Gusev1 1 Department of Biochemistry, School of Biology, Moscow State University, Moscow, Russia;2Imperial College School of Medicine at National Heart and Lung Institute, Dovehose Street
Trang 1Interaction of the small heat shock protein with molecular mass
25 kDa (hsp25) with actin
Olesya O Panasenko1, Maria V Kim1, Steven B Marston2and Nikolai B Gusev1
1
Department of Biochemistry, School of Biology, Moscow State University, Moscow, Russia;2Imperial College School of
Medicine at National Heart and Lung Institute, Dovehose Street, London, UK
The interaction of heat shock protein with molecular mass
25 kDa (HSP25) and its point mutants S77D + S81D (2D
mutant) and S15D + S77D + S81D (3D mutant) with
intact and thermally denatured actin was analyzed by means
of fluorescence spectroscopy and ultracentrifugation Wild
type HSP25 did not affect the polymerization of intact actin
The HSP25 3D mutant decreased the initial rate without
affecting the maximal extent of intact actin polymerization
G-actin heated at 40–45°C was partially denatured, but
retained its ability to polymerize The wild type HSP25 did
not affect polymerization of this partially denatured actin
The 3D mutant of HSP25 increased the initial rate of
poly-merization of partially denatured actin Heating at more
than 55°C induced complete denaturation of G-actin Completely denatured G-actin cannot polymerize, but it aggregates at increased ionic strength HSP25 and especially its 2D and 3D mutants effectively prevent salt-induced aggregation of completely denatured actin It is concluded that the interaction of HSP25 with actin depends on the state
of both actin and HSP25 HSP25 predominantly acts as a chaperone and preferentially interacts with thermally unfolded actin, preventing the formation of insoluble aggregates
Keywords: small heat shock protein; actin; thermal denaturation
Actin is the major component of the thin filaments of
muscle cells and of the cytoskeleton system of nonmuscle
cells It is therefore a very abundant protein, and its
concentration in smooth muscle is close to 800–900 lM[1]
Actin has a rather complex and labile tertiary structure [2,3]
Different types of stress can induce actin unfolding [4,5],
aggregation of partially folded actin [5,6] and redistribution
of actin inside the cell [7–9] Accumulation of partially
folded or aggregated proteins can induce significant damage
to cells This is especially important in the case of abundant
proteins, such as actin Therefore the cell has evolved
different mechanisms to prevent the formation of insoluble
aggregates, and heat shock proteins (HSPs) play an
important role in this process
The data in the literature indicate that the small heat
shock protein with molecular mass 25–27 kDa (HSP25)
plays an important role in actin remodeling, contractility of
different cell types and protection of the cytoskeleton under
different unfavorable conditions [7,8,10] Miron et al
[11,12] showed that avian HSP25 effectively inhibits actin
polymerization and prevents gelation of actin induced by filamin and/or a-actinin These observations were confirmed
by Benndorf et al [13], who showed that nonphosphoryl-ated monomers of HSP25 effectively inhibit actin polymerization, whereas phosphorylated monomers and nonphosphorylated multimers of HSP25 are ineffective in the regulation of actin polymerization The protein seg-ments of monomeric HSP25 involved in the inhibition of actin polymerization were determined recently [14] Although these data are of great interest, their application
to cell physiology is questionable as under physiological conditions HSP25 forms high molecular mass oligomers that are in equilibrium with low molecular mass oligomers [15,16], but practically do not dissociate to monomers The actin depolymerizing effect ascribed to HSP25 [11–14] contrasts with the stabilizing of microfilaments induced by HSP25 or its phosphorylated forms [7,17] Moreover, recently Butt et al [18] have shown that under in vitro conditions HSP25 either does not affect or even activates the polymerization of actin
To explain the contradictory results described in the literature we assumed that the mode of interaction is dependent both on the state of HSP25 and actin In this paper we analyze the effect of recombinant avian HSP25 and its mutants mimicking phosphorylation on the heat-induced aggregation and polymerization of intact and partially denatured actin
Materials and methods
Proteins HSP25 from chicken gizzard was purified by the procedure described previously [19] Chicken HSP25 was cloned,
Correspondence to N B Gusev, Department of Biochemistry,
School of Biology, Moscow State University,
Moscow 119992, Russia Tel./Fax: + 7 095 939 2747,
E-mail: NBGusev@mail.ru
Abbreviations: ANS, 8-anilinonaphtalene-1-sulfonic acid; HSP, heat
shock proteins; 1D mutant, chicken HSP25 with mutation S15D;
2D mutant, chicken HSP25 with mutation S77D + S81D;
3D mutant, chicken HSP25 with mutation S15D + S77D + S81D;
MAPKAP-2, mitogen-activated protein kinase-activated protein
kinase-2.
(Received 15 October 2002, revised 25 December 2002,
accepted 7 January 2003)
Trang 2expressed and purified as described by Bukach et al [19],
and Panasenko et al [20] Three point mutants of HSP25
with replacements S15D (1D mutant), S77D + S81D (2D
mutant), and S15D + S77D + S81D (3D mutant) were
obtained by the procedures published earlier [19]
Introduc-tion of extra negative charges in posiIntroduc-tions 15, 77 and 81
mimics phosphorylation of HSP25 by MAPKAP-2 kinase
that may occur in vivo [16] These mutations induce changes
in the quaternary structure of avian HSP25 similar to those
evoked by the corresponding mutations in mammalian
HSP27 [16,20] HSP25 may form oligomers of different sizes
[10,15,16], therefore for all calculations we used a molecular
mass for the monomeric proteins of 25 kDa
Rabbit skeletal actin was purified according to Pardee
and Spudich [21] G-actin in buffer G (5 mM Tris/HCl,
pH 8.2, 0.2 mMATP, 0.1 mMCaCl2, 0.5 mM
b-mercapto-ethanol, 1 mM NaN3) was stored on ice and used within
10 days of purification The purity of all proteins was
checked by SDS gel electrophoresis [22]
Thermal denaturation was achieved by incubation of
G-actin (usually 15 lM) in buffer G for the required period
of time at 43, 60 or 80°C
Limited proteolysis of intact and heated actin was
performed in buffer G at the weight ratio of
actin/N-tosyl-L-phenylalanine chloromethyl ketone-trypsin (Sigma) equal
to 100 : 1 or 50 : 1 After incubation at 25°C for 0.25–
20 min phenylmethanesulfonyl fluoride was added to a final
concentration of 0.5 mM The samples were mixed with the
sample buffer and after boiling were subjected to SDS gel
electrophoresis [22] After staining, the gels were scanned
and evaluated by theONEDSCANprogram The intensity of
the band of unhydrolyzed actin was plotted against the time
of incubation
Characterization of actin preparation quality
Fluorescence parameters were used for estimation of
nativity of actin preparations Corrected spectra of actin
fluorescence excited at 297 nm were recorded in the range
300–400 nm on the Hitachi F-3000 fluorescence
spectro-photometer Parameter A, introduced by Turoverov et al
[23] and equal to the ratio of intensities of fluorescence at
320 and 365 nm (I320/I365) was determined for different
preparations of actin Parameter A reflects the polarity of
the tryptophan environment Any changes in actin structure
influence this environment and affect parameter A
Prepa-rations of actin with A > 2.56 contain less than 2% of
inactivated actin, whereas parameter A for inactivated and
completely unfolded actin is equal to 1.3and 0.4,
respect-ively [5]
The interaction of intact and partially folded actin with
the hydrophobic probe 8-anilinonaphtalene-1-sulfonic acid
(ANS) (Sigma) was measured at 25°C in buffer G Samples
containing 2.3 lMof intact or heated actin and 140 lMof
ANS were excited at 390 nm, and spectra of fluorescence
were recorded in the range 400–600 nm as before
Actin aggregation
Light scattering, ultracentrifugation and size-exclusion
chromatography were used to follow the process of actin
aggregation After heating in buffer G under different
conditions G-actin (10–15 lM) was cooled to 25°C, and aggregation was initiated by the addition of KCl and MgCl2
up to 50 mMand 2 mM, respectively The increase of ionic strength promotes aggregation of thermally denatured actin [5] Aggregation was followed by light scattering, measured
at 560 nm, again using the Hitachi F-3000 fluorescence spectrophotometer
Ultracentrifugation was also used to follow aggregation
of partially unfolded actin In this case G-actin (15 lM) in buffer G was heated at 60°C for 1 h The samples were diluted with cold buffer G, cooled to 25°C, and mixed either with buffer H (20 mM Tris/acetate pH 7.6, 10 mM NaCl, 0.1 mM EDTA, 15 mM 2-mercaptoethanol, 10% glycerol) or with different quantities of HSP25 in buffer H and incubation for 20 min at 25°C Buffer S (50 mM imidazole pH 7.6, 750 mM KCl, 10 mM MgCl2, 1 mM ATP and 50 mM 2-mercaptoethanol) (1/5 of the sample volume) was added, and incubation was continued for
60 min at 25°C The samples obtained were then subjected
to ultracentrifugation at 100 000 g for 1 h The protein composition of the supernatant and pellet was determined
by quantitative SDS gel electrophoresis [22]
Size exclusion chromatography of intact and heated actin was performed on Acta-FPLC (Amersham-Pharmacia Biotech.) using Superdex 200 10/30 column The column was equilibrated and developed in buffer G The samples (90 lL) of intact or heated actin (15 lM) were loaded on the column and eluted with buffer G at the rate of 0.5 mLÆmin)1 Actin polymerization
The methods of fluorescent spectroscopy and ultracentri-fugation were used for registration of actin polymerization
In the first case F-actin was labeled by N-(1-pyrenyl)iodo-acetamide according to Kouyama and Mihashi [24] After the removal of insoluble N-(1-pyrenyl)iodoacetamide by low-speed centrifugation (10 min, 10 000 g), the modified F-actin was collected by ultracentrifugation (1 h, 100 000 g) The pellet of modified F-actin was dissolved in buffer G, dialyzed against buffer G for 48 h and subjected to ultra-centrifugation The supernatant contained G-actin with an extent of modification equal to 0.6–0.7 mol of N-(1-pyrenyl) iodoacetamide per mole of actin Size exclusion chroma-tography of modified G-actin revealed that the sample does not contain high molecular mass aggregates and is free of fluorescent label, unattached to the protein The sample of modified G-actin was stored on ice and used within 1 week
of purification Polymerization of pyrene-labeled actin was measured according to Pollard [25] and Miron et al [12] Briefly, polymerization was performed in buffer G and was initiated by the addition of KCl and MgCl2up to 50 mM and 2 mM, respectively Different quantities of actin nuclei (short fragments of F-actin) were added simultaneously with KCl and MgCl2 if the initial rate of polymerization was measured In the series of preliminary experiments we have shown that under the conditions used (1–4 lM of actin, containing 10–15% of pyrenyl-actin) there was no self-assembly of G-actin We also observed proportionality of the initial rate of polymerization to nucleus concentration
In addition, the increase in fluorescence induced by actin polymerization was hyperbolic in time, and that above the critical concentration the rate of polymerization was linearly
Trang 3dependent on actin concentration According to Pollard [25]
fulfilment of all these criteria is desirable for proper
measurement of the initial rate of actin polymerization
Polymerization of actin was performed at different actin
concentrations both in the presence and in the absence of
HSP25 or its mutant mimicking phosphorylation In the
series of separate experiments we measured the extent of
actin polymerization In this case, polymerization was
initiated by the addition of only KCl and MgCl2(50 mM
and 2 mM, respectively) In this case increase in fluorescence
was sigmoidal in time and was followed for 60–90 min until
it reached its maximal value
When ultracentrifugation was used for the measurement
of actin polymerization, G-actin (2–8 lMin buffer G) was
mixed with buffer H or with different quantities of HSP25
(or its mutants mimicking phosphorylation) in buffer H
The samples were incubated for 20 min at 25°C and
polymerization was initiated by addition of buffer S (1/5 of
the sample volume) After mixing, the samples were
subjected to ultracentrifugation (1 h, 100 000 g), and the
protein composition of both supernatant and pellet was
determined by quantitative SDS gel electrophoresis The
quantity of actin in the pellet was plotted against the total
quantity of actin in the sample
Results
Heat denaturation of actin
Before starting the investigation of the HSP25–actin
inter-action it was desirable to characterize the properties of intact
and heated actin We were mainly interested in the
irrever-sible changes in the structure of G-actin that were induced by
heating Therefore in all experiments the samples of actin in
G-buffer were heated for 1 h at the appropriate temperature
and after cooling different properties of actin were measured
at 25°C In the first series of experiments we analyzed the
effect of heating on intrinsic W fluorescence of actin After
recording corrected spectra of fluorescence, parameter A
(equal to I320/I365) was plotted against the temperature of
incubation (Fig 1) Parameter A was 2.65 for intact actin
and decreased to 1.3for actin heated at temperatures higher
than 55°C Further increase of the temperature up to 80 °C had no effect on parameter A The data presented agree with the results of Kuznetsova et al [6] and Turoverov et al [5] who showed that removal of Ca2+or addition of urea up to
4Mresults in partial unfolding of actin that is accompanied
by a decrease of parameter A from 2.5–2.6 to 1.2–1.3 Even prolonged heating at 80°C does not induce complete unfolding, which is achieved only in the presence of 6–8M urea or 4–5Mguanidine hydrochloride, and is characterized
by parameter A equal to 0.4 [5,6]
As already mentioned, for many proteins partial unfold-ing is accompanied by self-aggregation We used size-exclusion chromatography to follow heat-induced aggregation of actin Under the conditions used, intact actin was eluted as a symmetrical peak with the maximum at 8.65 mL (Fig 2) Heating of actin for 1 h at 43, 50 or 60°C resulted in the appearance of an additional peak at 7.65 mL
on the elution profile Increase of the temperature of incubation was accompanied by the simultaneous increase
of the peak eluted at 7.65 mL and decrease of the peak eluted
at 8.65 mL We were unable to determine the exact molecular mass of the protein species eluted in these two peaks because at low ionic strength of buffer G strongly acidic actin was partially excluded from Superdex 200 However, ovalbumin, having pI and molecular mass similar
to that of actin, was eluted as a symmetrical peak with a maximum at 8.65 mL (data not shown) Therefore, we may conclude that intact, unheated actin is eluted as a monomer Heating induces self-aggregation and the formation of actin oligomers that are eluted from the Superdex 200 column at 7.65 mL
Self-aggregation of actin can be due to the exposing of hydrophobic regions upon heating To check this suggestion
we analyzed the interaction of the hydrophobic probe ANS with intact and heated actin (Fig 3) Under the conditions used, free ANS had a rather low intensity of fluorescence with a broad maximum at 510–530 nm (Fig 3, curve 1) The addition of intact actin induced only a small increase in the fluorescence at 440–500 nm (Fig 3, curve 2) Much
Fig 1 Effect of heating on the parameter A (I 320 /I 365 ) of actin Actin
(15 l M ) in buffer G was heated at different temperatures for 1 h The
intensity of fluorescence at 320 nm (I 320 ) and 365 nm (I 360 ) excited at
297 nm was used for the determination of parameter A.
Fig 2 Size-exclusion chromatography of actin on a Superdex 200 col-umn Actin (15 l M ) was kept at 4 °C (1) or heated for 1 h at 43(2), 50 (3) or 60 °C (4) in buffer G After cooling, 90 lL of sample were loaded on the Superdex 200 column and eluted with buffer G at the rate of 0.5 mLÆmin)1 For clearance the elution profiles are shifted from each other by 8 mAu.
Trang 4higher fluorescence was observed if ANS was mixed with
actin heated at 43°C (Fig 3, curve 3), and in this case the
maximum fluorescence was shifted to 490–510 nm
The highest intensity of fluorescence, with a maximum at
465–475 nm, was observed when ANS was mixed with actin
heated at 60 or 80°C (Fig 3, curves 4,5)
The data presented indicate that heating is accompanied
by the change in the hydrophobic environment of W,
exposure of hydrophobic regions and self-aggregation of
actin Heating at any temperature higher than 55°C
induced similar effects on actin structure
Actin is fairly stable to trypsinolysis The main fragment
accumulated during incubation had an apparent molecular
mass of 33 kDa (Fig 4A) but even after prolonged
incu-bation with trypsin more than 40% of the actin remained
uncleaved (Fig 4B, curve 1) If actin heated at 60°C was
subjected to trypsinolysis under the same conditions, the
band of actin disappeared in the first 15–30 s (Fig 4B, curve
2) and a number of faint bands with different molecular
masses were accumulated in the incubation mixture
(Fig 4A) These results agree with other data in the
literature [6] and indicate that heat-induced unfolding
increased the susceptibility of actin to trypsinolysis In
contrast, if actin was heated for 1 h at 43°C, it becomes
more resistant to trypsinolysis than intact actin (Fig 4B,
curve 3) Three peptide bands with apparent molecular mass
of 29, 31 and 33 kDa were accumulated during the early
stages of trypsinolysis of actin heated at 43°C (Fig 4A)
During the late stages of trypsinolysis, predominantly one
major band with an apparent molecular mass of 33 kDa
was detected in the incubation mixture (Fig 4A) The data
for limited trypsinolysis indicate that after heating at 43°C
actin acquires a structure different from that of the intact
and thermally inactivated protein In this state, the
envi-ronment of W residues remains comparatively hydrophobic,
actin weakly interacts with ANS and only a small portion of
protein forms high molecular mass aggregates
Thermal unfolding can also affect actin polymerization
To investigate this possibility we heated actin containing
10% of pyrene-labeled protein for 1 h at 43and 60°C and analyzed polymerization and aggregation induced by the addition of KCl and MgCl2 (Fig 5) As expected, salt addition induced rapid polymerization of control unheated actin (Fig 5A, curve 1), that was accompanied by 14–16-fold increase in the fluorescence of the pyrene label attached
to C373 Actin heated at 43°C was also able to polymerize, although the rate and the extent of polymerization was slightly lower than in the case of unheated actin (Fig 5A, curve 2) Heating at 60°C completely prevented any increase in fluorescence induced by salt addition (Fig 5A, curve 3) This indicates that actin heated at temperatures higher than 60°C was not able to polymerize Addition of salt can induce not only polymerization of intact actin, but can also promote aggregation of partially unfolded actin [5] This process was followed by light scattering (Fig 5B)
Fig 3 Fluorescence spectra of 140 l M ANS in the absence of added
proteins (1) and in the presence of 2.25 l M of intact actin (2) or actin
heated for 1 h at 43 (3), 60 (4) or 80 °C (5) All measurements were
performed in G buffer at 25 °C and the fluorescence was excited at
350 nm.
Fig 4 Proteolytic fragmentation of intact actin and actin heated at 43
or 60 °C (A) Limited proteolysis of actin by trypsin (weight ratio actin/N-tosyl- L -phenylalanine chloromethyl ketone–trypsin, 50 : 1) Actin (15 l M ) in G-buffer was kept at 4 °C (gels marked 4 °C) or heated at 60 or 43 °C for 1 h (gels marked 60 °C and 43 °C, respect-ively) After dilution up to 2 l M , actin was subjected to trypsinolysis that was performed under identical conditions at 25 °C Aliquots were removed at the time indicated and the reaction was stopped by the addition of phenylmethanesulfonyl fluoride, followed by boiling in the sample buffer The samples obtained were subjected to quantitative SDS gel electrophoresis (B) Time course of the disappearance of the band of unhydrolyzed actin during limited trypsinolysis of intact actin (1) or actin preincubated at 60 °C (2) or 43 °C (3 ).
Trang 5Addition of the salts induced slow increase of the light
scattering of actin heated at 60°C (Fig 5B, curve 3) As this
sample of actin was unable to polymerize (Fig 5A), the
observed increase in light scattering can be due only to the
slow aggregation of partially folded protein In contrast,
addition of the salts to intact actin or to actin heated at
43°C induced rapid increase of the light scattering that
coincides in time with polymerization (Fig 5, curves 1,2)
Summing up, we may conclude that heating of actin at
43°C induced rather mild changes in the structure of actin
The samples of actin heated at 43°C have a more
hydrophilic environment of W residues than the intact
protein, weakly bind ANS, contain rather small quantities
of high molecular mass aggregates, are more resistant to
trypsinolysis than intact actin and retain the ability to
polymerize Actin samples heated at temperatures higher
than 55°C are partially unfolded, strongly interact with
ANS, are highly susceptible to trypsinolysis, contain high
quantities of high molecular mass aggregates and are unable
to polymerize We assumed that intact and heated actin would interact differently with HSP25 so we analyzed the effect of HSP25 on the polymerization of intact actin and aggregation of heated actin
Interaction of HSP25 with intact actin
In the first series of experiments we analyzed the effect of HSP25 on the apparent critical concentration and initial rate of actin polymerization (Fig 6A) In this case, G-actin containing 10% of pyrene-labeled protein was preincubated with HSP25 or its 3D mutant, and polymerization was initiated by the simultaneous addition of actin nuclei and salts Neither type of HSP25 had any significant effect on the critical concentration of actin (Fig 6A) Indeed the critical concentration of actin was equal to 0.25 ± 0.07; 0.21 ± 0.05, and 0.15 ± 0.06 lMin the absence of HSP25, and in the presence of the wild type HSP25 and its 3D mutant, respectively At the same time, both wild type and especially 3D mutant of HSP25 significantly decreased the initial rate of actin polymerization The extent of polymeri-zation was followed by means of fluorescence spectroscopy and ultracentrifugation In the first case, after preincubation with HSP25, polymerization of actin was initiated by salt addition As can be seen from Fig 6B, during the first
40 min the wild type recombinant HSP25 hardly affects actin polymerization, whereas the 3D mutant decreased the extent of polymerization by 20–25% Similar results were obtained by ultracentrifugation In this case, different quantities of actin were preincubated with HSP25 or its mutant, and immediately after salt addition the samples were subjected to ultracentrifugation at 100 000 g for 1 h The quantity of actin in the pellet was plotted against the total quantity of actin in the probe (Fig 6C) The wild type HSP25 has little effect on the extent of polymerization of intact actin, whereas both 2D and 3D mutants decreased the quantity of polymerized actin in the pellet It is worthwhile
to mention that the mutants of HSP25 affect the rate but not the maximal extent of intact actin polymerization If the samples before ultracentrifugation were incubated for 4 h at room temperature, the quantity of polymerized actin in the pellet was almost independent of the presence of HSP25 or its mutants (data not presented)
Summing up, we may conclude that the recombinant wild type HSP25 has almost no effect on the polymerization of intact actin 2D and 3D mutants mimic the phosphorylation
of HSP25 by MAPKAP-2 kinase and form oligomers with smaller molecular mass than the wild type HSP25 [20] These mutants decrease the initial rate of actin polymeriza-tion without affecting its critical concentrapolymeriza-tion (Fig 6A) Decrease of the initial rate results in decreased extent of polymerisation, measured during the first 40–60 min after initiation of polymerization (Figs 6B,C), but does not affect the final maximal extent of polymerisation, measured 4 h after initiation of polymerization
Effect of HSP25 on polymerization of actin heated
at 43 °C
As mentioned earlier, mild heating at 43°C results in some changes in actin structure, but this treatment does not
Fig 5 Effect of heating on the kinetics of polymerization (A) and
salt-induced increase of the light scattering (B) of actin Actin (15 l M )
containing 10% or pyrene-labeled actin in buffer G were incubated at 4
(1), 43(2), or 60 °C (3) for 1 h After cooling and diluting with buffer
G, so that the concentration of actin becomes equal to 10 l M , the
reaction was started by the addition of KCl and MgCl 2 up to the final
concentrations 50 and 2 m M , respectively Polymerization was
fol-lowed by an increase in fluorescence at 407 nm excited at 366 nm.
Light scattering (I/I o ) was followed at 560 nm.
Trang 6completely prevent actin polymerization In addition, this
heating regime resembles that occurring in vivo under
certain physiological conditions In preliminary experiments
we have shown that the increase of the time of heating at
43°C is accompanied by exponential decrease of parameter
A (I320/I365), that tends to a limit equal to 1.8 (data not
shown) This value is significantly higher than the
corres-ponding parameter obtained after heating at temperatures
higher than 55°C, which is equal to 1.3(Fig 1) Actin
(containing 10% of pyrene-labeled protein) was heated at
43°C for different periods of time, and samples of actin
having different parameter A (2.2–2.7) were analyzed for
their ability to polymerize In these particular experiments
we measured the initial rate of polymerization that was
initiated by simultaneous addition of actin nuclei and salts
In good agreement with earlier presented results, we found
that the wild type HSP25 has almost no effect on the initial
rate of polymerization of intact actin (A > 2.55), whereas
the 3D mutant of HSP25 slightly decreased the initial rate of
polymerization (15–25%, Fig 7) The effect of HSP25
became negligible when parameter A of actin was close to
2.4 Increase of the time of heating at 43°C leading to
decrease of parameter A up to 2.2–2.3was accompanied by
further decrease of the initial rate of polymerization When
parameter A was in the range of 2.20–2.35 the wild type
HSP25 had no effect on the initial rate of
polymeri-zation, whereas its 3D mutant activated the initial rate of
polymerization by 25–35% (Fig 7) This means that
depending on the state of actin the 3D mutant of HSP25
can either increase (if A < 2.4) or decrease (if A > 2.4) the
initial rate of polymerization
We also analyzed the effect of HSP25 and its mutants
on the extent of heated actin polymerization In this case,
actin (containing 10% of pyrene-labeled protein) was heated at 43°C until parameter A reached 2.2 Polymeri-zation was initiated by salt addition and was followed for
1 h Under the conditions used, wild type HSP25 did not affect actin polymerization, whereas the 3D mutant of HSP25 increased the rate of polymerization without affecting the maximal extent of polymerization (data not presented)
We suppose that this effect of the 3D mutant of HSP25
on the initial rate of actin polymerization can be explained
by the prevention of nonspecific aggregation of partially denatured actin that can trap intact actin The 3D mutant prevents aggregation and by this means increases the quantity of available actin monomers and therefore increa-ses the initial rate of polymerization
Fig 6 Effect of recombinant wild type HSP25 and its mutants
mimicking phosphorylation on polymerization of intact actin (A) Effect
of HSP25 and its 3D mutant on the kinetics of actin polymerization.
Samples containing 1–4 l M of actin (10% of pyrene-labeled protein)
were incubated for 5 min in the absence (1) or in the presence of 6 l M
of HSP25 (2) or its 3D mutant (3) Polymerization was initiated by the
simultaneous addition of actin nuclei, KCl and MgCl 2 up to the final
concentrations 0.5 l M , 50 m M and 2 m M , respectively The results
shown are representative of three experiments with three different
purified actin samples, and triplicate measurements of each
experi-mental point If not shown the error bars are smaller than the size
of symbol (B) Influence of HSP25 and its 3D mutant on the extent of
actin polymerization Samples containing 4 l M of actin (10% of
pyrene-labeled protein) were preincubated for 5 min at 25 °C in the
absence (1) or in the presence of wild type HSP25 (2) or its 3D mutant
(3) Polymerization was initiated by addition of KCl and MgCl 2 up to
50 and 2 m M , respectively The results shown are representative of
three independent experiments (C) Effect of HSP25 and its mutants on
actin polymerization measured by ultracentrifugation Samples
con-taining 0.12–0.48 nmol of unheated unmodified actin in 60 lL of
G-buffer were incubated for 5 min at 25 °C in the absence (1) or in the
presence of 10 l M of wild type HSP25 (2) or 10 l M of its 2D (3 ) or 3 D
(4) mutants One fifth of the volume of buffer S was added and after
mixing the samples were immediately subjected to ultracentrifugation.
The quantity of actin in the pellet is plotted against the total quantity of
actin in the sample The results are representative of five experiments
with four different preparations of actin.
Trang 7Effect of HSP25 on the aggregation of partially
unfolded thermally inactivated actin
As already shown, heating of actin at a temperature higher
than 55°C completely prevents its polymerization
(Fig 5A) After this type of heating actin formed small
oligomers (Fig 2), and an increase in ionic strength induced
further aggregation (Fig 5B) We analyzed the ability of
HSP25 and its mutants to prevent salt-induced aggregation
of partially unfolded actin heated at 60°C In the first series
of experiments actin was heated at 60°C for 1 h, cooled,
mixed with different species of HSP25, and after the
addition of salt subjected to ultracentrifugation Under
these conditions only aggregated actin was sedimented
Therefore, by measuring the quantity of actin in the pellet
we were able to estimate the chaperone activity of HSP25
and its mutants In the absence of HSP25 about 90% of
heated actin was found in the pellet after ultracentrifugation
(Fig 8A) Addition of the wild type recombinant HSP25
reduced the quantity of sedimented actin up to 60% Point
mutants mimicking phosphorylation of HSP25 by
MAPKAP-2 kinase were very effective in preventing
salt-induced aggregation of partially unfolded actin (Fig 8A)
Only 10–20% of the total quantity of actin presented in the
sample was precipitated in the presence of the 1D, 2D or 3D
mutant of HSP25 The data presented indicate that HSP25,
and especially its mutants mimicking phosphorylation,
effectively prevent salt-induced aggregation of partially
folded actin
To obtain more detailed information on the interaction of
HSP25 with partially unfolded actin, we analyzed the
effect of different quantities of HSP25 on the salt-induced
aggregation of actin In this case actin was either kept on ice
or heated at 60°C for 1 h The samples of actin were incubated with different quantities of HSP25 or its 3D mutant Polymerization (in the case of intact, unheated
Fig 8 Effect of HSP25 on the salt-induced aggregation of partially unfolded thermally inactivated actin (A) The influence of different HSP25 species on the salt-induced aggregation of thermally inactivated actin measured by ultracentifugation Actin (15 l M ) in buffer G was heated for 1 h at 60 °C After cooling and dilution to 2 l M , actin was mixed with different species of HSP25 (final concentration 4 l M ) and incubated for 20 min at 25 °C Aggregation was initiated by the addition of 1/5 of the sample volume of buffer S Samples were incu-bated for 1 h at 25 °C and subjected to ultracentrifugation (1 h,
100 000 g) Actin in the pellet was determined by quantitative SDS gel electrophoresis C, control without HSP25; WT, wild type recombin-ant HSP25; 1D, 2D and 3D, HSP25 mutrecombin-ants with replacement of one, two or three S residues (S15, S77 and S81) by D The results are representative of four independent experiments with three different preparations of actin and triplicate measurements of each experimental point (B) Concentration-dependent effect of the wild type HSP25 and its 3D mutant on polymerization of intact actin (1,2) and salt-induced aggregation of actin heated at 60 °C (3,4) 15 l M actin in buffer G was kept at 4 °C (1,2) or at 60 °C (3,4) for 1 h After cooling and dilution
to 2 l M , actin was mixed with different quantities of the wild type recombinant HSP25 (1,3) or its 3D mutant (2,4) and incubated for
20 min One fifth of the volume of buffer S was added and after incubation for 2 h at 25 °C the samples were subjected to ultracentri-fugation (1 h, 100 000 g) Actin in the pellet (percentage of the total actin in the sample) was determined by quantitative SDS gel electro-phoresis The results are representative of six independent experiments with three different actin samples.
Fig 7 Dependence of the rate of polymerization upon parameter A of
actin Actin (15 l M , containing 10% pyrene-labeled protein) in buffer
G was heated for 0–90 min at 43 °C and parameter A (I 320 /I 365 ) was
recorded The samples were cooled, diluted to a final actin
concen-tration of 4 l M and incubated in the absence (1) or in the presence of
6 l M of the wild type HSP25 (2) or its 3D mutant (3) for 5 min at
25 °C Polymerization was initiated by the simultaneous addition of
actin nuclei, KCl and MgCl 2 at final concentrations of 0.2 l M , 50 m M
and 2 m M , respectively The initial rate of polymerization was
deter-mined during the first 2 min of the reaction by increase in fluorescence
at 407 nm excited at 366 nm The results are representative of two
independent experiments with two different actin samples, with
trip-licate measurements of each experimental point.
Trang 8actin) or aggregation (in the case of heated actin) was
initiated by salt addition and was allowed to proceed for 2 h
at 25°C After ultracentrifugation the quantity of actin in
the pellet was determined by quantitative SDS gel
electro-phoresis Actin in the pellet (as a percentage of actin in the
total sample) was plotted against the HSP25 concentration
(Fig 8B) As described earlier, neither the wild type HSP25
nor its 3D mutant affected the final extent of polymerization
of intact unheated actin (Fig 8B, curves 1,2) Addition of
the wild type HSP25 reduced the quantity of aggregated
partially folded actin heated at 60°C (Fig 8B, curve 3)
However, even at a very high concentrations the wild type
HSP25 was not able to completely prevent aggregation of
partially unfolded actin In contrast, even much lower
quantities of the 3D mutant of HSP25 completely prevented
aggregation of actin heated at 60°C (Fig 8B, curve 4)
We may conclude that HSP25 effectively prevents
aggregation of partially folded actin Phosphorylation (or
mutations mimicking phosphorylation) increased the
chap-erone effect of HSP25 so that it becomes able to completely
prevent salt-induced aggregation of heated actin
Discussion
Heating induces significant changes in the structure of
G-actin Bertazzon et al [4] suggested that upon heating
native (N) actin is irreversibly converted to denatured (D)
(or partially folded) actin This first step of unfolding is
enthalpic and involves the denaturation of two independent
domains of approximately 11 and 31 kDa Addition of a
high concentration of guanidine hydrochloride or urea can
reversibly convert D (or partially folded) actin to the
completely unfolded (U) state This second step of unfolding
is reversible and purely enthropic Thus, the mechanism of
unfolding of G-actin was described by a simple scheme:
N-actin ! D-actin Ð U-actin
However, the first transition from N-actin to D-actin was
not a one-step process It has been shown [4] that the
calorimetric melting curve of actin was asymmetric, and that
the excess heat capacity curve of G-actin can be fitted into
two independent intermediate steps with Tm of 52 and
57°C, respectively Therefore, the scheme of actin unfolding
is more complex and can be represented in the form:
N-actin Ð D1-actin ! D2-actin Ð U-actin
where D1- and D2-actins represent two states of denatured
actin and the reversibility or irreversibility of transitions
between N, D1and D2are unknown Although D1and D2
states of actin were postulated, their properties and even
their existence was not confirmed experimentally
We propose that heating of actin at 40–45°C leads to a
slow transition from N-actin to D1-actin The D1 state of
actin is different from both native actin and from the
well-characterized denatured (or D2) state of actin Heating
under these mild physiologically relevant conditions results
in the accumulation of the protein with only a moderate
change in the W environment (Fig 1), diminished ability to
interact with ANS (Fig 3) and a small quantity of high
molecular mass aggregates (Fig 2) In addition, actin in this
state was more resistant to proteolysis than intact protein
and in this respect was completely different from denatured actin (Fig 4) Moreover, even after prolonged heating at
43°C actin retained its ability to polymerize (Figs 5 and 7) This property was completely lost by denatured actin (Fig 5) We may suppose that heating at 43°C induces unfolding of a small domain proposed by Bertazzon et al [4] At present it is difficult to locate this domain exactly in the crystallographic structure of actin However, it is known that trypsin predominantly cleaves actin at residues 62 and
68, forming fragments with apparent molecular masses of
33 and 9 kDa [26] Heating at 43°C partially protects actin from trypsinolysis (Fig 4) This fact may indicate that the above-mentioned small domain with molecular mass 9–11 kDa may include subdomains 1 and 2 of actin After heating at 43°C, actin turns into a state that is different from both the intact and denatured conformations This intermediate state may be of importance because under physiological conditions the body temperature of warm-blooded animals can rise up to 40–42°C
The denatured (D or D2) state of actin was analyzed in detail [4–6] This state is characterized by a very hydrophilic environment of W residues, exposed hydrophobic sites interacting with ANS and increased susceptibility to proteolysis Exposure of hydrophobic sites increases the probability of self-aggregation and therefore partially folded actin tends to aggregate Self-aggregation may be the reason for the irreversibility of transition from the native to the partially folded state [5] Transition of N-actin to the D (or
D2) state was observed after heating at temperatures higher than 55°C, after the removal of calcium or after the addition of low concentrations of urea or guanidine hydrochloride [5,6] Accumulation of denatured actin can
be dangerous for the cell as it tends to form high molecular mass aggregates
Let us analyze the interaction of HSP25 with the N-, D1 -and D2-forms of actin Miron et al [11,12] claimed that HSP25 effectively inhibits polymerization of N-actin by increasing its critical concentration Similar results were obtained by Benndorf et al [13], but only with the monomeric unphosphorylated form of HSP25 These results were obtained with HSP25 purified from avian or human tissues In both cases the starting steps of purifica-tion of HSP25 were performed according to Feramisco and Burridge [27] and an initial crude mixture contained a number of different proteins with molecular mass in the range of 20–80 kDa that were able to inhibit polymerization
of actin [28] The ability of HSP25 to inhibit actin polymerization was diminished or completely deteriorated
if the protein was purified on a hydroxyapatite column under special conditions [12]; recombinant HSP25 was also ineffective in the inhibition of actin polymerization [13,18] All of these facts can be explained by the suggestion that HSP25 purified from animal tissues contained trace amounts of a highly effective inhibitor of actin polymeriza-tion having a molecular mass of monomers close to that of HSP25 Cofilin, which has an apparent molecular mass of 20–22 kDa, could be one of the candidates for this role At substoichiometric concentrations cofilin inhibits actin poly-merization, induces depolymerization of actin and is able
to form oligomers with molecular mass in the range 22–100 kDa [29] As heat shock is accompanied by the simultaneous translocation of both cofilin and HSP25 to the
Trang 9nucleus [30], we may suppose that these two proteins can
interact with each other
Analyzing the interaction of native actin with HSP25
purified from avian tissues and with recombinant protein,
we found that the wild type HSP25 has little effect on the
rate or extent of actin polymerization At the same time, the
3D mutant of HSP25 slightly decreased the initial rate of
actin polymerization (Fig 6A) without affecting the
maxi-mal extent of polymerization Mutations mimicking
phos-phorylation induce partial dissociation of high molecular
mass oligomers of HSP25 and accumulation of dimers and
tetramers [20] Low molecular mass oligomers of HSP25
may interact with G-actin and in this way decrease the initial
rate of polymerization However, this interaction seems to
be weak and therefore HSP25 (or its 3D mutant) does not
affect the final extent of polymerization
Heating at 40–45°C leads to transition of native actin to
the D1form and is accompanied by a decrease in the rate
and extent of actin polymerization (Figs 5 and 7) This
could be due to the fact that at this heating regime some
actin becomes aggregated (Fig 2) and therefore is excluded
from polymerization The wild type HSP25 has little effect
on the polymerization of D1-actin (Fig 7), whereas the 3D
mutant of HSP25 increases the rate of polymerization
without affecting its maximal extent (Fig 7) This effect of
the 3D mutant may be explained by preventing aggregation
of actin leading to an increase in the concentration of
G-actin available for polymerization
Conversion of native actin to the D2-form completely
prevents polymerization (Fig 5A) Denatured actin
con-tains exposed hydrophobic sites (Fig 3) and tends to
aggregate upon addition of salt (Fig 5B) HSP25 prevents
salt-induced aggregation of denatured actin (Fig 8), and
HSP25 mutants mimicking phosphorylation possessed
higher chaperone activity than the wild type HSP25 The
chaperone activity of HSP25 strongly depends on the nature
of the target protein and on the state of HSP25
phosphory-lation For example, phosphorylation (or mutations
mimicking phosphorylation) decreases the chaperone
activity of human or murine HSP27 with citrate synthase,
insulin [16] and avian HSP25 with a-lactalbumin [20] At the
same time, phoshorylation (or mutations mimicking
phos-phorylation) increases the chaperone activity of HSP25 with
alcohol dehydrogenase [20] The same effect was observed in
the case of denatured actin Different types of stress induce
phosphorylation of HSP25 [10], thus converting it to the
form that effectively prevents aggregation of actin, and in
this way protect the cell from accumulation of large
quantities of insoluble material
Summing up we may conclude that depending on the
conditions HSP25 has multiple effects on polymerization
and aggregation of G-actin Monomers or low molecular
mass oligomers of HSP25 weakly interact with G-actin and
thereby slightly inhibit the initial rate of polymerization of
intact actin The HSP25 mutants, mimicking
phosphoryla-tion, stabilize partially denatured molecules of G-actin,
prevent formation of high molecular mass aggregates and in
this way increase the initial rate of polymerization of
partially denatured actin Finally HSP25, and especially its
mutants, effectively prevent salt-induced aggregation of
denatured actin, thereby protecting the cell from the
accumulation of insoluble proteins
Acknowledgements
The authors are grateful to Dr Alim S Seit-Nebi (V.A Engelhardt Institute of Molecular Biology, Russian Academy of Sciences) for the cloning and expression of recombinant forms of HSP25 and its mutants This investigation was supported by Russian Foundation for Basic Research and by the Wellcome Trust.
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