David Proby's Proteosomes & Chaperones Flashcards
Chaperones
- aka heat shock proteins
- Have affinity to exposedhydrophobic patches on incompletely folded proteins
• Bind andrelease protein substrates (in an ATP-‐ dependent manner), indirectly facilitating folding
• Conserved in bacteriaand eukaryotes
Hsp70
- The Hsp70 system interacts with extended peptide segments of proteins as well as partially folded proteins to prevent aggregation.
- When not interacting with a substrate peptide, Hsp70 is usually in an ATP bound state.
- A family of conserved ubiquitously expressed heat shock proteins (chaperones).
- As newly synthesized proteins emerge from the ribosomes, the substrate binding domain of Hsp70 recognizes sequences of hydrophobic amino acid residues, and interacts with them
When do Hsp70 proteins act?
Hsp70 acts cotranslationally, before peptide release from ribosomes
How is ATP used with Hsp70?
- When an Hsp70 protein is ATP bound, the lid is open and peptides bind and release relatively rapidly.
- When Hsp70 proteins are ADP bound, the lid is closed, and peptides are tightly bound to the substrate binding domain.
- ADP replacement with ATP triggers peptide release so that peptide can fold properly
- If the protein still isn’t folded correctly,Hsp70 can rebind
When do Hsp60 proteins act?
- After translation
* Once peptides are released from ribosomes
Hsp60
In eukaryotes the proteins Hsp60 and Hsp10 are structurally and functionally nearly identical to GroEL and GroES, respectively
Is the process of GroEL/ES mediated protein folding work on the first round usually?
A given protein will undergo multiple rounds of folding, returning each time to its original unfolded state, until the native conformation or an intermediate structure committed to reaching the native state is achieved.
How does GroEL (Hsp60) bind to the original protein?
- Unfolded substrate proteins bind to a hydrophobic binding patch on the interior rim of the open cavity of GroEL forming a binary complex.
- Binding of substrate protein in this manner, in addition to binding of ATP, induces a conformational change that allows association of the binary complex with a separate lid structure (GroES).
How does GroES (Hps 10) bind to the binary complex?
Binding of GroES to the open cavity of the chaperonin induces the individual subunits of the chaperonin to rotate such that the hydrophobic substrate binding site is removed from the interior of the cavity, causing the substrate protein to be ejected from the rim into the now largely hydrophilic chamber.
How does GroES get released?
Hydrolysis of ATP and binding of a new substrate protein to the opposite cavity sends an allosteric signal causing GroES and the encapsulated protein to be released into the cytosol.
What kind of experiment is this:
35S-‐Met labeling was performed for 15 seconds and then replaced with non-‐ radioactivemethionine.
This is a “pulse chase” type experiment
What happens when a protein cant be degraded in a timely manner?
Shit gets degraded
The Proteasom
> 50 protein subunit machine in cytoplasm and nucleus
Structure resembles a cylinder
Wheres the active site in a proteasom?
In the middle of the cylinder
Lee called it the “cellular chamber of doom”
The Proteasom is processive. What does this mean?
once it grabs on, doesn’t let go.
How does the proteasom use ATP?
- The unfoldase ring binds ATP which induces a confrontational change
- The strained ring structure pulls on the protein and unfolds it
Ubiquitin
- A small regulatory protein (76 amino acids and has a molecular mass of about 8.5 kDa) that has been found in almost all tissues of eukaryotic organisms.
- Key features include its C-terminal tail and the 7 lysine residues.
Ubiquitylation.
- The addition of ubiquitin to a substrate protein is called ubiquitination or ubiquitylation
- The ubiquitination reaction that targets a protein for degradation via the proteasome. The ubiquitination process covalently attaches ubiquitin, a short protein of 76 amino acids, to a lysine residue on the target protein.
How does Ubiquitination affect proteins?
- It can signal for their degradation via the proteasome.
- Alter their cellular location.
- Affect their activity.
- Promote or prevent protein interactions.
How many steps is Ubiquitination?
Ubiquitination is carried out in three main steps: activation, conjugation, and ligation,
How are the steps of ubiquination preformed?
performed by ubiquitin-activating enzymes (E1s), ubiquitin-conjugating enzymes (E2s), and ubiquitin ligases (E3s), respectively
Protein modifications by ubiquitin
- Protein modifications can be either a single ubiquitin protein (monoubiquitination) or a chain of ubiquitin (polyubiquitination).
- The ubiquitination bonds are always formed with one of the seven lysine residues as well as the very N-terminal methionine from the ubiquitin molecule.
How does polyubiquitination occur.
- First, a ubiquitin molecule is bonded by its C-terminus to a specific lysine residue on the target protein.
- Poly-ubiquitination occurs when the C-terminus of another ubiquitin, will be linked to one of the seven lysine residues, or the first methionine on the previously added ubiquitin molecule itself forming a chain.
- Catalyzed by E1 & E2.
E1 enzymes
- aka Ubiquitin-activating enzymes
- catalyze the first step in the ubiquitination reaction, which (among other things) can target a protein for degradation via a proteasome.
- This covalent attachment of ubiquitin to targeted proteins is a major mechanism for regulating protein function in eukaryotic organisms.
- The E1 enzyme along with ATP binds to the ubiquitin protein. The E1 enzyme then passes the ubiquitin protein to a second protein, called Ubiquitin carrier or conjugation protein (E2).
