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Rough Games and the Brain:The Structure and

Function of Proteins

Peggy BrickmanDepartment of Plant Biology

University of Georgia

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“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.

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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).

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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: http://www.youtube.com/watch?v=AmAML1-F2LE• 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.

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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?

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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?

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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?

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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?

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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?

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1. Triglycerides 4. Nitrogen 7. Steroids2. Amino acids 5. Nucleic Acids 8. Cellulose3. Glucose 6. Fatty Acid 9. Phosphates

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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 ____.

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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

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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.

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Structure of Amino Acids

• All amino acids have an amino and a carboxyl (acid) attached to a central carbon along with Hydrogen

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H

H

HN C

R

C

OH

O

side-chain

amino group

carboxylgroup

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Amino acids differ in the R group

• 20 Different R groups: Some• Non-polar (Hydrophobic)

• Polar (Hydrophilic)

• Some even contain sulfur

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phenylalanine

HH

HN C C

OH

O

CH2

HH

HN C

CH2

C

C

OH

O

H2N O

glutamine

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Amino Acids Form Proteins

• Condensation reactions create a covalent bond (Peptide Bond)

• Forms Polypeptides• DNA gene mRNA• Primary structure:

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HH

HN C C

OH

O

CH3

alanine

HH

HN C C

OH

O

CH2OH

serine

HH

HN C C

OH

O

H

glycine

H2O H2O

HH

HN C C

O

CH3

H H

N C C

O

CH2OH

H H

N C COH

O

H

met ala ser glygln thr phe glu leu lys tyr pro

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Each protein has a different pattern of amino acids

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gly cys cys alaile val glu gln ser val cys ser leuinsulin tyr

• The R-group of each amino acid is different, and thus imparts different qualities to the protein.• Hydrophilic amino acids are attracted to other hydrophilicsubstances, and hydrophobic are not.

asp his val serala glu phe arg gly tyr glu val hisB amyloid his

asp his phe serala glu phe arg gly tyr glu val hisB amyloidmutation

his

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III. How Proteins Differ:Function

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Structure

Metabolic Enzymes

Transport

Cell Signaling (Hormones)

Movement

Gene Expressionand Regulation

Channels& pumps

Protection

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How Can Proteins Have Many Different Functions? Scrabble Analogy

• Carbohydrates: Glucose

• Hard to make more than one word

• Proteins: 20 amino acids:• Glutamine• Isoleucine• Asparagine• Serine• Threonine• Lysine• Arginine

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G1

G1

G1

G1

G1

G1

G1

G1

G1

G1

G1

G1

E1

E1

I1I1

N1

N1

S1

S1

T1

T1

K5

K5

R1

R1

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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.

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CQ#2: What do proteins, lipids, and carbohydrates all have in common?

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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 serala glu phe arg gly tyr glu val hisß-amyloid his

asp his phe serala glu phe arg gly tyr glu val hisß-amyloidmutation

his

Why would changing one amino acid cause the whole protein to change shape?

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Secondary Structure

• Hydrogen bonds create shape.• http://www.stolaf.edu/people/giannini/flashanimat/proteins/

hydrophobic%20force.swf• Some examples of permanent structures: • -helix or -pleated sheet

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primary structure

secondary structure

amino acids

helix-sheet

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Tertiary Structure

• 3D packing of polypeptides. Often involve H-bonds.

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primary structure

secondary structure

amino acids

helix-sheet

tertiary structurehelix

-sheet

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Quaternary Structure

• Interactions between two or more polypeptide chains.

• Not found in all proteins.

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primary structure

secondary structure

amino acids

helix-sheet

tertiary structurehelix

-sheet

quanternary structure

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A. Peptide bonds linking the amino acids differ from one protein to another.

B. The two proteins have a different combination of amino acids along the chain of the polypeptide.

C. Presence of the element N is only found in one protein.D. The two proteins each contains their own unique types of

amino acids.E. The number of amino acids in the chains differ.

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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:

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IV. ßAPP Proteins and Alzheimer’s Disease

• ß-amyloid precursor protein (ßAPP) found in the phospholipid outer cell membranes of neurons.

ßAPP

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Outside the Cell

Inside the Cell

neurons

2626

CN COH

O

H

HH

CH

CH3CH3

CN COH

O

H

HH

CH3

CN COH

O

H

HH

C

C

H H

O-O

CN COH

O

H

HH

CH H

OH

A

C

B

D

valine alanine

aspartateserine

CQ#4: Which of the following amino acids might you NOT expect to find in the intra-membrane region of the ß-

APP protein?

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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.

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HH

HN C C

OH

O

CH3

alanine

HH

HN C C

OH

O

CH2OH

serine

HH

HN C C

OH

O

H

glycine

H2O

HH

HN C C

O

CH3

H H

N C C

O

CH2OH

H H

N C COH

O

H

met ala ser glygln thr phe glu leu lys tyr pro

H2O

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Brain Trauma Speeds Hydrolysis

• ß-amyloid precursor protein (ßAPP)

ßAPP

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Outside the Cell

Inside the Cell

ß-secretase

ß-amyloid

http://www.healthscout.com/animation/68/7/main.html

ß-amyloid mutant more sticky?

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A. Replacing an amino acid with a hydrophilic R-group with one that is hydrophobic.

B. Replacing an amino acid with a hydrophobic R-group with one that is even more hydrophobic.

C. Replacing an amino acid with a hydrophobic R-group with one that is hydrophilic.

D. Replacing an amino acid with a hydrophilic R-group with one that is even more hydrophilic.

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ß-amyloid

aqueous environmentoutside nerve cellCQ#5: Which amino acid

change in the ß-amyloid protein fragment would be most likely

to make it stick together?

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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.

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phenylalanine

HH

HN C C

OH

O

CH2

HH

HN C

CH

C

OH

O

CH3CH3

valine

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A. Peptide bonds B. Hydrogen bondsC. Non-polar covalent bonds D. Polar covalent

bonds

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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?

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A. Test for Alzheimer’s predisposition mutations as part of physicals prior to hiring.

B. Impose mandatory rest after head injuries.C. Fine or fire coaches breaking the rules.D. Change how the game is played so that concussions are

less likely to occur.E. 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.

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CQ#7: OK, so that drug didn’t work. What do you think the NFL should do for football players?

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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.

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A. It could change the amount of the protein produced.

B. It could change the protein’s 3-D shape and thus its ability to interact with other proteins.

C. It could change where the protein is located in the cell.

D. It could change the number of amino acids.

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Copies of ApoE 4

mutation

Average age of onset of Alzheimer’s

2 68

1 75

0 84

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?

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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.

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Interested in Reading More?• Scientific American: http://www.scientificamerican.com

has a great set of articles on the latest research, including one in March 2009 on prions and Alzheimers.

• Articles:– Medline search

http://www.nlm.nih.gov/medlineplus/alzheimersdisease.html – 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.

Image Credits

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Slide 3:Description: Photo of Chris Benoit.Source: From Wikimedia Commons, by dani nuestro from Bangkok, http://commons.wikimedia.org/wiki/File:BenoitInTheRing.jpgClearance: Creative Commons Attribution-Share Alike 3.0 Unported License(CC BY-SA 3.0).

Slide 4Description: Concussion illustration.Source: Adapted from Wikimedia Commons, http://commons.wikimedia.org/wiki/File:Concussion_Anatomy.png, original by Max Andrews.Clearance: Used in accordance with the Creative Commons Attribution-Share Alike 3.0 Unported license.

Slide 6Description: Neurofibrillary tangles in the Hippocampus of an old person with Alzheimer-related pathology.Source: Wikimedia Commons, http://commons.wikimedia.org/wiki/File:Neurofibrillary_tangles_in_the_Hippocampus_of_an_old_person_with_Alzheimer-related_pathology,_Gallyas_silver_stain.JPG, by Patho.Clearance: Used in accordance with the Creative Commons Attribution-Share Alike 3.0 Unported license.

Slide 9Description: Illustration comparing healthy brain and Alzheimer’s brain.Source: Alzheimer’s Association, http://www.alz.org/brain/09.asp Clearance: Used with permission.

Slide 11Description: Illustration of plaques.Source: Alzheimer’s Association, http://www.alz.org/brain/11.asp Clearance: Used with permission

Slide 18Description: 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 21-23Description: Illustration of four levels of protein structure. Source: Wikimedia Commons, http://commons.wikimedia.org/wiki/Image:Proteïen_struktuur.png.Clearance: Used in accordance with the Creative Commons Attribution-Share Alike 3.0 Unported license.

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