List the differences between the constitutive and regulated secretory pathways
Constitutive - Packaged proteins are released continuously by exocytosis Eg Serum albumin, collagen
Regulated - Packaged proteins are stored and only released when stimulus received Eg Insulin
Name a constitutively secreted protein?
Serum albumin, collagen
Named a regulated secreted protein?
Outline the secretory pathway in mammalian cells
1) Free ribosomes initiates protein synthesis from mRNA molecule
2) Hydrophobic N terminal signal sequence is produced
3) Signal sequence of newly formed protein is recognised and bound by the signal recognition particle (SRP)
4) Protein synthesis stops
5) GTP-bound SRP directs the ribosome synthesising the secretory protein to SRP receptors on the cytosolic face of the ER
6) SRP dissociates
7) Protein synthesis continues and the newly formed polypeptide is fed into the ER via a pore in the membrane (peptide translocation complex)
8) Signal sequence is removed by a signal peptidase once the entire protein has been synthesised
9) the ribosome dissociated
What protein modifications can occur in the ER?
- Signal cleavage (signal peptidase)
- Disulphide bond formation (protein disulphide isomerase)
- N-linked glycosylation (oligosaccharide-protein transferase)
What protein modifications can occur in the golgi?
- O-linked glycosylation (glycosyl transferase)
- Trimming and modification of N-linked oligosaccharides
- Further proteolytic cleavage
Where are proteins destined for secretion synthesised?
In the RER
What is the difference between glycosylation in the ER and the golgi?
ER - N-linked, oligosaccharide built up on a Dolichol phosphate carrier molecule and transferred to amine group of asparagine, uses N-acetylglucosamine phosphotransferase
Golgi - O-linked, oligosaccharide added to the OH group on a serine or threonine by glycosyl transferase
Describe N-linked glycosylation
Oligosaccharide built up on a Dolichol Phosphate carrier molecule sitting in the membrane. The oligosaccharide is then transferred to the amine group of an asparagine, using N-acetylyglucosamine phosphotransferase
Describe O-linked glycosylation
The modification of the hydroxyl on serine and/or threonine by glycosyl transferase
What type of glycosylation occurs in the golgi?
What type of glycosylation occurs in the ER?
Outline the formation of the mature insulin molecule
- PreProInsulin contains a signal sequence, A, B and C peptides Signal sequence cleaved by signal peptidase inside RER
- ProInsulin contains A, B and C peptides Endopeptidases cleave the C peptide
- Insulin contains A and B peptides Joined by 2 disulphide bridges, has 3 overall Active form
Note; C peptide is a good marker for measuring levels of endogenous insulin in diabetes
Describe the structure of collagen
- The basic unit of collagen is tropocollagen
- Primary sequence is (Glycine-X-Y)n
- Mostly proline or hydroxyproline in X and Y
- Glycine every third position Collagen made of 3 polypeptides
- 3 left handed helices
- Right handed alpha chains
- Non-extensible/compressible, high tensile strength
Outline production of collagen fibres
Within the cell
1) Two types of peptide chains are formed on ribosomes during translation, alpha-1 and alpha-2. These peptide chains (known as preprocollagen) have registration peptides on each end and a signal peptide
2) The preprocollagen is released into the lumen of the RER. Thereafter the signs peptides are cleaved inside the RER and the peptides are now called pro-alpha chains
3) Hydroxylation of lysine and proline amino acids occur inside the lumen. The process is dependent on ascorbic acid (vitamin C) as a cofactor. Further glycosylation of specific hydroxylysine residue occur
4) Triple helix structure formed in the ER from 2 alpha-1 chains and one alpha-2 chain. This is called pro collagen
5) Procollagen is transported into the golgi apparatus, where it is packaged and secreted by exocytosis
Outside the cell
1) Registration peptides are cleaved by pro collagen peptidase to form tropocollagen
2) Tropocollagen gather to form collagen fibrils, via covalent cross-linking by lysol oxides which link hydroxylysine and lysine residues. Multiple collagen fibrils form into collagen fibres
3) Collagen may be attached to cell membranes via several types of protein, including fibronectin and integrin
Outline nuclear targeting
1) A fully folded protein with a nuclear localisation signal is bound by importin α and β in the cytosol
2) The resulting complex binds to the nuclear pore and translocates into the nucleus in an energy-dependant mechanism
3) Once inside the nucleus the nuclear localisation signal is released and the importin bind to GTPase protein Ran
4) The importin α and protein complex is dropped off in the nucleus
5) Importins exported back out to cytoplasm to be recycled (leaving protein behind
6) Ran is transported back to the nucleus following hydrolysis of GTP
How can high rate of division of bacteria or cancer lead to cell resistance?
High rate of division in both bacteria and cancer cells means that there is a higher rate of mutation. Positive mutations, such as drug resistance will be positively selected for and breed a population of drug resistant cells.
How can decreased influx of drug in bacteria/cell lead to cell resistance?
For drugs, which require to be taken up by their target cells to take effect (Rifampicin, Tetracycline, Methotrexate), influx can be reduced. Cells achieve this by expressing a reduced amount or altered version that reduces affinity of the carrier protein that allows the drug through the cell membrane.
How can increased efflux of drug lead to cell/bacteria resistance?
P-Glycoprotein, or Multi-Drug Resistance Protein 1 (MDR1) is a protein similar in structure to CFTR that is responsible for the efflux of toxic products from a cell. In many cancers, expression of P-Glycoprotein is up-regulated. This allows the cells to increase the efflux of chemotherapy drugs, eg Methotrexate.
How can increased transcription of target lead to bacteria/cell resistance?
If the drug targets a specific product of transcription, eg a ribosome or enzyme, the cell can acquire resistance by increasing the transcription of the target to overwhelm the drug.
How can altering drug target lead to cell/bacteria resistance?
Specific target of drug acquires a mutation, lowering the affinity of the drug for it.
What is I cell disease?
- Lack of N-acetylglucosamine phosphotransferase
- Lysosomal hydrolases not targeted to lysosomes by addition of mannose-6-phosphate
- Mis-targeted for secretion so seen in elevated concentrations in the blood and urine
How are proteins destined for the lysosome targeted?
- Proteins destined for lysosomes are targeted for the addition of M6P groups by the presence of a signal patch, a sequence of several amino acids from different parts of the amino acid sequence. This reaction occurs in the golgi and involves 2 enzymes:
1) N-acetylglucosamine phosphotransferase
2) N-acetylglucosamine phosphoglycosidase
MP6 recognised by M6P receptors at the trans golgi and vesicles are pinched off for transport to the lysosome
Where are KDEL receptors found?
Cis face of the golgi
What is the purpose of KDEL receptors?
Bind to resident proteins of the ER that have been secreted by accident and return them to the ER in transport vesicles where the protein disassociates and the receptor is recycled back to the golgi
What is the mitochondrial signal sequence?
Amphipathic N-terminal signal sequence
What are the main protein complexes involved in mitochondrial destined secretions?
TOM and TIM
In the ER of what cells primarily would insulin be synthesised?
Beta Cells in the Islets of Langerhans
How is insulin stored in the cell before its release?
Stored in margination secretory granules
In what form is collagen synthesised?
How is secreted collagen converted to mature collagen?
Procollagen secreted which is converted to collagen by procollagen peptidases which cleave of the N and C terminal peptides Lysosyl oxidase then enables covalent crosslinks to form between tropocollagen molecules as lysine residues are converted to aldehyde derivatives which form aldol cross links
What is the repeating sequence of amino acids found in collagen?
Glycine-X-Y (usually proline/hydroxyproline)
What is the importance of vitamin C in the production of strong collagen?
Vitamin C and Fe2+ are required by the enzyme prolyl hydroxylase which converts proline to hydroxyproline enabling more hydrogen bonds to form which strengthens tropocollagen molecules