3:Protein modification Flashcards
What are some different types of modifications?
Proteolytic processing - Required for biological activity of some enzymes and blood factors.
Glycosylation - Can affect the solubility, half-life and biological activity of the protein
Phosphorylation - Regulates activity of various polypeptide hormones
Sulfation - Can affect biological activity and proteolytic processing
What is post translational modification?
Involves the alteration to the protein folding, binding and function It occurs after protein synthesis and folding.
Explain the process of protein glycosylation.
Glycosylation involves the transfer of precursor oligosaccharide by oligosaccharide transferase and the modification of this precursor. This modification increases the diversity of proteins and it is used as a tag to mark the state of a protein folding. It involves the polypeptide being glycosylated at the target asparagine (Asn) as it enters the endoplasmic reticulum lumen.
What are the roles and effects of Glycosylation?
- Protein folding
- Protein targetting/trafficking
- Ligand recognition/binding (i.e. antibody binding)
- Biological activity (i.e. the activation of gonadotrophin signal transductions)
- Stability (Increases the solubility, shields hydrophobic patches and protects it from proteolysis)
- Regulates the half-life (I.e. increases it)
- Immunogenicity
What are the two types of glycosylation and explain them. Give and example of an approved protein that is glycosyated.
O-linked = short (1-4 sugar residues) that are linked to serine and threonine. N-linked = Larger and are linked to asparagine. Involves the initial addition of large preformed molecules containing 14 sugar molecules, followed by the sequential addition and removal of sugar residues to produce the final product.
Examples of approved therapeutic proteins that are Glycosylated include: Activase, refacto, Rebif, Inductos etc. Human Erythropoietin (EPO) is a growth factor involved in the regulation/stimulation of red blood cell production. It consists of 3xN-linked oligosaccharides and 1xO-linked glycan.
Explain protein phosphorylation.
Protein Phosphorylation involves the transfer of a phosphate group to a protein substrate on a tyrosine (tyr), serine (ser) or threonine (Thr) amino acid residues.
Explain what is meant by the ‘Molecular switch’
The molecular switch is a protein activation/inactivation that is usually controlled by phosphorylation to activate and dephosphorylation to inactivate. This switch increases/decreases with other proteins such as transcription factors, receptors, enzymatic substrates, membrane proteins etc. It is also responsible for changing the subcellular localisation of proteins. In protein phosphorylation, cyclin dependent kinases(CDK) act as an integrating device.
Explain the role of proteolytic processing in insulin production
Insulin is produces as proinsulin that is cleaved after folding into a specific shape. Recombinant insulin involves the separation production of A + B chains that are combined to produce proinsulin that can be cleaved.
How do proteins bind to other molecules?
The conformation of a protein form specific binding sites on their surface. Ligands can bind to these sites by numerous weak non-covalent bond. The exact ligand to match the binding site can generate the tight bond arrangement.
What are the 3 interaction interfaces?
Surface-string: Enables a protein kinase to recognise proteins that it will phosphorylate.
Helix-Helix: Found in several gene regulatory protein families.
Surface-Surface: The most common interaction and is very specific.
Give an example of conformation dependent enzyme activity.
The active site of serine proteases is conformation dependent. The aspartic acid side chain induces the histidine to remove a proton from the serine. This activates serine to form a covalent bond with the substrate, hence hydrolysing a peptide bond.
Name the 3 aspects that can be considered when wanting to improve protein drugs
- Modifying primary sequence
- Mimic/modify native post-translational modifications
- Modify the final protein.
Explain the methods of mimicking post-translational modifications of proteins.
Convergent assembly - Modifying the group added after the protein of interest has been constructed
Linear assembly - Smaller peptides/amino acids that already bear desired groups are assembled into a larger protein.
What is the difference between protein drugs and peptide drugs?
Protein drugs - Commercial scale production is costly and difficult. They are excluded from compartments such as the blood brain barrier, hence there is limited penetration into tissues. They can be associated with severe side effects often caused by the bodies immune response.
Peptide drugs - Have a short half-life, limited access to intracellular space, low potency but is highly specific and can be stored at room temperature.
What are peptidomimetics?
Peptidomimetics are compounds whose essential elements mimic that of a natural peptide/protein. They retain the ability to interact with the biological target and hence produce the same biological effect.
