PB Final

nascent chain

AA chain in process of being synthesized

RQC

ribosome quality control complex: made of four proteins (Ltn1, CDC48, Rqc1, Rqc2)

goal of paper 1.2

determine mechanisms by which proteins Ltn1p, Rqc1p, Rqc2p recognize and rescue stalled nascent chains in 60s ribosome

UPS

ubiquitin proteasome system, serves to tag and degrade unwanted proteins

E1 of UPS

activates ubiquitin

E2 of UPS

conjugates ubiquitin

E3 of UPS

E3 ligase; bidns ubiquitin to target protein

structure of proteasome

2 19 S regulatory particles, 1 20 S core

Ltn1

type of E3 ligase, ubiquitinates stalled nascent chains

CDC48

uses ATP hydrolysis to get the ubiquitinated nascent chains from 60S subunit to deliver to the proteasomes

CryoEM

used to probe structure of biological molecules using electron microscopy

Cryo-EM key characteristics

samples are not fixed or stained (closer to native state), kept at cryogenic temperatures to minimize electron beam damage, thousands of images can be taken and recombined to make 3D reconstructions

contact regions of Rqc2

tRNA is A and P sites, Ltn1

method to find out which tRNA Rqc2 recruits

1. purify Rqc complexes and bound tRNAs

2. Extract tRNAs from purified complexes

3. reverse transcribe to get complimentary DNA

4. plot tRNA frequency

amino acids most commonly recruited by Rqc2p

alanine, threonine

3 predictions made by paper 1.2

1. Rqc2p dependent increase in the molecular weight of the nascent chain should be from the C-terminus

2. C terminus extension should be only Ala and Thr residues, starting at the stall sequence

3. Ala and Thr should not have a defined sequence

R12

stall region of reporter

TEV

cleavage site in reporter

prokaryote and archaea form of Hsp70

DnaK

eukaryote form of Hsp70

Hsp70

prokaryote and archaea form of Hsp40

DnaJ

eukaryote form of Hsp40

Hsp40

nucleotide exchange factor GrpE

reactivates DnaK by exchanging ADP for ATP, causing substrate release

firefly luciferase

type of protein that glows when properly folded

GuHCl

denaturing agent used to denature firefly luciferase

EC₅₀

half maximal effective concentration

endoplasmic reticulum (ER)

network of membranes important for protein trafficking, entry point for secretory pathwa

y

ER proteins

Proteins destined for the membrane, cell surface, or secretion (stressful, highly oxidizing environments), need special modifications to stabilize them

Unfolded Protein Response (UPR)

accumulation of misfolded proteins in the ER that causes stress

ER-Associated Degradation (ERAD)

degrades misfolded ER proteins; Misfolded substrates recognized in ER, delivered to the cytosol for degradation by proteasomes

polyglutamine expansion diseases (aka trinucleotide expansion disorders)

neurodegenerative diseases caused by an repeated expansion of the CAG nucleotide in specific genes

number of PolyQ repeats to be normal, unaffected

<28

number of PolyQ repeats to be intermediate, unaffacted

28-35

number of PolyQ repeats to be reduced penetrance, ± affected

36-40

number of PolyQ repeats to be full penetrance, affected

>40

HD brain atrophy regions

striatum (movement), cerebral cortex (memory, attention,…)

Htt protein expression

expressed everywhere, but highly in neurons

Htt protein function

not fully understood – likely acts as a “scaffold” to bring other proteins together for various jobs

type of mutation of Htt

toxic gain-of-function (dominant inheritance, increased severity with length)

3 questions asked by paper 3.1

1. is there a common mechanism of toxicity?

2. what are the primary mechanisms vs secondary?

3. Is there an early and specific event in toxicity?

N-end rule pathway

degradation pathway that uses the N-terminus amino acid Arginine to signal for degradation by 26s proteasome

ubiquitin fusion degradation (UFD) pathway

fuses ubiquitin to protein to mark it for degradation

PC12 cells

immortalized cell line for studying nerve cell development and neurotoxicity

UPRE

UPR element: a DNA sequence in the promoters of genes that are switched on as part of UPR

tunicamycin (TM)

induces the UPR

striatal cells

the neurons and glial cells that make up the striatum

Thapsigargin (TG)

drug used to induce ER stress

P97

mammalian homolog of yeast CDC48

P47

mammalian homolog of yeast Shp1

trapped complex from paper 3.1-predictions made by model

Restoring function of the p97/Npl4/Ufd1 ERAD complex should rescue toxicity

Preventing misfolded proteins from accumulating should rescue toxicity

central dogma of molecular biology

genetic information flows in one direction: DNA to RNA, and then proteins

prion

self-propagating, transmissible protein conformation

mammalian prion origin

Misfolding of PrP

fungal prion protein origin

several- not PrP though

PrP^C

normal form of prion protein, membrane associated, made of alpha helicies

PrP^Sc

misfolded form of prion protein, aggregates into amyloid fibers, made of beta sheets

three of ways of getting prions

acquired, familial, specific

Infectious prion

acquired from environment (e.g. mad cow disease)

familial prion

certain mutations in the gene for PrP increase the liklihood of PrP^C misfolding to form PrP^Sc

sporadic prion

PrP^C spontaneously misfolds to form PrP^SC

scrapie

prion disease of sheep and goats

Bovine Spongiform Encephalopathy (Mad Cow Disease)

prion disease in cows

variant Creutzfeldt-Jakob disease (vCJD)

prion disease in humans caused by eating beef infected with prions

Creutzfeldt-Jakob Disease (CJD)

most common human prion disease, caused usually by sporadic misfolding

PMCA

Protein Misfolding Cyclic Amplification

Main question from paper 3.2

Can PMCA be used to generate infectious prions in vitro?

astrogliosis

response of astrocytes to central nervous system (CNS) damage

loss of function mutation

protein stops functioning as normal due to mutation- aggregates or is degraded

cystic fibrosis

inherited chronic disease- affects lungs and digestive system and produces thick mucus which can obstruct pancreas

symptoms of CF

salty skin, persistent coughing and lung infections, wheezing and shortness of breath, failure to thrive

cause of CF

lack of functional form of CF transmembrane conductance regulator protein (CFTR)

inheritance pattern of CF

recessive

function of CFTR protein

acts as chloride ion channel- allows Cl^- to move across the cell membrane

regulatory mechanisms of CFTR

phosphorylation of regulatory domain by PKA opens channel

ATP binds to nucleotide binding domain to open channel

ATP hydrolysis closes channel

most common CF-causing mutation

F508del- causes CFTR to misfold in ER and be degraded by ERAD

adenovirus

vector used to introduce foreign DNA into cells; does not integrate into genome and requires readministration, as well as strong immune response

adeno-associated virus (AAV)

vector used to introduce foreign DNA into cells; integrates into genome and does not elicit strong immune response, though it can be very inefficient

CFTR potentiator

increases activity of defective CFTR at the cell surface (fixing channel gating)

CFTR correctors

overcome defective protein processing, minimize misfoling and increase trafficking to plasma membrane

goal of paper 3.3

discover CFTR correctors for F508del-CFTR that could advance to clinical studies

Fluorescence Resonance Energy Transfer (FRET)

cell culture assay technique- creates FRET pairs so the emission from one donor dye to an acceptor dye will excite the acceptor if they’re close together

VRT-768

identified drug from HTPS

VX-809

more portent derivative of VRT-768

Ussing chamber

measured Cl^- ion transport between epithelial cells by holding the current at 0 by applying an opposing current- the amount of current requires is the net ion movement across the epithelial layer

pulse-chase experiment

used to measure conversion of immature CFTR to mature CFTR; add radiolabeled AA (pulse), allow synthesis of proteins for defined period of time, add unlabeled AA (chase)

brefelden A (BFA)

drug that causes protein to be retained in ER

patch clamp

measures ion transport across membranes; detects opne/closed states and the fraction of time the ion channel spends open as “open probability” of P_O

name of VX-809 drug

Lumacaftor

name of VX-770 drug

Ivacaftor

VX-770

a potentiator

name of drug that is combination of lumacaftor and ivacaftor

orkambi

patients treated with Orkambi

patients homozygous for F508del-CFTR mutation

TRIKAFTA

triple combination therapy of elexacaftor/tezacaftor/ivacaftor

patients treated by TRIKAFTA

patients heterozygous for F508del allele

function and size of ribosomal small subunit

binds mRNA, 30S or 40S

function and size of ribosomal large subunit (aka peptidyl transferase)

catalyzes peptide bond formation, 50S or 60S

two sizes of ribosomes

70S or 80S

a-site

arrival site, where the AA comes in

P-site

where the peptide bond currently is- peptidyl-tRNA site

E-site

exit site