Protein Quality Control and Misfolded Proteins at the ER

What is the primary role of ER quality control?

To ensure only correctly folded proteins exit the ER to the Golgi

Why are misfolded proteins potentially harmful?

They can aggregate or interact incorrectly with other proteins and harm the cell or organism

Where are misfolded ER proteins typically retained?

In the endoplasmic reticulum

Which type of proteins are especially dangerous if misfolded and secreted?

Secreted proteins and cell-surface proteins

What GPCR was used as an example of ER quality control?

Opsin

Where does wild-type opsin localise in the cell?

Cell surface and ER during synthesis

Where does mutant opsin localise?

Endoplasmic reticulum only

What disease is caused by mutant opsin retention in the ER?

Retinal disease

Why were cells not permeabilised in the opsin experiment?

To label only cell-surface proteins with antibodies

Why is a cell-surface marker used as a control?

To confirm cells and antibody access to the plasma membrane

What proportion of newly made proteins fail to fold correctly?

About 20 percent

What is ERAD?

ER-associated degradation of misfolded ER proteins

Why can misfolded proteins not be degraded in the ER lumen?

The ER lumen lacks proteases for degradation

Where are ERAD substrates ultimately degraded?

In the cytosolic proteasome

What modification do many ER proteins receive during synthesis?

N-linked glycosylation

How many glucoses and mannoses are on the initial N-linked glycan?

Three glucoses and nine mannoses

What happens after two glucoses are removed from the glycan?

The protein becomes monoglucosylated

Which chaperones bind monoglucosylated glycoproteins?

Calnexin and calreticulin

What enzyme removes the final glucose to release the protein?

Glucosidase

What enzyme re-adds a glucose to misfolded proteins?

UGGT (glucosyl transferase)

What is the function of the calnexin/calreticulin cycle?

To give proteins multiple chances to fold correctly

What signals that a protein should exit the calnexin cycle?

Mannose trimming on the N-linked glycan

What enzymes perform mannose trimming?

ER degradation-enhancing alpha-mannosidases (EDEMs)

Why is mannose trimming called a timer?

Because it occurs slowly and allows time for folding attempts

Which mannose form prevents reglucosylation?

Man8

Which mannose form signals degradation?

Man7

What do calnexin and calreticulin act as in quality control?

Rescue chaperones

Are all ER proteins N-glycosylated?

No

What recognizes Man7 glycoproteins for degradation?

Quality control lectins

What is retrotranslocation?

Movement of misfolded proteins from the ER to the cytosol

Why must disulfide bonds be reduced during ERAD?

To allow proteins to pass back through the membrane

What small protein is added during ERAD to signal degradation?

Ubiquitin

What type of ubiquitination targets proteins for degradation?

Polyubiquitination

What enzyme catalyses ubiquitin addition?

E3 ubiquitin ligase

Name a candidate retrotranslocation channel

Sec61

What evidence supports Sec61 in ERAD?

Mutants allow import but block degradation

What other proteins may form retrotranslocation channels?

Derlins and E3 ligases

What dual role does Hrd1 play?

E3 ubiquitin ligase and retrotranslocation channel

What experimental system showed Hrd1 can retrotranslocate proteins?

In vitro reconstitution with proteoliposomes

How does Hrd1 initiate retrotranslocation?

By conformational change and autoubiquitination

What prevents backsliding of proteins during retrotranslocation?

Ubiquitination acting as a ratchet

What ATPase extracts ERAD substrates?

P97 (VCP)

What provides the energy for protein extraction?

ATP hydrolysis

Where are N-linked glycans removed before degradation?

In the cytosol

What alternative mechanism may extract hydrophobic proteins?

Lipid droplet budding from the ER

What triggers the unfolded protein response?

Accumulation of misfolded proteins in the ER

What is the goal of the UPR?

To restore ER homeostasis

How many UPR sensors exist in mammals?

Three

Name the three mammalian UPR sensors

IRE1 PERK and ATF6

Which UPR sensor is most evolutionarily conserved?

IRE1

What keeps IRE1 inactive under normal conditions?

Binding to BiP

How is IRE1 activated?

BiP dissociation and dimerisation or oligomerisation

What enzymatic activity does activated IRE1 have?

Ribonuclease activity

What mRNA does IRE1 splice?

XBP1 mRNA

What is the role of spliced XBP1?

It acts as a transcription factor for UPR genes

What other function does IRE1 RNase have?

Degradation of ER-targeted mRNAs

What is RIDD?

IRE1-mediated mRNA decay to reduce ER load

What structural change enhances IRE1 signalling?

Formation of higher-order oligomers

Are IRE1 oligomers permanent?

No

Which UPR sensor is a kinase?

PERK

What does PERK phosphorylate?

eIF2alpha

What is the effect of eIF2alpha phosphorylation?

Global reduction in translation

Why does PERK reduce translation?

To reduce protein load on the ER

Can any proteins still be translated during PERK activation?

Yes

How is ATF6 activated?

By proteolytic cleavage after ER stress

What happens if ER stress is prolonged or severe?

Apoptosis is triggered

What cell type dies in ER stress-related diabetes?

Pancreatic beta cells

What causes misfolding of insulin in the diabetes example?

Mutation preventing signal peptide cleavage

Why does insulin misfolding cause chronic ER stress?

Insulin is produced continuously in beta cells

What is the outcome of chronic UPR activation in this disease?

Beta cell death and diabetes