Deletions of the dystrobrevin protein were performed and the ability of the mutated forms to bind to dystrophin was tested both in vitroand in a two-hybrid assay, as well as their abilit
Trang 1Dystrobrevin requires a dystrophin-binding domain to function
Karine Grisoni, Kathrin Gieseler and Laurent Se´galat
CGMC, CNRS-UMR, Universite´ Lyon, Villeurbanne, France
Dystrobrevin is one of the intracellular components of the
transmembrane dystrophin–glycoprotein complex (DGC)
The functional role of this complex in normal and
patho-logical situations has not yet been clearly established
Dystrobrevin disappears from the muscle membrane in
Duchenne muscular dystrophy (DMD), which results from
dystrophin mutations, as well as in limb girdle muscular
dystrophies (LGMD), which results from mutations
affect-ing other members of the DGC complex These findaffect-ings
therefore suggest that dystrobrevin may play a pivotal role in
the progression of these clinically related diseases In this
study, we used the Caenorhabditis elegans model to address
the question of the relationship between dystrobrevin
binding to dystrophin and dystrobrevin function Deletions
of the dystrobrevin protein were performed and the ability
of the mutated forms to bind to dystrophin was tested both
in vitroand in a two-hybrid assay, as well as their ability to rescue dystrobrevin (dyb-1) mutations in C elegans The deletions affecting the second helix of the Dyb-1 coiled-coil domain abolished the binding of dystrobrevin to dystrophin both in vitro and in the two-hybrid assay These deletions also abolished the rescuing activity of a functional transgene
in vivo These results are consistent with a model according to which dystrobrevin must bind to dystrophin to be able to function properly
Keywords: dystrophin; dystrobrevin; nematode; Caeno-rhabditis elegans
Duchenne muscular dystrophy (DMD) is an inherited
muscular disease in which the patients’ muscles gradually
degenerate So far, no treatment exists for DMD The
disease is caused by mutations affecting the dystrophin gene,
which encodes a 3685-amino-acid protein (reviewed in [1])
Dystrophin is a submembrane protein associated with a
transmembrane dystrophin–glycoprotein complex (DGC)
comprising dystroglycans, sarcoglycans, sarcospan,
syntro-phins and dystrobrevins [1–3] DGC proteins have attracted
an increasing amount of attention over the last few years,
because they might help to explain the physiopathology of
the disease, and may also provide therapeutic clues
Dystrobrevins form a family of proteins that are unique
in that they are both dystrophin-associated proteins, and
homologous to the C-terminal region of dystrophin
Alpha-dystrobrevin was originally identified as a molecule that
copurifies with nicotinic acetylcholine receptors in sucrose
gradients [4,5] It was later recognized as one of the proteins,
which associates with dystrophin to form the dystrophin–
glycoprotein complex (DGC) [4,6,7] A second
dystro-brevin, b-dystrodystro-brevin, is mainly expressed in nerve tissues
[8,9] Mice carrying a knockout mutation of the a-dystrobrevin gene (adbn mice) suffer from a cardiac and skeletal muscle myopathy reminiscent of dystrophin (mdx) mutations [10]
a-Dystrobrevin binds to dystrophin via a coiled-coiled motif present in both proteins, and to the PDZ domain containing syntrophins [11,12] Indirect evidence suggests that dystrobrevin may also bind to other members of the DGC [13] Although no enzymatic activity has yet been assigned to dystrobrevins, there are several indications that they may play a role in signalling mechanisms First, dystrobrevins are tyrosine-phosphorylated proteins [5,14] Secondly, in the absence of a-dystrobrevin, the signalling molecule, neuronal nitric oxide synthase (nNOS) disappears from the muscular membrane [10]
In addition, two lines of evidence suggest that dystro-brevin may play a key role in the muscle degeneration observed in DMD and sarcoglycanopathies; first, dystro-brevin immunostaining decreases greatly in DMD and in several sarcoglycanopathies [15] Secondly, although the DGC components (with the exception of NOS) are not affected by the absence of dystrobrevin in adbn mice, muscle degeneration occurs
The nematode Caenorhabditis elegans has homologues of most of the DGC proteins (L Se´galat, unpublished results) There is one dystrophin- and one dystrobrevin-like gene in the genome of C elegans (dys-1 and dyb-1, respectively) [16,17] C elegans dystrophin and dystrobrevin are able to bind to each other in vitro [18] in the same way as their mammalian counterparts [12], and they also bind to syntrophin [18] dys-1 and dyb-1 mutants display a similar behavioural phenotype consisting of hyperactivity, exagger-ated bending of the head when moving forward, and a tendency to hypercontract [16,17] In addition, progressive muscle degeneration is observed when dys-1 or dyb-1
Correspondence to L Se´galat, CGMC, Universite´ Lyon1, 43 bld du 11
Novembre, 69622 Villeurbanne cedex, France.
Fax: + 33 4 72 44 05 55, Tel.: + 33 4 72 43 29 51,
E-mail: segalat@maccgmc.univ-lyon1.fr
Abbreviations: DGC, dystrophin–glycoprotein complex;
DMD, Duchenne muscular dystrophy; LGMD, limb girdle muscular
dystrophy; nNOS, neuronal nitric oxide synthase; AD, activation
domain; DNA-BD, DNA binding domain; SD, synthetic dropout
medium; SBR, syntrophin binding region.
(Received 16 October 2001, revised 10 January 2002, accepted
11 January 2002)
Trang 2its association with dystrophin First, we refined the
dystrophin-binding region on dystrobrevin (Dyb-1) by
performing deletion-mapping experiments in vitro We then
tested the ability of the truncated Dyb-1 proteins to bind to
dystrophin (Dys-1) in a yeast two-hybrid assay, as well as
their ability to rescue dyb-1 mutants
E X P E R I M E N T A L P R O C E D U R E S
Construction of deleted forms of Dyb-1
forin vitro binding experiments
Deletions were carried out on the dyb-1 coding sequence,
using clone AN450 [encoding Dyb-1 amino acids 390–543
fused in frame to the GST coding sequence; plasmid pGEX
3X (Pharmacia)] [18] AN450 DNA (500 ng) was cut with
the restriction enzyme MfeI The cut DNA was then
distributed among several tubes incubated with 0.05 lL of
BAL31exonuclease for various times (typically 0–10 min)
The reactions were stopped by adding EGTA to 4 mMand
heating at 65°C for 10 min DNA was purified on a Wizard
column (Promega) and the action of BAL31 was checked by
loading an aliquot of each tube onto an agarose gel column
The DNA corresponding to the deletions required was
treated by applying T4 DNA polymerase in the presence of
nucleotides to create blunt ends, which were ligated and the
plasmids were transformed in Escherichia coli DH5 Clones
were picked randomly and analysed using sequencing
procedures Any clones carrying a frame shift were rejected
Construct 6¢4 was built using similar procedures, but using
the enzyme HindIII instead of MfeI The amino acids
removed in the deletions were 489–499 (clone 2¢5), 487–513
(clone 5¢1), 489–528 (clone 5¢2B), 471–517 (clone 5¢5B), 478–
543 (clone 2¢1), and 391–450 (clone 6¢4) Clones 2¢1 and 6¢4
have been described previously [18], but clone 2¢1 was
erroneously reported to be deleted in amino acids 478–521
This correction makes no difference to the interpretation of
our previous data
In vitro interactions
Constructs were transformed into the E coli strain BL21
DE3 and the fusion proteins were produced as follows
After cell sonication and centrifugation, the supernatant
was loaded onto glutathione–Sepharose beads
(Pharma-cia) Approximately 20 lg of resin bound proteins were
washed in binding buffer [Hepes 20 mM pH 7.4, KOAc
110 mM, NaOAc 5 mM, Mg(OAc)22.5 mM, NP40 0.05%,
dithiothreitol 1 mM, leupeptin 10 mgÆmL)1, aprotinin
10 mgÆmL)1, pepstatin 10 mgÆmL)1,
phenylmethanesulfo-nyl fluoride (1 mM)] The 35S-labelled Dys-1 C-terminal
end was synthesized using a coupled in vitro transcription
and translation kit (Promega) with cDNA yk12c11 [16]
The preparations were incubated for 2 h at 4°C GST
controls were performed using 1–2 times the amount of
fusion protein After five washes with binding buffer, the
labelled proteins were eluted by boiling the preparation for
3 min in gel loading buffer Gels were dried, exposed
Constructs for the yeast two-hybrid assay The C-terminal end of Dys-1 (amino acids 2857–3674) was fused to the DNA binding domain (DNA-BD) of the Gal4 protein For this purpose, a 2,4 kb dys-1 cDNA fragment (yk12c11) was cloned into the polylinker of pAS2-1 (Clontech) with respect to the reading frame
Dyb-1 fragments and deletions were PCR amplified using clones AN 450, 2¢5, 5¢1, 5¢2B, 5¢5B and 6¢4 in pGEX 3X as templates (see below) and cloned into pACT2 (Clontech) in frame with the activation domain (AD) of the Gal4 protein and the HA epitope The resulting constructs were called AD-AN450, AD-2¢5, AD-5¢1, AD-5¢2B, AD-5¢5B, and AD-6¢4, respectively All DNA constructs were checked by performing DNA sequencing
Yeast two-hybrid analysis Construct DNA-BD-Dys-1 was transformed into the yeast strain CG 1945 using the LiAc transformation procedure (Clontech, Yeast protocols Handbook, PT 3024-1) Trans-formants were selected on synthetic dropout (SD) media (Clontech) minus tryptophan
A DNA-BD-Dys-1 expressing yeast strain was selected and transformed with plasmids AD-AN450, AD-2¢5, AD-5¢1, AD-5¢2B, AD-5¢5B, or AD-6¢4 Transformants were selected on SD media minus tryptophan and leucin Interactions between the DNA-BD-Dys-1 protein and the various forms of the AD-Dyb-1 fusion proteins were analysed on the basis of transactivation of the HIS3 reporter gene after 3 days of growth on SD medium devoid
of tryptophan, leucin and histidin A strain carrying both the DNA-BD-Dys-1 protein and the empty pACT2 plasmid was used as a negative control
Western blots with yeast protein extracts For Western blot analysis, yeast protein extracts were prepared from strains carrying both DNA-BD-Dys-1 plasmids and AD-Dyb-1 plasmids (AN450, AD-2¢5, AD-5¢1, AD-5¢2B, AD-5¢5B, or AD-6¢4) Overnight cul-tures (5-mL) were prepared in SD media minus trypto-phan and leucin The next day, 1 mL of overnight culture was transferred into 10 mL of YPD medium The diluted culture was incubated for several hours at 30°C until D600 ¼ 0.3 for 1 mL Cells (3 D600 units) were spun down and frozen at)70 °C for at least one hour The yeast pellet was resuspended in 60 lL of sample buffer [21] After boiling the mixture for 5 min, and centrifuging for 30 s at 13 000 g, 10 lL of supernatant was loaded onto each lane of a 0.1% SDS/10% polyacrylamide gel Proteins were transferred onto a BA83 nitrocellulose membrane (Schleicher & Schuell) in transfer buffer (Tris 25 mM, glycine 190 mM, SDS 0.01%, ethanol 20%) for 1 h at 100 V AD-Dyb-1 fusion proteins were detected using a rabbit polyclonal anti-(Dyb-1) Ig [19] at a dilution 1 : 500 Peroxidase-coupled anti-(rabbit IgG) Ig (Biorad) was used at a dilution of 1 : 3000 Blots
Trang 3were revealed using the ECL+ kit (Amersham) as
recommended by the supplier
Functional assay inC elegans
First, a dyb-1 functional construct was obtained by
modi-fying a previously built dyb-1:gfp construct [19] The
dyb-1:gfp construct, which has been previously described,
was shortened on the 5¢ end to leave 2.9 kb of upstream
sequence, and various restriction enzyme sites were removed
and added by performing synonymous point mutations to
yield the construct dyb-1:gfp VII, which has single AgeI and
MluI sites at codons 390 and 543 This construct encodes a
functional Dyb-1 gene as it can rescue dyb-1 mutations (data
not shown)
Secondly, Dyb-1 construct AN450 and the deletion
derivatives described above were transferred from pGEX
into dyb-1:gfp VII using PCR-amplifying procedures with
primers carrying AgeI and MluI sites, and cloned into the
single AgeI and MluI sites of dyb-1:gfp VII (Fig 5)
Positives clones were checked by determining their
seq-uence dyb-1:gfp VII and constructs carrying either AN450
or the deletions were injected at a concentration of
1 ngÆlL)1 along with the transformation marker KP13
[22] using standard procedures [23] into worms carrying the
putative null allele dyb-1(cx36) [17] Transgenic strains were
grown at 23°C
R E S U L T S
Mapping of the dystrophin-binding site on Dyb-1 The results of a previous study suggested that the Dys-1-binding region on Dyb-1 was located in the second helix of the predicted coiled-coil domain [18] We refined this analysis by creating additional deletions by random muta-genesis and testing their affinity for Dys-1 Clones 2¢5, 5¢1, 5¢2B, and 5¢5B were obtained by inducing exonuclease digestion of the reference clone AN450, which encodes the amino acids 390–543 of Dyb-1 fused to the GST protein [18] These four clones contain various breakpoints within the second helix of the predicted coiled-coil domain (H2) (Fig 1) The deleted amino acids were 489–499 (clone 2¢5), 487–513 (clone 5¢1), 489–528 (clone 5¢2B) and 471–517 (clone 5¢5B) Clone 2¢1, lacking amino acids 478–543, was used as a negative control [18] Clone 6¢4, lacking amino acids 391–450, was used as a second positive control [18] The constructs were used to produce Dyb-1–GST chimeric proteins in E coli, which were affinity purified on gluthati-one–Sepharose beads and subjected to in vitro binding with
35S-labelled Dys-1 Clones 2¢5 and 5¢2B bound to Dys-1 at levels that were not significantly different from those of the positive controls AN450 (Fig 2) and 6¢4 (gel not shown) In contrast, the binding activity of clones 5¢1 and 5¢5B was weaker (Fig 2) The difference between clones 2¢5, 5¢1 and
Fig 1 Deletions used in this study The ‘WT’ line represents the
amino-acid sequence of the wild-type Dyb-1 protein in the predicted
coiled-coil domain region The predicted helices forming the domain are
shown by hatched boxes Numbers above the wild-type sequence
indicate the amino-acid coordinates of the helices Deletions are shown
below the wild-type sequence Numbers indicate the coordinates of the
breakpoints Deletions were generated by exonuclease digestion Note
that the 6¢4 deletion extends on the left side further than shown on the
drawing For in vitro binding experiments, the corresponding DNAs
were cloned into the pGEX vector to produce Dyb-1–GST fusion
proteins [18] The right column gives the binding affinity of the various
constructs to35S-labelled Dys-1 in arbitrary units (mean ± SD) One
unit is defined as the autoradiogram intensity obtained with the
neg-ative control GST Asterisks indicate values significantly different from
wild-type Constructs 5¢1, 5¢5B and 2¢1 have significantly reduced
affinity to Dys-1.
Fig 2 In vitro binding of Dyb-1 (dystrobrevin) to Dys-1 (dystrophin) Representative example of in vitro binding experiments The same gel is shown in Coomassie staining (top) and autoradiography (bottom) The gel was loaded with various GST–Dyb-1 fusion proteins (and GST alone) after incubation with equal amounts of in vitro translated
35
S-labelled DYS-1 The signal intensity of the autoradiogram was quantitated with a radiographic analyser (Biorad) MW, Molecular mass markers T, aliquot of the in vitro translation product.
Trang 45¢2B is of interest because these clones have left breakpoints
differing by only two amino acids Although clones 2¢5 and
5¢2B (cutting at position 489) display a wild-type pattern of
binding behaviour, clone 5¢1 (cutting at position 487) does
not Therefore, the third heptad repeat of the second helix of
Dyb-1 (amino acids 484–490) seems to be critical for proper
Dys-1 binding to occur in vitro
Dys-1/Dyb)1 interactions in the yeast two hybrid assay
Next, we tested the ability of the various forms of Dyb-1 to
interact with Dys-1 in a two-hybrid assay The Dyb-1
control and mutant clones were fused to the activation
domain of the Gal4 yeast transcription factor and were
tested against the entire C-terminal end of Dys-1 (amino
acids 2857–3674) fused to the DNA-binding domain of
Gal4 The expression of wild-type and truncated proteins
was checked using the Western blotting procedure This
confirmed that all the fusion proteins were correctly
expressed and that the experiment was not biased by any
differences in the protein expression levels (Fig 3) Among
the six constructs tested, only the wild-type Dyb-1 fragment
and the 6¢4 fragment resulted in the growth of yeasts on
His-plates, which can occur only if Dyb-1 binds to Dys-1
(Fig 4) Similar results were obtained when a shorter Dys-1
fragment (amino acids 3402–3674) encompassing the
syn-trophin-binding domain and the coiled-coil domain
(cor-responding to BB810 in [18]) was used (not shown) These
results indicate that all the deletions affecting the second
helix of Dyb-1, including the shortest deletion (clone 2¢5),
greatly reduce the interactions between Dys-1 and Dyb-1 in
the yeast system
Functional complementation of Dyb-1 deletions
inC elegans
The only functional assay available for dystrobrevin resides
in functional complementation To test whether the
dele-tions of various parts of the coiled-coil domain had an effect
on the in vivo function of Dyb-1, we created transgenes
carrying the same deletions as those tested in vitro and in the yeast system We transferred the deletions into the vector dyb-1:gfp VII, which is a functional transgene consisting of genomic Dyb-1 sequences (Fig 5) Because the deletions are derivatives of clone AN450, a cDNA fragment that encompasses several exons, it was necessary first to check whether removing introns 7 and 8 had any effect on the rescuing activity of dyb-1:gfp VII When the 1.2-kb AgeI– MluI genomic fragment of dyb-1:gfp VII was replaced by the 450-bp AN450 cDNA fragment encoding the same amino acids (Fig 5), rescue of dyb-1(cx36) animals still occurred in two out of three lines transgenic lines (Table 1), which indicates that removing introns 7 and 8 did not impair the rescuing capacity of dyb-1:gfp VII Then we tested the various deletions Three out of the four lines obtained with deletion 6¢4 showed consistent, although only partial, rescue (Table 1) In these lines, the dyb-1 behavioural phenotype (head bending and hyperlocomotion) was intermediate between mutant and wild-type This indicates that, although deleting the syntrophin binding region (SBR) and the first helix reduces the activity of the protein, it remains partly functional Five lines were obtained with deletion 2¢5 (the shortest deletion affecting the second helix); only one out of the five lines tested showed a weak rescuing effect, which was far less conspicuous than that observed with construct 6¢4 (Table 1) Worms carrying the remaining constructs (5¢1, 5¢2B and 5¢5B) did not display any visible rescue All in all, these data indicate that deletions affecting the second helix
of the Dyb-1 coiled-coil domain strongly decrease or abolish the functional properties of Dyb-1
D I S C U S S I O N
The aim of this study was twofold: first, to confirm and refine the localization of Dys-1 binding sites on Dyb-1, and secondly to test whether there may exist a correlation
Fig 3 Western blot of Dyb-1 deletions produced in yeast Western blots
were prepared with protein extracts of yeast carrying DNA-BD-Dys-1
plasmids and empty pACT2 (lane 1), AD-AN450 (lane 2), AD-2¢5
(lane 3), AD-5¢1 (lane 4), AD-5¢2B (lane 5), AD-5¢5B (lane 6) and
AD-6¢4 (lane 7) The blot was probed with mouse monoclonal
antibodies directed against the HA epitope situated between the
C-terminal end of the activation domain of the Gal4 protein and
the various Dyb-1 proteins Molecular mass standards are shown on
the right.
Fig 4 Yeast two-hybrid assay A plate containing SD media minus leucin, tryptophan and histidine was seeded with yeast carrying DNA-BD-Dys-1 and various AD-Dyb-1 plasmids or empty pACT2 (as a negative control), and incubated at 30 °C for 3 days Growth on media devoid of histidine can occur only if Dys-1 and Dyb-1 interact and the HIS3 reporter gene is transactivated Only the wild-type construct AD-AN450 and the AD-6¢4 construct promoted growth in this assay The other constructs, all carrying deletions in the second helix of the Dyb-1 coiled-coil domain (H2), were unable to promote growth in this assay, which indicates that the H2 helix is a critical prerequisite for Dys-1/ Dyb-1 interactions to be possible.
Trang 5between the binding of dystrobrevin to dystrophin and
functional activity of dystrobrevin The C elegans model
organism was particularly well suited for the latter part
because transgenic animals can be quickly obtained in this
species As long as the catalytic, enzymatic, or other
functional activity of the dystrobrevin protein will remain
unknown, the only way of making functional
investi-gations will continue to be through complementation of
mutations
A preliminary in vitro study on Dys-1–Dyb-1 interactions
pointed to the second helix (H2) of the predicted coiled-coil
domain of Dyb-1 [18] Here, we refined this analysis by
studying additional deletions that subdivide the H2 domain
Our results confirm the previously published data and show
that H2 is involved in the interaction with Dys-1 in vitro
Within this domain, the first half seems to be particularly
critical as deletions infringing on this side lead to a decrease
in binding Constructs 2¢5, 5¢2B and 5¢1 are of particular
interest; the first two constructs break at amino acid 489 and retain binding to Dys-1, whereas the third one breaks at position 487 and its binding affinity is reduced two-fold Alternatively, this discrepancy might also be attributable to differences in the three dimensional structure of the helix imposed by amino acids on the C-terminal side of the breakpoint The predicted three-dimensional structure of the various constructs has not been investigated so far The two deletions of the H2 region that retained some
in vitrobinding activity (clones 2¢5 and 5¢2B) were unable to promote Dys-1–Dyb-1 interaction in the yeast two-hybrid system, which indicates that the yeast assay is more selective than the in vitro assay A possible explanation may reside in the differences in protein concentrations In the in vitro pull down experiment, the GST–Dys-1 moiety is in great excess
to Dyb-1, whereas Dys-1 and Dyb-1 are thought to be in the same range of concentration in the yeast assay In agree-ment with the yeast results, these two constructs (2¢5 and
Fig 5 Drawing of the constructs used for
in vivo complementation tests Top, map of the
dyb-1:gfp VII construct dyb-1:gfp VII is a
8-kb genomic fragment of the dyb-1 gene
cloned into a pGEM backbone, in which the
gfp coding sequence has been added
termin-ally to the dyb-1 coding sequence Bottom:
map of the constructs used for the in vivo
experiments The various deletions are
deri-vatives of the AN450 construct, a fragment of
dyb-1 cDNA cloned into the GST-containing
vector pGEX Deleted regions are indicated
in dark The deletions were transfered into
dyb-1:gfp VII by PCR using the unique AgeI
and MluI restriction sites of dyb-1:gfp VII As
a result, introns 7 and 8 of dyb-1:gfp VII were
removed in these constructs These construct
were injected in dyb-1(cx36) mutants to assay
their ability to rescue the mutant phenotype.
Table 1 Rescuing activity of Dyb-1 deletions Constructs were injected in dyb-1(cx36) animals along with the transformation marker KP13 [22] dyb-1(cx36) animals display a behavioral phenotype consisting of hyperactivity, exaggerated bending of the head when moving forward, and a tendency to hypercontract when moving backwards + + +, transgenic animals not distinguishable from wild-type animals; + +, transgenic animals resemble wild-type but remain slightly hyperactive and bend their head more than wild-type; +, transgenic animals remain hyperactive and bend their head, but can be distinguished from nontransgenic siblings in blind tests; ±, some transgenic animals show a slightly improved behavior but transgenics cannot be recognized with certainty in blind tests; –, no modification of the phenotype could be observed.
Construct
Number of transgenic lines
Number of rescuing lines
Rescue
in best line(s)
Trang 6results Although the 6¢4 deletion was still partly functional,
only very weak activity could be observed with deletion 2¢5
In conclusion, the results presented in this paper show
that the second helix of the coiled-coil domain of Dyb-1 is
necessary for binding to Dys-1 both in vitro and in vivo
These results are consistent with a model in which
dystrobrevin must bind to dystrophin to be able to function
properly
A C K N O W L E D G E M E N T S
We thank L Granger and P Morales for their excellent technical
assistance We wish to thank R Bourette, C Bourgin and V Corset for
helpful discussions, and A Dumont, P Me´rel, and S Que´tat for their
participation in this work during their training courses K G and L S.
were supported by a Rhoˆne-Alpes district grant and L S by an
Association Franc¸aise contre les Myopathies (A F M.) grant.
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