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Rough Games and the Brain: The Structure and Function of Proteins

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1 Rough Games and the Brain: The Structure and Function of Proteins
Peggy Brickman Department of Plant Biology University of Georgia 1

2 “Wrestler Chris Benoit Brain’s Forensic Exam Consistent with Numerous Brain Injuries” Science Daily (Sept 6, 2007) Sports Legacy Institute President, Chris Nowinski, a former Harvard football player and ex-professional wrestler, after hearing of Chris Benoit’s death, phoned Benoit’s father, Michael, with a ghoulish request: to borrow the remains of his son’s brain. “When Nowinski contacted me about conducting tests on Chris’ brain. I was extremely hesitant given the circumstances surrounding my son’s death,” said Michael Benoit. 2

3 Part I: The Case of Chris Benoit
Twice recognized by the World Wrestling Entertainment as the world heavyweight champion, Canadian Professional Wrestler, Chris Benoit, was booked to win his third championship the weekend of his death. Instead, that weekend he killed his wife and strangled his seven year-old son to death, and then hung himself using cords from a weight machine. Medical examiners concluded that the elevated testosterone levels in Benoit’s body (probably prescribed to remedy deficiencies resulting after prior steroid abuse) did not contribute to his violence. Nowinski believed that he did know the cause: repetitive head injuries Benoit and other athletes suffer in contact sports that result in Chronic Traumatic Encephalophathy (CTE). 3

4 Rough Games and the Brain…
Impact: when the head slams into a hard surface, the skull stops abruptly, while the brain, floating in cerebral fluid, continues to move and is shaken and sometimes bruised. Animation: Along with other damage, one result is that a nerve cell protein called ß-Amyloid Precursor Protein (ßAPP) is cut into pieces called ß-amyloid. Over time, neurofibrillary tangles containing tau protein fibers accumulate. 4

5 What are the functions of ß-APP, ß-amyloid and tau?
Results in axon growth and signaling to other neurons. (ß-APP increases the levels of cell structure protein, actin, for example. Tau is a protein found with cell structure microtubules.) Healing: ßAPP triggers inflammatory response and acts as anti-coagulant to prevent blood clots. May increase the expression of specific genes inside the cells. Why would releasing ß-APP cause harm? 5

6 Benoit’s Brain Neurofibrillary tangles (NFTs, like those seen below) have been found in patients suffering cognitive and intellectual dysfunction, including major depression. Similar NFTs were found in Chris Benoit’s brain. Did wrestling cause his death? 6

7 Proof needed to demonstrate link
The Sports Legacy Institute (SLI), which oversaw and coordinated the testing, is an independent medical research organization dedicated to studying the long-term effects of head injuries in sports. Most CTE occurs in boxers, but also in professional football players. Ten percent of retired pro-football players suffer from depression, the same as the general population. Before recommending drastic changes or additional rules for athletes, what type of evidence or experiments would you need to see linking concussions to CTE and depression? 7

8 Concussions and CTE Study of more than 2,500 former NFL players by the Center for the Study of Retired Athletes at UNC found that cognitive impairment, dementia, and depression rose proportionately with the number of concussions they had sustained (Guskiewicz et al,. 2003, 2005, 2007). Those who had sustained 3 or more concussions were more likely to experience “significant memory problems” and 5 times more likely to develop mild cognitive impairment. How might the NFL counter this data? 8

9 Dementia: CTE and Alzheimer’s Disease
Early pathologists stained sections of an Alzheimer’s brain with iodine and saw large brown regions. Named ß-amyloid mistaken for amylose Debate: Is it carb or lipid or protein? What are the differences? 9

10 CQ#1: Your answer should include the series of numbers in order from the choices below. Macromolecules are polymers composed of monomers. For example polysaccharides like ___ are made of the repeating monomer ___. Fats like ____ are made of the repeating monomer ____. Proteins are polymers made of repeating monomers called ____. 1. Triglycerides 4. Nitrogen 7. Steroids 2. Amino acids 5. Nucleic Acids 8. Cellulose 3. Glucose 6. Fatty Acid 9. Phosphates 10

11 II: Amino Acids: Specific composition/ comparison with
II: Amino Acids: Specific composition/ comparison with other biological molecules. Tau protein tangles like those in Benoit’s brain and ß-amyloid: 1984: Scientists purified protein from the tangled fibrils seen in Alzheimer’s brains. 1987: cloned the gene which coded for a 695 amino acid protein (ß-APP) which spanned the phospholipid bilayer. ß-amyloid are fragments of the protein that are composed of 28 amino acids strung together. 11

12 Not all early-onset dementia comes from brain trauma.
Most Alzheimer’s occurs in the elderly but about 1/2 million of the 5 million people who develop dementia or Alzheimer’s each year are under 65. They didn’t all have brain trauma, but some had a family history of the disease. Maybe these athletes’ dementia was inherited (5% of Alzheimer’s is caused by inherited dominant mutations.) People with the mutation develop symptoms much earlier than typical (~age 51). Some of these mutations (15%) are changes in the amino acids in the ß-APP protein. 12

13 Structure of Amino Acids
All amino acids have an amino and a carboxyl (acid) attached to a central carbon along with Hydrogen H N C R OH O side-chain amino group carboxyl 13

14 Amino acids differ in the R group
phenylalanine H N C OH O CH2 20 Different R groups: Some Non-polar (Hydrophobic) Polar (Hydrophilic) Some even contain sulfur H N C CH2 OH O H2N glutamine 14

15 Amino Acids Form Proteins
Condensation reactions create a covalent bond (Peptide Bond) Forms Polypeptides DNA gene mRNA Primary structure: H N C OH O CH3 H N C OH O H N C OH O CH2OH H alanine serine glycine H2O H2O H N C O CH3 CH2OH OH met ala ser gly gln thr phe glu leu lys tyr pro 15

16 Each protein has a different pattern of amino acids
gly cys ala ile val glu gln ser leu insulin tyr asp his val ser ala glu phe arg gly tyr B amyloid asp his phe ser ala glu arg gly tyr val B amyloid mutation The R-group of each amino acid is different, and thus imparts different qualities to the protein. Hydrophilic amino acids are attracted to other hydrophilic substances, and hydrophobic are not. 16

17 III. How Proteins Differ: Function
Structure Protection Metabolic Enzymes Channels & pumps Transport Gene Expression and Regulation Cell Signaling (Hormones) Movement 17 17

18 How Can Proteins Have Many Different Functions? Scrabble Analogy
Proteins: 20 amino acids: Glutamine Isoleucine Asparagine Serine Threonine Lysine Arginine Carbohydrates: Glucose Hard to make more than one word E1 I1 N1 S1 T1 K5 R1 G1 G1 G1 G1 G1 G1 18 18

19 CQ#2: What do proteins, lipids, and carbohydrates all have in common?
A: Type of reaction that links them covalently into large polymers of repeating monomers. B: Having more than a dozen monomers present in one polymer. C: Presence of the element N. D: Having monomers that can be either hydrophobic or hydrophilic. 19

20 IV. Proteins Fold into Active Shape
Primary Structure: sequence of amino acids in polypeptide. For each protein (ß-APP) the primary structure is always identical. asp his val ser ala glu phe arg gly tyr ß-amyloid asp his phe ser ala glu arg gly tyr val ß-amyloid mutation Why would changing one amino acid cause the whole protein to change shape? 20

21 Secondary Structure Hydrogen bonds create shape.
Some examples of permanent structures: a-helix or b-pleated sheet primary structure secondary structure amino acids ahelix b-sheet 21 21

22 Tertiary Structure 3D packing of polypeptides. Often involve H-bonds.
primary structure secondary structure amino acids ahelix b-sheet tertiary structure 22 22

23 quanternary structure
Quaternary Structure Interactions between two or more polypeptide chains. Not found in all proteins. primary structure amino acids ahelix b-sheet secondary structure b-sheet tertiary structure ahelix quanternary structure 23 23

24 CQ#3: Proteins such as the ß amyloid and the mutant ß amyloid that results in early-onset Alzheimer’s differ from one another because: Peptide bonds linking the amino acids differ from one protein to another. The two proteins have a different combination of amino acids along the chain of the polypeptide. Presence of the element N is only found in one protein. The two proteins each contains their own unique types of amino acids. The number of amino acids in the chains differ. 24

25 IV. ßAPP Proteins and Alzheimer’s Disease
neurons ß-amyloid precursor protein (ßAPP) found in the phospholipid outer cell membranes of neurons. ßAPP Outside the Cell Why would changing one amino acid in the mutation affect the proteins so much? Inside the Cell 25 25

26 CQ#4: Which of the following amino acids might you NOT expect to find in the intra-membrane region of the ß-APP protein? C N OH O H CH CH3 C N OH O H CH3 A B valine alanine C N OH O H C N OH O H O- C D serine 26 aspartate 26

27 ßAPP and Alzheimer’s typically polypeptides are converted back into amino acids (by proteases) by a reaction called hydrolysis. Also used to remove proteins when they are no longer needed, and to send signals. met ala ser gly gln thr phe glu leu lys tyr pro H N C O CH3 CH2OH OH H2O H2O H N C OH O CH3 alanine CH2OH serine glycine 27 27

28 Brain Trauma Speeds Hydrolysis
ß-amyloid mutant more sticky? ß-amyloid precursor protein (ßAPP) ßAPP Outside the Cell ß-secretase ß-amyloid ß-amyloid doesn’t tend to clump by itself, rather it starts to stick to existing clumps, and then these begin damaging nerve cells. Takes decades to start to see clumps, and then more to see damage as a result of clumps. Why most people who get Alzheimer’s are old. Inside the Cell 28 28

29 ß-amyloid aqueous environment outside nerve cell CQ#5: Which amino acid change in the ß-amyloid protein fragment would be most likely to make it stick together? Replacing an amino acid with a hydrophilic R-group with one that is hydrophobic. Replacing an amino acid with a hydrophobic R-group with one that is even more hydrophobic. Replacing an amino acid with a hydrophobic R-group with one that is hydrophilic. Replacing an amino acid with a hydrophilic R-group with one that is even more hydrophilic. 29 29

30 Shape and stickiness of protein dictated by amino acids
Changing amino acid 717 from valine to phenylalanine can produce ß-amyloid that sticks much better to other ß-amyloid fragments and causes plaques to form sooner in familial Alzheimer’s. H N C CH OH O CH3 valine phenylalanine H N C OH O CH2 30 30

31 Peptide bonds B. Hydrogen bonds
CA#6: Even if they don’t have the mutation, hard-hitting athletes may want to take some precautions. Perhaps taking a chemical designed to be more attractive to the basic R-groups on ß-amyloid fragments than the fragments were to each other thus preventing their aggregation. Phase III trials of such a drug (Alzhemed) in 2007 failed to show benefits in 1,000 Alzheimer’s patients. What kind of bonds in the ß-amyloid protein was Alzhemed meant to disrupt? Peptide bonds B. Hydrogen bonds C. Non-polar covalent bonds D. Polar covalent bonds 31

32 Impose mandatory rest after head injuries.
CQ#7: OK, so that drug didn’t work. What do you think the NFL should do for football players? Test for Alzheimer’s predisposition mutations as part of physicals prior to hiring. Impose mandatory rest after head injuries. Fine or fire coaches breaking the rules. Change how the game is played so that concussions are less likely to occur. It’s just part of the game like shot knees and back pain. Make sure the former players have great health insurance that includes psychiatric coverage and nursing home care. 32

33 NFL and Concussions Although continuing to support its policies on concussions and rejecting any link between concussions and depression & CTE, NFL has several initiatives: League and players union created a fund to help pay medical expenses of players suffering from dementia. New guidelines include: obligatory neuropsychological testing, “whistle-blower system” for anonymous reports of any coach’s attempt to override the wishes of concussed players or medical personnel. Handheld EEGs, and functional magnetic resonance imaging also in testing for early diagnosis. Soon may be new technique to replace the crude memory tests coaches and trainers routinely use to determine if a player can get back on the field. An experimental handheld device that uses electroencephalogram (EEG) to read the brain's electrical activity might help. Under development by Roy John, director of the Brain Research Laboratories at New York University.I t's currently being tested in the emergency rooms of three hospitals to determine if it can be used to quickly assess brain damage associated with motor accidents, stroke, seizure, and other sources of head trauma. Mark Lovell and his colleagues at the University of Pittsburgh School of Medicine used functional magnetic resonance imaging to measure brain activity in 200 high-school athletes with concussions, both right after injury and after the athlete had fully recovered. Athletes who had abtypical activity in the frontal cortex--the part of the brain that is likely to hit the skull when an athlete suddenly stops--initially scored lower on cognitive tests and had longer recovery times. When the patient's symptoms went away, brain activity returned to typical."These findings will help better define what recovery is," says Lovell. "If an athlete isn't reporting symptoms correctly, doctors could send someone back in who is in danger of having severe brain injury. 33

34 CQ#8: You might want to know if you have a mutation in another gene, ApoE4 that changes the amino cysteine to arginine, and affects age of onset of Alzheimer’s. How could changing the sequence result in a change in function? It could change the amount of the protein produced. It could change the protein’s 3-D shape and thus its ability to interact with other proteins. It could change where the protein is located in the cell. It could change the number of amino acids. Copies of ApoE 4 mutation Average age of onset of Alzheimer’s 2 68 1 75 84 34

35 Alzheimer’s Prevention
OK. You’re not a heavy-hitting athlete and don’t have any family history of Alzheimer’s, just like the 4.8/5 million people with Alzheimer’s over 65. So, there’s a 10% chance you’ll get it by age 65, and a 50% chance you’ll get it by 85. People who eat the so-called 'Mediterranean diet' (fruits, vegetables, bread, pasta, fish, olive oil and a little red wine, but low in dairy products and red meat) have a lower risk of Alzheimer's disease. So do people who exercise regularly, use (not lose) their minds, and have been taking non-steroidal anti-inflammatories like aspirin for least two years. Late-onset AD, the more common form, develops after age 65. In 1992, researchers found that certain forms of the apolipoprotein E (APOE) gene can influence AD risk: APOE ε2, a rarely occurring form, may provide some protection APOE ε3, the most common form, plays a neutral role; and APOE ε4, which is found in about 40 percent of people with AD; APOE ε4 lowers the age of onset and thus increases risk. (Having this gene form does not mean that a person will definitely develop AD; it only increases risk. Many people who develop AD do not have the APOE ε4 form. In a recent study, the mean age at AD onset was 68 years in patients with 2 E4 alleles, 75 years with 1 E4 allele, and 84 years in individuals with no E4 alleles. E3 is more similar to E4 than E2, as it carries only 1 amino acid substitution: Cys112 in E3 instead of Arg112 in E4. Conversely, E2 has 2 amino acid differences with E4: Cys112 instead of Arg112 and Cys113 instead of Arg113, as in E4 and E3.In the brain, the apoE proteins have been found associated with the characteristic AD plaques and with the tau protein in neurofibrillary tangles.[6] In vitro, apoE4 can increase phosphorylation of tau. Overexpression of human apoE4 or E3 in transgenic mice led to an increase in plaque formation, with the association of E4 with human amyloid precursor protein (hAPP), and with Abeta peptides in the plaques, a decrease in presynaptic terminals, and an increase in tau phosphorylation and in surrounding gliosis. Indeed among the 3 allelic forms, apoE4 folds in the least stable; E2 folds in the most stable conformation; and E3 shows an intermediate stability. Hence came the idea to find a small molecule able to bind E4 near the Arg61 residue to inhibit these domain interactions and induce more "spreading" in the molecule, and thus obtain an apoE3-like conformation. Instability may lead to Increased degradation; Alterations in cell signaling Increased binding to lipids; Modifications in protein-protein interactions; Increased membrane binding; Changes in transport through membranes; and Modified interactivity with cellular receptors 35

36 Interested in Reading More?
Scientific American: has a great set of articles on the latest research, including one in March 2009 on prions and Alzheimers. Articles: Medline search Lobo, I. (2008) Epistasis: Gene interaction and the phenotypic expression of complex diseases like Alzheimer's. Nature Education 1(1) McKee et. al. (2009) Chronic Traumatic Encephalopathy in Athletes: Progressive Tauopathy After Repetitive Head Injury. J. Neuropathol Exp Neurol.

37 Image Credits Slide 3: Description: Photo of Chris Benoit.
Source: From Wikimedia Commons, by dani nuestro from Bangkok, Clearance: Creative Commons Attribution-Share Alike 3.0 Unported License(CC BY-SA 3.0). Slide 4 Description: Concussion illustration. Source: Adapted from Wikimedia Commons, original by Max Andrews. Clearance: Used in accordance with the Creative Commons Attribution-Share Alike 3.0 Unported license. Slide 6 Description: Neurofibrillary tangles in the Hippocampus of an old person with Alzheimer-related pathology. Source: Wikimedia Commons, by Patho. Slide 9 Description: Illustration comparing healthy brain and Alzheimer’s brain. Source: Alzheimer’s Association, Clearance: Used with permission.

38 Slide 11 Description: Illustration of plaques
Slide 11 Description: Illustration of plaques. Source: Alzheimer’s Association, Clearance: Used with permission Slide 18 Description: Scrabble analogy for the greater complexity of proteins due to the greater number and type of monomers. Source: Peggy Brickman, adapted from “Nutrition” Insel, Turner, and Ross. Slides Description: Illustration of four levels of protein structure. Source: Wikimedia Commons, Clearance: Used in accordance with the Creative Commons Attribution-Share Alike 3.0 Unported license.

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