Nollen, Ellen Adriënne

English

In orrder to grow, divide, and to exert their specialized functions, our cells depend on a multitude of different proteins with specific roles in the cell. Proteins are characterized by the order of their building stones, the amino acids, the length of the amino acid chain, and the way this chain is folded into a three dimensional structure. This three-dimensional form determines the function of a protein. Proteins in their functional form are folded in such a way that the water repelling segments of the chains are not in contact with the surrounding aqueous environment in the cell. If these so-called hydrophobic domains would be exposed, they could, besides with hydrophobic domains within the same proteins, also non-functionally and irreversibly stick to hydrophobic domains of other proteins. These non-procluctive interactions could then lead to misfolding and inactivation of the proteins involved. ... Zie: Summar

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   University of GroningenHSP70 chaperone functions in stressed cells.Nollen, Ellen AdriënneIMPORTANT NOTE: You are advised to consult the publisher's version (publisher's PDF) if you wish to cite fromit. Please check the document version below.Document VersionPublisher's PDF, also known as Version of recordPublication date:2000Link to publication in University of Groningen/UMCG research databaseCitation for published version (APA):Nollen, E. A. (2000). HSP70 chaperone functions in stressed cells. s.n.CopyrightOther than for strictly personal use, it is not permitted to download or to forward/distribute the text or part of it without the consent of theauthor(s) and/or copyright holder(s), unless the work is under an open content license (like Creative Commons).The publication may also be distributed here under the terms of Article 25fa of the Dutch Copyright Act, indicated by the “Taverne” license.More information can be found on the University of Groningen website: https://www.rug.nl/library/open-access/self-archiving-pure/taverne-amendment.Take-down policyIf you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediatelyand investigate your claim.Downloaded from the University of Groningen/UMCG research database (Pure): http://www.rug.nl/research/portal. For technical reasons thenumber of authors shown on this cover page is limited to 10 maximum.Download date: 20-06-2022Su m maryI N .',rder to grow, divide, and to exert their specialized functions, our cellsI . -I depend on a multitude of different proteins u'ith specific roles in the cell.Proteins are characterized by t\e order of their building stones, the aminoacids, the length of the amino acid chain, and the u'ay this chain is folded intoa three dimensional structure. This three-dimensional form determines thefunction of a protein. Proteins in their functional form are folded in such a waythat the water repelling segrrrents of the chains arc not in contact r.r'ith the sur-rounding aqueous environment in the cell. If these so-called hydrophobicdomains would be exposed, they could, besides rvith hydrophobic domainswithin the sarne proteins, also non-functionally and irreversibly stick tohydrophobic domains of other proteins. These non-procluctive interactionscould then lead to misfolding and inactivation of the proteins involved.During a variety of processes in the cell, proteins need to be partially orcompletely unfolded, such as when the linear chain is transported from onecompartment in the cell to the other. To prevent tl-rat the temporally unfoldedproteins stick to each other, the cell contains chaperones. Chaperones are pro-teins that selectively bind to hydrophobic domains, prevent their aggregarion,and enable refolding of the unlblded proteins. In case proteins escape the pro-tection by chaperones and misfold, they will be removed by the degradationsystem clf the cell, the proteases.Some environmental factor or certain diseases cause an accumulation ofunfolded or misfolded proteins in the cell. This happens for example whencells are exposed to high temperatures or in protein misfolding diseases uchas Huntington's disease or Alzheimer's disease. In these cases, the balancebetween the amount of damaged proteins on one site and the chaperones andproteases on the other site is disturbecl in such as wav that there are nott ) 7enough chaperones for proteins ref<rlding and not enough proteases for degra-dation of the damaged proteins. The unfolded or misfolded proteins are notfunctional anvmore and accumulate in aggregates, which can have damagingeffects on cells and even can lead to cell death. How aggregates lead to celldeath is still unknorvn. Because it has been discovered only recently that pro-tein aggregates are a hallmark of protein misfolding diseases. currently muchresearch is done on the relation benveen protein aggregation and cell death.The finding that heat can cause ccll death is used in the treatment of can-cer (Chapter 1). Heat treatments, also called hyperthermia, are successfullyused in addition to radiotherapy or chemotherapy to increase the tumor cellkilling effect of these therapies. However, there are some limitations to the useof hyperthermia. Frequent use of heat treatmcnts within a too short time peri-od can cause a ternporal heat resistance of cancer cells. This phenomenon iscalled thermotolerance. It has been proposcd that thermotolerance is, at leastin part, caused by a rapid increase in the production of a specific group ofchaperones: the heat shock proteins. After a heat treatment, these heat shockproteins are prclduced and remain present in the celi.The level of resistance ofcells against heat strongll' correlates witl-r the expression levels of heat shockproteins. To prevent that tumors become heat resistant and to make optirnaluse of hyperthermia in the clinic, it is desirable to better understand the effectof heat on cells and the process of thermotolerance.In thermotolerant cells, several heat shock proteins (Hsp's) are present atincreased levels. One heat shock protein whose level strongly correlates withheat resistance is Hsp70, in which '70' stands for its molecular weight. 'fhisthesis concentrates on Hsp70. It describes how Hsp70 as a chaperone con-tributes to thermotolerance of cells (Chapter' 2 and Chapter 4) and how thechaperone activity of Hsp70 can be influenced by another, just recently iden-tif ied, non-heat shock protein Bagl, which binds to Hsp70 (Chapter 3 andChapter  4) .So far, the function of Hsp70 has been studied mainly in test tubes(Chapter l).These studies ha'n'e revealed that Hsp70 functions as a chaperone:Hsp70 protects unfolded proteins from irreversible aggregation and helpsthem to lefold to tireir native state. Although it is likely' that this refoidingprocess pla5's a role in thermoresistance of cells, it is not clear to what extentthe chaperone activity of Hsp70 contributes to thermotolerance of living cells.This has been investigated in Chapter 2. Hamster cells, rvhich under normalconditions do not produce Hsp70, rvere provided rvith a human gene thatcodes for Hsp70. The production of I{sp70 in tl-rese cells was controlled by the1 3 8SUMMA RYantibiotic tetracyclinc, which enabled a precise control of the production ofHsp70, independent of stress or heat. Chapter 2 describes the contribution ofHsp70 as a chaperone to the heat resistance of l iving cells. The effect of thepresence of Hsp70 alone is compared ro rhe effect of al1 Hsp's produced afterheat-stress. More production of Hsp70 increased the heat resistance of celis.This resistance, however, did not reach the level of resistance of cells that pro-duced all Hsp's. In addition, Hsp70 contributed to the protection of proteinsagainst the damaging effects of heat. This was investigated by measuring theextent of heat-denaturation of the model protein fircfly luciferase. The genecoding for this protein was adapted for expression in the nucleus or in thecytoplasm. Cells with the nuclear or cytoplasmic luciferase were heated andthe activity and aggregation of the protein were followed in time. Luciferasewas protected b5r the presence of Hsp70, both in the cytoplasm and in thenucleus. Furthermore it was investigated whether expression of Hsp70 alonewas sufficient for the protection of proteins similar to thermotolerant cells.Surprisingly, in the cytosol, Hsp70 could indeed protecr luciferase to the sameextent as all Hsp's together. This was not true for the nucleus. In the nucleusalso other Hsp's appeared to be involved in protection.Most cellular processcs require the activities of complex networks of inter-acting and collaborating proteins. To understand the function of newlv discov-ered proteins one therefore often searches for proteins with known functionsthat bind to the new protein. In this way it has becn found that Hsp70 bindsto a protein, Bagl that has been proposed to play a role in the protection ofcells against cell death after a varietl' of cellular stresses. Bagl levels are ele-vated in certain tumor cells, but the precisc function of Bagl is sti l l  unclear.In cells, four different forms of Bagl are expressed, which differ in the aminoacid chain length and are identical at one site of the amino acid chain. Thelongest isoform of Bagl is expressed only in the nucleus. Interestingly, all Baglisoforms bind to Hsp70. Experiments with purif ied Hsp70 and Bagl haveshown that the smallest isoform of Bagl inhibits the chaperone acriviry ofHsp70. Because in cells, the isoforms of Bagl bind to a variety of other, non-heat shock proteins, this study has investigated how these isoforms influencethe Hsp70 chaperone activity in cells (Chapter 3 and Chapter 4). It appearedthat when besides Hsp70 also the levels of individual Bagl isoforms wereincreased, Hsp70 was inhibited by all Bagl isoforms, both in the cytosol andin the nucleus. The location of the Bagl isoforms appeared to correspond tothcir influence on Hsp70: the longest isoform of Bagl selectively inhibited theHsp70 chapcronc activity in thc nucleus. The inhibitory cffcct of Hsp70 there-t39fore seems to be a general characteristic of Bagl proteins. It is clear from thesefindings that the influence of Hsp70 is not only determined by the levels ofHsp70. In addition, the concentration of Hsp70 binding proteins, such asBagl, appears to have an influence on the function of Hsp70.Chapter 2 and Chapter 3 described the influence of Hsp70 on the activi-ty of the model protein luciferase. It has remained unclear how and where theunfolded proteins are processed in the cell. In Chapter 4, a method has beendeveloped to directly follow the fate of unfolded proteins in living cells. Thenuclear from luciferase was fused to a green fluorescent protein (GFP).Thecoupling of GFP to luciferase enabled us to follow the changes in localizationof luciferase in a living cell under the microscope. \7hen cells with nuclearluciferase coupled to GFP were heated, the inactivated luciferase accumulatedin small foci throughout the whole nucleus. The unfolded proteins in thesesmall aggregates were not refolded if the cells were subsequently brought backto their normal growth temperature. If, however, the cellular levels of Hsp70were increased, the insoluble and inactive proteins accumulated in a few largeaggregates during heat shock. The localization of these aggregates appeared tooverlap with the localization of a sub-compartment of the nucleus: the nucle-olus. Surprisingly, from the nucleolus the proteins relocalized after return tonormal growth conditions, which was associated with the reactivation ofluciferase. How the unfolded proteins enter the nucleolus and what happensin the nucleolus to enable refolding of the proteins remains to be investigated.Although experiments in test tubes have provided important informationabout the function of Hsp70, the situation in a living cell appears to be morecomplex. In addition to the prevention of protein aggregation and proteinrefolding, Hsp70 appears also to be involved in transport and temporal stor-age of the unfolded proteins. It has been described that misfolded cytoplasmicproteins are transported to the centrosome of the cell, which is associated withtheir degradation. Transport and temporal storage at specific sites within thecell might therefore be a common event in the processing of damaged proteins.A possibie advantage of storage may be that is reduces damage to the ceil,because it minimizes the chance that damaged proteins aggregate with otherfunctional proteins or protein structures. A discussion about this possibil i tyand about the putative role of Hsp's in transport and storage processes is givenin Chapter 5. To understand more about the intcrplay between Hsp70 andother Hsp's in the protection of cells against protein damage, in the future,more studies in l iving cells wil l be required.1 4 0

HSP70 chaperone functions in stressed cells.

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HSP70 chaperone functions in stressed cells.

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