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HMS Chemical Biology and Biochemistry > Protein Quality Control > Flashcards

Flashcards in Protein Quality Control Deck (47)
1

There are ___ quality controls systems in the cell

2

one for ER proteins

one for cytoplasmic proteins

2

Transmembrane proteins are subject to ____ quality control

ER and cytoplasmic

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ER proteins are degraded by. . .

Pumping out to the cytoplasm!  There is no machinery for degradation within the ER itself.

 

Translocation to the cytoplasm is managed by the ER-associated degradation (ERAD) pathway.

4

Anfinson experiment

Anfinson showed that if you take a purified protein existing in its mature, folded state, then unfold it by adding a reagent that disrupts hydrogen bonding (urea), the protein will quickly recover its correct function when the urea is removed.

 

The conclusion: the three-dimensional structure of a protein is dictated by one thing, its amino acid sequence. No other information is necessary.

 

But. . . it was done on a simple protein that did not require chaperones.

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efficiency of the folding reaction is determined by competition between . . .

intramolecular interactions and intermolecular interactions

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Amyloid

Amyloid refers to the structure of a particular form of aggregate. Whereas aggregates are in general highly disordered, amyloid has a fibrillar morphology, which results from a partial β-sheet structure. The protein within an amyloid is partly ordered and partly disordered, but where ordered, the structure may not resemble the native state.

7

Key chaperone systems of the cytosol

Hsp70, Hsp90, and chaperonins

(Where HSP = heat shock protein)

 

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About ~___% of proteins require the chaperone system

About ~5-10% of proteins require the chaperone system

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Cytoplasmic Chaperone Pathways

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Hsp70 Mechanism

Hsp40 delivers an unfolded protein.

Hsp70 clamps around it by hydrolyzing its ATP, allowing Hsp40 to diffuse.

The protein may fold a bit.

ATP binding causes diffusion.

 

If the protein fails to fold, it will be once again sequestered by a chaperone until it does eventually fold.

11

Kinetic Partitioning of Chaperones

When Kon is greater than Kfold, chaperone systems function to sequester a bit of unfolded protein to prevent aggregation which stabilizes the unfolded state.

 

If folding is inefficient, the protein is targeted for proteasomal degradation.

12

Decision to Degrade or to Fold

It is not currently known what the trigger is, but Hip and Hop binding represents a decision to fold while BAG-1 and CHIP (CHIP being an E3) represents a decision to degrade via the proteasome.

13

Hsp90 Mechanism

14

Glucocorticoid Receptor Signaling

The glucocorticoid receptor, the protein remains inactive while bound to Hsp90, and only becomes folded and active when the steroid hormone cortisol binds to the receptor. It is then released from Hsp90, where it goes to the nucleus to activate transcription.

 

Takes advantage of the fact that Hsp90 has certain preferred substrates.

15

Chaperonin Mechanism

Effectively acts as a small chamber that allows small proteins to fold outside of influences from other cellular proteins.

16

ER chaperones

Bip (Binding Protein) is similar to Hsp70 and associates with proteins as they are translated into the ER, allowing them to fold via the same kinetic partitioning model.

17

ER "Dislocons"

Transporters that transit unfolded proteins out of the ER for proteasomal degradation in the cytoplasm.

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ER Quality Control

19

Proteins in the ER are glycosylated on ___ residues.

Aspararine (N)

20

Calnexin System

As long as the carbohydrate chain contains at least one glucose, calnexin associates with the protein.  Calnexin, a resident ER protein, anchors the protein to the ER while it is bound.

 

The mannosidases act as a sort of timer, cutting off one sugar at a time.  When the sugars run out, Calnexin can no longer bind, and so the unfolded protein is no longer anchored to the ER and will be exported for proteasomal degradation by a dislocon.

 

The calnexin system, thus, effectively operates as a timer for ER processing on the protein, and when it runs out, the protein is degraded.

21

In order to bind calnexin, the N-linked glycosyl chain needs at least. . .

one glucose.

 

Glucose is the key sugar for calnexin binding.

22

Retrotranslocation through a dislocon is _____-dependent.

Retrotranslocation through a dislocon is p97/Cdc48-dependent.

 

p97/Cdc48 provides the mechanical force to push the protein out of the ER with the aid of ATP hydrolysis.

23

Summary of plasma membrane permeability

24

Three major types of proteins responsible for transporting polar or charged molecules across membranes

Pumps

Channels

Transporters

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Pumps

Always operate against a concentration gradient and must utilize ATP or an equivalent energy source

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Channels

Allow for facilitated diffusion across a membrane and thus do not require ATP hydrolysis.

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Transporters

Move larger molecules by facilitated diffusion, usually aided by ion transit down a concentration gradient in the opposite direction.

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Types of transporter mechanism

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Protein Families of Transporters

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V-class pumps are important for. . .

Acidification of a compartment, such as the lysosome.

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P-class pumps are important for. . .

Acidifying an organ lumen (such as the stomach), sequestering calcium in the endoplasmic/sarcoplasmic reticulum, and generating a resting neuron potential.

32

F-class pumps are important for. . .

Generating ATP

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ABC Pumps are imporant for. . .

Transport of cholesterol, lipids, chloride, and other small molecules.

34

Background on Cystic Fibrosis

Current median survival ~37 years, obstructive lung disease responsible for ~80% of mortality.

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CFTR and CFTR expression regulators

Cystic Fibrosis Transmembrane conductance Regulator

 

Note that the role of the transporter in the sweat gland is to absorb chloride, hence the high chloride content in CF patient's sweat.

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CFTR membrane assembly

R must be phosphorylated by a cAMP-dependent kinase in order for the channel to be active.  May also serve a scaffolding role for other membrane channels.

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Transporter "conductance"

The number of ions that may cross through a channel when it is open.

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Transporter "gating"

Refers to whether or not the channel is open, and the amount of time it spends in the open vs closed state.

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CFTR Gating

One ATP molecule (green) is thought to be bound to the channel all the time. A second ATP molecule can bind the channel, to form the complex on the upper right. This can then lead to the opening of the channel (O1). ATP hydrolysis leads to the formation of O2, which is then converted to the closed state.

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post-translational folding

Whether or not domains of a protein associate together

41

Detailed Regulation of CFTR

See CBB7 final reading slide for full explanation.

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Delta F 508

Deletion of phenylalanine at the junction between cytoplasmic and transmembrane domains.  

 

Makes CFTR unable to fold on the cytoplasmic side efficiently, and so it is targeted for degradation via the Hsp -> E3 ubiquitin ligase -> proteasome pathway.

 

Also reduces conductance at the plasma membrane (~80% of WT)

43

G551D

Mutation in the binding pocket of one of CFTR's two ATPs.  Greatly reduces ATP binding affinity, locking the channel in a closed conformation indefinitely.  No problems with folding/protein trafficking.

44

Hsp70/90-associated transmembrane protein degradation is a form of _____.

Hsp70/90-associated transmembrane protein degradation is a form of ERAD.

 

It still applies to ER proteins and requires pumps to facilitate removal of transmembrane domains via dislocons.

45

Which chaperone systems utilize kinetic partitioning?

Hsps and BiP

 

NOT chaperonins.  They utilize physical partitioning instead,

46

ERAD Pathway

47

Translocation Mechanism