Stress proteins: HSP70

任碧琼 湖南省第二人民医院检验科

湖南中医药大学临床医学院检验教研室 Clinical lab of Hunan No.2 Provincial People's HospitalClinical Medicine Department Of Hunan Provincial Traditi

onal Chinese Medicine University

HSP

I am interested in

heat shock proteins

HSP

What are heat shock proteins

HSP

HSPs and HSR

Heat shock proteins (HSPs), are a group of stress-inducible proteins that are present in all cells in all life forms,and contribute to quality control by assisting the correct folding of both nascent and denatured proteins, and promoting the degradation of unrecoverable denatured proteins.

• 热休克蛋白（ HeatShockProteins ， HSPs ）是生物体（或离体培养的细胞）在不良环境因素作用下产生的具有高度保守性的应激蛋白，普遍存在于整个生物界。

• 热休克反应（ heatshockresponseHSR ）是一种生理性的快速短暂的细胞代谢调节，此期间细胞内一些正常基因的表达受到抑制，而一组特殊基因则被激活并表达，这组特殊基因就是热休克基因，所产生的蛋白质称为热休克蛋白。

• 热休克蛋白主要是通过帮助新生的或者变性的蛋白质正确折叠，促使变性的、无法修复的蛋白质降解等功能，对蛋白质的合成起质量控制作用。又叫做分子伴侣。　　

HSP

分类（ Classification ）• The different major heat shock proteins ar

e grouped, on the basis of their apparent molecular sizes (in kilodaltons, 千道尔顿 ), structure, and function, into different families.

• Those families include: Hsp100, Hsp90, Hsp70, Hsp60,HSP40 

and the small heat shock proteins family.

HSP

Functions of Hsp families

Family Major functions

Hsp100 Protein disaggregation, thermotolerance

Hsp90 Regulatory interactions with signaling proteins, stabilization of misfolded proteins

Hsp70 Protein folding, membrane transport of proteins

Hsp60 Protein folding (limited substrates in eukaryotic cytoplasm)

Hsp40 Protein folding, co-chaperone for Hsp70

Small Hsp

Stabilization of misfolded proteins, thermotolerance, eye lens structural proteins

HSP

Hsp70 Hsp70 的结构的结构•Size – 70kD

•Length – 645 AAs

•ATP dependent

•Single Monomer

• 3 Domains

•N –terminal - ATP Binding and hydrolysis (44 kDa)

Homologous to Actin and Hexokinase

•Substrate Binding – Hydrophobic regions (15-20 kDa)

•C-terminal –Lid for substrate binding (15 -20 kDa)

•Allosteric properties – conformational changes

HSP

Hsp70 Hsp70 的结构的结构 HSP

ATPase domain

(homology with actin

,

which also

binds ATP)

Polypeptide binding domain with bound peptide ‘substrate’

flexible linkage between ATPase and peptide-binding domains, and different conformations of molecule possible

polypeptide-binding domain consists of beta-sheet scaffold; loops possess hydrophobic residues that contact peptide

domain also has an alpha-helical ‘lid’ that is regulated by the ATPase activity

Jiang et al. (2005) Mol. Cell 20, 513-24.

Structural Basis of Interdomain Communication in the Hsc70 Chaperone

HSPHsp70 Hsp70 的结构的结构

History Of Heat Shock Proteins

• In the early 1960s, F. Ritossa first discovered the heat shock (HS) response while observing the salivary cells of Drosophila melanogaster (Ritossa, 1962).

• It was noted that heating these cells induced puffs to form at various regions of the polytene chromosomes. Further analysis revealed that these puffs were actually areas of localized transcription that correlated to the increase of several families of proteins.

HSP

Hsp70 的特点

Key Points:

•A Molecular chaperone

•Inducible during “stressful conditions” (heat, toxins, etc..)

•Ubiquitous expression - In all cells at all biological levels

•Highly conserved family (Hsp 40, 60, 90 etc..)

•Consists of stress-inducible and constitutive family members (Hsc 70, Hsp72)

Essential in Protein Folding Cellular stress protection通过大量的研究发现 HSR 具有以下特点：　　（ 1 ）普遍性： HSR 广泛存在于从原核到真核生物的生物界有机体内。　　（ 2 ）保守性： HSR 是存在于生物系统不同层次结构中的普遍现象。 HSR 所产生的 HSPs 从结构到功能都具有极端的保守性，其核酸序列在不同物种之间具有高度的同源性，如大肠杆菌的 DnaK 基因与真核生物 HSP70 基因有 40-60% 的同源性，真核生物间 HSPs 的同源性可达 60-80% 。　　（ 3 ）诱导的非特异性： HSR 不仅能为热损伤所诱导，而且可谓许多其它损伤因素及应激刺激，包括物理、化学因素乃至机械刺激（如葡萄糖缺乏、缺血、寒冷、创伤、中毒、重金属、饥饿、缺氧、氧自由基）所诱导，以及其它因素如感染（包括细菌、病毒和寄生虫感染）、恶性肿瘤等所诱导。

Factor of induced HSP productionFactor of induced HSP production

how do they work?

HSP

Functions of HSP70

• molecular chaperone

• immunomodulation

• quality control and thermotolerance

• danger signal

HSP

Molecular chaperone

• The primary function of HSP is to act as a “molecular chaperone” which facilitates the folding of both nascent, de novo synthesized proteins and proteins denatured by stress, helping those proteins reach their proper folded state.

HSP

Hsp70 - Molecular chaperone

•Recognizes & binds regions rich in hydrophobic residues

•Stabilizes & prevents misfolding by open conformation

•Prevents aggregation and degradation during synthesis

•Multiple Hsp70 complexes per polypeptide chain

•Releases or transfers to other chaperones/chaperonins (Hsp60,Hsp90)

HSP

HSP70 in protein folding

• The diagram shows the role of

heat-shock proteins and a cha

peronin in protein folding.  As t

he ribosome moves along the

molecule of messenger RNA,

a chain of amino acids is built

up to form a new protein molec

ule.  The chain is protected ag

ainst unwanted interactions wit

h other cytoplasmic molecules

by heat-shock proteins and a c

haperonin molecule until it has

successfully completed its foldi

ng.  

HSP

Protein Folding in Eukaryotes Involves Multi-Chaperone Systems

Hsp90 assisted

Hsp60 assisted

Quality control and thermotolerance

• HSP promote the degradation of unrecoverable and needless proteins.

• Thus HSP carry out “quality control” with regard to the production, recycling and disposal of proteins with in cells .

• It is known that cells expressing increased amounts of HSP, especially HSP70 and HSP27, are more resistant to stresses that induce apoptosis . This is known as “thermotolerance”.

HSP

• Cells subjected to mild stress induce

HSP which then protect them against

subsequent stress.

• However, in cells subjected to severe

stress, HSP promote apoptosis.

HSP

Hsp70 – Cellular Protection

•Stress Induced – Prevents protein denaturation and incorrect polypeptide aggregation during exposure to physiochemical insults

•Hsp70 expression linked in several different models of neurodegenerative diseases

HSP

The pattern of heat shock proteins in mammalian brain,either synthesized in a developmentally regulated manner or in response to stress,is non-random.This has been related to specific functions of different parts of the brain,including role of Hsps,particularly the Hsp70,in short- and long-term memory and making different parts of the brain more or less susceptible to stress-induced injuries.

HSP

• Besides these intracellular events, HSP also exist in extracellular fluids, and have been shown to contribute to immunomodulation.

• In innate immunity extracellular HSP, like various microbial substances, induce various proinflammatory cytokines.

HSP

HSP

Hsp70 - Additional Functions

Cancer

Prions

Clathrin cage disassembly

Dauer Stage

Apoptosis

Parkinson's Disease

Translocation

Involved in various cellular functions and diseases:

Different heat shock proteins are involved in a number of auto-immune disorders.Different heat shock proteins are involved in a number of auto-immune disorders.

HSP

• Given their role in antigen, HSPs are useful as immunologic adjuvantsin boosting the response to a viccine. Furthermore, some researchers speculate that HSPs may be involved in binding protein fragments from dead malignant cells and presenting them to the immune system.Therefore HSPs may be useful for increasing the effectiveness of cancer vaccines.

Cancer vaccine adjuvant

HSP

Anticancer therapeutics

• Intracellular heat shock proteins are highly expressed in cancerous cells and are essential to the survival of these cell types. Hence small molecule inhibitors of HSPs, especially Hsp90 show promise as anticancer agents.The potent Hsp90 inhibitor 17-AAG is currently in clinical trials for the treatment of several types of cancer.

HSP

How the induced HSP70 expression

and the function of extracellular HSP

HSP

Mechanisms of HSP release from cells

• Molecular chaperones can be released from cells into the body fluids by either physiological or accidental mechanisms

HSP

应激的细胞体液反应 Protectivesubstances

Acute phase

proteins Stress

proteinsCRP

α2-Ｍ

α1-ＡＴ ＨＰ PCT

？HSPs

HSP

Inactive heat shocktranscription factor(HSF)monomer

trimerization vialeucine zippers

Active trimer

cellular stress

stress-inducible geneHSEs

binding toHSEsreversal

to monomersfollowing stress

activation of transcription by HSF requires phosphorylation

monomer-trimer transition, and activity while bound to DNA is regulated by molecular chaperones

Regulation of the stress response:Regulation of the stress response:HSFHSF

HSF DNA bindingdomain (monomer)in complex withHSE sequence

HSF has a Winged Helix-Turn-Helix Motif

HSP

stressor

HSE HSPs

HSF

HSP70

• In acquired immunity they interact with antigenic polypeptides and assist in antigen presentation. The extracellular HSP are so-called adjuvant.

• Release of HSP from cells is triggered by stress and trauma, and is thus regarded as an immunological “danger signal”.

HSP

• Anti-HSP autoantibodies are found in patients with autoimmune diseases and inflammatory disorders, and these autoantibodies can modulate the “danger signal” triggered by extracellular HSP.

HSP

Danger signals

• Extracellular HSP have been shown to share immunomodulatory activities similar to adjuvants and proinflammatory cytokines.

• So they are sometimes referred to as “danger signals”, which signify certain invasion, and are a trigger for immune responses.

HSP

hsp70

1-6h 24-36h 60-90h0

10

20

30

40mildmedialseverecontrol

different phase

HS

P70 c

on

cen

trati

on

(ng

/ml)

*

**

Table1 HSP70 of serum of patient groups according ISS in different stage

1～ 6小时 24～ 36小时 60～ 90小时轻度伤组 15.01±4.49* 16.66±5.01* 16.74±3.94*

中度伤组 15.49±3.64* 17.22±4.03* 17.35±5.01*

重度伤组 20.01±7.48** 21.15±5.67** 21.34±7.38**

对照组 11.12±1.95 11.12±1.95 11.12±1.95

注：与健康对照组比较， *P<0.05、 **P<0.01。

**

*

****

* *

hsp70

1-6h 24-36h 60-90h0

10

20

30head injuredother injuredcontrol

different phase

HS

P70 c

on

cen

trati

on

(ng

/ml)

Table 4 HSP70 of patients of TBI and non- TBI in different stages

1～ 6小时 24～ 36小时 60～ 90小时

颅脑损伤组 20.37±4.32** 20.09±5.23** 19.19±4.74*

其他损伤组 15.12±4.08* 16.97±3.72* 17.27±4.59*

对照组 11.12±1.95 11.12±1.95 11.12±1.95

注：与健康对照组比较， *P<0.05、 **P<0.01。

**** *

** *

hsp70

1-6h 24-36h 60-90h0

10

20

30

40non-infectioninfectioncontrol

different phase

HS

P70 c

on

cen

trati

on

(ng

/ml)

Table 5 HSP70 level of patients of infection group and non-infection group in different stages

1～ 6小时 24～ 36小时 60～ 90小时感染组 20.01±8.08* 21.73±4.76** 23.07±6.75**

非感染组 16.79±4.55* 18.53±4.56* 16.93±3.46*

对照组 11.12±1.95 11.12±1.95 11.12±1.95注：与健康对照组比较， *P<0.05、 **

P<0.01。

*

*

*

* ****

How to make good use of

danger signals

HSP

HSP70 与WBC的对照研究

WBC

1-6h 24-36h 60-90h0

10

20

30mildmedialseverecontrol

different phase

WB

C(*

10^

9)

WBC

1-6h 24-36h 60-90h0

5

10

15

20

25non-infectioninfectioncontrol

different phase

WB

C(*

10^

9)

hsp70

1-6h 24-36h 60-90h0

10

20

30

40mildmedialseverecontrol

different phase

HS

P70

co

nce

ntr

atio

n(n

g/m

l)

hsp70

1-6h 24-36h 60-90h0

10

20

30

40non-infectioninfectioncontrol

different phase

HS

P70

co

nce

ntr

atio

n(n

g/m

l)

HSP

HSP70 与 PCT的对照研究

PCT

1-6h 24-36h 60-90h0

2

4

6

8mildmedialseverecontrol

different phase

PC

T c

on

cen

trat

ion

(ng

/ml)

PCT

1-6h 24-36h 60-90h0

2

4

6

8non-infectioninfectioncontrol

different phase

PC

T c

on

cen

trat

ion

(ng

/ml)

hsp70

1-6h 24-36h 60-90h0

10

20

30

40mildmedialseverecontrol

different phase

HS

P70

co

nce

ntr

atio

n(n

g/m

l)

hsp70

1-6h 24-36h 60-90h0

10

20

30

40non-infectioninfectioncontrol

different phase

HS

P70

co

nce

ntr

atio

n(n

g/m

l)

HSP

HSP70 在急诊检验中的应用前景

HSP

• 疾病的过程贯穿着各种应激，只是应激原不同，应激反应的存在意味着应激原的存在，因此外周血应激蛋白 HSP70 水平可以提示机体的应激状态以及应激程度，从而有效判断疾病的严重程度。

• 目前国内尚无专门的急诊病人病情严重程度评价系统，更无普遍适用于急诊病人的“潜在危重病”识别系统，对一些潜在危险性因素缺乏科学的认识和评判方法。建立一种适合于急诊病人和急诊医生的“急诊病人潜在危重病病情评价方法”有着非常紧迫的必要性。

• 用于急诊病人潜在危重病病情评价的实验室指标仍限于白细胞等血常规指标，有很大的局限性，因此，寻找适用于急诊病人潜在危重病病情评价的实验室参数、开发方便、快捷、有效的检测试剂有着巨大的研究空间和良好的社会意义。

Thank you for your attention !

HSP