338 #2

• Post-translational modifications


  Covalent and generally enzymatic modification of proteins following biosynthesis

  
• Post-translational modifications


  • Occur either at the same time as synthesis (technically co-translational), or after protein folding
  • Can be reversible depending on the nature of the modification

  
• Examples of post-translational modifications

  • Breaking peptide bonds
  • Removing the initiator methionine residue
  • Proteolytic cleavage to generate mature protein

  
• Disulfide bridges


  Covalent modification of proteins, where the protein sequence is not different to the gene sequence, but neither is it specified by the central dogma

  
• Role of disulfide bridges

  • Protein folding
  • Protein stability

  
• Formation of disulfide bridges

  1. Form under oxidative conditions

  2. Most cellular compartments are reducing environments, e.g. cytoplasm of cells

  3. Outside the cell, oxygen exerts a thermodynamic preference for formation

  4. Free cysteine thiols are usually in secreted proteins
• Amino acid residues that tend to have post-translational modifications

  • Hydroxyl groups of serine, threonine, and tyrosine
  • The amine forms of lysine, arginine, and histidine
  • The thiolate anion of cysteine
  • The carboxylates of aspartate and glutamate
  • The N- and C-termini
  • The amide of asparagine

  
• Phosphorylation


  • One of the most common forms of PTM, 30% of all proteins phosphorylated at any given time
  • Added by proteins called kinases

  
• Chemical effects of phosphorylation

  • Introduces a bulky group bearing two negative charges into a region of the target protein that was only moderately polar
  • The oxygen atoms of a phosphoryl group can H-bond with one or several groups in a protein
  • The negative charges can also repel neighbouring negatively charged residues (Asp or Glu)

  
• What can phosphorylation do to a protein

  • Direct interference (of ligand binding)
  • Creation of binding sites
  • Conformational change

  
• Protein kinases have specificity
• Phosphorylation is reversible
• The activity of kinases is tightly regulated

  • Reversible phosphorylation is the most prominent form of covalent modification in cellular regulation
  • Kinase/phosphatase activity is tightly regulated, by: Autophosphorylation, Binding of activator proteins or inhibitor proteins, Cellular location relative to substrates

  
• Example of the insulin receptor

  • Insulin receptor is a "receptor tyrosine kinase"
  • Binding of activator (insulin) initiates dimerization, this step activates the kinase activity, resulting in autophosphorylation
  • Creates a binding sites for downstream messaging
• Ubiquitination


  Addition of ubiquitin, a small polypeptide 76 amino acids long, to a substrate protein

  
• Effects of ubiquitination


  • Can mark for degradation via the proteasome
  • Alter their cellular location
  • Affect their activity
  • And promote or prevent protein interactions

  
• How ubiquitin is attached to a protein

  1. Ubiquitin most commonly linked between its last amino acid (glycine), and to a lysine residue on the substrate

  2. Can also be linked to cysteine residues through a thioester bond, serine and threonine residues through an ester bond, or the amino group of the protein's N-terminus via a peptide bond

  
• Ubiquitination requires three types of enzymes

  1. E1 activates Ubi independent of the substrate

  2. E2 can feed Ubi to many E3

  3. E3 ubiquitin ligases possess substrate recognition domains

  
• Ubiquitination diversity


  • The three-step E1/E2/E3 ubiquitination process is called the ubiquitination cascade
  • Humans possess 35 different E2 enzymes
  • Target protein selectivity resides with the E3 components
  • Human genome may contain as many as 1000 E3s
• Monoubiquitination


  The addition of one ubiquitin molecule to one substrate protein residue
• Multi-monoubiquitination


  The addition of multiple ubiquitin molecules to multiple residues on a single substrate
• Polyubiquitination


  The addition of a chain of ubiquitin molecules to a single residue on a substrate
• PROTACs capitalise on the natural system for clearing unwanted or damaged proteins, to destroy rather than inhibit proteins