Stress proteins: HSP70

1. HSP 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 Hospital Clinical Medicine Department Of Hunan Provincial Traditional Chinese Medicine University

2. HSP I am interested in heat shock proteins

3. HSP What are heat shock proteins

4. HSPs and HSR HSP • 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。　 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.

5. 分类（Classification） HSP • The different major heat shock proteins are 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.

6. HSP

7. Hsp70 的结构 HSP • 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

8. Hsp70 的结构 HSP

9. Hsp70 的结构 HSP 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 ATPase domain (homology with actin, which also binds ATP) Jiang et al. (2005) Mol. Cell 20, 513-24. Structural Basis of Interdomain Communication in the Hsc70 Chaperone

10. History Of Heat Shock Proteins HSP • 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.

11. Hsp70 的特点 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。 • 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)

12. Factor of induced HSP production

13. HSP how do they work?

14. Functions of HSP70 HSP • molecular chaperone • immunomodulation • quality control and thermotolerance • danger signal

15. Molecular chaperone HSP • 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.

16. Hsp70 - Molecular chaperone HSP • 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)

17. HSP70 in protein folding HSP • The diagram shows the role of heat-shock proteins and a chaperonin in protein folding.  As the ribosome moves along the molecule of messenger RNA, a chain of amino acids is built up to form a new protein molecule.  The chain is protected against unwanted interactions with other cytoplasmic molecules by heat-shock proteins and a chaperonin molecule until it has successfully completed its folding.  

18. Protein Folding in Eukaryotes Involves Multi-Chaperone Systems Hsp60 assisted Hsp90 assisted

19. Quality control and thermotolerance HSP • HSP promotethe degradation of unrecoverable and needless proteins. • Thus HSP carry out “quality control” with regard to theproduction, recycling and disposal of proteinswithin cells. • It is known that cells expressingincreased amounts of HSP, especially HSP70 and HSP27,are more resistant to stresses that induce apoptosis .This is known as “thermotolerance”.

20. HSP • Cells subjected to mild stress induce HSP which then protect them against subsequent stress. • However, in cells subjected to severestress, HSP promote apoptosis.

21. Hsp70 – Cellular Protection HSP • Stress Induced –Prevents protein denaturation and incorrect polypeptide aggregation during exposure to physiochemical insults • Hsp70 expression linked in several different models of neurodegenerative diseases

22. 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.

23. 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.

24. HSP

25. Hsp70 -Additional Functions HSP Involved in various cellular functions and diseases: Apoptosis Clathrin cage disassembly Parkinson's Disease Dauer Stage Translocation Prions Cancer Different heat shock proteins are involved in a number of auto-immune disorders.

26. Cancer vaccine adjuvant 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.

27. Anticancer therapeutics HSP • 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.

28. HSP How the induced HSP70 expression and the function of extracellular HSP

29. Mechanisms of HSP release from cells HSP • Molecular chaperones can be released from cells into the body fluids by either physiological or accidental mechanisms

30. 应激的细胞体液反应 HSP Protective substances Acute phase proteins Stress proteins CRP α2-Ｍ HSPs α1-ＡＴ ＨＰ PCT ？

31. Regulation of the stress response:HSF Active trimer cellular stress HSF has a Winged Helix-Turn-Helix Motif trimerization via leucine zippers binding to HSEs reversal to monomers following stress HSEs stress-inducible gene HSF DNA binding domain (monomer) in complex with HSE sequence HSP Inactive heat shock transcription factor (HSF) monomer • activation of transcription by HSF requires phosphorylation • monomer-trimer transition, and activity while bound to DNA is regulated by molecular chaperones

32. stressor HSP70 HSF HSE HSPs

33. HSP • 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”.

34. 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.

35. Danger signals HSP • 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.

36. Table1 HSP70 of serum of patient groups according ISS in different stage 注：与健康对照组比较，*P<0.05、**P<0.01。 ** ** ** * * * * * *

37. Table 4 HSP70 of patients of TBI and non- TBI in different stages 注：与健康对照组比较，*P<0.05、**P<0.01。 ** ** * * * *

38. Table 5 HSP70 level of patients of infection group and non-infection group in different stages 注：与健康对照组比较，*P<0.05、**P<0.01。 ** * ** * * *

39. HSP How to make good use of danger signals

40. HSP70与WBC的对照研究 HSP

41. HSP70与PCT的对照研究 HSP

42. 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。

43. HSP Thank you for yourattention !