1-45 Protein Misfolding and Disease

Question 1

What is the main pathway by which cells get ride of unwanted protein?

Answer 1

The ubiquitin-proteasome pathway.

1. Ubiquination: E1, E2, and E3 enzymes; thioester bonds between enzymes and ubiquitin; generate poly-ubiquitin chains
2. Proteasome: proteasome unfolds and degrades protein

Improper degradation is linked to many diseases, including cancer, neurodegeneration, CF, and autoimmune diseases.

Question 2

What is ubiquination?

Answer 2

The first step in the ubiquination-proteasome pathway to rid cells of unwanted protein.

• The E1 enzyme activates ubiquitin in an ATP-driven reaction, creating a high-energy, covalent, thioester E1-ubiquitin bond.
• One of several different E2s then transfers the activated ubiquitin to the target protein, which is bound to a specific E3 (again via a thioester E2-ubiquitin intermediate).
• E3 then catalyzes the final transfer to the epsilon amino group of one or more specific Lys residues on the target protein.
• This step is usually repeated to generate polyubiquitin chains of various lengths.

Question 3

How does the proteasome participate in protein degradation?

Answer 3

It is involved in the second step of the ubiquitin-proteasome pathway.

• Proteasome = 26S particle = 20S core particle (red) + 19S regulatory particle “caps” (blue/AAA-ATPase subunits and gold/non-AAA)
• Polyubiquitin binds to the gold 19S caps
• AAA-ATPase subunits (19S blue) unfold the bound protein
• Bound protein is threaded into the 20S cylinder, where it is cleaved

Peptides can be transported through the ER for antigen presentation by MHC class I or recycled to build new proteins. The ubiquitin is recycled.

Question 4

What are the major mechanisms by which protein structure and/or function can be perturbed?

Answer 4

Primarily by amino acid mutation, in the following ways:

• Direct knockout: A mutation of a residue that is essential for function (e.g., one involved in substrate binding or catalysis), altering a critical side chain though the structure and stability of the protein remain essentially unchanged
• Destabilization: A mutation pushes the equilibrium toward the unfolded state, and the protein cannot muster enough energy to fold (e.g., substantial change in a side chain in the tightly-packed, hydrophobic core)
• Toxic conformation: A mutation shifts the conformational equilibrium to an incorrectly folded state. It could be as simple as mutating a surface charged residue to a hydrophobic one, which causes the protein to aggregate (e.g., the Glu→Val mutation in sickle cell anemia)

Question 5

What is p53?

Answer 5

The “guardian of the genome.” A transcription factor activated by DNA damage (or some other problem) that triggers cell cycle arrest/apoptosis. It prevents the accumulation of chromosomal mutations, so it’s unsurprising that 50% of all tumors have point mutations in p53.

• Tetramer; 400 AAs/monomer
• Structure of the DNA binding domain is known (shown above).
• Extensive β-strands arranged in a β-clam fold
• Binding site = α-helix + loops
• β-clam provides scaffold for helix and loop (few interactions between clam and DNA)
• Single Zn²⁺ (NOT zinc finger motif) is necessary for site-specific DNA binding

Question 6

What are the three classes of p53 mutations?

Answer 6

1. DNA contact: Mutants alter side chains that directly bind to DNA, reducing binding without changing the overall protein structure/stability.
   Arg 248, Arg 273
2. Destabilizing: Decrease thermodynamic stability by disrupting the four noncovalent interactions, or cause proteins to aggregate.
   Tyr 220
3. Zinc binding: Arg 175

Question 7

How does p53 react to stability mutants?

Answer 7

Stability mutants decrease p53 thermodynamic stability, which should cause most proteins to degrade more quickly. p53, however, exhibits the opposite behavior due to negative feedback with MDM2, which accumulates mutant p53 in the cell to high levels.

Question 8

What are potential small molecule therapies for stability mutants?

Answer 8

• Crevice binders: Find a cavity/pocket to which a small molecule can be tailored to fit, both in terms of shape/size and chemical complementarity (hydrophobic to hydrophobic, (+) to (-), H-bond donor to H-bond acceptor). Since this nook/cranny is particular to the native structure of that protein, the small molecule will theoretically stabilize the folding of that protein only.
• MDM2 blockers: Block the interaction between p53 and MDM2 (E3 ubiquitin ligase that recognizes p53) with drugs designed to resemble the peptide and block its interaction with MDM2.

Question 9

What is a potential small molecule therapy for zinc binding mutants?

Answer 9

Zinc binding mutants weaken p53’s affinity for zinc. A synthetic metallochaperone can:

• Bind zinc
• Transport it through the plasma membrane (which is normally impermeable to ions)
• Buffers zinc conc. in the cell, maintaining it at a level high enough to allow binding to the correct site on p53, but not so high as to bind to the wrong sites on p53 and other proteins

Question 10

What mutation is the most common cause of cystic fibrosis?

Answer 10

70% of cases are caused by the deletion of Phe508 in the CF transmembrane conductance regulator (CFTR), a gated chloride channel of unknown structure. Though the structure and function of ΔF508 and “wild-type” are very similar, ΔF508 alters the fold/assembly pathway.

It is the most lethal mutation in the Caucasian population.

Question 11

What potential treatment options target ΔF508?

Answer 11

• 25° (less aggregation at lower temperatures)
• Small organic molecules: glycerol, myoinositol, benzoflavones
• Overexpressing chaperones
• Inhibiting degradation by the ubiquitin-proteasome pathway
• Stimulating CFTR function

Question 12

What are prion diseases? Name two prominent prion diseases.

Answer 12

Prion diseases are transmissible spongiform encephalopathies (TSEs). They are a group of fatal, progressive, degenerative CNS diseases. The infectious agent, a prion, is thought to be an alternate form of a normal brain protein.

1. Scrapie, bovine spongiform encephalopathy (BSE), elk/deer wasting disease: characterized by incessant rubbing, wasting, and loss of coordination. Amyloid fibers found in brain, nerve death. Invariably fatal.
2. Human Creutzfeld-Jakob disease (CJD), Kuru: nerve death, amyloid fibers in brain. Several means of transmission: genetic, cornea/dura mater grafts, iatrogens, human growth hormone/gonadotropin from cadavers