This investigation usedyeast to show that cells can be rendered hypersensitive to Hsp90 inhibitors by mutation to Hsp90 itself within the Hsp82 isoform of yeast Hsp90, the point mutation
Trang 1Sensitivity to Hsp90-targeting drugs can arise with mutation
to the Hsp90 chaperone, cochaperones and plasma membrane
ATP binding cassette transporters of yeast
Peter W Piper1, Stefan H Millson1, Mehdi Mollapour1, Barry Panaretou2, Giuliano Siligardi3,
Laurence H Pearl4and Chrisostomos Prodromou4
1
Department of Molecular Biology and Biotechnology, University of Sheffield, Firth Court, Western Bank, Sheffield, UK;
2
Division of Life Sciences and3Pharmaceutical Optical Spectroscopy Centre, Department of Pharmacy, King’s College London, Franklin-Wilkins Building, London, UK;4Section for Structural Biology, Institute of Cancer Research,
Chester Beatty Laboratories, London, UK
The Hsp90 molecular chaperone catalyses the final
activa-tion step of many of the most important regulatory proteins
of eukaryotic cells The antibiotics geldanamycin and
rad-icicol act as highly selective inhibitors of in vivo Hsp90
function through their ability to bindwithin the ADP/ATP
binding pocket of the chaperone Drugs based on these
compounds are now being developed as anticancer agents,
their administration having the potential to inactivate
sim-ultaneously several of the targets critical for counteracting
multistep carcinogenesis This investigation usedyeast to
show that cells can be rendered hypersensitive to Hsp90
inhibitors by mutation to Hsp90 itself (within the Hsp82
isoform of yeast Hsp90, the point mutations T101I and
A587T); with certain cochaperone defects and through the loss of specific plasma membrane ATP binding cassette transporters (Pdr5p, and to a lesser extent, Snq2p) The T101I hsp82 andA587T hsp82 mutations do not cause higher drug affinity for purified Hsp90 but may render the
in vivo chaperone cycle more sensitive to drug inhibition
It is shown that these mutations render at least one Hsp90-dependent process (deactivation of heat-induced heat shock factor activity) more sensitive to drug inhibition in vivo Keywords: Hsp90 inhibitor resistance; Hsp90 mutants; Sti1p; ATP binding cassette transporters; yeast
The Hsp90 molecular chaperone catalyses the final
activa-tion step of many of the most important regulatory proteins
of eukaryotic cells [1–3] Hsp90 is also a natural antibiotic
target, such that its activity can be inhibitedwith a high
degree of selectivity in vivo with the administration of the
antibiotics geldanamycin (GA; a benzoquinone ansamycin
produced by Streptomyces hygroscopicus [4]) andradicicol
(RD; a macrolactone produced by certain mycopathogenic
fungi [5]) GA andRD bindwithin the Hsp90 ADP/ATP
binding site, thereby inhibiting the ATP binding step of the
Hsp90 chaperone cycle [6–9]
Interest in Hsp90-targeting drugs as possible anticancer
agents was triggeredinitially with the identification of
GA andRD as compounds that couldreverse the
pheno-type of p60v–src-transformedcells in culture [10,11] GA
andRD act upon Hsp90, whose action is neededfor the
p60v–srctyrosine kinase to achieve an active state [12] In a variety of cell culture systems, GA administration leads to
a markeddestabilization of several of the most oncolog-ically relevant proteins such as p53, Erb-b, Raf-1 and steroidreceptors [12–15] Hsp90 inhibition can therefore simultaneously destabilize several of the key components
of multistep carcinogenesis [16] This destabilization is probably a result of these Hsp90 client proteins being unable to progress through the chaperone cycle Cells that lose Hsp90 function, as with GA/RD treatment, rapidly lose the ability to activate many signalling proteins and undergo retinoblastoma protein-dependent cell cycle arrest [17]
Antitumour effects of Hsp90 drugs have now been demonstrated using several animal model systems, the 17-allylamino derivative of GA (17-AAG) being more effective andless hepatotoxic in vivo than the parent GA [18] Although 17-AAG is now in clinical trials, its insolubility causes problems in administration It is also potentially a redox-cycling drug There is, therefore, an urgent need to identify or develop inhibitors of Hsp90 that are more selective andmore soluble than 17-AAG [16,19] It will be necessary to understand the factors that contribute to susceptibility or resistance to Hsp90 inhibitory compounds
To this end, we have investigated various mutants of the Hsp90 chaperone andpleiotropic drug resistance (PDR) systems of yeast, to help identify the factors that contribute
to sensitivity to Hsp90 inhibitor drugs
Correspondence to P.W Piper, Department of Molecular Biology and
Biotechnology, University of Sheffield, Firth Court, Western Bank,
Sheffield, S10 2TN, UK.
Fax: + 44 114 222 2850, Tel.: + 44 114 222 2851,
E-mail: peter.piper@sheffield.ac.uk
Abbreviations: GA, geldanamycin; RD, radicicol; ts,
temperature-sensitive.
(Received4 August 2003, revised28 September 2003,
accepted3 October 2003)
Trang 2Materials and methods
Strains and growth media
The Saccharomyces cerevisiae strains usedfor this study are
listedin Table 1 Deletion of the SBA1 and STI1 open
reading frames in W303-1a utilized PCR-generated
kan-MX4or HIS3MX6 [20] cassettes, respectively, these
dele-tions being confirmedby colony PCR [21] Cultures were
grown on YPDA medium (2% glucose, 2% bactopeptone,
1% yeast extract, 20 mgÆL)1adenine) GA was a gift from
the National Cancer Institute (Bethesda, MD, USA) RD
was purchasedfrom Sigma
Drug sensitivity assays
Cells were streakedon to 5 cm diameter YPDA plates
containing the indicated level of drug [22]
Western blot analysis
Total protein extracts were preparedandWestern blots
preparedas describedpreviously [23] using rabbit polyclonal
antisera raisedagainst the bacterially expressedHsp82 and
Sba1p of yeast
Hsp90 ATPase assays
Hsp90 ATPase assays useda regeneration coupledenzyme
assay [24], each 1 mL of assay using 2 lM of purified
recombinant Hsp82 as described previously [23]
Assays of HSF induction Heat shock factor activity was measuredusing cells transformedwith a URA3 plasmidcontaining a lacZ reporter under heat shock element control (HSE-lacZ [25])
A Beckmann BioMek robot was usedto add20 lL of minus uracil dropout medium (SD-ura), either with or without RD, to 16 replicate 25C 100 lL [26] cultures of each transformant Immediately thereafter, eight of these cultures were maintainedfor 1 h at 25C while the remaining eight were heat shockedto 39C for 1 h after which, 50 lL of 1Msodium carbonate was added The cells were then collectedby centrifugation andtheir lacZ activity measuredby a permeabilizedcell assay [25]
Results Certain point mutations in the native Hsp90 of yeast render cells much more sensitive to Hsp90 inhibitor drugs
To test whether mutations in the native Hsp90 chaperone of yeast influence sensitivity to Hsp90 inhibitors, we testedeight
S cerevisiaehsp82 mutants bearing point mutations in their single functional Hsp90 gene (HSP82) (Table 1) for their sensitivities to GA andRD (Fig 1) This set of mutants had originally been isolatedby Nathan andLindquist as corres-ponding to mutant forms of Hsp90 that cause temperature-sensitive (ts) yeast growth at 37C [27] They therefore represent mutations in the Hsp82 isoform of yeast Hsp90 that cause partial, rather than total, loss of the essential
Table 1 The yeast strains employed for this study and their sensitization to Hsp90 drugs.
Strain
Increase in GA/RD sensitivity Genotype
Strain origin P82a – W303–1a hsc82::LEU2 hsp82::LEU2 HIS3-GPD-HSP82a [27] T22I Slight (GA) b W303–1a hsc82::LEU2 hsp82::LEU2 HIS3-GPD-hsp82(T22I) a [27] A41V Nob W303–1a hsc82::LEU2 hsp82::LEU2 HIS3-GPD-hsp82(A41V)a [27] G81S Nob W303–1a hsc82::LEU2 hsp82::LEU2 HIS3-GPD-hsp82(G81S)a [27] T101I Yes b W303–1a hsc82::LEU2 hsp82::LEU2 HIS3-GPD-hsp82(T101I) a [27] G170D Slight (RD) b W303–1a hsc82::LEU2 hsp82::LEU2 HIS3-GPD-hsp82(G170D) a [27] G313S Slight (RD)b W303–1a hsc82::LEU2 hsp82::LEU2 HIS3-GPD-hsp82(G313S)a [27] E381K Slight (GA) b W303–1a hsc82::LEU2 hsp82::LEU2 HIS3-GPD-hsp82(E381K) a [27] A587T Yes b W303–1a hsc82::LEU2 hsp82::LEU2 HIS3-GPD-hsp82(A587T) a [27] W303–1a – MATa ura3–1 trp1–1 leu2–3112 his3–11 ade2–1 can1–100 ssd1-d2 Euroscarf
Dcpr6,Dcpr7 Moderate c W303–1a cpr6::URA3 cpr7::TRP1 [49] Dsti1,Dcpr6 Yesc W303–1a sti1DHIS3MX6 cpr6::URA3 This study Dsti1,Dsba1 Yesc W303–1a sti1DHIS3MX6 sba1DkanMX4 This study YPH500 – MATa, ura3–52, lys2–801 am , ade2–101 oc , trp1-D63,his3-D200, leu2-D1 [50]
Dpdr5,Dsnq2 (YYM4) Yes d YPH500 pdr5::TRP1 snq2::hisG [51]
a
This integratedwild-type (HSP82) or mutant (hsp82) gene for Hsp90 is the only functional Hsp90 gene in these strains andis expressed under the control of the constitutively active glyceraldehyde-3-phosphate (GPD1) gene promoter [27].bRelative to P82a (Fig 1A).crelative
to W303–1a parent (Fig 4A) d Relative to YPH500 parent (see Fig 4B).
Trang 3Hsp90 function, or that prevent the higher levels of Hsp90
activity needed for yeast growth at high temperatures [28,29]
Growth of these mutants relative to the strain expressing
the wild-type Hsp82 (p82a) on GA- or RD-containing plates
(Fig 1A) revealedthat a number of these hsp82 alleles render
the cells hypersensitive to GA (A587T, T101I; also to lesser
extent, T22I, G313S andE381K) Only two (T101I and
A587T) were associatedwith pronouncedsensitivity to RD
(Fig 1A) It was these latter two mutations, alleles causing
extreme sensitivity to both GA andRD, that we chose to
investigate further This is because the in vivo effects of GA
may not be limitedto its capacity to inhibit Hsp90 GA
possesses a benzoquinone ring, readily reduced in vivo by
NAD(P)H-dependent oxidoreductases It therefore has the
potential to cause oxidative stress RD, in contrast, is not a
redox-cycling compound[30] We were surprisedinitially by
the strong inhibitory action of RD on certain yeast mutants
(Fig 1A) as mammalian studies had indicated that RD
derivatives may not be very stable in vivo [16,30,31]
These increases in drug sensitivity could arise through
certain of the mutations causing loweredintracellular levels
of the drug target, Hsp90, itself This possibility could be
discounted as these hsp82 mutants all expressedsimilar
levels of Hsp82 (their sole Hsp90 isoform) Hsp82 levels in
the GA andRD-sensitive T101I andA587T hsp82 mutants
were essentially unalteredwith respect to the p82a cells
expressing the wild-type Hsp82 (Fig 1B)
The IC50for GA inhibition of the intrinsic ATPase
of purified Hsp82 is unaffected by the A587T mutation
We recently presentedtemperature/activity profiles for the
in vitroATPase activity of purifiedmutant forms of Hsp82,
the same mutant forms that are expressedin the hsp82 mutants in Fig 1 [32] There is no apparent correlation between this in vitro ATPase activity andthe in vivo sensitivity to Hsp90 drugs (Fig 1A), despite the fact that
GA andRD are both potent inhibitors of this ATPase [7,8]
Of the two Hsp82 mutations associatedwith high in vivo sensitivity to Hsp90 drugs, T101I dramatically reduces the
in vitroATPase activity of purifiedHsp82, whereas, A587T exerts little effect [32] Yet another inhibitor of the in vitro ATPase of Hsp90 is Sti1p, a cochaperone protein that also affects drug resistance (see below) When Sti1p, the functional equivalent of mammalian Hop, binds to Hsp90
in the GA/Hsp90 complex it displaces the bound GA [33]
If the increasedHsp90 drug sensitivity of the T101I and A587T hsp82 mutants (Fig 1A) was due to these mutations causing tighter drug binding to the chaperone, these mutations shouldrender the in vitro ATPase of Hsp82 more sensitive to drug inhibition We determined if A587T renders the ATPase of the purifiedchaperone more susceptible to inhibition by either GA or Sti1p in assays using wild-type andA587T mutant forms of Hsp82 (inhibition of the T101I mutant protein was not determined, its extremely low activity [32] making it much more difficult to obtain definitive data) The A587T mutation did not affect the
GA or Sti1p inhibitions of in vitro chaperone ATPase (Fig 2) Adenosine 5¢-(b,c-imino)triphosphate (AMP-PCP) binding to purified Hsp82 was also essentially unaffected by the A587T andT101I mutations (K values for AMP-PCP
Fig 1 GA and RDsensitivities of a collection of yeast strains
expres-sing either wild-type (p82a; w+) or mutant forms of Hsp82 (A) Strains
were streakedonto YPDA agar containing the
indicatedconcentra-tions of GA or RD The plates were then photographedafter 5 days of
growth at 20–22 C (B) Western blot measurement of the levels of
Hsp82 andSba1p (loading control) in these cells culturedat 22 C. Fig 2 GA and Sti1p inhibition of the intrinsic ATPase of purified
wild-type and A587T mutant Hsp82 Assays were conducted at 37 C as described previously [33], using 2 l M Hsp82 protein andeither the indicated level of GA (A) or zero, 2 l M and8 l M Sti (B) Activity at 100% 5000 pmol ATPÆmin)1Æmg)1for both protein samples; the
IC 50 for GA in this assay 3 l M [8,23].
Trang 4binding to the wild-type, T101I and A587T forms of Hsp82
measuredby CD spectroscopy being 33, 37 and37 lM,
respectively; G Siligardi, unpublished observation)
The T101I and A587T mutations allow RD to potentiate
the yeast heat shock response
Figure 2 reveals that the in vivo manifestation of increased
GA sensitivity in the A587T hsp82 mutant (Fig 1A) is not
due to increased drug affinity for the chaperone, suggesting
that it may require additional components of the Hsp90
chaperone machinery andpossibly an assembledHsp90/
cochaperone/client complex To seek evidence for whether
this is the case, we determined if, through the expression of
the T101I andA598T mutant forms of Hsp82, an
Hsp90-dependent process becomes more sensitive to Hsp90 drug
inhibition in vivo
In a variety of cell systems, Hsp90 inhibitor
administra-tion acts almost immediately to activate the heat shock
response [25,34,35] This reflects the requirement for Hsp90
in deactivation of the transcriptional activator of heat shock
genes, heat shock factor (HSF) When heat shockedto 37–
39C, the hsp82 mutants in Fig 1 all display considerably
higher levels of HSF activation relative to the wild-type
[25,35] They are therefore defective in this down-regulation
of HSF activity at these temperatures One of these mutants
(E381K hsp82) even displays a high HSF activity at low
temperatures of growth [25], indicating that Hsp90 is also
requiredin order to maintain HSF in its basal activity state,
the form present in unstressedcells
As mentionedabove, GA is potentially a source of
oxidative stress in vivo through its capacity to act as a
redox-cycling drug Oxidative stress is known to activate
HSF [36–38] We therefore usedRD, a non redox-active
compound, in investigating whether the expression of T101I
andA587T mutant forms of Hsp82 influences the capacity
of an Hsp90 inhibitor to activate the heat shock response In
the absence of heat stress, a 1 h, 1 lMRD administration
caused moderate increases in HSF activity, both in
wild-type, T101I hsp82 andA587T hsp82 cells (Fig 3) Heat
shock induced this activity still further, yet it was only in the cells expressing the T101I andA587T mutant Hsp82s, not cells expressing wild-type Hsp82, that such low amounts of
RD couldpotentiate this heat-inducedincrease in HSF activity (Fig 3) Heat shockedcells of these two hsp82 mutants are therefore more responsive to RD administra-tion (responsiveness measuredas HSF activity)
Losses of Hsp90 system cochaperones and plasma membrane ATP binding cassette (ABC) transporters can sensitize cells to Hsp90 drugs
Hsp90 works in association with a number of cochaperone proteins These may, in many cases, stabilize discrete multiprotein complex intermediates of the Hsp90 chaperone cycle, thereby improving the overall efficiency of client protein activation by Hsp90 At least nine such Hsp90 system cochaperones have now been identified in yeast {Sti1p(Hop), Cdc37p, Cns1p, Sba1p(p23), Cpr6p, Cpr7p, Sse1p, Hch1p, Aha1p [39–42]}
We testedwhether Hsp90 drug resistance is affectedby the loss of several of the cochaperones that are nonessential for viability of yeast (Sti1p, Sba1p, Cpr6p, Cpr7p, Hch1p andAha1p) Sse1p andessential cochaperones such as Cdc37p and Cns1p were not included in this screen At
22C there were no appreciable effects of the loss of Sba1p, Cpr6p (Fig 4A), Hch1p or Aha1p (not shown) on drug sensitivity, whereas the loss of Sti1p increasedsensitivity to both GA andRD (Fig 4A) With loss of Cpr7p, the cyclophilin whose loss causes the most markedphenotype
in yeast [43], drug sensitivities were slightly increased (an increasedsensitivity of cpr7 cells to GA hadbeen reported previously [44]) However, in these cells with a W303-1a genetic background, these effects on drug sensitivity due to the loss of Cpr7p were appreciably smaller than those due to the loss of Sti1p (Fig 4A)
An increasedGA sensitivity of sti1D cells was notedin an earlier study, work that also identified increased GA sensitivity with the loss of the Sse1p cochaperone [41] We have since foundthese effects of the sti1D mutation on drug sensitivity to be influencedstrongly by the isoform of Hsp90 that is expressed(at a similar level) in the cells Increased
GA andRD sensitivity with the loss of Sti1p was most markedin S cerevisiae expressing the Candida albicans Hsp90, less in cells expressing solely the native S cerevisiae Hsp82 andnegligible in cells expressing solely the S cere-visiaeHsc82 [22] These differences are quite remarkable as the two isoforms of S cerevisiae Hsp90 (Hsc82 andHsp82) share no less than 97% sequence identity [28]
It is probable that yeast cells use plasma membrane pumps to catalyse a cellular efflux of Hsp90-targeting drugs, just as they actively efflux very many other xenobiotics and antitumour agents [45,46] We therefore investigatedwhe-ther the pleiotropic drug resistance (PDR) system contri-butes to Hsp90 inhibitor resistance Strains lacking two of the major plasma membrane ATP-binding cassette (ABC) transporter determinants of drug resistance (Pdr5p and Snq2p [46]) were streakedonto plates containing GA and
RD This revealedthe Dpdr5 mutant to be hypersensitive to both drugs and the Dsnq2 mutant to be slightly sensitive to
RD (Fig 4B) Pdr5p is a broad-specificity ABC transporter that provides resistance to a wide range of hydrophobic and
Fig 3 Expression of a HSE-LacZ reporter of HSF activity in cells
expressing the wild-type (p82a), or T101I and A587T mutant forms of
Hsp82 Basal andheat-induced(1 h 39 C) HSE-LacZ activity (open
andsolidbars, respectively) was determinedboth in the absence (–)
andpresence (+) of 1 l M RD Data represents the mean andSD of
eight assays.
Trang 5cationic compounds in yeast Its substrate specificity is
remarkably similar to that of the human ABC transporter
(Mdr1) overexpressed in a number of multiple
drug-resistant tumours [45]
Discussion
This study is the first to reveal that an increased sensitivity to
Hsp90 drugs can arise with mutations to Hsp90 itself
(Fig 1) andwith specific ABC transporter defects (Fig 4B)
Previously, an increasedGA sensitivity hadbeen shown to
result from the loss of certain cochaperones [41,44]; results
that have been partly confirmedandextendedin this work
(Fig 4A) The IC50for inhibition of the in vitro ATPase of
purifiedHsp90 is around3 lMfor GA and1 lMfor RD
[8,23] (Fig 2) The effects of short-term exposure of yeast
cells to the latter low RD concentration also are quite
readily monitored (Fig 3) In contrast, on Petri dishes
where cells are growing for extended periods, levels of these
drugs in excess of 100 lMcan still permit the growth of
wild-type cells, though certain mutants are clearly inhibited
[22](Figs 1 and4) This resistance to long-term Hsp90 drug
exposure is attributable partly to the actions of the
membrane drug pumps (Fig 4B) These drug efflux
activ-ities cause yeast to be remarkably resistant to a wide range
of inhibitory compounds and can limit the effectiveness of
yeast-based drug screens [45] Instability of the Hsp90 drug
compounds themselves may be another factor in this
resistance to long-term Hsp90 drug exposure (GA is readily
oxidized; while RD possesses dienone and epoxide groups that are potentially reactive anda lactone ring that presents possibilities for esterase action [30])
The sensitization of yeast to Hsp90 drugs, whether through expression of the T101I or A587T mutant forms of the native Hsp82 (Fig 1) or through heterologous expres-sion of the human Hsp90b [22], is not a reflection of a higher binding affinity of the chaperone for the drug The IC50for
GA inhibition of the in vitro ATPase of purifiedyeast Hsp82
is unaffectedby the A587T mutation (Fig 2) andsimilar for both yeast andhuman Hsp90s [47] Are the T101I and A587T hsp82 mutations acting selectively to sensitize the assembledHsp90/cochaperone/client protein complex to drug inhibition of progression through the chaperone cycle,
or is it simply that these mutations are reducing Hsp90 activity in vivo, which in turn leads to increased sensitivity to Hsp90 drugs in a general way? A number of indicators suggest the former Yeast needs higher levels of Hsp90 for high temperature growth [28,29], so that mutations causing
a substantially reduced Hsp90 activity should all present ts phenotypes Nevertheless there appears to be no correlation between the degrees of drug sensitivity and temperature sensitivity displayed by these hsp82 strains (compare Fig 1A of this report with Fig 2B of [27]) The mutant with the most severe ts phenotype (T22I hsp82 [27]) is not the most drug-sensitive (Fig 1A) There is also no corre-lation between the in vivo drug sensitivity of each hsp82 mutant andthe in vitro ATPase of the corresponding purifiedchaperone [32] Glucocorticoidreceptor activity
Fig 4 Analysis of cochaperone and ABC transporter mutants for Hsp90 drug sensitivity (A) RD andGA sensitivities of strains bearing deletions in genes for Hsp90 system cochaperones Wild-type cells (w + ), Dsti1, Dsba1, Dcpr6 or Dcpr7 single mutants, also Dsba1,Dsti1 and Dcpr6,Dsti1 double mutants, all from a W303–1a genetic background, were photographed after 2 days of growth at 30 C on YPDA in the absence or presence of the indicated concentrations of GA or RD (B) RD and GA sensitivities of strains bearing deletions of the Pdr5 and Snq2 plasma membrane ABC transporters Wild-type cells (wt), Dpdr5 or Dsnq2 single mutants, anda Dpdr5,Dsnq2 double mutant, all of YPH500 genetic background, were photographedafter 2 days of growth at 30 C on YPDA in the presence of the indicated concentrations of GA or RD.
Trang 6measurements in these strains indicate that the different
hsp82alleles, rather than all simply lowering Hsp90 activity,
exert diverse in vivo pleiotropic effects on Hsp90 client
protein activation/deactivation processes [27] Furthermore
the deactivation of heat-induced HSF activity is more
sensitive to drug inhibition in cells expressing the T101I or
A587T mutant forms of Hsp82 (Fig 3) This though is only
an indication, not formal proof, that these two specific
mutant Hsp82s may allow the drug to exert stronger
inhibitory effects on the Hsp90 chaperone cycle
In yeast expressing wild-type Hsp90s, increased drug
sensitivity is generally apparent with the loss of the Sti1p
(Hop) cochaperone (Fig 4A) Sti1p binding to Hsp90 may
help stabilize the ATP/ADP-free state of Hsp90 [33], ready
for its loading with a fresh substrate client protein [the latter
probably as a complex with Hsc70 andYdj1(Hsp40)] ATP
binding to the Hsp90 N-terminal domains in the Hsp90
dimer then causes these N-domains to associate [32] This
ATP-induced conformational change may also be the signal
for Hsc70, Ydj1 and Sti(Hop) to be displaced from the
complex andfor other cochaperone proteins, including
Sba1(p23), to bindso as to produce the later multiprotein
complexes of the Hsp90 chaperone cycle Hsp90 drugs
inhibit ATP binding [6–8], therefore progression to these
later stages of the chaperone cycle It may be the progression
to these later complexes that is more sensitive to Hsp90
drugs in yeast expressing the T101I or A587T mutant forms
of Hsp82 (Fig 1) or the human Hsp90b [22] Although
Sti1p contacts the C-terminus of Hsp90 [48], its binding also
displaces bound GA, indicating that there is also an
interaction of Sti1p with the ADP/ATP binding site of the
chaperone [33] The increaseddrug sensitivity of sti1D
mutant cells (Fig 4A) might therefore be attributable to the
absence of a protein that limits access of the drug to its
binding site on Hsp90 Such a model is probably
oversim-plistic as we have foundthe increaseddrug sensitivity with
the loss of Sti1p to be strongly dependent on the form of
Hsp90 being expressedin the yeast [22]
Though this study has focussed on mutations that cause
an increasedsensitivity to Hsp90 drugs, it is probable that
increasedresistance can also arise (e.g through
gain-of-function PDR mutations leading to the overproduction or
overactivation of membrane pumps catalysing drug efflux
from the cell) Mdr1p, the human ABC transporter
overexpressedin a number of multiple drug-resistant
tumours, has a spectrum of diverse substrates that overlap
quite remarkably with those of Pdr5p (a major yeast ABC
transporter determinant of Hsp90 drug resistance; Fig 4B)
[45] It remains to be establishedwhether
increasedresist-ance to Hsp90 drugs can arise with mutational alteration to
the Hsp90 chaperone machine
Acknowledgements
We are indebted to Susan Lindquist, Didier Picard, Richard Gaber,
andKarl Kuchler for gifts of strains Part of this work was supported
by the Wellcome Trust.
References
1 Csermely, P., Schnaid er, T., Soti, C., Prohaszka, Z & Nard ai, G.
(1998) The 90kDa molecular chaperone family: structure, function
andclinical applications A comprensive review Pharmacol Ther.
79, 1–39.
2 Pearl, L.H & Prodromou, C (2000) Structure and in vivo function
of Hsp90 Curr Opin Struct Biol 10, 46–51.
3 Picard, D (2002) Heat-shock protein 90, a chaperone for folding andregulation Cell Mol Life Sci 59, 1640–1648.
4 DeBoer, C., Meulman, P.A., Wnuk, R.J & Petersen, D.H (1970) Geldanamycin, a new antibiotic J Antibiot (Tokyo) 23, 442– 447.
5 Wicklow, D.T., Joshi, B.K., Gamble, W.R., Gloer, J.B & Dowd, P.F (1998) Antifungal metabolites (monorden, monocillin IV, and cerebrosides) from Humicola fuscoatra traaen NRRL 22980, a mycoparasite of Aspergillus flavus sclerotia Appl Environ Microbiol 64, 4482–4484.
6 Stebbins, C.E., Russo, A.A., Schneid er, C., Rosen, N., Hartl, F.U.
& Pavletich, N.P (1997) Crystal structure of an Hsp90-geldana-mycin complex: targeting of a protein chaperone by an antitumor agent Cell 89, 239–250.
7 Prodromou, C., Roe, S.M., O’Brien, R., Ladbury, J.E., Piper, P.W & Pearl, L.H (1997) Identification and structural char-acterization of the ATP/ADP-binding site in the Hsp90 molecular chaperone Cell 90, 65–75.
8 Roe, S.M., Prodromou, C., O’Brien, R., Ladbury, J.E., Piper, P.W & Pearl, L.H (1999) Structural basis for inhibition of the Hsp90 molecular chaperone by the antitumor antibiotics radicicol andgeldanamycin J Med Chem 42, 260–266.
9 Schulte, T.W., Akinaga, S., Murakata, T., Agatsuma, T., Sugi-moto, S., Nakano, H., Lee, Y.S., Simen, B.B., Argon, Y., Felts, S., Toft, D.O., Neckers, L.M & Sharma, S.V (1999) Interaction of radicicol with members of the heat shock protein 90 family of molecular chaperones Mol Endocrinol 13, 1435–1448.
10 Uehara, Y., Hori, M., Takeuchi, T & Umezawa, H (1986) Phe-notypic change from transformedto normal inducedby benzo-quinoidansamycins acccompanies inactivation of p60src in rat kidney cells infected with Rous sarcoma virus Mol Cell Biol 6, 2198–2206.
11 Kwon, H.J., Yoshida, M., Fukui, Y., Horinouchi, S & Beppu, T (1992) Potent andspecific inhibition of p60v-src protein kinase both in vivo and in vitro by radicicol Cancer Res 52, 6926–6930.
12 Schneider, C., Sepp-Lorenzino, L., Nimmesgern, E., Ouerfelli, O., Danishefsky, S., Rosen, N & Hartl, F.U (1996) Pharmacologic shifting of a balance between protein refolding and degradation mediated by Hsp90 Proc Natl Acad Sci USA 93, 14536–14541.
13 Schulte, T.W., Blagosklonny, M.V., Ingui, C & Neckers, L (1995) Disruption of the raf-1-Hsp90 molecular complex results in destabilisation of raf-1 and the loss of raf )1–ras association.
J Biol Chem 270, 24585–24588.
14 Whitesell, L., Sutphin, P., An, W.G., Schulte, T., Blagosklonny, M.V & Neckers, L (1997) Geldanamycin-stimulated destabili-sation of mutatedp53 is med iatedby the proteasome in vivo Oncogene 14, 2809–2816.
15 Bagatell, R., Khan, O., Paine-Murrieta, G., Taylor, C.W., Akinaga, S & Whitesell, L (2001) Destabilisation of steroid receptors by heat shock protein 90-binding drugs: a ligand-independent approach to hormonal therapy of breast cancer Clin Cancer Res 7, 2076–2084.
16 Workman, P & Maloney, A (2002) HSP90 as a new therapeutic target for cancer therapy: the story unfolds Expert Opin Biol Ther 2, 3–24.
17 Srethapakdi, M., Liu, F., Tavorath, R & Rosen, N (2000) Inhibition of Hsp90 function by ansamycins causes retino-blastoma gene product-dependent G1 arrest Cancer Res 60, 3940–3946.
18 Supko, J.G., Hickman, R.L., Grever, M.R & Malspeis, L (1995) Prelinical pharmacological evaluation of geldanamycin as an antitumour agent Cancer Chemother Pharmacol 36, 305–315.
Trang 719 Aherne, W., Maloney, A., Prodromou, C., Rowlands, M.G.,
Hardcastle, A., Boxall, K., Clarke, P., Walton, M.I., Pearl, L &
Workman, P (2003) Assays for HSP90 andinhibitors Methods
Mol Med 85, 149–161.
20 Brown, A.J.P & Tuite, M.F (1998) Yeast Gene Analysis
Aca-demic Press, San Diego.
21 Ling, M., Merante, F & Robinson, B.H (1995) A rapidand
reliable DNA preparation methodfor screening a large number of
yeast clones by polymerase chain reaction Nuceic Acids Res 23,
4924–4925.
22 Piper, P.W., Panaretou, B., Millson, S.H., Truman, A.,
Molla-pour, M., Pearl, L.H & Prodromou, C (2003) Yeast is selectively
hypersensitisedto heat shock protein 90 (Hsp90)-targetting drugs
with heterologous expression of the human Hsp90b, a property
that can be exploitedin screens for new Hsp90 chaperone
inhibi-tors Gene 302, 165–170.
23 Panaretou, B., Prodromou, C., Roe, S.M., O’Brien, R., Ladbury,
J.E., Piper, P.W & Pearl, L.H (1998) ATP binding and hydrolysis
are essential to the function of the Hsp90 molecular chaperone
in vivo EMBO J 17, 4829–4836.
24 Ali, J.A., Jackson, A.P., Howells, A.J & Maxwell, A (1993) The
43-kilodalton N-terminal fragment of the DNA gyrase B protein
hydrolyzes ATP and binds coumarin drugs Biochemistry 32,
2717–2724.
25 Harris, N., MacLean, M., Hatzianthis, K., Panaretou, B & Piper,
P.W (2001) Increasing the stress resistance of Saccharomyces
cerevisiae, by the overactivation of the heat shock response that
results from Hsp90 defects, does not extend replicative life span
but can be associatedwith a slower chronological ageing of
non-dividing cells Mol General Genomics 265, 258–263.
26 Adams, A., Gottschling, D.E., Kaiser, C.A & Stearns, T (1997)
Methods in Yeast Genetics., ColdSpring Harbor Laboratory
Press, ColdSpring Harbor, New York.
27 Nathan, D.F & Lindquist, S (1995) Mutational analysis of Hsp90
function: interactions with a steroidreceptor anda protein kinase.
Mol Cell Biol 15, 3917–3925.
28 Borkovich, K.A., Farrelly, F.W., Finkelstein, D.B., Taulien, J &
Lindquist, S (1989) hsp82 is an essential protein that is required in
higher concentrations for growth of cells at higher temperatures.
Mol Cell Biol 9, 3919–3930.
29 Morano, K.A., Santoro, N., Koch, K.A & Thiele, D.J (1999) A
trans-activation domain in yeast heat shock transcription factor is
essential for cell cycle progression during stress Mol Cell Biol 19,
402–411.
30 Piper, P.W (2001) The Hsp90 chaperone as a promising drug
target Curr Opin Invest Drugs 2, 1606–1610.
31 Smith, D.F., Whitesell, L & Katsanis, K (1998) Molecular
cha-perones: biology andprospects for pharmacological intervention.
Pharmacol Revs 50, 493–514.
32 Prodromou, C., Panaretou, B., Chohan, S., Siligardi, G., O’Brien,
R., Ladbury, J.E., Roe, S.M., Piper, P.W & Pearl, L.H (2000)
The ATPase cycle of Hsp90 drives a molecular clamp via
tran-sient dimerization of the N-terminal domains EMBO J 19, 4383–
4392.
33 Prodromou, C., Siligardi, G., O’Brien, R., Woolfson, D.N.,
Regan, L., Panaretou, B., Ladbury, J.E., Piper, P.W & Pearl, L.H.
(1999) Regulation of Hsp90 ATPase activity by tetratricopeptide
repeat (TPR)-domain co-chaperones EMBO J 18, 754–762.
34 Zou, J., Guo, Y., Guettouche, T., Smith, D.F & Voellmy, R.
(1998) Repression of heat shock transcription factor HSF1
acti-vation by HSP90 (HSP90 complex) that forms a stress-sensitive
complex with HSF1 Cell 94, 471–480.
35 Duina, A.A., Kalton, H.M & Gaber, R.F (1998) Requirement for Hsp90 anda CyP-40-type cyclophilin in negative regulation of the heat shock response J Biol Chem 273, 18974–18978.
36 Bonner, J.J., Carlson, T., Fackenthal, D.L., Paddock, D., Storey,
K & Lea, K (2000) Complex regulation of the yeast heat shock transcription factor Mol Biol Cell 11, 1739–1751.
37 Lee, S., Carlson, T., Christian, N., Lea, K., Kedzie, J., Reilly, J.P.
& Bonner, J.J (2000) The yeast heat shock transcription factor changes conformation in response to superoxide and temperature Mol Biol Cell 11, 1753–1764.
38 Ahn, S.G & Thiele, D.J (2003) Redox regulation of mammalian heat shock factor 1 is essential for Hsp gene activation andpro-tection from stress Genes Dev 17, 516–528.
39 Chang, H.C., Nathan, D.F & Lindquist, S (1997) In vivo analysis of the Hsp90 cochaperone Sti1 (p60) Mol Cell Biol 17, 318–325.
40 Dolinski, K., Muir, S., Cardenas, M & Heitman, J (1997) All cyclophilins and FK506 binding proteins are, individually and collectively, dispensable for viability in Saccharomyces cerevisiae Proc Natl Acad Sci USA 94, 13093–13098.
41 Liu, X.D., Morano, K.A & Thiele, D.J (1999) The yeast Hsp110 family member, Sse1, is an Hsp90 cochaperone J Biol Chem.
274, 26654–26660.
42 Siligardi, G., Panaretou, B., Meyer, P., Singh, S., Woolfson, D.N., Piper, P.W., Pearl, L.H & Prodromou, C (2002) Regulation of Hsp90 ATPase activity by the co-chaperone Cdc37p/p50cdc37.
J Biol Chem 26, 26.
43 Duina, A.A., Marsh, J.A & Gaber, R.F (1996) Identification of two CyP-40-like cyclophilins in Saccharomyces cerevisiae, one of which is requiredfor normal growth Yeast 12, 943–952.
44 Dolinski, K.J., Cardenas, M.E & Heitman, J (1998) CNS1 encodes an essential p60/Sti1 homolog in Saccharomyces cerevisiae that suppresses cyclophilin 40 mutations andinteracts with Hsp90 Mol Cell Biol 18, 7344–7352.
45 Kolaczkowski, M., van der Rest, M., Cybularz-Kolaczkowska, A., Soumillion, J.P., Konings, W.N & Goffeau, A (1996) Anticancer drugs, ionophoric peptides, and steroids as substrates
of the yeast multidrug transporter Pdr5p J Biol Chem 271, 31543–31548.
46 Bauer, B.E., Wolfger, H & Kuchler, K (1999) Inventory and function of yeast ABC proteins: about sex, stress, pleiotropic drug and heavy metal resistance Biochim Biophys Acta 1461, 217–236.
47 McLaughlin, S.H., Smith, H.W & Jackson, S.E (2002) Stimula-tion of the Weak ATPase Activity of Human Hsp90 by a Client Protein J Mol Biol 315, 787–798.
48 Scheufler, C., Brinker, A., Bourenkov, G., Pegoraro, S., Moroder, L., Bartunik, H., Hartl, F.U & Moarefi, I (2000) Structure of TPR domain-peptide complexes: critical elements in the assembly of the Hsp70-Hsp90 multichaperone machine Cell
101, 199–210.
49 Duina, A.A., Chang, H.C., Marsh, J.A., Lindquist, S & Gaber, R.F (1996) A cyclophilin function in Hsp90-dependent signal transduction Science 274, 1713–1715.
50 Sikorski, R.S & Hieter, P (1989) A system of shuttle vectors and yeast host strains designed for efficient manipulation of DNA in Saccharomyces cerevisiae Genetics 122, 19–27.
51 Mahe´, Y., Parle-McDermott, A.G., Nourani, A., Delahodde, A., Lamprecht, A & Kuchler, K (1996) The ATP-binding cassette multidrug transporter Snq2 of Saccharomyces cerevisiae: a novel target for the transcription factors Pdr1 and Pdr3 Mol Microbiol.
20, 109–117.