Hsp90

Hsp90 (heat shock protein 90) is a molecular chaperone and is one of the most abundant proteins in unstressed cells. It is a ubiquitous molecular chaperone found in eubacteria and all branches of eukarya, but it is apparently absent in archaea. Whereas cytoplasmic Hsp90 is essential for viability under all conditions in eukaryotes, the bacterial homologue HtpG is dispensable under non-heat stress conditions.

In mammalian cells, there are two genes encoding cytosolic Hsp90 homologues, with the human Hsp90α showing 85% sequence identity to Hsp90β. There is also high homology to Hsp90 from lower eukaryotes and prokaryotes: yeast Hsp90 is 60% identical to human Hsp90α and HtpG is still 34% identical to human Hsp90α. Hsp90 is one of the heat shock proteins, and is upregulated in many cells in response to stress.

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Introduction

Heat shock proteins, as a class, are among the most prolific cellular proteins across all species. As their name implies, heat shock proteins respond to a cell becoming stressed by an increase in heat. They account for 1–2% of total protein in unstressed cells. When heated, Hsp90 increases to 4–6% of cellular proteins.^[1] Heat shock protein 90 (Hsp90) is among the most common heat related protein. It is called HSP for obvious reasons, while the 90 comes from the fact that it weighs roughly 90 kiloDaltons. A 90 kD size protein is considered a fairly large non-fibrous protein. The role of Hsp90 covers many things, including: signaling, protein folding and tumor repression. In each role, Hsp90 works in a different way than the last, which has allowed it to remain under constant study since its 1980s discovery through mutant observation and drug treatment among many methods. This protein was first isolated by stressing a cell and then extracting from the cell. They stressed the cell either by heating, dehydrating or a number of other means of causing a cell’s proteins to begin to denature.^[2] Later, researchers realized that HSP90 might have other, much more specific roles in the cell that were engaged even when the cell was not in stress. These roles will be addressed later.

Isoforms

The human cell contains four isoforms: cytosolic Hsp90β, which is constitutively expressed, the inducible α-form, GRP94/gp96 in the endoplasmatic reticulum, and the mitochondrial TRAP1/hsp75. The α- and the β-form show 85% sequence identity "and are thought to be the result of a gene duplication event that occurred millions of years ago".^[3] Recently, a membrane associated variant of cytosolic Hsp90, lacking an ATP-binding site, has been identified and was named Hsp90N.^[4] 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.

Structure

Common features

The structure of HSP90 is like every other protein and has all of the common structures associated with all proteins: alpha helixes, beta pleated sheets and random coils. Being a cytoplasmic borne protein essentially determines 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 helixes and eight anti-parallel beta pleated sheets that are folding into various alpha/beta sandwiches, the 3-10 helixes make up around 11% of the proteins amino sequences which is much higher than the average 4% in other proteins.^[5] Three areas, the ATP binding, protein binding and dimerizing regions, all in particular are highly important to its function.

Domain structure

Hsp90 consists of three structural domains:

• a highly conserved N-terminal domain of ~25 kDa,
• a middle domain of ~40 kDa,
• a C-terminal domain of ~12 kDa and
• a "charged linker" region, that connects the N-terminus with the middle domain.^[6][7][8]

Crystal structures are available for the N-terminal domain of yeast and human Hsp90,^[9][10][11] for complexes of the N-terminus with inhibitors and nucleotides,^[9][10] and for the middle domain of yeast Hsp90.^[12] Recently the structure of a full-length yeast Hsp90-complex was elucidated.^[13] There exists no structural information of the entire protein or for the C-terminal domain. The N-terminal domain shows high homology not only amongst members of the Hsp90 chaperone family, but also to members of the GHKL superfamily.^[7]

Hsp90 constitutively forms homodimers whereas the contact sites are localized within the C-terminus.^[14]

N-terminal domain

  The binding pocket for ATP resp. the inhibitor geldanamycin is situated in the N-terminal domain.^[9][10] Involved amino acids in the ATP interaction are Asp79, Leu34, Asn92, Gly121, Phe124, Asn37, and Lys98. Mg²⁺ and a few water molecules that establish some hydrogen bonds between the interaction partners are important for the ATP recruitment. Glu33 is necessary for ATP hydrolysis.

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, and
• a 6-turn α-helix.^[7]

It is likely to be involved in client binding, i.e. some proteins interacting with that region are PKB/Akt1, eNOS^[15][16], Aha1, Hch1. The ATPase activity is activated by the binding of Aha1 resp. Hch1.^[12][17]

C-terminal domain

The C-terminal domain is supposed to possess an alternative ATP-binding site, which becomes accessible when the N-terminal Bergerat pocket is occupied.^[18][19]

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.^[20][21]

Current studies

The ATPase binding region of HSP90 is currently under a great degree of study, because of the interest of its role in cancer and protein maintenance. This area of the protein is near the N-terminus and has a high affinity site to bind ATP at an uncharacteristically bent manner compared to other proteins, thus, tumor related experiments involving this section of HSP90 are commonly conducted with an antibacterial drug geldanamycin^[5][22] – other molecules affecting Hsp90 function are herbimycin, radicicol, deguelin^[23], and derrubone.^[24] The aforementioned region is a sizable cleft in the side of protein which is measured to be 15 Å deep, the opening has a high affinity for ATP, and when given a suitable substrate, cleaves the ATP into ADP and P_i, where an allosteric inhibitor in relation to the ATPase activity can bind and prevent function.^[5] Since protein folding and regulation are ATP reliant, these functions are effectively put to an end when the ATP site is blocked.

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.^[25] This lid has been studied with artificial mutants that replace the 107Ala with asparagine so as to interact with the polar, groups to which it interacts with when “closed” and has been found to leave the AMP+PnP conformation unchanged, yet, greatly increased the ATPase activity.^[25]

Cancerous cells

Cancerous cells allow massive overproduction of products, such as Her-2 (p185erbB2), whose inhibition can induce apoptosis [2]. HSP90's function in the regulation and correct folding of at least 100 proteins^[26] allows it to refold and/or degrade these products before they trigger cell death, in this way, tumors are allowed to grow relatively unchecked for longer before the body begins to combat the cancerous cells, geldanamycin has been used as an anti-tumor agent with great success, 50% reduction of tumor growth has been realized with doses of geldanamycin.^[5] The drug was originally thought to be a kinase inhibitor and has since been proven to be an HSP90 ATP binding site inhibitor, uses a compact conformation, and inserts itself in to the binding site attaching strongly with Van der Waals forces and partially with a few hydrogen bonds.^[5] Needless to say, it provides a durable bond that will markedly reduce HSP90 function in cells.

Another important role of Hsp90 in cancer is the stabilization of mutant proteins such as v-Src, the fusion oncogene Bcr-Abl, and p53 that appear during cell transformation. This means that Hsp90 can probably act as a "buffer" for mutated proteins bred by several inactivated DNA damage repair pathways.^[27] However HSP 90 can be play a pernicious role in cancer. This has been demonstrated in breast cancer[3], and apart from geldanamycin and its derivatives, many other pharmacological inhibitors[4] of HSP 90 are being tested. Hsp90 is inevitable for induction of vascular endothelial growth factor (VEGF) and nitric oxide synthase (NOS).^[16] Both are important for de novo angiogenesis that is required for tumour growth beyond the limit of diffusion distance of oxygen in tissues.^[27] It also promotes the invasion step of metastasis by assisting the matrix metalloproteinase MMP2.^[28] Together with its co-chaperones it modulates tumour cell apoptosis "mediated through effects on AKT^[15], tumor necrosis factor receptors (TNFR) and nuclear factor-κB (NF- κB) function."^[29]

Finally one can say that hsp90 participates in many key processes in oncogenesis such as self-sufficiency in growth signals, stabilization of mutant proteins, angiogenesis, and metastasis. Targeting Hsp90 with drugs like the geldanamycin derivative 17-N-Allylamino-17-demethoxygeldanamycin (17-AAG) has shown promising effects in clinical trials.

Protein binding

  The protein binding region of HSP90 is located towards the C-terminus of the amino sequence. The two conformational states in which HSP90 appear are called the ATP-bound state and the ADP-bound state, which drive what is commonly referred to as a “pincer type” active site, in which, the conformational change is between open and closed, respectively.^[30] 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.^[31] When a substrate is in place, the ATPase function near the N-terminal forces the shape changes that clamps the protein down on the substrate.^[25] In a reaction similar to that of other molecular clamp proteins like GyrB and MutL, this site performs virtually all of the protein folding functions that HSP90 plays a role in, while MutL and GyrB function in the topoisomerase strand-passage reaction and use a clamp with a high amount of positively charged sidechains that acts on the negative backbone of DNA.^[32] Naturally, the ability to clamp onto protein allows it do several functions such as protein maintenance (hence its chaperonin status) and protein transport.

HSP90's role of chaperonin and transporter can be described well by its interaction with transforming cellular signal molecules and the proteasomes that may or may not degrade them. The S26 proteasome and all of its subsequent subunits are an integral part of proteolysis as well as the regulation in the cell and not only has been found to cease functioning, but also break up into its constituent subunits without the constant supply of functional HSP90 needed to maintain its tertiary structure.^[33] HSP90 is a major helper in assembling and causing the ATP-dependent folding of S26, the importance of this is found in the fact that the S26 proteasome targets virtually all eukaryotic proteins for degradation and are usually marked for destruction through the polyubiquitation pathway.^[34][35] Furthermore, experiments done with heat sensitive HPS90 mutants and the S26 proteasome have indicated that, most likely, HSP90 was responsible for most, if not all, of the ATPase activity of the proteasome.^[34] As previously stated, the S26 proteasome performs proteolysis on virtually all ubiquinated proteins which includes some tyrosine kinases, such as Her-2 (p185erbB2) which is commonly overproduced in cancerous tumors and p60v-src which is the transforming agent coded for by the Rous sarcoma virus.^[36] In the cases of both Her-2 (p185erbB2) and p60v-src studies using benzoquinone ansamycin antibiotics (BA) have indicated that HSP90's ATPase active site is being blocked in a way similar to geldanamycin would and therefore the chaperonin is unable to adequately complex the aforementioned tyrosine kinases.^[36] As a result of HSP90 inability to bind to the kinases, and preventing their imminent ubiquitination by complexing the kinase to HSP90's transmembrane homolog GRP94^[37] and are left to be subsequently tagged and degraded by proteasomes.^[38] As stated, HSP90's plays a role in many of the facets of all types of cellular processes.

Summary

It is clear that HSP90 plays a Janus-like role in the body. It is both everywhere and, yet, plays specific roles in the cell. The ability for the chaperonin to both make the S26 proteasome stable in vivo so as to allow the cell to degrade unwanted and/or harmful proteins in a timely manner and to be responsible for allowing tumor causing kinases to persist in the cytoplasm that would normally be broken down by the same proteasome confirms these specific roles and at the same time show its functional diversity. First stage cancer treatment drug tests such as those with geldanamycin and its variations have put HSP90's importance into focus and have highlighted the need for full scale research into HSP90 related pathways. Naturally, with cancer being such a prevalent problem it, in particular, will encompass a good portion of future experimentation. Combined with the interest in HSP90's in vivo protein folding functions by proteomics researchers, this chaperonin will have a wide array of research completed in the near future.

Genes

• HSP90AA1, HSP90AA2, HSP90AB1, HSP90B1

References