Seminar III: Biological MoleculesSue Werness, PhD

•Proteins•Sickle Cell Anemia

What Do You Know About Hemoglobin?

What kind of biomolecule is it? Carbohydrate? Nucleic Acid? Protein? Lipid?

What does it do?What kind of cells have hemoglobin molecules?

Manufacturing a Protein?

How do you manufacture a protein?

Step 0: Where does the blueprint for a protein come from?

Step 1: How are the basic components of proteins put together? The components of proteins are synthesized into long chains (polypeptides) with the help of an enzyme (helper, catalyst).What are these basic components of proteins?

Step 2: How does a fully functional protein get made from simple chains of these components?

Manufacturing a Protein?

How do you manufacture a protein?

Step 0: Where does the blueprint for a protein come from? Central Dogma: DNA -> RNA -> Protein

Step 1: How are the basic components of proteins put together? The components of proteins are synthesized into long chains (polypeptides) with the help of an enzyme (helper, catalyst).What are these basic components of proteins?

Step 2: How does a fully functional protein get made from simple chains of these components?

Manufacturing a Protein?

How do you manufacture a protein?

Step 0: Where does the blueprint for a protein come from? Central Dogma: DNA -> RNA -> Protein

Step 1: How are the basic components of proteins put together? The components of proteins are synthesized into long chains (polypeptides) with the help of an enzyme (helper, catalyst). What are these basic components of proteins? Amino acids

Step 2: How does a fully functional protein get made from simple chains of these components?

Manufacturing a Protein?

How do you manufacture a protein?

Step 0: Where does the blueprint for a protein come from? Central Dogma: DNA -> RNA -> Protein

Step 1: How are the basic components of proteins put together? The components of proteins are synthesized into long chains (polypeptides) with the help of an enzyme (helper, catalyst).What are these basic components of proteins? Amino acids

Step 2: How does a fully functional protein get made from simple chains of these components? Assemble the pieces (polypeptides) togetherAdd finishing touches such as sugars or phosphatesFold into the correct shape

What Is an Amino Acid?The carboxyl (COOH) and amino (NH2) groups are

components of every amino acid. Wolfe described an amino acid as consisting of a central C (carbon) with 4 components attached.

What are the other 2 components attached to the central carbon?

What Is an Amino Acid?The carboxyl (COOH) and amino (NH2) groups are also

components of every amino acid. Wolfe described an amino acid as consisting of a central C (carbon) with 4 components attached.

What are the other 2 components attached to the central carbon?H atomR group that depends on the specific amino acid20 different amino acids

R|

NH2 - C – COOH

|H

Functions of ProteinsList several functions or roles of proteins in cells.

Protein FunctionsList several functions or roles of proteins in cells. Enzymes – to catalyze reactions Support – collagen, keratin Contraction - muscle Hormones - insulin Receptors – recognizes signals Storage – milk, egg white Defense - antibodiesTransport - hemoglobin

Protein StructureFor proteins, shape is key to their proper functioning.

Explain the 4 levels of protein structure (shape):Primary?

Secondary?

Tertiary?

Quaternary?

Protein StructureFor proteins, shape is key to their proper functioning.

Explain the 4 levels of protein structure (shape):Primary? Unique sequence of amino acids in the polypeptide,

determined by DNA

Protein StructureFor proteins, shape is key to their proper functioning.

Explain the 4 levels of protein structure (shape):Secondary? results from hydrogen bonding between amino

acids within the polypeptide chain and leads to regular patterns such as alpha-helixes and beta-pleated sheets

HN

NH

HN

O

O

O

NH

HN

HNHN

O

O

O

NH

HN

HO

O

O

O

H2N

O

Beta Pleated Sheet

Alpha Helix

Hydrogen Bonds

What is Hydrogen Bonding?YOU are experts in hydrogen bonding!

Have you ever seen a drop of water?Have you ever seen ice?

Protein Structure, Polar Molecules and Hydrogen Bonding

Polar molecules are formed when atom of high electronegativity bonds with atoms of smaller electronegativity

Molecule forms locally charged areas

Oxygen

HydrogenHydrogen

Oxygen

Negative Pole

Positive Pole

Hydrogen bond

Opposite charges attract!

Protein StructureFor proteins, shape is key to their proper functioning.

Explain the 4 levels of protein structure (shape):Tertiary? 3-D shape of a protein caused by interactions

between R groups3-D Shape achieved by folding via proteins called Chaperonins

Polypeptide enters

Chaperonin

Protein exits Chaperonin

1 2 3

Protein StructureFor proteins, shape is key to their proper functioning.

Explain the 4 levels of protein structure (shape):Quaternary? three-dimensional shape of a protein caused by

linking together of multiple polypeptides

http://commons.wikimedia.org/wiki/File:Hemoglobin_structure.JPG

What Happens if the Protein Has a Single Amino Acid in Error?

What Happens if the Protein Has a Single Amino Acid in Error?

DNA point mutation causes 1 error in primary structureIn general, primary structure errors can devastate

secondary, tertiary, and quaternary structureProteins are like tools; they have to be the correct shape

for the job or they don’t work at all!

Using the right tool Using the wrong tool

Any Questions So Far?

How Does Sickle Cell Disease Occur?How do you “catch” the disease?What is the difference between sickle cell disease (SCD or

SCA) and sickle cell trait (SCT)?

NormalSC Trait SC DiseaseSC TraitNormalCarrier Carrier

What happens to the protein hemoglobin in SCD?

What does it have to do with protein structure?Just 1 amino acid substitution

What amino acid is replaced with what amino acid on -hemoglobin? Normal :

Sickled:

Impact on secondary, tertiary, quaternary structure?Impact on overall properties of hemoglobin?

Val HisLeu

Thr Pro Glu Glu……

Val HisLeu

Thr Pro Val Glu……

Hemoglobin in Sickle Cell DiseaseHemoglobin consists of 4 polypeptides, 2 -hemoglobins and 2 -

hemoglobins interlocked together in quaternary protein structureThe 4 polypeptides stick to each other in hemoglobin complex and do not

NORMALLY interact with one another In SCD, -hemoglobin has 1 wrong amino acid and this affects primary,

secondary and tertiary structure

Normal hemoglobin does not interact with

each other

Abnormal hemoglobin sticks together and

forms crystals when in low oxygen

It Gets Even Worse!Hemoglobin polymerizes into sticky strings and

distorts red blood cell shape into a sickled shapeSickling causes potassium and water to flow out of cell

Causes cell dehydrationCell sickles even more

Sticky stringy hemoglobin does not carry nitric oxide readilyNitric oxide has recently been discovered to be important

blood gas that RELAXES the capillaries and lets the RBCs though*

Normal hemoglobin carries nitric oxide as well as oxygenOxygen doesn’t get to tissues -> causes MORE polymerization

of Hemoglobin (happens in low O2 environment) *John R. Pawloski, Douglas T. Hess, and Jonathan S. Stamler, Impaired vasodilation by red blood cells in sickle cell disease.

PNAS 2005 102:2531-2536; published online before print February 7, 2005, doi:10.1073/pnas.0409876102

RBC(with HbS)

GardosPotassium Ion Channel

↑ Dense / sickled cells↑ Hemolysis

↓ Hemoglobin (Anemia)

Clogging of arteriesand capillaries in tissues= PAIN and ORGAN Damage

H2OK+

Dense/Sickled RBC

K+

H2O

Dehydrated, hypersicklable RBC

Deoxygenation

↑Ca++

Stiff Capillaries

History of Sickle Cell Trait/DiseaseAny questions so far?Get ready for a 5 minute “field trip”http://www.pbs.org/wgbh/evolution/library/01/2/l_012_02.html

1.Click on Information2.Open new browser window

1. When http address is pasted into chat, select it, and then control-C to copy

2. Control - v (paste) into address bar of new browser windowWhen you get to the Evolution page, click on QuickTime OR

Real Player -> video in upper right corner

USA - 2,000,000 African Americans have SCT (8%)Africa – 20% have SCT

http://en.wikipedia.org/wiki/File:Malaria_distribution.jpg

Historical distribution of malaria (no longer endemic in Europe) shown in green

http://en.wikipedia.org/wiki/File:Sickle_cell_distribution.jpg

Distribution of the sickle-cell trait shown in pink and purple

http://en.wikipedia.org/wiki/File:Paludisme_-_Frequence_statistique.png

Consequences to Body of Hemoglobin Mis-folding?

Two kinds of things happen to red blood cells (RBCs) as a result of the “bad” hemoglobinSickled cells lyse (break apart) and die after only 10 – 20 days –

consequences?Sickled shape cells clog and stick to stiffened blood vessels –

consequences?What organs are affected by these maladies and how are

they affected?What symptoms occur?

Consequences of Early RBC Lysis and Death

Anemia – Low Red Blood Cell (RBC) countsNot enough RBCs to carry oxygen to tissuesSymptoms: fatigue, dizziness, weakness, fast pulseConsequences: Slow growth (children), risk of pulmonary hypertension

Bone marrow tries to keep making more red blood cellsBone marrow can sometimes quit (aplastic anemia) after viral infection

Several organs are overwhelmed by job of cleaning up after destroyed red blood cellsSpleen can become clogged with debris and eventually destroyed

RBCs blocked from getting to organs, extreme weakness, can result in death Consequence: Spleen is part of immune system; ability to fight infections

compromised -> Leads to Acute Chest Syndrome (infections) Most common cause of death is infection

Liver can become jaundiced and blocked with RBC debrisKidneys can become overloadedGall bladder can form gallstones, can become inflamed

From Mayo clinic web site: Sickle Cell Anemia (2009). Retrieved September 4, 2009 from http://www.mayoclinic.com/health/sickle-cell-anemia/DS00324From Harvard web site: Sickle Cell Disease(2007). Retrieved August 24, 2009 from http://sickle.bwh.harvard.edu/menu_sickle.htmlFrom WebMD web site: Sickle Cell Disease (2008). Retrieved September 4, 2009 from http://www.webmd.com/a-to-z-guides/sickle-cell-disease-topic-overview

Consequences to Body of Hemoglobin Mis-folding?

Two kinds of things happen to red blood cells (RBCs) as a result of the “bad” hemoglobinSickled cells lyse (break apart) and die after only 10 – 20 days –

consequences?Sickled shape cells clog and stick to stiffened blood vessels –

consequences?What organs are affected by these maladies and how are

they affected?What symptoms occur?

Consequences of Clogged Stiff Capillaries and Arteries

Brain - strokes (kill children), aneurysmsEye retinas - retinal bleeding, blindnessJoints – frequent sites of PainBones – death of bone tissue, deformity, pain, osteoporosisHeart – damage to heart tissueLungs

Acute chest syndromePulmonary hypertension

Genitals – priapism (long lasting erection)Hand-foot syndrome – swelling, pain in hands, feet from

joint damageLegs – skin ulcers

From Mayo clinic web site: Sickle Cell Anemia (2009). Retrieved September 4, 2009 from http://www.mayoclinic.com/health/sickle-cell-anemia/DS00324From Harvard web site: Sickle Cell Disease(2007). Retrieved August 24, 2009 from http://sickle.bwh.harvard.edu/menu_sickle.htmlFrom WebMD web site: Sickle Cell Disease (2008). Retrieved September 4, 2009 from http://www.webmd.com/a-to-z-guides/sickle-cell-disease-topic-overview

Can You Be Sick and Die With Sickle Cell Trait?

People with sickle cell trait usually have no symptomsBut - Athletes have died because of intense exercise

inducing sickle cell crises (low oxygen -> death of spleen tissue, rapid breakdown of muscle tissue and renal failure)Risk of sudden death during exercise is ~15% higher with SCTSome not aware they have sickle cell trait2010 Fall – All Division I athletes will undergo screening for

sickle cell trait Worry that false positives won’t be followed up Worry that athletes won’t receive genetic counseling Will they get discrimination because of positive results?

Should there be such a screening program?Johns Hopkins Medical Institutions (2010, September 21). New sickle cell screening program for college athletes comes with serious

pitfalls, experts say. ScienceDaily. Retrieved September 22, 2010, from http://www.sciencedaily.com/releases/2010/09/100908171120.htm

Any Questions So Far?

Risk/Benefit Analysis of Established Treatments

Symptom ManagementPain medication for painful crisesAntibiotics for infectionVaccines to prevent infectionsTransfusions for short term “correction”Oxygen for breathing difficulties

Disease Modifying DrugsHydroxyurea

CuresBone marrow transplant

Risk/Benefit Analysis of Established Treatments

Symptom ManagementPain medication for painful crises; possible addiction, possible

under-medication?Antibiotics for infection; possible antibiotic resistance?Vaccines to prevent infections; possible reactions to vaccines?Transfusions for short term “correction” and prevention; possible

iron overload, risk of hepatitis?Oxygen for breathing difficulties

Disease Modifying DrugsHydroxyurea

CuresBone marrow transplant; risk, lack of siblings with matching

tissue, cost

Hydroxyurea TreatmentTreatment Effects

Boosts production of fetal hemoglobin from bone marrowIncreases destruction of sickled cells

Side EffectsLow white blood count, damage to bone marrowPossible leukemiaVomitingBreaks chromosomes

from Hydroxyurea for Sickle Cell Patients (2008). Retrieved from Web site of News-Medical.Net - Latest Medical News and Research from Around the World. http://www.news-medical.net/news/2008/02/28/35770.aspx http://www.rxlist.com/droxia-drug.htm

Hydroxyurea TreatmentTreatment Effects and Benefits

Boosts production of fetal hemoglobin from bone marrowIncreases destruction of sickled cells

Side Effects/RisksLow white blood count, damage to bone marrowPossible leukemiaVomiting and other digestive disordersRashesChromosome damage

from Hydroxyurea for Sickle Cell Patients (2008). Retrieved from Web site of News-Medical.Net - Latest Medical News and Research from Around the World. http://www.news-medical.net/news/2008/02/28/35770.aspx http://www.rxlist.com/droxia-drug.htm

Experimental TreatmentsGene therapy

Putting correct gene in bone marrow Turning off “bad” gene and activating gene to generate only fetal hemoglobin

Butyric Acid – food additive that may boost fetal hemoglobinL-Glutamine – amino acid keeps sickled cells from sticking to veinsClotrimazole – anti-fungal medicine that may prevent dehydration

and sickling of red blood cellsNitric Oxide – may reduce sickle cell clumping, may help Hb bind

oxygen Medical College of Georgia (2010, October 19). Inhaling nitric oxide eases pain crises in sickle cell patients, researchers find. ScienceDaily. Retrieved October 20, 2010, from http://www.sciencedaily.com/releases/2010/10/101019121758.htm

Nicosan – herbal treatment from Nigeria, mixture of 4 Nigerian plants, nontoxic, company in bankruptcy, not approved in the US

From Mayo clinic web site: Sickle Cell Anemia (2009). Retrieved September 4, 2009 from http://www.mayoclinic.com/health/sickle-cell-anemia/DS00324

A Phase III, 52-Week, Randomized, Double-Blind,

Placebo-Controlled Study of the Clinical Efficacy and Safety

of ICA-17043 with or without Hydroxyurea Therapy in

Patients with Sickle Cell Disease

Efficacy and safety of the Gardos channel blocker, senicapoc (ICA-17043), in patients with sickle cell anemia, Ataga, Kenneth, et. al. BLOOD, Volume 111( 8): 3391-3397, 2008.

SURPRISING RESULT

Potential of Ion Channel Blocking Drug• Hypothesis: Blocking of Gardos potassium ion

channel with a novel ion channel blocker drug can reduce dehydration, hemolysis, and sickling of RBCs in sickle cell disease (SCD) and lead to clinical improvement.

New drug should reduce anemia, reduce clogging

Fewer painful crises

RBC(with HbS)

GardosPotassium Ion Channel

↑ Dense / sickled cells↑ Hemolysis

↓ Hemoglobin (Anemia)

Clogging of arteriesand capillaries in tissues= PAIN and ORGAN Damage

H2OK+

Dense/Sickled RBC

K+

H2O

Dehydrated, hypersicklable RBC

Deoxygenation

↑Ca++

↑ Hemoglobin (Improvement in anemia)

↓ Dense / sickled cells

↓ Hemolysis

RBC(with HbS)

KK++

Normal Density RBC

Blocks Gardos potassium channel

Prevents dehydration and formation of dense / sickled cells

Hopefully less SCD PainIt works

It works

It didn’t work!!

Conclusions Concerning Novel Ion Channel Blocker Drug

Ion Channel Blocking Drug Caused a reduction in indicators of hemolysis (cells breaking down)Caused the red blood cells to have a normal round shapeCELLS WEREN’T SICKLED; DIDN’T BREAK DOWNArteries and capillaries shouldn’t have clogged!

Ion Channel Blocking Drug failed to reduce crisis rates in patients with SCDPATIENTS STILL IN PAIN

CLINCAL TRIAL WAS STOPPED

Why didn’t it work?

Why Ion Channel Blocker May Have FailedIon channel blocker LOWERS nitric oxideSo, even though Ion channel blocker un-sickles the red

blood cells, they may NOT be able to “smooth” their way through the small arteries ---- Clogging still occurs

Painful crises still occurIf the ion channel blocker INCREASED nitric oxide, would

it have worked?Books describing sickle cell anemia do not

portray entire picture

Alternative Treatment of Sickle Cell Disease

Arginine (amino acid) used by the body to make nitric oxidePossible diet treatment for sickle cycle disease

Arginine found in peanuts, cashews, walnuts, brazil nuts, coconut, seafood, chocolate, soybeans, chickpeas

Arginine found to be helpful for some cardiovascular conditions such as angina

Ongoing clinical trial testing arginine in acute chest syndrome Arginine Treatment of Acute Chest Syndrome (Pneumonia) in Sickle Cell

Disease Patients Clinical Trial NCT00029731 Retrieved September 6, 2009 from NIH Clinical Trials Web site at

http://clinicaltrials.gov/ct2/show/NCT00029731

Lifestyle Management of SCD?

Lifestyle Management of SCD?Take folic acid (bone marrow needs folic acid to keep

manufacturing red blood cells)Drink lots of water – keeps blood dilute and reduces chance of

sicklingAvoid stressModerate exercise but NOT heavy exercise!Avoid low oxygen environments such as high altitudes and

non-pressurized airplanes, cigarette smokeAvoid OTC medicine – especially pseudoephedrine or anything

that constricts blood vesselsPrevent infections with vaccines, hand washing, careful food

preparation, chronic use of antibiotics in children

Prevention of SCD?

Prevention of SCD?Genetic diseaseGenetic counselingPreimplantation genetic diagnosis.

Eggs from the mother fertilized with sperm from father.Fertilized eggs are tested for sickle cell gene and those free

of the sickle cell gene are implanted into the mother’s womb

Expensive , always successful.

From Mayo clinic web site: Sickle Cell Anemia (2009). Retrieved September 4, 2009 from http://www.mayoclinic.com/health/sickle-cell-anemia/DS00324