Macromolecular quality control

Question 1

What is macromolecular quality control?

Answer 1

Cellular processes that ensure that macromolcules, such as proteins, are properly folded, guaranteeing proper functioning

Question 2

What are the cellular risks associated with proteins not properly folded?

Answer 2

-Dilution of active components (functioning proteins)
-Dominant-negative mutations
-Aggregation and interference with cellular functions

Question 3

What are the two central mechanisms of macromolecular quality control?

Answer 3

1) RNA level: preventing synthesis of abnormal transcripts
2) Protein level: destroying aberrant proteins

Question 4

What are chaperones?

Answer 4

Proteins that assist the folding of macromolecular structures (e.g., proteins), guiding them along the productive folding pathways

Question 5

Explain how heat shock protein genes are regulated through heat-shock response elements.

Answer 5

Heat-shock response elements (HSE) are regulatory elements located upstream of Hsp70 in the nucleus. HSF1 is a transcription factor of HSE bound to Hsps in the cytoplasm, preventing the localization of the TF in the nucleus. Upon heat shock, proteins denature, unfold, and get targeted by Hsps, thereby freeing HSF1 and allowing its translocation to the nucleus. Once in the nucleus, HSF1 activates HSE, upregulating the expression of Hsps, allowing to control productive folding for more proteins.

Question 6

Why is refolding taxing in energy?

Answer 6

Chaperones are ATPases

Question 7

What are the two major classes of Hsps? What functions do they carry?

Answer 7

Hsp60-like: forming chamber complexes where conditions are appropriate for protein refolding
Hsp70-like: bind to hydrophobic residues of aa. chains, clamping down through ATP hydrolysis, shielding hydrophobic patches from unfavourable interactions

Question 8

How do chaperones identify misfolded proteins?

Answer 8

Recognize exposed hydrophobic patches.

Question 9

What process directs misfolded proteins to degradation? How does it work?

Answer 9

Mutliubiquitination: E1 enzyme, shared by all ubiquitin ligases, that uses ATP to activate ubiquitin for conjugation and transfers it to an E2 enzyme. The E2 enzyme interacts with a specific E3 partner and transfers the ubiquitin to the target protein. The E3 (ubiquitin ligase), which may be a multi-protein complex, is, in general, responsible for targeting ubiquitination to specific substrate proteins

Question 10

Why could there be multiple isoforms of E3?

Answer 10

Would recognize different degradation signals.

Question 11

What is a proteasome? What are its two main structural components?

Answer 11

Macromolecular machines degrading multiubiquitinated target proteins

Cap: binds ubiquitin chain of targeted proteins, removes ubiquitin tag, unfolds them (ATP), and pushes down in the central cylinder
Central cylinder: stack of inward-facing protease subunits

Question 12

Why is it bad to let misfolded proteins accumulate?

Answer 12

They form aggregates and can impede on cell functions

Question 13

True of false: many neurological diseases are associated with abnormal misfolded protein aggregates.

Answer 13

True.

Question 14

What is parkin?

Answer 14

E3 ubiquitin ligase, plays a role in the destruction of damaged mitochondria through autophagy.

Question 15

Describe one type of structure that can form from an aggregate of misfolded proteins. What is particular about its structure when it comes to degradation?

Answer 15

Amyloid filament, formed from the stacking of beta-sheet elements

Very resilient to proteasome degradation

Question 16

What is characteristic of the cellular propagation of the Prion disease?

Answer 16

Abnormally folded prion protein PrP catalyzes the conversion of normally folded to abnormally folded protein, forming bigger and bigger stacks of amyloid

Question 17

What are the two main response pathways of en endoplasmic reticulum against misfolded proteins?

Answer 17

1. Chaperone-mediated export of misfolded proteins (back) into cytoplasm for ubiquitination-degradation
2. UPR: unfolded protein response -> unfolded ER protein activates a transmembrane protein kinase/ ribonuclease that specifically cleaves the (nonfunctional) mRNA for a transcription factor involved in ER chaperone gene expression. The gene regulatory protein is translated in the cytoplasm and translocated into the nucleus, where it activates the transcription of genes encoding chaperones = upreg. of chaperones

Question 18

What is the advantage of lysosomal degradation in comparison to proteasomal degradation?

Answer 18

Lysosomes can degrade aggregates of misfolded proteins, while proteasomes cannot.

Question 19

What are the steps leading to lysosomal degradation?

Answer 19

Induction of autophagy: membrane forms around the aggregate of misfolded proteins, forming the autophagosome (double-membrane structure)

Fusion with lysosome: outer membrane of the autophagosome fuses with the single membrane of the lysosome -> leading to a vesicle that contains a vesicle (that contains the misfolded protein aggregate) with in it

Lysosomal enzymes get through the inner membrane of the autophagosome and chew up the misfolded protein aggregate

Question 20

Where do originate the lysosomal enzymes?

Answer 20

From the trans Golgi network + characteristic membrane proteins of the lysosome

Question 21

Is some of the material from the late endosome preserved in the lysosome?

Answer 21

No, all degraded and replaced by material coming from the trans Golgi network

Question 22

What is mitophagy? How is parkin involved in the case of Parkinson’s disease?

Answer 22

Mitophagy = mitochondrial autophagy (lysosomal degradation)

Parkin is tagging and recruiting ubiquitin to damaged mitochondrion -> autophagosome formation -> lysosome degradation

Parkin is mutated in Parkinson’s disease

Question 23

What are the three major autophagy pathways?

Answer 23

Macroautophagy
Chaperone-mediated autophagy: direct transfer of individual protein molecules through the lysosomal membrane
Microautophagy: invagination of lysosome, endosome, or any membrane

Question 24

What are the 3 leading hypotheses as to why aggregated proteins cause cell death?

Answer 24

1. Sequestration of essential functional proteins
2. Overload of ubiquitination/proteasome mechanisms
3. Apoptosis threshold for cells with irrevocable amounts of aggregated proteins: phosphorylation of JNK (kinase), hsp70 stimulates JNK phosphatases, so if all bound to misfolded proteins -> balance of phosphorylation/dephosphorylation lost and apoptosis activated by high rate of JNK kinase activation

Question 25

Detail how the chaperone Hsc70 is involved at the synapse?

Answer 25

Hsp70-related constitutive chaperone Hsc-70 plays a key role in uncoating clathrin-coated synaptic vesicles during synaptic vesicle recycling

Participates in SNARE complex assembly -> complex is moving a lot, easy for proteins to misfold

Hsc70 directs misfolded synaptic proteins to a microautophagy pathway and is important for maintaining “rejuvenation” of the synaptic protein pool