tutorial stuff

The protein induces a misfolding cascade in other proteins, causing them to become dysfunctional and accumulate in the brain

Problems: mutation, old age, (prion) infection • Genetic amyloid-related disease: single changes in nucleotides change amino acid sequence leads to unstable intermediates that can form fibrils • Peptide fragments of a protein that are not folded into the stable globular whole - fibrils form • Breakdown of chaperone system to prevent intermediates that tend to result in fibril formation • Infection by misfolded fibril structures

Hereditary disorders: Cystic fibrosis Emphysema Alzheimer’s disease Prion-based diseases: Mad Cow disease - cows Creutzfeld-Jakob disease - in humans

Fatal transmissible neurodegenerative diseases (spongiform encephalopathies) Lengthy incubation period with no conventional immune response Modes of acquisition: * Spontaneous * Heritable * Infectious

Progressive neurological decline: *Rapidly progressive dementia (within months) *Ataxia/ uncoordinated movements *Visual problems *Akinetic mutism (awake/aware but cant speak)

Abnormal form of a normal protein i.e. PrPSc is an abnormal form of PrPc that causes prion disease

PrPc - rich in alpha helices - soluble - PK sensitive - no aggregation PrPsc - rich in beta sheets - insoluble - PK resistant - aggragation

normal conformation + Prion conformation (high β sheet content) are near each other and this way the normal protein in transient unfolded state may be more susceptible to conversion into prion protein then nucleation causes amyloid fibrils to break into multiple new seeds

Cell surface signals of PrPC are decreased in prion infected cells

Most PrPSc is accumulated throughout endosomal compartments and are particularly abundant in recycling endosomes with little in the Golgi apparatus

PrPC proteins can be found in the plasma membrane with a GPI anchor PrPSc accumulates near PrPC leading to potential for conversion PrPC proteins are taken into the cells through endosomes Conversion can also occur in endosomes or lysosomes We observe a redistribution of PrPC when there is PrPSc present

Canadian Blood Services – Original Criteria People were ineligible to donate blood if: *They had spent 3 or more months in the UK from January 1980 –December 31, 1996 *They had spent 5 or more years in France or the Republic of Ireland between January 1980 and December 31, 2001 because they discovered possible transmission of variant Creutzfeldt-Jakob disease by blood transfusion now: change in eligibility criteria made by Canadian Blood Services (CBS) in November 2023 to allow these people to donate blood

What we believe in theory: There are rules for folding a protein into its lowest stable energy state. What really happens In the cell: Internal and external stressors that promote unfolding and misfolding.

Anfinsen’s dogma 1) Every sequence of amino acids auto assembles into a unique, defining conformation. 2) Due to thermodynamic molecular forces, polypeptides automatically assume unique, stable conformations. Protein folding is: * Spontaneous * Reversible * Unique

The Anfinsen dogma in the cell: “The probability that such a polypeptide will fold correctly after removing the denaturant increases at low protein concentration (which limits interpolypeptide interactions) and low temperatures (which attenuates hydrophobic interactions).” The slide gives the Anfinsen dogma as context. This dogma states that a protein’s amino acid sequence alone determines its final folded structure (its native conformation). In other words, if you unfold (denature) a protein and then remove the denaturant, it should, in theory, refold itself correctly.

All proteins will try to fold into a low energy, thermodynamically stable state Chaperones are at the crux of the battle

Are found in all organisms from bacteria to humans * Their importance is highlighted by the observation that many are evolutionarily conserved * Facilitate proper folding and prevent aggregation of nascent proteins General mechanism: * To prevent inappropriate interactions between amino acid residues and increase the efficiency of protein folding

Molecular chaperones: * Bind to a short segment of a protein substrate and stabilize unfolded proteins, thereby preventing these proteins from aggregating and being degraded Chaperonins: * Form folding “chambers” into which all or part of an unfolded protein can be sequestered, giving it time and an appropriate environment to fold properly - ATP binding triggers lid closure and substrate release into central chamber

Can have 8 to 9 homomeric or heteromeric subunits in each ring * The “lid” function is incorporated into the subunits * ATP hydrolysis triggers the closing of the lid

Two models: 1) The ‘‘mechanical force’’ model: chaperonin holds on to protein. The chamber holds onto the protein and physically pushing it into the right shape. 2) Release of protein into a closed environment. The lid closes and the chaperonin lets go of the protein. Inside the chamber, the chemical properties of that environment allow the protein to fold.

Dr. Frydman’s group purified extracts of a protein called rhodanese, and the chaperonins. Together, these complexes were then incubated in the presence or absence of ATP for 10 min. They were then analyzed using native gels with lid on: cheperonin holds protein with lid off: chaperonin releases protein (seen with western blot, Protein(Rho) is with chaperonin when there is a lid, vs Protein (Rho) is free from chaperonin when there is no lid.

In a modified version of the Chaperonin, , called Cpn-rls. Loop 11 conformation has been modified so that the substrate protein cannot be released. Red arrow indicates shift in Loop 11. In Cpn-rls, the lid closes, but the substrate is not released from the edges of the chaperonin - no folding occurred.