SC235: General Biology I Human Perspectives

1. SC235: General Biology I Human Perspectives Seminar III: Biological Molecules Sue Werness, PhD

2. AGENDA Proteins Sickle Cell Anemia

3. 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?

4. 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?

5. 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?

6. 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?

7. 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) together Add finishing touches such as sugars or phosphates Fold into the correct shape

8. 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?

9. What Is an Amino Acid? R | NH2 - C – COOH | H 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 atom R group that depends on the specific amino acid 20 different amino acids

10. Functions of Proteins List several functions or roles of proteins in cells.

11. Protein Functions List 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 - antibodies • Transport - hemoglobin

12. Protein Structure For proteins, shape is key to their proper functioning. Explain the 4 levels of protein structure (shape): Primary? Secondary? Tertiary? Quaternary?

13. Protein Structure For 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

14. Protein Structure Hydrogen Bonds Beta Pleated Sheet Alpha Helix For 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

15. What is Hydrogen Bonding? YOU are experts in hydrogen bonding! Have you ever seen a drop of water? Have you ever seen ice?

16. Protein Structure, Polar Molecules and Hydrogen Bonding Negative Pole Polar molecules are formed when atom of high electronegativity bonds with atoms of smaller electronegativity Molecule forms locally charged areas Oxygen Oxygen Hydrogen Hydrogen Positive Pole

17. Hydrogen Bonds Hydrogen bond Opposite charges attract!

18. Protein Structure 3-D Shape achieved by folding via proteins called Chaperonins Polypeptide enters Chaperonin Protein exits Chaperonin 1 2 3 For 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 groups

19. Protein Structure http://commons.wikimedia.org/wiki/File:Hemoglobin_structure.JPG For 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

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

21. What Happens if the Protein Has a Single Amino Acid in Error? • DNA point mutation causes 1 error in primary structure • In general, primary structure errors can devastate secondary, tertiary, and quaternary structure • Proteins 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

22. Any Questions So Far?

23. Sickle Cell Anemia

24. 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)?

25. Genetics of Sickle Cell Disease/Trait SC Trait Normal Normal SC Trait SC Disease Carrier Carrier

26. 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 b-hemoglobin? • Normal : • Sickled: • Impact on secondary, tertiary, quaternary structure? • Impact on overall properties of hemoglobin? Val Val His His Leu Leu Thr Thr Pro Pro Glu Val Glu Glu …… ……

27. Hemoglobin in Sickle Cell Disease • Hemoglobin consists of 4 polypeptides, 2 a-hemoglobins and 2 b- hemoglobins interlocked together in quaternary protein structure • The 4 polypeptides stick to each other in hemoglobin complex and do not NORMALLY interact with one another • In SCD, b-hemoglobin has 1 wrong amino acid and this affects primary, secondary and tertiary structure b Abnormal hemoglobin sticks together and forms crystals when in low oxygen b b b b b b b b b b Normal hemoglobin does not interact with each other b b b b b

28. It Gets Even Worse! • Hemoglobin polymerizes into sticky strings and distorts red blood cell shape into a sickled shape • Sickling causes potassium and water to flow out of cell • Causes cell dehydration • Cell sickles even more • Sticky stringy hemoglobin does not carry nitric oxide readily • Nitric 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 oxygen • Oxygen 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

29. Gardos Potassium Ion Channel K+ RBC (with HbS) Gardos Ion Channel Role in Pathophysiology ↑ Dense / sickled cells ↑ Hemolysis Deoxygenation K+ H2O H2O ↑Ca++ ↓ Hemoglobin (Anemia) Dense/Sickled RBC Dehydrated, hypersicklable RBC Stiff Capillaries Clogging of arteries and capillaries in tissues = PAIN and ORGAN Damage

30. History of Sickle Cell Trait/Disease Any questions so far? Get ready for a 5 minute “field trip” http://www.pbs.org/wgbh/evolution/library/01/2/l_012_02.html • Click on Information • Open new browser window • When http address is pasted into chat, select it, and then control-C to copy • Control - v (paste) into address bar of new browser window • When you get to the Evolution page, click on QuickTime OR Real Player -> video in upper right corner

31. Co-occurrence of Malaria and Sickle Cell Trait http://en.wikipedia.org/wiki/File:Sickle_cell_distribution.jpg http://en.wikipedia.org/wiki/File:Malaria_distribution.jpg Distribution of the sickle-cell trait shown in pink and purple Historical distribution of malaria (no longer endemic in Europe) shown in green • USA - 2,000,000 African Americans have SCT (8%) • Africa – 20% have SCT

32. Frequency of Malaria in 2006 http://en.wikipedia.org/wiki/File:Paludisme_-_Frequence_statistique.png

33. Consequences to Body of Hemoglobin Mis-folding? • Two kinds of things happen to red blood cells (RBCs) as a result of the “bad” hemoglobin • Sickled 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?

34. Consequences of Early RBC Lysis and Death • Anemia – Low Red Blood Cell (RBC) counts • Not enough RBCs to carry oxygen to tissues • Symptoms: fatigue, dizziness, weakness, fast pulse • Consequences: Slow growth (children), risk of pulmonary hypertension • Bone marrow tries to keep making more red blood cells • Bone marrow can sometimes quit (aplastic anemia) after viral infection • Several organs are overwhelmed by job of cleaning up after destroyed red blood cells • Spleen 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 debris • Kidneys can become overloaded • Gall 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/DS00324 From Harvard web site: Sickle Cell Disease(2007). Retrieved August 24, 2009 from http://sickle.bwh.harvard.edu/menu_sickle.html From 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

35. Consequences to Body of Hemoglobin Mis-folding? • Two kinds of things happen to red blood cells (RBCs) as a result of the “bad” hemoglobin • Sickled 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?

36. Consequences of Clogged Stiff Capillaries and Arteries • Brain - strokes (kill children), aneurysms • Eye retinas - retinal bleeding, blindness • Joints – frequent sites of Pain • Bones – death of bone tissue, deformity, pain, osteoporosis • Heart – damage to heart tissue • Lungs • Acute chest syndrome • Pulmonary hypertension • Genitals – priapism (long lasting erection) • Hand-foot syndrome – swelling, pain in hands, feet from joint damage • Legs – skin ulcers From Mayo clinic web site: Sickle Cell Anemia (2009). Retrieved September 4, 2009 from http://www.mayoclinic.com/health/sickle-cell-anemia/DS00324 From Harvard web site: Sickle Cell Disease(2007). Retrieved August 24, 2009 from http://sickle.bwh.harvard.edu/menu_sickle.html From 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

37. Can You Be Sick and Die With Sickle Cell Trait? • People with sickle cell trait usually have no symptoms • But - 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 SCT • Some not aware they have sickle cell trait • 2010 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

38. Any Questions So Far?

39. Risk/Benefit Analysis of Established Treatments • Symptom Management Pain medication for painful crises Antibiotics for infection Vaccines to prevent infections Transfusions for short term “correction” Oxygen for breathing difficulties • Disease Modifying Drugs Hydroxyurea • Cures Bone marrow transplant

40. Risk/Benefit Analysis of Established Treatments • Symptom Management • Pain 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 Drugs • Hydroxyurea • Cures • Bone marrow transplant; risk, lack of siblings with matching tissue, cost

41. Hydroxyurea Treatment • Treatment Effects Boosts production of fetal hemoglobin from bone marrow Increases destruction of sickled cells • Side Effects Low white blood count, damage to bone marrow Possible leukemia Vomiting Breaks 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

42. Hydroxyurea Treatment • Treatment Effects and Benefits • Boosts production of fetal hemoglobin from bone marrow • Increases destruction of sickled cells • Side Effects/Risks • Low white blood count, damage to bone marrow • Possible leukemia • Vomiting and other digestive disorders • Rashes • Chromosome 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

43. Experimental Treatments • Gene 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 hemoglobin • L-Glutamine – amino acid keeps sickled cells from sticking to veins • Clotrimazole – anti-fungal medicine that may prevent dehydration and sickling of red blood cells • Nitric 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

44. SURPRISING RESULT 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.

45. 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

46. Gardos Potassium Ion Channel K+ RBC (with HbS) Gardos Channel Role in Pathophysiology ↑ Dense / sickled cells ↑ Hemolysis Deoxygenation K+ H2O H2O ↑Ca++ ↓ Hemoglobin (Anemia) Dense/Sickled RBC Dehydrated, hypersicklable RBC Clogging of arteries and capillaries in tissues = PAIN and ORGAN Damage

47. Prevents dehydration and formation of dense / sickled cells Blocks Gardos potassium channel K+ Normal Density RBC RBC (with HbS) Hopefully less SCD Pain ↓ Dense / sickled cells ↓Hemolysis Clinical Results It works ↑ Hemoglobin (Improvement in anemia) It works It didn’t work!!

48. 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 shape • CELLS WEREN’T SICKLED; DIDN’T BREAK DOWN • Arteries and capillaries shouldn’t have clogged! • Ion Channel Blocking Drug failed to reduce crisis rates in patients with SCD PATIENTS STILL IN PAIN • CLINCAL TRIAL WAS STOPPED • Why didn’t it work?

49. Why Ion Channel Blocker May Have Failed • Ion channel blocker LOWERS nitric oxide • So, 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 occur • If the ion channel blocker INCREASED nitric oxide, would it have worked? • Books describing sickle cell anemia do not portray entire picture

50. Alternative Treatment of Sickle Cell Disease • Arginine (amino acid) used by the body to make nitric oxide • Possible 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