Huntington’s Disease

1. Huntington’s Disease Parmvir Singh 4/14/2010

2. What is Huntington’s Disease? • Huntington’s Chorea or Huntington’s Disorder • A genetic disorder[5] • 1-3% of patients have no familial history of the disease[7] • Progressive neuro-degenerative disorder • 30,000 impacted Americans • Age of onset varies: usually 30-50 yrs • Before age 20: Juvenile(10%)[5] • Very “individualized” disorder (10-20yrs)

3. What Causes Huntington’s Disease? • Autosomal dominant mutation on chromosome 4 with complete penetrance[5] • Does not skip generations • Huntingtin gene encodes huntingtin protein • Normal huntingtin protein becomes mutant huntingtin protein • PolyQ expansion from CAG repeats[5] • Genetically programmed death (apoptosis) of neurons in the brain

4. Symptoms of Huntington’s Disease • Emotional instability • Personality change • Loss of intellectual capabilities (learning, memory, etc.) • Uncontrolled movements (chorea) • Dementia • Loss of motor skills • Anxiety • Paranoia • Etc.[5][7]

5. How is Huntington’s Disease Diagnosed? • Pre-natal testing is available (chronic villus) • Look at family history to determine who is at risk • Physical and neurological exam after symptoms start to occur[3] • Cannot predict when onset will occur • A blood test • DNA from blood is analyzed to determine # of repeats • <30 repeats will not develop disease • 35-40 may or may not develop • >70 likely to develop juvenile-onset form of the disease[3]

6. Social Impact of Huntington’s Disease • Costs associated with care-giving • Decision to get tested • Inevitability • What do you think when you see someone with a neuro-degenerative disorder? • Risk of having kids • Coin toss • Age of onset

7. Nancy Wexler and Discovery of CAG repeats • Mother died of disease • Clinical psychology, 1 formal course in biology • 1969: becomes head of Hereditary Disease Foundation, founded by her father • Maracaibo, Venezuela for 20 years • Currently researching for a cure • 64 years old[13] • Mapping Fate: A Memoir of Family, Risk, and Genetic Research by Alice Wexler[1] http://ethosgeek.files.wordpress.com/2009/04/f_r31_nancywexler1.jpg

8. Woodrow Wilson Guthrie • Most famous person to ever suffer from the disease • Wrote over 1400 songs • Mother died at age 41 • Two of his daughters have died from the disease as well (6 children) • “Huntington’s Chorea Blues” • “I got this thing called chorea in my head wanna walk but I fall down instead folks say "Woody, he's just drunk again" but I haven't had a drink since I don't know when besides...I only drink when I'm alone...or with somebody My arms felt funny moving all the time and sometimes my head didn't feel like mine kept telling myself it was the Ballantine Ale and them jugs of wine on the writing trail I prefer a disease you can sober up from”[4] 7/14/1912 – 10/3/1967

9. Huntingtin • Normally a 350 kDa cytoplasmic protein • Exact function is unknown but may be involved in signaling, transportation, protein-protein interactions and prevention of apoptosis[9] • PolyQ expansion >36 near amino terminus causes Huntington’s (exon 1) • Age of onset and severity depend on # of repeats • At least 9 neuro-degenerative diseases are known to be caused by polyQ repeats[10] • Gain of function mutation • Aggregation, altered gene transcription, apoptosis, proteosome function, ubiquitination, axonal transport, endocytosis, AP transmission, and Ca2+ signaling[9] • What conformation does the expanded polyQ region adopt? • Beta sheet (aggregates), alpha helix (not involved in many protein interactions), random coil (relatively inert, but many weak interactions, polyQ believed to increase random coil length and lead to abnormal protein interactions and aggregation) • Conformation flexibility and amyloid diseases (Alzheimer’s, Parkinson’s, Prion disease) • Length of poly Q is one of the determinants of conformation

10. Amino-terminal Region of Huntingtin (PDB ID 3IOT: Maltose Binding Fusion Protein Figure 1: The three chains of maltose binding fusion protein are shown in in cyan, red, and magenta. Calcium ions are shown in yellow and zinc ions are shown in blue.

11. Amino Terminal Region of Huntingtin Figure 2: Location of the amino terminal region that is bound with maltose binding fusion protein. Huntingtin amino terminal region is shown in red. MBFP is shown in cyan. Calcium and zinc ions have been omitted for clarity. 9

12. A Closer Look at the Amino Terminal Region Figure 3: Ribbon structure of the amino terminal region of Huntingtin. The variable N-terminus region (residues 371-387) is shown in cyan. The Poly Q region (residues 388-404, equilibrates between a-helix and random coil but sometimes goes into the extended conformation as in the last few polyQ residues) is shown in red. The Poly P region (residues 405-415, induces the polyQ into extended configuration and might stabilize aggregates) is shown in yellow. The P, Q region (residues 416-419 here, but actually 15 AA) is shown in blue. Figure 4: Stick structure of the amino terminal region of Huntingtin. Residue 371 M (residue 1 in Huntingtin) is shown in orange. Residue 387 (residue 17 in Huntingtin) is shown in Magenta. The polyP region can also exist as a-helix, random coil, or extended conformations.

13. Huntingtin Interacting Protein 1- PDB ID 2QA7 • Protein family is important in endocytosis • Marker for colon, prostate, and brain cancer • “Proapoptotic” protein, leads to death of medium spiny neurons • Huntington’s disease occurs when mutant Huntingtin does not interact with HIP 1 (>36 glutamine greatly reduces interaction) • Normal huntingtin locks the HIP1 into a “rigid, extended conformation” [12]. • HIP1 protein interactor (HIPPI) recruited to form a complex with HIP1 • The two proteins interact at their pseudo-death factor domains (pDED) • Recruits procaspase-8, which activates caspase-3apoptosis[12]

14. HIP1Asymmetric Unit and Biological Assembly Figure 5: Ribbon structure of the asymmetric unit of the HIP1 coiled coil at 2.8 angstrom. Each dimer is made up of 2 parallel helices. Notice the anti-parallel configuration of the dimers. Residues 371-481 are shown. • Figure 6: Biological assembly of HIP1. Notice the 2 right handed helices twisted into a left-handed coiled coil. Helix 1 is shown in red. Helix 2 is shown in cyan. Residues 441-481 are not visible in Helix 2.

15. The Opened Region of the Coiled Coil Figure 7: The biological assembly showing the opened region near the C-terminus. The helix becomes slightly distorted due to the excessive negative charge. This region is conserved in HIP1 and HIP1 related. It is also conserved in many closely related species such as rabbits and mice. The repulsive forces are thought to make the protein more flexible[9]. Figure 8: A close-up of the opened region of HIP1. The residues E445, D446, E448, E456, E458, and E465 are thought to be crucial in this region. This region is highly negatively charged at physiological pH. This creates significant repulsive tension in the highly acidic region. The unfavorable juxtaposition of like charges in a coiled-coil motif is an important feature for kinesin activity[9].

16. Disulfidebond at Cys 430 Figure 9: A close up view of the disulfide bond between the Cys 430(yellow) of Helix 1(red) and Helix 2(cyan). It brings together repulsive forces and distorts HIP1 [9].

17. HIP1 and HIPPI Interaction Region Figure 10: Biological assembly of HIP1 showing the region that interacts with HIPPI in yellow. The residues that interact with HIPPI are F432-Y474, but the tyrosine was not available in the structure. The surface is 58 angstroms in length. It is highly positively charged and highly basic[9]. Figure 11: Zoomed in view of Phenylalanine 432(yellow). When F432 is replaced with glycine, the cell toxicity is reducedThis residue is conserved in the death effector domain of other proteins as well. K474 is unique to HIP1 and HIPPI. . K474, which was not shown in the structure is important for recruiting HIPPI [9].

18. Caspase-3 (PDB ID 1QX3) • Recall: Huntingtin with >36 Q fails to bind with HIP1This allows HIP1 and HIPPI to form a complex HIP1/HIPPI complex recruits procaspase-8 to activate caspase-3Apoptosis • Cysteine proteases; proteolytic cleavage of cellular proteins(zymogen) leading to apoptosis • Implicated in cancers and neurodegenerative disorders[11]

19. Caspase- 3 Figure 12: The biological assembly of caspase-3. The protease is composed of 2 identical catalytic domains. Each catalytic domain is a heterodimer. The 1st subunit of each heterodimer is shown in cyan. The 2ndsubunit of each heterodimer is shown in red. The two catalytic domains are anti-parallel to each other. There are 2 active sites, located at opposite ends of the 2 catalytic domains. [11]

20. Active Site of Caspase-3 Figure 13: The active site of caspase-3, delineated by binding with Ac-DEVD-CHO, is shown. The side chains of the active site make direct contact with proteins. The side chains of Tyr204, Phe256 and Trp206 delineate a hydrophobic pocket within the narrow binding cleft at the surface of caspase-3. The active site:Cys 163 (yellow), His 121 (cyan), Ser 205 (blue), Gln 160 (pink), Arg 64 (white), Arg 207 (forest green), Ser 209 (grey), Phe 250 (gold), Asn 208 (sky blue), Lys 210 (salmon), Asp 211 (green) [2]

21. Current treatments Tetrabenazine • No treatment or cure • Drugs only to help symptoms • Most drugs have side-effects like fatigue, restlessness, and over-exicatability[7] • People who exercise more during their lifetime tend to fight better [7] • Tetrabenazine • 1st FDA approved drug for Huntington’s • 8/15/2008 • Fights symptoms of Chorea[8] • Huntexil • 2/3/10 • Improves motor function[6] http://dailymed.nlm.nih.gov/dailymed/archives/image.cfm?archiveid=9363&type=img&name=xenazine-figure-01.jpg Pridopidine http://upload.wikimedia.org/wikipedia/commons/3/35/Pridopidine.png

22. Antibody MW1 (PDB ID 2GSG): Hope for a Cure? • MW1 Fv recognizes poly Q of all lengths and binds to it • Soluble polyQ are a potential target for therapeutic drugs • MW1 is a heterodimer • Multimeric MW1 has higher affinity for longer polyQ regions than shorter polyQ regions because the longer polyQ regions contain more binding sites(epitopes) • Study comparing binding with 10, 16, 25,39,46 Gln • Antibody can only bind to proteins in their pre-aggregation state • A dimeric MW1 binds 46Q with 27X greater affinity than monomeric • A tetrameric MW1 binds a 46 Q region with 39-52 x greater affinity than a monomeric MW1mutant huntingtin can have many more repeats than this • No significant increase in binding affinity for 10 and16 Q regions • A tetrameric MW1 binds shorter polyQ repeats with much lower affinity than it binds longer poly Q repeats helpful in distinguishing normal polyQ from mutated polyQ[10]

23. A Look at Antibody MW1 • Figure 14: A look at the asymmetric unit of MW1, an antibody for polyQ. Chain A of each heterodimer is shown in cyan. Chain B of each heterodimer is shown in red. MW1 recognizes polyQ via hydrogen bonds and van der waals interactions. [10] Figure 15: The biological assembly of MW1. Chain A is shown in blue. Chain B is shown in yellow. The affinity for poly Q increases as the length of the polyQ segment increases. [10]

24. There is Hope http://www.brain.riken.jp/labs/cagrds/JPEG/polyQ_e.jpg

25. References • "Alice Wexler- Mapping Fate." Hereditary Disease Foundation- Alice Wexle. Web. 10 Apr. 2010. <http://www.hdfoundation.org/bios/alicew.php>. • Chou, Kuo-Chen, David Jones, and Robert L. Heinrikson. "Prediction of the Tertiary Structure and Substrate Binding Site of Caspase-8." FEBS Letters 419.1 (1997): 49-54. • "Diagnosis of Huntington's Disease." Neurological Disorders - Dementia, Alzheimer's Disease, Parkinson's Disease, Multiple Sclerosis, Neuropathy. Web. 10 Apr. 2010. <http://neurology.health-cares.net/huntingtons-disease-diagnosis.php>. • Flannery, Tom. "Talkin' Woody Guthrie Huntington's Chorea Blues." Songaweek. 2004. Web. 10 Apr. 2010. <http://www.songaweek.com/woody/songs/huntington.html>. • "HUNTINGTON DISEASE." National Center for Biotechnology Information. Johns Hopkins University. Web. 27 Mar. 2010. <http://www.ncbi.nlm.nih.gov/entrez/dispomim.cgi?id=143100>. • Huntington's Disease Drug Works. Web. 27 Mar. 2010. <http://hddrugworks.org/>. • "Huntington's Disease Information Page." National Institute of Neurological Disorders and Stroke (NINDS). Web. 27 Mar. 2010. <http://www.ninds.nih.gov/disorders/huntington/huntington.htm>. • "Huntington's Disease: Treatments and Drugs." Mayo Clinic Medical Information and Tools for Healthy Living. 8 May 2009. Web. 10 Apr. 2010. <http://www.mayoclinic.com/health/huntingtons-disease/DS00401/DSECTION=treatments-and-drugs>. • Kim, Mee W., Yogarany Chelliah, Sang W. Kim, Sbyszek Otwinowski, and Ilya Bezprozvanny. "Secondary Structure of Huntingtin Amino-terminal Region." Structure 17.9 (2009): 1205-212. • Li, Pingwei, Kathryn E. Huey-Tubman, Tiyu Gao, Xiaojun Li, Anthony P. West, Melanie J. Bennett, and Pamela J. Bjorkman. "The Structure of a PolyQ–anti-polyQ Complex Reveals Binding According to a Linear Lattice Model." Nature Structural and Molecular Biology 14 (2007): 381-87. • Ni, Chao-Zhou, Chenglong Li, Joe C. Wu, Alfred P. Spada, and Kathryn R. Ely. "Conformational Restrictions in the Active Site of Unliganded Human Caspase-3." Journal of Molecular Recognition 16 (2003): 121-24. • Niu, Quian, and Joel A. Ybe. "Crystal Structure at 2.8Å of Huntingtin-interacting Protein 1 (HIP1) Coiled-coil Domain Reveals a Charged Surface Suitable for HIP-protein Interactor (HIPPI)." Journal of Molecular Biology 373.5 (2008): 1197-205. • "WIC Biography - Nancy Wexler." Welcome to WIC - Breaking News and Opinion, Womem. Web. 10 Apr. 2010. <http://www.wic.org/bio/nwexler.htm>.