Transcript Spinal Muscular Atrophy

SPINAL MUSCULAR ATROPHY
A Detailed Look at the Mechanism of a Neurodegenerative
Disease That Causes Muscle Deterioration
By: Afif Hossain
INTRODUCTION TO SMA
 Atrophy-
•
•
•
•
•
•
the medical term for wasting or shrinkage,
which is what generally happens to muscles when they’re
not active
Autosomal recessive disease that affects about 1 in every
6000 individuals
Linked to the survival of motor neuron gene, SMN1
SMN protein discovered in 1995
Cause:
loss or degeneration of nerve cells in the spine
known as motor neurons
Loss of motor neurons is a result of a deficiency in a motor
neuron protein called survival of motor neurons, or SMN
for short
SMN plays an integral role in normal motor neuron
function, and may even directly affect muscle cells
themselves1
INTRODUCTION TO SMA
•
•
•
•
•
Mechanism:
motor neurons,
located in the spinal cord, signal
muscle contraction by carrying a
signal from the spinal cord, down
long, wire-like projections, to muscles
Without ample motor neurons, signal
transduction is hindered and muscles
cannot function, leading to atrophy
Specifically, mutations on
chromosome 5 debilitate the
production of SMN protein
Greater levels of SMN protein lessen
the severity of the disease, which is
loosely related to age (if onset is
later, then degeneration of motor
neurons will occur later)1
SMA is entirely genetic
TYPES OF SMA

•
•
•

•
•
•
•
•
Type 1 SMA:
Acute and infantile- from birth to 6 months of age
Children often have trouble breathing, swallowing, and sucking
and suffer from hypotoniaa
High fatality rates: mean survival is 5.9 months, 95% of cases die
by 18 months
Type 2 SMA:
Most common type of SMA, often related with developmental
motor delay
Chronic and infantile- onset between 6 and 18 months after birth
Muscles closer to the center of the body (proximal muscles) are
usually more affected or earlier (thighs as opposed to the lower
legs)1
Respiratory issues and spinal curvature (scoliosis) also present
major problems for future health
Survival into young adulthood or even later can be expected (2 to
30 years)2
a progressive muscle weakness and flaccid or reduced muscle tone
TYPES OF SMA
 Type
•
•
Chronic and juvenile- onset of SMA 18 months after birth
Muscular functioning is much more developed and life
expectancies are often normal, but respiratory issues and
spinal curvature still need to be monitored
 Type
•
•
•
3 SMA:
4 SMA:
Milder form of SMA that affects adults (normal life
expectancies)
There are forms of SMA that are not related to SMN and
do not originate from mutations of chromosome 51, but
those disorders will not be the focus of my presentation
Mental and emotional development and sensation are
entirely normal in SMA
FREQUENCY AND DIAGNOSIS
•
•
•
•
•
•
Second most common autosomal-recessive inherited
disorder after cystic fibrosis
SMA types I and III each account for about one fourth of
cases, whereas SMA type II accounts for one half of all
cases by itself
SMA I: Weakness is greater in proximal than distal
muscles and may mimic muscle disease (myopathy) and
limbs and joints may be deformed at birth
SMA II: Infants cannot get to a sitting position on their
own but may retain that position if placed into it;
proximal muscle weakness still present
All patients with spinal muscular atrophy retain at least
1 copy of SMN2, which generates only 10% of the amount
of full-length SMN protein (compare to SMN1)
Possible therapeutic pathway is to promote SMN2 to
function like the missing SMN1 gene2
SOCIAL IMPLICATIONS AND FUNDING
Bioethical Issues




Disease can be detected in prenatal testing
Sharing genetic information between family members
Family planning- in vitro fertilization
Although SMA cannot be prevented and the
symptoms cannot be mitigated, (since it is entirely
genetic), testing still allows individuals and families
to cope with the disease6
Economic Aspects



Over $60 million available for funding by the
Spinal Muscular Atrophy Foundation
$400 for a carrier screen and $260,000 for the
lifetime cost of a child with severe disease
Researchers have concluded that 11,000 women
would have to be screened to prevent one case of
SMA, at a cost of $4.7 million per case averted7
INTRODUCTION TO SMN TUDOR DOMAIN, 1MHN
•
•
•
•
•
The SMN protein plays an important role in the assembly of
the spliceosomal small nuclear ribonucleoproteinb complexes.
snRNPs are essential to the removal of introns from premRNA, a critical aspect of post-transcriptional modification of
RNA
The SMN protein is found in both the nucleus, where it
localizes near snRNPs, and the cytoplasm, where the SMN
protein plays an important role in the assembly of snRNPs
In order for snRNPs to assemble, they must interact with Sm
proteinsc
In the Tudor Domain of the SMN protein, the SMN proteins
must bind to arginine-glycine (RG) rich C-terminal tails of the
Sm proteins in vitro3
b RNA-protein complexes that combine with unmodified pre-mRNA and various other
proteins to form a spliceosome, a large RNA-protein molecular complex upon which splicing
of pre-mRNA occurs
C A protein that belongs to a group of seven core components of splicing small nuclear
ribonucleoprotein particles
α1
Figure 1
Figure 1. Structural
depiction of the SMN
Tudor Domain. One
alpha helix is shown
between the fourth and
fifth beta sheet. The five
beta sheets are shown in
orange, connected by
three loops, shown in
blue. The N and C
terminals are
emphasized with cyan.
The antiparallel beta
sheets form a beta barrel
structure .
SMN TUDOR DOMAIN AND RG TAILS
•
•
•
•
•
Recent studies show that the Sm proteins, which
contain symmetrically dimethylated arginine residues
(sDMA), modify sDMAs in vivo
Arginine methylation increases the affinity for the
interaction of the SMN Tudor domain with RG repeats,
shown by the saturation of the binding site at a much
lower RG:protein ratio
The SMN Tudor domain uses the same binding pocket
for the interaction with methylated and non-methylated
RG tails
Differences in the saturation end points indicate amides
(Trp102, Tyr109, Tyr127 and Tyr130) close to sDMA
methyl groups, which form the binding pocket
Glycine residues in the methylated RG repeats is
postulated to provide conformational stability required
for RG-Tudor Domain interactions3
Figure 2. Crystal Structure of the
SMN Tudor Domain. (a) Ribbon
representation of the SMN Tudor
Domain in blue, highlighting the
binding pocket in yellow (aromatic
side chains include Tryptophan 102
and Tyrosine 109, 127, and 130).
Glutamate 134 is shown in green.
(b) Close up view of the binding
pocket.
Figure 2a
Figure 2b
SMN TUDOR DOMAIN, 1MHN
•
•
•
Modification of sDMA proteins is important
because it strongly enhances the affinity of the
SMN/Sm interaction and has been implicated in
the regulation of U snRNP assembly (similar to
methylation, involves Gemin2-7)
Glu134, which when mutated to lysine is linked to
type I SMA, is located close to the sDMA binding
pocket and abolishes Sm binding in vitro and
interferes with snRNP assembly in vivo3
The point mutation E134K hinders proper
recognition of the RG-repeats by the SMN
protein and the SMN Tudor domain, which
causes SMA I
INTRODUCTION TO THE GEMIN6-GEMIN7
HETERODIMER FROM THE HUMAN SMN COMPLEX
•
•
•
•
•
•
As mentioned before, the SMN complex’s function is assembly of
small nuclear ribonucleoproteins (snRNPs), which are major
components of spliceosomesd
SnRNPs consists of one U snRNA molecule, a core composed of
seven highly conserved Sm proteins, and several snRNP-specific
proteins
The major U snRNAs are exported to the cytoplasm where the Sm
protein core is assembled on the Sm site of the U snRNA and the
5′-cap is hypermethylated (modification leads to snRNP
formation)4
The SMN complex contains several proteins called Gemins2–7
Gemin6 and Gemin7 have a two-fold-symmetric heterotetramer
symmetry and similar folds, with a five-stranded bent β sheet
flanked by α helices, which form a hydrophobic pocket and only
allow for highly specific interactions
Since reduced levels of SMN, as a result of deletions or “loss-offunction” mutations in the SMN gene cause SMA, the role of
Gemin6/7 is vital to SMN protein production
specialized RNA and protein subunits that removes introns from a transcribed pre-mRNA
segment
d
STRUCTURAL VIEW OF GEMIN6 AND GEMIN7
Figure 3.
Gemin6 in cyan
and Gemin7 in orange.
Chains highlighted, N and C
termini shown in their
respective colors. SMN
protein binding sites are
shown in gray. Having
similar folds, with a fivestranded bent β sheet flanked
by α helices, the two form a
heterodimer connected by β4
of Gemin6 and β5 of Gemin7,
resulting in the formation of a
continuous 10-stranded β
sheet. The longer N-terminal
helix of Gemin7 packs tightly
into a hydrophobic pocket
formed by α1, β2-β4, and α2 of
Gemin6, which is flanked by a
network of hydrogen bonds
formed between α1 side
chains in Gemin7 and pocket
residues of Gemin6. This
corroborates the high
specificity to which Gemin6
and Gemin7 interact.4
Figure 3
SIMILARITIES BETWEEN GEMIN6/7 AND SM
PROTEINS
•
•
•
•
•
Amino acid sequences in Gemin6 and Gemin7 contain folds
that are similar to those observed in the spliceosomal Sm
proteins
For this reason, Gemin6/7 could serve as an Sm dimer
surrogate, binding to individual Sm proteins or to Sm
subcomplexes to form a ring-like structure in preparation
for snRNA loading
Therefore, Gemin6 and Gemin7 have been shown to
interact in vitro with the human Sm proteins, binding and
organizing Sm proteins in preparation for snRNP assembly.
This correlates to both the proposed theory that human Sm
proteins form heptameric rings when assembled on the Sm
sites of U snRNAs and the function of symmetrically
dimethylated arginine residues in the RG-rich tails to
recognize Sm proteins
Gemin6/7 facillitates Sm/SMN protein interactions4
SM PROTEIN AND GEMIN6/7 INTERACTIONS
(b) Specific residues of
interaction. In SmB/B’, Asn39
hydrogen bonds with Gly74 and
Asp35, linking the C-terminal
segments of strands β2 and β4
with the N terminus of β3. In
Gemin6, Asn43, Gly62, and Asp38
form an identical hydrogen
bonding network. Similarly, SmD3
residues Asp37, Asn40, and Tyr62
and Gemin7 residues Asp96,
Asn101, and Tyr103 form the
same hydrogen bonding patterns4.
Figure 4b
Figure 4a
Figure 4. Mechanism that Sm proteins interact
with Gemin 6/7. (a) There are two binding surfaces
that Gemin6/7 could bind to Sm proteins: The β5
surface of Gemin6 and the β4 surface of Gemin7 are
both exposed and available for interaction with Sm
proteins in the Gemin6/7 complex. Sm Proteins
represented with arrows.
HUMAN DCPS (3BL9) AND ITS MECHANISM
•
•
•
•

Although SMA is determined by a mutation or deletion
in both copies of the SMN1 gene, the severity of SMA is
modified by the second gene, SMN2, that produces an
mRNA that is incorrectly spliced with the deletion of the
last exon
When SMN2 has a point mutation, it can only produce
10% of the SMN protein necessary for normal muscular
functioning
DcpS modulates gene expression at the level of mRNA
turnover and pre-mRNA splicing
DcpS functions in the last step of mRNA decay to
hydrolyze the mRNA cap structure (m7GpppN) following
3′to 5′ exonucleolytic decay and 5’ end decapping, which
causes mRNA decay (dephosphorylates GDP to GMP in
the residual cap structure, reducing the energy
necessary for the mRNA to undergo translation)5
DcpS continues to decap mRNAs that lack a stop codon,
causing SMN2 protein levels to decrease
(d)
(a)
Figure 5. (a) Active site His277Asn mutation in
complex with m7GpppG substrate and inhibitor
(b)
(b)
m7GpppG
base
occupies a narrow
pocket staking
between Leu206 and
Trp175, and also forms
hydrogen bonds to the
Glu185 side chain and
the carbonyl oxygen of
Pro204
In order for inhibition to be effective, both
the open and closed active sites must be
inhibited. Since D157493 can only bind to
the closed active site, a point mutation
from Histadine to Asparanine inhibits the
open active site.
(c)
(c,d) The open
active site is
conformationally
“open” because it
is surrounded by
α helices,
whereas the
closed active site
is
conformationally
“closed”, because
it is tangled in β
sheets5.
POSSIBLE TREATMENTS: D157493
(C5-QUINAZOLINES)
•
•
•
•
Although the exact role and mechanism of DcpS in relation
to the SMN protein is not clearly understood, there is a
general consensus that DcpS is the therapeutic target of
SMA because its role in inhibiting the production of the
SMN protein by closing the SMN protein’s substrate-bound
complex and opening its product-bound complex
Therefore, a group of drugs called C5-quinazolines,
commonly referred to as D157493, are currently being
tested for their therapeutic value in treating SMA
C5-quinazolines are experimental drug compounds that are
in vitro potent DcpS inhibitors, causing increases in
SMN mRNA levels in SMA and corresponding increases in
SMN protein production (by inhibiting DcpS at its active
sites)
Other drugs, like histone deacetylase inhibitors, including
valproic acid have also been shown to increase
transcription of the SMN2 gene and thus increase levels of
the SMN protein5
REFERENCES
Video Clip: Fight Spinal Muscular Atrophy. FightSMA.
http://www.youtube.com/watch?v=aZUVFRAyl_I
1.
2.
3.
Muscular Dystrophy Association. “Facts About Spinal
Muscular Atrophy (SMA).” MDA Publications. 2010.
Tsao, Bryan and Armon, Carmel. “Spinal Muscular
Atrophy.” eMedicine. 14 Jan 2009.
Sprangers, Remco, Groves, Matthew R., Sinning,
Irmgard and Sattler, Michael. “High-resolution X-ray
and NMR Structures of the SMN Tudor Domain:
Conformational Variation in the Binding Site for
Symmetrically Dimethylated Arginine Residues.”
Science Direct. March, 2003.
REFERENCES CONT’
4.
5.
6.
7.
Ma, Yingli, Dostie, Josée, Dreyfuss, Gideon and Van
Duyne, Gregory D. “The Gemin6-Gemin7
Heterodimer from the Survival of Motor Neurons
Complex Has an Sm Protein-like Structure.” Science
Direct. July 2005.
Singh, Jasbir, Salcius, Michael, Liu, Shin-Wu, and
Staker, Bart L. “DcpS as a Therapeutic Target for
Spinal Muscular Atrophy.” National Institute of
Health Public Access. November 2008.
Norrgard, Karen. “Medical Ethics: Genetic Testing
and Spinal Muscular Atrophy.” Scitable. Web. 2008.
Preidt, Robert. “Screening for Spinal Muscular
Atrophy Not Cost-Effective: Study.”
MedicineNet.com. Web. 2 Februrary, 2010.