RER/glycosylation

Question 1

Which end is the signal sequence located on the newly made peptide?

Answer 1

N-terminal end

It targets the peptide to the RER

Question 2

In the signal hypothesis…

The role of the SRP

Answer 2

Binds to the signal sequence of the peptide and stalls translation

Also…binds to the SRP receptor in the RER membrane

Question 3

The SRP receptor

Answer 3

Associated with the closed translocon channel in the RER membrane

Question 4

Binding of the SRP to its receptor —>

Answer 4

1. Mediates the binding of GTP to both the SRP and its receptor

Question 5

GTP hydrolysis in the signal hypothesis

Answer 5

GTP is bound to SRP and its receptor

Promotes the opening of the translocon…and the dissociation of the ribosome to the SRP

The SRP also dissociates from its receptor

Question 6

After the SRP is dissociated from its receptor and the ribosome…

Answer 6

SRP is recycled

Ribosome becomes associated with the open translocon

Translation continues again…and the peptide is translated through the open channel

Question 7

Co-translation/translocation

Answer 7

Refers to the continuation of translation of the peptide through the open translocon and into the RER lumen

(Signal hypothesis)

Question 8

Signal peptidases in the signal hypothesis

Answer 8

As signal peptide is translocated into the RER lumen

It is cleaved by signal peptidases

Question 9

As translation finishes….

Answer 9

Ribosome falls off the mRNA and RER surface

Ribosome dissociates into its respective 40S and 60S

Translocon channel closes and peptide folds into its native structure

Question 10

Soluble matrix proteins vs. integral proteins

Answer 10

Soluble = translocated through ER membrane and into lumen or matrix

Integral = inserted directly into ER membrane (have hydrophobic nature of amino acids that form the transmembrane spanning

Question 11

Glycosylation (general)

Answer 11

Enzymatic addition of carbohydrates (glycans) to proteins

Question 12

Glycation (general)

Answer 12

NON-enzymatic addition of carbs (glycans) to proteins

Ex: glycation of Hgb (Hemo A1c) —> relevant in diabetes

Question 13

Glycosylation of proteins aids in…

Answer 13

Primarily protein folding and targeting

Also…protein stability (if go to E.C leaflet of plasma membrane)

Also…cell-to-cell contact

Question 14

Lectins

Answer 14

Carbohydrate-binding proteins

Can interact with other glycosylated proteins

Question 15

ABO blood typing

Answer 15

Results of EC glycosylation (antigenic)

Question 16

2 types of glycosylation

Answer 16

O-linked

N-linked

Question 17

O-linked glycosylation

Answer 17

Carbs added individually

Sequentially to -OH groups (O-linked) of serine and threonine

Enzyme: gylcosyl transferase

Example: adding carbs to proteins to generate ABO blood groups

Question 18

N-linked glycosylation

Answer 18

Carbs added as a performed oligosaccharide complex to Asparagine (Asn) residues

…in an Asn-X-(Ser/Thr) consensus sequence

Occurs in ER as the peptide is translocated through the ER membrane

Complex is further processed in ER and Golgi….and can serve as a mechanism for targeting of the protein to its final cellular destination

Question 19

Where does synthesis of oligosaccharide complex start

Answer 19

Cytosol

Then further in ER lumen/Golgi after added to peptide in N-linked gly.

Question 20

Dolichol phosphate

Answer 20

Membrane bound molecule that the oligosaccharide precursor is added upon

Question 21

Activated nucleotide-sugar donors

Answer 21

UDP-sugar or GDP-sugar

Add sugars to the dolichol phosphate sequentially until the oligosacc. Precursor is complete

Question 22

Tunicamycin

Answer 22

Analog for the first nucleotide sugar substrate (UDP-GluNAc)

Inhibits first reaction of oligosacc precursor formation

Prevents formation of complex

Dramatic effects in protein folding, stability, targeting, and secretion

***used to study eukaryotic protein folding…NOT a clinical drug

Question 23

Oligosaccharyl transferase

Answer 23

Enzyme that transfers the complex from the dolichol phosphate to the Asn residue of the consensus sequence

Once attached the complex is further processed by glycosidases

Question 24

Disulfide bonds primary role

Answer 24

Primarily found in secreted proteins or in proteins found in the EC leaflet of the plasma membrane

Also aid in proper folding and provide a strong covalent structure and stability to the protein

Question 25

Where are disulfide bonds formed?

Answer 25

ONLY in the ER lumen

Only under oxidative conditions as the protein is translocated through the ER membrane

Question 26

Protein disulfide isomerase (PDI)

Answer 26

Transfers oxidized disulfide bonds, found in the enzyme structure to the reduced protein substrate

In doing so…

Cysteine residues in PDI = reduced

Cys in substrate = oxidized

PDI can also rearrange disulfide bonds in the substrate protein to allow the correct conformation for foldiing

Question 27

Alpha-1-antitrypsin deficiency (A-1AT)

General info

Answer 27

Made and secreted from the liver

Genetically inherited

Most common genetic cause of emphysema in adults (familial emphysema) and liver disease in children (jaundice, cirrhosis)

Question 28

A-1AT deficiency mechanism and role of neutrophils

Answer 28

Neutrophils secrete elastase in order to move through the lung tissue matrix…

A-1AT is needed to inhibit typsin and unregulated elastase —> otherwise tissue damage in lungs

Mechanism = point mutation in protein that prevents proper folding….crystalline aggregates in ER lumen

—> improper secretion of A-1AT and other proteins from lumen —> damages liver tissue

Question 29

A-1AT deficiency treatment

Answer 29

Goal to minimize symptoms

Immunizations for flu and pneumonia to prevent symptoms

No smoking/pollution

A-1AT replacement therapy = no persuasive data

Broncodilators and steriods to open up airways

O2 if lungs really fucked

Liver transplants (most common cause of this in peds)