SESSION 9 Flashcards
Define proteolytic cleavage
Breaking peptide bonds to remove part of the protein
Define chemical modification
Addition of functional groups to amino acid residues, e.g. Phosphorylation
Define protein targeting
Protein targeting is the biological mechanism by which proteins are transported to the appropriate destinations in the cell or outside it
Proteins can be targeted to the inner space of an organelle, different intracellular membrane, plasma membrane, or to exterior of the cell via secretion
Contrast the constitutive and regulated secretory pathways
Constitutive secretion
- continuous process
- proteins packaged into vesicles and release continuously by exocytosis
- e.g. Serum, albumin, collagen
Regulated secretion
- proteins released in response to a signal, e.g. Hormone
- proteins packaged into vesicles but not released until stimulus received, e.g. Insulin
- endocrine cells, exocrine cells and neuroscience cells
Provide an overview of the secretory pathway mammalian cells
1) free ribosome 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 to 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 dissociates and is recycled
List protein modifications which can occur in the ER and Golgi complex
Modifications in the ER:
- signal clevage (signal peptidase)
- disulphide bond formation (protein disulphide isomerase)
- N- linked glycosylation (oligosaccharide- protein transferase)
Modifications in the Golgi:
- O- linked glycosylation (glycosyl transferase)
- trimming and modification of N- linked oligosaccharide
- further proteolytic processing (some proteins only)
Distinguish between N- linked and O- linked glycosylation of proteins
N- linked glycosylation
The oligosaccharide is built on a Dolichol Phosphate carrier molecule sitting in the membrane
Dolichol Phosphate is a special long chain hydrocarbon molecule that inserts into the membrane with its phosphate group protruding
The oligosaccharide is then transferred to the Amide groups of Asparagine (Asn)
O-linked glycosylation
The modification of the hydroxyl groups on serine and threonine
Glycosyl transferase builds up a sugar chain from Nucleotide Sugar Substrates
Describe the role that proteolytic processing plays in the formation of important secreted proteins
This is similar to that of mRNA maturation, rather than using specific nucleases endoproteases (sequence specific) and exoproteases (amino peptidase, carboxypeptidase)
Proteolytic removal of the N- terminal signal sequence is a very early event occurring in the ER, this is also known as the removal of the ‘pre- segment’
Further processing can occur in some proteins and does so in the Golgi, this is the removal of the ‘pro- segment’
E.g. Preproalbumin -> Proalbumin -> Albumin
Outline the formation of the mature insulin molecule
Preproinsulin- contains signal sequence; A,B and C peptides
Signal sequence is cleaved by signal peptidase
Proinsulin- contains A, B and C peptides
Endopeptidases cleave C peptide
Insulin:
- contain A and B peptides
- joined by 2 disulphide bridges
- active form
- NB. C peptide is a good marker for measuring levels of endogenous insulin in diabetics
What is required for protein sorting?
- a signal, intrinsic to the protein
- a receptor that recognises the sing also gives and directs it to the correct membrane
- a translocation machinery
- energy to transfer the protein to its new place
Outline protein targeting to peroxisomes
Peroxisome targeting sequence is usually present on the C- terminus of the protein
PTS is recognised by the PTS receptor PeX5
Translocation machinery (across peroxisomal membrane) -> 13 proteins make up a transport channel across the peroxisomal membrane -> basically forming a pore
Energy -> ATP hydrolysis needed to allow recycling of the PTS receptor -> back out to the cytosome to collect more
What happens if targeting to peroxisome goes wrong?
Peroxisome biogenesis disorders, e.g. Zellweger syndrome (1 of 4 peroxisome disease)
The diseases are caused by defects in any one of 13 genes, termed PEX genes, required for the normal formation and function of peroxisome
Zellweger syndrome may include poor muscle tone, poor feeding and distinctive facial features
Also develop life threatening problems with other organs, e.g. Liver, heart and kidney
Do not usually survive past the age of 1
Define the signal recognition particle
- receptor needed to bind to the signal peptide on proteins destined for the ER
- made of proteins and RNA
- part recognises the signal sequences and another part recognises the ribosome
What are the functions of the endoplasmic reticulum?
- insertion of proteins into membranes
- specificity of proteolytic cleavage
- glycosylation
- formation of S- S bonds
- proper folding of proteins
- assembly of multi- subunit proteins
- hydroxylation of selected lys and pro residues
Why is glycosylation of proteins important?
- correct protein folding
- protein stability
- facilitates interactions with other molecules
- deficiencies in N- linked glycosylation lead to serve inherited human disease: congenital disorders of glycosylation (CDG)
