Week 2 Flashcards
What do heat shock protiens do
Serve to protect cells from diverse phsyical and chemical insults
Who discovered heat shock response
Ferruccio Ritossa
What happened in the heat shock discovery
Inccaubator was turned up and chromatin puffs form heat induced activation of gene transcrption localized in that specific regions of the cchromosomes
What temperature are heat shock protiens triggered by
Just a few degrees
What are some examples of changes in cell morphology and organization
Remodelling cytoskelton,
fragmentation and disassemble of the golgi and ER, swelling of the nucleoli
What happens in the fibroblasts of a chinese hamster that is treated with hyperthermia
Disruption of the fine structure of the actin filaments but stabilization of the cell cortex (MORE ROUNDED)
What are the 7 classes of proteins for cells to cope with stress
Chaperons
DNA/RNA repair
Metabolic enzymes
Regulatory proteins
Proteolytic system
Transport and detoxification
Cell organization
What are chaperons
Specific proteins that interact with, stabilize or help other protiens to acquire its functionally active conformation without being present in its final structure
What do RNA and DNA enzymes
Necessary to repair DNA damage and processing failures that occur during stress
What are metabolic enzymes
Needed to reorganize and stabilize the energy supply of the cell
What are regulatory protiens
Transcrption factors or kinases needed to further initiate stress response pathways or to inhibit expression cascafes
What is the proteolytic stystem enzymes
Needed to clear misfolded and irreversibly aggregated protiens from the cell
What are cell organization proteins
Required in sustaining cellular structures such as the cytoskeleton
What are transport and detoxifying proteins for
needed to maintain or restore membrane stability and function
What can detect up-regulation of heat shock protein
Western blot technique
What else is important for protein folding pathway
Chaperone HSPS like HSP70
What is the oil drop model
Some proteins are capable of self-assembly into a single stable conformation hydrophobic inside hydrophilic outside
Where do chaperones bind
Hydrophobic patches to prevent misfolding
Do chaperons determine by the three dimensional state
NO amino acid sequences does chaperons just helps them get there
What are the five chaperone families
Hsp100
Hsp90
Hsp70
Hsp 60
sHsps
What chaperone family is energy independent
sHSPs
What are the two general families of chaperones
Molecular chaperones and Chaperonins
What are molecular chaperones
Proteins that bind and stablize unfolded proteins, and facilitate their folding through exposed peptide sequences of hydrophobic amino acids prevent them from aggregating
What are chaperonins
Ring shaped chaperons that form small folding chambers that encapsulate non native protiens in an ATP dependent manner
What is the structure of Hsp100
Two hexametric ring complexes with a channel
How does Hsp100 work
By actively pulling misfolded protiens through a central chanel using ATP which is then unfolded
What happens once the protien is relased from HSP100
it can refold properly
Is Hsp100 in humans
NO
What organisms encode HSP90
Everything except archea
What are the five subfamiles of HSP90
Cytosolic
ER
Chlorplast
Mitochondrial TNFR
Bacterial high temperature
What are the three domains of Hsp90
N-terminal ATP binding domain
Middle domain
C-terminal domain
What does Hsp90 always funciton as
Dimer
What is the process of ATPase of Hsp90
- Inactive substrates binds to the middle domain when the chaperone is in the open confirmation
- ATP then binds to the NTD conformational change and closes the lid
- After lid closure NTD dimerize and twists
- NTD dissociated anre the release of activated substrate (folded protien)
What is Hsp90 known as
A sensor of heat shock response
What does Hsp90 inhibit
HSF1 transcrptions factors which induces many stress sensitivite genes
What is happening under normal conditions for Hsp 90
HSF1 is bound to Hsp90 in the cytoplasama
What happens when the cell is stress in regards to Hsp90
accumulation of unfolded protiens occur which comepte for HSF1 but HSF1 binds more readily these monomers rapidly trimerize and transloacte to the nucleuse
What else is required for HSF-1
Postranslational modification
How does HSF1 upregulate transcrption of target genes
By binding the heat shock element present in the target gene promoters
What does induction of heat shock protiens do
Inhibits apoptosis and promotes cell survival
What is the DBD
DNA binding domain it is resposbinsle for binding HSF1 to the shock element N terminal domain
What does the trans activation domain do
C terminal domain required for binding the transcription factor to the pre-initiation complex
What makes up the preinitiation complex
RNA polymerase II and general transcption factors
What is the regulatory domain responsible for
Regulating HSF1 activity
How does the Regulatory domain regulate HSF1 in the absence of stress
RD restraians the HSF1 TAD negatvie regulation of the trans activating capacity of HSF1
What is HR
Specific domains called hydrophobic heptad repeat regions mediate oligomerization/trimerization through formation of three stranded coiled-coil structure
How is trimerization negatively regulated
Intermolecular interactions between the HR-A/B and HR-C domains under non-stress conditions
What are heat shock elements
Specific sequences in DNA found in genes
What is the pentameric sequence
Inverted of nGAAn
How many repeats serve as a binding site for HSF1 trimer
3
What is a prominent feature of HSF1 to convert it to a trimer
Extensive hyperphosphorylation of serine residues in the RD
Can posttranslational modification of HSF1 stimulatory or inihibitory
BOTH
What is sumolyation
Covalent attachment of small SUMO proteins similar to ubiquitin doesn’t direct proteins for degradation
What always represses HFS1 activity
Phosphorylation-dependent sumolyation that serves as a stress sensititve barrier that restains HSF1 activity upon moderate stress
What heat shock protien does not work is association
HSF3
What else is HSF1 responsible for
Immune responses and cancer
What are HSF1 and HSF2 known for
Developmental process, oogenesis, spermatogensis and corticogensis
What is HSF4 involved in
Development of different sensory orgrans
Exposure of cells to heat shock can be detected by what
Fluorescence microscopy
Where are nuclear stress bodies found
Located close to the nucleoli or nuclear envelope
What was used to detecet HSF1
monoclonal antibodies
Where are HSF1 and HSF2 found
Co-localized
What are Hsp70 and Hsp90 known as
Foldases
What are some properties of Hsp70
Highly conserved
Located in many organelles
Stress inducible
ATP dependent
Function as a monomer
Regulated by two co-chaperones
What are the two domains of Hsp 70
N terminal nucleotide binding domain
C terminal substrated binding domain (SBD)
What does the NBD bind too
ATP and hydrolyzes it to ADP
What is the SBD divided into
Beta-sheet base and an alpha helical lid
What is the lid consit of
5 helices A-E
What follows the SBDa
Unstructured region
What happens when Hsp70 is ATP bound
The SBD-a is open and peptides bind and release realtivitly rapidly there is a low affinity with the help of HSP40
What is BiP
Principal Hsp70 chaperone of ER stress in the ATP bound state
What happens with the hydrolysis of ATP in HSP 70
Accelerated by HSP40 closes the a-helical lid and a tight binding of substraate by HSP70 (high affinity) proper protien folding is facilitated
What happens once the protien is folded correctly by Hsp70
Dissociation of ADP and binding of ATP by NEF to have the open confirmation
What are chaperonins
Large double ring complex and form barrel shaped structures with two folding chambers
What are group 1 chaperonins
Have 7 subunits in each ring expressed in bacteria, mitochondria and chlorplasts and cooperate with Hsp10 protiens which form the lid of the cage
What are examples of Group 1 chaperonins
Hsp60 in eukaryotes and GroEL in bacteria
What are group II chaperonins
Expressed in archaea and the eukaryotic cytosol and have 8-9 subunits in each ring and no lid is required so independent of Hsp10
Can each 7 subunit bind ATP
YES
What are the four steps of chaperonin-mediated protien folding
- Mis-folded protein is captured by hydrophobic residues near the entrance of the upper folding chamber (the lower chamber is blocked by the lid)
- ATP binding then induces a conformational change of the chamber that triggers the GroEL lid to seal the upper chamber containing the encapsulated protien
- Protien folding in chamber due to ATP hydrolosis once it is hydroloyzed another ATP can bind and a new lid binds to the other chamber
- The binding causes the lid to dissociate from the upper chamber and release the folded protien
What is the highly conseverd domain in sHsp
ACD
What does the ACD structurally composed off
Beta sandwhich of two anti baraelle sheets of three or four beta strands
What else might ACD do
Mediate strong protein interactions and contribute to oligomerization of sHsps
Do sHsp domains contain ATP binding site
NO
What are the other domains of sHsps and are they important
NTR and CTR and yes for oligomerization and function
What are sHsps been orginally termed as
holdases as they bind and hold many unfolded proteins
What type of recations work with sHsp
Hydrophobic interactions
Is it a spontaneous recation of refolding non-native protiens in sHsp
NO you need the cooperation of ATP-dependent chaperones
What does the current model suggest
- sHsps populate at equilibrium a wide variety of inter-converting oligomers
- Under stress conditions substrate proteins are destabilized and being to unfold
- The sHsp ensemble becomes activated by remodelling the large ensemble in favor of smaller oligomers with more substrate binding sites
- Activates sHsps bind unfolded protiens in an energy-independent manner
- sHsp/substrate complexes are stabilized in different forms
- Bound substrates may recative spontaneously or are refloded by the ATP dependent Hsp70 chaperone system
What else can sHsps do
Matian the stability of actin filaments under stress conditions
The regulation depends of actin depends on what
phosphorylation of sHss which occuers under stress condtions
What happens to actin in unstressed cells
unphosphorylated sHsps monomers (HSBP1) cap the plus end so it doesn’t polymerizaie
What happens to actin in stressed cells
sHsps are phosphorylated lareg oligomers are dissocated and phosphorylate sHsps bind to the surface of actin filaments and protect them from depolymerization and aggregation
What chaperones are in E.coli
Hsp60 (GroE), Hsp70, sHsps and Hsp90 Hsp100
What chaperones are in S.cerevisiae
Hsp70 sHsps, Hsp100,HSP 90 Chaperonins are abundant under phsiological conditions but downregulated in heat stress
What is present in A.fulgidus
The chaperone system up-regulated upon heat shock consists only of sHsps and Hsp60
What is present in H.sapiens
Hsp70 Hsp90 sHsps are upregulated Hsp60 is not Hsp100
