spooner (L8-10) Flashcards
protein synthesis location
Most protein synthesis in a eukaryotic cell starts on free cytosolic ribosomes (exceptions – mitochondrial and plastid translation)
protein synthesis steps
In cytosol, there is a common pool of ribosome subunits
They assemble on an mRNA and remain free in the cytosol
Once the first ribosomal subunit moves away from the start of translation, it reveals an empty site for another ribosome can bind
There is a biological problem with the cytosol
We get a lot of polysomes
At end of translation, protein is released, folded, activated
Ribosomes fall apart, and feed back into the common pool in the cytosol
Cyclic turn
macromolecular crowding
Macromolecular crowding favours aggregation of proteins
So conc of proteins is dramatically increased and there is a limited solubility of proteins
Because everything is so much more concentrated, so conc for substrates are much higher than expected, so the reaction rates are very very quick(faster than in vitro)
Crowding drives the cellular activity, which helps us survive
But this env still causes damage in protein structure
define nascent proteins and how they are also crowded
Nascent proteins are in a non-native, aggregation-prone conformation (not active)
Since a single mRNA is translated at the same time by multiple ribosomes, nascent proteins are extruded in close proximity.
They are therefore in danger of aggregation.
how are newly synthesised proteins are non-functional?
they can be unfolded or misfolded. so they are protease-sensitive, non-functional and prone to aggregation, so they are destroyed and removed from cells
Folded proteins are stable, resistant to proteases and are functional
they have these protease sensitive sites inside the structure, so protected, stable and resistant
types of chaperones while the protein folds
- Hydrophobic patches on nascent/unfolded proteins are recognised by Heat shock protein 40 family members (Hsp40 co-chaperone), which…
- …deliver the substrate to ATP-bound (OPEN conformation) Heat shock cognate protein 70 (Hsc70 chaperone) and stimulate the ATPase activity of Hsc70…
- …resulting in ADP-bound (CLOSED conformation) Hsc70 shielding the hydrophobic patches of the substrate, preventing aggregation, and allowing time for the hydrophilic parts of the substrate to fold.
- Upon nucleotide exchange, Hsc70 adopts its open conformation, releasing the substrate, with folded soluble structures: this partly folded protein may now snap into its final conformation.
How do we study chaperone interactions?
In vitro recapitulation
① Heat target protein (PrX) at 45C for 15 min and separate aggregated (P, pellet) and soluble (S) fractions by centrifugation. SDS-PAGE, silver stain and quantify.
② Heating in the presence of Hsp40 increases the S fraction
③ Heating in the presence of Hsc70 has a larger effect (there are multiple Hsc70s)
④ Hsp40 and Hsc70 together have even more effect
⑤ Maximal solubilisation requires Hsp40, Hsc70 and ATP
LOOK AT L8S10 PICTURES
Role of Hsc70
Hsc70 does not actively FOLD a protein, but binds and shields its hydrophobic regions and thus prevents AGGREGATION of nascent or unfolded proteins.
A partially-folded Hsc70 client protein may be:
released, and find its stable conformation
passed on to other chaperones for further folding and/or assembly into multimeric complexes
released, in preparation for transport into an organelle (more later)
or passed to proteasomes (more later) for degradation
How are protein clients released from Hsc70?
A nucleotide exchange factor (NEF) binds the Hsc70:client complex and removes ADP from the nucleotide-binding site of Hsc70.
This promotes NUCLEOTIDE EXCHANGE, allowing entry of ATP into the nucleotide binding site of Hsc70.
Hsc70:ATP adopts an OPEN conformation, releasing the client protein.
Some Hsc70 co-chaperones:
There are multiple NEFs for Hsc70 (e.g. BAG-1, BAG-2, HSPBP1).
Hsp40 family members
CHIP (E3 ubiquitin ligase)
Hsc70 chaprones decide on when and where the protein client is released
Hsc70 chaperones make a decision on when the client should be released and to which of the protein systems or location
Straight forward release of client protein and nucleotide exchange
Cochaperone transport it to another chaperone (hsp90) which only accepts partially folded clients (to allow it to finish folding or to incorporate other proteins to build multimeric complexes)
Or direct a partially folded client to an actin or tubulin subunit to direct them to a cage called chaperonin
function of chaperonins
Chaperonins provide a cage that isolates small (<70 kDa) folding proteins (e.g. tubulin, actin) from the cytosol. Residence time ~10s
How is the protein’s fate determined?
There is no absolute control over a protein’s fate, it’s a competition between cochaperones
A fully formed client could be sent for destruction and vice versa
Balance of chaperones doesnt seem to have control
We always generate proteins that are unusable
Summary - cytosolic molecular chaperones can:
prevent aggregation of unfolded proteins:
e.g. Hsc70 binds hydrophobic regions of a client, delaying folding of these regions until the hydrophilic parts of the target protein have gained structure
provide a controlled environment for folding:
e.g. chaperonins form a cage that encloses the target protein, allowing folding in a protected environment, away from the concentrated cytosol – they may even aid folding directly
permit assembly of multimeric complexes:
e.g. assembly of histone complexes, clathrin cages, etc.
AND… can direct proteins with folding problems for destruction:
e.g. the Hsc70 co-chaperone BAG-1 can engage a Hsc70:client complex with the proteasome
Architecture of the proteasome
The 20S core particles are cylinders with three proteolytic activities: chymotrypsin-like, trypsin-like and peptidylglutamyl-peptide hydrolysing (caspase-like). The active sites are inside the barrel, encoded by the β subunits.
Each 20S core has 19S caps, regulatory particles (RP) at one or both ends.
Central hollow core of 4 stacked heteromeric rings
B subunits encode catalytic activity (they can encode a chymotrypsin, trypsin or caspase like activity)
- So any proteasome that is 20S is capped at one end
- If it is 30S then it is capped at both ends
How is a protein targeted to the proteasome?
Ubiquitin (Ub) is a conserved 76 amino acid protein found in all eukaryotic cells.
Cytosolic proteins destined for proteasomal degradation are usually marked for destruction by covalent addition of a chain of Ub molecules (polyubiquitylation) allowing them to be bound by the 19S RP.
A chain of four Ub proteins means a doomed protein: tetra-Ub is a degradation signal.
what happens to a protein that has failed Hsc70-mediated folding?
STEPS:
- Ub is activated by an E1 ubiquitin-activating enzyme
- Activated Ub is transferred to an E2 ubiquitin-conjugating enzyme
- The E2-Ub conjugate associates with an E3 ubiquitin ligase
- The E3-E2-Ub conjugate binds the target protein…
- …and transfers Ub to the target
(READ NOTES AND LOOK AT DIAGRAMS IN L8S18)
Specificity of E1s, E2s and E3s
There are ~9 E1s in mammalian cells: Vital enzymes.
There are >30 E2s: Each can select their own E3s, so they ultimately provide some substrate specificity.
There are 100s of different E3s: Each type selects its target proteins by recognising some specific feature (E3s effectively control the stability of proteins involved in key cellular processes)
how and where is ubiquitin added to a portein?
Ubiquitin is normally added covalently to the side chain of an available LYSINE residue on the target molecule.
The process is repeated using side chains of lysine residues in ubiquitin, until a chain of at least 4 Ub is completed.
This multi-ubiquitin (4 or more) tag is a degradation signal.
Polyubiquitylated proteins can be bound by the proteasomal 19S RP.
Targeting the proteasome and destruction STEPS
- Polyubiquitylated proteins bind the 19S regulatory particle of the proteasome
- The RP uses ATP to generate energy to unfold the target protein and feed it into the 20S core. Deubiquitylases (DUBs) remove Ub molecules and return them to a common pool for recycling
- Three proteolytic activities are encoded by the β subunits of the 20S core
- The target protein is degraded into small peptides (typically 7–9 amino-acid residues long, though they can range from 4 to 25 residues), which are ejected from the proteasome
(THE UPS CYCLE IN L8S24 !!! )
how is the proteasome not just a destructive machine?
JUDGE/JURY/EXECUTIONER
There is a fail-safe mechanism.
It can also re-fold proteins.
One of the RP subunits acts as a chaperone that DIRECTS some clients for destruction and RE-FOLDS others back to their native conformation
The UPS is relevant for:
- proteins that fail to fold correctly
- normal turnover of cytosolic proteins (each at their own rate)
- proteins whose concentrations must change rapidly (e.g. cyclins )
- viral proteins
- misfolded proteins ejected from the ER
When proteasomes or E3s fail:
- Proteins that would normally be destroyed accumulate instead.
- This can lead to the formation of aggregates e.g. in neurons of people with Parkinson’s and Alzheimer’s.
- If cell cycle proteins are not degraded properly, it can lead to cell proliferation (as in cancer).
Overactive proteasomes:
have been implicated in autoimmune diseases including systemic lupus erythematosus and rheumatoid arthritis.
different types of cytosolic post-translational modifications
- proteolytic cleavage (to activate proteins)
- addition of lipids to permit membrane targeting
- phosphorylation
- ADP ribosylation
- methylation
proteolytic cleavage (to activate proteins)
The effector proteases of apoptosis are stored in an inactive condition. Activation is by proteolytic cleavage and subunit rearrangement
addition of lipids to permit membrane targeting
Many regulatory proteins (e,g. Rabs that regulate membrane traffic) are modified by the addition of lipids. Rabs are doubly prenylated (prenylated lipids are either a 15C farnesyl or a 20C geranylgeranyl).
When the prenyl groups are masked by GDI (GDP dissociation inhibitor), Rab-GDP is cytosolic.
Following nucleotide exchange, GDI dissociates and the prenyl groups of Rab-GTP enter the target membrane
