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BIO319 - CELLULAR SIGNALLING > Inositol Signalling > Flashcards

Inositol Signalling Flashcards

1
Q

What are phosphatidylinositols (PIs)?

A

Phospholipids with the head group inositol (a carbohydrate group).

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2
Q

In what forms are PIs most commonly seen?

A
  1. PIs - these are most common
  2. Phosphorylated PIs (PPIs) - they can be phosphorylated on the 3/4/5 OH group in different combinations. They are not as common but have important functions.
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3
Q

Name some functions of PPIs.

A

Are involved in membrane structure, cell signalling, cytoskeletal regulation and membrane trafficking.

  • Are markers for membrane identities.
  • Regulate membrane ion channels.
  • Act as substrates of 2 TM signalling systems.
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4
Q

Why is PI more common than PPI if it is known that PPIs have more functional roles?

A

It is not exactly known, however, it is thought that PIs are in abundance to make PPIs.

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5
Q

How was it initially determined that PIs have a key role, and are not just structurally important?

A

It was found that secretory tissues produced increased PI upon stimulation.
Experiment:
1. The pancreas of pigeons was stimulated with acetylcholine.
- This caused proteolytic hormones to be secreted.
2. This was incubated with radioactive inositol.
3. It was observed that there is only a rapid turonver of PI, not all PLs.

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6
Q

From the 60s to now, what 5 have key things been found about PIs?

A
  1. That there are many PIs, and are all found in eukaryotes.
  2. That they are involved in the PLC/InsP3 and DAG signalling events.
  3. That these PIs have different functions.
  4. That there are PI-3-kinases which are involved in signalling, membrane trafficking and disease.
  5. That some have a central role in a spectrum of cell signalling events.
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7
Q

What are the 2 main functions of PIs? Are there other emerging roles?

A

Cell signalling and membrane trafficking.

Yes there are more emerging roles, e.g. immune cell development.

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8
Q

How is the function of PIs mediated?

A

Through interactions with protein with specific domains binding to PIs.
- e.g. PH/PX, FYVE, ENTH domains.

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9
Q

What is the source of inositol?

A

Diet and is made in tissues.

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10
Q

Why was it initially thought that inositol was vitamin B8?

A

It was found that a diet supplemented with inositol cured a fatty liver and type 2 diabetes symptoms.
- Not a vitamin as it is made in tissues too.

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11
Q

What is inositol?

A

A stable, sugar like molecule.

- It has a hexachlorocyclohexane structure (6 carbon saturated ring, each C has a H and OH)

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12
Q

What form of inositol is used in nature and discussed?

A

myo-inositol.

- This appears in its preferred shape: chair shape.

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13
Q

What is the chair shape of inositol?

A

On the 2nd carbon, the OH group is axial (outside the ring plane) and all others are equitorial (inside ring plane).

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14
Q

How many isomers are there of inositol?

A

9

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15
Q

What form of inositol is only made directly in the tissues?

How and where is this done?

A

Myo-inositol, made in most tissues but is best made in the testes.

  1. Glucose-6-phosphate is rearranged into a ketone derivative by MIPS and NAD+.
    - NAD+ accepts the hydrogens off glucose and donates them back to give a different configuration.
    - This gives inositol-3-phosphate.
  2. Inositol monophosphatase then removes the phosphate from inositol-3-phosphate to give inositol.
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16
Q

Where is it thought that MIPS originated from and why?

A

From archaea.

  • All MIPS enzymes are related, and have the same catalytic core.
  • MIPS is found in some bacteria (actinobacteria), but is in all hydrophobic archaea (v hydrophillic ones can’t tolerate it).

This suggests that an archaeal cell merged with a bacterial cell, where it was then passed onto eukaryotes.

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17
Q

Name the 8 main PIs (combination of P on 3/4/5 OH)

A
  • PI
  • PI3P
  • PI4P
  • PI5P
  • PI-3,4-P2
  • PI-4,5-P2 (PIP2)
  • PI-3,5-P2
  • PI-3,4,5-P3 (PIP3)
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18
Q

Describe the structure of a PI, noting where the different phosphorylated parts are seen.

A

FA tails - glycerol part - phosphate - glucose derivative part

Phosphorylated parts are seen on the glucose derivative part, replacing the OH groups on carbon 3/4/5.

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19
Q

How is interconversion between PIs and PPIs mediated?

A

By kinases and phosphatases.

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20
Q

Name some forms of inositol found in eukaryotes and their main functions.

A
  • PPIs (phosphorylated phosphatidylinositols): many functions, e.g. involved in signalling and membrane trafficking.
  • Inositol sphingolipids (inositol based phospholipids): anchor proteins to the cell surface.
  • PI and Inositol sphingolipid anchors: anchor proteins and carbohydrates to the cell surface.
  • Inositol polyphosphates (inositol breakdown product): supply phosphate to plants, found in soil.
  • Inositol pyrophosphates (same as above but more phosphates): role in gene regulation.
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21
Q

Name the form of inositol found in all kingdoms and its main function.

A

Stabilising solutes
- These are highly hydrated and make inositol and inositol dervatives as solutes in the cytosol to stabilise cells found in unfavourable conditions, e.g. high salt areas.

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22
Q

Name the form of inositol in actinobacteria and its main function.

A

Mycothiol

- This is used in a similar manner as we use glutathione; as a protective agent against ROS.

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23
Q

Where are PIs seen?

A

In mycobacteria, plants, yeast and animals.

There is an archaeal homologue called ArcIns.

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24
Q

Describe the PI:PPI content of different organelles.

A

In the ER and mitochondria, there are many PIs but v few PPIs. The difference is much larger in ER than mitochondria
- This is the opposite for all other organelles, which have much more PPIs in various forms.

25
Q

Describe the PI:PPI content of different tissues/cells.

A

In most cells, the PPIs are no more than a tenth of the PI.
- RBCs are an exception, as they only have a plasma membrane, which contains a lot of PPIs.

Wet tissues, e.g. liver, have low PIs compared to PPIs.

26
Q

Where are the main PPIs found in a eukaryotic cell and what are their functions?

A

PI:
- Is abundant in cells, does not have specific functions.

PI3P:

  • Found in endosomes which transfer cargo within the cell and process material.
  • Membrane traffic regulator.

PI4P:

  • Mostly in the trans Golgi and in vesicles going to the plasma membrane.
  • Is a PI-4,5-P2 precursor and is an ER/trans-Golgi traffic regulator.

PI5P:

  • Found in cytoplasmic membranes and the nucleus.
  • Functions not yet known, suggested to be involved in nuclear signalling.

PI-3,4-P2:

  • In vesicles from endocytosis.
  • Needed for endocytosis.

PI-4,5,-P2:

  • Found in the plasma membrane, converted from PI4P.
  • Is involved in signalling through PLC and type I PI3K pathways. Is also a cytoskeletal and ion channel regulator and is needed for exo- and endo-cytosis.

PI-3,5-P2:

  • In lysosomes.
  • Is a membrane traffic regulator.

PIP3:

  • Found in membranes.
  • Involved in signalling. It is split into DAG and InsP3 by PLC/PIC.
27
Q

What is special about PIP3 function evolution?

A

Seen to emerge only after multicellular animals evolved, suggesting that it evolved late in eukaryotic diversification.

28
Q

How is it thought that PIs cause a response which is involved in signalling pathways?
From 50s to 80s.

A

Thought that PIs respond to a stimulated receptor, and causes an increased calcium level inside the cell.

  1. It was seen that a variety of secretory tissues, including lymphocytes, cause an increased PI turnover when stimulated.
    - > Therefore hypothesised that the PI turnover is linked to receptor signalling.
  2. It was seen that acetylcholine stimulates PI hydrolysis.
    - > Therefore hypothesised that PI hydrolysis causes an increase in intracellular calcium.
    - > This hypothesis was confirmed to be true.
29
Q

What is PIC and what does it do?

A

It is a family of enzymes, like phospholipase C (PLC) but specific to inositols: phosphoinositide PLC.

  • They hydrolyse PIs.
  • It mainly hydrolyses PI-4,5-P2 into DAG and InsP3.
30
Q

What does DAG and InsP3 do?

A

InsP3 goes from the plasma membrane to the ER, where it acts on calcium channels selective to InsP3 to release calcium stores from the ER into the cytosol. This increases the calcium levels intracellularly.

DAG stays in the plasma membrane where it activates PKC, which drives regulation.

31
Q

What is now known as the main pathway of how PIs activate downstream cell signalling?

A
  1. PI in the plasma membrane is turned into PIP2 (PI-4,5-P2) by certain kinases.
  2. An agonist stimulates a receptor, e.g. an agonist stimulates a Gq GPCR.
  3. This active receptor causes PIP2 to be split into DAG and InsP3 by PIC.
  4. DAG and InsP3 have their downstream effects.
32
Q

What domains do PICs have?

A

Common domains:

  • TIM barrel, which makes the catalytic core.
  • EF hand which binds the small amount of calcium required for activity.
  • A C2 domain, this binds to the membrane as it has hydrophobic myo-acids.

Most enzymes have it:
- PH domain, this binds to phosphorylated head groups of PI lipids.

33
Q

What are the 3 main families of PICs and their functions/regulation?

A

PIC beta:
- These are coupled to GPCRs through the Gq or G11 subunit.

PIC gamma:
- These are coupled to RTKs and growth factors through SH2 domains.

PIC delta:
- Function and regulation is unknown.

PIC epsolom:
- Activated by Ras.

34
Q

Which family of PIC is most common?

A

delta - in all eukaryotes.

The other families are not seen in plants.

35
Q

What is a sideman function of InsP3?

A

Can be converted into InsP6 which is involved in nuclear mRNA export.

36
Q

What is the second signalling pathway that PIs are involved in?

A

Production of PIP3 with PI-3-kinase (PI3K).

37
Q

What observations were made regarding PIP3 and what was the conclusion made?

A
  1. PI kinase (PIK) is associated with the Src oncogene, and this kinase makes PI3P.
  2. Neutrophils make PIP3 in response to a peptide MLF which mimics stimulation.
  3. PIP3 is made in RTK stimulated fibroblasts and by GPCR stimulated neutrophils.
  4. A PI3K stimulated by PDGF makes PIP3.

Conclusion: PIP3 is a membrane associated messenger with many effectors, including GPCRs and RTKs.

38
Q

Describe the PI3K family.

A

They are in 3 families which differ in the domains present on their regulatory subunit.

39
Q

Describe the PI3K class I family.

A
  • Involved in PIP3 synthesis.
  • Split into further classes (alpha/beta/gamma).
  • Are regulated by many proteins and have 3 in vitro substrates (PI, PI4P, PI-4,5-P2).
  • Can have SH2 domain and GAP domain.
40
Q

Describe the PI3K class II family.

A
  • Involved in PIP-3,4-P2 synthesis.
  • Not known what they are regulated by and have 2 in vitro substrates (PI, PI4P).
  • No specific domains.
41
Q

Describe the PI3K class III family.

A
  • Involved in PI3P synthesis.
  • Thought to be constitutively regulated but not sure. Have one substrate in vitro, PI.
  • No Ras binding domain but can have heat repeats.
42
Q

Which class of PI3K is most common?

A

Class I (a/b)

43
Q

Discuss the PI3K class I family (activation etc).

A

PI3K Ia/b:

  • Activated by RTKs through the SH2 domains on their regulatory subunit.
  • Also activated by Ras.

PI3K I-gamma:

  • Activated by GPCRs through the G-beta-gamma subunit.
  • Also activated by Ras.
44
Q

Discuss the importance of PI3K-Ia in cancer.

A

It is constitutively activate in many cancers through mutations. Therefore, it causes non-regulated growth and therefore metastasis.

  • Many mutations are post-natal and are seen in stem cell tissue.
  • In these cancers, targeted drugs which inhibit this kinase can be used, e.g. wortmannin
45
Q

What is a limit to using wortmannin as an anti-cancer drug in those which have PI3K mutations?

A

It has a high toxicity level, meaning that the dosage must be minimised.
- However, it is still seen to be effective.

46
Q

What is PTEN and why is it important?

A

Phosphatase and tensin homolog.

  • It was identified as a tumour suppressor gene which encodes a PIP3 phosphatase.
  • It was found to be mutated to be inactive in many cancers, suggesting that it is involved in preventing increased cell growth and proliferation.
47
Q

What do findings about PTEN and PI3K show about the function of PIP3?

A

They confirm that PIP3 causes proliferation.

  • When PTEN is inactive (usually inactivates PIP3), tumours grow.
  • When PI3K is overactive (usually makes PIP3 active), tumors grow.
48
Q

What are other known processes which are mediated by PIP3? (cells, glucose, others)

A
  • Cancer
  • Cell survival
  • Cell cycle progression and cell growth
  • Stimulation of glucose transport (PIP3 recruits GLUT4 to vesicles)
  • Glycogen synthesis
  • Motility/chemotaxis, e.g. in neutrophil migration
  • Protein synthesis (via IP6)
  • Calcium mobilisation (PIC activation)
  • Longevity
  • Superoxide generation
49
Q

How does PIP3 interact with proteins?

A

Mainly via PH domains

50
Q

Name some key PH domains which are specific to PIP3

A
  • Ras-GAPs.
  • PDK1
  • Tek Tyr-kinases (Btk, Itk etc).
51
Q

Are there other targets for PIP3 other than PH domains? If so, what are they? Are they common?

A

Yes, not as common as PH domains.

  • FYVE domains
  • SEC14 homology domains
  • Others which are not yet defined.
52
Q

Where are FYVE domains usually seen?

A

In the endosomal protein EEA1. This binds to the membrane via PI3P (FYVE domain) and Rab5.

53
Q

What is the main function of PX domains and where are they found?

A

When found in ‘sorting nexins’, they are involved in many membrane interactions and fusion events.
When found in the phox protein, they are involved in killing bacteria via neutrophils.

54
Q

What is a common function of PI3P binding proteins? What domains are usually seen on these proteins?

A

Vesicle trafficking in and between the Golgi, endosome, lysosome and plasma membrane. They are key for the membrane cycling within these organelles.
They usually have FYVE or PX domains.

55
Q

What domains are usually seen in Class III PI3Ks (for binding to PI3P)?

A

FYVE domains and PX domains.

56
Q

What is a role of Class II PI3Ks (make PI-3,4-P2)?

A

Clathrin-dependent endocytosis.

57
Q

Describe clathrin-mediated endocytosis, noting the role of PI-3,4-P2.

A
  1. Cell surface receptors cause a series of elements to gather around a patch.
  2. This causes the recruitment of clathrin.
  3. The membrane invaginates and forms a vesicle with cargo in.
    - At these sites, PI-4,5-P2 is made by kinases.
  4. Budding of the vesicle occurs.
    - As vesicles form, PI-4,5-P2 is reduced to PI4P.
  5. The neck of the vesicle starts to form.
    - Here, Class II PI3Ks make PI-3,4-P2 from PI4P.
    > This PI recruits dynamin, causing the vesicle to pinch off.
  6. Vesicle is now inside the cell and cargo is released. The clathrin coat falls off and is recycled.
    - Here, PI4P is in the membrane and the vesicle is labelled with PI3P for endocytic pathway progression.
  • PI4P is in the membrane throughout all the steps.
58
Q

Where is PI-3,5-P2 found and when is it synthesised?

A

In yeast, it is synthesised during hyper-osmotic stress.

- Also found in mammalian fibroblasts and all eukaryotes.

59
Q

How is PI-3,5-P2 synthesised?

A
  1. PI3P is made from Class I PI3K
  2. PI3P is phosphorylated on the 5- position to give PI-3,5-P2.

The enzyme which does the second step is FAB1 (yeast) and PIKfyve (eukaryotes)

BIO319 - CELLULAR SIGNALLING (15 decks)

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