heat shock cognate 70 kDa; hsc70 members, which actively participate in a number of vital cellular functions such as protein folding, translocation, degradation and the acquisition of ce
Trang 1hsc70 chaperone function
Athanasia Salma, Apostolos Tsiapos and Ioannis Lazaridis
Laboratory of General Biology, Medical School, University of Ioannina, Greece
The heat shock protein 70 (hsp70) family of
molecu-lar chaperones consists of stress inducible (e.g hsp70)
and constitutively expressed (e.g heat shock cognate
70 kDa; hsc70) members, which actively participate in
a number of vital cellular functions such as protein
folding, translocation, degradation and the acquisition
of cell thermotolerance [1–4] In order to perform
their functions, the hsp70s cooperate with a wide
range of divergent proteins collectively known as
co-chaperones Among them, the DnaJ family
mem-bers seem to be the major and critical partners in
regulating at least the folding activity of the hsp70
chaperone machine [5]
DnaJ proteins exert their function by binding to
hsc70 and stimulating its ATPase activity ATP
hydro-lysis converts hsc70 from an open to a closed state
with low exchange rates, therefore promoting a cycle
of substrate binding and release [6] In mammals, more
than 20 DnaJ homologues have been reported and
classified into three groups on the basis of their
domain characteristics [7] All members of the DnaJ
family contain the J domain, which is a 70-amino acid sequence structured in four helices with a loop between helices II and III containing the highly conserved tri-peptide HPD, which is necessary for binding to hsc70 [8] The type I DnaJs, in addition to the J domain, contain a Gly⁄ Phe-rich region followed by a stretch of cysteine repeats Type II DnaJs lack the cysteine repeats and type III DnaJs lack both the G⁄ F region and the cysteine repeats Type I and Type II proteins seem to have similar functions and they bind non-native substrates, in contrast to type III proteins which may not bind denatured polypeptides and thus proba-bly function as ‘specialized’ molecular chaperones [9] dj2 is the best characterized type I mammalian DnaJ and it has been defined as the mammalian homologue of the bacterial DnaJ and the yeast Ydj1 [10–12] It is mainly cytosolic and acts as a cochaperone to hsc70 assisting the folding of denatured proteins and facilitat-ing the protein import into the mitochondria [13–15] As
a critical member of the hsc70 chaperone machine, dj2 was found, when overexpressed, to suppress aggregate
Keywords
DnaJ; hsc70; molecular chaperone; protein
folding; T antigen
Correspondence
I Lazaridis, Department of Biology,
Faculty of Medicine, University of Ioannina,
453 32 Ioannina, Greece
Fax: +302 6510 97863
Tel: +302 6510 97752
E-mail: ilazarid@cc.uoi.gr
(Received 2 April 2007, revised 9 July 2007,
accepted 30 July 2007)
doi:10.1111/j.1742-4658.2007.06019.x
Simian virus 40 large T antigen is a J-domain-containing protein with mul-tiple functions Among its numerous activities, T antigen can bind heat shock cognate 70 (hsc70) but the biological significance of this interaction has not been fully understood Here, we show that T antigen can act as an hsc70 co-chaperone enhancing the protein-folding ability of the hsc70 chap-erone machine We also show that T antigen exerts its function in colla-boration with the mammalian homologue of DnaJ Moreover, we show that the participation of T antigen in the hsc70 chaperone machine has cell-type-specific characteristics
Abbreviations
dj2, mammalian homologue of DnaJ; GST, glutathione S-transferase; hsc70, heat shock cognate 70 kDa; hsp, heat shock protein;
mutTAg, mutant TAg; SV40, simian virus 40; TAg, SV40 large T antigen; wtTAg, wild-type TAg.
Trang 2formation of androgen receptor and huntingtin caused
by expanded polyglutamine tracts [16,17]
Simian virus 40 (SV40) large T antigen (TAg) is a
multifunctional oncoprotein which is able to induce
transformation in multiple cell types [18] It has been
shown that T antigen can act as a molecular chaperone
because it contains a functional J domain [19] It has
also been shown that the J domain is essential for
T antigen to exert its functions related to
transforma-tion, enhancement of cell division, release of Rb⁄ E2F
complexes and viral DNA replication [20–23] T antigen
was found to bind hsc70 in an ATP-dependent manner
that requires the ATPase activity to be provided by
hsc70 [24] Although the biological significance of the
above binding is not yet clear, the prevailing view states
that upregulation of the hsc70 chaperone machine is
required for several viral functions [25]
In this report, we show that T antigen binds to
hsc70 both directly and indirectly via dj2 We also
show that the above binding is ATP independent and
contributes significantly to the folding activity of the
hsc70 chaperone machine Finally, we show that the
formation of the heterotrimeric complex T antigen–
dj2–hsc70 is cell-type-specific, because it is observed in
cells nonpermissive for SV40 viral lytic infection and
not in cells permissive to lytic infection
Results
In order to investigate the interactions of T antigen
with the hsc70 chaperone machine, we purified hsc70,
dj2 and T antigen wild-type and mutant proteins as
described in Experimental procedures Our efforts to
express the full-length forms of wild-type and mutant
T antigen fused to glutathione S-transferase were not
successful, despite the fact that we tested extensively
both the bacterial and the insect cell expression
systems We believe that the difficulty in purifying full-length GST-tagged T antigen forms was due to the large size of the resulting proteins Therefore, we decided to purify truncated forms of T antigen which included the J domain, the Rb-binding domain and the DNA-binding domain, but lacked the ATPase domain
of the C-terminus Given that the ATPase domain of the T antigen has been shown to be dispensable for hsc70 binding [24], we reasoned that the truncated T-antigen proteins would mimic the function of their full-length counterparts
T antigen binds directly to hsc70 Upon performing GST pull-down experiments, we observed that the wild-type TAg (wtTAg) bound to hsc70 in an ATP-dependent manner with a stoichiome-try of 1 : 1, as reported previously [24] We also con-firmed the inability of the mutTAg protein to bind hsc70, even in the presence of ATP (data not shown)
It is interesting to note that hsp70 exhibited the same characteristics as hsc70, in that it was found to bind wtTAg in an ATP-dependent fashion, but with signifi-cantly reduced affinity (data not shown)
T antigen and dj2 cooperate in binding to hsc70 Having established the assay conditions, we tested whether the presence of a typical hsc70 co-chaperone, namely dj2, interfered or altered the TAg⁄ hsc70-bind-ing characteristics The first findhsc70-bind-ing was that dj2 bound TAg in an ATP-independent manner (Fig 1A, lanes 3 and 4) Although functional dimerization of DnaJs has been reported [31–33], our results clearly indicate that binding between type I and type III DnaJs is possible
in an ATP-independent manner Moreover, it was shown that unlike hsc70, dj2 bound mutTAg indicating
Fig 1 dj2 participation in the TAg ⁄ hsc70 complex Pull-down assays were performed using GST-tagged wtTAg and mutTAg as described in Experimental procedures Lanes 1, 2 and 8 represents purified pro-teins (A) GST–wtTAg was incubated with the indicated proteins in the presence or absence of ATP Bound proteins were detected by SDS ⁄ PAGE, stained with Coo-massie Brilliant Blue (upper), followed by western blotting (IB) (B) The same experi-ment as in (A) was performed using GST– mutTAg instead of GST–wtTAg.
Trang 3once again that dj2 and T antigen, although both
J-domain-containing proteins have clearly distinct
hsc70-binding properties (Fig 1B, lanes 3 and 4) The
above data also show that dj2 and hsc70 have different
binding properties to wtTAg and mutTAg Most
importantly, we observed that the presence of dj2
dis-rupted the stoichiometric balance of hsc70⁄ wtTAg
complex reducing the amount of bound hsc70 and
allowing hsc70 to bind wtTAg even in the absence of
ATP (Fig 1A, lanes 5 and 6) Similar results were
obtained when we used mutTAg instead of wtTAg in
our pull-down assays More specifically, we observed
that in the presence of dj2, hsc70 bound mutTAg in an
ATP-independent fashion (Fig 1B, lanes 5 and 6)
These findings led us to hypothesize that dj2 binds
T antigen in an ATP independent manner and the
resulting heterodimer complexes with hsc70 via dj2
To verify this hypothesis we proceeded in a
pull-down experimental scheme in which wtTAg was
sequentially incubated with hsc70 and dj2 As shown
in Fig 2, formation of the dj2⁄ TAg complex prior to
hsc70 addition resulted in the recruitment of increased
amounts of hsc70 to the glutathione Sepharose-bound
GST–wtTAg (compare lanes 1 and 2) Densitometry
analysis revealed that there is a 2–2.5-fold increase in
the amount of hsc70 collected in the Tag⁄ dj2 complex
In contrast, addition of dj2 to the already formed
hsc70⁄ TAg complex decreased the amount of bound
hsc70 by 50% (compare lanes 1 and 3) Once again the
absence of ATP in the binding reactions did not
influ-ence hsc70 recruitment to the dj2⁄ TAg complex,
confirming our conclusion that the participation of the
T antigen in this complex is mainly mediated by dj2 The reduced amounts of hsc70 bound to TAg prior to dj2 addition (lanes 1 and 5) are probably due to the fact that the binding affinity of dj2 to hsc70 is stron-ger The possibility of an antagonistic effect between dj2 and TAg for hsc70 binding cannot be excluded, but the finding that formation of the dj2⁄ TAg complex recruits more hsc70 (compare lanes 1, 3 and 5 with lanes 2, 4 and 6) indicates that the order of inter-actions is important for the final composition of the complex
T antigen enhances the folding activity of the hsc70 chaperone machine
Because one of the main functions of the dj2⁄ hsc70 chaperone complex is the folding of denatured or par-tially folded proteins [29] we investigated the involve-ment of T antigen in the above process Therefore,
we performed luciferase-refolding experiments and observed that indeed T antigen facilitated hsc70 and dj2 in recovering of activity for denatured luciferase (Fig 3) We also found that T antigen can act as an hsc70 co-chaperone, albeit with much reduced activity when compared with dj2 However, we observed that the presence of wtTAg significantly enhanced the fold-ing activity of the dj2⁄ hsc70 chaperone machine This was verified by the finding that the maximal refolding
of luciferase achieved by the hsc70⁄ dj2 dimer, was fur-ther increased with the addition of wtTAg Impor-tantly, the mutTAg had no effect on the function of hsc70 alone or hsc70 with dj2 suggesting that the effect
of wtTAg is dependent on its co-chaperone activity
Fig 2 dj2 recruits increased amounts of hsc70 to the
heterotrimer-ic complex Pull-down experiments were performed using GST–
wtTAg and in the assay mixture hsc70, dj2 and ATP were added
sequentially as indicated The composition of the complexes
formed was analysed by SDS ⁄ PAGE followed by Coomassie
Bril-liant Blue staining.
Fig 3 wtTAg enhances the folding activity of the hsc70 ⁄ dj2 chap-erone pair Refolding assays were performed as described and luciferase activity was monitored at several time points ranging from 0 to 60 min This diagram depicts values taken after 60 min incubation with the indicated proteins and expressed as percentage
of the native luciferase activity.
Trang 4The ATP independence of the heterotrimeric
TAg–dj2–hsc70 complex formation is cell type
specific
The in vitro binding data raised the question of how
T antigen, dj2 and hsc70 may interact in the cellular
context In order to investigate these interactions we
chose two transformed cell lines, namely Cos7 and
SVTT1, which constitutively express T antigen but
dif-fer in that the Cos7 cells are permissive for SV40 lytic
infection, whereas the SVTT1 are not Cell extracts
were immunoprecipitated with an anti-TAg coupled to
protein G beads (as described in Experimental
proce-dures) and the immunoprecipitates were analysed for
the presence of TAg, dj2 and hsc70 In accordance with
previous findings [30], we detected the formation of
the hsc70⁄ TAg complex only in nonpermissive cells
(Fig 4B, +ATP) The amount of hsc70 bound to TAg
was quantitated as 50% of the input We also detected
the presence of dj2 in the immunoprecipitates of both
cell types in the presence of ATP and our quantification
measurements showed that 15–20% of the input was
retained in the complex However, to our surprise, we
found that in the absence of an ATP regeneration
sys-tem, both dj2 and hsc70 were not able to associate with
T antigen in the extracts of non permissive cells
(Fig 4B, –ATP) In contrast under the same conditions
the association of T antigen with dj2 persisted in
per-missive cells (Fig 4A, ±ATP) in accordance with our
in vitro findings that this binding is ATP independent
Collectively, the above results led us to conclude that
the interactions between T antigen, dj2 and hsc70 are
cell-type dependent
Discussion
Although the ability of T antigen to bind hsc70 was
extensively studied, the exact mechanism by which this
binding contributes to the T-antigen-mediated cellular transformation remains elusive [34,35] However, a model has been suggested according to which the J-domain-dependent association of T antigen with hsc70 stimulates chaperone activity, which in turn releases E2F from its pRb complex allowing the ‘free’ E2F to transactivate the genes required for cell prolif-eration [20,22,36] Our results, without disputing the above model, indicate that T antigen might not exert its hsc70-related functions alone but in combination with dj2, a classical hsc70 co-chaperone This sugges-tion is based on our findings that, in vitro, T antigen associates with dj2 in an ATP-independent manner and this binding persists even in the presence of hsc70 (Fig 1A) As a result, T antigen is recruited to the hsc70 chaperone complex via dj2 Therefore, the pres-ence of T antigen allows hsc70 to utilize yet another J-domain-containing protein and enhance its chaperon-ing function It is interestchaperon-ing to note that even a mutated form of T antigen unable to bind hsc70 can
be recruited to the complex via dj2 (Fig 1B)
Our interpretation of the above data is that besides the formation of the expected dj2⁄ hsc70 (dimer : monomer) complexes, the presence of T anti-gen leads to the formation of novel heterotrimeric complex comprising of monomeric forms of TAg⁄ dj2⁄ hsc70 The formation of these complexes does not exclude the possibility of direct binding between
T antigen and hsc70 On the contrary, given the abun-dance of hsc70, one can envisage the presence of all three types of complex in the cellular context This interpretation is supported by the finding that associa-tion of T antigen with dj2 prior to the addiassocia-tion of hsc70 leads to the recruitment of increased amounts of hsc70 to the complex In contrast, addition of dj2 to the already formed TAg⁄ hsc70 dimer does not increase the levels of hsc70 present in the complex, indicating that probably dj2 associates with hsc70 in a separate
A
Fig 4 Cell-type-specific interaction of T antigen with hsc70 Immunoprecipitations of Cos7 cell extracts (A), SVTT1 extracts (B), and NIH 3T3 transfected with mutTAg extracts, as a control (C), were performed using an anti-(T antigen) serum column Bound proteins were detected
by western blotting The presence or absence of ATP regeneration system in the lysates is indicated FL, analysis of the flow through mate-rial; ctrl, the control IgG-protein G beads; Inp, the whole cellular lysate (10% of total).
Trang 5complex which does not include T antigen
Interest-ingly, the increased amounts of hsc70 required by the
TAg⁄ dj2 heterodimer do not seem to be affected by
the presence of ATP, indicating once more that the
ATP-independent dj2 binding to hsc70 is the driving
force for the creation of the heterotrimeric complex
(Fig 2) Therefore, based on our results, we believe
that T antigen can bind hsc70 in two distinctly
differ-ent ways First, it can bind directly in an
ATP-depen-dent fashion, and second, it can bind indirectly via dj2
in an ATP-independent manner
The functional significance of our findings was
investigated by monitoring the chaperone-folding
activity of the detected complexes Indeed, we found
that the presence of T antigen enhanced the ability of
the hsc70⁄ dj2 chaperone pair to refold denatured
lucif-erase We believe that this increased folding activity
was due to the function of the newly formed
heterotrimeric complex (TAg⁄ dj2 ⁄ hsc70) In contrast,
the presence of the mutated form of T antigen was
not able to change the folding activity of the hsc70⁄ dj2
chaperones In other words, despite the fact that the
trimeric complex mutTAg⁄ dj2 ⁄ hsc70 can be clearly
formed (Fig 1B), mutTAg is unable to further
enhance luciferase folding, probably due to its inability
to stimulate the hsc70 ATPase activity This last
assumption is supported by the finding that although
wtTAg can act as a weak hsc70 cochaperone, mutTAg
seem to be unable to positively contribute to the
enhancement of the hsc70 folding activity Collectively,
we concluded that T antigen facilitates the hsc70
chaperone machine by enhancing its folding function
In order to detect the existence of the described
complexes in vivo, we performed immunoprecipitations
with an anti-(T antigen) serum in cellular extracts of
Cos7 and SVTT1 cells As shown in Fig 4, our
approach was quite effective in that our antibody
col-umn depleted most of the T antigen from the cell
lysates As expected, based on our previous data [30],
formation of the TAg⁄ hsc70 complex was observed
only in nonpermissive cells, reinforcing our suggestion
that this binding has cell-type-specific characteristics
Moreover, we detected the presence of dj2 in both the
TAg⁄ hsc70 complex (nonpermissive cells) and in the
T antigen immunoprecipitate (permissive cells)
How-ever, to our surprise, we observed that in the absence
of ATP the dj2 and hsc70 did not associate with TAg
in SVTT1 cells, in contrast to the Cos7
immuno-precipitates where the binding of T antigen to dj2
persisted (Fig 4) Given that the dj2⁄ TAg binding is
ATP independent, as shown above, we suspect that
the activation of an additional, yet unidentified,
cellular factor is responsible for the cell type specific
dissociation of the heterotrimeric complex in the absence of ATP or conversely enabling dj2 to bind TAg in Cos7 cells Overall, our data using cellular extracts clearly suggest the existence of a cell type specific organization of the hsc70 chaperone machine, which is probably related to the T antigen J-domain-mediated viral functions
Experimental procedures
Cell lines
The Cos7 and SVTT1 (NIH 3T3 cells expressing wtTAg) cell lines established from monkey or mouse embryos, respectively, were maintained in minimal essential medium supplemented with 10% fetal bovine serum at 37C in a humidified 5% CO2 atmosphere Sf9 insect cells were grown in Sf900II medium (Gibco, Invitrogen, Carlsbad, CA) containing 10% fetal bovine serum at 27C and used for the production of baculoviruses
Cloning, expression and purification of recombinant proteins
The pQE-32-Mydj2 construct [13] was used to overexpress Mydj2–6·His in Escherichia coli SG13009 Briefly, over-night cultures of E coli carrying the pQE-32-Mydj2 plasmid were diluted 10-fold and cultured for 1 h After isopropyl thio-b-d-galactoside induction (1 mm) for 2 h at
37C, cells were collected by brief centrifugation at 6000 g using an Avanti J-25 centrifuge with JA-14 rotor (Beckman Coulter, Fullerton, CA) and cell lysates were prepared by sonication The recombinant protein was purified using a
Ni⁄ nitrilotriacetic acid column and imidazole elution (50 ± 250 mm) as described by the manufacturers (Qiagen, Valencia, CA)
The SV40 large T antigen was subcloned from pSG5-T (a generous gift from J A DeCaprio, Harvard Medical School, MA) into the BamH1 site of pGEX-4T-1 (AMRAD)
to create the pGEX-4T-1–wtTAg construct The resulting plasmid was digested with EcoR1 and HindII, treated with Klenow and religated to create the pGEX-4T-1–wtTAg282 construct which was used to express the first 282 amino acid residues of the wild-type T antigen The mutated form
of T antigen was subcloned from pSG5-T-H42Q (a gener-ous gift from J A DeCaprio) to pGEM and subsequently inserted to the EcoR1 and Sal1 sites of pGEX-4T-1 The resulting plasmid was digested with Not1 and HindIII, blunt ended with Klenow and religated to create the pGEX-4T-1–mutTAg268, which was used to express the first 268 amino acid residues of T antigen with a single amino acid substitution (H42Q) The described constructs, named GST–wtTAg and GST–mutTAg, respectively, were expressed in BL21(DE3) bacterial cells and the corresponding
Trang 6proteins were purified from lysates according to standard
procedures [26]
Baculovirus expressed His6·-tagged hsc70 and hsp70
were prepared according to Bac-To-Bac baculovirus
expres-sion system procedure (Invitrogen)
Pull-down assays
GST fusion proteins (3 lg; wtTAg, mutTAg) or GST alone,
were incubated for 30 min at room temperature with 30 lL
of glutathione–agarose beads and ‘blocked’ in 1% fish
gela-tin in assay buffer (150 mm NaCl, 20 mm Tris⁄ HCl pH 7.5,
2 mm MgCl2, 250 mm sucrose, 1% Triton X-100, 0.1 mm
EGTA, 1 mm phenylmethanesulfonyl fluoride, 1 mm DTT)
After washing three times with the same buffer, the beads
were combined with equal amounts of purified His–hsc70
and⁄ or His–dj2 and further incubated for 1 h at room
tem-perature in the presence or absence of ATP regeneration
buffer (1 mm ATP, 30 mm creatine phosphate, 0.25 mg
phosphokinase per mL, 2 mm MgCl2, 1 mm dithiothreitol)
The beads were washed six times with assay buffer, before
eluting the bound proteins with hot Laemmli buffer [27]
Western blots were performed using the following
anti-bodies: T antigen-specific mAb PAb419, which have been
produced by hybridoma cells Hsc70-specific antibody
(SPA-815, StressGen), Hsp70-specific antibody (SPA-810,
StressGen) and Dj2-specific antibody (HDJ-2⁄ DNAJ Ab-1,
NeoMarkers) Quantitation of the pulled down proteins
was performed using the quantity one densitometry
software provided by Bio-Rad Inc (Hercules, CA)
Immunoprecipitation
Whole-cell extracts were prepared from COS and SVTT1
cells in lysis buffer containing 150 mm NaCl, 20 mm
Tris⁄ HCl pH 7.5, 2 mm MgCl2, 250 mm sucrose, 1%NP40,
0.1 mm EGTA, 1 mm phenylmethanesulfonyl fluoride,
1 mm dithiothreitol and 1 lgÆmL)1each of protease
inhibi-tors aprotinin, leupeptin and pepstatin The extracts were
centrifuged at 13 000 g for 15 min using a Sigma 1-14
microcentrifuge with 12094 rotor (Sigma-Aldrich, Munich,
Germany), precleared with 20 lL of IgG–protein G (3 lg)
for 30 min at room temperature and then incubated with
20 lL of anti-TAg–protein G beads (40 lg of anti-TAg
serum) or with 20 lL IgG–protein G beads (40 lg of
anti-[mouse IgG] serum) overnight at 4C, in the presence or
absence of ATP regeneration buffer The antibodies were
covalently coupled to the beads using DMP (Pierce,
Rock-ford, IL) as a cross-linker After incubation, the beads were
washed three times with lysis buffer Proteins present in the
immune complexes were eluted with lysis buffer
supple-mented with 1 m NaCl The eluates were trichloroacetic
acid precipitated and the pellets were resuspended in 30 lL
Laemmli buffer for subsequent analysis by SDS⁄ PAGE and
western blotting
Luciferase assays
Luciferase activity was measured as described [28] with minor modifications Briefly, 1 mg luciferase (Promega, Madison, WI) was chemically denatured by diluting 3-fold in buffer containing guanidinium-HCl (6 m) 25 mm Hepes (pH 7.6), 50 mm KC1, 5 mm MgCl2 and 1 mm dithiothreitol for 40 min at room temperature Refolding reactions were performed in 125 lL volumes by diluting denatured luciferase (1 lL) into refolding buffer contain-ing 25 mm Hepes (pH 7.6), 50 mm KC1, 5 mm MgCl2, purified chaperones, and ATP regeneration buffer The samples were incubated at 30C and at the indicated times, 1 lL of each reaction was diluted into 60 lL of luciferase assay mixture (Promega) Activity was measured using a luminometer (Junior LB9509 EG & G Berthold, Bad Wildbad, Germany) Native luciferase activity was measured after first diluting the stock solution threefold into refolding buffer and then diluting 125-fold into the same buffer All activities were calculated as a percent of the native luciferase activity and all data points are the average of at least two replicates
Acknowledgements
We thank M Cheetham and H.H Kampinga for critically reading the manuscript We also thank
Dr P Kouklis for helpful advice This work was sup-ported by grants from the EU (QLRT-1999, #30720) and the Greek Ministry of Education (Hrakleitos)
References
1 Hendrick JP & Hartl FU (1993) Molecular chaperone functions of heat shock proteins Annu Rev Biochem 62, 349–384
2 Georgopoulos C & Welch WJ (1993) Role of the major heat shock proteins as molecular chaperones Annu Rev Cell Biol 9, 601–634
3 Hartl FU (1996) Molecular chaperones in cellular pro-tein folding Nature 381, 571–579
4 Patterson C & Cyr D (2002) Welcome to the machine Circulation 106, 2741–2746
5 Takayama S, Xie Z & Reed JC (1999) An evolutionarily conserved family of hsp70⁄ hsc70 molecular chaperone regulators J Biol Chem 274, 781–786
6 Young JC, Agashe VR, Siegers K & Hartl FU (2004) Pathways of chaperone-mediated protein folding in the cytosol Nat Rev Mol Cell Biol 5, 781–791
7 Cheetham ME & Caplan AJ (1998) Structure, function and evolution of DnaJ: conservation and adaptation of chaperone function Cell Stress Chaperones 3, 28–36
8 Tsai J & Douglas MG (1996) A conserved HPD sequence of the J-domain is necessary for Ydj1
Trang 7stimulation of hsp70 ATPase activity at a site distinct
from substrate binding J Biol Chem 271, 9347–9354
9 Kelley WL (1998) The J domain family and the
recruit-ment of chaperone power Trends Biochem Sci 23,
222–227
10 Qiu X-B, Shao Y-M, Miao S & Wang L (2006) The
diversity of the DnaJ⁄ hsp40 family, the crucial partners
for hsp70 chaperones Cell Mol Life Sci 63, 2560–2570
11 Chellaiah A, Davis A & Mohanakumar T (1993)
Clon-ing of a unique human homologue of the Escherichia
coliDNAJ heat shock protein Biochim Biophys Acta
1174, 11–13
12 Oh S, Iwahori A & Kato S (1993) Human cDNA
encoding DnaJ protein homologue Biochim Biophys
Acta 1174, 114–116
13 Bozidis P, Lazaridis I, Pagoulatos G & Angelidis CE
(2002) Mydj2 as a potent partner of hsc70 in
mamma-lian cells Eur J Biochem 269, 1553–1560
14 Terada K & Mori M (2000) Human DnaJ homologs
dj2 and dj3 and bag-1 are positive cochaperones of
hsc70 J Biol Chem 275, 24728–24734
15 Terada K, Kanazawa M, Bukau B & Mori M (1997)
The human DnaJ homologue dj2 facilitates
mitochon-drial protein import and luciferase refolding J Cell Biol
139, 1089–1095
16 Kobayashi Y, Kume A, Li M, Doyu M, Hata M,
Oht-suka K & Sobue G (2000) Chaperones hsp70 and hsp40
suppress aggregate formation and apoptosis in cultured
neuronal cells expressing truncated androgen receptor
protein with expanded polyglutamine tract J Biol Chem
275, 8772–8778
17 Wyttenbach A, Carmichael J, Swartz J, Furlong RA,
Nazain Y, Rankin J & Rubinsztein DC (2000) Effects
of heat shock, heat shock protein 4 (HDJ-2) and
protea-some inhibition on protein aggregation in cellular
mod-els of Huntington’s disease Proc Natl Acad Sci USA
97, 2898–2903
18 Saenz-Robles MT, Sullivan CS & Pipas JM (2001)
Transforming functions of simian virus 40 Oncogene
20, 7899–7907
19 Kelley WL & Georgopoulos C (1997) The T⁄ t common
exon of simian virus 40, JC and BK polyomavirus T
antigens can functionally replace the J-domain of the
Escherichia coliDnaJ molecular chaperone Proc Natl
Acad Sci USA 94, 3679–3684
20 Srinivasan A, McClellan AJ, Vartikar J, Marks I,
Cantalupo P, Li Y, Whyte P, Rundell K, Brodsky JL &
Pipas JM (1997) The amino-terminal transforming
region of simian virus 40 large T and small t antigens
functions as a J domain Mol Cell Biol 17, 4761–4773
21 Studbal HJ, Zalvide J, Campbell KS, Schweitzer C,
Roberts TM & DeCaprio JA (1997) Inactivation of
pRB-related proteins p130 and p107 mediated by the J domain of simian virus 40 large T antigen Mol Cell Biol 17, 4979–4990
22 Sullivan CS, Cantalupo SP & Pipas JM (2000) The molecular chaperone activity of simian virus 40 large T antigen is required to disrupt Rb-E2F family complexes
by an ATP-dependent mechanism Mol Cell Biol 20, 6233–6243
23 Peden KW & Pipas JM (1992) Simian virus 40 mutants with amino-acid substitutions near the amino terminus
of large T antigen Virus Genes 6, 107–118
24 Sullivan CS, Gilbert SP & Pipas JM (2001) ATP-depen-dent simian virus 40 T-antigen–hsc70 complex forma-tion J Virol 75, 1601–1610
25 Sullivan CS & Pipas JM (2001) The virus–chaperone connection Virology 287, 1–8
26 Sambrook J, Fritsch FF & Maniatis TM (1989) Molecu-lar Cloning A Laboratory Manual, 3rd edn Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY
27 Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4 Nature 227, 680–685
28 Levy EJ, McCarty J, Bukau B & Chirico WJ (1995) Conserved ATPase and luciferase refolding activities between bacteria and yeast hsp70 chaperones and mod-ulators FEBS Lett 368, 435–440
29 Kelley WL (1999) Molecular chaperones: how J domains turn on hsp70s Curr Biol 9, R305–R308
30 Sainis I, Angelidis C, Pagoulatos GN & Lazaridis I (2000) Hsc70 interactions with SV40 viral proteins differ between permissive and non permissive mammalian cells Cell Stress Chaperones 5, 132–138
31 Langer T, Lu C, Echols H, Flanagan J, Hayer MK & Hartl FU (1992) Successive action of DnaK, DnaJ and GroEL along the pathway of chaperone-mediated pro-tein folding Nature 356, 683–689
32 Li J, Qian X & Sha B (2003) The crystal structure of the yeast hsp40 Ydj1 complexed with its peptide sub-strate Structure 11, 1475–1483
33 Nemethova M, Smutny M & Wintersberger E (2004) Transactivation of E2F-regulated genes by polyomavi-rus large T antigen Evidence for a two-step mechanism Mol Cell Biol 24, 10986–10994
34 Brodsky JL & Pipas JM (1998) Polyomavirus T anti-gens: molecular chaperones for multiprotein complexes
J Virol 72, 5329–5334
35 De Caprio JA (1999) The role of the J domain of SV40 large T in cellular transformation Biologicals 27, 23–28
36 Zalvide J, Stubdal H & DeCaprio JA (1998) The J domain of simian virus 40 large T antigen is required to functionally inactivate pRb family proteins Mol Cell Biol 18, 1408–1415