Exam 3, second half

Question 1

How to generate an antibody?

Answer 1

Take sample of protein to study, inject antigen into foreign organism, causing antibody production for the antigen. Collect organism’s blood and purify. Can be used with a secondary antibody that contains a radioactive, chemiluminescent, or fluorescent label to bind to the primary antibody, making it glow and illuminate. Antibodies can be used for techniques like Western blot, Immunofluorescence, and immunoaffinity column chromatography.

Question 2

Western/Immuno Blot

Answer 2

Technique to analyze presence of a specific protein using electrophoresis. Run protein on gel with SDS, transfer to nitrocellulose membane, add primary and secondary antibodies, and observe the bands that result. The brighter the bands, the more of the target protein contained in the sample. Example, muscle proteins are more concentrated in heart cells than in kidneys. Is protein present, and if so how much

Question 3

Immunofluorescence

Answer 3

Technique used to detect protein in fixed cells using confocal fluorescent or fluorescent microscopy. Cell culture is added to fixative like formaldehyde to prevent degradation. Add 1’, 2’ antibodies, generate slide, observe sample. Able to observe multiple proteins and their general area by using different colored tracers with multiple ABs, colocalization. If colors are close together, suggests that the proteins MAY interact.

Question 4

Immunoaffinity column chromatography

Answer 4

Technique used to collect specific protein for further analysis. Agarose beads are attached to 1’ AB, placed in a column. Cell lysate added to bind AB with protein of interest. Light salt wash rinses excess protein, medium salt wash yields target protein. Used to learn sequence of protein amino acids and structure, and other proteins interacting with target will stick to it and precipitate with it, coimmunoprecipitation. Both proteins will wash out, and other protein can be identified with a Western Blot.

Question 5

Secretory system

Answer 5

Major pathway for mRNA associated with attached and free ribosomes. Nucleus, mitochondria, chloroplasts, peroxisomes are made by cytoplasmic ribosomes, NOT ROUGH ER. PM, secretory vesicles, endosomes, lysosomes are made by ribosomes that attach to the Rough ER

Question 6

Endoplasmic Reticulum ER

Answer 6

Continuous with outer nuclear membrane. Extensive network of membrane, tubules, cisternae(sacs). Interior is known as ER lumen, contains large variety of proteins, enzymes, carbohydrates. Rough and Smooth sections with different function

Question 7

Rough ER

Answer 7

Ribosomes attach to RER membrane, functions in protein synthesis. Proteins in PM, those secreted, endosomes, and lysosomes made by fixed RER ribosomes. Contains SRP receptor, translocator, associated proteins that are not present in the SER.

Question 8

Smooth ER

Answer 8

Functions in lipid synthesis and detoxification of chemicals.

Question 9

Transitional ER

Answer 9

vesicles leaving the ER, proteins from RER and lipids from SER meet here before leaving ER entirely to move to Golgi

Question 10

Cotranslational translocation

Answer 10

Targeting proteins to the ER, DURING translation. Examples are RTK, GCPR, LIGC, NaK pump, secretory vesicles and ligands. Protein being made has an N-terminal signal sequence NTSS, which causes Signal Recognition Particle SRP to take it to an SRP receptor in the ER membrane. Protein with NTSS moves through protein translocator in the ER, SRP released. Signal peptidase in lumen cleaves NTSS.

Question 11

Post-translational translocation

Answer 11

Uncommon in mammals, translocation after TLN is already finished. Chaperone proteins in cytoplasm bind to the new protein, preventing folding. Protein has internal signal sequence ISS, causing BiP protein to pull the C terminal end of the new protein through the translocator into the ER lumen. If Stop transfer sequence STS is present, hydrophobic alpha helix, the translocator will open while processing it, embedding the STS in the membrane. C terminal will be inside ER lumen, N terminal will remain outside in cytoplasm

Question 12

Single Pass transmembrane protein (Nt in lumen, Ct in cytoplasm)

Answer 12

NTSS causes SRP to pull Nterminal into translocator, where signal peptidase cleaves NTSS. Protein contains STS, which embeds the protein in the membrane, leaving N-terminal inside lumen and C-terminal outside.

Question 13

Single pass transmembrane protein (Nt in cytoplasm, Ct in lumen)

Answer 13

ISS causes BiP to pull C terminal in, STS embeds in membrane. C terminal is left in lumen, N terminal is in cytoplasm.

Question 14

Multi-pass transmembrane protein

Answer 14

If N-terminal is in lumen, starts with NTSS, otherwise ISS is used to pull C terminal in. STS will embed a portion of the protein in the membrane, alternates between ISS and STS after the first sequence. NTSS is not used after the first step, if used at all.

Question 15

Protein modifications (general types)

Answer 15

Folding, covalent modifications to protein portions, and cleavage

Question 16

Protein Folding modifications

Answer 16

Proteins can be folded into 2’, 3’, 4,’ structures. Chaperone proteins in the lumen facilitate proper folding. Example are Heat Shock Proteins like BiP, which heats proteins to biologically activate them. Another example are protein disulfide isomerases, which help to generate disulfide bridges between sulfhydryl groups in methionine and cystine.

Question 17

Protein cleavage

Answer 17

Signal peptidase cleaves NTSS in proteins with N terminal inside

Question 18

Protein Glycosylation modification

Answer 18

Generation of a glycoprotein via the addition of an oligosaccharide to protein, allowing for cell to cell recognition and interaction.

Question 19

Process of Glycosylation and glycoprotein folding process, calreticulin pathway for quality control

Answer 19

Protein is being made with co-translational translocation, N terminal in lumen. Dolichol, a lipid carrier that carries oligosaccharides, will transfer the oligosaccharide to the N terminal end of the protein using oligosaccharyl transferase. Then 2 of 3 glucose residues on the oligosaccharyl will be removed from the glycoprotein. Chaperone protein calreticulin will then folds the protein. After folding, 3rd glucose is removed. A Folding Sensor protein checks that the Glycoprotein is properly folded. If proper, sends Glycoprotein to transitional ER, and if improper, either prompts retrotranslocation or refolding by calreticulin. Calreticulin pathway is exclusively for GLYCOPROTEINS.

Question 20

Retrotranslocation

Answer 20

Malformed protein is moved into the cytoplasm, tagged by ubiquitin, causing the proteosome, a group of proteases, to degrade the protein.

Question 21

Glycoproteins’ function in neutrophils example

Answer 21

Outside of neutrophil membrane has glycoproteins with specific oligosaccharides to allow for interactions with other cells. Example Endothelial cell has receptors that recognize the oligosaccharides, allowing the neutrophil to interact with and ultimately adhere to the endothelial cell and push in between endothelial cells to areas where it is needed.

Question 22

Addition of protein to a glycolipid

Answer 22

A protein in the ER can move to a membrane-embedded GPI anchor, a glyco(sugar)lipid, which cleaves a new C terminal on the protein and attaches to it.

Question 23

Quality control in the ER

Answer 23

Occurs in rough ER, makes sure proteins are properly folded and modified. Unfolded protein response and calreticulin pathway are used to perform these checks.

Question 24

Smooth ER detoxification function and application

Answer 24

Liver cells contain large concentration of smooth ER for detoxification. Smooth ER increases based on more frequent use of the liver. SER intakes and modifies toxic chemicals to make them water soluble for urine excretion. Takes 5 days to reduce Smooth ER to normal levels.

Question 24

Unfolded Protein Response UPR

Answer 24

Can be used on any protein in the rough ER. Regularly, some chaperone proteins are folding proteins while others inhibit signaling molecules from stopping TLN. If there is an excess of unfolded proteins, all or most of the chaperones will switch to folding exclusively. Signaling molecules are no longer inhibited by chaperones and signals degradation of mRNA and terminating TLN in the cytoplasm. Proteins that are irreparable are sent to cytoplasm and are retrotranslocated.

Question 24

Smooth ER lipid synthesis function for steroid production

Answer 24

Smooth ER also makes steroids, example cholesterol, estradiol, testosterone, etc. Testosterone and estradiol are mainly produced by SER in the gonads, testes or ovaries’ cells. SER also makes ceramide for use in the Golgi body.

Question 24

Smooth ER

Answer 24

Functions in lipid synthesis and Chemical detoxification. Lipids are sent out to transitional ER to form vesicles, meeting proteins from RER.

Question 24

Smooth ER lipid synthesis function for phospholipid production

Answer 24

Cytoplasmic enzymes generate phospholipids on the cytoplasmic side of the smooth ER membrane, which is a phospholipid bilayer, adding to it. Uses scramblase enzymes to make transverse movement of the phospholipids, flipping some of the phospholipids to the Smooth ER lumen side. Vesicles are made of phospholipid bilayers, so they are constantly generated.

Question 24

ER resident proteins homing process

Answer 24

If proteins such as BiP that need to return to the ER to do their function, they will be sent back after they are processed in the Golgi body. ER residents contain ER homing signals, KDEL or KKXX, that signal their need to be returned. Other examples of residents are calreticulin, peptidase, protein translocators, and any others with ER function.

Question 24

Export from ER, Transitional ER

Answer 24

Proteins from the RER and phospholipids from the SER move to and meet at the transitional ER. Lumenal Proteins are transported by the fusion of phospholipids and transmembrane proteins, which forms the Transport vesicle that moves to the ER Golgi Intermediate Complex ERGIC, and then into the Cis-face of Golgi cisternae, then medial, then trans-face.

Question 24

ER export, signals

Answer 24

Lumenal proteins will bind to the transmembrane proteins in the ER, which contains an ER export signal. GPI-anchored proteins will also bind to transmembrane proteins with a signal. Transmembrane proteins have their own signal for export. Export signals are required to leave the ER.

Question 24

Cholesterol function

Answer 24

Steroid that modulates membrane fluidity, 4-fused ring carbon structure. Modulates based on temperature, decreasing fluidity at high temperatures and increasing it at low temperatures. Prevents over and under-packing in membranes.