1. Purvee Patel

2.  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.

3. 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.

4. "I might have had a tough break, but I have an awful lot to live for." -Lou Gehrig

5. 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].

6.  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.

7.  It is a homodimer that is responsible for catalyzing the detoxification of superoxide anion (free radical), O 2 -, 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].

8.  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.

9. [2]

10. Cys 111 A Cys 111 B

11.  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].  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. This image shows the stable disulfide bond between Cys 57 and Cys 146. It allows for the structural stability of the gene.

14.  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, O 2 -, towards the catalytic copper site [3].  The zinc loop (Loop IV) contains all of the zinc-binding residues [3].

15.  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 1 st β strand of the beta barrel.  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.

17.  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.

18.  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].  S134N and H46R ◦ Both mutations result in defective metal binding [3].

19.  TDP-43 is a pathogenic protein with many functions: ◦ It acts as a splicing factor that binds to the intron8- exon9 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.  About 14 mutations related to ALS are found on TDP-43 [10].

20.  The gene contains 2 RNA- recognition motifs- RRM1 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.

21.  While most RRM have 4-stranded β-sheets, RRM2 has 5 strands. It has a αβ sandwich, which the 5- stranded β in between 2 α helices [7]. ◦ This 5 th 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.  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].

22. This is a monomer subunit of the dimer.

25. 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

26. Extra β strand in the RRM2 domain Atypical dimerization Pathogenic Inclusions of C-terminus fragments in brain cells ALS

27. [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): 6980- 6985. [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.

28. [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): 2031- 2041. [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.