HSPs heat shock proteins HSPs chaperones.ppt

heat shock proteins

1. Heat shock Proteins (HSPs)

Heat shock proteins (HSP) are expressed in
response to various biological stresses,
including heat, high pressures, and toxic
compounds. It is also one of the most
abundant cellular proteins found under non-
stress conditions

Hsp90 is part of a family of proteins known as
"chaperones," which are solely dedicated to
helping other proteins fold and assume their
proper functions.

The chaperones Hsp70 and Hsp90 together
with co-chaperones function to fold proteins in
the cytoplasm. Sometimes Hsp70 and Hsp90
function sequentially to fold the same protein

Cells are vigilant about getting these folds
right because misfolded proteins can change
the normal life of the cell. In some cases
change is good, in others deadly.

When HSP90 is compromised the number of
morphological changes increases, which lead
to formation of inactive or abnormally active
polypeptides.
Domain structure of HSP90.

2. Mad Cows, People & yeast
What do "mad cows," people with
neurodegenerative diseases and an unusual
type of yeast have in common? They are all
experiencing the effects of misfolded proteins,
according to HHMI investigator Susan
Lindquist of the University of Chicago.
Her research identified a role for HSP90 in
the process of evolution.
They have reported that fruit flies that make
too little of the Hsp90 protein develop
dramatic deformities, such as an extra
antenna, additional bristles, notched wings or
malformed eyes.
The defects result from multiple hidden
variations in the genome.
When affected flies are interbred, these
factors are enriched and subsequent
generations have the same deformities, even
though they have normal levels of Hsp90.
Susan Lindquist
University of Chicago
"My view is that
molecular chaperones
are a way of changing
the traits of an
organism that arose
very early in evolution.
They might be as old as
RNA and DNA."
 Source:http://www.hhmi.org/annual98/research/madcow.html

3. HSPs in protein folding

The diagram shows the role of heat-
shock proteins and a chaperonin in
protein folding.  As the ribosome moves
along the molecule of messenger RNA,
a chain of amino acids is built up to
form a new protein molecule.  The
chain is protected against unwanted
interactions with other cytoplasmic
molecules by heat-shock proteins and a
chaperonin molecule until it has
successfully completed its folding.  
Source: (http://www.cs.stedwards.edu/chem/Chemistry/CHEM43/CHEM43/HSP/FUNCTION.HTML)

4. HSP90-alpha protein sequence

HSP90alpha 732 aa
  HSP90beta 724 aa
 

>HSP90alpha
MPEETQTQDQPMEEEEVETFA FQAEIAQLMSLIINTFYSNKEIFLRELISNSSDALDKIRYESLTDPSKLDSGKELHINLIPNKQDRTLTIVDTGIGMTKADLINNLGTIAKSGTKA FM
EALQAGADISMIGQFGVGFYSAYLV AEKVTVITKHNDDEQYAWESSAGGSFTVRTDTGEPMGRGTKVILHLKEDQTEYLEERRIKEIVKKHSQFIGYPITLFVEKERDKEVSDDE
AEEKEDKEEEKEKEEKESEDKPEIEDVGSDEEEEKKDGDKKKKKKI KEKYIDQEELNKTKPIWTRNPDDITNEEYGEFYKSLTNDWEDHLAVKHFSVEGQLEFRALLFVPRRAPF
DLFENRKKKNNIKLYVRRVFIMDNCEELIPEYLNFIRGVVDSEDLPLNISREMLQQSKILKVIRKNLVKKCLELFTELAEDKENYKKFYEQFSKNIKLGIHEDSQNRKKLSELLRYYTS
ASGDEMVSLKDYCTRMKENQKHIYYITGETKDQVANSAFVERLRKHGLEVIYMIEPIDEYCVQQLKEFEGKTLVSVTKEGLELPEDEEEKKKQEEKKTKFENLCKIMKDILEKKVE
KVVVSNRLVTSPCCIVTSTYGWTANMERIMKAQALRDNSTMGYMAAKKHLEINPDHSIIETLRQKAEADKNDKSVKDLVILLYETALLSSGFSLEDPQTHANRIYRMIKLGLGIDED
DPTADDTSAAVTEEMPPLEGDDDTSRMEEVD
Green bases = Geldanamycin-Binding Domain

Brown bases = Transmembrane segments as
predicted by Tmap.
Hydrophobic: A,C, I,L, M, F, V,P, Y,W
Hydrophilic: R,N, H,D, E,Q, K, S,T Either G
GREASE output
TMAP output

5. BLASTp

With the HSP90 sequence in hand we used Blastp to find homologous sequences

We were surprised to find a lot of homologous sequences across many species like
Humans, Chicken,Pig, Mouse,Horse,Fish, Coral,fruit fly, mosquito, nematode,& even
crops like rice, maize & tobacco.

The first 100 matches had e-values ranging from 0 to e-153, so they were *very*
strong matches indicating a high degree of conservation of the protein through
evolution.
ID Name Score Evalue
304882 heat shock 90kDa protein 1, alpha [Homo sapiens] N... 1247 0.0
352285 heat shock protein 1, alpha [Mus musculus] NP_0346... 825 0.0
761972 heat shock protein 86 [Rattus norvegicus] NP_78693... 825 0.0
341493 heat shock protein 90A [Cricetulus griseus] AAA369... 817 0.0
609431 heat shock protein 90 - chicken 816 0.0
609432 heat shock protein 84 - mouse 745 0.0
449511 (Q9W6K6) Heat shock protein hsp90 beta [Salmo sala... 731 0.0
459017 heat shock protein hsp90 [Oncorhynchus tshawytscha... 730 0.0
446434 heat shock protein hsp90beta [Danio rerio] AAC2156... 729 0.0
361999 heat shock protein 90 [Rattus sp.] AAB23369.1 [S45... 724 0.0
460597 heat shock protein 90 [Pleurodeles waltl] AAA92343... 719 0.0
738604 90-kDa heat shock protein [Bombyx mori] BAB41209.1... 712 0.0
146263 Heat shock protein 83 CG1242-PA [Drosophila melano... 669 0.0
755572 heat shock protein 90 [Dendronephthya klunzingeri]... 662 0.0
226533 (P33126) Heat shock protein 82 [Oryza sativa (Rice)] 612 e-174
1888761 heat shock protein 82 - common tobacco (fragment) 612 e-174
252633 heat shock protein [Arabidopsis thaliana] CAA72513... 600 e-170
236351 (Q9XGF1) HSP80-2 [Triticum aestivum (Wheat)] 598 e-169
283559 (Q08277) Heat shock protein 82 [Zea mays (Maize)] 593 e-168
152674 heat shock protein 86 [Plasmodium falciparum] AAA6... 591 e-167
1899880 (Q8LLI6) Heat shock protein Hsp90 [Achlya ambisex... 579 e-164
245912 heat shock protein 90 [Lycopersicon esculentum] AA... 544 e-153

6. Multiple sequence alignment

Multiple
sequence
alignments
were done
using ClustalW
using different
species & the
following
unrooted
phylogenetic
tree was
generated.

7. 
Rooted
phylogenetic
tree.

8. Structure analysis
Comparison between Open & Closed conformations of
human HSP90 alpha
Transmembrane segments in yellow
We found ‘open’ and
‘closed’ conformations
for the Geldanamycin-
Binding Domain of the
Human Hsp90 protein &
decided to study their
differences. The parts in
yellow are the selected
residues which are also
the transmembrane
segments. The residues
‘gtia’ in the sequence
viewer show where the
structures differ. The
same region is depicted
as the little grey segment
at one end of the
transmembrane
segments

9. Comparison of HSP90 structures in Yeast & Human
We compared the HSP90 structures in yeast & human & found that the protein structures were very similar. The
picture below shows the 2 structures superimposed with the highlighted portion showing an additional residue in the
yeast sequence.
Structures compared:
 1YES Human Hsp90 Geldanamycin-Binding Domain, "open" Conformation [mmdbId:7483]
1AMW : Atp Binding Site In The Hsp90 Molecular Chaperone,Saccharomyces cerevisiae
 (yeast)
[mmdbId:7950]
Red : identical residues
Blue : similar residues
Yellow: selected residues

10. Microarray Data
We found microarray data on the Cancer Genome Anatomy Project web site

http://cgap.nci.nih.gov/Genes/GeneFinder
HSP90-alpha
This screen shot
shows the
microarray data
for HSP90 alpha
expression in
various types of
cancers.

11. HSP90-beta
This screen shot
shows the
microarray data for
HSP90 beta
expression in
various types of
cancers.

12. Current Studies on HSP90
Changes in protein conformation are involved in some of the most
devastating and intractable diseases.
“Studies in yeast may help us decipher the fundamental nature of these
disorders, including Creutzfeldt-Jakob, Alzheimer’s, Huntington’s, and
Parkinson’s disease in humans and mad cow disease in cattle. Several of the
protein culprits are being imported into yeast, which allows for the
manipulation & study of their folding transitions & testing of therapeutic
strategies.
(http://www.wi.mit.edu/nap/pdfs/Directors_Report/dir_lindquist02.pdf) 
Conclusion:
 HSP90 is a powerful evolutionary mechanism that ensures apparent genetic
stability at physiological conditions and at the same time allows the mutations that
could rapidly become manifest under stress.     
References:
http://www.stanford.edu/class/gene211/hsp90_search
http://www.chemie.tu-muenchen.de/biotech/en/hsp90-e.html
http://www.hhmi.org/annual98/research/madcow.html
www.ashland.edu/~kstine/Research/Stress%20proteins.pdf