Hsp90 molecular chaperone: structure, functions and participation in cardio-vascular pathologies

The review is devoted to the analysis of structural and functional properties of molecular chaperon Hsp90. Hsp90 is a representative of highly widespread family of heat shock proteins. The protein is found in eubacteria and all branches of eukarya, but it is apparently absent in archaea. It is one of key regulators of numerous signalling pathways, cell growth and development, apoptosis, induction of autoimmunity, and progression of heart failure. The full functional activity of Hsp90 shows up in a complex with other molecular chaperones and co-chaperones. Molecular interactions between chaperones, different signalling proteins and proteinpartners are highly crucial for the normal functioning of signalling pathways and their destruction causes an alteration in the cell physiology up to its death.

In tro duc tion.When var i ous phys i cal and chem i cal fac tors af fect the cells, their most fre quent tar get are pro teins; they dam age the pro teins struc ture and change their func tions.In or der to cope with pro tein dam age the cells should ac ti vate a mech a nism of specific de fence.The in ves ti ga tion on the pro cesses of cell func tion ing and fold ing of pro teins in vivo revealed a se ries of cell com po nents, in volved ei ther in the ca tal y sis of pro tein fold ing or in the reg u la tion of dis tri bu tion of newly syn the sized pro teins among alter na tive path ways of fold ing and ag gre ga tion [1].
Ellis [2] for mu lated an as sump tion that though pro tein fold ing is a spon ta ne ous pro cess there are crit i cal stages, at which the par tic i pa tion of cell fac tors may be cru cial.The role of these fac tors, called mo lec u lar chaperones, con sists in pro vid ing op ti mal con di tions for the pro cess of pro tein fold ing via re moval of "hindrances" or "in ad e quate con tacts", which re sults in pre ven tion of non-spe cific ag gre ga tion of pro teins, syn the sized anew, and pro vid ing a trans port of these pro teins to those intracellular com part ments, where they are usu ally lo cal ized and func tion.The fact that chaperones act as traf fic con trol lers is also sup ple -mented by their par tic i pa tion in pen e tra tion of proteins through mem branes.
Chaperones are a part of a large fam ily of heat shock pro teins (hsp), the syn the sis of which in the cell is con sid er ably in creased in re sponse to heat shock or other kinds of cell stress.At the same time most proteins of this fam ily are syn the sized rather in ten sively even at nor mal con di tions [1].
Mo lec u lar chap eron Hsp90 be longs to a widely spread fam ily of heat shock pro teins; they were found in eubacteria and all branches of eukarya, but they are ap par ently ab sent in archaea [3].Hsp90 is one of molec u lar chaperones, which is abun dantly pres ent in the cell at nor mal con di tions.Bac te rial chaperone is known as HtpG.A hu man cell con tains four isoforms of chaperones: cy to plas mic form Hsp90b, syn thesized con sti tu tively in the cells, and in duc ible form -Hsp90a; Hsp90 an a logue -GRP94, the ex pres sion of which is reg u lated by glu cose -was found in endoplasmic re tic u lum; and mi to chon drial TRAP1/hsp75 [3].Forms Hsp90a and b are 85% iden ti cal in their amino acid con tent which may be explained by du pli ca tion of genes more than 300 mil lion years ago.There is a high homology among Hsp90 of lower eukaryotes, hu mans, and prokaryotes: the identity of Hsp82 of yeast to hu man Hsp90a and HtpG is 60% and 34%, re spec tively [4].
Hsp90 struc ture.The crys tal struc ture of a full-length pro tein Hsp90 was de scribed about 10 years ago [4].At the same time qual i ta tive crys tals of a full-sized pro tein Hsp90, suit able for X-ray structural anal y sis, are yet to be stud ied, there are crys tals of spe cific do mains.Hsp90 pro tein is a dimer of two iden ti cal sub units.Each mono mer has three in de pendent do mains: from highly con ser va tive » 25 kDa N-ter mi nal do main; » 40 kDa M-do main, and » 12 kDa C-ter mi nal do main [5] (Fig. 1).N-ter mi nal domain is con nected to M-and C-re gion via so called charged linker which is vari able by both its con tent and length in dif fer ent types and isoforms of the protein.The charged linker is not es sen tial for Hsp90 func tion and is en tirely ab sent in Hsp75/TRAP1 and HtpG [6].
N-ter mi nal do main.The firs sig nif i cant prog ress in the study on Hsp90 struc ture was the de ter mi na tion of N-ter mi nal do main by lim ited pro te ol y sis [5].The in ves ti ga tion of the do main crys tal struc ture of hu man and yeast Hsp90 re vealed that it has a two-layer ab-sand wich struc ture [7] with a densely folded anti-par al lel b-layer, con sist ing of eight stripes, closed by nine a-coils on one side.There is a deep pocket, formed by ATP/ADP-bind ing site, in the centre of a-coil part.This pocket in N-do main of hu man Hsp90 was re vealed to be an oblig a tory site of bind ing geldanamycin (GA) which pre vents Hsp9 form ing com plexes with pro tein kinase and ste roid hor mone re cep tors [8].The true func tion of N-do main and its pocket be came ev i dent only by the com par i son of amino acid se quence of Hsp90 and two ATP-de pendent pro teins -topoisomerase II (DNA gyrase B, GyrB) and pro tein MutL.Con ser va tive amino acid res i dues, in volved in ATP bind ing and its hy dro ly sis, were de fined [9].The iden ti fi ca tion of key res i dues in N-ter mi nal struc ture of Hsp90 by the method of mu tagen e sis re vealed that N-ter mi nal do main con tains ATP-bind ing site (Fig. 2, a) [5,6].Sub se quently, crys tal struc tures have been de ter mined for N-ter minal do mains of the mam ma lian endoplasmic re tic u lum Hsp90 isoform GRP94 [10] and HtpG for Esch e richia coli [11].
M-do main.Af ter the struc tural anal y sis of N-termi nal do main of hu man and yeast Hsp90, the crys tal struc ture of proteolytically re sis tant, so called mid dle seg ment -M-do main of yeast was in ves ti gated [12].Its struc ture con sists of a large aba-sand wich fixed to N-ter mi nal site of the pro tein, and a small aba-sandwich, bound to C-ter mi nal do main of Hsp90.aba-sand wiches are con nected to each other via a solid ring of a-coil (Fig. 2, b).A big ger aba-sandwich has a fold (curve) sim i lar to the equiv a lent region of GyrB and pro tein MutL, al though there is a con sid er able dif fer ence in the re gion in volved in the in ter ac tion of MutL and GyrB with DNA.A smaller aba-do main is unique, though it has ar chi tec tural sim i lar ity to the big ger, clas sic aba-do main.The anal ogy be tween MutL and GyrB was stud ied us ing site-spe cific mu ta gen e sis [9].
It was re vealed that the mid dle seg ment con tains a ma jor site of in ter ac tion with "cli ent" pro teins.This is a small con ser va tive hy dro pho bic site with a Trp 300 as the cen tre and an un usual amphipatic pro tu ber ance, formed by amino acid res i dues 327-340.The cen tral b-sheet of aba-do main con tains a ly sin, in ter act ing with g-phos phate of ATP, bound to N-do main Hsp90 [5].The method of site-spe cific mu ta gen e sis also revealed con ser va tive Arg 380 , which is im por tant for ATP-ac tiv ity of Hsp90 of yeast both in vi tro and in vivo.M-do main of Hsp90 is also a fac tor, re spon si ble for dif fer en ti a tion be tween dif fer ent types of cli ent pro teins [5].
C-ter mi nal do main.The crys tal struc ture of C-ter mi nal do main of Hsp90 homologue, iso lated from E. coli -HtpG , was firstly an a lyzed (Fig. 2, c) [13].C-ter mi nal do main is es sen tial for chap eron dimerization.C-do main of HtpG is a dimer, con sisting of small ab-do mains.The com par i son of pri mary and sec ond ary struc tures of C-ter mi nal do mains of Hsp90 of prokaryotes and eukaryotes re vealed that the most char ac ter is tic fea ture of C-do main of HtpG is the ab sence of more than 35 amino acid res i dues in the ter mi nal seg ment, namely -in MEEVD-mo tive, involved in bind ing with TPR-do main (tetratricopeptide re peat do main) [14].Nev er the less, the gen eral structure of C-do main of Hsp90 of both eukaryotes and bac te ria is very sim i lar and as the crys tal struc ture for eukaryotic Hsp90 is cur rently ab sent, the bac te rial struc ture of HtpG may serve as an ex am ple of a good work ing model.Tak ing the above men tioned into consid er ation, we may con clude that the com plete structure of mo lec u lar chaperone Hsp90 is known in general, how ever only on the ba sis of its sep a rate domains.There is one more site, the struc ture of which has not been stud ied yet.It is the site, lo cated be tween N-and M-do mains (Fig. 1, a), which is no ta ble for weak con ser va tism.Both bac te rial HtpG and mi tochon drial pro tein do not have this site.Its size is in the range of 30-70 amino acid res i dues in dif fer ent types of Hsp90 and isoforms.The ex per i ments in vi tro [15] and in vivo [16] proved that this seg ment is not cru cial for Hsp90 func tion ing.
Conformational changes and ATP-bind ing.The in hi bi tion of Hsp90 by GA is known to in duce its deg ra da tion within proteasome.As the chaperone activ ity of Hsp90 is de ter mined by the bal ance of ATP and ADP and the pres ence of pro tein-as sis tants [9], there is a ques tion how its ATPase ac tiv ity in flu ences the in ter ac tion with cli ent pro teins.This in ter ac tion was con sid ered by the au thors of [15] to be conformational change in the struc ture of Hsp90 -the tran si tion from the open form into the closed one.The elec tron mi cros copy dem on strated that while in ter acting with ATP N-ter mi nal do main causes conformational changes in C-do main: the for ma tion of a dimer struc ture in which mono mers are ori ented anti-parallelly to each other in the ab sence of ATP and the for ma tion of a ring struc ture in the pres ence of ATP.At the same time the method of X-ray struc tural anal y sis of a full-sized HtpG mol e cule [14] dem onstrated the pres ence of a V-shaped struc ture, form ing dimers, which is no ta ble for par al lel lo ca tion of monomers.The dif fer ence in con for ma tions shown by electron mi cros copy of the crys tal struc ture may re flect con sid er able flex i bil ity of Hsp90 mol e cule in the absence of ATP-bind ing.It is doubt ful whether this chaperone mol e cule has the only fixed con for ma tion.The as so ci a tion of N-ter mi nal do mains was dem onstrated by sev eral meth ods for Hsp90 from yeast, chicken, and other eukaryotes.The model of conformational tran si tions of Hsp90 from the open form into the closed one in the pro cess of ATP bind ing tes ti fies to the fact that the for ma tion of a dimer is neces sary for the hy dro ly sis of ATP, and two halves of Hsp90 co op er ate with each other to achieve dimer con for ma tion, ca pa ble of hy dro lys ing ATP [17].The stages of achiev ing this con for ma tion are a com plex struc tural pro cess, in which sev eral ar eas of Hsp90 are sub ject to in ter de pen dent re lated trans po si tions, required for any of two ac tive sites in the dimer to be cat a lyt i cally ac tive.
These data are in good agree ment with the re sults of ki netic anal y sis of Hsp90 of hu mans, which did not re veal the co op er a tion in clas sic sense be tween protomers in Hsp90 dimers at ATP hy dro ly sis [18].The no tion of "func tion ally re lated ATP cy cle" is the def i ni tion of the pro cess, in which ATP bind ing sta bilizes so called "tense" conformational and func tional state of Hsp90, de pend ent on the pres ence of g-phosphate of ATP, and the sub se quent hy dro ly sis to ADP destabilizes the con for ma tion and al lows Hsp90 dimer to re lax [5].The gen er ally ac cepted model of the ATP-cy cle is sim i lar to that of dimers GyrB and MutL, in which N-ter mi nal do mains are lo cated very close to each other, and each of them is packed tightly in the di rec tion to wards the mid dle seg ment of the same chain.In ATP-bound state DNA-gyrase B forms so called mo lec u lar clamp, which is sup posed to close around the DNA chain, and dur ing ATP hy dro ly sis it re leases DNA af ter its supercoiling [19,20].The peptide binds to the Hsp90 ATP-bind ing cen tre in a sim ilar way: it moves in side the chaperone and re leases at the mo ment of ATP hy dro ly sis [5].
Point mu ta tions in Hsp90, slow ing down ATP hydro ly sis but not in flu enc ing ATP-bind ing, also decrease the ef fi ciency of ATP-de pend ent com plex [21].This raises the ques tion: how is the sub strate pro tein kept in the ATP-cen tre? Al though a space en cir cled by the Hsp90 dimer is prob a bly too small for an en tire pro tein sub strate, a siz able do main could be ac commo dated.There is also as sump tion that Hsp90 does not en close its sub strate but rather that a substrate-bind ing face is op ti mally ex posed by Hsp90 in the ATP clamp state.
Re cent in ves ti ga tions have proved the oblig a tory pres ence of one more nu cle o tide-bind ing site at C-termi nal do main of Hsp90 [22].The de le tion of 20 amino acid res i dues in C-ter mi nal do main (660-680) destroys bind ing of both ATP and novobiocin.C-domain nu cle o tide-bind ing site may get ac ti vated ei ther when N-ter mi nal site is oc cu pied by a nu cle o tide or is in ac ti vated, and also when N-site is de leted.On the other hand, the bind ing of an ti bi otic novomicin to C-site blocks bind ing of ATP on N-site as well.Therefore, these two sites, re quired for ATP bind ing, work in good co or di na tion, which is im por tant for reg u lation of Hsp90 conformational state.A role of this addi tional site is not yet clear, but its in hi bi tion by novoboicin and cis-plat i num is in ter est ing for fur ther in ves ti ga tions of its func tion [22].
Hsp90 func tions.Hsp90 func tions in vivo as the main com po nent of the ma jor ity of multi-pro tein complexes; along with other fac tors it is one of the key reg u la tors of nu mer ous sig nal ing path ways, it par tic ipates in pro tein deg ra da tion and per forms also ad ditional func tions of con trol, ro ta tion, and trans fer of var i ous pro teins and sub strates [5].
Nu clear ex port.Many sig nal re sponses of a cell are the rea son of changes in lo cal iza tion of sig nal ing pro teins or pro tein kin ases which move and then ac tivate tran scrip tion fac tors, lo cated in the nu cleus.There is very lit tle data on the move ment of mol e cules in side the nu cleus.Pro tein mo bil ity in side the nu cleus may be dif fusely lim ited, but it might be at trib uted to a spe cific frac tion of res i dent nu clear pro teins only.As the func tions of nu clear pro teins are of ten lim ited by spe cific subnucleus com part ments, the sys tem of their mo bil ity should be fa cil i tated by a pur pose ful de livery.Hsp90 reg u lates the ac tiv ity of over 100 pro teins, in volved in the cell sig nal transduction [23].These are so called "cli ent" pro teins of Hsp90.The cell sig nal transduction also in volves a multi-pro tein com plex Hsp90/Hsp70, form ing heterocomplexes of "cli ent" pro teins and Hsp90 in the cy to plasm and nu cleus.
In case of sig nal ing pro teins, act ing as tran scription fac tors, the com plexes of "cli ent" pro teins and Hsp90 also con tain one or sev eral TPR-do mains of immunophilins or their homo logues and bind to TPR-do mains on Hsp90.Dy namic as sem bly of heterocomplexes and Hsp90 is re quired for fast movement through the cy to plasm to the nu cleus along microtubular tracks.In this sys tem the role of immunophilins con sists in con nect ing the "cli ent" pro tein/Hsp90 com plex to the cy to plas mic dynein -a mo tor pro tein, pro vid ing the move ment to wards the nu cleus.Fa cil i tated dif fu sion al lows the move ment of re cep tor/Hsp90/immunophilin com plexes into the nucleus through the nu clear pore com plex (NPC).Then non-bound re cep tors spread in the nu cleus, reach ing the dis crete nu clear cen tres to bind with chromatin.Ac tive as sem bly of the heterocomplex with Hsp90 is re quired for the move ment to the nu clear cen tre and for the ac tive ex change of tran scrip tion fac tors between chromatin and nucleoplasma.
Ex port of 60S ri bo somal sub unit.Tran si tion of large sub strates through NPC re quires sig nif i cant conformational changes in both NPC and a sub strate.Hsp90 sup ports these struc tural changes [24].
It was re vealed that nu clear ex port is pre ferred to nu clear im port at the con di tions of cel lu lar hy per trophy.In hy per tro phied cells, for in stance, in cardiomyocytes, the amount of mRNA, de liv ered to the cy to plasm for pro tein syn the sis, is con sid er ably in creased [25], the pro tein syn the sis is en hanced, which should re sult in the in ten si fied syn the sis of ribo somes.It is note wor thy that cardiotrophin-1 (CT-1), which is a fac tor, pro mot ing the stim u la tion of cardiomyocyte hy per tro phy (it makes the cell grow rather than dif fer en ti ate), stim u lates the syn the sis of Hsp70 and Hsp90 as well.The en hanced syn the sis of Hsp90 in hy per tro phied cells seems to al low them to sup port the free nu clear ex port of large sub strates [26].The fol low ing model is sug gested for the nu clear ex port of 60S ri bo somal sub units.The tran si tion of 60S ri bo somal sub unit through NPC re quires the maxi mal di la tion of their pores, fa cil i tated by Hsp90 due to its chaperone func tion.Then Hsp90 fa cil i tates the tran si tion of 60S ri bo somal sub unit from NPC to cytoskeleton, "lead ing" the ri bo somal sub unit through the cell via so called trans port cytoskeleton route to its fi nal des ti na tion place.It was dem onstrated ear lier that Hsp90 pro motes the for ma tion of ri bo somal crys tals in side the nu cleus [28], and the tran si tion of 60S ri bo somal sub unit through NPC takes place with the me di a tion of Hsp90.The lat ter helps the sub strate "to dis solve" in the hy dro pho bic en vi ron ment of NPC.Tak ing into con sid er ation a huge size of 60S ri bo somal sub units, their trans port is a more com pli cated mech a nism, in volv ing more factors and var i ous in ter ac tions be tween 60S ri bo somal sub units and NPC than the same pro cesses for such small sub strates as pro teins and tRNA.
Hsp90 and sig nal transduction.Nu mer ous proteins, which are transduction sig nals in a cell, are main sub strates of Hsp90.These pro teins de pend on Hsp90, which keeps them in ac tive con for ma tion.Dis or ders in Hsp90 func tions due to mu ta tions or treat ment with such in hib i tors as ansamycins cause nu mer ous changes in the cel lu lar cy cle, which cor re spond to the con tri bu tion of Hsp90 into the sig nal ing path ways of the cell.For in stance, point mu ta tion in Hsp90 dur ing the reg u la tion of cell di vi sion re sults in the change in many stages of mi totic sig nal ing cas cade, the centrosomal cy cle in the course of mi to sis [28].In vivo ex per i ments re vealed the sig nif i cance of Hsp90 for the func tion ing of ste roid hor mone re cep tors [29], some ty ro sine and serine/threonine kin ases, such as pp60/v-src, Wee-1, Cdk4, Raf, and Akt [30] as well as some pro teins -synthase of ni tro gen ox ide and calcineurin [31].These are large and multi-do main pro teins, the func tion ing of which re quires sta bi liz ing re la tions with other fac tors, such as lig ands for ste roid hor mone re cep tors or cyclins for cyclin-de pend ent kin ases.As sig nal ing pro teins, hav ing nu mer ous reg u -la tory func tions, are of ten un der the im pact of conformational changes, their in ter ac tion with Hsp90 may be the re sult of struc tural changes in these proteins, which fa cil i tate their rec og ni tion by Hsp90.
Hsp90-de pend ent sig nal ing path way of ste roid hor mone re cep tors is char ac ter ized the most [30].A glucocorticoid re cep tor mono mer (GR) and a progesteron re cep tor are loaded on Hsp90 via Hsp70/Hor-de pend ent mech a nism, bind to Hsp90 and achieve their hor mone-bind ing con for ma tion.As soon as the folded monomeric re cep tor is free from the chaperone, it binds to a cor re spond ing ste roid hormone im me di ately, form ing a dimer and ac ti vat ing it.A dimer be comes non-sta ble and is rec og nized by a chaperone again.
The progesteron re cep tor and GR are used for the re con struc tion of typ i cal multi-chaperone com plexes with pu ri fied Hsp90, Hsc70, Hop, and p23 of mammals [32].Hsp90/Hop/Hsp70/Hsp40 com plex was called a foldosome.A foldosome con verts GR from the folded state, in which a ste roid-bind ing site is closed and thus is not ca pa ble of in ter act ing with the ste roid, into the open state, ac ces si ble for the ste roid [33].While form ing a foldosome Hop binds to Hsp90 and Hsp70 via TPR-do mains.Hop binds a dimer Hsp90 mol e cule, and chaperone Hsp40 is a co-chaperone of Hsp70, which is also pres ent in the tetracomplex and ex e cutes ATP/Mg 2+ -and K + -depend ent open ing of ste roid-bind ing site.While the ste roid-bind ing site is open, Hsp90 con verts into ATP-bound con for ma tion, dy nam i cally sta bi lized by p23 pro tein, which re sults in re leas ing Hop from Hsp90.The com plex formed is as sem bled in such a way that sev eral changes in it are oc cur ring in dynamic fash ion.Immunophilins are also con nected to Hsp90 via TPR-do mains, and when Hop leaves the com plex, the immunophilin may bind a sin gle TPR-ac cep tor site on the re cep tor-bind ing Hsp90 dimer.Some Hsp70 pro teins may also form an in terme di ate com plex.
Al though the func tion of Hsp90, de ter mined by ATP hy dro ly sis in the course of ste roid hor mone recep tor mat u ra tion, was dem on strated, it is doubt ful in case of in vi tro con di tions.There is an as sump tion regard ing v-src and other kin ases that Hsp90 sta bi lizes un pro tected cat a lytic do mains prior to as sem bling the kinase into the fi nal sig nal ing com plex.This mech anism is con cep tu ally re lated to the ste roid re cep tor path way.The vari ant of the sig nal ing path way of ecdysone ste roid re cep tor (EcR -re cep tor of fam ily of heterodimeric retinoid X) was de scribed for Drosophila.Along with the pro tein part ner USP, the iso lated re cep tor is ready to bind to EcR rather than to DNA, and Hsp90 con verts a hor mone-bound re cep tor into the ac tive DNA-bind ing state [34].
There is one more mech a nism of in ter ac tion, which al lows hu man Hsp90 reg u late its own ex pression by means of iso la tion of monomeric in ac tive form of HSF1 (heat shock tran scrip tion fac tor) at non-stress con di tions [35], when dis or derly placed pro teins, formed as a re sult of stress ful con di tion, may com pete with HSF1 for bind ing to Hsp90, thus re leasing the tran scrip tion fac tor for trimerization and ini tia tion of heat shock re sponse.
GR is an exellent model for the study on nucleus-cy to plas mic trans fer of the tran scrip tion fac tor from the cy to plasm to the nu cleus, as it is usu ally located in the cy to plasm of cells, free from the hor mone, and its fast tran si tion to the nu cleus is hor mone-depend ent.It was re vealed that GRs were con stantly mov ing be tween the cy to plasm and the nu cleus, thus they sup port nu cleus ac cu mu la tion of GR/Hsp90 com plex via ligand-de pend ent trans for ma tion [36].The tran si tion of GR takes place along with Hsp90 move ment.The trans for ma tion, de pend ent on the ligand, con verts the re cep tor from the state of form ing a "sta ble" com plex with Hsp90 into the state when GR/Hsp90 com plex is more ac tive in the cy cle of its ac cu mu la tion and dissimilation.A more ac tive complex is re quired for fast move ment of the re cep tor.The model of ret ro grade tran si tion of GR with the help of dynein along cytoskeleton tracks was first sug gested in 1993 [37].Re cent years have wit nessed a con sid erable im prove ment of this model.As of date, it is known that some Hsp90-bound immunophilins are bound by peptidyl-prolyl-isomerase (PPIase) domains with the cytosplasmatic dynein.The com plex was iso lated from the cells and re con structed in a cell-free sys tem [38].The tran si tion of GR and sev eral other tran scrip tion fac tors from the cy to plasm into the nu cleus is ei ther slowed down or blocked by Hsp90 in hib i tors, co-ex pres sion of the frag ment of PPIase-do main, block ing the bind ing of immunophilin to dynein and co-ex pres sion of dynamitin, which causes the dis so ci a tion of dynein and its "load".There fore, the ob vi ous proofs were obtained in vivo to sup port the model of GR tran si tion.
Con trary to the pen e tra tion of pro teins into mi tochon dria and other organelles, where pro teins should be un folded to pass, their tran si tion through NPC does not in flu ence the na tive state due to a con sid er able size of pores enough for rather large multimeric complexes -(1-3)•10 6 Da [38].As soon as sig nal ing proteins ap proach the nu cleus mem brane, they get into the pores via fa cil i tated dif fu sion.Cur rently the prefer ence is given to the model of re ceptor/Hsp90/immunophilin com plex for trans fer through nu clear pores [39].In fact nei ther Hsp90 nor Hsp90-bound immunophilins have their own nu clear lo cal iza tion sig nals (NLS).They are pres ent in the nucleus due to their at ten dant trans fer via nu mer ous NLS-con tain ing "cli ent" pro teins.
The in ter ac tion of nu clear pro teins with sol u ble part ners or the ones in ag gre gate state re sults in a consid er able im pair ment of their mo bil ity [40].Spe cific nu clear com part ments of ten gov ern the func tions of nu clear pro teins.In par tic u lar, the au thors of [41,42] dem on strated the tran si tion of pro teins from the nucleus along the tracks through the pores, but the na ture of threads (who plays a role of nu clear actin?) and poten tial mov ing mo tor pro teins (a role of nu clear my osin?) is yet to be dis cov ered.The ovary cells of Chinese ham ster were used to dem on strate that ex pressed nu clear GR FKBP52 and hor mone-free GR are located in the same loci of the nu cleus.Sim i lar lo cal ization is in good agree ment with the as sump tion that FKBP52 moves to the tar get along the sug gested "stag ing ar eas" in side the nu cleus at least.
There are ev i dences to the fact that GR/Hsp90 com plex is nec es sary for the hor mone fix a tion, that is also proven by the abil ity of the re cep tor to re cy cle in the GR/Hsp90 com plex in side the nu cleus.The same cy cle in the nu cleus was dem on strated in DeFranko lab o ra tory [43].It was re vealed that GR, re leased from chromatin, may re peat edly be used n in the process of bind ing the hor mone with out exit out of the nu cleus.Geldanamycin in hib its re peated use of the hor mone and thus in hib its the re lease of GR from chromatin.It is in agree ment with the role of Hsp90/Hsp70 com plex in the ter mi na tion of tran scription ac tiv ity in case of de crease in the level of the free hor mone.In situ sys tem was used to dem on strate the dis place ment of the re cep tor within the nu cleus on the model of pu ri fied Hsp90/Hsp70 com plex.Af ter the dis so ci a tion of the hor mone [H] GRs are re leased from bind ing sites on chromatin.How ever, there is still a ques tion on the time of trans for ma tion of GR dimer into a mono mer: does it hap pen while the re ceptor is bound to chromatin or when it makes a com plex with Hsp90?The stehiometry of the fi nal heterocomplex is one GR mol e cule, bound to Hsp90 dimer [44].Hop co-chaperone or ga nizes all the chaperones, par tic i pat ing in this pro cess, into one com plex -Ísp90/Hop/Ísp70/Ísp40.When ATP-bind ing cen tre of GR/Hsp90 com plex is in active con for ma tion, GR re turns to its bind ing state, and p23 in ter acts with Hsp90 to sta bi lize the com plex.Nuclear GR, used re peat edly in GR-Hsp90-p23 com plex, may in ter act with the hor mone not leav ing the nu cleus and then par tic i pate in bind ing again.
p23 com po nent is of im por tant in stru ment in the inves ti ga tion on the role of Hsp90 in the course of transcrip tion com plex dis as sem bly and in the study on repeated bind ing of the re cep tor in the nu cleus.In vi tro ex per i ments dem on strated two mech a nisms of p23 action [24].The ex per i ments on GR/Hsp90 as sem bly dem on strated that p23 acts as co-chaperone Hsp90 and in ter acts with GR/Hsp90 com plex as soon as it is formed, thus sta bi liz ing it.p23 may also act as a chaperone, in hib it ing the ag gre ga tion of denaturated pro teins and keep ing them in folded state.In vivo exper i ments showed the par tic i pa tion of p23 in the sta bili za tion of chaperone com plex Hsp90/Hsp70 [44].There are also nu mer ous ev i dences to the fact that as a part of heterocomplex Hsp90 is in volved in the pro cess of tran si tion of transcriptional fac tors, which are "cli ent" pro teins of Hsp90, along the cy to plasm and nu cleus.
Hsp90 and pro tein deg ra da tion.A visit card of the ac tiv ity of ansamycin in liv ing cells is the in duction of deg ra da tion of sub strate pro teins by Hsp90 chaperone us ing ubiquitin-proteasome path way.Though his tor i cally ansamycins were dis cov ered as in hib i tors of ty ro sine kin ases, they are cur rently known as in hib i tors of Hsp90.Ansamycins are ca pa -ble of in hib it ing chaperone-me di ated fold ing of Hsp90 sub strates, thus block ing their dis as sem bly from Hsp90.On the other hand, some sub strate proteins are re leased by ansamycin from the com plex with Hsp90 [45,46].
In what way do Hsp90-bound pep tides serve as a deg ra da tion tar get?TPR co-chaperone CHIP, ca pa ble of rec og niz ing both chaperones (hsp90 and Hsc70), con tains a so called U-do main, which is ho mol o gous to E4-ubiquitin fac tors, and may pres ent chaperone-bound non-placed pro teins for ubiquitination.There fore, super-ex pres sion of CHIP in cul ti vated cells re sults in the in crease in ubiquitination and deg ra da tion of GR and transmembrane reg u la tor of mucoviscidosis of CFTR, which are the sub strates of Hsp90 and Hsc70.CHIP ac tiv ity is re quired for the ki net ics of pro tein fold ing and deg ra da tion.Due to this ac tiv ity the sub strates inter act with Hsp90 lon ger, for in stance as a re sult of inhib it ing ATP-cen tre for their fur ther ubiquitination.The ex is tence of CHIP al lows the as sump tion that sim i larly to pro tein fold ing the deg ra da tion of proteins is reg u lated by ad di tional fac tors [45].
Re cent stud ies dem on strated [47] that Hsp90 is bound di rectly to ri bo somal pro tein S3 (rpS3).Due to the for ma tion of Hsp90/rpS3 com plex the ri bo somal pro tein rpS3 is pro tected from ubiquitination and proteasome deg ra da tion, which al lows it to keep its func tion in biogenesis of the ri bo some later.Hsp90 inhib i tors in flu ence the dis as sem bly of rpS3 from Hsp90, fur ther as so ci a tion of the pro tein and Hsp70 and the in duc tion of its deg ra da tion.It was also revealed that the ri bo somal pro tein S6 (rpS6) in ter acts with Hsp90 as well, and chaperone in hib i tors have sim i lar in flu ence on its dis as sem bly from Hsp90 and fur ther deg ra da tion.There fore, there are grounds for the as sump tion that Hsp90 reg u lates the func tion of ribo somes via sup port ing the sta bil ity of 40S ri bo somal pro teins, for in stance of rpS3 and rpS6.
Cardioprotection and phar ma co log i cal property of Hsp90.The ex per i ments on the cul ture of cardiomyocytes dem on strated that Hsp90 sta bi lizes the pro tein ErbB2 [48,49] which is a vi tal com po nent of GPCR-sig nal ing path way of cardiomyocytes.The ex per i ments on mice, per formed to in ves ti gate the influ ence of doxorubicin, the in hib i tor of can cer cells, dem on strated that Hsp90 is re spon si ble for the sta bi liza tion of ErbB2 in the heart.The in crease in the quantity of ErbB2 in the heart in re sponse to some dam age may be an im pulse to the sur vival of cells.How ever, the dam age ex ceed ing the thresh old level (high concen tra tions of doxorubicin) can not be pre vented by the ac ti va tion of ErbB2 or Akt.It is note wor thy that the in crease in the con cen tra tion of ErbB2 (along with fur ther ac ti va tion of GRCR-sig nals) takes place prior to ob vi ous im pair ments of sys tolic func tion of the heart.Such change in ErbB2 con tent may be used in the di ag nos tics of pa tients, who re ceive the treat ment with anti-ErbB2 in the case of breast can cer, and who have a risk of de vel op ing heart symp toms.Doxorubicin is the in hib i tor, en hanc ing the dis as sembly of ErbB2 and Hsp90, which re sults in the deg ra dation of ErbB2 pro tein.The treat ment of can cer of suck ling gland leads to the in creased level of the expres sion of Hsp90 and Hsp70 in the heart.Ox i da tive dam age of the myocardium, in duced by the stress, influ ences the ex pres sion of Hsp90 in vivo.Be sides doxorubicin, cyclosporin A [50] also in duces in vivo ex pres sion of Hsp90 in the heart, which is re lated to the mod u la tion of en do the lial NO-synthase sig nal ing path way.It is ev i dent that the search for novel anticancer treat ment should in clude the es ti ma tion of their pos si ble side ef fects in vivo, namely, their in fluence on the level of Hsp90 in the cell, in par tic u lar, in cardiomyocytes.
The superexpression of Hsp90 in the ischemic stage of car diac in farc tion may pro tect the car diac muscle from ischemia-reperfusion di la tion due to the stimu la tion of the NO en do the lial path way [51].The culture of en do the lial cells was used to dem on strate the asso ci a tion of cardio-pro tec tive ac tiv ity of Hsp90 and its abil ity to in ter act with the kinase Akt and calcineurin-phosphatase as an adapter, pro mot ing both phosphorilation of Ser 1177 and de-phosphorilation of Thr 495 eNOS-synthase (en do the lial ni tric ox ide synthase).Hsp90 is a prom is ing tar get for in creas ing the for ma tion of eNOS in vivo, which may be ef fi cient in re spect of de creas ing the level of myo cardial/reperfusion in jury.
A role of Hsp90 in the reg u la tion of apoptosis is rather am big u ous and de pends on apoptotic stim uli, usu ally ex pos ing anti-apoptotic ac tiv ity.The for ma -tion of Akt-Hsp90 com plex in vivo sta bi lizes the activ ity of Akt kinase and pro tects the cells from apoptosis, pre vent ing the dephosphorylation of Akt [30].Kin ases Raf-1 and MEK par tic i pate in such impor tant cel lu lar pro cesses as pro lif er a tion, dif fer en ti ation, and apoptosis, and are vi tal par tic i pants of pathogenesis of cardiohypertrophy and car diac de ficiency [52].Hsp90 was found in the as so ci a tion with many com po nents and reg u la tors of the Raf-1/MEK sig nal ing in Hsp90 mol e cule of Drosophila sup press the ac tiv ity of Raf-1 and de crease the af fin ity be tween Hsp90 and Raf-1.Geldanamycin also in hib its the forma tion of Raf-1/Hsp90 com plex and causes the deg rada tion of Raf-1.The ac ti va tion of Raf-1 kinase prevents the apoptosis of cardiomyocytes [52].Hsp90 alone or in the com plex with Hsp70 binds Apaf-1 and pre vents the for ma tion of Apaf-1 com plex with cytochrome C, thus be ing a neg a tive reg u la tor of CytC-me di ated apoptosis.
Tak ing the abovementioned into con sid er ation, one may as sume that Hsp90 takes an ac tive part in pre vent ing the apoptosis of cardiomyocytes.
The pro cess of for ma tion of new myofibrils oc curs dur ing the en tire pe riod of or gan ism de vel op ment.It is well known that cardiomyocytes do not di vide, but they are ca pa ble of re spond ing to in creas ing hemodynamic load, en larg ing the weight and ca pac ity of the con trac tile ap pa ra tus.Such hy per tro phy of the heart may oc cur both in nor mal con di tions (for instance, in sports men with high phys i cal strain) and at heart dis eases of dif fer ent eti ol ogy.In the most compli cated forms the growth of heart tis sue re sults in the de crease in the size of its cham bers, de crease in the vol ume of pumped out blood and is dan ger ous for the life of a pa tient [54].
It is known that Hsp90 and Hsc70 are bound by my o sin, form ing a multimeric com plex of my o sin threads.The half-life of this com plex is short.It is accu mu lated in case of in hib it ing Hsp90 with geldanymicin or due to other rea sons, post pon ing the as sem bly of myofibrils.The cardiomyocyte seems to launch the same mech a nism of hy per tro phy as in the case of em bry onic pe riod when the syn the sis of myofibrils takes place de pend ing on reg u la tory proteins.The in hi bi tions of biosynthesis of these reg u latory com po nents stops the as sem bly of myofibrils.
As it was al ready men tioned above, the ac tiv ity of Hsp90 (sim i lar to other chaperones) is reg u lated and mod u lated via the in ter ac tion with adap tor pro teins and co-chaperones, for in stance, p23 or re cently discov ered Sgt1 [55] and melusin [56,57].It was found out that Sgt1 is pres ent in var i ous cells and tis sues of mam mals, in clud ing the skel e tal and mus cle parts, and melusin is syn the sized only in the skel e tal and heart tis sues.The in ter ac tion of Sgt1 and Hsp90/Hsp70 is mod u lated by S100A6 pro tein via Ca 2+ -de pend ent path way, which may be a sig nif i cant mo ment in the de vel op ment of apoptosis, de pend ing on intracellular con cen tra tion of cal cium.
Re cent stud ies have shown that melusin is a new com po nent -co-chaperone of Hsp90, ca pa ble of protect ing the ci trate synthase (CS) from ag gre ga tion, pos si bly due to heat shock [58].It is also note wor thy that in hib i tors of Hsp90 are inductors of chap er ons syn the sis, in par tic u lar, of Hsp90 it self.The syn the sis of chaperones in the cell is in duced by HSF-1.Hsp90 dis so ci ates from HSF-1 at the mo ment of its ac ti va tion due to com pet ing bind ing of un folded pro teins [59].The in hib i tors of Hsp90 pro mote the tran scrip tion activ ity of HSF-1 due to the de struc tion of Hsp/HSF-1 com plex, as a re sult HSF-1 re leases and translocates into the nu cleus, phos phory lates and binds to HSE, thus in duc ing the tran scrip tion of genes hsp40, hsp70, and hsp90.In par tic u lar, due to bind ing of Hsp90 the in hib i tor radicicol ac ti vates the tran scrip tion of genes of chaperones in the cells of neo na tal cardiomyocytes of rats.
There fore, Hsp90 mod u lates the level of cardioprotection of cardiomyocytes, thus reg u lat ing the ex pres sion of Hsp90 di rectly via its abil ity to bind HSF-1 [60].In cardiomyocytes the ex pres sion of Hsp90 and cardioprotection are mod u lated by the amount of Hsp90.The ap pli ca tion of non-toxic de riva tives of radicicol may be come one of the stages of devel op ing ther a peu tic means for the treat ment of cardio vas cu lar dis eases.
The in tro duc tion of the gene hsp90 into the myocardium [51] pre vents ischemic cardiomyopathy in pigs and stim u lates the syn the sis of eNOS by chaperone Hsp90.It is well-known that NO has a cardio-pro tec tive func tion, but at the same time the increase in the level of its syn the sis re sults in ap pear -ance of cytotoxic peroxynitrites with superoxide rad icals, which dam age tis sues.Pro longed pro duc tion of NO, de ter mined by the ac cu mu la tion of tu mour growth sup pres sor p53, re sults in apoptosis.
Re gard less of the fact that superoxide an ion also in duces apoptosis, the bal ance of NO and superoxide an ion re sults in cross-pro tec tive ef fect and the pro cess of apoptosis is con sid er ably in hib ited [61].The in vesti ga tion of the func tions of mo lec u lar chaperone Hsp90, one of which is cardioprotection, and the es tima tion of pos si ble role of in hib i tors of Hsp90 in this func tion, makes this pro tein a prom is ing tool of cardio-vas cu lar ther apy.