Question

6. Why do misfolded proteins tend to aggregate with one another (0.5 pt)?

Answer 1

Protein aggregation is a biological phenomenon in which mis-folded proteins aggregate (i.e., accumulate and clump together) either intra- or extracellularly. These protein aggregates are often correlated with diseases. In fact, protein aggregates have been implicated in a wide variety of disease known as amyloidoses, including ALS, Alzheimer's, Parkinson's and prion disease.

Protein structures are stabilized by non-covalent interactions and disulfide bonds between two cysteine residues. The non-covalent interactions include ionic interactions and weak van der Waals interactions. Ionic interactions form between an anion and a cation and form salt bridges that help stabilize the protein. Van der Waals interactions include nonpolar interactions (i.e. London dispersion force) and polar interactions (i.e. hydrogen bonds, dipole-dipole bond). These play an important role in a protein's secondary structure, such as forming an alpha helix or a beta sheet, and tertiary structure. Interactions between amino acid residues in a specific protein are very important in that protein's final structure.

When there are changes in the non-covalent interactions, as may happen with a change in the amino acid sequence, the protein is susceptible to misfolding or unfolding. In these cases, if the cell does not assist the protein in re-folding, or degrade the unfolded protein, the unfolded/misfolded protein may aggregate, in which the exposed hydrophobic portions of the protein may interact with the exposed hydrophobic patches of other proteins.There are three main types of protein aggregates that may form: amorphous aggregates, oligomers, and amyloid fibrils.

Misfolded proteins can form protein aggregates or amyloid fibrils, get degraded, or refold back to its native structure.

The mechanisms and intermediates of Protein Misfolding

Misfolding is produced by an incorrect folding process that results in the formation of a protein with a different conformation from its native fold. Protein misfolding can occur by several reasons (i) somatic mutations in the gene sequence leading to the production of a protein unable to adopt the native folding; (ii) errors on the processes of transcription or translation leading to the production of modified proteins unable to properly fold; (iii) failure of the folding and chaperone machinery; (iv) mistakes on the post-translational modifications or trafficking of proteins; (v) structural modification produced by environmental changes or (vi) induction of protein misfolding by seeding and cross-seeding mechanisms.

The most frequent destiny for misfolded proteins is self-aggregation, because the mistaken exposure of fragments to the solvent that are normally buried inside the protein, lead to a high degree of stickiness. The β-sheet structural motif offers the most favorable organization for these intermolecular aggregates and can accommodate an almost unlimited number of polypeptide chains . As a result misfolded proteins exist as a large and heterogenous continuum of polymeric sizes, which are usually classified in broad and not very well defined categories, such as oligomers, protofibrils and fibrils .Soluble oligomers are small assemblies of misfolded proteins that are present in the buffer soluble fraction of tissue extracts and usually include structures ranging in size from dimers to 24-mers . Recent compelling evidence coming from several independent studies of different proteins indicates that oligomers might be the most toxic species in the misfolding and aggregation pathway . Indeed, both synthetic and natural oligomers have been shown to induce apoptosis in cell cultures at very low concentrations , block long term potentiation in brain slice cultures and impair synaptic plasticity and memory in animals . Protofibrils are larger aggregates that can be seen using electron microscopy as curvilinear structures of 4–11 nm diameter and <200 nm long . Protofibrils increase in size with increased time and protein concentration and are elongated by growth on their ends . Annular protofibrils are pore-like assemblies that form in the cell membrane and may contribute to cell death . Protofibrils and annular protofibrils have also been shown to be highly toxic in various in vitro studies . Amyloid fibrils are long, straight and unbranched structures of around 10 nm diameter and usually several μm lengths . They bind the dies Congo red and thioflavin and show a typical “cross-β” X-ray diffraction pattern consisting of two major reflections at 4.7 Å and 10 Å found on orthogonal axes . Fibrils can also elicit toxicity in cultured cells, but usually at much higher concentrations than oligomers and protofibrils .

The mechanism of protein misfolding and aggregation follows the so-called “seeding-nucleation” model . In this process, the initial steps of misfolding are thermodynamically unfavorable and progress slowly, until the minimum stable oligomeric unit is formed that then grows exponentially at a fast speed. There is two kinetic phases in the seeding-nucleation model of polymerization. Firstly, during the lag phase, a low amount of misfolded and oligomeric structures are produced in a slow process, generating seeds for the next step. Once nuclei are formed, the elongation phase takes place and results in fast growing of the polymers. The addition of pre-formed seeds can reduce the length of the lag phase, accelerating the exponential phase. Oligomers are perhaps the best seeds to propagate the misfolding process in an exponential manner. However, larger structures as fibrils could be as well important to propagate this event in vivo, due to their higher resistance to biological clearance than smaller aggregates.