Protein Aggregation

Competing Pathways: Folding vs Aggregation

• some proteins can take off-pathway routes, leading to misfolding and aggregation

• exposed hydrophobic regions and β-sheet structures in partially folded or misfolded intermediates can non-specific interactions leading to promote aggregation

• aggregates typically occupy low-energy states in the protein energy landscape (highly stable)

• molecular chaperones avert aggregation by shielding hydrophobic residues and preventing kinetically stable non-functional structures (misfolded intermediates)

Proteostasis

• proteostasis = protein homeostasis

• the protein homeostasis network encompasses a large number of proteins that maintain the proteome functional by directing protein synthesis, folding, trafficking and degradation

Protein Aggregation & Diseases

• there are over 30 human pathologies associated to protein aggregation

  • e.g. Alzheimer’s (AD), Amyotrophic lateral sclerosis (ALS), Huntington, Parkinson’s, prion diseases

• these diseases are characterized by the presence of amyloid protein aggregates in the affected tissues

  • specific proteins are found at unusually high concentrations inside the aggregates compared to their normal levels.

• in most cases, the cause of the disease is unclear and no cure exists

• there are many types of amyloid diseases

Amyloid Fibrils vs Amorphous fibrils

• amorphous aggregates: disordered protein clumps, usually not associated to disease

• amyloid aggregates: highly ordered fibrils with a characteristic cross-β structure, and are associated to disease, insoluble protein assemblies

  • β-strands aligned perpendicularly to the fibril axis

  • cross-β structure refers to the stacked β-sheet arrangement, which provides fibrils w/ high stability, and that an be arranged in 3 manners

    • antiparallel β-sheets, parallel β-sheets, β-solenoid

  • high stability => resistant to degradation or resolubilization

Amyloid Fibrils: Amyloid Organization

• amyloids are organized hierarchically

  • starts w/ individual proteins/peptides that form β-sheets (D)

  • β-sheets stack to form protofilaments (C),

  • which twist together into amyloid fibrils (B), creating highly stable and structured aggregates (A)

Amyloid Fibrils: Insolubility

• amyloid insolubility arises from extensive intermolecular H-bonding between β-strands, which stabilizes the stacked β-sheet structure

• and…

• hydrophobic interactions, VDW forces and electrostatic interactions further stabilize fibril packing, making amyloids highly resistant to degradation and solubilization

Assembly of Amyloid Fibrils

• amyloid fibril formation starts w/ misfolded protein or peptide monomers assembling into oligomers (multiple copies of the same proteins)

  • oligomers act as seeds for further aggregation

• once fibrils form, they can accelerate growth by recruiting more monomers and amplifying fibril formation thru fragmentation and/or secondary nucleation, where new fibrils emerge from fibril surfaces

Assembly & Propagation Amyloid Fibrils: Prion

• a prion (protein infection) is an amyloid fibril that can convert the native conformation of the PrP prion protein (PrP^C) into the infectious nonnative structure (PrP^Sc) following the formation of a seed

• very rare, can be promoted by mutations

• only prions are considered infection agents; aggregates formed by other amyloideogenic proteins display prion-like behavior, meaning they can cause template misfolding and spread within and across diff brain regions

Assembly & Propagation Amyloid Fibrils: Creutzfeldt-Jakob Disease (sCJD)

• spontaneous conversion is extremely rare and can cause sCJD disease (infection disease caused by a protein; infection agent = prion protein)

• once converted, the nonnative form can propagate thru the brain, leading to rapid dementia

• cannabilism and inappropriate handling of surgical tools can transmit the disease, making Prp^Sc an infection agent that can also infect other mammals

Toxicity of Amyloid Fibrils (diseases)

• whether a protein aggregate is toxic or protective has been debated; general agreement is that the small fibrils/protofibrils are cytotoxic

• small fibrils: perturbs PM, mitochondrial function (accumulation of reactive oxygen species in cells), physical trafficking by disrupting ER netowrk

• can spread from one cell to another (potentially spreading disease over the tissues)

• as aggregates get bigger, they start binding and trapping proteins (eg. proteosome)

  • proteosome normal function: clears misfolded proteins

    • if trapped by aggregate, it can’t function properly

Amyloid Fibrils: Sporadic Aggregation

• sporadic aggregation is rare but many elements can accelerate aggregation (happens spontaneously, w/o a trigger)

• Protein aggregation usually requires overcoming an energy barrier (misfolding + nucleation)

Nucleation

• first step and rate-limiting step in amyloid fibril formation

• several protein molecules come together to form a small oligemeric nucleus

  • this nucleus is a rare and short-lived intermediate that must reach a critical size before stable fibril growth can begin

• once this nucleus forms, additional proteins can rapidly add to the growing fibril. At this point, the process if non-reversible

• in some cases, secondary nucleation can occur; occurs when the surface of existing fibrils promotes the formation of new nuclei, accelerating the production of additional aggregates, including smaller toxic proto-fibrils

Tafamidis

• drug to treat transthyretin-mediated amyloid cardiomyopathy

  • progressive heart condition caused by build-up of transthyretin-containing amyloids in the heart

• the small molecule stabilizes the tetramer, reducing the ability of the monomer to misfold and aggregate (need monomer to allow formation of fibrils)