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.

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 LindquistUniversity 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

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)

HSP90-alpha protein sequence

HSP90alpha 732 aa  HSP90beta 724 aa  

>HSP90alphaMPEETQTQDQPMEEEEVETFAFQAEIAQLMSLIINTFYSNKEIFLRELISNSSDALDKIRYESLTDPSKLDSGKELHINLIPNKQDRTLTIVDTGIGMTKADLINNLGTIAKSGTKAFMEALQAGADISMIGQFGVGFYSAYLVAEKVTVITKHNDDEQYAWESSAGGSFTVRTDTGEPMGRGTKVILHLKEDQTEYLEERRIKEIVKKHSQFIGYPITLFVEKERDKEVSDDEAEEKEDKEEEKEKEEKESEDKPEIEDVGSDEEEEKKDGDKKKKKKIKEKYIDQEELNKTKPIWTRNPDDITNEEYGEFYKSLTNDWEDHLAVKHFSVEGQLEFRALLFVPRRAPFDLFENRKKKNNIKLYVRRVFIMDNCEELIPEYLNFIRGVVDSEDLPLNISREMLQQSKILKVIRKNLVKKCLELFTELAEDKENYKKFYEQFSKNIKLGIHEDSQNRKKLSELLRYYTSASGDEMVSLKDYCTRMKENQKHIYYITGETKDQVANSAFVERLRKHGLEVIYMIEPIDEYCVQQLKEFEGKTLVSVTKEGLELPEDEEEKKKQEEKKTKFENLCKIMKDILEKKVEKVVVSNRLVTSPCCIVTSTYGWTANMERIMKAQALRDNSTMGYMAAKKHLEINPDHSIIETLRQKAEADKNDKSVKDLVILLYETALLSSGFSLEDPQTHANRIYRMIKLGLGIDEDDPTADDTSAAVTEEMPPLEGDDDTSRMEEVD 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

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... 12470.0

352285 heat shock protein 1, alpha [Mus musculus] NP_0346... 8250.0

761972 heat shock protein 86 [Rattus norvegicus] NP_78693... 8250.0

341493 heat shock protein 90A [Cricetulus griseus] AAA369... 8170.0

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... 7310.0

459017 heat shock protein hsp90 [Oncorhynchus tshawytscha... 7300.0

446434 heat shock protein hsp90beta [Danio rerio] AAC2156... 7290.0

361999 heat shock protein 90 [Rattus sp.] AAB23369.1 [S45... 7240.0

460597 heat shock protein 90 [Pleurodeles waltl] AAA92343... 7190.0

738604 90-kDa heat shock protein [Bombyx mori] BAB41209.1... 7120.0

146263 Heat shock protein 83 CG1242-PA [Drosophila melano... 6690.0

755572 heat shock protein 90 [Dendronephthya klunzingeri]... 6620.0

226533 (P33126) Heat shock protein 82 [Oryza sativa (Rice)] 612e-174

1888761 heat shock protein 82 - common tobacco (fragment) 612 e-174

252633 heat shock protein [Arabidopsis thaliana] CAA72513... 600e-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... 591e-167

1899880 (Q8LLI6) Heat shock protein Hsp90 [Achlya ambisex... 579e-164

245912 heat shock protein 90 [Lycopersicon esculentum] AA... 544e-153

Multiple sequence alignment

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

Rooted phylogenetic tree.

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

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

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.

HSP90-beta

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

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_searchhttp://www.chemie.tu-muenchen.de/biotech/en/hsp90-e.htmlhttp://www.hhmi.org/annual98/research/madcow.htmlwww.ashland.edu/~kstine/Research/Stress%20proteins.pdf