Lecture 10

• Chaperones


  Proteins that assist in the folding of other proteins
• Protein Synthesis in Cytosol

  1. Assembly of ribosomal subunits in the cytosol with mRNAs encoding cytosolic proteins

  2. Ribosomes remain free within the cytosol, forming polysomes
• Macromolecular Crowding

  The cytoplasm of a eukaryotic cell is crowded, which influences the behavior of molecules differently than in dilute solutions
• Role of Molecular Chaperones

  Ensure newly synthesized proteins fold correctly by shielding hydrophobic patches and facilitating correct folding
• Hsc70 and Hsp40 chaperones


  • Assist in folding by shielding hydrophobic patches on proteins, preventing improper interactions and facilitating correct folding
• Mechanism of Action of Chaperones

  1. Chaperone in ATP-bound open state binds to nascent protein, shields hydrophobic regions, and upon ATP hydrolysis to ADP, changes to closed conformation that secures the protein during folding

  2. Release of folded protein is triggered by exchange of ADP for ATP, reverting the chaperone to open state
• Folding and Assembly

  Chaperones assist in the assembly of multimeric protein complexes, and chaperonins provide an isolated environment where small proteins can fold away from cytosolic interactions
• Regulation and Fate of Proteins

  Network of chaperones and co-chaperones decide if a protein will be refolded or directed towards degradation pathways like proteasomes or lysosomes
• Experimental methods can demonstrate the role of chaperones in preventing aggregation and promoting solubility
• Further research has shown that chaperones can also disaggregate pre-formed protein aggregates, indicating their potential in treating diseases characterized by protein aggregation
• Proteasome

  Complex protein structure abundant in the cytosol and nucleoplasm, comprising a 20S core particle surrounded by 19S regulatory particles
• 20S core of Proteasome


  • Contains a cylinder with three proteolytic activities: chymotrypsin-like, trypsin-like, and peptidylglutamyl-peptide hydrolysing (caspase-like)
• Ubiquitin

  Small, 76 amino acid protein found universally in eukaryotic cells, used to tag proteins for degradation by the proteasome
• Ubiquitin-Proteasome Pathway

  1. Activation and Conjugation: Ubiquitin is activated by E1, transferred to E2, and then transferred to target protein by E3 ligase

  2. Targeting and Degradation: Ubiquitylated protein is recognized by proteasome, unfolded by 19S RP, and degraded in 20S core
• Specificity of Enzymes in Ubiquitination

  Primarily provided by the variety of E3 ubiquitin ligases, which recognize particular features of proteins
• Role of the Proteasome Beyond Degradation

  Certain RP subunits can refold some client proteins back to their native conformation instead of directing them for destruction
• Failure of the UPS can lead to accumulation of misfolded proteins, which is a hallmark of several neurodegenerative diseases, and dysregulation in the degradation of cell cycle proteins can contribute to uncontrolled cell proliferation, seen in cancers
• Proteolytic Cleavage

  Crucial for activating many proteins, including the effector proteases of apoptosis
• Lipid Modification

  Used to target proteins to membranes, such as the addition of prenyl groups to Rabs to regulate membrane traffic
• Phosphorylation


  Critical post-translational modification that can either activate or deactivate proteins, often mediated by kinases and phosphatases
• Phosphorylation Inhibitors


  • SCH772984 (inhibitor of ERK)
  • U0126 (inhibitor of MEK)
• Phosphorylation can be highly complex, as seen with the p53 protein, which has 24 known phosphorylation sites and plays a role in responding to cellular stress
• ADP-Ribosylation

  Involves the addition of ADP-ribose residues to proteins, affecting functions like cell signaling, DNA repair, and apoptosis
• Methylation


  Typically occurs on arginine or lysine residues and is crucial for regulating protein function and gene expression, such as in histone methylation
• Understanding these protein modifications is critical for grasping how proteins function and are regulated within the cell