Investigating the mechanism of amyloid gain of toxicity Flashcards
how do heterogeneous oligomers cause gain of toxicity
they expose hidden hydrophobic patches which drive aberrant protein:protein interactions, sequestering proteins that are usually in a specific cellular location
how do misfolded proteins cause gain of toxicity
they saturate chaperones and interfere with QC allowing defective proteins to slip through
how does s-syneuclin oligomers and ab(1-40) oligomers interfere with lipid membranes
form pores
when investigating the mechanism of gain of toxicity which proteins should be used
proteins which have no biological function so any effects are solely a result of aggregation
briefly describe the approach used by West et al to design proteins for use to investigate the mechanism of gain of toxicity
designed denovo proteins which were all-beta with 6 beta strands. he created a library and introduced random variation at polar sites by incorporation His, Lys, Asp,Glm, Glu or Asn and at non-polar sites by introducing His, leu, Iso, Val or Phe. The alternating pattern of hydrophobic/hydrophyllic residues leads to formation of amphiphilic beta strands. he then screened for the most aggregate prone proteins.
how did west et al check whether the proteins formed the structures we believed they did
measured the amount of secondary structure
how did west et al show they formed amyloid fibres
he plotted fluorescence vs wavelength using ThT and NIAD-4 which are dyes that only fluoresce in the presence of amyloid fibres and ANS which fluoresces upon binding to solvent exposed hydrophobic patches. The proteins were found to be NAID4 positive. confocal immunofluorescence microscopy also should that the proteins expressed in cells had collapsed shape and accumulated in aggregates in the perinuclear space
what did the MTT assay show about these proteins in human embryonic kidney cells
they reduced cell viability after 3 days and also found that toxicity is proportional to aggregate propensity
how was it determined whether the proteins where forming fibres or oligomers in vivo
oligomeric aggregation intermediates were detected by dot blots using the A11 antibody
what approach was used to determine what causes gain of toxicity
SILAc was used to identify proteins which bind aggregates. 3 experiments occurred in the same “pot” and were distinguished via light, medium and heavy isotope labelling so all exits occurred under the same conditions. this found that 60% of b17 and b4 proteins interact with b23, in some cases over 50% of total cellular proteins were sequestered by b aggregates. the majority of proteins pulled down were cytoplasmic, nucleus or mitochondrial proteins and spun a range of cellular roles however there was no observable cytosolic response
why is there no cytosolic stress response observed in the presence of b aggregates
they may impair the cels ability to respond to stress and Hsp70 and Hsp110 were both pulled down in lilac experiments
what are the 2 categories of proteins which interact with b aggregates
old interactors and new interactors
what is meant by old interactors
they have more and larger stretches of disordered residues and regions which are less hydrophobic and expose aggregation prone regions in the folded state
what is meant by new interactors
the have normal hydrophobicity and lower disorder but are considerably larger. due to size, during translation hydrophobic patches are exposed so are aggregation prone during folding
