Session 6 Flashcards
(47 cards)
0
Q
When do ribosomes remain cytosolic?
A
- Protein is destined for cytosolic or posttranslational import into organelles
1
Q
When do ribosomes attach to the ER membrane?
A
- Protein is destined for membrane or secretory pathway via co-translational insertion
2
Q
What is required for protein sorting?
A
- Signal intrinsic to the protein
- Receptor that recognises the signal and directs it to the correct membrane
- Translocation mechanism
- Energy to transfer the protein to a new place
3
Q
Describe the constitutive secretory pathway
A
- Continuous process
- Proteins packaged into vesicles and released continuously by exocytosis
- eg serum albumin, collagen
4
Q
Describe the regulated secretory pathway
A
- Proteins release in response to a signal eg hormone
- Proteins packaged into vesicles but not released until stimulus is received eg insulin
5
Q
Describe protein synthesis in the secretory pathway
A
- Free ribosome initiates protein synthesis from mRNA molecule
- Hydrophobic N-terminal signal sequence is produced
- Signal sequence of newly formed protein is recognised and bound by the signal recognition particle (SRP)
- Protein synthesis stops
- GTP-bound SRP directs the ribosome synthesising the secretory protein to SRP receptors on the cytosolic face of the ER
- SRP dissociates
- Protein synthesis continues and the newly formed polypeptide is fed into the ER via a pore in the membrane (peptide translocation complex)
- Signal sequence is removed by a signal peptidase once the entire protein has been synthesised
- The ribosome dissociates and is recycled
6
Q
What protein modifications can take place in the ER?
A
- Signal cleavage (signal peptidase)
- Disulphide bond formation (protein disulphide isomerase)
- N-linked glycosylation (oligosaccaride-protein transferase)
7
Q
What protein modifications can take place in the Golgi?
A
- O-linked glycosylation (glycosyl transferase)
- Trimming and modification of N-linked oligosaccarides
- Further proteolytic processing (some proteins only)
8
Q
What is N-linked glycosylation?
A
- Oligosaccaride is built up on a Dolichol Phosphate carrier molecule that sits in the membrane
- Dolichol Phosphate is a special long chain hydrocarbon molecule that inserts into the membrane with its phosphate group protruding
- The oligosaccaride is transferred to the amide group of Asparagine (Asn) via a N-glycosyl link
9
Q
What is O-linked glycosylation?
A
- The modification of the Hyroxyl groups of serine and threonine
- Glycosyl transferase builds up a sugar chain from nucleotide sugar substrates
- Carbohydrate added via glycosidic link to hydroxyl of Serine or threonine
10
Q
What role does proteolytic processing have in the formation of secreted proteins?
A
- Exoproteases (eg amino peptidase, carboxypeptidase) are used
- Removal of the pre-segment takes place in the ER and involves the proteolytic removal of the N-terminal signal sequence
- Removal of the pro-segment occurs in some proteins and takes place in the Golgi
- eg preproalbumin -> proalbumin -> albumin
11
Q
How is the mature insulin molecule formed?
A
- Preproinsulin contains a signal sequence, and A, B and C peptides
- The signal sequence is cleaved by the signal peptidase enzyme leaving proinsulin
- Proinsulin contains A, B and C peptides
- Endopeptidases cleave C peptide leaving insulin (active form)
- Insulin contains A and B peptides joined by disulphide bridges
- C peptide is a good marker for measuring levels of endogenous insulin in diabetes
12
Q
What is the basic unit of collagen?
A
- Tropocollagen
13
Q
What is the primary sequence of collagen?
A
- Glycine-X-Y
- Mostly Proline or Hydroxyproline in X and Y positions
14
Q
What is collagen made up of?
A
- 3 polypeptides: 3 a-chains in a left handed triple helix (non-extensible/compressible, high tensile strength)
15
Q
What is the function of Proline residues in collagen?
A
- Give correct geometry for extended a-chain conformation
- Prevents the peptide assuming another shape eg a-helix, B-sheet
16
Q
What is the function of Hydroxyproline residues in collagen?
A
- Increases amount of inter-chain H-bonds
17
Q
How is Hydroxyproline formed?
A
- From Proline residues by the enzyme Prolyl Hydroxylase
18
Q
What does Prolyl Hyrdroxylase need and why is it important?
A
- Requires Vitamin C and Fe2+ for activity
- Scurvy is due to low Vitamin C as there is weak tropocollagen triple helices
19
Q
How are Tropocollagen subunits synthesised?
A
- Synthesised first as preprocollagen (pre-hydrophobic signal sequence marks protein for secretion; pro-subunits synthesised with N- and C-terminal peptides to prevent the formation of collagen fibres inside cells)
- Procollagen is secreted from cells by exocytosis
- Procollagen Peptidases cleave N and C terminal peptides (extracellular)
- Collagen subunits (Tropocollagen) form covalent cross-links
~Lysine residues -> aldehyde derivates by enzyme lysyl oxidase (requires vitamin B6 and Cu2+ ions)
~ Aldehyde derivates then spontaneously form Aldol cross-links
20
Q
Where do the proteins in the nucleus come from?
A
- Made on ribosomes in the cytoplasm and are transported into the nucleus
21
Q
How do proteins from the cytoplasm enter the nucleus?
A
- Proteins pass through nuclear pores (protein channels that span nuclear membrane)
- Proteins that need to enter the nucleus contain a Nuclear Localising Sequence (NLS) (4-8 basic amino acids in primary sequence)
- Needs proteins to shuttle between cytosol and nucleus (eg importin) to import nuclear proteins
22
Q
What is the process of importing nuclear proteins from the cytosol into the nucleus?
A
- Fully folded protein with a NLS is bound by importin a and B in the cytosol
- Resulting complex binds to the nuclear pore and translocation unto the nucleus in an energy dependent mechanism
- Once inside the nucleus, the nuclear protein is released and the importins bind to a small GTPase protein know as Ran
- Importins are exported from the nucleus and can recycled to transport more nuclear proteins
- Ran is transported back to the nucleus following hydrolysis of GTPASE
23
Q
Where are mitochondrial proteins synthesised?
A
- In the cytosol
24
In what state are mitochondrial proteins transported into the mitochondria?
- Unfolded state
25
What do proteins destined for the mitochondria contain?
- Amphipathic N-terminal signal sequence of 10-80 amino acids
26
What are the unfolded mitochondrial proteins stabilised by in the cytosol?
- Interaction with molecular chaperones such as Mitochondrial Import Stimulating Factor (MSF)
27
What recognises the signal sequence of mitochondrial proteins?
- Recognised by TOM proteins in the outer membrane that form a protein import channel (Translocase of the Outer Membrane)
- Proteins destined for the matrix are transported across the inner mitochondrial membrane by TIM proteins (Translocase of the Inner Membrane) (requires ATP and a membrane potential)
- Proteins of the inner mitochondrial membrane also contain additional signals that stop them entering the matrix
28
What happens to the protein once across the inner mitochondrial membrane?
- N-terminal sequence is removed by mitochondrial processing peptidase (MPP)
- The protein folds into its native conformation in an ATP-dependent process helped by chaperones eg mitochondrial Hsp70
29
How are lysosomal hydrolases targeted to lysosomes?
- Addition of Mannose-6-phosphate (M6P) signal to N-linked oligosaccarides on the protein
- Occurs in the Golgi
- Involves enzymes: N-acetylglucosamine phosphotransferase;
N-acetylglucosamine phosphoglycosidase
30
How are proteins destined for lysosomes targeted for the addition of the M6P groups
- Presence of a signal patch (sequence of several amino acids from different parts of the amino acid sequence)
31
What are M6P groups on the targeted proteins recognised by?
| What happens to the receptors?
- M6P receptors at the trans Golgi
- Vesicles are pinched off for transport to the lysosome
- Once at the lysosome, the acid pH causes dissociation of the protein and receptor
- The receptor is recycled back to the Golgi for further use
32
How is the protein prevented from returning to the Golgi with the receptor from the lysosome?
- Phosphate group is removed from the M6P group on the protein
33
What is I-Cell disease and what is it caused by?
- Genetic defects in the N-acetylglucosamine phosphotransferase enzyme result in a lack of M6P addition to lysosomal targeted proteins
- In I-cell disease this results in lysosomal hydrolases being mistargeted for secretion -> large amount of these proteins present in the blood and urine
34
What proteins and how can they be lost from the ER?
- Proteins like protein disulphide isomerase and signal peptidase that are resident within the lumen of the ER
- Lost when vesicles are pinched off and transported to the Golgi
34
What happens to lost proteins?
- There is a selective method that allows ER-resident proteins that have escaped to be retrieved
35
What is the selective method of retrieval of ER proteins?
- ER resident proteins have the sequence Lys-Asp-Glu-Leu (KDEL) near the C-terminus
- These interact with KDEL receptors in the Golgi (enhanced by low pH)
- ER proteins bound to the receptor are returned to the ER in transport vesicles
- ER resident proteins dissociate from the receptor in the neutral condition within the ER
- KDEL receptor is transported back to the Golgi
36
What drug targets bacterial cell walls and how?
- Penicillin
- Inhibits transpeptidase enzyme that forms cross-links in the cell wall
- Osmotic pressure causes cell lysis
37
What drug affects bacterial transcription and how?
- Rifampicin
| - Binds to bacterial RNA polymerase preventing transcription
38
What drug affects bacterial protein synthesis and how?
- Tetracycline
| - Competes with tRNA at A site of bacterial ribosome
39
What drugs act as anti-folates in cancer therapy and how?
- Methotrexate
- Impairs synthesis of tetrahydrofolate (which is essential for DNA synthesis) from folic acid
- Competitively inhibits dihydrofolate reductase (DHFR)
40
What general mechanisms can cause cells to become resistant to an an antibiotic or drug?
- High rate of division
- Decreased influx
- Increased efflux
- Increased transcription of target
- Altered target
41
How does a high rate of division cause resistance in cells?
- Causes a higher rate of mutation in bacteria in cancer cells
- Positive mutations (eg drug resistance) will be positively selected for and breed a population of drug resistant cells
42
How does a decreased influx cause resistance in cells?
- Drugs that require to be taken up by their target cells to have an effect can have their influx reduced (eg rifampicin, tetracycline, methotrexate)
- Cells express a reduced or altered amount version that reduces the affinity of the carrier protein that allows the drug through the cell membrane
42
How does increased efflux cause resistance in cells?
- P-Glycoprotein (Multi-Drug Resistance Protein 1 (MDR1) - similar in structure to CFTR a) is responsible for the efflux of toxic products from a cell
- Expression of P-Glycoprotein is up-regulated in many cancers, allowing the cells to increase the efflux of chemotherapy drugs eg Methotrexate
44
How does increased transcription of target cause resistance in cells?
- Increasing the transcription of the target product, eg ribosome or enzyme, can overwhelm the drug
46
How can an altered target cause resistance in cells?
- If the target acquires a mutation, the affinity of the drug for it is reduced
