Lecture 14 - Protein Degredation and Autophagy

Question 1

Three instances when proteins need to be degraded?

Answer 1

1. Incomplete/mis-sense.
2. Damaged.
3. Unwanted.

Question 2

What does mis-sense mean?

Answer 2

Proteins not required.

Question 3

When do incomplete/missense proteins occur?

Answer 3

1. Cellular errors, e.g. incorrect AA sequence.
2. Proteins with disulphide mutations.
3. Products of premature termination.

Question 4

Why do damaged proteins occur?

Answer 4

1. Mis-folded.
2. Protein ageing.
3. Denaturation.

Question 5

Why can some proteins be unwanted?

Answer 5

1. Inactive/’used’
2. Excess of a specific subunit.
3. Made in excess.

Question 6

There are two degradation routes. Name them.

Answer 6

Proteosome and lysosome.

Question 7

Difference between a proteosome and a lysosome?

Answer 7

Proteosome = cytosolic structure, soluble proteins degraded here. 
Lysosome = membrane-bound organelle, membrane proteins (e.g. those that enter by endocytosis), autophagy involved.

Question 8

How are proteins targeted to the lysosome and proteosome for degradation?

Answer 8

Ubiquitin.

Question 9

Structure of ubiquitin?

Answer 9

76 AA polypeptide, mainly added to lysine side chains.

Question 10

Ubiquitin is added to proteins via 3 Ub enzymes. Name them.

Answer 10

E1, E2 and E3.

Question 11

Function of the 3 Ub enzymes?

Answer 11

E1 = activates Ub. ATP-dependent. Ub now bound to E1. 
E2 = receives Ub from E1 and either uses E3 Ub ligase to transfer the Ub directly to the substrate, or uses an E3 HECT domain. 

E3 either has a ring domain or a HECT domain.

Question 12

Difference between a ring domain and a HECT domain?

Answer 12

Ring domain = ligase; Ub goes directly from E2 –> substrate.
HECT domain = goes from E2, to E3, to the substrate.

Question 13

Two types of ubiquitination?

Answer 13

Mono- or poly-

Question 14

Ub molecules attach to lysine resides via?

Answer 14

Isopeptide linkage.

Question 15

Explain the different types of polyubiquitination.

Answer 15

Can either have a chain structure or can be branched. Branched structure forms because Ub molecules have seven lysine residues.

Question 16

Is ubiquitination reversible? Explain your answer.

Answer 16

YES. De-ubiquitinases (DUBs) can remove Ubs from proteins and recycle them.

Question 17

How are different Ub proteins targeted to different regions of degradation?

Answer 17

Different Ub conformations = different molecular tags.

Question 18

Proteosome uses two types of receptor for protein degradation. Name them.

Answer 18

Intrinsic receptor = possesses Ub binding domains to bind ubiquitinated cargo. Bound to the proteosome.
Shuttling receptor = Ub binding domain AND a proteosome protein binding domain.

Question 19

The lysosome can perform autophagy and endocytosis which both use a different type of receptor. Name them.

Answer 19

Autophagy - receptor with a Ub binding domain and a domain that targets it to the autophagosome membrane.
Endocytosis - two domains, one targets the membrane and the other binds Ub.

Question 20

Structure of the proteosome?

Answer 20

Central cylinder and cap proteins.

Central cylinder contains the proteases. Ub protein is targeted to the cap proteins where ATP is used to unfold the protein. Protein then spread through the proteosome where it is divided into its AA’s.

Question 21

Structure of cap proteins?

Answer 21

Hexameric AAA-ATPase structure.

Question 22

An example of proteosome-mediated degradation: the tumour suppressor protein p53.

Answer 22

• p53 is ubiquitinated.
• p53 causes the expression of Mdm2, an E3-Ub ligase.
• p53 is polyubiquitinated and targeted to the proteosome.
• Mdm2 levels decrease. Autoregulatory.
• MdmX inhibits transcription of p53. Mdm2 regulatesthe levels of Mdmx via ubiquitination.

Question 23

When receptors enter an early endosome for degradation in a lysosome, they can techically still signal. ALL PARTS OF THE RECEPTOR MUST BE INTERNALISED. How does this occur?

Answer 23

Invaginations of the endosomal membrane to form a multivescular body (MVB).

Question 24

Which family of proteins are responsible for the formation of MVBs?

Answer 24

ESCRT proteins.

ESCRT = Endosomal Sorting Complexes Required for Transport.

Question 25

In simple terms, what is autophagy?

Answer 25

Self eating. Cells completely surround an organelle/part of their cytoplasm using a membrane and deliver

Question 26

How is autophagy stimulated?

Answer 26

Stress, starvation and exercise.

Question 27

Types of autophagy?

Answer 27

1. Macroautophagy
2. Mitophagy - degredation of mitochondria.
3. Pexophagy - perioxisomes.

Question 28

Autophagy regulates energy homeostasis. Explain this.

Answer 28

Since autophagy is a starvation response, material is degraded to its building blocks to provide energy.

Question 29

What happens if autophagy is impaired?

Answer 29

• Lack of nutrients.
• Damaged mitochondria cannot be removed - ROS accumulation.
• Bacterial infection cannot be cleared = chronic infection.