T9: Protein synthesis and degradation

Question 1

3 components that make up the ER

Answer 1

1. RER
2. SER
3. nuclear envelope

Question 2

Structure and locations of SER

Answer 2

-series of tightly interconnected membrane tubules
-enzymes found in lumen
-large abundance in hepatocytes, muscle cells and purkinje cells

Question 3

What do proteins in the lumen of SER regulate (4)

Answer 3

-lipid synthesis
-glycogen metabolism
-detoxification of xenobiotics
-Ca2+ ion storage

Question 4

Functions of the RER (4)

Answer 4

-protein synthesis
-protein folding
-protein secretion
-glycosylation of polypeptide chains

!!! all transmembrane proteins regardless of final localisation are formed and folded in RER

Question 4

Structure and location of RER

Answer 4

-flattened cisternae with ribosomes associated to the external membrane
-abundant in cells involved in secretion, eg. B cells in production of antibodies

Question 5

Structure of nuclear envelope

Answer 5

-subcompartment of the RER because of the continuity between them
-lumen of nuclear envelope and RER are also connected
-contain nuclear pores (macromolecular molecules that allow molecule movement between nucleus and cytoplasm)

Question 6

Function of nuclear envelope (2)

Answer 6

-hold DNA in place
-allow substance movement through nuclear pores

Question 7

2 areas where proteins can be synthesised and the differences between the proteins made

Answer 7

1. synthesis on cytosolic polysomes (activated aggregates of free ribosomes):
   -nuclear proteins (TFs/histones)
   -soluble proteins (final destination being the cytoplasm)
   -mitochondrial proteins
2. synthesis on RER bound ribosomes:
   -secreted proteins (antibodies)
   -integral membrane proteins (ion channel subunits)
   -ER, Golgi and lysosome lumen resident proteins

Question 8

purpose of proteins containing a signal peptide

Answer 8

-signal peptide contained in the NH3 amino terminal of a short aa sequence
-this signals that the protein is headed to be secreted to extracellular environment
-signal peptide recognized by SRP (signal recognition particle)
-this translocates ribosomal complex with newly forming protein to the RER membrane where it can bind to SRP receptor
-polypeptide chain association to translocation complex
-removal of signal peptide and completion of protein synthesis
-secretion of protein inside lumen of RER to start folding process

Question 9

The folding cycle (4 stages)

Answer 9

1. N linked glycosylation: addition of carb groups on asparagine residues - ends long before exit of protein from ER lumen
2. signal peptide cleavage (removal of signal peptide) - ends after termination of translation
3. disulphide bond formation - ends immediately before exit of protein from ER
4. Oligomerization: formation of oligomers to acquire 4ary structure

!!! ONLY 4 STARTS PURELY POST TRANSLATION (1,2,3 start during translation)

Question 10

What does it mean when we say that protein folding is a set of parallel events?

Answer 10

folding occurs all throughout protein synthesis cycle: hence modifications can be both translational AND post translational

Question 11

details of N glycosylation of asparagine

Answer 11

1. N glycosylation of asparagine: occurs due to OST enzyme (oligosaccharide protein transferase) which removes a carb group from the DOLICHOL donor and transfers it to the polypeptide

Question 12

details of signal peptide cleavage

Answer 12

-achieved by SPC (signal peptidase complex) that is associated to the ER membrane
-occurs in very early translation
-can help assess whether translation is occurring with an ER membrane, because when the cleavage occurs the molecular weight of the protein decreases and this decreases can be picked up by biotechniques (electrophoresis)

Question 13

folding enzymes def

Answer 13

-Enzymes that catalyse the rearrangement of covalent bonds between cysteine residues (mainly disulfide formation).
- most folding enzymes are part of the PDI (protein disulfide isomerase) family

!! pro-isomerization of proline is also possible though

Question 14

2 main classes of enzymes that mediate folding

Answer 14

1. chaperones
2. folding enzymes

Question 15

chaperones def

Answer 15

enzymes that assist protein folding without modification of covalent bonds

Question 16

3 main chaperone proteins

Answer 16

1. BiP (binding protein)
2. Calnexin (3ary structure)
3. glucose regulated protein 94 (GRP94)

Question 17

why are chaperones and folding enzymes important?

Answer 17

Modify protein correctly.
If a protein is incorrectly folded, it is retained in the lumen of the ER (ER quality control) which ensures the viability of cells

Question 18

action of chaperones/folding enzymes as translation begins

Answer 18

-association of BiP and PDI with the proteins just entering the ER lumen
-this happens parallel to N glycosylation and cleavage of signal protein

RESULT: cumulative work of all these molecules cause polypeptide chain to acquire its ‘native conformation’

Question 19

definition of the native and non native conformation of a protein

Answer 19

native conformation: arrangements of protein in its correct and stable configuration

non-native conformation: arrangements of protein in its incorrect and unstable configuration

Question 20

What happens upon the formation of a protein in either its native or non-native conformation post translationally?

Answer 20

NATIVE: correctly folded:
-protein recruited to COPII coated vesicles and exit towards Golgi apparatus

NON-NATIVE: incorrect folding:
-recognition as a misfolded protein, meaning that protein is retained within ER (quality control system) and a degradation process is induced such as ERAD and autophagy

Question 21

difference between post translational membrane insertion and cotranslational translocation mechanisms

Answer 21

1. post translational: conventional protein folding for proteins that are to be released extracellularly: translocated to the RER at the BEGINNING of translation
2. cotranslational: occurs for proteins that are synthesised on free ribosomes in cytoplasm: translocated either to RER or mitochondrial/peroxisomes POST translation

This causes some differences in how proteins are processed

Question 22

what are the 2 mechanisms in the response to protein misfolding?

Answer 22

1. UPR - unfolded protein response
2. ERAD - ER associated degradation

Question 23

UPR trigger and detailed sequence

Answer 23

TRIGGER: accumulation of misfolded proteins in ER which causes disequilibrium (the amount of proteins entering ER are not the same as the ones exiting it) PROCESS: 1. Sensors that were inactive under normal conditions (BiP bound) transfer BiP to misfolded proteins which activates them. Sensors = Perk, ATF6, IRE1alpha 2. Response of sensors: -increased translation of chaperones -decreased translation of general proteins -increased translation of genes involved in ERAD -increase in ER membrane surface 3. RESULTS EITHER IN: -cell survival if the ER is able to re establish equilibrium -apoptosis if the stress cannot be reverted and it poses too great a threat to the cell

Question 24

Specific stress signals that are picked up by sensors in UPR (6)

Answer 24

1. difference in composition of ER membrane 2. difference in REDOX potential within ER lumen 3. difference in amount of polypeptide glycosylation in lumen 4. increased flux in translocome 5. decrease in activity of ERAD 6. decrease in Ca2+ concentration in ER lumen

Question 25

ERAD trigger and detailed sequence

Answer 25

TRIGGER: removal of mannose residues from misfolded proteins PROCESS: 1. misfolded protein is sent to retrotranslocation complex which causes retrotranslocation of the proteins from the ER lumen to cytoplasm 2. proteins are polyubiquitinated during the retrotranslocation and end up in cytoplasm to be degraded by the proteasome system

Question 26

Structure of ubiquitin and of proteasome

Answer 26

ubiquitin: small protein of 65 amino acids weight 8.4 kiloDaltons proteasome: big protein complex --> empty cylinder made of 3 subunit proteins

Question 27

Ubiquitin/proteasome degradation sequence

Answer 27

1. ubiquitin proteins are activated through activity of ubiquitin ligases. Hence they are activated, conjugated and ligated to target proteins that need to be degraded 2. Polyubiquitinated molecules are recognised by proteasome 3. Movement of misfolded protein along the cylinder and cleaving into smaller peptides that are then released into cytoplasm

Question 28

types of proteins that proteasome vs lysosome degradation targets

Answer 28

PROTEASOME: soluble proteins that are present in cytoplasm without being confined within a membrane bound organelle LYSOSOME: proteins confined within a membrane bound organelle

Question 29

Lysosome protein degradation sequence

Answer 29

!! controlled by autophagy and so triggered by intrinsic extracellular stressors PROCESS: 1. formation of phagophore - membrane intermediate which encircles intracellular components 2. formation of autophagosome - completely closed membrane intermediate 3. Fusion of autophagosome with lysosome causes formation of autophagolysosome 4. lytic enzymes within lysosome digest molecular components in the lumen of autophagosome 5. Release of components into cytoplasm where they can be recycled for cell metabolism

Question 30

What is the difference between autophagy/lysosome degradation and other methods of degradation

Answer 30

autophagy/lysosome degradation can break down: proteins, nucleic acids, lipids and sometimes entire organelles. All other methods can only degrade proteins

Question 31

3 stressors that trigger autophagy/lysosome degradation

Answer 31

EXTRACELLULAR STRESSES: 1. hypoxia 2. starvation 3. activation of protein kinases (activated upon energy depletion)

Question 32

a key difference between phagocytosis and autophagy

Answer 32

phagocytosis is triggered by a foreign pathogen which needs to be digested whereas autophagy is triggered by extracellular stresses like starvation