Lecture 3: Post-translational Modifications Flashcards
Describe protein glycosylation.
Glycosylation is the addition of a sugar molecule to the protein. Each protein has it’s own pattern of glycosylation (sugar type, location of sugar molecule etc).
As a protein is being produced, it passes into the lumen of the ER, where it undergoes multi-step initial glycosylation. As the polypeptide passes through transport proteins into the ER, oligosaccharyl transferase transfers a sugar residue from carrier lipid in ER membrane to an Asn residue on the polypeptide chain.
The protein buds off from the ER in a vesicle, binds to the Golgi apparatus where it may be further modified, and is then packaged for secretion or transported to the membrane for incorporation or sorting into a lysosome.
The precursor polysaccharide undergoes modification such as trimming or processing.
Most cytosolic and nuclear proteins are not glycosylated.
What are post translational modifications?
Occur after protein synthesis and folding.
Mature protein only becomes functional after covalent modification. These modifications are necessary for binding to receptors or substrates.
Examples include protein glycosylation, phosphorylation.
What is the role of glycosylation, and what are the effects of glycosylation?
Marks state of protein folding.
Tag for protein targeting or trafficking.
Ligand recognition or binding.
Biological activity, eg activation of gonadotrophin signal induction.
Stability: increases solubility, shields hydrophobic regions, protection from proteolysis
Regulates half life
Immunogenicity
Describe the two types of glycosylation.
O-linked: 1-4 sugar residues are linked to Ser or Thr. Dehydration synthesis attaches sugar residue to oxygen atom of hydroxyl group on side chain.
N-linked: initial addition of large, preformed molecule containing 14 sugar molecules to Asparagine. Some of the initial residues are then removed via trimming in the Golgi, or more added to obtain the final structure.
Precursor oligosaccharide has a core region which is often the only part remaining after trimming process in the Golgi.
Only asparginines in the sequence Asn-X-Ser, and Asn-X-Thr are glycosylated.
OH group of sugar binds to the N in Asparagine side chain.
Approved glycosylated biopharmaceuticals include EPO, hormones, antibodies and blood factors
Describe protein phosphorylation and the role of cyclin-dependent kinases.
CDK complexes with cyclin to phosphorylate substrates appropriate for particular phases of the cell cycle.
Phosphate group binds to protein at Thr, Tyr or Ser residues. Acts as a molecular switch: phosphorylation = activation, dephosphorylation = deactivation.
Phosphorylation may increase or decrease interaction with other proteins, for example enzyme substrates, membrane proteins, transcription factors and receptors.
May lead to change in subcellular location, eg protein movement from cytosolic to nucleus.
Describe protein binding, and the three types of interaction surfaces.
The conformation of the folded protein determines the specific binding sites on the protein surface. Ligand bind to binding sites through weak, non-covalent bonds. For a tight bond to be achieved between the ligand and the binding site, the ligand must be an exact match for the binding site.
SURFACE-STRING
Enables a protein kinase to recognise proteins that it will phosphorylate.
HELIX-HELIX
found in several families of gene regulatory proteins.
SURFACE-SURFACE
most common and very specific.
Discuss the aim and considerations of improving protein drugs.
AIM: to improve the pharmacological properties of naturally occurring proteins for use as pharmaceuticals (eg half life, receptor-ligand interactions, stability).
Must consider at which stage the protein will be modified (primary sequence, post translational modifications or modification of the final protein)
Describe the two methods of mimicking post-translational modifications.
- CONVERGENT ASSEMBLY: modifying group is added after protein of interest has been constructed.
- LINEAR ASSEMBLY: smaller peptides or amino acids that already bear desired group are assembled into the larger protein.
Compare protein and peptide drugs.
PROTEIN DRUGS commercial production is costly and difficult Excluded from compartments in the body (eg do not cross BBB) Limited penetration into tissues Severe side effects, eg immune response PEPTIDE DRUGS shorter half life Limited access to Intracellular space High specificity Lower potency than protein drugs Cn be stored at room temperature
Discuss peptidomimetics.
Compounds whose essential components mimic a natural peptide or protein, and retain the ability to interact with the biological target and produce the same biological effect.
Unstable or low potency peptides are modified by:
- introduction of non-natural AAs
- formulation of peptide heterodimers
- incorporating rigid structures (cyclic, alpha helix, beta sheet)
- conjugation
- making cell-penetrating peptides
