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Thermolability of mutant MMACHC protein in the vitamin B12-responsive cblC disorder.
[homocystinuria without methylmalonic aciduria]
Methylmalonic
aciduria
and
homocystinuria
,
cblC
type
,
is
the
most
common
inborn
error
of
cellular
vitamin
B
12
metabolism
.
We
previously
showed
that
the
protein
carrying
the
mutation
responsible
for
late-onset
cblC
(
MMACHC
-R
161
Q
)
,
treatable
with
high
dose
OHCbl
,
is
able
to
bind
OHCbl
with
wild-
type
affinity
,
leaving
undetermined
the
disease
mechanism
involved
[
Froese
et
al
.
,
Mechanism
of
responsiveness
,
Mol
.
Genet
.
Metab
.
(
2009
)
.
]
.
To
assess
whether
the
mutation
renders
the
protein
unstable
,
we
investigated
the
thermostability
of
the
wild-
type
and
mutant
MMACHC
proteins
,
either
unbound
or
bound
to
different
cobalamins
(
Cbl
)
,
using
differential
scanning
fluorimetry
.
We
found
that
MMACHC
-wt
and
MMACHC
-R
161
Q
are
both
very
thermolabile
proteins
in
their
apo
forms
,
with
melting
temperatures
(
T
(
m
)
)
of
39
.
3
+
/
-
1
.
0
and
37
.
1
+
/
-
0
.
7
degrees
C
,
respectively
;
a
difference
confirmed
by
unfolding
of
MMACHC
-R
161
Q
but
not
MMACHC
-wt
by
isothermal
denaturation
at
35
degrees
C
over
120
min
.
However
,
with
the
addition
of
OHCbl
,
MMACHC
-wt
becomes
significantly
stabilized
(
Delta
T
(
m
max
)
=
8
degrees
C
,
half
-maximal
effective
ligand
concentration
,
AC
(
50
)
=
3
microM
)
.
We
surveyed
the
effect
of
different
cobalamins
on
the
stabilization
of
the
wild-
type
protein
and
found
that
AdoCbl
was
the
most
stabilizing
,
exerting
a
maximum
increase
in
T
(
m
)
of
approximately
16
degrees
C
,
followed
by
MeCbl
at
approximately
13
degrees
C
,
each
evaluated
at
50
microM
cofactor
.
The
other
cobalamins
stabilized
in
the
order
(
CN
)
(
2
)
Cbi
>
OHCbl
>
CNCbl
.
Interestingly
,
the
AC
(
50
)
's
for
AdoCbl
,
MeCbl
,
(
CN
)
(
2
)
Cbi
and
OHCbl
were
similar
and
ranged
from
1
-
3
microM
,
which
compares
well
with
the
K
(
d
)
of
6
microM
for
OHCbl
[
Froese
et
al
.
,
Mechanism
of
responsiveness
,
Mol
.
Genet
.
Metab
.
(
2009
)
.
]
.
Unlike
MMACHC
-wt
,
the
mutant
protein
MMACHC
-R
161
Q
is
only
moderately
stabilized
by
OHCbl
(
Delta
T
(
m
max
)
=
4
degrees
C
)
.
The
dose-response
curve
also
shows
a
lower
effectivity
of
OHCbl
with
respect
to
stabilization
,
with
an
AC
(
50
)
of
7
microM
.
MMACHC
-R
161
Q
showed
the
same
order
of
stabilization
as
MMACHC
-wt
,
but
each
cobalamin
stabilized
this
mutant
protein
less
than
its
wild-
type
counterpart
.
Additionally
,
MMACHC
-R
161
Q
had
a
higher
AC
(
50
)
for
each
cobalamin
form
compared
to
MMACHC
-wt
.
Finally
,
we
show
that
MMACHC
-R
161
Q
is
able
to
support
the
base-off
transition
for
AdoCbl
and
CNCbl
,
indicating
this
mutant
is
not
blocked
in
that
respect
.
Taken
together
,
our
results
suggest
that
protein
stability
,
as
well
as
propensity
for
ligand-induced
stabilization
,
contributes
to
the
disease
mechanism
in
late-onset
cblC
disorder
.
Our
results
underscore
the
importance
of
cofactor
stabilization
of
MMACHC
and
suggest
that
even
small
increases
in
the
concentration
of
cobalamin
complexed
with
MMACHC
may
have
therapeutic
benefit
in
children
with
the
late-onset
,
vitamin
responsive
cblC
disease
.
Diseases
Validation
Diseases presenting
"type counterpart"
symptom
esophageal squamous cell carcinoma
homocystinuria without methylmalonic aciduria
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