Sickle molecular

Question 1

sickle cell mutation

Answer 1

• chromosome 11
• pt mutation A to T
• glu to val

Question 2

HbS

Answer 2

• reside 6 is now Val
• deoxygenation HbS forms 14 stranded polymers
• when well oxygenated returns to normal tetramers
• some cells remain irreversible

Question 3

hallmarks

Answer 3

• cycling b/n biconcave and sickled shape-weakens PM
• resulting RBC hemolysis (anemia) and vasoocclusion-ischemia, pain
• some sickle cells locked in sickle shape even when cell has o2 and in monomer
• 2-40% of circulating RBCs in homozygotes

Question 4

vasooculsion

Answer 4

• disease is polygenic on 2,6, and 11
• varies extent of inflammation and vasculopathy
• vasoocclusion is altered RBCs, leukocytes, BV endo cells and plasma factors
• RBCs and WBCs latch to endo and then to each other

Question 5

sickle cell crises

Answer 5

• those with less than 1 per year have a much longer life span than those with 1-3 or more than 3 per year
• 15 year difference
• molecular basis and biomarkers?

Question 6

expression of globins

Answer 6

• alpha is on 16, beta on 11
• erythropoiesis yolk sac in embyonic development, midway through 1st trimester to liver, then bone marrow at birth
• LCR for beta cluster
• embryonic to fetal gamma and alpha gamma at switch to liver
• feta gamma and alpha gamma to beta at birh
• beta genes are linearly arranged from 5’-3’ with a distal LCR directing expression

Question 7

HbF and sickle cell severity

Answer 7

• 3 loci associated with HbF levels and B globin disorder severity
• b cluster itself (cis acting haplotype on 11), HBS1l-MYB, and BCL11A
• bind to other TFs and complexes switch gamma to beta
• variants of the TFs that cause high HbF levels are clinically less severe

Question 8

BCL11A

Answer 8

• TF
• trans acting
• gamma globin silencer
• chromosome 2

Question 9

HBS1L-MYB

Answer 9

• intergenic interval on chromosome 6
• MYB influences HbF expression
• TF that represses gamma globin

Question 10

SNPs

Answer 10

• SNPs on the TFs on chromosomes 2 and 6 can increase HbF

- if around 20%, less severe form of disease

Question 11

haplotypes in the beta globin gene cluster

Answer 11

• HbS on 5 different B globin like gene cluster haplotypes which contain a polymorphism acting cis that regulates HbF
• 5 different mutations have different regulations og HbF
• Africa, Middle East, and India
• severe disease order goes Bantu, Benin, Senegal, Arab Indian
• Amount of HbF goes opposite way-most in Arab Indian

Question 12

Inflammation and vasculopathy

Answer 12

• ischemia reperfusion cycle
• vasoocclusion and ischemia cause a metabolic change, which leads to a burst of ROS when blood flow restored
• during ischemia, XO increased, upon reperfusion XO converts o2 to superoxide radical
• burst of ROS from endo cells (RBCs), adherent leukocytes and XO on endo surface
• ROS leads to NFkB, IF and cytokines, activation of leukocytes, increased adhesion molecules, further plugging, decreased NO–>abnormal endo dependent vasodilation

Question 13

sickle cell in double jeopary

Answer 13

• sickle cell RBCs contain 3x as much o2 radicals compared to normal RBCs
• they also have very low levels of reduced glutathione, particularly in highest density RBCs
• highest density, most damaged, lease antioxidants

Question 14

ROS and antioxidants

Answer 14

• RBC susceptible to oxidative damage because always carrying o2 at various po2s
• o2 radical (which comes from Fe II-III, which happens faster in sickle cells, with o2 on- superoxide) changed to h2o2 by super oxide dismutase
• h2o2 degraded by catalase, glutathione peroxidase (GPX), perioxiredoxin2, or reoxidation of Fe (makes OH radical-bad)
• normally radicals degraded to below toxic levels
• RBCs also sink for o2 radicals produced by other cell types

Question 15

metHb

Answer 15

• lower affinity for heme
• released hemin and free iron on cytoplasmic surface
• hemin or free FE can convert h2o2 into OH radical by catalyzing Fenton reaction or in concert with o2 radical by Haber Weiss reaction
• this is in sickle cell not as much in normal because Fe II to Fe III slower

Question 16

glutathione

Answer 16

• reduced and becomes oxidized when degrading h2o2

- non enzymatic

Question 17

Increased ROS and RNS in sickle cell

Answer 17

• increased antioxidation of HbS into metHb and o2 radical
• h2o2 in contact with met causes release of heme and iron more readily-catalyzed to OH radical by Fenton and Haber-Weiss
• superoxide binds NO
• released cell free HbS binds NO, limits is vasodilitory, anti IF, and antithrombotic properties–>ince in BV endo activation and ROS release
• ischemia-reperfusion-increased XO-inc NADPH oxidase-ince superoxide
• PMNs produced ROS in inc IF in NADPH oxidase dependent resp burst

Question 18

reduced antioxidant activity in SCD

Answer 18

• increased SOD–inc h2o2 and OH rad
• GPX and catalase activity reduced
• GSH reduced and inversely proportional to density-highest density are most damaged and have least GSH

Question 19

cysteine modifications and oxidative stress

Answer 19

• in presence of h2o2, thiolate can be converted to sulfenic acid (SOH)
• SOH will react with neighboring cys or GSH and create SS bonds, or make a sulfenamide
• under increased stress SOH tp sulfinic acid (SO2H) or sulfonic acid (SO3H)-irreversible damage

Question 20

scramblase

Answer 20

• works with flipase to keep phospholipids on the right side
• lose phosphatidyl symmetry across membrane because scramblase is damaged and therefore flipase unopposed
• patches of neg charged head group on outer leaflet
• associates with clotting factors and lead to hypercoag and thrombosis
• also recognition site for macrophage association

Question 21

RBC membrane skeleton

Answer 21

• spectrin responsible for shape and elasticity
• spectrin is antiparallel ab heterodimer forms tetramer by head to head linkage
• tails attach to actin protofilaments (14 g actin monomers long)
• protein 4.1 binds to tails of b spectrin
• adducin binds to B spectrin and F actin-both to reinforce spectrin/actin
• attached to lipid bilayer by protein 4.1 to glycophorin C
• and ankryin from B spectrin to transmembrane proteins B and 3

Question 22

bonds in skelton

Answer 22

• spectrin binds to actin
• adducin binds to spectrin and actin to bridge
• 4.1 binds to spectrin and membrane and actin
• ankyrin binds to spectrin and membrane

Question 23

normal actin vs ISC actin

Answer 23

• disulfide bridge b/n cys 284 and cys 373
• post translation modification
• polymerize and depolymerize more slowly than normal, can still bind spectrin
• just disassemble slowly

Question 24

spectrin

Answer 24

• self ubiquitinator- E2/E3
• normally puts 2 ub on 20 cys and links to 21 K
• SCA-no ub- E2/3 doesn’t work because oxidation of cysteines
• makes tighter RBC skeleton, can’t dissociate
• cysteine locations correspond to B2 and B1, which contain protein 4.1 and adducin binding domains
• no ub, tighter, no room for adducin/ protein 4.1 to move, no dissociation (normal spectrin/adducin (4.1)/actin binding is roomy)

Question 25

Defects on ISC membrane

Answer 25

- skeleton disassembles and reassembles slowly because: 1. B actin SS bridge-actin dissembles slowly or not at all 2. diminished Ub-tighter spectrin/actin/4.1 and spectrin/adducin/actin - locked in sickle shape

Question 26

Dense?

Answer 26

- deoxygenation of RBCs (dehydration and inc corpuscular Hg)-->HbS polymerization and sickling-->sickling induced leak channel - Ca++ in-->K+channel open - K+ loss from channel and from leak itself - Mg+ loss-->KCl cotransport activation - all leads to even more dehydration

Question 27

two step model (dense and lock) step 1

Answer 27

1. decreasing GSH levels-->dehydration (K+leak) and dense -NAC blocks lower GSH induced sickle cell K+ efflux and dehydration -oxidative damage to K+Gardos pathway (which causes leaking and is from increased ROS due to low GSH) can be reversed with NAC, which decreases GSSG:GSH (more GSH, less dehydration) -highest density have <1% of the GSH found in low density cells following events: 1. lowering intracellular GSH keads to increased [ ] of O2 radicals and reversible oxidative damage to Gardos channel 2. oxidative damage to Gardos leads to increased K+ loss and dehydration 3. NAC raises GSH and is antioxidant, can reverse oxidative damage

Question 28

two step model step 2

Answer 28

2. second step is locking of dense cells - occurs due to oxidative damage of actin and lack of ub on spectrin- both caused by diminished GSH (SS on actin and SS on spectrin) - both partially reversible with NAC- antioxidant that raises GSH- no SS on actin or spectrin - in vitro reduces SS on actin

Question 29

NAC

Answer 29

- 2400 mg/day - inc GSH - dec dense cells - dec ISC - dec crises - *may have just found that NAC may stop scramblase, which would bring serine symmetry back to bilayer

Question 30

other treatments

Answer 30

- antibiotics - analgesics - hydroyxurea - blood and bone marrow stem cell therapeutics

Question 31

future treatments

Answer 31

- effect K leakage from RBSc-senicapoc - effect NO levels-statins - effect oxidative stress-NAC - effect adhesion-statins - effect IF-lovastatin - effect HbF levels-decitabine - replace defective gene

Question 32

biomarkers

Answer 32

- identification of 20 monocyte proteins whose levels are highly correlated with 5 year crisis rates - decreased apolipoprotein A1 in plasma correlated with sickle cell associated pulm hypertension and acute pain episodes - increased microRNA mir144 leads to low antioxidant capacity, increase oxidative stress, and more sever phenotype as measured by increase hemolysis, lower Hb [ ] and higher % reticulocytes - use in longitudinal trials to see how early they can predict severity - personalized medicine