Hsp90
Encyclopedia
Hsp90 is a molecular chaperone and is one of the most abundant protein
s expressed in cell
s. It is a member of the heat shock protein
family, which is upregulated in response to stress. Hsp90 is found in bacteria
and all branches of eukarya, but it is apparently absent in archaea
.
Whereas cytoplasmic Hsp90 is essential for viability under all conditions in eukaryote
s, the bacterial homologue HtpG is dispensable under non-heat stress conditions.
Heat shock proteins, as a class, are among the most highly expressed cellular proteins across all species. As their name implies, heat shock proteins protect cells when stressed by elevated temperatures. They account for 1–2% of total protein in unstressed cells. However, when cells are heated, the fraction of heat shock proteins increases to 4–6% of cellular proteins.
Heat shock protein 90 (Hsp90) is one of the most common of the heat-related proteins. The protein is named "HSP" for obvious reasons, and the "90" comes from the fact that it weighs roughly 90 kiloDaltons
. A 90 kDa protein is considered fairly large for a non-fibrous protein.
The function of Hsp90 includes assisting in protein folding
, cell signaling
, and tumor
repression. This protein was first isolated by extracting proteins from stressed cells. These cells were stressed by heating, dehydrating or by other means, all of which caused the cell’s proteins to begin to denature
. As discussed in more detail below, researchers later realized that Hsp90 has other essential functions in unstressed cells.
Hsp90 is 60% identical to human Hsp90α.
In mammal
ian cells, there are two or more genes encoding cytosol
ic Hsp90 homologues, with the human Hsp90α showing 85% sequence identity to Hsp90β. The α- and the β-forms are thought to be the result of a gene duplication
event that occurred millions of years ago.
The five functional human genes encoding Hsp90 protein isoform
s are listed below:
There are 12 human pseudogene
s (non-functional genes) that encode additional Hsp90 isoforms that are not expressed as proteins.
A membrane-associated variant of cytosolic Hsp90, lacking an ATP-binding site, has recently been identified and was named Hsp90N. This HSP90α-Δ-N transcript is a chimera, with the first 105 bp of the coding sequence derived from the CD47
gene on chromosome 3q13.2, and the remaining coding sequence derived from HSP90AA1
. However, gene-encoding Hsp90N was later proven to be non-existent in human genome. It is possibly a cloning artifact or a product of chromosomal rearrangement occurring in a single cell line.
(i.e., alpha helix
es, beta pleated sheets
, and random coils). Being a cytoplasm
ic protein requires that the protein be globular in structure, that is largely non-polar on the inside and polar on the outside, so as to be dissolved by water. Hsp90 contains nine helices and eight anti-parallel beta pleated sheets, which combine to form several alpha/beta sandwiches. The 310 helices make up approximately 11% of the protein's amino acid residues, which is much higher than the average 4% in other proteins.
s:
Crystal structure
s are available for the N-terminal domain of yeast
and human Hsp90, for complexes of the N-terminus with inhibitors and nucleotide
s, and for the middle domain of yeast Hsp90. Recently structures for full length Hsp90 from E. coli , yeast , and the dog endoplasmic reticulum were elucidated.
Hsp90 forms homodimers where the contact sites are localized within the C-terminus in the open conformation of the dimer. The N-termini also come in contact in the closed conformation of the dimer.
not only among members of the Hsp90 chaperone family but also to members of the ATPase/kinase GHKL
(Gyrase
, Hsp90, Histidine Kinase
, MutL) superfamily.
A common binding pocket for ATP and the inhibitor geldanamycin
is situated in the N-terminal domain. Amino acids that are directly involved in the interaction with ATP are Leu34, Asn37, Asp79, Asn92, Lys98, Gly121, and Phe124. In addition, Mg2+ and several water molecules form bridging electrostatic and hydrogen bond
ing interactions, respectively, between Hsp90 and ATP. In addition, Glu33 is required for ATP hydrolysis
.
The MD is also involved in client protein binding. For example, proteins known to interact this the Hsp90 MD include PKB/Akt1
, eNOS
, Aha1
, Hch1
. Furthermore, substrate binding (e.g., by Aha1 and Hch1) to the MD is also known to increase the ATPase
activity of Hsp90.
At the very C-terminal end of the protein is the tetratricopeptide repeat (TPR) motif recognition site, the conserved MEEVD pentapeptide, that is responsible for the interaction with co-factors such as the immunophilins FKBP51
and FKBP52
, the stress induced phosphoprotein 1 (Sti1/Hop), cyclophilin-40
, PP5
, Tom70
, and many more.
-binding, protein-binding, and dimerizing domain, each of which playing a crucial role in the function of the protein.
(1.5 nanometres) deep. This cleft has a high affinity for ATP, and when given a suitable protein substrate, Hsp90 cleaves the ATP into ADP
and Pi
. Direct inhibitors of ATP binding or allosteric inhibitors of either ATP binding or ATPase activity can block Hsp90 function. Another interesting feature of the ATP-binding region of Hsp90 is that it has a “lid” that is open during the ADP-bound state and closed in the ATP-bound state, in the open conformation, the lid has no intraprotein interaction, and when closed comes into contact with several residues. The contribution of this lid to the activity of Hsp90 has been probed with site-directed mutagenesis
. The Ala107Asp mutant stabilizing the closed conformation of the protein through the formation of additional hydrogen bonds substantially increases ATPase activity while leaving the AMP+PnP conformation unchanged.
The ATPase
-binding region of Hsp90 is currently under intense study, because it is the principal binding site of drugs targeting this protein. Antitumor drugs targeting this section of Hsp90 include the antibiotics geldanamycin
, herbimycin
, radicicol
, deguelin
, derrubone
, and macbecin
.
Hsp90, while in the open conformation, leaves some hydrophobic residues exposed, to which unfolded and misfolded proteins that have unusual hydrophobic regions exposed are recruited with high affinity. When a bound substrate is in place, the energy-releasing ATP hydrolysis by the ATPase function near the NTD forces conformational changes that clamp the Hsp90 down onto the substrate. In a reaction similar to that of other molecular clamp proteins like GyrB and MutL, this site drives virtually all of the protein folding functions that Hsp90 plays a role in. In contrast, MutL and GyrB function as topoisomerase
s and use a charge clamp with a high amount of positively charged sidechains that is electrostatically attracted to the negative backbone of DNA.
The ability of Hsp90 to clamp onto proteins allows it perform several functions including assisting folding, preventing aggregation, and facilitating transport.
, intracellular transport, maintenance, and degradation of proteins as well as facilitating cell signaling.
pathway. These ubiquitinated proteins are recognized and degraded by the 26S proteasome
. Hence the 26S proteasome is an integral part of the cell's mechanism to degrade proteins. Furthermore a constant supply of functional Hsp90 needed to maintain the tertiary structure
of the proteasome. Finally experiments done with heat sensitive Hsp90 mutants and the 26S proteasome suggest that Hsp90 is responsible for most, if not all, of the ATPase activity of the proteasome.
(GR) is the most thoroughly studied example of a steroid receptor whose function is crucially dependent on interactions with Hsp90. In the absence of the steroid hormone cortisol
, GR resides in the cytosol
complexed with several chaperone proteins including Hsp90 (see figure to the right). These chaperones maintain the GR in a state capable of binding hormone. A second role of Hsp90 is to bind immunophilins (e.g., FKBP52
) that attach the GR complex to the dynein
protein trafficking pathway, which translocates
the activated receptor from the cytoplasm into the nucleus. Once in the nucleus, the GR dimerizes and binds to specific sequences of DNA and thereby upregulates
the expression of GR responsive genes. Hsp90 is also required for the proper functioning of several other steroid receptors, including those responsible for the binding of aldosterone
, androgen
, estrogen
, and progesterone
.
and AKT
(Inhibition of these proteins may trigger apoptosis
). Hsp90 stabilizes various growth factor receptors and some signaling molecules including PI3K and AKT proteins, hence inhibition of Hsp90 may induce apoptosis
through inhibition of the PI3K/AKT signaling pathway and growth factor signaling generally.
Another important role of Hsp90 in cancer is the stabilization of mutant proteins such as v-Src, the fusion oncogene
Bcr/Abl, and mutant forms of p53
that appear during cell transformation. It appears that Hsp90 can act as a "protector" of less stable proteins produced by DNA mutations.
Hsp90 is also required for induction of vascular endothelial growth factor (VEGF
) and nitric oxide synthase
(NOS). Both are important for de novo angiogenesis
that is required for tumour growth beyond the limit of diffusion distance of oxygen in tissues. It also promotes the invasion step of metastasis
by assisting the matrix metalloproteinase
MMP2. Together with its co-chaperones, Hsp90 modulates tumour cell apoptosis "mediated through effects on AKT
, tumor necrosis factor receptor
s (TNFR) and nuclear factor-κB (NF-κB) function." Also, Hsp90 participates in many key processes in oncogenesis such as self-sufficiency in growth signals, stabilization of mutant proteins, angiogenesis, and metastasis.
-like role in the cell, where it is essential for the creation, maintenance, and destruction of proteins. Its normal function is critical to maintaining the health of cells, whereas its dysregulation may contribute to carcinogenesis
. The ability for this chaperone to both stabilize the 26S proteasome (which enables the cell to degrade unwanted and/or harmful proteins) and stabilize kinases against the same proteasome demonstrates its functional diversity. The use of Hsp90 inhibitors
in cancer treatment highlight Hsp90's importance as a therapeutic target.
Targeting Hsp90 with drugs has shown promising effects in clinical trials. For example, the Hsp90 inhibitor geldanamycin
has been used as an anti-tumor agent. The drug was originally thought to function as a kinase
inhibitor but was subsequently shown to be an Hsp90 inhibitor where it uses a compact conformation to insert itself into the ATP binding site.
HSP90 beta has been identified as one of the autoantigenic biomarker and target involved in human ovarian autoimmune disease leading to ovarian failure and thereby infertility.
Prediction and validation of the immunodominant epitope/s of HSP90 beta protein has been demonstrated using sera from infertile women having anti-HSP90 autoantibodies. The decapeptide EP6 (380-389)is a major immunogenic epitope of HSP90 followed by EP1 (1-12) and EP8 (488-498). Knowledge of binding epitopes on the autoantigen is necessary to understand the subsequent pathologic events. Predicted 3D structures of these peptides demonstrated that they exist in the loop conformation, which is the most mobile part of the protein. Also, analysis of the sequences of HSP90 beta across several species reveals that EP6 peptide forms a part of a well-conserved motif. A polyclonal antibody generated to the immunodominant epitope- EP6 confirms similar biochemical and cellular immunoreactivity as seen with the patients' sera with anti-HSP90 autoantibodies. The study might generate new tools for the detection of disease-inducing epitopes and a possible therapeutic intervention.
Protein
Proteins are biochemical compounds consisting of one or more polypeptides typically folded into a globular or fibrous form, facilitating a biological function. A polypeptide is a single linear polymer chain of amino acids bonded together by peptide bonds between the carboxyl and amino groups of...
s expressed in cell
Cell (biology)
The cell is the basic structural and functional unit of all known living organisms. It is the smallest unit of life that is classified as a living thing, and is often called the building block of life. The Alberts text discusses how the "cellular building blocks" move to shape developing embryos....
s. It is a member of the heat shock protein
Heat shock protein
Heat shock proteins are a class of functionally related proteins involved in the folding and unfolding of other proteins. Their expression is increased when cells are exposed to elevated temperatures or other stress. This increase in expression is transcriptionally regulated...
family, which is upregulated in response to stress. Hsp90 is found in bacteria
Bacteria
Bacteria are a large domain of prokaryotic microorganisms. Typically a few micrometres in length, bacteria have a wide range of shapes, ranging from spheres to rods and spirals...
and all branches of eukarya, but it is apparently absent in archaea
Archaea
The Archaea are a group of single-celled microorganisms. A single individual or species from this domain is called an archaeon...
.
Whereas cytoplasmic Hsp90 is essential for viability under all conditions in eukaryote
Eukaryote
A eukaryote is an organism whose cells contain complex structures enclosed within membranes. Eukaryotes may more formally be referred to as the taxon Eukarya or Eukaryota. The defining membrane-bound structure that sets eukaryotic cells apart from prokaryotic cells is the nucleus, or nuclear...
s, the bacterial homologue HtpG is dispensable under non-heat stress conditions.
Heat shock proteins, as a class, are among the most highly expressed cellular proteins across all species. As their name implies, heat shock proteins protect cells when stressed by elevated temperatures. They account for 1–2% of total protein in unstressed cells. However, when cells are heated, the fraction of heat shock proteins increases to 4–6% of cellular proteins.
Heat shock protein 90 (Hsp90) is one of the most common of the heat-related proteins. The protein is named "HSP" for obvious reasons, and the "90" comes from the fact that it weighs roughly 90 kiloDaltons
Atomic mass unit
The unified atomic mass unit or dalton is a unit that is used for indicating mass on an atomic or molecular scale. It is defined as one twelfth of the rest mass of an unbound neutral atom of carbon-12 in its nuclear and electronic ground state, and has a value of...
. A 90 kDa protein is considered fairly large for a non-fibrous protein.
The function of Hsp90 includes assisting in protein folding
Protein folding
Protein folding is the process by which a protein structure assumes its functional shape or conformation. It is the physical process by which a polypeptide folds into its characteristic and functional three-dimensional structure from random coil....
, cell signaling
Signal transduction
Signal transduction occurs when an extracellular signaling molecule activates a cell surface receptor. In turn, this receptor alters intracellular molecules creating a response...
, and tumor
Tumor
A tumor or tumour is commonly used as a synonym for a neoplasm that appears enlarged in size. Tumor is not synonymous with cancer...
repression. This protein was first isolated by extracting proteins from stressed cells. These cells were stressed by heating, dehydrating or by other means, all of which caused the cell’s proteins to begin to denature
Denaturation (biochemistry)
Denaturation is a process in which proteins or nucleic acids lose their tertiary structure and secondary structure by application of some external stress or compound, such as a strong acid or base, a concentrated inorganic salt, an organic solvent , or heat...
. As discussed in more detail below, researchers later realized that Hsp90 has other essential functions in unstressed cells.
Isoforms
Hsp90 is highly conserved and expressed in a variety of different organisms from bacteria to mammals – including the prokaryotic analogue htpG (high-temperature protein G) with 40% sequence identity and 55% similarity to the human protein. YeastYeast
Yeasts are eukaryotic micro-organisms classified in the kingdom Fungi, with 1,500 species currently described estimated to be only 1% of all fungal species. Most reproduce asexually by mitosis, and many do so by an asymmetric division process called budding...
Hsp90 is 60% identical to human Hsp90α.
In mammal
Mammal
Mammals are members of a class of air-breathing vertebrate animals characterised by the possession of endothermy, hair, three middle ear bones, and mammary glands functional in mothers with young...
ian cells, there are two or more genes encoding cytosol
Cytosol
The cytosol or intracellular fluid is the liquid found inside cells, that is separated into compartments by membranes. For example, the mitochondrial matrix separates the mitochondrion into compartments....
ic Hsp90 homologues, with the human Hsp90α showing 85% sequence identity to Hsp90β. The α- and the β-forms are thought to be the result of a gene duplication
Gene duplication
Gene duplication is any duplication of a region of DNA that contains a gene; it may occur as an error in homologous recombination, a retrotransposition event, or duplication of an entire chromosome.The second copy of the gene is often free from selective pressure — that is, mutations of it have no...
event that occurred millions of years ago.
The five functional human genes encoding Hsp90 protein isoform
Protein isoform
A protein isoform is any of several different forms of the same protein. Different forms of a protein may be produced from related genes, or may arise from the same gene by alternative splicing. A large number of isoforms are caused by single-nucleotide polymorphisms or SNPs, small genetic...
s are listed below:
| family | subcellular location |
subfamily | gene | protein |
|---|---|---|---|---|
| HSP90A | cytosol Cytosol The cytosol or intracellular fluid is the liquid found inside cells, that is separated into compartments by membranes. For example, the mitochondrial matrix separates the mitochondrion into compartments.... ic |
HSP90AA (inducible) |
HSP90AA1 HSP90AA1 Heat shock protein HSP 90-alpha is a protein that in humans is encoded by the HSP90AA1 gene.-Interactions:Heat shock protein 90kDa alpha , member A1 has been shown to interact with TGF beta receptor 2, FKBP5, TGF beta receptor 1, Glucocorticoid receptor, AKT1, Hop, Peroxisome proliferator-activated... |
Hsp90-α1 |
| HSP90AA2 HSP90AA2 Heat shock protein 90kDa alpha , class A member 2, also known as HSP90AA2, is a human gene. The protein encoded by this gene belongs to the Hsp90 family of heat shock proteins.... |
Hsp90-α2 | |||
| HSP90AB (constitutively expressed) |
HSP90AB1 HSP90AB1 Heat shock protein HSP 90-beta is a protein that in humans is encoded by the HSP90AB1 gene.-Further reading:... |
Hsp90-β | ||
| HSP90B | endoplasmic reticulum Endoplasmic reticulum The endoplasmic reticulum is an organelle of cells in eukaryotic organisms that forms an interconnected network of tubules, vesicles, and cisternae... |
HSP90B1 HSP90B1 Heat shock protein 90kDa beta member 1 , known also as endoplasmin, gp96, grp94 and ERp99, is a chaperone protein that in humans is encoded by the HSP90B1 gene.... |
Endoplasmin/ GRP-94 |
|
| TRAP | mitochondrial Mitochondrion In cell biology, a mitochondrion is a membrane-enclosed organelle found in most eukaryotic cells. These organelles range from 0.5 to 1.0 micrometers in diameter... |
TRAP1 TRAP1 Heat shock protein 75 kDa, mitochondrial is a protein that in humans is encoded by the TRAP1 gene.-Interactions:TRAP1 has been shown to interact with EXT2, EXT1 and Retinoblastoma protein.-Further reading:... |
TNF Receptor- Associated Protein 1 |
There are 12 human pseudogene
Pseudogene
Pseudogenes are dysfunctional relatives of known genes that have lost their protein-coding ability or are otherwise no longer expressed in the cell...
s (non-functional genes) that encode additional Hsp90 isoforms that are not expressed as proteins.
A membrane-associated variant of cytosolic Hsp90, lacking an ATP-binding site, has recently been identified and was named Hsp90N. This HSP90α-Δ-N transcript is a chimera, with the first 105 bp of the coding sequence derived from the CD47
CD47
CD47 is a protein that in humans is encoded by the CD47 gene.- Function :CD47 is a membrane protein, which is involved in the increase in intracellular calcium concentration that occurs upon cell adhesion to extracellular matrix...
gene on chromosome 3q13.2, and the remaining coding sequence derived from HSP90AA1
HSP90AA1
Heat shock protein HSP 90-alpha is a protein that in humans is encoded by the HSP90AA1 gene.-Interactions:Heat shock protein 90kDa alpha , member A1 has been shown to interact with TGF beta receptor 2, FKBP5, TGF beta receptor 1, Glucocorticoid receptor, AKT1, Hop, Peroxisome proliferator-activated...
. However, gene-encoding Hsp90N was later proven to be non-existent in human genome. It is possibly a cloning artifact or a product of chromosomal rearrangement occurring in a single cell line.
Common features
The overall structure of Hsp90 is similar to that of other proteins in that it contains all of the common secondary structural elementsSecondary structure
In biochemistry and structural biology, secondary structure is the general three-dimensional form of local segments of biopolymers such as proteins and nucleic acids...
(i.e., alpha helix
Alpha helix
A common motif in the secondary structure of proteins, the alpha helix is a right-handed coiled or spiral conformation, in which every backbone N-H group donates a hydrogen bond to the backbone C=O group of the amino acid four residues earlier...
es, beta pleated sheets
Beta sheet
The β sheet is the second form of regular secondary structure in proteins, only somewhat less common than the alpha helix. Beta sheets consist of beta strands connected laterally by at least two or three backbone hydrogen bonds, forming a generally twisted, pleated sheet...
, and random coils). Being a cytoplasm
Cytoplasm
The cytoplasm is a small gel-like substance residing between the cell membrane holding all the cell's internal sub-structures , except for the nucleus. All the contents of the cells of prokaryote organisms are contained within the cytoplasm...
ic protein requires that the protein be globular in structure, that is largely non-polar on the inside and polar on the outside, so as to be dissolved by water. Hsp90 contains nine helices and eight anti-parallel beta pleated sheets, which combine to form several alpha/beta sandwiches. The 310 helices make up approximately 11% of the protein's amino acid residues, which is much higher than the average 4% in other proteins.
Domain structure
Hsp90 consists of four structural domainProtein domain
A protein domain is a part of protein sequence and structure that can evolve, function, and exist independently of the rest of the protein chain. Each domain forms a compact three-dimensional structure and often can be independently stable and folded. Many proteins consist of several structural...
s:
- a highly conserved N-terminal (NTD) domain of ~25 kDa
- a "charged linker" region, that connects the N-terminus with the middle domain
- a middle domain (MD) of ~40 kDa
- a C-terminal domain (CTD) of ~12 kDa.
Crystal structure
Crystal structure
In mineralogy and crystallography, crystal structure is a unique arrangement of atoms or molecules in a crystalline liquid or solid. A crystal structure is composed of a pattern, a set of atoms arranged in a particular way, and a lattice exhibiting long-range order and symmetry...
s are available for the N-terminal domain of yeast
Yeast
Yeasts are eukaryotic micro-organisms classified in the kingdom Fungi, with 1,500 species currently described estimated to be only 1% of all fungal species. Most reproduce asexually by mitosis, and many do so by an asymmetric division process called budding...
and human Hsp90, for complexes of the N-terminus with inhibitors and nucleotide
Nucleotide
Nucleotides are molecules that, when joined together, make up the structural units of RNA and DNA. In addition, nucleotides participate in cellular signaling , and are incorporated into important cofactors of enzymatic reactions...
s, and for the middle domain of yeast Hsp90. Recently structures for full length Hsp90 from E. coli , yeast , and the dog endoplasmic reticulum were elucidated.
Hsp90 forms homodimers where the contact sites are localized within the C-terminus in the open conformation of the dimer. The N-termini also come in contact in the closed conformation of the dimer.
N-terminal domain
The N-terminal domain shows high homologyHomology (biology)
Homology forms the basis of organization for comparative biology. In 1843, Richard Owen defined homology as "the same organ in different animals under every variety of form and function". Organs as different as a bat's wing, a seal's flipper, a cat's paw and a human hand have a common underlying...
not only among members of the Hsp90 chaperone family but also to members of the ATPase/kinase GHKL
GHKL domain
The GHKL domain is an evolutionary conserved protein domain.This family represents the structurally related ATPase domains of histidine kinase, DNA gyrase B and HSP90...
(Gyrase
DNA gyrase
DNA gyrase, often referred to simply as gyrase, is an enzyme that relieves strain while double-stranded DNA is being unwound by helicase. This causes negative supercoiling of the DNA...
, Hsp90, Histidine Kinase
Histidine kinase
Histidine Kinases are multifunctional, typically transmembrane, proteins of the transferase class that play a role in signal transduction across the cellular membrane. The vast majority of HKs are homodimers that exhibit autokinase, phosphotransfer, and phosphatase activity. HKs can act as...
, MutL) superfamily.
A common binding pocket for ATP and the inhibitor geldanamycin
Geldanamycin
Geldanamycin is a benzoquinone ansamycin antibiotic that binds to Hsp90 and inhibits its function. HSP90 client proteins play important roles in the regulation of the cell cycle, cell growth, cell survival, apoptosis, angiogenesis and oncogenesis.Geldanamycin induces the degradation of proteins...
is situated in the N-terminal domain. Amino acids that are directly involved in the interaction with ATP are Leu34, Asn37, Asp79, Asn92, Lys98, Gly121, and Phe124. In addition, Mg2+ and several water molecules form bridging electrostatic and hydrogen bond
Hydrogen bond
A hydrogen bond is the attractive interaction of a hydrogen atom with an electronegative atom, such as nitrogen, oxygen or fluorine, that comes from another molecule or chemical group. The hydrogen must be covalently bonded to another electronegative atom to create the bond...
ing interactions, respectively, between Hsp90 and ATP. In addition, Glu33 is required for ATP hydrolysis
Hydrolysis
Hydrolysis is a chemical reaction during which molecules of water are split into hydrogen cations and hydroxide anions in the process of a chemical mechanism. It is the type of reaction that is used to break down certain polymers, especially those made by condensation polymerization...
.
Middle domain
The middle domain is divided into three regions:- a 3-layer α-β-α sandwich
- a 3-turn α-helix and irregular loops
- a 6-turn α-helix.
The MD is also involved in client protein binding. For example, proteins known to interact this the Hsp90 MD include PKB/Akt1
AKT1
RAC-alpha serine/threonine-protein kinase is an enzyme that in humans is encoded by the AKT1 gene. Multiple alternatively spliced transcript variants have been found for this gene.- Function :...
, eNOS
Nitric oxide synthase
Nitric oxide synthases are a family of enzymes that catalyze the production of nitric oxide from L-arginine. NO is an important cellular signaling molecule, having a vital role in many biological processes...
, Aha1
AHSA1
Activator of 90 kDa heat shock protein ATPase homolog 1 is an enzyme that in humans is encoded by the AHSA1 gene.-Interactions:AHSA1 has been shown to interact with Heat shock protein 90kDa alpha , member A1.-Further reading:...
, Hch1
AHSA2
AHSA2 also known as AHA1, activator of heat shock 90kDa protein ATPase homolog 2 is a human gene which encodes a protein which acts as co-chaperone of Hsp90...
. Furthermore, substrate binding (e.g., by Aha1 and Hch1) to the MD is also known to increase the ATPase
ATPase
ATPases are a class of enzymes that catalyze the decomposition of adenosine triphosphate into adenosine diphosphate and a free phosphate ion. This dephosphorylation reaction releases energy, which the enzyme harnesses to drive other chemical reactions that would not otherwise occur...
activity of Hsp90.
C-terminal domain
The C-terminal domain possesses an alternative ATP-binding site, which becomes accessible when the N-terminal Bergerat pocket is occupied.At the very C-terminal end of the protein is the tetratricopeptide repeat (TPR) motif recognition site, the conserved MEEVD pentapeptide, that is responsible for the interaction with co-factors such as the immunophilins FKBP51
FKBP5
FK506 binding protein 5, also known as FKBP5, is a protein which in humans is encoded by the FKBP5 gene.- Function :The protein encoded by this gene is a member of the immunophilin protein family, which play a role in immunoregulation and basic cellular processes involving protein folding and...
and FKBP52
FKBP52
FK506-binding protein 4 is a protein that in humans is encoded by the FKBP4 gene.This protein contains TPR repeats and has a PPlase domain.-Further reading:-External links:...
, the stress induced phosphoprotein 1 (Sti1/Hop), cyclophilin-40
PPID
Peptidylprolyl isomerase D , also known as PPID, is an enzyme which in humans is encoded by the PPID gene.- Function :...
, PP5
TFPI2
Tissue factor pathway inhibitor 2 is a protein that in humans is encoded by the TFPI2 gene.-Further reading:...
, Tom70
TOMM70A
Mitochondrial import receptor subunit TOM70 is a protein that in humans is encoded by the TOMM70A gene.-Further reading:...
, and many more.
Mechanism
The Hsp90 protein contains three functional domains, the ATPAdenosine triphosphate
Adenosine-5'-triphosphate is a multifunctional nucleoside triphosphate used in cells as a coenzyme. It is often called the "molecular unit of currency" of intracellular energy transfer. ATP transports chemical energy within cells for metabolism...
-binding, protein-binding, and dimerizing domain, each of which playing a crucial role in the function of the protein.
ATP binding
The region of the protein near the N-terminus has a high-affinity ATP-binding site. The ATP binds to a sizable cleft in the side of protein, which is 15 ÅÅngström
The angstrom or ångström, is a unit of length equal to 1/10,000,000,000 of a meter . Its symbol is the Swedish letter Å....
(1.5 nanometres) deep. This cleft has a high affinity for ATP, and when given a suitable protein substrate, Hsp90 cleaves the ATP into ADP
Adenosine diphosphate
Adenosine diphosphate, abbreviated ADP, is a nucleoside diphosphate. It is an ester of pyrophosphoric acid with the nucleoside adenosine. ADP consists of the pyrophosphate group, the pentose sugar ribose, and the nucleobase adenine....
and Pi
Phosphate
A phosphate, an inorganic chemical, is a salt of phosphoric acid. In organic chemistry, a phosphate, or organophosphate, is an ester of phosphoric acid. Organic phosphates are important in biochemistry and biogeochemistry or ecology. Inorganic phosphates are mined to obtain phosphorus for use in...
. Direct inhibitors of ATP binding or allosteric inhibitors of either ATP binding or ATPase activity can block Hsp90 function. Another interesting feature of the ATP-binding region of Hsp90 is that it has a “lid” that is open during the ADP-bound state and closed in the ATP-bound state, in the open conformation, the lid has no intraprotein interaction, and when closed comes into contact with several residues. The contribution of this lid to the activity of Hsp90 has been probed with site-directed mutagenesis
Site-directed mutagenesis
Site-directed mutagenesis, also called site-specific mutagenesis or oligonucleotide-directed mutagenesis, is a molecular biology technique in which a mutation is created at a defined site in a DNA molecule. In general, this form of mutagenesis requires that the wild type gene sequence be known...
. The Ala107Asp mutant stabilizing the closed conformation of the protein through the formation of additional hydrogen bonds substantially increases ATPase activity while leaving the AMP+PnP conformation unchanged.
The ATPase
ATPase
ATPases are a class of enzymes that catalyze the decomposition of adenosine triphosphate into adenosine diphosphate and a free phosphate ion. This dephosphorylation reaction releases energy, which the enzyme harnesses to drive other chemical reactions that would not otherwise occur...
-binding region of Hsp90 is currently under intense study, because it is the principal binding site of drugs targeting this protein. Antitumor drugs targeting this section of Hsp90 include the antibiotics geldanamycin
Geldanamycin
Geldanamycin is a benzoquinone ansamycin antibiotic that binds to Hsp90 and inhibits its function. HSP90 client proteins play important roles in the regulation of the cell cycle, cell growth, cell survival, apoptosis, angiogenesis and oncogenesis.Geldanamycin induces the degradation of proteins...
, herbimycin
Herbimycin
Herbimycin is a benzoquinone ansamycin antibiotic that binds to Hsp90 and alters its function. HSP90 client proteins play important roles in the regulation of the cell cycle, cell growth, cell survival, apoptosis, angiogenesis and oncogenesis.It was originally found by its herbicidal activity, and...
, radicicol
Radicicol
Radicicol, also known as monorden, is a natural product that binds to Hsp90 and alters its function. HSP90 client proteins play important roles in the regulation of the cell cycle, cell growth, cell survival, apoptosis, angiogenesis and oncogenesis.-Further reading: Review of the chemistry and...
, deguelin
Deguelin
Deguelin is a derivative of rotenone. Both are compounds classified as rotenoids of the flavonoid family and are naturally occurring insecticides...
, derrubone
Derrubone
Derrubone is an isoflavone, a type of flavonoid. It bears a prenyl acetylation. It was originally isolated from the Indian tree Derris robusta. Recent research indicates that it acts as an inhibitor of Hsp90 to its function as a chaperone protein....
, and macbecin
Macbecin
Macbecin belongs to the ansamycin family of antibiotics and was first isolated from actinomycete bacteria. Macbecin possesses potent antitumor properties.-Structure:Macbecin has an unusual macrocyclic lactam structure...
.
Protein binding
The protein-binding region of Hsp90 is located toward the C-terminus of the amino sequence. The Hsp90 protein can adopt two major conformational states. The first is an open ATP-bound state and the second is a closed ADP-bound state. Thus, ATP hydrolysis drives what is commonly referred to as a “pincer-type” conformational change in the protein binding site.Hsp90, while in the open conformation, leaves some hydrophobic residues exposed, to which unfolded and misfolded proteins that have unusual hydrophobic regions exposed are recruited with high affinity. When a bound substrate is in place, the energy-releasing ATP hydrolysis by the ATPase function near the NTD forces conformational changes that clamp the Hsp90 down onto the substrate. In a reaction similar to that of other molecular clamp proteins like GyrB and MutL, this site drives virtually all of the protein folding functions that Hsp90 plays a role in. In contrast, MutL and GyrB function as topoisomerase
Topoisomerase
Topoisomerases are enzymes that regulate the overwinding or underwinding of DNA. The winding problem of DNA arises due to the intertwined nature of its double helical structure. For example, during DNA replication, DNA becomes overwound ahead of a replication fork...
s and use a charge clamp with a high amount of positively charged sidechains that is electrostatically attracted to the negative backbone of DNA.
The ability of Hsp90 to clamp onto proteins allows it perform several functions including assisting folding, preventing aggregation, and facilitating transport.
Normal cells
In unstressed cells, Hsp90 plays a number of important roles, which include assisting foldingProtein folding
Protein folding is the process by which a protein structure assumes its functional shape or conformation. It is the physical process by which a polypeptide folds into its characteristic and functional three-dimensional structure from random coil....
, intracellular transport, maintenance, and degradation of proteins as well as facilitating cell signaling.
Protein folding and role as chaperone
Hsp90 is known to associate with the non-native structures of many proteins, which has led to the proposal that Hsp90 is involved in protein folding in general. Furthermore Hsp90 has been shown to suppress the aggregation of a wide range of "client" or "substrate" proteins and hence acts as a general protective chaperone. However Hsp90 is somewhat more selective than other chaperones.Protein degradation
Eukaryotic proteins that are no longer needed or are misfolded or otherwise damaged are usually marked for destruction by the polyubiquitationUbiquitin
Ubiquitin is a small regulatory protein that has been found in almost all tissues of eukaryotic organisms. Among other functions, it directs protein recycling.Ubiquitin can be attached to proteins and label them for destruction...
pathway. These ubiquitinated proteins are recognized and degraded by the 26S proteasome
Proteasome
Proteasomes are very large protein complexes inside all eukaryotes and archaea, and in some bacteria. In eukaryotes, they are located in the nucleus and the cytoplasm. The main function of the proteasome is to degrade unneeded or damaged proteins by proteolysis, a chemical reaction that breaks...
. Hence the 26S proteasome is an integral part of the cell's mechanism to degrade proteins. Furthermore a constant supply of functional Hsp90 needed to maintain the tertiary structure
Tertiary structure
In biochemistry and molecular biology, the tertiary structure of a protein or any other macromolecule is its three-dimensional structure, as defined by the atomic coordinates.-Relationship to primary structure:...
of the proteasome. Finally experiments done with heat sensitive Hsp90 mutants and the 26S proteasome suggest that Hsp90 is responsible for most, if not all, of the ATPase activity of the proteasome.
Interaction with steroid receptors
The glucocorticoid receptorGlucocorticoid receptor
The glucocorticoid receptor also known as NR3C1 is the receptor to which cortisol and other glucocorticoids bind....
(GR) is the most thoroughly studied example of a steroid receptor whose function is crucially dependent on interactions with Hsp90. In the absence of the steroid hormone cortisol
Cortisol
Cortisol is a steroid hormone, more specifically a glucocorticoid, produced by the adrenal gland. It is released in response to stress and a low level of blood glucocorticoids. Its primary functions are to increase blood sugar through gluconeogenesis; suppress the immune system; and aid in fat,...
, GR resides in the cytosol
Cytosol
The cytosol or intracellular fluid is the liquid found inside cells, that is separated into compartments by membranes. For example, the mitochondrial matrix separates the mitochondrion into compartments....
complexed with several chaperone proteins including Hsp90 (see figure to the right). These chaperones maintain the GR in a state capable of binding hormone. A second role of Hsp90 is to bind immunophilins (e.g., FKBP52
FKBP52
FK506-binding protein 4 is a protein that in humans is encoded by the FKBP4 gene.This protein contains TPR repeats and has a PPlase domain.-Further reading:-External links:...
) that attach the GR complex to the dynein
Dynein
Dynein is a motor protein in cells which converts the chemical energy contained in ATP into the mechanical energy of movement. Dynein transports various cellular cargo by "walking" along cytoskeletal microtubules towards the minus-end of the microtubule, which is usually oriented towards the cell...
protein trafficking pathway, which translocates
Protein targeting
Protein targeting or protein sorting is the mechanism by which a cell transports proteins to the appropriate positions in the cell or outside of it. Sorting targets can be the inner space of an organelle, any of several interior membranes, the cell's outer membrane, or its exterior via secretion...
the activated receptor from the cytoplasm into the nucleus. Once in the nucleus, the GR dimerizes and binds to specific sequences of DNA and thereby upregulates
Downregulation and upregulation
Downregulation is the process by which a cell decreases the quantity of a cellular component, such as RNA or protein, in response to an external variable...
the expression of GR responsive genes. Hsp90 is also required for the proper functioning of several other steroid receptors, including those responsible for the binding of aldosterone
Mineralocorticoid receptor
The mineralocorticoid receptor , also known as the aldosterone receptor or nuclear receptor subfamily 3, group C, member 2, is a protein that in humans is encoded by the NR3C2 gene that is located on chromosome 4q31.1-31.2.MR is a receptor with high affinity for mineralocorticoids...
, androgen
Androgen receptor
The androgen receptor , also known as NR3C4 , is a type of nuclear receptor that is activated by binding of either of the androgenic hormones testosterone or dihydrotestosterone in the cytoplasm and then translocating into the nucleus...
, estrogen
Estrogen receptor
Estrogen receptor refers to a group of receptors that are activated by the hormone 17β-estradiol . Two types of estrogen receptor exist: ER, which is a member of the nuclear hormone family of intracellular receptors, and the estrogen G protein-coupled receptor GPR30 , which is a G protein-coupled...
, and progesterone
Progesterone receptor
The progesterone receptor also known as NR3C3 , is an intracellular steroid receptor that specifically binds progesterone...
.
Cancerous cells
Cancerous cells over express a number of proteins, including growth factor receptors, such as EGFR, or signal transduction proteins such as PI3KPhosphoinositide 3-kinase
Phosphatidylinositol 3-kinases are a family of enzymes involved in cellular functions such as cell growth, proliferation, differentiation, motility, survival and intracellular trafficking, which in turn are involved in cancer. In response to lipopolysaccharide, PI3K phosphorylates p65, inducing...
and AKT
AKT
Akt, also known as Protein Kinase B , is a serine/threonine protein kinase that plays a key role in multiple cellular processes such as glucose metabolism, cell proliferation, apoptosis, transcription and cell migration.-Family members:...
(Inhibition of these proteins may trigger apoptosis
Apoptosis
Apoptosis is the process of programmed cell death that may occur in multicellular organisms. Biochemical events lead to characteristic cell changes and death. These changes include blebbing, cell shrinkage, nuclear fragmentation, chromatin condensation, and chromosomal DNA fragmentation...
). Hsp90 stabilizes various growth factor receptors and some signaling molecules including PI3K and AKT proteins, hence inhibition of Hsp90 may induce apoptosis
Apoptosis
Apoptosis is the process of programmed cell death that may occur in multicellular organisms. Biochemical events lead to characteristic cell changes and death. These changes include blebbing, cell shrinkage, nuclear fragmentation, chromatin condensation, and chromosomal DNA fragmentation...
through inhibition of the PI3K/AKT signaling pathway and growth factor signaling generally.
Another important role of Hsp90 in cancer is the stabilization of mutant proteins such as v-Src, the fusion oncogene
Oncogene
An oncogene is a gene that has the potential to cause cancer. In tumor cells, they are often mutated or expressed at high levels.An oncogene is a gene found in the chromosomes of tumor cells whose activation is associated with the initial and continuing conversion of normal cells into cancer...
Bcr/Abl, and mutant forms of p53
P53
p53 , is a tumor suppressor protein that in humans is encoded by the TP53 gene. p53 is crucial in multicellular organisms, where it regulates the cell cycle and, thus, functions as a tumor suppressor that is involved in preventing cancer...
that appear during cell transformation. It appears that Hsp90 can act as a "protector" of less stable proteins produced by DNA mutations.
Hsp90 is also required for induction of vascular endothelial growth factor (VEGF
Vascular endothelial growth factor
Vascular endothelial growth factor is a signal protein produced by cells that stimulates vasculogenesis and angiogenesis. It is part of the system that restores the oxygen supply to tissues when blood circulation is inadequate....
) and nitric oxide synthase
Nitric oxide synthase
Nitric oxide synthases are a family of enzymes that catalyze the production of nitric oxide from L-arginine. NO is an important cellular signaling molecule, having a vital role in many biological processes...
(NOS). Both are important for de novo angiogenesis
Angiogenesis
Angiogenesis is the physiological process involving the growth of new blood vessels from pre-existing vessels. Though there has been some debate over terminology, vasculogenesis is the term used for spontaneous blood-vessel formation, and intussusception is the term for the formation of new blood...
that is required for tumour growth beyond the limit of diffusion distance of oxygen in tissues. It also promotes the invasion step of metastasis
Metastasis
Metastasis, or metastatic disease , is the spread of a disease from one organ or part to another non-adjacent organ or part. It was previously thought that only malignant tumor cells and infections have the capacity to metastasize; however, this is being reconsidered due to new research...
by assisting the matrix metalloproteinase
Matrix metalloproteinase
Matrix metalloproteinases are zinc-dependent endopeptidases; other family members are adamalysins, serralysins, and astacins. The MMPs belong to a larger family of proteases known as the metzincin superfamily....
MMP2. Together with its co-chaperones, Hsp90 modulates tumour cell apoptosis "mediated through effects on AKT
AKT
Akt, also known as Protein Kinase B , is a serine/threonine protein kinase that plays a key role in multiple cellular processes such as glucose metabolism, cell proliferation, apoptosis, transcription and cell migration.-Family members:...
, tumor necrosis factor receptor
Tumor necrosis factor receptor
A tumor necrosis factor receptor , or death receptor, is a trimeric cytokine receptor that binds tumor necrosis factors . The receptor cooperates with an adaptor protein , which is important in determining the outcome of the response A tumor necrosis factor receptor (TNFR), or death receptor, is a...
s (TNFR) and nuclear factor-κB (NF-κB) function." Also, Hsp90 participates in many key processes in oncogenesis such as self-sufficiency in growth signals, stabilization of mutant proteins, angiogenesis, and metastasis.
Clinical significance
Hsp90 plays a JanusJanus (mythology)
In ancient Roman religion and mythology, Janus is the god of beginnings and transitions, thence also of gates, doors, doorways, endings and time. He is usually a two-faced god since he looks to the future and the past...
-like role in the cell, where it is essential for the creation, maintenance, and destruction of proteins. Its normal function is critical to maintaining the health of cells, whereas its dysregulation may contribute to carcinogenesis
Carcinogenesis
Carcinogenesis or oncogenesis is literally the creation of cancer. It is a process by which normal cells are transformed into cancer cells...
. The ability for this chaperone to both stabilize the 26S proteasome (which enables the cell to degrade unwanted and/or harmful proteins) and stabilize kinases against the same proteasome demonstrates its functional diversity. The use of Hsp90 inhibitors
Hsp90 inhibitors
Among heat shock proteins the focus on HSP90 has increased due to its involvement in several cellular phenomenon and more importantly in disease progression. Zhao and Houry, in 2005, demonstrated that HSP90 keeps the death proteins in an apoptosis resistant state by direct association...
in cancer treatment highlight Hsp90's importance as a therapeutic target.
Targeting Hsp90 with drugs has shown promising effects in clinical trials. For example, the Hsp90 inhibitor geldanamycin
Geldanamycin
Geldanamycin is a benzoquinone ansamycin antibiotic that binds to Hsp90 and inhibits its function. HSP90 client proteins play important roles in the regulation of the cell cycle, cell growth, cell survival, apoptosis, angiogenesis and oncogenesis.Geldanamycin induces the degradation of proteins...
has been used as an anti-tumor agent. The drug was originally thought to function as a kinase
Kinase
In chemistry and biochemistry, a kinase is a type of enzyme that transfers phosphate groups from high-energy donor molecules, such as ATP, to specific substrates, a process referred to as phosphorylation. Kinases are part of the larger family of phosphotransferases...
inhibitor but was subsequently shown to be an Hsp90 inhibitor where it uses a compact conformation to insert itself into the ATP binding site.
HSP90 beta has been identified as one of the autoantigenic biomarker and target involved in human ovarian autoimmune disease leading to ovarian failure and thereby infertility.
Prediction and validation of the immunodominant epitope/s of HSP90 beta protein has been demonstrated using sera from infertile women having anti-HSP90 autoantibodies. The decapeptide EP6 (380-389)is a major immunogenic epitope of HSP90 followed by EP1 (1-12) and EP8 (488-498). Knowledge of binding epitopes on the autoantigen is necessary to understand the subsequent pathologic events. Predicted 3D structures of these peptides demonstrated that they exist in the loop conformation, which is the most mobile part of the protein. Also, analysis of the sequences of HSP90 beta across several species reveals that EP6 peptide forms a part of a well-conserved motif. A polyclonal antibody generated to the immunodominant epitope- EP6 confirms similar biochemical and cellular immunoreactivity as seen with the patients' sera with anti-HSP90 autoantibodies. The study might generate new tools for the detection of disease-inducing epitopes and a possible therapeutic intervention.