Sept 10 - Protein folding, assembly, and quality control in the endoplasmic reticulum

Question 1

Challenges to protein folding in the cell

Answer 1

1. High concentration of stuff in the cell - favors non-productive intermolecular interactions due to constant collisions
2. Protein folding must occur cotranslationally - bits that emerge first may have to wait to interact with bits that emerge later.
3. Post-translational modifications that occur in vivo may affect protein folding

Question 2

Chaperones

Answer 2

Enablers of protein folding - cover up hydrophobic regions that are eventually buried in the interior of the protein or disrupt hydrophobic interactions that have occurred, giving the protein another chance, or sequester proteins to allow other chaperones to work

Question 3

Hsp70/DnaK family

Answer 3

Cover up hydrophobic regions that are eventually buried in the interior of the protein

Cochaperones include DnaJ/hsp40 (brings substrates to hsp70s and stimulates ATPase activity) and GrpE (facilitates exchange of ADP for ATP - analogous to GEFs for GTPases)

007 agents are involved in cover-ups.
40 has 4 which is like the sail - like a boat bringing substrates to hsp70.
GrpE - groupie - exchanging things when it gets there.

Question 4

Hsp60 family, Hsp100

Answer 4

Disrupt hydrophobic interactions that have occurred, give the protein another chance to fold (hsp60, hsp100 families)

60 - devils are disruptive - then you give the protein another chance

Question 5

BiP/GRP78

Answer 5

Most abundant chaperone in the ER. The ER member of the Hsp70/DnaK family. Binds hydrophobic regions as soon as they start emerging from the lumen of the ER. There is a large ATPase domain and then a substrate binding domain.
Lid-like structure which opens and closes.

Hsp7 - ER - 7 looks like an Ear.

Question 6

GrpE

Answer 6

Facilitates exchange of ADP for ATP, analogous to GEFs for GTPases. For the Hsp70 family.

Not all hsp70s have GrpE protein due to INTRINSIC EXCHANGE ACTIVITY - none known for BiP

Question 7

Hsp90 family

Answer 7

Also an ATPase- orthologues in several compartments.
Involved in maintaining cytosolic proteins in receptive state - binds nuclear hormone receptors in absence of ligand.
Functions with cochaperones - hsp70, HOP

90 - The 9 looks like the nuclear hormone receptor sticking up, it binds to this. (that’s all I got)

Question 8

Hsp60/GroEL family

Answer 8

Major importance in bacteria.
GroEL - capped with a GroES/cpn10 family member

Not in ER - limited to cytosol and mitochondria. Participates in folding of a large but limited set of proteins. Bounces peptide around, permits it to stretch, alter conformation, break hydrophobic interaction. Traps folding intermediates in inner chamber of cylindrical structure.

The devil Growels and Grows

Question 9

Hsp40/J domain proteins

Answer 9

Several are present in the ER, facilitate BiP function

Question 10

Grp94

Answer 10

Hsp90 family member, limited clients, including Ig light chain, growth factors, not required for cell viability, but essential in metazoan development

Question 11

Peptidyl proline isomerase

Answer 11

Enzyme that facilitates isomerization of proline - functional analogs in the cytosol include cyclophilin and FKBP - chaperone/foldase in ER

Question 12

Calnexin/calreticulin

Answer 12

ER specific chaperone that functions to retain glycoproteins specifically, allows them time to fold.

Question 13

Protein disulfide isomerase

Answer 13

ER specific enzymes that facilitate correct formation of disulfide bonds, breakup incorrect disulfide bonds.

Question 14

VSV-G associates with what and then what?

Answer 14

First transiently with BiP and then more stably with calnexin. Calnexin is an ER specific chaperone that retains glycoproteins specifically, allowing them to fold.

Question 15

N-linked versus O-linked oligosaccharides

Answer 15

In the secretory pathway, proteins are modified by N-linked and O-linked oligosaccharides.
N-linked - conjugated to amide group of asparagine - these play a role in quality control, monitoring whether glycoprotein is properly folded. N linked sugars are added cotranslationally to Asn residues in the sequence Asn-x-Ser/Thr
O-linked - conjugated to hydroxyl group of serine or threonine. Different classes of O-linked oligosaccharides are added to cytosolic proteins. They are built by sugar post-translationally.

Question 16

Do chaperones interact concomittantly or sequentially with substrate proteins?

Answer 16

They do either.

Question 17

How does cotranslational addition of carbohydrate chains help deal with folding in cells?

Answer 17

It positions regions of the protein on the exterior surface - hydrophobic regions collapse into the interior. Carbohydrates are very hydrophilic.

Question 18

Asn-X-Ser/Thr

Answer 18

N-linked sugars are added co-translationally to Asn residues in this sequence in order to help with monitoring whether the protein is properly folded

Question 19

Are carbohydrates hydrophobic or hydrophilic?

Answer 19

Hydrophilic

Question 20

How do sugars get added to the protein?

Answer 20

Basically the sugars get added and then progressively clipped away. Each of the cleavages serves a function in quality control to ensure fidelity of protein folding.

Question 21

Calnexin and calreticulin

Answer 21

calnexin - Membrane protein
Calreticulin - soluble homologue

These recognize monoglucosylated proteins (proteins in which the first 2 glucose residues have been trimmed by glucosidase I and II

If Glucosidase II trims the 3rd glucose residue off then calnexin and calreticulin fail to bind and release the protein for secretion.

Question 22

UDP glucosyl transferase (UGGT)

Answer 22

Recognize incompletely folded proteins, puts the glucose back on

Question 23

What’s the calnexin cycle?

Answer 23

Involved with degrading proteins which still have one glucose on them - improperly folded proteins. Retains glycoproteins specifically, giving them additional time to fold.

Question 24

Castanospermine

Answer 24

Glucosidase I inhibitor

Question 25

What happens if you immunoprecipitate calnexin from cells with and without castanospermine?

Answer 25

With castanospermine - you get

Without castanospermine - You get a lot of … I don’t understand this experiment at alllllll

Question 26

Calreticulin interactions

Answer 26

Make folding more efficient

Question 27

UDP-glucose:glycoprotein glucosyltransferase

Answer 27

The folding sensor. Recognizes both oligosaccharide and protein moiety of misfolded proteins. Binds misfolded proteins via the first GlcNAc residue (not accessible in folded molecules) or to hydrophobic regions of the protein.

Question 28

Tunicamycin

Answer 28

Blocks N-glycosylation completely - competitive acceptor of core glycan from dolichol intermediate

Question 29

Deoxynojirimycin or castanospermine

Answer 29

Glucosidase inhibitors, block downstream trimming and Golgi modification

Question 30

Deoxymannonojirimycin or swainsonine

Answer 30

Inhibit mannosidases and hence downstream modifications.
Inhibitor of mannosidase I
This inhibitor interferes with mannose I, indicating that ERAD substrates must have a glycan structure of GlycNAc2Man8

Question 31

How do you know if protein is modified by N-glycosylation?

Answer 31

Change in Mr after treatment with inhibitors

Change in Mr after treatment of cell lysates or immunoprecipitates with deglycosidase enzymes

Question 32

Endoglycosidase H

Answer 32

Cleaves only high mannose N-glycans (ER forms)

Question 33

Protein N-glycanase F

Answer 33

Cleaves all N-linked glycans, including those modified in golgi

Question 34

Cysteine

Answer 34

The amino acid

Question 35

Cystine

Answer 35

The double bond

Question 36

How does the ER promote disulfide bond formation?

Answer 36

• Careful regulation of redox potential - the ER is mildly oxidizing, whreas the cytosol is reducing
• Presence of enzymes - protein disulfide isomerases - that promote formation of correct disulfide bonds and break incorrect ones

Question 37

What is the ER? oxidizing or reducing?

Answer 37

Oxidizing - it si conducive to disulfide bond formation. Formation of disulfide bond results in the loss of 2 electrons, or oxidation. These are transferred via hydrogen to a terminal acceptor.

Oxidized glutathione (with S-S dimer) is 100:1 in cytoplasm, but 3:1 in the ER.

Question 38

What is the cytoplasm - oxidizing or reducing?

Answer 38

Quite reducing - it donates electrons.

Oxidized glutathione (with S-S dimer) is 100:1 in cytoplasm, but 3:1 in the ER.

Question 39

Glutathione

Answer 39

Oxidized glutathione (with S-S dimer) is 100:1 in cytoplasm, but 3:1 in the ER.

Question 40

Protein disulfide isomerase

Answer 40

Soluble ER protein that has 2 binding domains that recognize misfolded or unfolded proteins. Most PDI in the ER is in its oxidized form (with 2 CC bonds), oxidized by the Ero1 complex.

PDI catalyzes the formation of disulfide bonds by accepting electrons from 2 cys residues in the target protein enabling them to form disulfide bond.
It also isomerizes disulfide bonds - breaks some and puts them together.

Question 41

Which migrates faster? disulfide bonded VSVG or non-disulfide bonded?

Answer 41

Disulfide bonded because it is less floppy.

EArliest forms of BiP lack all or some native disulfide bonds and migrate slower.

Question 42

How do we study oligomerization? (Formation of oligomers)

Answer 42

1. Sizing - velocity gradient sedimentation (sucrose gradient), gel filtration chromatography
2. Chemical cross-linking
3. Resistance to SDS

Question 43

Where do misfolded proteins go?

Answer 43

They are retained in the ER or retrieved from early Golgi and given a second chance to fold correctly. Proteins that do not fold correctly are sequestered and/or degraded.

Question 44

What proteins are subject to quality control?

Answer 44

1. Proteins that are slow to fold
2. Excess subunits
3. Misincorporated aas
4. Mutations interfere with folding
5. Misfold due to environmental features

Question 45

What happens to proteins that fold badly?

Answer 45

1. They can form stable aggregates in ER

2. They can be degraded by ERAD

Question 46

ERAD

Answer 46

ER-associated degradation

ER recognizes terminally misfolded or unassembled proteins
Misfolded protein is targeted to ERAD machinery and exported to the cytosol by reverse translocation
Retronslocated proteins are ubiquitylated, deglycosylated, and degraded by the proteasome.

Question 47

ERAD-L, ERAD-M, ERAD-C

Answer 47

Lumenal, membrane, and cytoplasmic ERAD

Question 48

Unfolded Protein Response

Answer 48

UPR - Increase in levels of incompletely folded or unfolded proteins results in up-regulation of chaperone expression as part of a stress response. This can result from viral infection, exression of mutated protein, hypoxia, cancer…

The signal and response system used to sense levels of misfolded proteins is known as the UPR, or unfolded protein response, or alternatively the ER stress response

Question 49

What does the cell do to deal with unfolded proteions?

Answer 49

1. PERK –> phosphorylation of eIF2alpha - translational control
2. ATF6 –> ERSE - protein refolding
3. IRE1 –> UPRE element - protein degradation

Associated with the duration of stress.

Question 50

IRE1

Answer 50

Signaling molecule that splices pre-formed mRNA. Resident ER transmembrane protein kinase with endonuclease activity.
Oligomerization of IRE1 results in endonuclease activity, specific for an RNA - HAC1 in yeast, or XBP in mammals.
This results in altered splicing of HAC1 or XBP1 mRNA, which encodes a UPR-specific transcription factor