Regulation of hemoglobin synthesis

Question 1

What proportion of all heme is synthesized by mature RBCs in the bone marrow?

Answer 1

85%

Question 2

name 3 pathways/steps of hgb synthesis, and where they happen.

Answer 2

1. Iron acquisition by transferrin
   a. Transferrin binds to transferrin receptor on cell surface
2. Synthesis of protoporphyrin IX
   a. Made in mitochondria
   b. Iron is inserted in protoporphyrin IX to form heme
3. Globin synthesis
   a. Made in ribosomes
   b. Goes with heme to form hemoglobin

Question 3

What is the difference between heme synthesis in nonerythroid cells vs erythroid cells?

Answer 3

• Heme synthesis in NONERYTHROID cells (Extracellular iron - Fe-Tf ? heme catabolism? ferritin?);
• ERYTHROID cells are dependent of transferrin iron;

Question 4

What happens in Tf deficiency?

Answer 4

o Not enough Tf-iron = erythroid precursors of BM cannot produce optimum amount of Hgb.
 RBCs are less red (hypochromic)
 Smaller than usual (microcytic) – defect in differentiation and proliferation

Question 5

How many enzymes are involved in heme synthesis? Where are they located?

Answer 5

Involves 8 enzymes in total

• 4 in mitochondria
• 4 in cytoplasm

1. MITO: Condensation of succinyl-CoA + glycine to form d-aminolevulinic acid; By ALA synthase (ALAS)
2. CYTO by ALAD
3. CYTO by PBGD
4. CYTO by UCeS
5. CYTO by UROD
6. CYTO –> MITO: by CPO
7. MITO by PPO
8. MITO: Insertion of Iron (ferrous iron; Fe2+) in protoporphyrin IX to form heme
   a. Enzyme: Ferrochelatase

Question 6

How many TfR do erythroid precursors have on their cells?

Answer 6

around 1,000,000 receptors per ell

Question 7

Differentiate TfR1 from TfR2

Answer 7

• In erythroid cells, Tf-Fe(III) complex in plasma is transported into cells principally through transferrin receptor 1 (TfR1);
• TfR2 is expressed primarily in the liver and binds to Tf-Fe(III) complex at a much lower affinity than TfR1.
(More of a regulatory function; involved in the hepcidin pathway)

Question 8

How does the TfR bring iron into the cell?

Answer 8

by ENDOCYTOSIS
- To be internalized, iron (as Fe3+) is delivered by transferrin to the transferrin receptors (1 transferrin = 2 irons)
- Tf-R bound to Tf with iron is internalized by endocytosis; ferric iron bound to Tf is then released and reduced to Fe2+ by enzyme STEAP on the membrane of the endosome.
*Endosome also have DMT1 on their membrane which will pump Fe2+ out in the cytosome
*Then a proton pump on their membrane will met H+ ions enter the endosome and acidify the inside
Affinity of Tf is pH-dependant thus the acidity of the endosome will decrease its affinity to iron
Once iron is out, endosome goes back to the cell surface where transferrin is released for another cycle of iron transport
Tf molecules can go about this process about 100-200x during their lifespan

Question 9

What happens to the iron pumped out of the endosome?

Answer 9

After Iron is released from endosome, process is not well understood.
It is believed that, in erythroid cells, iron comes in contact with the mitochondria, to be used by ferrochelatase, inserted into propoporphyrin IX to form heme in the mito.

Question 10

Explain the step of ferrochelatase in heme synthesis

Answer 10

Ferrochelatase is a protein with a Fe-S cluster
It inserts the iron in the heme before it is transported out of the mitochondria to be assembled into hemoglobin with globin proteins

Question 11

Explain a difference between erythroid cells and non-erythroid cells in terms of getting iron

Answer 11

Erythroid cells

• Rely exclusively on iron from Tf for heme synthesis
• Rate of acquisition of iron from Tf controls the rate of heme synthesis
• Tf-R is expressed differently

Non-erythroid cells
- Acquire iron for heme from different sources (Extracellular (Tf), Intracellular (ferritin), heme catabolism)

Question 12

Explain a difference between erythroid cells and non-erythroid cells in terms regulation of TfR

Answer 12

Non-erythroid cells

• Low iron: IRP-1 binds to IRE on 5’ UTR to impair translation so ferritin is not translated
• Low iron: IRE located in 3’ UTR region; higher Tf-R levels
• High iron: IRP-1 leaves IRE –> ferritin translation active
• High iron: IRP leaves IRE and mRNA unstable, decreased translation.

In erythroid cells,
- Transferrin receptor mRNA levels are only slightly affected by high iron concentrations
o Compensate disability in transferrin receptor RNA by increasing transcription rate of the gene
- Ferritin mRNA translation is massively impaired during terminal differentiation of erythroid progenitors
o CFUEs (late stage of terminal differentiation of erythroid cells) are synthesizing a lot of heme and hgb. Instead of having a lot of ferritin. Unknown mechanism.

Question 13

Explain the “kiss and run” model

Answer 13

When feeding erythroid precursors with radioactive iron, most of the iron is incorporated in heme in a short amount of time. This strongly suggests iron is directly brought to the mitochondria, bypassing the cytosol
Bafilomycin (inhibits proton pumps of endosomes/acidification of endosome): dramatic decrease in amount of iron in heme with only small amount.
A lot of evidence supports that in erythroid cells, Tf-iron is directly linked to mitochondria by kiss and run mechanism
It is believed that there are docking molecules on the wall of the mitochondria and of the endosome that facilitate interaction. Help for iron from DMT1 to get to ferrochelatase. However, those docking molecules have not yet been identified.

Question 14

What is ALA-S? What are its 2 types?

Answer 14

Enzyme that catalyses the first step of heme synthesis in the mitochondria
ALA-S1 is present in most tissues for formation of heme
ALA-S2 is only present in erythroid cells

Question 15

How are ALA-S1 and 2 regulated?

Answer 15

ALA-S1 is negatively regulated by heme. Heme inhibits the transcription of ALA-S1, inhibits the translocation from the cytosol to the mitochondria, and inhibits the activity of ALA-S1 over its substrate.

In non-erythroid cells, the rate limiting step of hgb synthesis is the one mediated by the enzyme ALA-S1.

ALA-S2 in erythroid cells is not inhibited by heme. It has an IRE located I the 5’ UTR region. Low iron = IRE binded by IRP = lower translation of ALA-S2 = less protporphyrin IX translated.
–> Rate limiting step is iron acquisition

Question 16

What is the rate limiting step of heme synthesis in non-erythroid cells? In erythroid cells

Answer 16

In non-erythroid cells, the rate limiting step of hgb synthesis is the one mediated by the enzyme ALA-S1.

In erythroid cells, Rate limiting step is iron acquisition

Question 17

Which chromosome has the alpha globin gene in humans? beta globin gene?

Answer 17

• Alpha = chromosome 16

- Beta = chromosome 11

Question 18

How are the globin genes regulated?

Answer 18

Since they are clustered, they are regulated by promoter region (Locus control region (LCR))- genes are under the same regulation during development

Question 19

Explain the “globin switch”

Answer 19

E globins (Gower 1, gower 2 and Portland) are down regulated in early embryonic life
Expression of alpha and gamma genes increase
At birth, alpha and beta genes are up-regulated, gamma is down regulated

Question 20

Name an important transcription factor for globin, other genes it helps transcript and the effect it has on alpha vs. beta globin

Answer 20

Bach1
Bach1 is also important in the transcription of heme oxygenase
Bach1 has the same effect on alpha and beta globin

Question 21

Explain how Bach1 works as TF

Answer 21

When it is bound to the promoter of alpha and beta, it suppresses the expression of their genes
Increased intracellular heme –> binds Back1 –> leaves the cell and is degraded in the cytoplasm –> transcriptional level of alpha and beta increases
Thus, heme and globin are synthesized in similar quantities

Question 22

How is globin translation regulated in erythroid cells?

Answer 22

Globin translation is regulated in erythroid cells by the eIF2α kinase heme-regulated inhibitor (HRI)

Question 23

Name examples of serine-threonine kinases in the family of elF2alpha

Answer 23

o	PERK (PKR-like ER kinase)
o	PKR (protein kinase double-stranded RNA-dependent) 
o	GCN2 (general control non-derepressible-2)
o	HRI

Question 24

What happens when eIF2alpha is phosphorylated?

Answer 24

• Phosphorylation of eIF2α reduces general rate of protein synthesis.

Question 25

What are the 2 main roles of HRI (heme regulatory inhibitor) in erythroid cells?

Answer 25

• The HRI kinase has two key roles in erythroid cells:
  (1) it serves to couple the synthesis of globins genes to the amount of heme present in the cell
  (2) it promotes the survival of erythroid precursors when iron levels are low

Question 26

Where is HRI expressed?

Answer 26

• HRI is only expressed in erythroid cells

Question 27

How does HRI regulate globin synthesis?

Answer 27

HRI phosphorylates eIF2alpha when heme is low —> repression of globin synthesis in erythroid cells
When heme is high (such as in thalassemia) –> HRI is inactivated by heme –> increase of globin synthesis to avoid toxic accumulation of heme

Question 28

Name 3 possible causes of hypochromic microcytic anemia

Answer 28

 Iron deficiency (most common) (can also be d/t Chronic inflammation or malignancy)
 Defect in propoporphyrin synthesis (hence heme synthesis)
 Defect in globin synthesis

Question 29

What is sideroblastic anemia?

Answer 29

Sideroblastic anemia is a group of disorders characterized by a variable population of hypochromic red cells in the peripheral blood and by ringed sideroblasts in the bone marrow.
cannot make heme due to defect in ALAS
Causes iron overload in the mitochondria

Question 30

What is the most frequent form of inherited sideroblastic anemia?

Answer 30

X-linked sideroblastic anemia (XLSA)

Question 31

Name 3 features of XLSA

Answer 31

o (1) detectable disease in new borns but not always present clinically until mid-life or, rarely, later;
o (2) death from hemachromatosis at a relatively young age;
o (3) abundance of siderocytes in peripheral blood.
Give Vit B6 for Tx

Question 32

What do male mice have when they have XLSA?

Answer 32

• Hemizygous males have microcytic anemia and iron overload (ALAS2 misense mutations, ROS production)

Question 33

What are iron-sulfur clusters?

Answer 33

• ISC are prosthetic groups in proteins that play an essential role in cell metabolism;
• ISC proteins are among the most important electron carriers in nature and are found in mitochondria, the cytosol and nucleus;
• They are integral components of the respiratory and photosynthetic electron transfer chains.

Question 34

What is X-linked sideroblastic anemia with ataxia?

Answer 34

• X-linked sideroblastic anemia with ataxia (XLSA/A) is a rare syndromic form of inherited sideroblastic anemia of early onset;
• It is associated with non- or slowly progressive, predominantly truncal, spinocerebellar ataxia and severe, selective cerebellar hypoplasia (mild hypochromic microcytic anemia);
  Give Vit B6 for Tx

Question 35

What causes XLSA/A? what does it do?

Answer 35

• This form of sideroblastic anemia is due to defects in ABCB7 (inner mitochondrial membrane);
• This deficiency induces a disruption in the maturation of cytosolic ISC, indicating the ABCB7 is an essential component of the ISC export machinery.
• Iron overload

Question 36

What is GLRX5? what does a mutation in GLRX5 cause?

Answer 36

• GLRXs are small ubiquitous disulfide oxidoreductases known to be use reduced glutathione (GSH) as a electron donor and are part of the ISC assembly machinery;
• The patient described with this kind of sideroblastic anemia has a homozygous mutation in the GLRX5 gene;
• The patient suffers with mild microcytic anemia associated with iron overload and a low number of ringed sideroblasts (hemoglobin progressively decreases).

Question 37

What is porphyria? what are the types?

Answer 37

Defect of heme biosynthesis pathway
Caused by partial enzyme deficiency in heme biosynthetic pathway besides ALAS2 (e.g. ferrochelatase)
2 types:
-	Congenital erythropoietic porphyria
-	Erythropoietic protoporphyria

Question 38

Name 2 types of porphyrias

Answer 38

2 types:

• Congenital erythropoietic porphyria
• Erythropoietic protoporphyria

Question 39

What is Congenital erythropoietic porphyria?

Answer 39

Deficiency in uroporphyrinogen III cosynthase
CEP is transmited as an autossomal recessive trait;
The associated hemolytic anemia is generally not microcytic.

Question 40

What is erythropoietic protoporphyria? what symptoms does it cause?

Answer 40

Ferrochelatase deficiency or decreased activity

• EPP is characterized clinically by photosensitivity to visible light commencing in childhood, and biochemically by elevated red cell PPIX levels;
• In most of the cases, the inheritance of EPP is described as an autosomal dominant disorder with incomplete penetrance;
• FECH deficiency leads to accumulation of PPIX in normoblasts, erythrocytes, plasma, skin, and liver, causing life long acute photosensitivity and, in approximately 2% of patients, severe liver disease.
• Microcytic anemia occurs in 20-60% of patients.