Lecture #7 - Post-translational modifications Flashcards
What’s a proteome?
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PTM provide a way to increase complexity of life without…
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Two categories of protein PTM - what are they?
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Phosphorylation
- Can occur on the side chains of what? Sometimes what aa’ are phosphorylated?
- What enzyme catalyses phosphorylation?
- What enzyme catalyses dephosphorylation?
- The addition of the larger charged phosphate to the OH group induces what?
- This affects function - give two examples
- Explain his theory of cyclic type activity
Quite often protein phosphorylation/dephosphorylation is used to switch between protein conformation states to enable a biological process in a cell
Phosphorylation example #1: Insulin receptor
Elevated blood glucose promotes the release of ______ into the blood stream
Insulin binds to the ______ protein subunits of the insulin receptor
This causes a conformation change that is communicated to the intracellular side protein subunits, and activates _____ _____ domains
Specific _____ are phosphorylated on the __-______ which then leads to phosphorylatioon of ‘____ _____ ____’ proteins, which act as second messengers in the cell, and result in transfer of ______ glucose transporter proteins to the cell membrane to enable uptake of glucose
Elevated blood glucose promotes the release of INSULIN into the blood stream
Insulin binds to the EXTRACELLULAR protein subunits of the insulin receptor
This causes a conformation change that is communicated to the intracellular side protein subunits, and activates TYROSINE KINASE domains
Specific TYR are phosphorylated on the Β-SUBUNITS which then leads to phosphorylation of ‘INSULIN RECEPTOR SUBSTRATE’ proteins, which act as second messengers in the cell, and result in transfer of GLUT4 GLUCOSE transporter proteins to the cell membrane to enable uptake of glucose
Phosphorylation example #2: The sodium/potassium ion pump is phosphorylated
The _______ membrane located sodium/potassium ion pump has evolved to pump Na+ ____ and K+ ____ a cell to maintain intracellular high [K+] and low [Na+] relative to outside the cell
When the ion pump protein complex is phosphorylated a protein conformation change enables……..
When the ion pump is dephosphorylated a protein conforma8on change enables……….
The phosphorylation is achieved as a result of the hydrolysis of a……..
The INTEGRAL membrane located sodium/potassium ion pump has evolved to pump Na+ OUT and K+ INTO a cell to maintain intracellular high [K+] and low [Na+] rela8ve to outside the cell
When the ion pump protein complex is phosphorylated a protein conformation change enables THREE NA+ TO BIND AND BE TRANSLOCATED OUT OF THE CELL
When the ion pump is dephosphorylated a protein conformation change enables TWO K+ TO BIND AND BE TRANSLOCATED INTO THE CELL
The phosphorylation is achieved as a result of the hydrolysis of a HIGH ENERGY PHOSPHATE BOND IN ATP
An illustration of how protein phosphorylation can alter protein conformation:
Phosphorylation (- charged) of a _____ side chain amino acid in a protein…
…promotes recruitment of (+ charged) amino acid side chains (eg arginine) from other parts of the protein amino acid sequence to form…….
…this results in movement of parts of the protein amino acid sequence, which alters…….
…which results in alteration of protein _______
Phosphorylation (- charged) of a HYDROXYL side chain amino acid in a protein…
…promotes recruitment of (+ charged) amino acid side chains (eg arginine) from other parts of the protein amino acid sequence to form STABILISING ELECTROSTATIC INTERACTIONS…
…this results in movement of parts of the protein amino acid sequence, which alters THE 3- DIMENSIONAL CONFORMATION OF THE PROTEIN…
…which results in alteration of protein FUNCTION
Gamma-carboxy glutamic acid (Gla)
An additional carboxly group is _____ linked to ______ ______ in some blood coagulation proteins
____ to _____ Glu aa’ are modified to Gla, which enables the formation of __-____ Ca2+ binding sites. This enables blood coagulation proteins to interact with ______ as part of the blood-clot-forming process
The formation of Gla requires what?
-The formation of gamma-carboxy glutamic acid (Gla) is a specialised form of PTM carboxylation that occurs in some blood coagulation proteins. This generates a malonyl-type two carboxyl Gla side chain structure that enables the bidentate chelation of divalent metal ions, in particular Ca2+. The precursor forms of the enzymes prothrombin and profactor IX and X are modified in this way. Ten to 12 glu residues in a localised ~40 amino acid sequence within each protein are gamma-carboxylated, which, in the presence of Ca2+ enable the proteases to associate with platelet surfaces, forming protein complexes and activating downstream components of the blood coagulation cascade. THIS CARBOXYLATION PTM HAS A REQUIREMENT FOR THE DIHYDRO FORM OF VITAMIN K AND O2 AS PART OF THE CO2 FIXATION PROCESS TO FORM GLA.
Vitamin K and blood coagulation
‘Vitamin K’ comprises a number of compounds
varying in the number of _____ units
Vitamin K deficiency is quite rare but results in bleeding disorders
_____, a structural analogue, interferes with vitamin K _____ during γ-carboxylation of blood cloing proteins which inhibits clot forma8on
Warfarin (and also aspirin derivatives) are used clinically to _____ clot formation risk in patients
Why is Vit K important?
‘Vitamin K’ comprises a number of compounds
varying in the number of isoprenoid units
Vitamin K deficiency is quite rare but results in bleeding disorders
Warfarin, a structural analogue, interferes with vitamin K chemistry during γ-carboxyla8on of blood cloing proteins which inhibits clot forma8on
Warfarin (and also aspirin deriva8ves) are used clinically to reduce clot forma8on risk in pa8ents
Vit K stabilises the enzyme used to modify the prothrombin to attach the carboxly group
Extrinsic and intrinsic pathways of blood coagulation
What three things are gamma-carboxylated and where?
This forms a local high density of what?
This assist the association of the proteins with platelet surfaces leading to the formation of _____ _____ and activation of proteases as part of the coagulation pathway
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Conversion of fibrinogen to a soft clot of fibrin
Fibrinogen (340kDa) consists of ________ subunits linked by disulfide bonds
_______ hydrolyses parts of the proteins, releasing fibrinopeptides
The hydrolysed fibrin monomer is able to aggregate to form a s___ _____
The fibrin clot mesh-traps red blood cells and together with platelet adhesion, seals the wound
Note also
(i) the hydrolysis of fibrinogen by thrombin is an example of ________ ____ _______ ______
(ii) the formation of _____ ______ in fibrinogen is another example of a PTM
Fibrinogen (340kDa) consists of 2 α, 2 β and 2 γ subunits linked by disulfide bonds
Thrombin hydrolyses parts of the proteins, releasing fibrinopeptides
The hydrolysed fibrin monomer is able to aggregate to form a soY clot
The hydrolysed fibrin monomer is able to aggregate to form a soft clot
The fibrin clot mesh-traps red blood cells and together with platelet adhesion, seals the wound
Note also
(i) the hydrolysis of fibrinogen by thrombin is an example of peptide bond hydrolysis PTM
(ii) the formation of disulphide bonds in fibrinogen is another example of a PTM
Hydroxylation
- The hydroxylation happens at what position of proline (can also happen on the what carbon?). What about another aa’ that can be hydroxylated at 5’?
- What is highly abundant and is significant;y hydroxylated?
- What does this PTM provide?
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Vit C required for hydroxylation PTM
- What enzyme hydroxylates the proline and lysine residues in cologne? And what do they require?
- What is Vit C required to maintain?
- A decrease in hydroxylation results in what?
- Can we synthesise Vit C?
- What does scurvy result from?
- How exactly does it affect collagen?
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