Dynamic regulation of protein

Question 1

What are the means of regulating protein activity in the cell?

Answer 1

• protein levels
• protein location and concentration
• ligand binding
• cofactor requirements (Ca2+ or GTP)
• protein phosphoyrlation and dephosphorylation
• proteolytic cleavage

Question 2

when do proteins get degraded?

Answer 2

Normal protein turnover

1) housekeeping proteins get damaged and need to be replaced
2) misfolded proteins need to be recycled - damaged proteins upset normal cell processes and are removed by degradation

dynamic process

3) levels of proteins tightly regulated to control function ex) cyclins control entry into or exit from a cell process are synthesized to promote entry into cell cycle and are degraded when they want to turn off
4) in the course of being activated some proteins get cleaved and need to be replaced

Question 3

how are proteins degraded?

Answer 3

1) lysosomal degradation - degrades proteins imported into the cell
2) proteasomal degradation - degrades cytosolic proteins marked for destruction

Question 4

how do lysosomes work?

Answer 4

They are highly acidic (have proton pumps that pump H+ ions in to lower pH) - they have acid hydrolases that are only active at this low pH (protect cytosolic proteins from activites if they leak). Lysosymes will degrade any biological material - protein, nucleic acid, carbohydrates and lipids

Question 5

What is autophagy’s role in degradation?

Answer 5

Autophage- proteins and organelles in the cell that are damaged and need to be recycled are packaged into autophagosomes (membranous organelles formed from the ER)

phagocytosed or endocytosed material also targeted to lysosme for degradation

Question 6

what is the key was to get rid of ddangers to the cell?

Answer 6

autophagy

• implicated in many chronic inflammatory diseases-
• defects in any of the enzymes can lead to lysosomal storage diseases and accumulation of unwanted biomolecules -

Question 7

what does ubiquitination do?

Answer 7

Ubiquitination = target for degradation

ubiquitin is a compact protein added to lysine residues on proteins

Question 8

what is the Ubiquitin-proteasome system? (UPS)

Answer 8

stability of many proteins is determined by whether they can be ubiquitinated -

• addition of ubiquitin to lysine resides in a protein is the first step of this system
• subsequent additions of ubiquitins
• the poply-ubiquitinated protein is recognized by the proteasome

Question 9

describe the process of ubiquitination

Answer 9

1. ubiquitin is activated by being attached to the ubiquitin-activating enzyme (E1)
2. ubiquitin is transferred to the ubiquitin conjugating enzyme (E2)
3. final transfer of ubiquitin to target protein mediated by E3 ligase
4. E3 ligases recognize the target/substrate and so confer specificity on the pathwy a

Question 10

What ubiquitination enzyme is associated with parkinson’s disease?

Answer 10

parkin- E3 ligase

• loss of function mutation associated with the autosomal recessive form of parkinsons

loss of parkin’s E3 ligase activity is thought to play a pathogenic role in both inherited and sporadic PD

The exact mechanism is unknown,

Question 11

What ubiquitination enzyme is associated with tumorigenesis?

Answer 11

MDM2- E3 ligase

targets the tumour suppressor p53 for degradation

upregulated in many cancers

Question 13

Describe the structure of the proteasome

Answer 13

proteasome is a barrel shaped complex found in the nucleus or cytoplasm - the core complex is composed of 7 alpha type proteins and 7 beta type proteins

Question 14

Describe the proteasome function

Answer 14

• have three different protease activities
  
  • Trypsin - after arginine/lysine
  • chymotrypsin- after tyrosine or phenylalanine
  • caspase-like - after aspartic or glutamic acid
• proteins fed in through opening at top and enter into the center of the proteasome - cleaved by the action of the proteases - peptides exit the bottom

Question 15

where does the proteasome get the energy to unfold proteins and remove ubiquitin chains?

Answer 15

it gets its energy from ATP

Question 16

Describe the clinical use of Bortezomib

Answer 16

it is an inhibitor of the UPS system - treat patients with multiple myeloma (a cancer of plasma cells)

the drug blocks the proteolytic action of the proteasome

this is a good thing because many of the transcription factors that are regulated by the inhibitor IkB - often in myeloma, this IKB inhibitor is degraded by the proteasome before it inhibitors the transcription factor=leading to proliferation

If we turn off the degradation, we can allow this IkB to inhibit the transcription factor NFKB as it should

Question 17

What common post-translational modifications do we use for protein regulation?

Answer 17

• ligand binding
• cleavage to produce active protein/enzyme
• phosphorylation
• ubiquitiniation
• fatty acid modification

Question 18

Describe the post translational modification of ligand binding

Answer 18

ligand binding - changes shape of protein often to active form- release of inhibitory subunits, or change cell loaction

ex) steroid hormone receptors bind their ligand and get a conformational change - dimerise and enter the nucleus

Question 19

describe the post-translational modification of proteolytic cleavage

Answer 19

zymogens- pro-enzymes

cleavage of pro-enzymes activates it

Question 20

Describe the post-translational modificaiton of reversible phosphorylation of proteins

Answer 20

phosphate group added to Serine, threonine, and tyrosine residues

tyrosine is least abundant but most effective

phosphorylation alters protein function, marks for degradation, alters localization or promotes interactions

• kinases add the phosphate
• phosphatases remove it

Question 21

What is the purpose of Fatty Acid modification of proteins?

Answer 21

for membrane anchoring or protein-protein interactions