David Proby's Proteosomes & Chaperones

Question 1

Chaperones

Answer 1

• 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

Question 2

Hsp70

Answer 2

• 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

Question 3

When do Hsp70 proteins act?

Answer 3

Hsp70 acts cotranslationally, before peptide release from ribosomes

Question 4

How is ATP used with Hsp70?

Answer 4

• 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

Question 5

When do Hsp60 proteins act?

Answer 5

• After translation

* Once peptides are released from ribosomes

Question 6

Hsp60

Answer 6

In eukaryotes the proteins Hsp60 and Hsp10 are structurally and functionally nearly identical to GroEL and GroES, respectively

Question 7

Is the process of GroEL/ES mediated protein folding work on the first round usually?

Answer 7

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.

Question 8

How does GroEL (Hsp60) bind to the original protein?

Answer 8

• 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).

Question 9

How does GroES (Hps 10) bind to the binary complex?

Answer 9

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.

Question 10

How does GroES get released?

Answer 10

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.

Question 11

What kind of experiment is this:

35S-­‐Met labeling was performed for 15 seconds and then replaced with non-­‐ radioactivemethionine.

Answer 11

This is a “pulse chase” type experiment

Question 12

What happens when a protein cant be degraded in a timely manner?

Answer 12

Shit gets degraded

Question 13

The Proteasom

Answer 13

> 50 protein subunit machine in cytoplasm and nucleus

Structure resembles a cylinder

Question 14

Wheres the active site in a proteasom?

Answer 14

In the middle of the cylinder

Lee called it the “cellular chamber of doom”

Question 15

The Proteasom is processive. What does this mean?

Answer 15

once it grabs on, doesn’t let go.

Question 16

How does the proteasom use ATP?

Answer 16

• The unfoldase ring binds ATP which induces a confrontational change
• The strained ring structure pulls on the protein and unfolds it

Question 17

Ubiquitin

Answer 17

• 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.

Question 18

Ubiquitylation.

Answer 18

• 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.

Question 19

How does Ubiquitination affect proteins?

Answer 19

• It can signal for their degradation via the proteasome.
• Alter their cellular location.
• Affect their activity.
• Promote or prevent protein interactions.

Question 20

How many steps is Ubiquitination?

Answer 20

Ubiquitination is carried out in three main steps: activation, conjugation, and ligation,

Question 21

How are the steps of ubiquination preformed?

Answer 21

performed by ubiquitin-activating enzymes (E1s), ubiquitin-conjugating enzymes (E2s), and ubiquitin ligases (E3s), respectively

Question 22

Protein modifications by ubiquitin

Answer 22

• 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.

Question 23

How does polyubiquitination occur.

Answer 23

• 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.

Question 24

E1 enzymes

Answer 24

• 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).

Question 25

Overview of ubiquitination

Answer 25

• 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 conjugation protein (E2).
• The E2 protein complexes with an Ubiquitin protein ligase (E3).
• This Ubiquitin protein ligase recognizes which protein needs to be tagged and catalyzes the transfer of ubiquitin to that protein.
• This pathway repeats itself until the target protein has a full chain of ubiquitin attached to itself

Question 26

E2 enzymes

Answer 26

• aka Ubiquitin-conjugating enzymes
• perform the second step in the ubiquitination reaction.
• E1 passes the ubiquitin protein to E2
• Once conjugated to ubiquitin, the E2 molecule binds one of several ubiquitin ligases (E3) via a structurally conserved binding region

Question 27

Proteasome specificity

Answer 27

• Once a protein has been tagged with one ubiquitin molecule, additional rounds of ubiquitination form a polyubiquitin chain that is recognized by the proteasome’s 19S regulatory particle.
• This triggers the ATP-dependent unfolding of the target protein that allows passage into the proteasome’s 20S core particle, where proteases degrade the target into short peptide fragments for recycling by the cell.

Question 28

How many E1, E2, and E3 killers do humans have?

Answer 28

• A particular cell usually contains only a few types of E1 molecule, a greater diversity of E2s, and a very large variety of E3s.
• The E3 molecules responsible for substrate identification and binding are thus the mechanisms of substrate specificity in proteasomal degradation.
• Each type of E2 can associate with many E3s

Question 29

E3

Answer 29

• a protein that recruits an E2 ubiquitin-conjugating enzyme that has been loaded with ubiquitin, recognizes a protein substrate, and assists or directly catalyzes the transfer of ubiquitin from the E2 to the protein substrate.
• The ubiquitin is attached to a lysine on the target protein by an isopeptide bond.
• E3 ligases interact with both the target protein and the E2 enzyme, and so impart substrate specificity to the E2.
• Ubiquitination by E3 ligases regulates diverse areas such as cell trafficking, DNA repair, and signaling
• The human genome encodes over 600 putative E3 ligases, allowing for tremendous diversity in substrates.[

Question 30

Can functional,properly foldedproteins be controlled in expression by degradation?

Answer 30

yeah boi

“functional, properly folded proteins can also be controlled in expression by regulated degradation”