Week 1-3

Question 1

List all the stressor groups and what belongs in them

Answer 1

Environmental stress:
Heat shock
Oxidants
Hypoxia
Osmotic insult
Heavy metals
Mechanical stress

Physiological stress: cell life cycle
Cell cycle
Development
Differentiation
Growth factors
exercise

Pathological Stress:
Cancer
Diabetes
Fever
Infection
Inflammation
Ischemia
Myocardial infarction
Parkinson’s

Question 2

What’re the types of cell responses in order to maintain homeostasis, can they coexist

Answer 2

1. Stress-specific signaling pathways (cell survival if successful)
2. Common(non stress-specific) cell responses (cell death):
   a)Global reduction of protein translation
   b) Chaperons
   c) O=GlcNAc
   d) Cytoskeleton and more

Question 3

components of adaptive stress response pathway

Answer 3

1. Transcription Factors
   - Under normal conditions the TF is negatively regulated through degradation and capture/store thru sensor interactions
2. Enzymatic Transducers
   - When STRESSED, transducers (kinase, hydroxylases etc) are triggered and covalently modify sensors/TF
3. Cytoprotective Genes
   - Modification activates nuclear translocation of TF = enables cytoprotective genes (overcomes stress)

summary:
normal needs TF to be degraded by interactions with SENSORS

stress needs TRANSDUCERS to modify SENSORS = TF goes into nucleus and starts up cytoprotective genes that overcomes stress or apoptosis

Question 4

What were the KO results in transgenic mice

Answer 4

• Loss of TF vary from impairments to lethality
• Loss of sensors is always Lethal
• Loss of both can rescue embryonic lethal phenotype, in these pathways:
  1) oxidative stress
  2)DNA damage

Question 5

Explain Global Reduction in Protein Synthesis Under Stress

Answer 5

a) Normally eIF2-GTP delivers initiator Met-tRNA binds with small ribosome 40S

b) but Stress-induced phosphorylation of eIF2 causes
- global reduction in normal translation but - - increases translation of ATF, a TF that makes stress-sensitive genes

Question 6

How many overlapping reading frames are possible at once, what’s an example of one

Answer 6

3 are possible

the AUG start codon of ATF4 is not recognized during translation of uORF2

Question 7

How does translation of ATF increase when global reduction is suppose to hinder overall translation

what’re the steps

Answer 7

Delayed Translation Reinitiation and uORFs in the mRNA seq of stress sensitive genes (ATF)
(Train Theory - ribosome is the train)

1. uORFs are blockers that stop ribosomes from getting to the protein that needs translating (ATF), ribosome usually translates uORF1, picks up an elf2-GTP then translate uORF2 which overlaps the coding seq (CDS) of the protein we want translated (no ATF)
2. when stressed there’s no elf2-GTP cause of the phosphorylated elf2-P meaning that after ribosome translates uORF1, elf2-GTP pickup is DELAYED and misses the uORF2 stop. no uORF2 means no overlap with the ATF CDS = it gets translated

summary: low lvls of elf2-GTP caused by stress-induced phosphorylation delays ribosomes after translating uORF1 and allows translating to start at the ATF4 CDS

Question 8

What is the Global Heat-shock-induced changes in cells, how does the cell try and fix these problems

Answer 8

Heat shock o f3-5 degrees leads to structure damage (unfolded or aggregated proteins)

this could lead to one of the following:
1. morphological changes (cytoskeleton)
2. transcriptional up-regulation of HSPs (Chaperons)
3. Stop transcription/translation to conserve energy

Question 9

property and function of HSP100

Answer 9

ATP dependent

Hexameric pore ring that using ATP to pull misfolded proteins thru and untangles it (dissolves aggregates)

good in yeast/bacteria, none seem in humans

Question 10

property and function(give steps) of HSP90

Answer 10

• ATP dependent scissors FOLDASSES
• Found in high concentrations in almost all cell compartments (exp archaea)
• acts as a heat shock response sensor, inhibiting TFs Called HSF1 that can induce stress-sensitive genes that encode chaperones

three domains
1. (N-term ATP binding domain)
2. middle domain
3. C-term dimerization domain
all parts can interact with clients (unfolded/native forms)

ATPase cycle of HSP90 Chaperone system
1. unfolded protein binds to MD when in open conformation

2. ATP binds to n-term and conformation change to close lid
3. after lid closes, n-term dimerizes (molecular clamp) with TWISTED subunits making the HSP90 closed dimer
4. This mestabtable conformation goes thru ATP hydrolysis which leads to n-term dissociation and release of the folded protein

Question 11

What’re the HSP substrates (Client proteins): How do they work

Answer 11

Signal transducers like TF/kinases

HSF1 (heat shock factor):
induces expression of stress-sensitive genes encoding chaperons

norm: inactive state is bound to HSP90 @cytoplasm

Stressed:
1. dissociation of HSF1/HSP90 complex (too many unfolded proteins want HSP90) causes it to trimerize and translocate to nucleus

2. post translational modification like phosphorylation or binding to HSEs (in promoter region of genes like HSP70/HSP27 which make HSPS)

Question 12

property and function(steps) of HSP70 chaperone cycle

Answer 12

ATP dependent and crucial for maintaining homeostasis FOLDASSES

regulated by co-chaperones/co-factors (HSP40 and NEF)
BiP is a Hsp70 chaperone of ER stress in ATP-bound state

a) N-term Nucleotide Binding domain (hydrolyses ATP)

B) c-term Substrate binding domain (divided into beta sheets followed by alpha-helical (A-E) lid)

c) Unstructed region

1. Low Affinity:
   - ATP-bound with help of HSP40 is open lid and loosely binds peptides (bind/release rapidly)
2. High Affinity:
   Hsp40 helps get it to ADP-bound = closed and tightly binds peptides (highest affinity to substrate binding)
3. Release: NEF helps replace ADP with a fresh ATP to get it to open and release

Question 13

property and function of HSP60 and the steps of the GroEL-GroES folding cycle

Answer 13

ATP dependent double barrel folding chamber
Group I Chaperonins in mitochondria of eukaryotes

HSP60 (GroEL in bacteria): is chambers
- Consist of 7 subunits per ring.

• Hsp10 (or GroES in bacteria) is lid

GroEL-GroES cycle steps:
1. GroES lid blocks bottom chamber so unfolded protein enter thru top GroEL chamber

2. ATP binding causes conformational change sealing protein inside (GroES lid closes off top chamber too)
3. ATP hydrolysis inside slow folds protein (~10 sec). While another ATP activating the lower chamber.
4. lid comes off upper chamber, releasing the folded protein. Misfolded proteins can re-enter

Question 14

Property and function of Small HSPs - how does it protect actin filaments

Answer 14

only one not that is energy independent (NO ATP)

alpha-crystallin domain(ACD), a compact beta-sandwich with anti-parallel sheets of 3-4 strands

Normal: regulates actin by capping it

stressed:

Holds onto misfolded proteins until other HSP70 etc can fix it by

forming monomers, dimers, and large complexes that when stressed(phsophorylated) becomes active and break down into smaller forms to bind actin filaments preventing their breakdown (Actin)

Question 15

Damaging effects of heat shock

Answer 15

1. cytoskeletal reorganization (actin filaments -> stress fibres, and aggregation of others like microtubuli)
2. fragments Golgi and ER
3. decreases quantity and quality of mitochondria/lysosomes
4. swelling of nucleoli
5. protein aggregates in cytosol
6. increased membrane fluidity

all this causes cell cycle arrest

Question 16

What’re the classes of Heat Shock Proteins

Answer 16

1. Chaperones
   helps other proteins become active w/out being in the final form
2. DNA/RNA modifying enzymes
   (repairs errors during stress)
3. Metabolic enzymes
   Reorganize and stabilize the energy supply

4.Regulatory proteins (TFs, kinases)
Promote more stress response pathways or inhibit expression cascade (ribosome assembly pathways)

4. Proteolytic system (degradation system)
5. Transport and detoxification
   maintain/restore membrane stability
6. Cell organization (cytoskeleton)

Question 17

Explain kinetics of Protein up-regulation to heat stress

Answer 17

Log2 is common as double is common, HSP quantities grow rapidly at peaks of up to an hr and expression decreases when problems are fixed

to detect HSP expression changes, you must consider time-dependent patterns

Question 18

How do normal proteins fold? what’s wrong with it? How is it managed?

Answer 18

hydrophilic/polar side are on the outside

but with typical protein concentration being 300mg/ml, newly folded proteins may expose the hydrophobic side which risks misfolding/aggregation

chaperones bind to hydrophobic patches and guide them on favourable paths (GUIDES not determine how it folds)

Question 19

Type of Chaperone families

Answer 19

molecular chaperones bind and stabilize exposed patches and stop them from aggregating

chaperoning are ring-shaped chambers and folds them properly

Question 20

What’s the relationship between Chaperones, HSP, HSE and HSF

Answer 20

HSPs are types of chaperones (molecular or chaperonins)

HSFs bind to HSEs in promoter regions of HSP70/27 which makes HSPs

Question 21

Go into detail about how Post-Translational modifications of HSF-1 Happens and give all benefits

Answer 21

1. shit tone of serines get Hyper-phosphorylated in RD + more acetylation and phosphorylation in other regions
2. Sumoylation is: during mild stress, phosphorylation dependent SUMO represses HSF1 activation (sumo and ubiquitin are the same but sumo doesn’t send to degradation, just represses HSF1)

pros:

inhibitory and stimulatory
inhibits apoptosis (stopping cytochrome C release the trigger, apoptosome formation the machinery start button, and caspase activity the machine operator)

and promotes cell survival (protein refold, increasing GSH the protection, modulating actin and aKT activity)

immune responses (Hsp1 cancer regulation, HSP 2/3 is developmental processes oogenesis/spermatogenesis regulation, hsp4 sensory organs)

Question 22

What role does HSF1, HSF2, HSF 3, and HSF4 play in cellular response

Answer 22

HSF1: Main regulator of the heat shock response, and immune responses.

HSF2: Works with HSF1 in stress response and development.

HSF3: Functions independently from other HSFs.

HSF4: Involved in the development of sensory organs, not in heat shock response.

Question 23

What’re nuclear stress bodies and when do they form

Answer 23

When cells are exposed to heat shock, HSF1 and HSF2 gather in nucleoili or nuclear envelope

They are transient and visible under fluorescence microscopy and co-localize to help regulate the stress response

Question 24

What’re the key domains of HSF1 and their roles

Answer 24

DNA-binding domain (DBD): N-terminal (~100 amino acids), binds to heat shock elements (HSEs) in target gene promoters after HSF1 trimerizes.

Trans-activation domain (TAD): C-terminal, binds to the preinitiation complex to initiate transcription.

Regulatory domain (RD): Controls HSF1 activity, suppresses HSF1 in non-stress conditions, and is regulated by post-translational modifications.

Question 25

What is a group II chaperonin, why is it different than HSP60 which is a Group I

Answer 25

Group II Chaperonins (TRiC/CCT in eukaryotic cytosol, thermosome in archaea):
Have 8-9 subunits per ring.
Do not require Hsp10 as a lid, they are self-sufficient.
GroEL-GroES Folding Cycle (E. coli):

Question 26

How do chaperone networks differ across organisms during heat shock?

Answer 26

E. coli (Bacteria): All classical chaperones are activated under stress.

(Yeast): relies primarily on Hsp100, 70, and sHsps, and a limited subset of Hsp90 co-chaperones.
Hsp60 (CCT/TriC) is downregulated during heat stress.

A. fulgidus (Archaea): only of sHsps and Hsp60 (CCT/thermosome), both present in high concentrations during stress.

H. sapiens (Humans):

Hsp70, Hsp90, and sHsps are upregulated by heat shock, while Hsp60 (CCT/TriC) is not. Humans lack Hsp100 (ClpB) homologs.