ELŐADÁS KIVONAT CLASSROOM LECTURE HANDOUT financed … · CLASSROOM LECTURE HANDOUT financed by the program „Practice-oriented, student-friendly modernization of the biomedical

TÁMOP-4.1.1.C-13/1/KONV-2014-0001 projekt

Biological Research Centre Address: H-6726 Szeged, Temesvári krt. 62. Mail: H-6701 Szeged, POB 521. www.brc.hu

„Az élettudományi-klinikai felsőoktatás gyakorlatorientált és hallgatóbarát korszerűsítése a vidéki képzőhelyek nemzetközi versenyképességének erősítésére”

program keretében finanszírozott

ELŐADÁS KIVONAT

CLASSROOM LECTURE HANDOUT

financed by the program

„Practice-oriented, student-friendly modernization of the biomedical education for strengthening the international competitiveness of the rural Hungarian universities”

Dátum / Date:

2016. OKTÓBER 19. / OCTOBER 19, 2016

Helyszín / Place:

MTA SZBK BIOFIZIKAI INTÉZET, TANÁCSTEREM / LECTURE ROOM, INST. OF BIOPHYSICS, BIOLOGICAL RESEARCH CENTRE

SZEGED, TEMESVÁRI KRT. 62.

Az előadás címe / Title of the presentation:

HEAT SHOCK PROTEINS AND THEIR ROLE IN HUMAN DISEASES

Előadók / Speakers:

MELINDA E. TÓTH, MIKLÓS SÁNTHA

M.E. Tóth, M. Sántha October 19, 2016

1

Heat shock proteins and their role in human diseases

part I.The heat shock proteins

Melinda E. TóthInstitute of Biochemistry

October 19, 2016

„Practice-oriented, student-friendly modernization of the biomedical education for strengthening the international competitiveness of the rural Hungarian universities”TÁMOP-4.1.1.C-13/1/KONV-2014-0001

Heat shock proteins (Hsps)

Ubiquitously expressed, evolutionarily conserved chaperone proteins.

Heat shock response was first observed in the Drosophila buscii salivary gland chromosomes by Ritossa in 1962.

Hsps can be induced by heat, heavy metal or ethanol treatment, hypoxia, ischemia, and they are also upregulated in several diseases and infections.

Their most important function is to protect cells from the toxic effects of stress.

Ferruccio Ritossa

De Maio et al. Cell Stress and Chaperones (2012) 17:139–143

Preconditioning: a mild, sublethal heat-stress can induce the expressionof Hsp and increase cell survival after a subsequent, normally lethal heat treatment.

Chaperone functions of Hsps

Muchowski and Wacker (2005) Nat Rev Neurosci. 6:11-22.

Hsps play roles in normal cellular homeostasis and development.

They regulate the biosynthesis, folding/unfolding, transport and assembly of cellular proteins.

They facilitate the degradation of certain abnormal proteins.

They participate in antigen presentation.

They are also implicated in differentsignal transduction pathways.


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During stress conditions, Hsps help to prevent the change of the conformation of other proteins.

They protect uncorrectly folded proteins against aggregation.

Upon recovery they facilitate the refolding of misfolded proteins.

They assist in the proteasomal degradation of peptides that can not be refolded.

Hsps also have roles in membrane protection during stress conditions, and they have anti-apoptotic functions


Heat shock protein families

- small Hsp (sHsp, HSPB)- Hsp70 (HSPA)- Hsp40 (DNAJ)- Hsp110 (HSPH)- Hsp90 (HSPC)- chaperonin families (Hsp60)

Rutherford (2003)Nat Rev Genet. 4:263-74.

Small heat shock (HspB) protein family

Gusev et al. (2002)Biochemistry 67:511-519.

Molecular weights between 16-40 kDa.

α-crystallin domain : a conserved C-terminal domain of 100 amino acids.

α-crystallin domain

Mammalian small Hsp family consists of 11 members.

They usually form homo- or heterooligomeric complexes up to ~700 kDa.

Post-translational modifications: phosphorylation on serine residues.


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Functions of sHsps

Wilhelmus et al. (2007)Mol Neurobiol. 35:203–216

Stabilize the cytoskeleton and membranes during stressconditions.

Protect cells from apoptosis and oxidative stress.

Bind to partially denatured proteins and prevent their aggregation, maintaining them in a refolding competent state.

ATP independent chaperones.

Hsp70 (HspA) protein family

Protein Alternativenames

Homology toHspA1A

Cellular localization Stress-induced

HspA1A Hsp70, Hsp72, Hsp70-1

100 Cytosol, nucleus, lysosomes

Yes

HspA1B Hsp70, Hsp72, Hsp70-2

99 Cytosol, nucleus, lysosomes

Yes

HspA1L Hsp70-Hom 90 Cytosol, nucleus No

HspA2 Hsp70-3 84 Cytosol, nucleus No

HspA5 BiP, GRP78 64 ER No

HspA6 Hsp70B’ 85 Cytosol, nucleus Yes

HspA8 Hsc70, Hsp73 86 Cytosol, nucleus No

HspA9 GRP75, mtHsp75, mortalin

52 Mitochondria No

Based on Daugaard et al. (2007)FEBS Letters 581:3702–3710

Functions of Hsp70

Nollen and Morimoto (2002)Journal of Cell Science 115:2809-2816

During stress conditions one Hsp70 participates in the refolding of the denatured proteins.

Hsp70 contains two functional units:


4

Csermely and Yahara (2002)Molecular Pathomechanisms and New Trends in Drug Research, Taylor and Francis, London and New York, pp 67-75.

Catalytic cycle of Hsp70

Hsp40 (DnaJ) protein family

Largest human Hsp family (50 members,three subgroups) Type I (DnaJA)

Type II (DnaJB)

Type III (DnaJC)

J domain Gly/Phe-rich region Cys repeats

Based on Qiu et al. (2006) Cell. Mol. Life Sci. 63:2560–2570and Kampinga et al. (2009) Cell Stress Chaperones 14:105–111

Conserved,N-terminal J-domain, through which they bind to Hsp70 proteins

Qiu et al. (2006) Cell. Mol. Life Sci. 63:2560–2570

Hsp40 proteins can bind substrate peptides and transfer them to Hsp70, while the J-domain promotes ATP hydrolysis.



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Hsp110 (HspH) protein family

Protein Alternative names Cellularlocalization

HspH1 Hsp105 Cytosol

HspH2 HSPA4, APG-2, HSP110 Cytosol

HspH3 HSPA4L, APG-1 Cytosol

HspH4 Grp170, ORP150, HSP12A

ER

Based on Kampinga et al. (2009)Cell Stress Chaperones 14:105–111 Liu and Hendrickson (2007) Cell 131:106-120

Functions:- ‘holdases’, they recognize and bind denatured proteins maintaining them in a

refolding-competent state- nucleotid exchange factors for Hsp70, removing ADP after ATP hydrolysis.

Protein Alternative names Cellularlocalization

HspC1 HSP89, HSP90 Cytosol

HspC2 HSP90α Cytosol

HspC3 HSP90B, HSP90β Cytosol

HspC4 GRP94, endoplasmin ER

HspC5 TRAP1, HSP75

Hsp90 (HspC) protein family

Based on Kampinga et al. (2009) Cell Stress Chaperones 14:105–111and Csermely et al. (1998) Pharmacol. Ther. 79:129–168

Csermely et al. Pharmacol. Ther. 79:129–168, 1998

1-2% of total cellular proteins

Functions of Hsp90

Whitesell and Lindquist (2005)Nature Reviews Cancer 5:761-772

- suppress the aggregation of unstable proteins

- disaggregate loose protein aggregates

- regulate cellular signalling


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HSF

HSE

Hsps

Transcriptional regulation of Hsps

Heat shock factors (HSFs)- special transcription factors

Heat shock elements (HSEs) are located in the promoter region of the Hsps

Based on Morimoto (1998) Genes Dev. 12:3788-3796

HSF

HSFHSF

Organisms Expression

HSF1 Human, mouse, chicken ubiquitous

HSF2 Human, mouse, chicken ubiquitous

HSF3 Chicken, mouse ubiquitous

HSF4 Human tissue-specific: heart, skeletal muscle, brain

Based on Morimoto (1998) Genes Dev. 12:3788-3796and Westerheide et al. (2012) Current Protein and Peptide Science 13:86-103

DNA bindingdomain

Transactivationdomains

Trimerizationdomains

Regulatory domain

Westerheide et al. (2012) Current Protein and Peptide Science 13:86-103



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Vígh et al. (2007) Trends Biochem Sci. 32:357-63

Role of Hsps in human diseases:Chaperone function

Native proteinMisfolded protein

Aggregates

sHsp oligomeric complexes with misfolded proteins

Hsp70-Hsp40substrate complex

Proteasoma

Hsp70Hsp40

sHsp

Vígh et al. (2007) Trends Biochem Sci. 32:357-63

Membrane quality control


8

Membrane quality control

Based on Nakamoto and Vígh (2007) Cell. Mol. Life Sci. 64:294–306

Oxidative stress

Oxidative stress

Aging

Neurodegenerativedisorders Stroke

Myocardialinfarction

Diabetes

Cancer

Oxidative stress

Protein oxidationLipid peroxidation DNA damage Cytoskeletaldamage

ROS

GSH

Protein aggregation

Oxidative stress


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Oxidative stress

Protein oxidationLipid peroxidation DNA damage Cytoskeletaldamage

ROS

GSH

Protein aggregation

sHsps

Proteasomaldegradation

Oxidative stress

Apoptosis

Ischia and So 2013 Nature Reviews Urology 10:386-395

Ischemia/reperfusion injury

Ethanol induced cytotoxicity

Ageing and neurodegenerative disorders

Obesity and diabetes

Cancer

Role of Hsps in human diseases:


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Ischemia

ATP production

Anaerobicglycolysis

Calciumaccumulation

Loss of membrane

integrity

Mitochondrialdysfunctions

ROS generation

Reviewed in Nishizawa and Nagata 2000

HSF1

Hsp70

HSF1

HSF1HSF1

Hsp90Hsp27


Role of Hsps in post-ischemic recovery in heart

Currie et al. 1988:

Source:http://www.meditec.hia.rwth-aachen.de/

42 °C

24 hours

Ischemic perfusion

improved myocardial recovery

reduced mithocondrial damageincreased Hsp70 expression


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Treatment Organism Effect Ref.Heat shock pretreatment

Rat heart Reduced infarct size Donnelly et al. 1992

Sublethal ischemic or heat pretreatment

Rabbit heart Reduced infarct size Marber et al. 1993

Hsp70 overexpression Primary rat cardiocytes Increased cell survival Cumming et al. 1996

Hsp27 or αB-crystallin overexpression

Rat cardiomyocytes Increased cell survival Martin et al. 1997

Hsp70 overexpression Mice heart Improved post-ischemic myocardial recovery, decreased cellular injury

Plumier et al. 1995

Hsp27 overexpression Mice heart Preserved contractile function, decreased oxidative stress

Hollander et al. 2004

Bimoclomol (Hsp inducer) treatment

Rat heart Antiischemic, antiarrhythmic effect

Vígh et al. 1997

Exercise training Rat heart Decreased myocardial lipid peroxidation

Demirel et al. 1998

Role of Hsps in post-ischemic recovery in heart

Treatment Organism Effect Ref.

Ischemic pretreatment Gerbil brain Decreasedhippocampal cell death

Kitagawa et al. 1990

Transgenicoverexpression of Hsp70

Mice brain Reduced neuronaldamage

Plumier et al. 1997

Viral overexpression of Hsp70

Rat brain Improved neuronsurvival

Yenari et al. 1998

Geldanamycin (Hspinducer) treatment

Rat brain Reduced infarctvolume and cell death

Lu et al. 2002

Role of Hsps in ischemic brain damage

Increased membrane fluidity

Protein denaturation

Cytotoxic effects of ethanol

ROS

Apoptotic cell death

Cytoskeleton


12

Ethanol induced apoptotic cell death

Zhou et al. (2001) Am J Pathol. 159:329–338.

Ikonomidou et al. (2000) Science 287:1056-60.

Inhibition of cytochrome c release

Inhibition of caspase 3and caspase 9 activation

Hsp

Increased membrane fluidity

Protein denaturation

hsp gene

mRNA

HSE

HSF

HSF activation

Alcohol stress

Maintenance of membrane stability Prevention of protein denaturation/ aggregation

Protective effects of Hsps

Tóth et al. (2014) Cell Stress Chaperones 19:299-309.

Aging and neurodegenerative diseases




Proteasoma

Hsp70

Hsp40

sHsp

Normal protein homeostasis


13

Aging and neurodegenerative diseases




Proteasoma

Hsp70

Hsp40sHsp

Aging

Aggregates

Protein-misfolding disorders

Disease Protein Localization of aggregates

Alzheimer’s disease (AD) Amyloid-β Intra- and extracellular

Alzheimer’s disease (AD) Hyperphosphorylatedtau

Intracellular

Parkinson’s disease (PD) α-synuclein Intracellular

Huntington’s disease (HD) Huntingtin Intracellular

Amyotrophic lateralsclerosis (ALS)

Superoxide dismutase(SOD)

Intracellular

Prion disease Prion protein Extracellular

Mattson (2004)Nature 430:631-9.


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Aggregation of amyloid



Disease model Treatment Effect Ref.APPsw mouse model of AD Hsp70 overexpression Improved cognitive function,

reduced plaque formationand neuronal loss

Hoshino et al. 2011

APPsw/Psen1 mousemodel of AD

Hsp27 overexpression Improved cognitive and synaptic functions, reducedplaque formation

Tóth et al. 2013

SOD1-G93A mouse modelof ALS

Arimoclomol treatment Improved muscle function, increased life span

Kieran et al. 2004

Rat model of PD GRP78 overexpression Reduced α-syn neurotoxicityand apoptosis

Gorbatyuk et al. 2012

R6/2 mouse model of HD DNAJB2a overexpression Reduced mutant huntingtinaggregation, improved neurological performance

Labbadia et al. 2012

R6/2 mouse model of HD HSF1 overexpression Increased life span Fujimoto et al. 2005

Role of Hsps in neurodegenerative diseases

Reviewed in Kakkar et al. (2014) Dis Model Mech. 7:421-34.


15

Obesity and diabetes

Diabetes

Chronicstress state

Disturbedprotein

homeostasis

Oxidativestress

ER stress

Impairedstress

response

Protein aggregation

Hsps

Protein glycation

Heat shock response and diabetes

Hooper 1999:

Hot tub treatment for 30 min daily for 3 weeks

Improved fasting glucose

Relief ofneuropathic symptoms1 % drop in HbA1

Trend toward weight loss

Treatment Model Effect Ref.

Far infrared lighttherapy

diabetic mice(db/db)

Improved obesity related insulinresistance

Kokura et al. 2007

Electric heating blanket Fat fed mice Prevented diet inducedhyperglycemia, hyperinsulinemiaand insulin resistance

Chung et al. 2008

Hot water immersion Fat fed rats Improved glucose tolerance, insulin-stimulated glucosetransport

Gupte et al. 2009

BGP-15 treatment Leptin-deficient(ob/ob) mice

Reduced fasting levels of glucoseand insulin

Chung et al. 2008

BGP-15 treatment Insulin-resistantpatients

Increased insulin sensitivity and glucose utilization

Literáti-Nagy et al. 2009

Bimoclomol treatment Streptozoicintreated diabeticrats

Improved wound healing Vígh et al. 1997

Heat shock response and diabetes


16

Cancer

Poor blood supply

Inadequate glucose, oxygen and pH level

Chemotherapy, radiotherapy

Hsps

Good prognosis Poor prognosis No correlation

Hsp27 expression endometrialadenocarcinomas, oesophageal cancer,malignant fibrous histiocytomas

ovarian, gastric, liverand prostate cancer, and osteosarcomas

head and necksquamous cancer, bladder, renal cancer, leukemia

Hsp70 expression oesophageal cancer, pancreatic cancer, renal cancer,and melanoma

breast, endometrial, uterine cervicalcancer, transitional cell carcinoma of the bladder

ovarian, oral, headand neck squamous cancer, gastric and prostate cancer,leukemia

Hsp90 expression endometrial cancer breast cancer ovarian and oralcancer

Prognostic implications of Hsps

Reviewed by Ciocca and Calderwood (2005) Cell Stress Chaperones 10:86–103

Response to anticancer therapies

Hspoverexpression

Preventing apoptotic celldeath of the tumor cells(doxorubicin treatment)

Refolding/stabilizingdenatured proteins

(herceptin treatment)

Improving DNA repair

Inhibition or downregulation of Hspsseems to be a possible treatment

additional to traditionalchemotherapy (quercetin, OGX-427)


17

Tumor immunotherapy based on Hsps

Zhang and Zheng (2013) Oncology Letters 6:1543-1549

Part II.

The cardio- and neuroprotective roles of a smallheat shock protein, Hsp27.

(in vivo studies)

Miklos SanthaInstitute of Biochemistry

October 19, 2016

„Practice-oriented, student-friendly modernization of the biomedical education for strengthening the international competitiveness of the rural Hungarian universities”TÁMOP-4.1.1.C-13/1/KONV-2014-0001

Production of Hsp27 transgenic mice

(Based on the protocol of UC San Diego Health System)


18

vektorCMV Hsp27 bGHpA

Physical map of the transgene

c

(source: http://card.medic.kumamoto-u.ac.jp/)

Fertilized mouse oocytes

(source: http://acces.ens-lyon.fr/biotic/procreat/clonage/html/TechniquesTransgenese.htm)

Pronucleus microinjection


19


(Based on the protocol of UC San Diego Health System)


Two independent transgenic lines (24 and 33) wereidentified using PCR

Hsp27

Analysis of Hsp27 expression in transgenic offsprings using Western blot

• Total protein from brain and heart tissues was purified

Hsp27


20

Doxorubicin

source: http://elifesciences.org/

Patel and Kaufmann, 2012. eLife 2012;1:e00387

source:http://chem257.pbworks.com/

Effect of Doxorubicin on cardiomyocytes

source: http://biomedfrontiers.org/

Dimitrakis et al. 2012. Cell and tissue research, 350(2):361-372.

Cardioprotective role of Hsp27 after doxorubicin treatment

Tunnel assay(DNA fragmentation)


21

Expression of Hsp27 in the brain of transgenic mice

Tóth et al. 2010. Cell Stress Chaperons 15. (6):807-17

Is there a neuroprotective role of Hsp27?

Acut alcohol treatment(20% ethanol ip.)

Improved motor coordinationfootprint analysisbalance –beam walkinginverted screen testswimming test

Tóth et al. 2010. Cell stress Chaperons 15. (6):807-17

Behavioural tests• To investigate the effect of acute ethanol administration

by analysing ataxia, muscle strength and motor coordination

• Four groups : EtOH inj. wild-type EtOH inj tg, saline inj wild-type saline inj tg ,n=10 mice / group

• Before test mice were trained three times

• 2 g/kg i.p. injection of 20% EtOH,

• In three out of the 5 tests one-way ANOVA revealed a significant difference between the transgenic and wild type mice


22

Footprint analysis• Stepping patterns were compared• 60 cm long 7 cm wide runner • Lined with white paper• the fore- and hind-paws of the animals were covered with different colors

of non-toxic paints• middle steps of a series of steps were analysed (6 step)• For quantitative comparsion of footprint patterns, stride length and width

of front and back leg and front/hind paw overlap were measured

Results of footprint assay• EtOH injected wild type

mice tend to walk on whole paw

• and produced toe dragging

• footprint of EtOH injected transgenic mice were much similar to the pattern of saline injected mice

• Significant difference in the stride length• significantly impaired forelimb-hindlimb coordination in the EtOH injected wild

type group

• There is no significant difference in the results of females


23

Balance beam test• Coordination and balance

• 1m long 10mm round horizontal wooden beam

• Time taken to run along the beam, slips and falling-off were recorded

• Three trials

Time taken to run along the beam

Performance in balance beam test

• In this test 86% of wild type mice fell off at least once from the beam, while in the transgenic group only 53%.

• In the wild type group mice fell off more then two times in average, while in the transgenic group less then once


24

Inverted screen test

• coordination and strength

• 25x30 cm wire mesh elevated 50 cm above the ground

• Mice not climbing to the top were considered to have reduced strength or coordination

Result of inverted screen test

• Almost all EtOH injected wild type mice fell off the screen

• 43% of the EtOH treated transgenic mice could climb over the edge of the screen (50% of males and 26% of females)





Less neuronal deathFluoroJade staining of cortical and hippocampalslices


Chronic alcohol consumption (20% ethanol for 5 weeks)


25

Neuronal death after chronic ethanol consumptionhippocampus cortex

Tóth et al. 2010. Cell Stress Chaperons 15. (6):807-17

Wt EtOH

Wt control

Tg EtOH

Tg control

cerebellum





Alzheimer’s disease(APPSwexPse1 (AD) x Hsp27 mice)

Improved learning and memoryBarnes mazeMorris water maze

Improved presynaptic functionExcitabilityLTP

Less amyloid plaquescortex hippocampus

Apoptosis was not influenced cleaved caspase-3 stainingFluoroJade staining

Tóth et al. 2013. Cell stress Chaperons 18.(6):759-71

Less neuronal deathFluoroJade staining of cortical and hippocampalslices


Chronic alcohol consumption (20% ethanol for 5 weeks)


26

(source: http://omrf.org/about-omrf)

(source: http://www.webmd.com/alzheimers/)

Alzheimer’s disease

(source: https://halcyonorganics.com/)

Azheimer’s disease


27

(source: http://www.bmb.leeds.ac.uk/staff/nmh/amy.html)

(source:http://scitechdaily.com/)

(source: http://www.emoryhealthsciblog.com/)


28

X

APPSwexPse1dE9AD model mice

Hsp27 overexpressing

(source: http://sfari.org/news-and-opinion)

AD model X Hsp27 transgenic mice

(sources: http://jaxmice.jax.org/)

Expression of Hsp27 protein in the brain

Tóth et al. 2013. Cell Stress Chaperons 18.(6):759-71

Morris water maze

(source: http://neuroamer.wordpress.com/)


29

Barnes maze

(source: http://blog.163.com/)

Effect of Hsp27 overexpressionon behavior of AD mice


Effect of Hsp27 overexpression on presynaptic plasticity in AD mice

LTP

Neuronal excitability

Paired-pulsefacilitation (PPF)



30

hippocampus

cortex

Effect of Hsp27 overexpression in AD miceon amyloid plaque formation

on neuronal death


Thank you for your attention!

This work is supported by the European Union, co-financed by the European Social Fund, within the framework of " Practice-

oriented, student-friendly modernization of the biomedical education for strengthening the international

competitiveness of the rural Hungarian universities " TÁMOP-4.1.1.C-13/1/KONV-2014-0001 project.

Documents

ELŐADÁS KIVONAT CLASSROOM LECTURE HANDOUT financed … · CLASSROOM LECTURE HANDOUT financed by the program „Practice-oriented, student-friendly modernization of the biomedical