Transcript Amyotrophic Lateral Sclerosis

Purvee Patel
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Imagine being at the peak of your life, getting
accomplishment after accomplishment, and
being in great shape.
Imagine beating numerous records in baseball,
collecting hundreds of trophies and awards,
achieving win after win, having stadiums filled
with fans screaming your name, and being called
a hero and a legend in the sport of baseball [3].
Now imagine falling and tripping through the
field that you were the master of, not being able
to catch a single ball, and losing control of your
own body as your muscles slowly deteriorate.
Imagine being told you can never play baseball
ever again.
This is the story of the
legendary baseball player, Lou
Gehrig. He was a man who played
besides Babe Ruth and broke many
records. He was loved and admired
by millions of fans and his fellow
players. But, at the age of 36,
Gehrig was diagnosed with
Amyotrophic Lateral Sclerosis- a
disease that would cause the
deterioration of his body and lead
to his death in 1941, when he was
only 37. The disease is often
referred to as Lou Gehrig’s disease
in his honor.
"I might have had a tough break, but I have
an awful lot to live for."
-Lou Gehrig
After Gehrig’s death, his wife, Eleanor
decided to dedicate her life to research to
find out more about such neurodegenerative
diseases. She supported the Muscular
Dystrophy Association, helping it become one
of the world’s leading private research
organizations and service providers for
people suffering from ALS [1].
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A neurodegenerative disease characterized by the
deterioration of the motor neurons of the primary motor
cortex, the spinal cord, and the brainstem [9].
Symptoms include muscle weakness in the upper and
lower limbs, involuntary muscle twitching, and cramps;
respiratory symptoms, new bladder dysfunction, sensory
symptoms, cognitive symptoms, and multi-system
involvement such as dementia and parkinsonism.
A person with this disease is left disabled, and often will
show symptoms of depression. Many patients with ALS
even seek physician-assisted suicide [8].
Two types of ALS- familial amyotrophic sclerosis (fALS)
and sporadic amyotrophic sclerosis (sALS) [4].
Can be used by mutations on a few different genes, but I
will focus on the two.
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It is a homodimer that is responsible for catalyzing
the detoxification of superoxide anion (free radical),
O2-, converting it to molecular oxygen and hydrogen
peroxide [5].
Each of the subunits has a 8-stranded beta barrel,
that is responsible for binding one copper and one
zinc [5].
Each subunit contains 1 zinc atom, 1 copper atom, 2
free cysteines, and 2 cysteines in a stable disulfide
bond [11].
The stability of the disulfide bond allows for the high
tertiary and quaternary structural stability of the gene
[11].
Over 120 mutations found on the SOD1 gene are
responsible for ALS [7].
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SOD1 is an enzyme that plays a key role in the cell’s
defense against harmful free radicals such as
superoxide, a reactive oxygen species [4].
It detoxifies superoxide through cyclic reduction and
the reoxidation of Cu [4].
Mutations have been found in many different regions
of the protein, including the beta barrel, the loop
regions, and at the zinc and copper binding sites [4].
These alterations can cause a conformational change
and SOD1 is unable to perform its functions.
The superoxide can, then prove to be toxic to cells,
especially the motor neurons. This degeneration of
motor neurons within the brain leads to Amyotrophic
Lateral Sclerosis or Lou Gehrig’s Disease.
[2]
Cys 111 A
Cys 111 B
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Cysteine 146 and 57 are linked
by a stable disulfide bond and
Cysteine 6 is also nonreactive.
However, Cysteine 111 may be
reactive [11].
In mutated forms of SOD1, the
disulfide bond is more vulnerable
to reduction and this reduces
stability of the molecule causing
the dimer to dissociate to two
monomers [11].
This image shows the stable disulfide bond
between Cys 57 and Cys 146. It allows for the
structural stability of the gene.
Cys 111 is the binding site of the copper ion. The copper
must be bound at the active in order to produce reactive
oxygen. Mutations in the copper binding site can lead to
ALS [11]. Solvent exposure to Cys can also cause ALS,
specifically neurodegeneration.
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Throughout the SOD1 there are loops that
connect the beta strands. Two of these loops
have significant functions.
The electrostatic loop (Loop VII) contains
charged residues that carry the superoxide,
O2-, towards the catalytic copper site [3].
The zinc loop (Loop IV) contains all of the
zinc-binding residues [3].
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Mutations at the Alanine 4 (A4) residue account for
a rapid form of ALS [5]. The A4 is the most
common mutation in SOD1 resulting in fALS [9].
◦ There are 3 specific positions- A4V, A4T, and A4S.
◦ Ala4 is located on the 1st β strand of the beta barrel.
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Mutations at the Alanine 93 (G93) residue
◦ There are 6 specific positions- G93A, G93C, G93D, G93R,
G93S, and G93V.
◦ Ala 93 is located at the short loop between the antiparallel
strands 5 and 6.
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Mutations at these site cause:
◦ Structural change in the zinc loop and electrostatic loop
(if it’s the metal bound form of A4 or G93)
◦ Destabilization of both the monomer and dimer for of
SOD1. The dissociation of the dimer into monomers due
to the reduction of the intrasubunit disulfide bond is a
factor contributing to the development of ALS [5].
◦ Destabilization of the beta barrel  small displacements
of surrounding residues that have a huge impact on the
destabilization of the monomeric forms [5].
All these mutations in A4 and G93 lead to an
accumulation of unfolded and unstable SOD1
genes that become toxic and harmful to motor
neurons.
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T54R and I113T
◦ The oligomerization rates drastically differ in the
two mutations. T54R has a slower rate compared to
SOD1 and I113T has a rate that’s more than twice
that of SOD1. This causes in stability and
conformational changes in the structure [2].
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S134N and H46R
◦ Both mutations result in defective metal binding [3].
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TDP-43 is a pathogenic protein with many
functions:
◦ It acts as a splicing factor that binds to the intron8exon9 region of the cystic fibrosis transmembrane
conductance regulator, causes exon 9 to be skipped and
this leads to an inactive form of the CFTR protein, found
in patients with cystic fibrosis [7].
◦ Pathogenic TDP-43 in cellular cytoplasmic and
intranuclear inclusions are hyperphosphorylated,
ubiquitinated and cut into C-terminal fragments. The
inclusion of these fragments into brain motor neurons
leads to neurodegenerative diseases such as ALS.
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About 14 mutations related to ALS are found on
TDP-43 [10].
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The gene contains 2 RNA- recognition motifsRRM1 and RRM2 and a C-terminal domain that
contains many glycines.
The RRM1 and RRM2 bind to TAR DNA and RNA
sequences.
The C-terminal glycine-rich domain bind to
hnRNP proteins to form complexes that play a
role in splicing inhibition (Ex. Exon-9 skipping)
[7].
Not much is known about the structure of this
molecule as it causes the degeneration of motor
neurons.
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While most RRM have 4-stranded β-sheets, RRM2
has 5 strands. It has a αβ sandwich, which the 5stranded β in between 2 α helices [7].
◦ This 5th strand (S4) is atypical and, although still not
clear its function, it is thought that this can be related to
the pathogenic inclusions of TDP-43 C-terminal
fragments in the brain cells of ALS patients.
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In order to maintain the dimeric structure, there
are 2 hydrogen bonds that form between the
atoms of Asp 247 on each subunit and 2
hydrogen bonds that form between the Glu245
from one subunit and Ile249 from the other [7].
Three 5’-end nucleotides, T2, T3, and G4, play a
key role in the interaction of RRM2 and the
single-stranded DNA strand [7].
This is a monomer
subunit of the dimer.
Mutations
Conformational
Change in Zinc
Loop and
Electrostatic
Loop
Dissociation of
Dimers and
Destabilization
of β Barrel
Accumulation
of Unstable and
Unfolded SOD1
in Motor
Neurons
ALS
Extra β strand
in the RRM2
domain
Atypical
dimerization
Pathogenic
Inclusions of
C-terminus
fragments in
brain cells
ALS
[1] “About Lou: Biography.” The Offical Website of Lou Gehrig. Web.
<http://www.lougehrig.com/index.php>.
[2] Banci, Lucia, Ivano Bertini, Mirela Boca, Vito Calderon, Francesca Cartini, Stefania
Girotto, and Miguela Vieru. “Structural and dynamic aspects related to
oligomerization of apo SOD1 and its mutants.” Proceedings of the National
Academy of Sciences of the United States of America. 106(2009): 69806985.
[3] Elam, Jennifer Stine, Alexander B. Taylor, Richard Strange, Svetlana Antonyuk, Peter
A. Doucette, Jorge A. Rodrigues, S. Samar Hasnain, Lawrence J. Hayward,
Joan Selverstone Valentine, Todd O Yeates, and P. John Hart. “Amyloid-like
filaments and water-filled nanotubes formed by SOD1 mutant proteins
linked to familial ALS.” Nature Structural Biology. 10(2003): 461-467.
[4] “Eleanor Gehrig was Champion for MDA.” MDA: ALS Division. 2010. Web.
<http://www.als-mda.org/gehrig.html>.
[5] Galaleldeen, Ahmad, Richard W. Strange, Lisa J. Whitson, Svetlana V. Antonyuk,
Narendra Narayana, Alexander B. Taylor, Jonathon P. Schuermann, Stephen
P. Holloway, S. Samar Hasnain, and P. John Hart. “Structural and biophysical
properties of metal-free pathogenic SOD1 mutants A4V and G93A.” Archives
of Biochemistry and Biophysics. 492(2009): 40-47.
[6] Hough, Michael A., J. Gunter Grossmann, Svetlana V. Antonyuk, Richard W. Strange, Peter A.
Doucette, Jorge A. Rodriguez, Lisa J. Whitson, P. John Hart, Lawrence J. Hayward, Joan
Selverstone Valentine, and S. Samar Hasnain. “Dimer destabilization in superoxide
dismutase may result in disease-causing properties: Structures of motor neuron disease
mutants.” Proceedings of the National Academy of Sciences of the United States of
America. 101(2004): 5976-5981.
[7] Kou, Pan-Hsein, Lyudmilla G. Doudeva, Yi-Ting Wang, Che-Kun James Shen, and Hanna S.
Yuan. “Structural insights into TDP-43 in nucleic-acid binding and domain interactions.”
Nucleic Acids Research. 37(2009): 1799-1808.
[8] Mitchell, J. D., and G. D. Borasio. “Amyotrophic lateral sclerosis.” The Lancet. 369(2007): 20312041.
[9] Rowland, Lewis P., and Neil A. Sheider. “Medical Progress: Amyotrophic Lateral Sclerosis.” The
New England Journal of Medicine. 344 (2001): 1688-1700.
[10] Wijesekera, Lokesh C. and P. Nigel Leigh. “Amyotrophic lateral sclerosis.” Orphanet Journal of
Rare Diseases.4(2009): 1-22.
[11] You, Zheng, Xiaohang Cao, Alexander B. Taylor, P. John Hart, and Rodney L. Levine.
“Characterization of a Covalent Polysulfane Bridge in Cu, Zn Superoxide Dismutase.”
Biochemistry. 49(2010): 1-20.