ddC, another inhibitor.

Why?

41

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.

42

STRUCTURE OF HIV PROTEASE

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

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

SUCCESS AND FAILURE OF STRUCTURAL

GENOME PROJECT

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 -

49

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.

PRESCRIPTION OF HERCEPTIN

50

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.

STRUCTURE OF ANTIBODY

51

HUMANIZED ANTIBODY

52

ADVANCEMENT OF ANTIBODY

PRODUCTION

53

54

IMPORTANCE OF PRODUCTION TECHNOLOGY

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

supplied sufficiently.

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

H2O H

H C C

H

CONH2 Acrylamide Acrylnitrile

COMPARISON OF ACRYLAMIDE PRODUCTION

PROCESS Cupper Catalyst

Method

Bio-Catalyst

Method

Hydrolysis Removal

of Cu ion Concentration

Acrylamide

Removal

of catalyst

Acrylnitrile

H2O

Catalyst

Preparatio

n

Acrylamide

Unreacted AN

Acrylnitrile

H2O

v Acrylnitrile

H2O Acrylamide

Hydrolysis Removal

of catalyst

COMPARISON OF CO2 PRODUCTION

0

2

3

4

5

1

Raw

Materials

Steam

Electric

Power

CO

2 P

rod

ucti

on

[kg-C

O2/ k

g-A

Am

]

Catalytic

Process

Enzymatic

New-Process

Enzymatic

Old-Process

Environmental Information Science (1996) 25(3) 61

SCREENING MICROBES TO

PRODUCE ACRYLAMINDE

R-CN R-CO2H

R-CONH2

Nitrilase

Nitrile hydratase Amidase

NH3R-CN R-CO2H

R-CONH2

Nitrilase

Nitrile hydratase Amidase

NH3

Activity

Measuremen

t

Soil Sample

Subcultur

e

Plate Subculture

Inoculatio

n

HISTORY OF MICROBES USED FOR

ACRYLAMIDE PRODUCTION

Nitrile Hydratase

1st Generation

Rhodococcus sp.N-774

2nd Generation

Pseudomonas chlororaphis B23

3rd Generation

Rhodococcus rhodochrous J-

1

CH2=CHCN + H2O CH2=CHCONH2

HISTORY OF INDUSTRIAL ACRYLAMIDE PRODUCTION

Sulfuric acid

catalyst

1954

Copper catalyst

1969

1974

A.A. production by

Bio-plants

Nitto Chem. Co.

1985

N774

4,000/year 1985

B23

6,000/year 1988

J1

15,000/year20,000/year 19911995

SNF Co. France was licensed to use

Bio-plants.

1999

Discovery of acrylamide producing microorganisms

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

AA production by

Sulfuric acid catalyst

Nitto Chem. Co.

1957

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

5-cyanovaleramide

Nicotineamide

Adiponitrile

Rhodococcus rhodochrous J1

Pseudomonas chlororaphis B23

3-Cyanopyridine

PRODUCTION OF NICTINEAMIDE

BY NITRILE HYDRATASE

0 : Material (3-Cyanopyridine) with NHase from Rhodococcus rhodochrous

J1

1 : Reaction intermediates

6 : Product (Acrylamide)

18: Product (acrylamide)

NHASE IS USED FOR INDUSTRIAL

ACRYLAMIDE PRODUCTION

NHase

Chemical catalyst

0

5

10

15

20

25

30

35

40

45

50

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

Acr

ylam

ide

Pro

duct

ion

(kilo

-to

n)

Bio process

Copper catalyst

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.

Large Scale

Low Activity

Small Scale

High Activity

PHOTOACTIVATION OF NHASE

0 10 20 30 40 50 0

10

20

30

40

50

Time [h]

NH

ase

Act

ivit

y [

un

its/

mg

-dry

-cel

l]

Dark Inactivation

Light Activation

The reaction of NHases in a crystal can be simultaneously

started by light activation

Fe a b NO

Inactive NHase

(Nitrosylated)

NO

NO

hu

dark

RCONH2 RCN H2O

Fe a b

Active NHase

Photoreactivity of NHase is

Regulated by NO

DIFFERENCE FTIR SPECTRA OF NHASE

BETWEEN BEFORE AND AFTER

PHOTOACTIVATION

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

A: Natural

B: N15 Label

PHOTOREACTIVITY OF NHASE IS REGULATED BY NO

1900 1880 1860 1840 1820 1800

Wavenumber (cm-1)

1865 1844

1853

0.002 A. Inactive form

Activity [U/mg]

6.5

B. Active form 831

C. Active NO 0

D. Active NO

Light 718

NO

h

h

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

REGULATION OF NHASE BY NO

NO

NO

RCONH2 RCN H2O

Fe a b NO

Inactive

NO Bound Form

Fe a b

hu

Dark

hu

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)

Nitriles Amides

R-CN R-CO-NH2

OH2

Catalytic center

Fe (III) or Co(III)

Hetero-dimer , (MW=23kDa)

NHase is used for the industrial production of

acrylamide and nicotinamide.

CRYSTAL STRUCTURE OF FE-TYPE NHASE

(NO-BOUND FORM) AND ITS METALLOCENTER

subunit Active Center

subunit

Nitrosylated form

(inactive)

1,4-dioxane

Arg56

Arg141

Cys112

Cys114 NO

Fe

STRUCTURE OF FE-CENTER OF NHASE

FeIII

Ser113

Cys109-SH

Oxygen

Nitrogen

Carbon

Sulfur

Cys112-SO2H Cysteine sulfinic acid

NO Cys114-SOH

Cysteine sulfenic

acid

(Nagashima et al., Nat. Struct, Biol. (1998) 5, 347)

2 main chain amide notrogen as

coordinated. oxidation state of 3 Cys sulfures are different.

NHase is the first

example having post-

translationally modified

cysteine ligands.

Amido

nitoroge

n

METHOD FOR TIME RESOLVED

X-RAY CRYSTALLOGRAPHY

1. Crystals of NO bound NHase was soaked

with tBuNC.

2. NHasetBuNC complexes were photo-

activated.

3. At each time, the crystals were flash cooled

with N2 gas at 95K (Reaction stop).

293K

0 min

293K

Flash-Cooling

293K

293K 293K 95K

0 min X min

h

tert-butylisonitrile

(tBuNC)

120 min

NO

0 min 18

min

> 440

min 440 min

TIME RESOLVED CRYSTALLOGRAPHY

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 Anfinsens Experiment

1972 Nobel Prize in Chemistry

Raw Egg Fried Egg Boiled Egg

Heat

Cool, but

irreversible

BOILED EGG PROBLEM

Denature

Renature

Aggregation

Boiled Egg

DISEASE OF PROTEIN

-IF IT IS ISOLATED, OK. BUT ---

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

Denature

DISEASE OF PROTEIN

-RESCUE BY CHAPERONE-

By Chaperone

Isolation Cure

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 C

hap

eron

es

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

70 kDa DanK HspA group proteins,

Hsp70, Grp 78

Protein folding or

preventing protein

folding

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)

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.

CdS or CdSe

Several nm

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

Group I chaperonins Group II chaperonins

Are found in eubacteria and in

endosymbiotic organelles (mitochondria

and chloroplasts)

Have a detachable lid (GroES)

Exist in eukaryotic cytosol (CCT)

and in archaea (thermosome)

Are independent of GroES - Built-in

lid

GroES

GroEL

ATP

binding

ATP

hydrolysis

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

ATP

binding

ATP

hydrolysis

?

How does it work without co-chaperonin ?

Features of group I and group II

chaperonins

Exhibits high protein folding

activity

Is composed of two highly

homologous subunits (a, b)

Each subunit forms homo-

oligomer and functions as a

chaperonin. Crystal structure of aG65C/I125T mutant

Shomura et al. (2004) J Mol Biol

Chaperonin from a hyperthermophilic

archaeum, Thermococcus strain KS-1

Helical protrusion of group II

chaperonin

Subunit structure of group II

chaperonin

The protrusion is thought to

seal off the central cavity

(Built-in lid).

Helical protrusion The region is also assumed

to be involved in the binding

of substrate proteins.

However, the exact role

is quite unclear.

Model for functional cycle of

T. KS-1 chaperonin

Non-native

polypeptide

ATP

Pi

Native

polypeptide

Nucleotide-free form

ATP-bound form

ADP-bound form

H

A

I

E

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

C. R. Booth et al. (2008)

Nature Struct. Mol. Biol

lid-closure with twisting

motion

Cryo-EM, single particle

reconstruction

Kanzaki et al. (2008) J Biol Chem

TO INVESTIGATE PROTEINS DYNAMICS

Specialized Method for Protein Dynamics

Observed Rotational motion of F1-ATPase Nature 386:299(1997)

labeling Labeling is also valid for synchrotron radiation analysis

Tracer: nanocrystal Diffracted X-ray Tracking

120

protein

nanocrystal

DIFFRACTED X-RAY TRACKING (DXT)

Structural

Change

Laue spot

moves

Laue spot

gold nanocrystal

(20-50 nm)

protein

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

X-rays

THE METHOD FOR MONITORING TWISTING MOTION:

DIFFRACTED X-RAY TRACKING (DXT)

Au

DIFFRACTED X-RAY TARCKING: DXT

direction = 1 mrad (0.057)

White

X-ray

White

X-ray

Tilting Twisting

direction = 6 mrad (0.34)

Resolution of DXT

polyimide film

aqueous

solution

polyimide film

gold

X-rays

protein

gold nano

crystal

INSTRUMENTATION OF DXT

PF-AR NW14A

(35-60mA)

Sample

heater

Image

Intensifier V7739P (Hamamatsu

Photonics)

White X-

ray

Sample CCD:

C4880-80(Hamamatsu

photonics)

Chaperonin

KS-1

(D263C/C366S)

Thermococcus chaperonin

D263C

50 m

TWISTING MOTION WAS DETECTED

2=

0.2rad

2

Sekiguchi et al. (2013) PLoS One

TRACES OF DIFFRACTION SPOTS 60 , 2mM ATP 60, 0mM ATP RT, 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

time (ms) time (ms)

MSD

(mrad2)

MSD

(mrad2)

25

20

15

10

5

0

60

40

2

0

0

80

100

120

140 60, 2mM ATP 25, 2mM ATP 60, 0mM ATP

0 100 200 300 400 0 100 200 300 400

THE RING TWISTS CW OR CCW ?

128

caged ATP

(inactive ATP)

transparent teflon

film

gold

X-rays

protein

Au-crystal

70 m thick

Laser

355nm

ATP

UV

irradiation

UV-triggered DXT :

To confirm the twisting direction of

chaperonins ring when ATP binds to the ring

Sekiguchi et al. (2013) PLoS One

CAGED ATP: TWISTS CCW AFTER UV FLASH

129

An

gu

lar

dis

pla

cem

en

t

THANK YOU VERY MUCH