Variant actin proteins overexpressed in mamma-lian cell lines fail to incorporate into actin filaments in a manner correla-ting with the degree of misfolding observed in the cell-free ass
Trang 1cardiomyopathy cause inefficient protein folding and
perturbed filament formation
Søren Vang1, Thomas J Corydon2, Anders D Børglum2, Melissa D Scott3, Judith Frydman3, Jens Mogensen4, Niels Gregersen1and Peter Bross1
1 Research Unit for Molecular Medicine, Aarhus University Hospital and Faculty of Health Sciences, Denmark
2 Institute of Human Genetics, University of Aarhus, Denmark
3 Department of Biological Sciences and BioX Program, Stanford University, CA, USA
4 Department of Cardiology, Aarhus University Hospital, Denmark
Hypertrophic cardiomyopathy (HCM) is inherited by
autosomal dominant transmission with a prevalence of
approximately 1 : 500 The condition is defined by the
presence of unexplained myocardial hypertrophy and
myocardial histology is characterized by myocyte
dis-array [1] HCM may be caused by missense mutations
in any one of eight known sarcomeric genes These
genes encode proteins of the cardiac sarcomere,
com-ponents of thick and thin filaments with contractile,
structural or regulatory functions (thick filament:
MYH7, MYL3, MYL2, MYBPC3; thin filament:
ACTC, TNNT2, TNNI3, TPM1 [2]) It has been hypo-thesized that the mutant protein (poisonous peptide) causes a dominant negative inhibition of the protein produced from the normal allele, impairing the sarco-meric contractile performance [3] This is thought to eventually lead to a compensatory hypertrophy of the heart [4] A few mutations in the MYBPC3 gene are believed to give rise to haplo-insufficiency [5]
Dilated cardiomyopathy (DCM) is the most com-mon cause of heart failure and cardiac transplantation
in the young DCM is usually transmitted in a
domin-Keywords
a-cardiac actin; chaperone; dilated
cardiomyopathy; hypertrophic
cardiomyopathy; protein folding
Correspondence
S Vang, Research Unit for Molecular
Medicine, Aarhus University Hospital,
Skejby Sygehus, Brendstrupgaardsvej,
DK-8200 A ˚ rhus N, Denmark
Fax: +45 89496018
Tel: +45 89495150
E-mail: vang@ki.au.dk
(Received 12 January 2005, revised 24
February 2005, accepted 25 February 2005)
doi:10.1111/j.1742-4658.2005.04630.x
Hypertrophic cardiomyopathy (HCM) and dilated cardiomyopathy (DCM) are the most common hereditary cardiac conditions Both are frequent causes of sudden death and are often associated with an adverse disease course Alpha-cardiac actin is one of the disease genes where different mis-sense mutations have been found to cause either HCM or DCM We have tested the hypothesis that the protein-folding pathway plays a role in dis-ease development for two actin variants associated with DCM and six asso-ciated with HCM Based on a cell-free coupled translation assay the actin variants could be graded by their tendency to associate with the chaperonin TCP-1 ring complex⁄ chaperonin containing TCP-1 (TRiC ⁄ CCT) as well as their propensity to acquire their native conformation Some variant pro-teins are completely stalled in a complex with TRiC and fail to fold into mature globular actin and some appear to fold as efficiently as the wild-type protein A fraction of the translated polypeptide became ubiquitinated and detergent insoluble Variant actin proteins overexpressed in mamma-lian cell lines fail to incorporate into actin filaments in a manner correla-ting with the degree of misfolding observed in the cell-free assay; ranging from incorporation comparable to wild-type actin to little or no incorpor-ation We propose that effects of mutations on folding and fiber assembly may play a role in the molecular disease mechanism
Abbreviations
ACTC, a-cardiac actin gene; CCT, chaperonin containing TCP-1; DCM, dilated cardiomyopathy; DMSO, dimethylsulfoxide; HCM, hypertrophic cardiomyopathy; TRiC, TCP-1 ring complex; VLCAD, very-long chain acyl-CoA dehydrogenase.
Trang 2ant fashion; however, recessive, X-linked and
mito-chondrial inheritances have also been reported More
than 20 disease genes have been reported so far,
enco-ding a wide variety of proteins expressed in cardiac
myocytes [6] Recently DCM mutations in several
sar-comeric genes have been identified [2]
The a-cardiac actin gene (ACTC) was the first gene
identified to harbor both HCM and DCM mutations,
with six mutations leading to HCM and two mutations
leading to DCM (Fig 1) [7–10] Based on both the
clinical findings in the patients carrying the different
actin mutations and the putative protein–protein
inter-actions deduced from the X-ray structures, the
pre-vailing hypothesis proposes that while actin mutant
variants are incorporated into thin filaments in both
cases, in HCM an altered structure of the region
inter-acting with myosin impairs force generation, whereas
in DCM disturbance of the interactions with proteins
of the Z-disk impair force transmission from the
sarco-mere to the surrounding syncytium [7,9,11]
Clinical diversity or ‘phenotypic heterogeneity’ is a
hallmark of HCM and implies that factors other than
the underlying major gene defect modify the impact of
the mutant gene at the cellular and clinical level An
increasing interest in the modulating factors that result
in the lack of correlation between genotype and
pheno-type has evolved in recent years [12,13] These
modify-ing factors may be genetic, in which case a second
gene may regulate the expression of the primary defect
differently in different patients The variation may also
be due to environmental factors such as age, diet,
exer-cise, pharmaceutical agents and the efficiency of the cellular protein folding machinery
The protein folding machinery of the cell is known
to respond to cellular stress and changes in physico-chemical conditions [14] The mechanism by which chaperones influence other cellular processes include increasing de novo folding efficiency, assisting refold-ing of proteins denatured by stress, and modulatrefold-ing the balance between folding and degradation of mis-folded proteins [15,16] It is the quality control func-tion of chaperones that we are most interested in, because molecular chaperones and proteases may act
in conjunction to determine the fate of the variant protein [17–19] Three pathological scenarios can be pictured Firstly, decreasing the negative dominance
of a poisonous peptide, thereby suppressing the sever-ity of the disease; secondly, complete or nearly complete degradation of the mutant allele product resulting in haploinsufficiency as the disease mechan-ism; and thirdly, the inability to eliminate misfolded proteins leading to accumulation of cell toxic aggre-gates In all three cases degradation and folding path-ways of the gene products are likely to be important factors
Studies from several groups indicate that multiple chaperone complexes work to assist the folding of actin [20–23] During actin synthesis, prefoldin binds and stabilizes the incompletely folded nascent polypep-tide and releases it for further folding to the cytosolic chaperonin, referred to as TCP-1 ring complex (TRiC)
or chaperonin containing TCP-1 (CCT) It has previ-ously been shown that variations in a-skeletal actin impair folding and polymerization [24], and that actin variants that are unable to fold are degraded by the ubiquitin-proteasome pathway [20]
This study has investigated the folding and stability
of variant a-cardiac actins leading to HCM and DCM Using a cell-free system of coupled in vitro transcrip-tion⁄ translation, we have studied the folding of the actin variant proteins and their different interaction affinities for chaperones We have also used immunostaining and confocal laser scanning microscopy of transfected cells
to study the ability of the variant actin protein to incor-porate into filaments in the cytoskeleton
Results Some mutant actin proteins display perturbed interaction kinetics with the TRiC chaperonin leading to delayed folding
The folding pathway of wild-type and mutant actin polypeptides was studied using a cell-free
transcrip-Met 305
Arg 312
Ala 331
Ala 295
Glu 99
Tyr 166 Fig 1 Missense variations in the actin structure Ribbon
represen-tation of the actin monomer based on the crystal structure of rabbit
b-actin (PDB accession number 1ATN) The residues mutated in
HCM are colored in blue and the residues mutated in DCM are
white Figure prepared with the MOLMOL program [44] Amino acid
substitutions shown in bold lead to DCM.
Trang 3tion⁄ translation system Full length wild-type actin
and eight variant actin cDNA constructs containing
disease-causing mutations were transcribed and
trans-lated in the presence of [35S]methionine at 37C
(Table 1) Samples were taken at different time points
up to 60 min and analyzed by both SDS⁄ PAGE and
native PAGE Separation by SDS⁄ PAGE and
visual-ization by phosphorimaging showed a single band of
42 kDa with similar intensity for all actin variants,
indicating homogeneous production of the nascent
polypeptide for all constructs (not shown) In this and
other experiments, we observed the transcription⁄
translation system to have a maximum activity at
t¼ 0–20 min
In the initial experiment cDNAs encoding Rattus
norvegicuswild-type, human wild-type, six
HCM-caus-ing, and two DCM-causing a-cardiac actin mutations
were expressed (Fig 1) The rat actin has the same
sequence at the protein level but minor changes at the
DNA level, and thereby served as an alternative
wild-type control The native PAGE analysis showed that
the actin polypeptides are mainly found in three
com-plexes: one distinct band with slow mobility, a fainter
band with intermediate mobility, and a diffuse, rapidly
migrating band (Fig 2A) This pattern of complex
for-mation over time varied between wild-type and mutant
actin polypeptides Of all the mutants, the Arg312His
actin had the largest percentage of the protein retained
in the slowly migrating complex This retention did
not reduce over time, as seen for wild-type actin as a
consequence of the reduced protein production rate
Furthermore, the rapidly migrating smear was almost
absent in samples containing Arg312His actin This
phenomenon was temperature sensitive and less
pro-nounced at 30C (not shown) than at 37 C, a trait
often seen with proteins with a defective folding
path-way [25]
Previous studies have shown that the TRiC
chapero-nin binds actin in a high molecular mass complex, and
is involved in its biogenesis [21,22] The slowly
migra-ting band was shown through immunoblotmigra-ting of native PAGE gels to comigrate with the TCP-1 subunit
of TRiC (Fig 2B, left) Additionally, anti-(TCP-1) IgG was able to immunoprecipitate actin from reticulocyte lysate reactions in contrast to a negative control anti-body raised against very-long chain acyl-CoA dehy-drogenase (VLCAD) (Fig 2B, right) The reticulocyte extracts contain a large amount of hemoglobin, which forms nonspecific interactions with radioactive material and migrates as the intermediate band (Fig 2C,D) The broad, rapidly migrating smear corresponds to the folded actin monomer This was confirmed upon addi-tion of 2 lg DNase I to a pulse-chase reacaddi-tion of wild-type actin, followed by analysis of the mobility shift of this band by native PAGE and radiography (Fig 2C) DNase I binds the actin monomer and condenses the smear to a distinct band of characteristic mobility The smear was totally absent in the Arg312His actin lanes and therefore no actin–DNase I band appeared after addition of DNase I (not shown)
Phosphorimager scanning of the TRiC band as a fraction of the whole lane in Fig 2A gave a measure
of the percentage of actin bound to TRiC at different time points This shows the Arg312His and Glu99Lys variant proteins to have a sevenfold and threefold increased relative amount of actin–TRiC complex, respectively, when compared to wild-type after
60 min of translation at 37C (not shown) The results indicate that these mutant proteins have diffi-culty folding to assume their native form and remain TRiC-bound Actin molecules that fail to reach a native state upon release from TRiC may rebind for
a second round of folding, possibly by interaction with other chaperones as a transitional transferring step [20,26]
Pulse-chase labeling experiments with initial biosyn-thesis of [35S]actin at 37C for 30 min and subse-quent termination of translation with cycloheximide emphasized the difference in the folding kinetics between wild-type and the Arg312His variant
Table 1 Primers used for the mutagenesis of the a-cardiac actin (ACTC) gene The mutated bases are highlighted in bold.
Trang 4(Fig 2D) Reticulocyte lysate contains 20 lm hemin,
which is required for translation initiation in this
sys-tem Hemin has inhibitory effects on the
ubiquitin-proteasome degradation system [27] and was therefore
removed by a desalting step prior to the chase period
to asses the proteolytic capabilities of the extract
Samples were taken at different time points and transferred to native loading buffer in the presence of EDTA to inhibit the TRiC ATPase, and kept on ice until separation by native PAGE At t¼ 0 after the protein synthesis termination, the ratio between TRiC-bound actin and monomeric actin is
approxi-Wild T
ype Rat Wild type Glu99L
ys
Met305LeuAla331Pro
Tyr166Cys
y
TRiC
Hb
N-actin
10 20 40 60 10 20 40 60 10 20 40 60 10 20 40 60 10 20 40 60 10 20 40 60 10 20 40 60 10 20 40 60 10 20 40 60 10 20 40 60
A
P
IP
TRiC
B
0
TRiC
N-actin
180 120 90 60 120
90 60 30
D
C
Hb
*
Min.
Min.
Wild Type
N-actin Actin-DNaseI complex
Hb
0 2 4 0 2 4 Hours
- - - + + + 2µg DNase I
Fig 2 Some actin mutations have reduced folding efficiency and prolonged chaperone interaction In vitro protein biosynthesis carried out using coupled transcription ⁄ translation systems Wild-type and mutant full length cDNA in the pcDNA3.1 vector and radioactive labeling was performed using 10 lCiÆlL)1[ 35 S]methionine (A) The transcription ⁄ translation reactions were incubated at 37 C and put on ice in a native loading buffer containing 5 m M EDTA at the times indicated, until separation by native PAGE on 4–15% gel and visualization by radiography (B) Following in vitro translation of wild-type cDNA for 30 min protein synthesis was stopped with cycloheximide at a final concentration of
150 lgÆmL)1and processed by native PAGE One gel was processed by radiography (R) and one was visualized by immunoblotting (IB) with TRiC antibodies Additionally, the sample was precipitated by Sepharose beads coupled to either antibodies against TRiC or VLCAD for 1 h The washed pellet (P) or supernatant (S) was analyzed by SDS ⁄ PAGE and radiography (C) In vitro translation of wild-type cDNA for 30 min followed by termination of protein synthesis using cycloheximide DNase I (2 lL) was added to each sample and incubated at 37 C for the times indicated before separation by native PAGE and analyzed by radiography (D) Experimental pulse chase conditions as in (C) Samples were incubated 0–240 min at 37 C and analyzed by native PAGE and autoradiography An unidentified band is indicated by an asterisk N-actin, native actin; Hb, hemoglobin; R, radiography; IB, immunoblot; IP, immunoprecipitation Amino acid substitutions shown in bold lead
to DCM.
Trang 5mately sevenfold higher for Arg312His actin
com-pared to wild-type The strong Arg312His actin–TRiC
complex band persists for up to two hours This can
be explained by a tight actin–TRiC complex or by a
dynamic dissociation⁄ reassociation equilibrium shifted
towards formation of complex In addition, a new
actin-containing band of unknown nature is now
visi-ble in the wild-type lanes, denoted by an asterisk in
Fig 2D
Impaired folding leads to decreased formation
of compact native actin structures
To assess the fraction of folded to unfolded actin, the
wild-type and mutant actins were subjected to a mild
protease treatment The cDNAs were expressed in vitro
incorporating [35S]Met for 30 min followed by
termin-ation of protein transltermin-ation with cycloheximide The
labeled protein products were treated with
protein-ase K, separated by SDS⁄ PAGE and analyzed by
phosphorimaging Actin in its native monomer
confor-mation has a compact, protease-resistant structure [20]
and when subjected to mild digestion with
protein-ase K it is cleaved at the peptide bond between Met47
and Gly48, producing a globular C-terminal 35 kDa
fragment [28] The array of mutant proteins tested
exhibited different resistance to the digestion, all
pro-ducing the 35 kDa fragment and some being further
degraded This suggests that the variant actins
Glu99Lys, Pro164Ala, Met305Leu, Arg312His, and
Glu361Gly are stalled in a partly folded conformation
having reduced resistance to proteolytic degradation
(Fig 3A, upper panel)
We also assessed the fraction of folded to unfolded
actin by measuring the fraction of folded actin
mono-mer Only full length and correctly folded actin can
form a high affinity complex with DNase I [20] By
immobilizing DNase I on Sepharose beads, folded
actin can be pulled down from solution and quantified
by SDS⁄ PAGE [20] The assay was performed on the
samples mentioned above, and again the variants
Glu99Lys, Pro164Ala, Met305Leu, Arg312His and
Glu361Gly was shown to be strongly impaired in
reaching the native conformation (Fig 3A, lower
panel)
There was a clear correlation between the assay of
protease resistance and the assay of actin folding
(Fig 3B), indicating that the fraction of variant actin
that is not able to reach its native structure has a loose
structure and is accessible to nonspecific proteases
The variants not impaired in these assays appeared to
fold even better than the wild-type actin The reason
for this is unknown
Variant actin aggregates in a ubiquitin-dependent manner
Unfolded or misfolded proteins are often recognized and eliminated by the quality control system [18,19]
In the cytosol, substrates are marked by covalent modification with multiubiquitin chains, followed by degradation by the proteasome to protect the cell from exposed hydrophobic polypeptide segments [29] To test whether the misfolded variant actins are processed
by the proteasome, in vitro pulse-chase assays were performed for the wild-type and Arg312His mutant protein Wild-type and variant actin were expressed
in vitro at 37C for 30 min At 30 min, subsequent translation was terminated by the addition of cyclohexi-mide Degradation was measured in the presence of the proteasome inhibitor MG132, methylated ubiqu-itin, EDTA or dimethylsulfoxide (DMSO) during the chase period (Fig 4) Samples were taken at 0, 2 and
4 h and kept on ice until electrophoretic analysis At
4 h, a sample was centrifuged at 16 000 g for 20 min and washed in 1% (v⁄ v) Triton X-100 to detect the formation of detergent-insoluble aggregates All sam-ples were boiled in SDS loading buffer and separated
by SDS⁄ PAGE followed by quantification by phos-phorimaging (Fig 4) Wild-type actin was stable dur-ing the four hour chase period, and produced only a small amount of SDS soluble aggregate and no detect-able high molecular mass ubiquitinated actin How-ever, the amount of soluble Arg312His actin decreased over time and after four hours only approximately 25% remained After 4 h, a large fraction of SDS sol-uble actin and high molecular mass ubiquitinated actin was detected in the Triton X-100 insoluble pellet Concordantly, treatment with the specific proteasome inhibitor MG132 did not have any effect on either the wild-type or the Arg312His actin This indicates that following polyubiquitination, the protein aggregates and cannot be degraded by the proteasome Incubation with methylated ubiquitin inhibited the formation of high molecular mass ubiquitin conjugates Methylated ubiquitin can be efficiently ligated to protein sub-strates, but terminates elongation of polyubiquitin chains because its reactive lysine residue is blocked [30] Ubiquitination can be completely blocked by treatment with EDTA The E1 catalyzed activation of ubiquitin is Mg2+- and ATP-dependent and is there-fore blocked by the addition of EDTA [29] By inhibit-ing ubiquitin conjugation either partly or completely, the formation of Triton X-100 insoluble aggregates was also inhibited, suggesting the aggregation to be ubiquitin-dependent Results of the quantification of full length actin in the SDS⁄ PAGE are shown in
Trang 6Fig 4B,C Quantification of all radioactive material
having higher molecular mass than actin, and thus
rep-resenting ubiquitin conjugates, is shown in Fig 4D
Some actin variants show perturbed filament
formation
We investigated the ability of the mutant proteins to
form filamentous actin in cell culture To test the
sub-cellular localization of mutant actins and their ability
to polymerize, we expressed full length wild-type and
mutant actin using the mammalian expression vector pcDNA3.1⁄ Myc-His A, producing a C-terminally c-Myc-tagged chimeric protein The tagged versions of the actin variants behaved like the untagged versions when tested in the in vitro system (data not shown) Transfected HEK-293 or COS-7 cells were stained with primary antibodies against c-Myc prior to labeling with green fluorescent secondary antibodies and costained with Texas Red fluorescent phalloidin, a standard mar-ker for filamentous actin The in situ immunostaining was visualized by confocal laser scanning microscopy
Glu361Gl y
Ala331Pr p Met305Leu Ala295Ser Tyr166Cys Pro164Ala Glu99L ys Wild T
ype
Glu361Gl y
Ala331Pr p Met305Leu Ala295Ser Tyr166Cys Pro164Ala Glu99L ys Wild
Type
Wild
type
Glu99L ys Pro164Ala Tyr166Cys Ala295Ser Met305L
ys
Ala331ProAr g312His Glu361Gl
y
0,0
0,5
1,0
1,5
2,0
2,5
3,0
DNase I rescued Proteinase K digested
A
B
Fig 3 Some actin variations lead to a non-native loose protein structure In vitro translation for 30 min at 37 C followed by termination of protein synthesis using 150 lgÆmL)1cycloheximide (A) Resistance to protease treatment was assessed by digestion with 20 lgÆmL)1 pro-teinase K for 15 min at 20 C and inhibition by 10 m M PMSF for 10 min on ice Actin binding to DNase I was measured by incubation with Sepharose beads coupled to DNase I for 1 h at 4 C followed by stringent washing and elution of actin by SDS loading buffer containing 40% (v ⁄ v) formamide (B) The samples were analyzed by SDS ⁄ PAGE and quantified by phosphorimager scanning Each sample was normal-ized to its respective untreated sample and relative difference to the wild-type was calculated Error bars indicate standard deviations Amino acid substitutions shown in bold lead to DCM.
Trang 7SDS gel
A
B
Wild type
Hours
4 3 2 1 0 0,0
0,2
0,4
0,6
0,8
1,0
1,2
Arg312His
Hours 0,0
0,2 0,4 0,6
4 3 2 1 0
0,8 1,0 1,2
Wild T ype Arg312HisWild T
ype Arg312HisWild T
ype Arg312HisWild T
ype Arg312His
0 10 20 30 40
Wild T
ype Arg312HisWild T
ype Arg312HisWild T
ype Arg312HisWild T
ype Arg312His
0
5
10
15
20
Fig 4 Insoluble misfolded actin accumulates in a ubiquitin dependant manner In vitro translation for 30 min followed by termination of pro-tein synthesis using cycloheximide at a final concentration of 150 lgÆmL)1 Samples taken at 0, 2 and 4 h after termination were briefly spun
at 16 000 g dissolved in SDS buffer on ice until gel separation Samples at 4 h were spun at 16 000 g for 20 min, washed in 1% Triton
X-100 and dissolved in SDS buffer All samples were analyzed by SDS ⁄ PAGE and quantified by phosphorimager scanning (A) Autoradiogram
of SDS ⁄ PAGE gel containing wild-type and Arg312His actin with DMSO (0.1% v ⁄ v; vehicle control), the proteasome inhibitor MG132 (25 l M ), Met-Ub (1 mg mL)1), and EDTA (8 m M ) The numbers are the chase period in hours and P is the pelleted non-Triton X-100 soluble fraction at t ¼ 4 h (B) Quantified phosphorimager scanning of the full length soluble actin band calculated relative to t ¼ 0 values DMSO (d), MG132 (s), Met-Ub (,) and EDTA (.) (C) Bar representation of Triton X-100 insoluble actin aggregates relative to wild-type DMSO val-ues Only the 42 kDa actin band was scanned and the wild-type DMSO fraction was set to 1 (D) Scanning of high molecular mass ubiquiti-nated actin relative to the wild-type DMSO soluble sample at 4 h Ubiquitiubiquiti-nated actin was defined as the scanning of all radioactive material running slower than 42 kDa in the SDS gel Triton X-100 soluble samples (unfilled bars) and Triton X-100 insoluble samples (filled bars).
Trang 8and images were processed using Adobe photoshop.
Because neither HEK-293 cells nor COS-7 cells are
muscle cells, and do not contain sarcomeres, one can
question the physiological relevance of actin expression
in these cells However, as wild-type a-cardiac actin is
capable of incorporating into cytoskeletal actin fibers,
they may mimic the development of HCM and DCM
at a sufficient level to distinguish and grade the actin variants
The red fluorescent phalloidin stained the actin fila-ments, composed of either endogenous actin only or a combination of endogenous and exogenous actin In the cell lines used, this gave rise to a discrete staining
of the outer cell boundaries and only to a lesser extent the actin cables in the cytosol (Fig 5) The c-Myc tar-geted green fluorescence stained the actin coded by the transfected actin cDNA only – both as part of the actin filament and as nonincorporated actin molecules
By merging the red and green signal, the intracellular localization of mutated actin as well as its ability to be incorporated in actin filaments could be evaluated c-Myc-tagged wild-type actin mostly colocalized with phalloidin-stained actin filaments, indicating correct processing and filament formation of tagged actin The Tyr166Cys, Ala295Ser and Ala331Pro mutant actin proteins colocalized with actin filaments similar to the wild-type with a clear c-Myc staining in the cell cyto-skeleton but also some diffuse staining juxtaposed to the nucleus The Glu99Lys, Pro164Ala, Met305Leu, Arg312His and Glu361Gly mutant proteins were distri-buted evenly throughout the cytosol with no apparent colocalization with the phalloidin stained actin These findings are in agreement with the in vitro results above, showing that variant actins, which are impaired
in folding and have an increased tendency to become insoluble, also fail to be incorporated in filaments It is even noticeable that variants having a less severe fold-ing deficiency in vitro like the Met305Leu actin also have slightly better filament incorporation in vivo Culturing cells transfected with wild-type or mutant actin with the proteasomal inhibitor MG132 overnight before fixation caused neither any further accumula-tion of actin in the cytosol, nor any formaaccumula-tion of aggregates, as judged by the immunofluorescence ana-lysis (data not shown)
Fig 5 Only correctly folded actin proteins can incorporate into fibers COS-7 cells transiently transfected with c-Myc tagged actin wild-type and mutant variants and stained for nuclei (Hoechst
33258, blue), filamentous actin (phalloidin, red) and transfected actin (c-Myc antibodies, green) followed by confocal laser scanning microscopy Colocalization of filamentous actin and transfected actin lead to yellow color Plus and minus symbols indicate colocali-zation with the cell cytoskeleton Mock transfected cells showed
no cross reactivities from the c-Myc antibody Transfection with a vector expressing the green fluorescent protein (GFP) served as a positive control for the transfection procedure giving a transfection frequency of approximately 60% Amino acid substitutions in bold type face lead to DCM The results are representative of three sep-arate experiments.
Trang 9Mutations in the a-cardiac actin gene may lead to
re-modeling of the heart and cause either HCM or DCM
The prevailing hypothesis states that the mutated gene
product can be incorporated into actin fibers and
exerts a negative effect on the gene product from the
normal allele [3] However, an initial requirement for
fiber incorporation is proper folding to a native-like
actin structure maintained by the protein quality
con-trol system In addition to a stable structure, the actin
variant must still be able to bind its many regulatory
binding proteins for proper polymerization [23]
Sev-eral examples exist of disease-related missense
varia-tions leading to incomplete folding with a variety of
consequences for the cell To test these events, we
expressed the wild-type and eight actin variant proteins
in an in vitro translation system using rabbit
reticulo-cyte extracts Using this system, we found a subset of
the mutant proteins to have altered chaperone
inter-action kinetics, as visualized on native nondenaturing
PAGE The most severe folding-defective variant
pro-tein and the one with the highest tendency to remain
in complex with the TRiC chaperonin was Arg312His
actin, which is found in one patient with DCM For
this mutant, the amount of soluble TRiC-bound actin
decreased over time This suggests that actin folding is
an iterative process in which each cycle of binding and
release from the chaperonin can fold a subset of the
mutant actin proteins; however, with reduced efficiency
compared to the wild-type (Fig 2C)
The fraction of unfolded mutant protein was
assessed by performing two types of folding assays
First, a mild digestion with proteinase K, which
pro-duces a 34 kDa C-terminal fragment There are drastic
differences in the protease susceptibility of different
mutant actins, suggesting different degrees of folding
A common trait is the size of the product, indicating
the eventual formation of a wild-type-like structure
The second assay involves binding of native actin by
DNase I conjugated beads Through this assay we saw
that folded, full length wild-type, but not specific
mutants of actin, bind to DNase I Both assays taken
together indicate that some mutants have an impaired
folding pathway Our results show that all actin
mutants can be folded to the mature form, but with
widely varying efficiency (Fig 3)
Increased turnover of mutant proteins is
character-ized in several hereditary diseases, exemplified by the
elimination of missense variations in phenylalanine
hydroxylase leading to phenylketonuria and
short-chain acyl-CoA dehydrogenase (SCAD) missense
vari-ant proteins, leading to SCAD deficiency [25,31,32], as
well as deficiencies in other metabolic and tumor sup-pressor genes [33–36] These conditions are inherited
by autosomal recessive transmission whereby misfolded polypeptides are eliminated, leading to loss of function and disease development In cardiomyopathies caused
by mutations in the ACTC gene, the elimination of the actin variant proteins before their incorporation into fibers could either function as a positive modulator and reduce the severity of the clinical phenotype, or as
a negative modulator to increase a haploinsufficiency effect A different pathology could arise from failure
to degrade misfolded actin variants and accumulation
of cell-toxic aggregates as seen in desmin-related cardio-myopathy [37] Indeed, the presence of filament oligo-mers, the cytotoxic precursor of aggresomes, has been identified in cardiomyocytes from both DCM and HCM patients [38] The genetic characterization for these cardiomyocytes is, however, not accounted for During various cell stress situations, the protein qual-ity control system may fail in processing the misfolded proteins leading to further increase in accumulation Recent studies have shown that TRiC interacts with
a number of non-native proteins containing b-sheets, including the von Hippel-Lindau tumor suppressor protein [34] and the WD (tryptophan-aspartate) repeat proteins [39] A broader specificity towards b-sheet containing proteins suggests that TRiC may suppress aggregation of polyglutamine containing proteins in neurodegenerative diseases [40] It is possible that the ability of TRiC to prevent aggregation of actin vari-ants plays a role in the development of the disease, and may underlie differences in the expression of a dis-ease phenotype in different patients having the same actin mutation
In contrast to the fate of the above-mentioned and many other misfolded proteins, the misfolded Arg312His actin is not degraded by the ubiquitin-pro-teasome system; at least not within the time scope and experimental conditions in this study Arg312His actin rather accumulates in a Triton X-100 insoluble fraction Inhibition of the ligation of ubiquitin monomers to actin (either partly or completely) increased the percentage of actin found in the soluble fraction This suggests that polyubiquitinated but not unmodified actin mutants are prone to become detergent-insoluble and aggregate This is consistent with examples of colocalization of ubiquitin and aggregates in cells [41] Extending the experiment to all eight mutants showed a correlation between the degrees of misfolding as judged by the pro-teinase K resistance and DNase I binding assay, and tendencies to form detergent-insoluble fractions (data not shown) Calculating the theoretical change in aggre-gation rate as a consequence of the mutations using the
Trang 10method described by Chiti et al [42] gives aggregation
rates corresponding to the experimentally observed
aggregation tendency The variant actins that are
impaired in folding and showed tendencies to become
insoluble in detergent in our analyses give an increased
calculated aggregation rate, with the Arg312His having
the highest rate increase (data not shown) This
correla-tion suggests a similarity between the actin-containing
aggregates and the ones found in amyloid deposits
in neurodegenerative diseases, rather than amorphous
unstructured aggregates [42]
Examination of mutant actin in intact cells reveals a
correlation between the degree of protein misfolding
and impairment of incorporation into filaments This
suggests that diminished ability to form filaments is
caused by folding impairment The nonfilamentous
mutant actin proteins appear diffuse throughout
the cytosol, apparently without being degraded by the
ubiquitin proteasome system, as treatment with the
potent proteasomal inhibitor MG132 did not result in
increased accumulation of misfolded actin (not shown)
The inability of misfolded actin monomers to form
filaments raises the question whether the negative
dom-inance of the disease is evolving from the filaments
with incorporated variant actin proteins as previously
suggested [7–10] or from negative effects of misfolded
actin molecules in the cytosol Genetic heterogeneity
and different environmental factors may modify the
residual folding efficiency of the mutant proteins, and
thereby exert modulation on the clinical expression To
further define such modifying factors, inbred rodent
models with controlled genetic and environmental
backgrounds will be imperative
A recent HCM study showed no specific phenotype
associated with ACTC mutations [8] Reviewing the
clinical data, there seems, interestingly, to be a
correla-tion between the severity of the disease and the ability
of the variant actin to incorporate into filaments In
patients carrying mutations giving rise to actin proteins
capable of incorporating into fibers (Tyr166Cys,
Ala295Ser, and Ala331Pro), only five out of 19 were
symptomatic, whereas eight out of nine were
sympto-matic when the actin protein had reduced ability to
incorporate (Glu99Lys, Pro164Ala and Met305Leu;
P< 0.005, Fisher’s Exact test) This may arise from
the reduced amount of actin filament
(haploinsuffi-ciency), or from stressful cellular situations resulting
from the accumulation of misfolded and⁄ or aggregated
actin protein Based on data from this study and the
fact that toxic oligomers previously have been
described in HCM [38], the latter seems to be the most
likely These findings, however, must be interpreted
with caution because of the small number of patients
In general, distinct mutations in sarcomeric proteins cause either dilated or hypertrophic cardiomyopathy;
no mutation can lead to both DCM and HCM, disre-garding end stage dilation in HCM This suggests that the mutations initiate different series of events that remodel the heart We show that both DCM-causing ACTC mutations encode actin variants with inefficient folding and perturbed filament formation, whereas only half of the HCM-causing ACTC mutations (three out of six) encode actin proteins that appear misfolded
by the criteria employed here It is therefore tempting
to speculate that the inability to form myofilaments and⁄ or the accumulation of aggregates as a result of protein misfolding could be one of the cellular patho-logical effects of a mutation that influence the direc-tion of cardiac remodeling towards either HCM or DCM Previous mouse models and myotube assays have been used to study the effect of sarcomeric varia-tions; however, the causal molecular mechanisms underlying these effects have never before been stud-ied The effect of impaired protein folding precedes the potential effect of the malfunctioning variant protein The presence of misfolded proteins may influence the cellular stress level and impair the Ca2+ sensitivity of the myocyte
The finding that protein misfolding and the influence from the protein quality control system may have effects
on clinical progression of DCM and HCM is a para-digm shift for these types of cardiomyopathies Research into this new avenue may give a basis to design novel therapeutic strategies and new categories of model systems like those pursued in amyloidosis diseases inclu-ding Alzheimer’s and Parkinson’s disease [17]
Experimental procedures Plasmids
The complete ACTC cDNA sequence was obtained using the IMAGE clone CM147-m7 as template in a PCR reac-tion using Pfu Turbo polymerase from Stratagene (La Jolla,
CA, USA) The forward primer contains a KpnI site and an optimized Kozak sequence (GGTACCGCCACCATG), and the reverse primer contains an XhoI site The PCR product was purified and ligated into the KpnI⁄ XhoI site of the pcDNA3.1 vector (Invitrogen, Carlsbad, CA, USA) Muta-genesis was performed using the Quick change kit (Strata-gene) All clones were checked for mutagenesis and the absence of PCR-induced errors For constructing a c-Myc His-tagged chimeric actin, a PCR product from the same forward primer and a reverse primer bearing an XhoI restriction site substituting the stop codon was inserted into the KpnI⁄ XhoI site in the pcDNA3.1 ⁄ Myc-His A vector