Proteasomes and Exosomes Flashcards
(39 cards)
What are the two main degradation systems in eukaryotes?
Lysosomes and proteasomes
What are lysosomes and for what are they responsible?
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).
What are proteasomes and for what are they responsible?
These are large, compartmentalised ATPase/proteases complexes. They are selective and degrade cytosolic and nuclear proteins. ERAD substrates.
What other, minor proteolysis systems exist in eukaryotes?
There are other systems, such as mitochondrial proteases, Ca2+ activated (cell injury) and caspases (programmed cell death).
What is protein degradation important for?
- 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
Which protein folding disease causes blindness?
Retinitis Pigmentosa: mutant rhodopsin leading to photoreceptor loss
What is autophagy?
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,.
What stimulates autophagy?
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.
How conserved are ATP-dependent protease machines?
These are found in all three domains of life; eukaryotes, archaea and bacteria. For example, the simplest is probably CipP in bacteria.
What is the structure of the bacterial CipP ATP-dependent protease?
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.
What protease system is found in tuberculosis bacteria?
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).
What is the structure of the proteasome?
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.
What happens to the ubiquitin tag in proteolysis?
It is recycled.
What is the function of the protease lid supercomplex?
The lid superstructure regulates access to the ATPase and recognises ubiquitin tags.
How are proteins targeted to the proteasome?
The proteasome is a highly specific targeting system directs proteins to proteasome for degradation, using covalent attachment of the small protein ubiquitin.
What are the common substrated of proteasomes?
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.
What are the products of proteasomes and what is their fate?
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.
What is the structure of the proteasome?
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.
What is the role of the base complex?
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.
What is the role of the lid complex?
The 19S lid is responsible for deubiquitination, as it is an Rpn11-metalloprotease.
What is the structure of the 20S core?
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.
What do the outer alpha subunits of the 20S core do?
- 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.
What are the inner beta subunits of the 20S core do?
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.
What are the protease actvities of the inner beta subunits of the 20S core?
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
