L11 - protein processing and targeting Flashcards
What is the constitutive secretory pathway?
A secretory pathway from the golgi to outside the cell by exocytosis. In constitutive secretion proteins are secreted from a cell continuously, regardless of external factors or signals. No external signals are needed to initiate this process.
What is an endosome?
endocytic vesicle
On a histological slide, how can you tell the difference between lysosome and peroxisomes?
Lysosomes are darker
What types of proteins are synthesised by ribosomes attached to the ER membrane?
Proteins destined for membrane or secretory pathway
What types of proteins are synthesised by ribosomes free in the cytoplasm?
Protein destined for cytosol, or post-translational import into organelles
If a protein synthesised by a ribosome in the cytoplasm doesn’t remain in the cytoplasm which organelles can it be imported into?
Peroxisome
Mitochondrion
Nucleus (via nuclear pores)
If a protein synthesised by a ribosome attached to the ER doesn’t remain in the ER but goes onto the golgi complex, where can it be directed to after the golgi?
Plasma membrane
Lysosome
Secretory vesicle -> extracellular
How is a protein synthesised by a ribosome attached to the outside membrane of the ER imported into the lumen of the ER?
Co-translational insertion
Which general four things are required for proteins to be sorted?
- Signal (address) - intrinsic to the protein
- Receptor - recognises signal&directs to correct membrane
- Translocation machinery - to transport across membranes
- Energy - for protein transfer
Where can the signal peptide be found in proteins?
N-terminus
C-terminus
Internal (but available)
Describe the 5 stages of import of proteins into the mitochondrial matrix
- Protein with signal peptide kept unfolded by chaperones
- Signal binds receptor
- Protein fed through pore in outer membrane
- Protein moves through channel in adjacent inner membrane
- Targeting signal cleaved
Describe how proteins are imported into the nucleus
- Cargo protein (with nuclear localisation sequence) binds the carrier protein importin
- Cargo+ importin travel through nuclear pore
- Cargo displaced inside nucleus by binding of Ran-GTP
- Ran-GTP+ cargo recycled into cytoplasm
Which localisation signal can be mutated in Pyruvate dehydrogenase deficiency?
Mitochondrial targeting sequence
Which localisation signal can be mutated in Swyer syndrome (XY genotype but outwardly female due to malfunction in localisation of the sex determining Y protein (SRY required for testis differentiation)
Nuclear localisation signal
SRY is a transcription factor
Which localisation signal can be mutated in Leri-Weill dyschondrosteosis and Langer mesomelic dysplasia which leads to a short stature because SHOX protein (required for skeletal develpoment) is not localised to the correct area?
Nuclear localisation signal
SHOX is a transcription factor
Describe how proteins are imported into the peroxisome
- Cytosol peroxisomal import receptor binds cargo with PTS (peroxisome targeting sequence)
- Peroxisomal protein remains folded and import receptor integrates into translocation channel - opening it
- Import receptor and PTS dissociate
- Receptor returned to cytosol (Energy requiring step)
What is a regulated secretion?
In regulated secretion, proteins are packaged as described in the constitutive pathway, but they are only secreted in response to a specific signal, such as neural or hormonal stimulation.
List some cells which do regulated secretion?
- Endocrine cells
- Exocrine cells
- Neurocrine cells - secreting neurotransmitters
What is meant by cell polarity?
These cells have an apical-basal polarity defined by the apical membrane facing the outside surface of the body, or the lumen of internal cavities, and the basolateral membrane oriented away from the lumen.
What are the features of cells which do regulated secretion?
Specialised cells
Polarised
Very organised ultrastructure
How does co-translational insertion of proteins into the ER occur?
- Ribosome translating protein
- SRP binds signal peptide and ribsome
- SRP mediates binding to SRP receptor on ER membrane, stopping translation
- GTP binding to SRP receptor triggers transfer of ribsomome and peptide to translocon, which opens
- Peptide transported through translocon
- Signal peptide cleaved
- Channel closes, ribosome disconnect and protein folds
Which protein mediates a 3-way association with an ER receptor, ribosome and signal peptide so that co-translational insertion of proteins into the ER can occur?
Signal-recognition particle (SRP) - multi-domain riboprotein
Which proteins other than secretory proteins need to be inserted into ER membrane?
Membrane proteins for plasma membrane or internal membrane of secretory pathway
How is a membrane protein inserted into the ER membrane?
Same mechanism as secretory proteins but the protein has a stop transfer anchor sequence (a second hydrophobic sequence) which prevents is transferring any further into the lumen of the ER
What is hydroxylated in the ER?
selected lysine and proline residues
What conformation changes to proteins occur in the ER?
Formation of S-S bonds
Proper folding of proteins
Assembly of multisubunit proteins
What alterations to the peptide chain occur in the ER?
Specific proteolytic cleavage
Glycosylation
Formation of S-S bonds
Hydroxylation of selected Lys Pro residues
What occurs to some membrane proteins in the ER?
Insertion of proteins into membranes
Why is glycosylation of proteins important?
- Correct protein folding
- Protein stability
- Facilitates interactions with other molecules
- Deficiency in N-linked glycosylation -> severe inherited diseases: congenital disorders of glycosylation (CDG)
What is N-linked glycosylation?
Sugars are added on an asparagine side chain (contains an amino group, hence N-linked)
How is the oligosaccharide transferred to the asparagine side chain in N-linked glycosylation?
It is preassembled on a lipid carrier and then transferred as the peptides crosses the ER membrane into the lumen
What happens to the oligosaccharide side chain in the ER and Golgi?
It is modified extensively by trimming and addition of further sugars
What do peptidyl-prolyl isomerases do?
Accelerate the interconversion of cis and trans isomers of proline residues.
This needs to occur during the folding of a number of proteins (especially IgGs)
What is the role of protein disulphide isomerase (PDI)?
It contains two cysteine residues which are bonded together (oxidised). It comes along and catalyses a disulphide bond forming (oxidation) between two reduced cysteines in a substrate molecule - being reduced itself in the process
How is PDI (an ER resident protein) retained in the ER and not secreted?
It has a KDEL region which binds to a KDEL receptor in the cis-Golgi cisternae, triggering retrograde transport from golgi back to ER
Why can folding problems occur?
- Proteins may be trapped in mis-folded conformation
- Protein contains mutation resulting in mis-folding
- Protein may be incorrectly associated with other subunits
What happens if there are folding problems of proteins?
ER chaperone proteins attempt to correct the problem
Name 3 chaperone proteins?
BIP - binding immunoglobulin protein
Calnexin
Calreticulin
What does the chaperone protein BIP bind to?
It binds to exposed amino acid sequences that would normally be buried in the interior of a folded protein
What do the chaperone proteins Calnexin and Calreticulin bind to?
They bind to OLIGOSACCHARIDES on incompletely folded proteins
What does binding of chaperone protein due to misfolding achieve?
- Retain unfolded proteins in the ER
- Act as sensors to “monitor” extent of protein mis-folding:
(i) mediate increased transcription of chaperones
(ii) mediate reduction in translation
What happens if misfolding can not be corrected?
Protein may return to cytosol for degradation
Protein may accumulate to toxic levels in the ER, leading to a disease (may arise due to single mutations)
What modifications to proteins can occur in the Golgi apparatus?
Modification of sugars:
i) phosphorylation
(ii) sulfation (of tyrosine too
(iii) addition
(iv) removal
Other than modifications, what role does the Golgi apparatus have?
Sorting of proteins into vesicles for the:
lysosome
plasma membrane
secretory vesicles
What signal is added in the Golgi apparatus to lysosomal proteins so that they can be delivered to the lysosome?
mannose-6-phosphate signal
Which receptor mediates the delivery of lysosomal enzymes from the trans-Golgi network to the lysosomes?
mannose-6-phosphate receptor
Where does O-linked glycosylation occur?
Golgi apparatus
Where does N-linked glycosylation occur compared to O-linked glycosylation?
ER lumen vs Golgi apparatus
What does O-linked glycosylation entail?
Attachment of sugar to -OH group of serine or threonine
What type of molecules is O-linked glycosylation important for?
Proteoglycans - component of extracellular matrix and mucus secretions
Name a molecule which undergoes consitutive secretion
Collagen
Name some molecule which undergo regulated secretion
Insulin Also these peptides derived from the precursor protein POMC: Beta-endorphin ACTH alpha-MSH beta-lipotropin
What can be a signal for regulated secretion?
Hormones
neurotransmitters
How does an extracellular signal result in regulated secretion?
Binds to a membrane receptor triggering an intracellular signalling pathway which stimulates secretory vesicles (which are storing secretory proteins) to fuse with the cell’s plasma membrane and release their proteins into intercellular space
What is the most abundant protein in the body?
Collagen (35-30%)
Where is collagen found?
Connective tissue:
tendons, ligaments, cartilage, bone, loose connective tissue (providing structure to internal organs)
Which cell secretes collagen?
Fibroblasts in connective tissue
What is the basic unit of collagen?
tropocollagen
Describe the structure of tropocollagen
300nm rod-shaped protein
characteristic right-handed triple helix
3 polypeptide (alpha-chains), each 1000ish aa long
glycine in every third position in each alpha-chain
What are the properties of the triple helix?
non-extensible
non-compressible
high tensile strength
What should the triple helix of collagen not be confused with?
The protein secondary structure - alpha-helix
Apart from glycines in every third position in the alpha-chains, what other amino acids tend to be in the chains?
Mostly proline of hydroxyproline
What stabilises the triple helix?
H-bonds between alpha chains
There are different types of alpha-chains of collagen. What does this allow?
Different combination of alpha chains make different types of collagen.
Different tissues have different distributions of collagen types
What characteristic feature of collagen fibres can be seen with high resolution electron microscopy?
Regular repeating grooves in the fibres
Why does assembly of collagen occur outside of fibroblasts?
Collagen fibres are very long. They would cause damage to the cell if assembled intracellularly.
What is the pneumonic to help remember the steps of the biosynthesis of collagen?
CHADPOGRL
What does C stand for in the pnemonic CHADPOGRL?
CLEAVAGE of signal peptide (as enters rER lumen)
What does H stand for in the pnemonic CHADPOGRL?
HYDROXYLATION of selected proline and lysine residues
What does A stand for in the pnemonic CHADPOGRL?
Addition of N-linked oligosaccharides and galactose to hydroxylysine residues
What does D stand for in the pnemonic CHADPOGRL?
DISULPHIDE bonds - between chains at C-terminus -> chain alignment
What does P stand for in the pnemonic CHADPOGRL?
PROCOLLAGEN - triple helix formed
What does O stand for in the pnemonic CHADPOGRL?
O-LINKED - oligosaccharide chains are completed by addition of glucose (in Golgi)
What does G stand for in the pnemonic CHADPOGRL?
GOLGI- transports vesicles out of cell
What does R stand for in the pnemonic CHADPOGRL?
REMOVAL - of N-and C-terminal propeptides
What does L stand for in the pnemonic CHADPOGRL?
LATERAL association - of collagen molecules followed by covalent cross-linking -> aggregation of fibrils into collagen fiber
What event changes prepro alpha-chains into pro-alpha chains?
Cleavage of signal peptide by signal peptidase as enter ER lumen
Why are proline and lysine of collagen alpha-chains hydroxylated in the rER?
Increase the available h-bonding between alpha-chains in the triple helix
Why does lack of Vitamin C cause scurvy?
Vitamin C (ascorbate) is a cofactor of the enzyme prolyl hydroxylase which hydroxylates proline and lysine residues of collagen alpha-chains. A lack of Vit C therefore decreases the number of hydroxylated prolines and lysine in the alpha-chain, decreasing the number of h-bonds that can form between each other in the triple helix. Therefore the triple helix conformation is not as stable and the collagen is not as strong.
Collagen is found in all types of connective tissue: skin, bone, tendons, cartilage, loose connective tissue which gives organs their form. These tissues can breakdown in scurvy giving the following symptoms:
- bleeding and swelling of the gums
- muscle and joint pain
- tiredness
- the appearance of red dots on the skin
What is the role of prolyl hydroxylase?
- Associated with PDI (protein disulphide isomerase) in the ER
- Requires Vit C and Fe2+ irons for activity
- Allows H-bonding to stabilise triple helix
- Scurvy is due to weak tropocollagen triple helices
Which parts of the alpha-chains do not form triple helices?
Propeptides : regions of amino acids at the N and C-terminus (removed outside the cell)
Disulphide bonds between alpha-chains at the C-terminus allow for what?
Chain alignment
Why does collagen display regular banding?
Collagen molecules associate laterally in a regular arrangement. Gaps between tropocollagen molecules stain darker with a heavy metal stain. This creates a light and dark banding pattern, where light regions have no gaps and dark regions do.
What is the role of lysyl oxidase?
It coverts lysine residues into aldehyde derivatives which can form cross-links between tropocollagens in the collagen fiber
Where is lysyl oxidase found? What are its cofactors? Function?
Extracellularly
Vitamin B6 and Cu2+
Covalent cross-links
What disease does a deficiency in lysly oxidase cause?
Ehlers-Danlos syndrome
Apart from a deficiency in lysly oxidase, what else can cause Ehlers-Danlos syndrome?
Mutation in collagen type V
Describe the structure of proinsulin
Single polypeptide
What modification happens to proinsulin in the ER?
Disulphide bonds are formed, stabilising the folding
What modifications to proinsulin occur post-Golgi?
Connecting peptide removed (C peptide), leaving complete two-chain insulin molecule (A and B peptide held together by disulphide bonds)
How many enzymes does the proteolytic processing of insulin require?
3
Where does proteolytic processing of pro-insulin occur?
Inside the secretory vesicle, after it has bud from the Trans Golgi Network
Proteolytic processing can be very complex. How can it yield different products in different cell locations?
Different cell locations have different amounts of processing enzymes
What type of secretory protein is commonly proteolytically processed?
Hormones
Why is the precursor protein POMC protelytically processed into:
(i) ACTH and beta-lipotropin, in the anterior lobe of the pituitary
BUT
(ii) alpha-MSH, gamma-lipotropin, beta-MSH and beta-endorphin, in the intermediate lobe of the pituitary?
Different processing enzymes are compartimentalised to different parts of the pituitary
Why is proteolytic processing so common in the secretory pathway?
- Can give rise to very small products that would be too short to enter the ER via co-translational mechanism.
- Some secreted protein (e.g. hydrolytic enzymes) would be destructive if activated inside the cell.
- Multiple bioactive products can be produced at the same time
- Avoid activation of insulin receptors in secretory pathway
