NDD 1 - protein folding and prions Flashcards
What was Anfinsen’s dogma (1950s)?
used urea to disrupt the non-covalent bonds in ribonuclease enzyme, and β-mercaptoethanol (a reducing agent) to disrupt the disulfide bonds denatured the enzyme. Then removed the urea & β-mercaptoethanol enzyme was functioning again; the polypeptide chain had refolded - basically said that the most important thing is the primary of the polypeptide chain Concluded in 1972 - Native confirmation of a protien occurs bc this particular shape is thermodynamically most stable in the intracellular environment
Levinthal 1969
Levinthal’s paradox In 1969, Cyrus Levinthal noted that, because of the very large number of degrees of freedom in an unfolded pp chain, the molecule has an astronomical number of possible conformations. If you consider an 100 AA peptide chain which has two angles per aa bond, and each angle has three different conformations - this gives you 3^100 possible folding conformations. Levinthal said it would take you 10^27 years to try all of these conformations. Every life system in practice does this in a matter of ms. In 1969 proposed that protein folds so rapidly bc its constituent aas interact locally, thus limiting the conformational space the protein has to explore and forcing the protein to follow a funnel-like energy landscape that allows it to fold into the most stable configuration
Jumper 2021
Released the entirety of the source code - will likely form a fundamental part of computational biology. The paper explains the AI system and how it was created.
Kakade 2022
- PINK1 mutation is second most frequent cause of EOPD
- Had previously been shown that PD-causing mutations were in the kinase domain
- Question was about mechanisms of mutations lying outside the kinase domain
- They used AlphaFold2 for PINK1 – saw an interaction between a N-terminal a-helix extension and C-terminal a-helix extension
- Confirmed using mutagenesis that mutations within this interface lead to disrupted PINK1 stabilisation and activation in response to mitochondrial damage
- Looked at fibroblasts from one pt w EOPD due to homozygous PINK mutations in this region – also had defective PINK1 stabilisation
Limitation - the last bit of evidence is only correlative – direct causality is not shown and this could a mutation secondary to disease development - would be good to induce this mutation in an animal model and see phenotypic effects
Auluck 2002 science
Demonstrated the importance of protein chapersones. They directed HUMAN ASYN (a-synuclein) overexpression in Drosophila –> progressive loss of dopaminergic (DA) neurons and DA neurons stained positive for alpha-synuclein (remarkably similar to the Lewy-bodies found in PD). So drosophila ‘get PD’ - get ~50% loss of DA neurons by day 20. If you co-express HSP70 with ASYN, you don’t lose the DA neurons and you have the same number of neurons as at day 1. Therefore was able to rescue phenotype in drosophila. Before this, we didnt understand the importance of chaperones. Caveats - When looked at neurons, protection of the DA neurons was completely independent of Lewy body pathology or distribution of HSP70 - doesn’t really make sense - we don’t have a good explanation Explanations - Chaperones detoxify the aggregates (don’t prevent their formation but do prevent their toxicity), if you deplete chaperone levels in a cell the cell becomes much more vulnerable to external stressors - maybe when youre upregulating the chaperone = you have more of a reserve so when all of the baddness with a-syn still happens youve boosted chaperone levels a bit (more robust to being hit with other environmental things)
Cooley et al 2014
The did an in vitro cellular model of peripheral amyloidosis. They upregulated UPR (unfolded protein response) using UPR-associated transcripton factors (XBP1 and ATF6). By driving UPR, increase of chaperone levels. When they did they, they found reduced secretion of amyloid clumps of protein (they found reduced extracellular aggregation ofamyloidogenic Ig light chain into soluble aggregates) Caveats - They used a peripheral amyloidosis disease (might not be the same for AD) and this was in vitro (big jump to human brain)
raju et al 018
Mini-chaperone is a peptide derived from αA-crystallin,, and functions like a molecular chaperone similar to full-length αA-crystallin. This group generated a synthetic minichaperone that was able to cross the cell membrane and was found to be able to prevent beta-amyloid fibril formation and suppress B-amyloid toxicity in vitro
Lee et al 2010
“Found 1st drug-like molecule that we could find that could activate proteasome function. The group found an enzyme that can inhibit the degradation of ubiquitin-protein conjugates - Ups14 Did a high-throughput screen with over 60,000 compounds that could inhibit USP14 – found 1. Applied this USP14 inhibitor in in vitro cell model (MEFs expressing tau) enhanced degradation of several proteasome substrates that have been implicated in neurodegenerative disease, accelerates proteolysis and enhanced resistance to oxidative stress”
Spilman et al 2010
Show that long-term inhibition of mTOR by rapamycin prevented AD-like cognitive deficits and lowered levels of Abeta42, a major toxic species in AD in the PDAPP transgenic mouse model. Further found that autophagy was increased in the neurons of rapamycin-treated transgenic, but not in non-transgenic, PDAPP mice (expected as non-transgenic dont have AD).
Does this mean we could give it prophylactically? Probably not bc of immunosuppressive effect
Stanley Prusiner 1982
Coined the term prion. Was invetsigating TSE. In 1982 he and his team produced a preparation from a hamster brain that contained an infectous agents comprise only of a single protein.Prusiner had anticipated finding that the purified scrapie agent would be a viru (however his prepaprtions contained protein but no nucleic acid) fundamental paper establishing the β-pleated sheets. The group eventually established using infrared spectroscopy that when prions had formed the PrPSC type, it had a high content of β-pleated sheets, when PrPC were helical He won the Nobel Prize in Physiology or Medicine in 1997 for his discovery of prions Has been much criticism - Injected brain tissue homogenates into experimental animals and when neurological symptoms appear they say they had transmitted BSE - some say they were producing experimental allergic encephalomeylitis
Gajdusek et al 1966
Innoculated material from the brains of infected pts followed by serial transfer to the brains of other primates. Demonstrated the virus had an unusually long incubation period of several years. This was the first example of the infectious spread of a noninflammatory degenerative disease in humans.Once he had connected the practice of funerary cannabalism to Kuru, cannabalism was eliminated and kuru disappeared within a generation. Prusiner’s work led to the identification of prions causing these diseases.
Büeler et al 1993
They generated Prn-p0/0 mice that lacked PrPc. When inoculated with mouse scrapie prions, these mice remained free of scrapie symptoms for at least 13 months, while WT controls all died within 6 months. . This shwoed that PrPc is required for usual susceptibility to scrapie. This held promise as it suggested it would be possible to breed sheep and cattle resistant to the disease.
Mallucci et al 2007
PrP gene in Cre/LoxP transgenic mice flanked by 2 LoxP sites; inoculated with prions (bad protein) into brain (they were expressing human PrP anyway) mouse version of CJD. o 9 weeks after prion infection, they caused Cre-mediated PrP deletion (floxed the Cre-mediated PrP site with viral vector to decrease any expression of PrP) o These mice recovered the burrowing behaviour that was initially lost – i.e. they recovered o But control mice got progressively worse + died
Castilla et al 2005
PMCA – protein misfolding cyclic amplification – amplify (PCR) until you by chance misfold a protein. Did this with PrP to produce PrPSC (infectious [scrapie] version of PrP; NB PrPC = normal) in vitro. Inoculated wild-type hamsters with PrPSC – all hamsters suffered from Scapie disease ~170 days post-inoculation. o Caveats: used whole-brain homogenates from healthy hamsters as a source for this cycling process, rather than just taking proteins + cycling this. So anything could be in it.
Minikel et al 2016
Analysed 16,000 prion disease cases, 60, 000 control exomes and genomwide sequencing data from ~530,000 people. It was found that missense variants in PRNP previously reported to be pathogenic are at least 30 times more comon in the population than expected on the bases of genetic prion disease prevalence. While several variants of may in fact represent benign or low-risk variants, these data suggest the existence of non-genetic factors that affect disease manifestation. It is also possible that some healthy subjects with PRNP mutations have developed mechanisms that protect them from developing disease (e.g. PrPsc-specific antibdoies that shield them against the pathogenic effects of PRNP mutations)
