Stress Proteins: Nomenclature, Division and Functions

Prokaryotic and eukaryotic cells react to exposition unfavourable conditions of the outer environment by increased synthesis of the stress proteins. The structures and functions of these proteins are evolutionary highly conserved and they are present in different variations in the cells of all living organisms. Increased synthesis of the stress protein apparently correlates with an organism's resistance to stress and with a level of own stress 1. Ritossa described cellular stress response for the first time in 1962. He observed the characteristic puffs that indicated a transcriptional activity on discreet chro-mosomal loci in salivary glands of Drosophila mela-nogaster at an increased temperature 2. Proteins coded by the genes in described loci were named by Tissieres et al. as the heat shock proteins (hsp), because their increased synthesis was reached by a sudden increase in temperature 3. Other studies showed that an expression of hsp is induced by many various factors in the cell. These factors include: a) changes in: temperature, pH, osmolarity, and radiation, and b) higher concentration of heavy metals, ethanol, antibiotics, fatty acids and reactive oxygen forms. The stress response can be invoked by a change of partial gas pressure in the atmosphere, by absence of nutrients or by infection in higher organisms. Thus, hsp resp. stress proteins enable cells to survive when there are unfavourable conditions in the outer environment 4, 5, 6, 7. There are two main functions of stress proteins which are essential for reparation of each living cell which is damaged by stress. They are: 1) the participation in protein folding into their correct tertiary structure, incorporation of polypeptides into intracellular membranes or in transport of proteins across those membranes 8, 9 , 2) the function of some the stress proteins in ubiquitin-dependent protein degradation; ub-iquitin itself also belongs to the hsp super-family 10, 11, 12, 13, 14. Although during stress the synthesis of the stress proteins has increased considerably, a lot of the stress proteins are expressed as the constitutive proteins, and they play the significant role even in the cells which are not exposed to the stress factors 15. The stress proteins have also been important models for these studies, not only of stress response in the last years 16. The study of Tissieres et al. (1974) introduced the term " heat shock proteins " , and it belongs to the beginning of research on the stress proteins 3. In a context …

Prokaryotic and eukaryotic cells react to exposition unfavourable conditions of the outer environment by increased synthesis of the stress proteins.The structures and functions of these proteins are evolutionary highly conserved and they are present in different variations in the cells of all living organisms.Increased synthesis of the stress protein apparently correlates with an organism's resistance to stress and with a level of own stress 1 .
Ritossa described cellular stress response for the first time in 1962.He observed the characteristic puffs that indicated a transcriptional activity on discreet chromosomal loci in salivary glands of Drosophila melanogaster at an increased temperature 2 .Proteins coded by the genes in described loci were named by Tissieres et al. as the heat shock proteins (hsp), because their increased synthesis was reached by a sudden increase in temperature 3 .Other studies showed that an expression of hsp is induced by many various factors in the cell.These factors include: a) changes in: temperature, pH, osmolarity, and radiation, and b) higher concentration of heavy metals, ethanol, antibiotics, fatty acids and reactive oxygen forms.
The stress response can be invoked by a change of partial gas pressure in the atmosphere, by absence of nutrients or by infection in higher organisms.Thus, hsp resp.stress proteins enable cells to survive when there are unfavourable conditions in the outer environment 4,5,6,7 .
There are two main functions of stress proteins which are essential for reparation of each living cell which is damaged by stress.They are: 1) the participation in protein folding into their correct tertiary structure, incorporation of polypeptides into intracellular membranes or in transport of proteins across those membranes 8,9 , 2) the function of some the stress pro-teins in ubiquitin-dependent protein degradation; ubiquitin itself also belongs to the hsp super-family 10,11,12,13,14 .
Although during stress the synthesis of the stress proteins has increased considerably, a lot of the stress proteins are expressed as the constitutive proteins, and they play the significant role even in the cells which are not exposed to the stress factors 15 .The stress proteins have also been important models for these studies, not only of stress response in the last years 16 .

NOMENCLATURE AND THE BASIC DIVISION OF STRESS PROTEINS
The study of Tissieres et al. (1974) introduced the term "heat shock proteins", and it belongs to the beginning of research on the stress proteins 3 .In a context of current knowledge, the term "protein heat shock proteins", especially in eukaryotes, is used rather as a historical name.Particularly, it still overlaps with the logically evidently more correct term "stress proteins".This name specifies all the group of proteins generally where expression has increased due to an incidence of the stress factors.An abbreviation for the stress protein(s) "hsp" already remains in use.Above all, there are some proteins in prokaryotes where synthesis is affected by the heat shock, and they are denoted like heat shock proteins only, although they may be produced due to another stress factors than the heat stress as well 29 .Sometimes we can see another abbreviation, "hsc" (heat shock cognates), which has been used for the constitutive forms of hsp.Those forms of hsp are also present at non-stressed cells, and in contrast to the majority of other proteins, their intracellular concentrations have been increased during the heat shock.
In eukaryotic cells, different stress factors induce the synthesis of another, even if similar proteins or the same proteins are localised in another cellular compartment 30 .This has contributed to the introduction of a great number of trivial names and abbreviations, mostly derived from initial letters of the English terms.They describe stress factors or functions of the certain proteins themselves.For example, grp78 is the abbreviation for the stress protein that is induced by an exhaustion of glucose or by an abundance of calcium ions in the outer environment.Grp78 can be present under normal (nonstress) conditions and is denoted as hsc78, otherwise known as hsp78.The identical protein is found underneath the abbreviation BiP (binding protein), because it binds heavy chains of immunoglobuline molecules 31 .Another example can be the DnaK protein in Escherichia coli, which is linked with its participation upon DNA replication 32 .
The term "chaperone" is used very often.This term points out the function of the protein directly.It concerns the stress as well as non-stress proteins, which accompany unfolded polypeptides during their cellular transport, and they make passage of proteins through the membranes possible or their integration into cellular organelles.A similar, well-known term, "chaperonin" is the alternative name for the GroEL protein.It is abbreviated as "cnp60" or "hsp60".The chaperonins are present in bacteria as well as in mitochondria and in chloroplasts, denopting the organelles probably originated from the endosymbiosis 33,34 .An identical term is also used for GroES (cnp10), which has some sequence similarity with GroEL and also for some other proteins e. g. hsp58 35 , P 1 36 , 65kDa antigen 37 etc.Chaperonins, according to Coates et al. 38 would be divided into two "sub-families", GroE and TCP1.
This increased knowledge we have about stress proteins has lead to division of the hsp super-family into specific families 39 .The significant determinant criteria are the molar masses of proteins 40 .The important sequence and functional homologies were observed among the members of individual hsp families as well 41,42 .The division of hsp into the families is not standardised precisely yet.Earlier dividing of families: hsp90, hsp70, hsp60 and small hsp 43 has been extended step by step to hsp110, hsp100, hsp90, hsp70, hsp60, hsp40, hsp10 and small hsp families 1,44 .Numeric indexing represents the protein molecular masses in kDa.The stress proteins are registered into appropriate families according to their approximate molecular masses, their functions in the cells and their homologies in the primary structures.
Currently, we have the largest amount of information about the stress proteins hsp90, hsp70, hsp60 and small hsp families.That is why, the major attention will be dedicated to them in the following text.

hsp90
Hsp90 (hsp83) is the most abundant cytosolic protein in the eukaryotic cells.Its homologues were found in the endoplasmic reticula (ER) of higher eukaryotes (grp94 also called endoplasmin) and in prokarytic cells (HtpG).Hsp90 exists in vivo, obviously as a dimer and it co-exists as a cytosolic protein in higher eukaryotes in two homologue isoforms.Those are indicated as alpha and beta and are produced in the same quantity.In E. coli the HtpG (68 kDa) differs from hsp90 by the absence of a charged area of approximately 50 amino acids, which is characteristic for the other hsp90 homologues 45 .
Under physiological conditions, hsp90 was found in association with several intracellular proteins including calmodulin, actin, tubulin, several kinases, and some receptor proteins 46,47,48,49 .Significant interest is dedicated to the pivotal role of hsp90 into the regulation of hormonal receptors 50,51,52,53 .Receptors what aren't bounded to their hormones are probably bound to hsp90 shortly after their translation 54 .In case of the glucocorticotropic receptor, binding of hsp90 leads to an enhancement capability of the receptor to bind to the steroid hormone 55 .The C-terminal region of the hsp90 molecule is responsible for the receptor binding 56 .
Formation of the complex of hsp90 with non-receptor tyrosine kinases was also described as viral oncoprotein v-Scr 57,58 .Hsp90 has even chaperone function 59 that is comparable to GroEL function and can suppress an aggregation of proteins during their repeated assembling into their tertiary structures 60 .Cytosolic hsp90 aggregates with hsp70 under the stress conditions and it is suggested that interaction occurs of both hsp(s) with unfolded proteins 49 .

hsp70
Proteins in the hsp70 family are known for their ability to bind peptide chains.They act in: 1) a protection of the nascent proteins, 2) a protein transport across the membranes, 3) repeated assembling of unfolded proteins, and 4) the protein degradation 61,62,63,64 .
The structure of hsp70 consists of a domain with ATPase activity and a domain capable to bind peptides 65 .By roentgenographic analysis it was found that the ATPase domain has high structural homology with ATPase domains of hexokinases 66 and actin 67 .The domain that is capable to bind peptide was modelled on the structure of MHC I antigen 68 .MHC I antigen selection can also be connected by localisation of the genes coding for some hsp70 inside a gene region of the MHC complex 69 .The localisation above mentioned was found in man experimentally 70,71 .For instance BiP/grp78 was localised on the 9q34 position 72 .These genes were also identified in other species, e.g.rat 73 , goat 74 , cattle 75 , pig 76 , and frog 77 .The genes coding for hsp70 are also located in eukaryotes in the different loci resp.chromosomes and form the multigene families 69 .The high grade of structural homology exists in the hsp70 family, e.g. in man, hsp70-1 gene is distanced from gene hsp70-2b by P. Kopeček, K. Altmannová, E. Weigl only 8 kb, and both coding for the protein with the same primary structure 71 .
There is approximately 50 percent homology in eukaryotic hsp70 and prokaryotic DnaK protein in E. coli 78 .This protein performs its functions frequently in co-operation with DnaJ protein and GroE in E. coli 79,80,81,82,83,84 .Genetic and biochemical study results support the hypotheses that proteins of DnaJ type co-operate with certain hsp70 proteins in all organisms 85 .For instance, the proteins Kar2p (homologue DnaK) and Sec63p (DnaJ homologue) participate on a protein transport to the ER in Saccharomyces cerevisiae 86 .
This confirms a considerable evolutionary conservancy of the entire hsp super-family 62 .Thus it could be expected that an immune system will recognise the stress proteins as the body's own antigens and therefore, the immune responses against hsp will be rare.Nevertheless, if the contrary is true 87,88,89,90 and the stress proteins from hsp60 and hsp70 families belong to the group of immunodominant antigens 91,92,93 .

hsp60
In 1981 the essential functions of GroEL were described in bacterial growth in head morfogenesis of lambda and T4 phages and in the tail formation of T5 phages 94 .The chaperonins (the group of stress proteins belonging to the hsp60 family) have significant roles in polypeptide folding and in protein transport in the cells as well 8 .These proteins are not only in bacterial cells, but also in mitochondria and in chloroplasts of eukaryotic cells, and the genes coding for hsp60 form a part of the nuclear genome 35,95,96 .Similar structural motifs were observed among mitochondrial hsp60, plastid hsp60, and hsp60 in E. coli 35,97 .Although hsp60 forms a twolayer heptamer ring in chloroplasts and in the majority of mitochondria 98 , the ovary cells of the Chinese hamster 99 and moth sperms 100 have the single heptamer rings of hsp60 in their mitochondria.Chloroplasts in contrast to mitochondria and E. coli, which contain only one type of hsp60 polypeptide 95,96 , have roughly identical quantity of the two diverse forms of hsp60 polypeptides alpha and beta 101,102 .The chloroplast hsp60 was originally identified like a component that has appeared in biosynthesis of ribulose biphosphate karboxylase (Rubisco), where it binds the unfolded polypeptides before they form the holoenzyme 103 .
Generally, chaperonins are able to form stable complexes with proteins, which are imported to chloroplasts and to mitochondria 98 .They perform their chaperone function also in co-operation with the other molecules, e. g. cnp10 and hsp70 81,104 .Hsp60 also has other important functions in an immune response due to its already mentioned immunodominant properties 91,92,93 .Furthermore, bacterial hsp60 and hsp70 take a part in regulation of gene transcription coding for the stress proteins 105 .

TRANSCRIPTION OF GENES CODING FOR HSP
A significant correspondence in the stress response of different organisms exists.Structural and functional similarities were described among members of hsp families 1 .Certain similarities can be seen in the regulation of stress gene expression as well.Why is considerable attention given to the regulation of transcription of the genes coding for the stress proteins at all?During the heat shock, the transcription of those genes is increased as much as 100 times 106 that offers the following opportunities: 1) detection of specific regulation components and quantitative evaluation of an expression in time, and an understanding to their role in transcription as well 18,107 , 2) creation of the model on an investigation of kinetics and mechanisms of these processes; because genes coding for the stress proteins are activated rapidly by proteins, which are present in non-inducted cells 17 , 3) obtaining the information about the primary structures of DNA elements and protein factors, which take a part on transcription control and about their localisation into the chromatin 108 , 4) an application of the knowledge on hsp gene transcription that has a general significance for the study of transcription control, because some promoters of hsp genes are assembled from the elements, which can be found in the promoters of other genes 17 .
From the presented specifications, a common significance of the investigation of transcription of the genes coding for the stress proteins and their exploitation for better understanding to mechanisms of transcription to make a general image of the stress protein expression.Due to the different mechanisms in prokaryotes and eukaryotes, they will be discussed separately in this text.

Transcription of genes for the stress proteins in prokaryotes
Most of the informations about the transcription of genes for the stress proteins were obtained from the studies using bacterium E. coli.The cells respond to increased levels and activity of the transcription factor -sigma32 (32 indicates the molecular mass in kDa) by increased transcription in the genes coding for the stress proteins.Sigma32 factor is coded by the gene rpoH and controls transcription of about 20 hsp, e.g.DnaK, DnaJ, GroEL and GroES (Fig. 1).These proteins were identified by 2-D electrophoresis 105 .Major transcription factor is sigma70 in the prokaryotic cells in addition to the afore mentioned sigma32 109 .If the level of sigma70 is reduced, hsp expression increases and leads to the conception of competition for RNA polymerase between sigma70 and sigma32 factors 105 .Regulation function of the sigma32 is, above all, based on its synthesis, activa-Stress proteins: nomenclature, division and functions tion and degradation 110 .Under normal conditions, sig-ma32 levels are very low, but its half-life is getting longer at the increased temperature 111 .Quantity of the sigma32 is influenced by negative feedback as well e.g. by DnaK.Without the presence of DnaK the lifetime of sigma32 is 10-30 times longer.Similar effect on the sigma32 level was also demonstrated with the presence of DnaJ and GrpE.Furthermore sigma32 activity is dependent on temperature 81 .
The gene coding for sigma32 (rpoH) has at least 4 promoters.Three of them, P1, P4 and P5 are transcribed in attendance of sigma70 112 , while P3 is transcribed by RNA polymerase that contains sigma24 factor, which is more active under very high temperature 113 .P1 is the most distant promoter and also the strongest one.Its activity is not evidently regulated, but its -35 region coincides with the neighbour ftsX gene.Therefore, there is a hypothesis of the possible connection with transcription of fts gene, which is perhaps mediated by DnaA protein 114,115,116 .P1 and P4 promotors are responsible for the majority of the transcription of rpoH gene during vegetative conditions.In addition P4 embodies 2-3 times higher activity under elevated temperature 112 .P3 and P4 promotors bind the DnaA protein, which specifically inhibits the transcription from those promoters 117 .P5 is a relatively weak promoter, its effect boosts in the absence of glucose, or with ethanol addition.This promoter requires the presence of cAMP and of cAMP receptor protein in vitro 118 , but their presence is not necessary during an absence of carbon 119 .P3 is mostly active in extreme conditions, e. g. at lethal temperatures (over 50 °C), when the other transcription factors are apparently inhibited 158 .Several bacterial promoters have the sequence similarities to rpoHp3.One of the bacterial promoters is the htrA gene coding for degP protease (it acts under the higher temperature).This is transcribed with sigmaE (sigma24) in vitro 120,121 .These results imply that the second regulone for hsp expression controlled by sigmaE is co-existed in E. coli.This regulone can intensify or supplement the effect of sigma32 regulone, which is expressed on the basis of another stress.

Transcription of genes for the stress proteins in eukaryotes
Eukaryotic stress genes are generally controlled by HSE (heat shock elements) and hsf (heat shock factors), which are bound to them closely 20 .All of the HSE contain a basic motif of sequence 5'-NGAAN-3'.These sequences go subsequently in reversing orientation and their number is different, e. g. 4 HSE were found, and each of them contains 3 or 4 basic units in the hsp70 gene of D. melanogaster.On the other hand, 7 or 8 basic units were found in the hsp83 gene, which are only in one HSE.An insertion of 5bp can occur among repeated units in HSE as well 122 .The basic unit of 5bp can have a different primary structure.This is significant for the strength of the HSE region.The second position (G) is absolutely conserved, while the positions 3 and 4 (A) are less conserved.Substitutions were also found on the first position.Mostly, there is appeared (A) there, (T) is rare.Such substitution -(T) instead (A) leads to the significant decrease of hsp70 expression.Chemical modifications of (A), and also (T) have caused in both cases a lower affinity of hsf to the modified HSE regions.Position 5 is the least conserved, but it also affects activity of transcription 17 .
Hsf are functionally conserved in an entire eukaryotic empire.They are present as monomers in non-stressed cells, while they form trimers in the stressed cells 18 .The trimer is distinguished by a higher asymmetry compared to the monomeric form 123 .Different numbers of genes may code for hsf, e. g. one gene coding for hsf is in Drosophila melanogaster 124 , with vertebrates having several similar genes for hsf 18 .Heat shock response is mediated by hsf1 in man 107 .Hsf structures in different species are diverse with an exception of two conserved regions on their N-ends, which represent DNA binding and trimerisation domains 124 .
Interaction hsf-HSE can contain a various number of the basic HSE units.The smallest detectable part of HSE bounded to hsf is 10 bp long, and it exists in configuration "tail-to-tail" or "head-to-head".Hsf covers these parts in same manner.Hsf are also constitutive proteins in monomeric forms in eukaryotes (with the exception of yeasts) 125 .They are activated during the heat shock and form the trimers 17,107 .Hsf are redistributed from non-specific places on chromatin to separated chromosomal targets.A subpopulation of hsf molecules is transferred from the cytoplasmatic compartments into the nuclei during heat shock as well 18 .Thus, hsf and HSE interact if the hsf is in trimer form 126 .In yeasts (S. cerevisiae), the trimer form of hsf is present P. Kopeček, K. Altmannová, E. Weigl in its constitutive state, and remains bound on HSE during both normal and stress conditions.The stimulation of transcriptional activity of hsf in S. cerevisiae is apparently connected with phosphorylation in its serine and threonine residues.Binding activity of hsf is induced by various agents, which affect protein structure.These agents include: heat, slightly acidic pH (6,5) or presence of detergents 107,127 .
Original hypothesis of hsf activation in vitro is based on direct formation of an oligomeric state (trimer) 128 .The impossibility of dissociation of trimers to the monomeric forms in normal conditions shows that it cannot be a reversible one-component system.This leads to an idea of another intracellular component, which can act in the folding of nascent hsf polypeptide during its synthesis, and during disintegration of hsf trimer, when the cell is returned back to the normal conditions 17 .This offers also an idea of possible participation of molecular chaperones including some other hsp 129 (Fig. 2).

BIOLOGICAL FUNCTION OF THE STRESS PROTEINS
Principal roles of the stress proteins are reparation, protection, or elimination of damaged proteins in the cells.In this context there are generally two important biological functions: 1) the function of molecular chaperones 8,9 , and 2) the function of the stress proteins in protein degradation 10,11,12,13,14 .

Molecular chaperones
The significance of the molecular chaperones is in their assessment in a protein folding or refolding to their native conformations by stabilising of their partly denatured states 9,130,131 .Chaperones contain no specific information for polypeptide folding, but they are able to prevent production of aggregates from the nascent polypeptides 34,132 .It could be noticed that other heat induced proteins like peptide-isomerases and disulphid--isomerases act directly in a formation of higher protein structures 133 , and they are called "foldases" trivially 134 .
Chaperones from the hsp70 family have a dominant role in complex formation with nascent polypeptides on the ribosomes and thus inhibit their premature folding since a translation is terminated 37 .Hsp70 has not only function in the production of mentioned complexes, but it also participates in a transport function and in polypeptide folding 135 .Basic function of hsp70 is the binding of unfolded proteins and their release.It proceeds in an ATP dependent cycle 136,137 , in which: 1) partially unfolded protein associates with C-terminal domain hsp70 138,139 , 2) binding of co-chaperone hsp40 started a dissociation of ATP with N-terminal domain of hsp70 140 , which leads to a conformational change (hsp70/ADP complex), 3) hsp40 dissociates, 4) BAG -1 protein binds, 5) ADP/ATP exchange is initiated, 6) BAG-1 dissociates, 7) bounded proteins are released finally (fig.3

.).
Mostly, the proteins require an accomplishment of their correct native conformation to pass for several times by the described cycle 34 .This cycle is called the hsp70 chaperone machine, in which hsp70 co-operates with hsp40 and BAG-1 proteins 141 .
Complex of the stress proteins with already synthesised protein enables transport of this protein into cellular organelles 9,142 .Chaperones are able to stabilise the proteins that are damaged by influence(?) of the stress factors, and they participate into their renaturation.This function was described in detail for GroEL protein 55,143 .

Stress proteins: nomenclature, division and functions
GroEL recognises hydrophobic residues on the surface of partly denatured proteins 143 and introduces them into the central cavity of its heptameric ring that is formed by 57-kDa units 144,145,146 .Proteins bound in this GroEL complex are protected against the effects of proteolytic enzymes 144 .This "cage" model suggests a protein folding through a creation of the inner microenvironment, in which a protein folding can continue since the protein is protected against aggregation 147 .Another interactive alignment model was found on the limiting number of the steps in ATP dependent cycle 148 and on an intramolecular reorganisation of protein segments 149 .Its principle is more similar to the mechanism of hsp70 chaperone function.Both models do not exclude each other.Both models include co-operation with a co-chaperone GroES 150,151 , resp.co-operation of cnp60 with cnp10 152 in folding of partly unfolded proteins 98,146,153 .

Protein degradation
Molecular chaperones take a part in degradation of irreversibly damaged proteins 43 .The proteins, which are determined to degradation, have to be in a soluble state to be recognised by proteolytic enzymes.hsp preserves such a state and therefore the chaperone function of hsp is essential not only for the repair of damaged proteins, but also for their degradation 154 .
Hsp have other functions in the degradation process.The stress protein -DegP has not only a chaperone function, which dominates under lower temperatures, but also has proteolytic activity under higher temperatures 155 .
The pivotal function of ubiquitin in protein degradation is a little distinct from the function of molecular chaperones.Ubiquitin is ranked among the hsp and was found in all of the eukaryotic organisms 10,11,12,13,14 .Ubiquitin mRNA contains several subsequently repeating copies of the ubiqutin gene.The level if this mRNA can raise 2-50 times during the stress 156 .Ubiquitin is formed by 76 amino acids and it is probably the most conserved protein in evolution 157 .The difference between ubiquitin molecules in man and in nematodes was only one amino acid 156 .
The C-part of ubiquitin binds by isopeptide bound to etha-amino groups of lysine residues in substrate proteins.By the same way, it repeatedly binds to the already bounded ubiquitin molecules 11 .Thus it forms the side chains on the substrate proteins, which are recognised by multi-subunit protease, known as 26 S proteasome.When the "marked" substrate proteins, including ubiquitine, are broken down the ubiquitin monomers are ready for the further cycle 156 .

Fig. 1 .
Fig. 1.The scheme of the regulation of hsp expression in prokaryotes 158 .