Proteasomes and Exosomes

Question 1

What are the two main degradation systems in eukaryotes?

Answer 1

Lysosomes and proteasomes

Question 2

What are lysosomes and for what are they responsible?

Answer 2

These are membrane-bound organelles which are full of hydrolases. They have an internal pH of 4-6. They are non-selective and degrade membrane and endocytosed proteins (receptor-mediated endocytosis is selective, however).

Question 3

What are proteasomes and for what are they responsible?

Answer 3

These are large, compartmentalised ATPase/proteases complexes. They are selective and degrade cytosolic and nuclear proteins. ERAD substrates.

Question 4

What other, minor proteolysis systems exist in eukaryotes?

Answer 4

There are other systems, such as mitochondrial proteases, Ca2+ activated (cell injury) and caspases (programmed cell death).

Question 5

What is protein degradation important for?

Answer 5

• Cell regulation
• Adaptation to environmental conditions – fasting
• Cell cycle progression – cyclins are degraded at very specific points in the cell cycle
• Inflammatory signalling: phosphorylation of IκB -> it is targeted for degradation -> NF-κB enters the nucleus -> inflammatory response
• Clearance of misfolded, mutated or damaged proteins, prevention of aggregation

Question 6

Which protein folding disease causes blindness?

Answer 6

Retinitis Pigmentosa: mutant rhodopsin leading to photoreceptor loss

Question 7

What is autophagy?

Answer 7

The cytoplasm or organelles are engulfed by autophagosome vesicles, which are a double-membrane vesicle. How these chose what to envelop and take away is not completely clear.

These fuse with the lysosome for degradation,.

Question 8

What stimulates autophagy?

Answer 8

This action is upregulated when nutrients are low, or in the fasting state, or when there are low levels of insulin. This generates nucleotides, amino acids and fatty acids for new synthesis or energy metabolism.

Question 9

How conserved are ATP-dependent protease machines?

Answer 9

These are found in all three domains of life; eukaryotes, archaea and bacteria. For example, the simplest is probably CipP in bacteria.

Question 10

What is the structure of the bacterial CipP ATP-dependent protease?

Answer 10

Two heptameric rings enclose a cavity in which there is proteolytic activity, with a narrow channel by which to enter. Both sides of the CipP are enclosed by CipA (hexameric rings), which are ATPases and direct the protein in and unfold it. Appropriate recognition must take place on the outside of this complex, as you don’t want to degrade just anything.

Question 11

What protease system is found in tuberculosis bacteria?

Answer 11

In TB, there is a system that looks like the archaeal protease. Instead of recognising a ubiquitin tag, the tag is Pup (protein is tagged by pupulation rather than ubiquitination).

Question 12

What is the structure of the proteasome?

Answer 12

The proteasome is found in eukaryotes. It is very abundant, making up ~1% of the total cell protein.

It has a double protease ring in the middle, regulatory domains and then the ATPase, then a lid structure supercomplex on both ends.

Question 13

What happens to the ubiquitin tag in proteolysis?

Answer 13

It is recycled.

Question 14

What is the function of the protease lid supercomplex?

Answer 14

The lid superstructure regulates access to the ATPase and recognises ubiquitin tags.

Question 15

How are proteins targeted to the proteasome?

Answer 15

The proteasome is a highly specific targeting system directs proteins to proteasome for degradation, using covalent attachment of the small protein ubiquitin.

Question 16

What are the common substrated of proteasomes?

Answer 16

Substrates are mainly endogenous cytoplasmic or nuclear proteins, e.g. transcription factors. For example, cyclins are degraded at precise times, which is essential for progression through the cell cycle. Other substrate proteins may be those encoded by viruses or other intracellular parasites, or misfolded proteins.

Question 17

What are the products of proteasomes and what is their fate?

Answer 17

The products of the proteasome are small peptides. In the immune system, peptide antigens are formed by a version of the proteasome for presenting on the surface of cells.

Otherwise, the proteasome products get chopped up by peptidases into amino acids for recycling.

Question 18

What is the structure of the proteasome?

Answer 18

The 20S core particle (CP) is made of 2 copies of each of 14 different proteins, assembled in rings of 7. The 4 rings are stacked on each other.

There are two identical 19S regulatory particles (RP), one at each end of the core particle. Each is made of 18 different proteins, including 6 ATPases. Some of the subunits have sites that recognise the small protein ubiquitin.

Question 19

What is the role of the base complex?

Answer 19

Recognition, unfolding and translocation of ubiquitinated substrates is done by the 19S base.

Six highly conserved AAA subunits form the ATPase ring in the base (hetero-hexameric ring). Rpt2 opens the gate of the 20S core, and Rpt5 binds ubiquitin chains.

Question 20

What is the role of the lid complex?

Answer 20

The 19S lid is responsible for deubiquitination, as it is an Rpn11-metalloprotease.

Question 21

What is the structure of the 20S core?

Answer 21

The x-ray structure of the 20S core particle complex in yeast has been solved at a 2.4Aͦ resolution.

It has dimensions of 148 Aͦ x 113Aͦ and consists of 28 subunits in stacked rings, α subunits in the exterior rings and β in the interior rings. The 20S core particle is ~700kDa.

The channel in the α ring is 13Aͦ: too narrow for folded proteins, but can be opened up by activators.

Question 22

What do the outer alpha subunits of the 20S core do?

Answer 22

• Contain nuclear localisation signal
• Are catalytically inert
• Form ring structure essential in proteasome assembly
• Enable binding of adaptor proteins: 19S (PA700), 11S (PA28), Blm20 (PA200).

There is a slightly different set of adaptor proteins for the version of the proteasome which creates peptides for antigen presentation.

Question 23

What are the inner beta subunits of the 20S core do?

Answer 23

The active sites of the β subunits are N-terminal nucleophilic hydrolases. Active β subunits are responsible for autocatalytic cleavage of the propeptide during assembly (provide the catalytic sites for protein degradation).

In eukaryotic proteasomes, only six of the 14 β subunits (3 in each ring) actually possess functional catalytic activity.

Question 24

What are the protease actvities of the inner beta subunits of the 20S core?

Answer 24

Each of the three β subunits possesses a different proteolytic activity, with a different specificity in each. Thus, each cavity has three different protease;

• Chymotrypsin-like: cleaves after hydrophobic residues
• Trypsin-like: cleaves after basic residues
• Caspase-like: cleaves after acidic residues

Question 25

How are AAA proteins involves in the protease pathway?

Answer 25

Hsp100 proteins can totally unfold their substrate proteins, in order to deliver them to associated proteases, or they can dissolve large aggregates, in cooperation with the Hsp70 system.

Question 26

How is the unfolded protein pulled into the proteasome??

Answer 26

Substrate-induced rearrangement of the ATPase subunits creates a widened pore and a continuous central channel throughout the enzyme.

The ATPase subunits have a spiral action, acting sequentially to pull the protein down by weakly binding to protein via their conserved tyrosine loops. This has yet to be observed, however.

Question 27

What is the mechanism of ubiquitination?

Answer 27

E1 activates ubiquitin at the C-terminal glycine residue.

E2 transfers activated ubiquitin to E3 ligase, to which substrate protein is specifically bound.

E3 catalyses covalent attachment of Ub to the substrate.

The C-terminal glycine of ubiquitin forms an isopeptide bond with the ε-amino of a lysine residue on the substrate.

Question 28

What role does each enzyme in the ubiquitination pathway play in the specificity of the process?

Answer 28

There is a huge repertoire of combinations of the three ubiquitin ligases. There is one ubiquitin-activated enzyme (E1) per cell, although homologs exist for activating ubiquitin-like proteins. There are more ubiquitin-conjugating enzymes (E2), and then far more ubiquitin-protein ligases (E3): 500-1000 in mammals, which are specific for substrates and E2s.

Most of the specificity of proteasome targeting comes from the E3 step, but combination with multiple E2s greatly increases the number of possible combinations for specific recognition.

Question 29

How does polyubiquitination target the protein for degradation?

Answer 29

At the 26S proteasome, four ubiquitin tags are recognised by proteasomal 19S subunit Rpn10 and Rpt5. The 19S subunit Rpn11 then deubiquitinates the substrate, which is then unfolded by the 19S AAA hexamer.

The substrate is subsequently degraded by the 20S proteasome into peptides 3-22 amino acids in length.

Question 30

What structures are found in ubiquitin ligase E3 enzymes?

Answer 30

E3 enzymes can be monomeric or oligomeric; for example, the SCF complex is an example of an oligomeric RING finger E3, whereas there are also monomeric RING finger E3s.

Non-RING finger monomeric E3s may also have HECT domains.

Question 31

What proteolysis inhibiting drug is used in cancer treatements?

Answer 31

The drug bortezomib (Velcade), approved in the US in 2003, blocks the proteolytic action of the proteasome and is effective in some cases of multiple myeloma, a cancer or plasma cells. It results in a failure to carry out inflammatory signalling.

Question 32

What effect does bortezomib have upon the cell?

Answer 32

The failure to degrade IκB as a result of inhibition of the proteasome blocks the signalling action of the transcription factor NF-κB. Dozens of genes needed for proliferation and adhesion of myeloma cells are turned off.

The failure to degrade cyclins inhibits completion of the cell cycle and thus stops the proliferation of cancer cells. The drug is given intermittently and its action is reversible. Cancer cells are killed while normal cells are spared, as cancer cells are hit first because they’re dividing so rapidly.

The drug may inhibit the ability of the cancer cell to protect itself against the damage caused by chemotherapy drugs. Inhibition of Bcl-2 leads to death of the cell by apoptosis. Angiogenesis and metastasis are also inhibited.

Question 33

What is the exosome?

Answer 33

The exosome is a nuclease for degradation, processing and quality control of a wide variety of RNAs. It is conserved in all eukaryotic and most archaeal organisms.

Question 34

What regulatory RNAs is the exosome involved in producing?

Answer 34

• miRNA (microRNA): 21-22 nucleotides
• siRNA (small interfering RNA) 20-25 nucleotides
• snRNA (small nuclear RNA)
• snoRNA (small nucleolar RNA)

Question 35

What mRNA quality control mechanisms is the the exosome involved in?

Answer 35

• nonsense-mediated decay: detection and degradation of mRNA with nonsense mutations
• non-stop decay: detection and degradation of mRNA lacking a stop codon
• no go decay: degradation of mRNA with strong secondary structure stalling the ribosome

Question 36

What is the conserved subunit composition of the exosome?

Answer 36

Exosomes are multi-protein complexes. They have a common structural core composed of nine subunits. There is a hexameric RNase PH-like ring consisting of 3 of each of the two different kinds of subunits, and a trimeric cap. There are additional nuclease subunits only present in eukaryotes: Rrp44 (cytoplasm and nucleus) and Rrp6 (nucleus).

Question 37

What is the archaeal exosome structure and how does it function?

Answer 37

In the archaeal exosome, 3 of the 6 core subunits are active. There is a pore through which single-stranded RNA is passed, and a processing chamber is found within the ring. In this way, the RNase activity is not exposed to the cytoplasm, which would result in the RNA being randomly degraded.

Question 38

What is the structure of the eukaryotic exosome, and how does it function?

Answer 38

In the eukaryotic exosome, there are 10 subunits and the catalytic activity is in the additional subunit, Rrp44. The RNase-like ring is no longer the site of RNase catalytic activity in eukaryotes.

The ssRNA is passed through the cap and then the channel through the ring to the exonucleolytic site in Rrp44.

Question 39

What did the crystal structure of the eukaryotic exosome unexpectedly show?

Answer 39

There is also an endonuclease activity, exposed to the solvent, which is not part of the internal channel pathway.