Transcript Testing the Effect of Changing Heat Shock Protein 90:

Heat Shock Proteins, Hsp90
Inhibitors, and Protein
Degradation
By: Vince Centioni
Paper: “A high affinity conformation
of Hsp90 confers tumour selectivity
on Hsp90 inhibitors”
I. Background
A. Protein Degradation
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Protein molecules are continuously synthesized
and degraded in all living organisms. The
concentration of individual cellular proteins is
determined by a balance between the rates of
synthesis and degradation, which in turn are
controlled by a series of regulated biochemical
mechanisms.
The only way that cells can reduce the steady
state level of a particular protein is by degradation.
Thus, complex and highly-regulated mechanisms
have been evolved to accomplish this degradation.
A. Protein Degradation cont.
General principles: Peptide bond: Planar
amide bond between α-carboxyl group & αamino group of 2 adjacent amino acids.
 Proteolysis: Biochemical degradation of
protein through hydrolysis of peptide bonds.
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A. General Principle Diagram:
Peptide + Water
Acid + Amine
A. Protein Degradation cont.
(Intracellular Proteolytic Systems)
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Lysosomal and Non-lysosomal
Lysosomal: Steps= Uptake (Autophagy) into
lysosome: Secretory vesicles; Cytoplasm;
Organelles, and enzymatic degradation .
Non-lysosomal: Steps= Tagging of protein to be
degraded (by ubiquitnation). Recognition of
proteolytic system: exposure of peptide sequence
or distinction of unfolded protein segments
Example : Proteasome
Ubiquitin-Proteasome Degradation:
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A. Protein Degradation cont.
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Cells which are subject to stress such as
starvation, heat-shock, chemical insult or
mutation respond by increasing the rates of
degradation.
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Selective degradation of particular proteins
may occur in response to internal and
external signals.
B. Heat Shock Proteins
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Heat Shock proteins: Family of proteins found in
all cells that are expressed in response to cold,
heat and other environmental stresses.
Some serve to stabilize proteins in abnormal
configurations, and play a role in folding and
unfolding of proteins, acting as molecular
chaperones.
There are four major subclasses: Hsp90, Hsp70,
Hsp60, and small Hsps
B. Heat Shock Proteins cont.
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Heat shock proteins are induced when a cell
undergoes various types of environmental stresses
like heat, cold, and oxygen deprivation.
Heat shock proteins are also present in perfectly
normal conditions where they act as chaperones
making sure that the cell’s proteins are in the right
shape and in the right place at the right time.
HSPs also help shuttle proteins from one
compartment to another inside the cell, and
transport old proteins to “garbage disposals”
inside the cell.
B. Heat Shock Proteins cont.
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Heat shock proteins and the immune system:
under normal conditions HSPs are found outside
the cell. But if a cancerous or infected cell has
become so sick that it dies and its membrane
bursts, all of cell’s contents spill out, including
Hsps that are bound to peptides.
These extracellular Hsps send a very strong
“danger r signal” to the immune system,
instructing it to destroy the other diseased cells.
C. Hsp-danger signal to the immune
system:
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A sick cell dies and ruptures, spilling the Hsppeptide complexes.
These extracellular complexes of HSPs and
peptides are detected by circulating immune
system cells called APCs (antigen-presenting
cells).
The Hsp complexes bind the CD91 receptor on the
APC cell surface. The APC can then take in the
Hsp complexes, and then they travel to the lymph
nodes.
C. Hsp-danger signal cont.
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In the lymph nodes, the APCs take the peptides
that were associated with HSPs and re-represent
them on the cell surface.
Specialized immune cells called T cells “see”
these peptides and are then programmed to seek
out the cells bearing these specific, abnormal
peptides.
Because every person and every cancer is
different, the unique repertoire of antigenic
peptides represents that individual specific
cancer’s “fingerprint.”
D. Heat Shock Protein 90
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Cellular chaperone protein required for the
activation of several eukaryotic protein kinases,
including the cyclin-dependant kinase (CDK4).
Geldanamycin, and inhibitor of the proteinrefolding activity of Hsp90, has been shown to
have antitumor activities.
Hsp90 is the most abundant heat shock protein
under normal conditions.
D. Hsp90 cont.
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The protein exists as two major isoforms, Hsp90alpha and Hsp90-beta.
The protein’s activity has been shown to be ATP
dependant with a unique pocket located in the Nterminal region.
This pocket is the site where ATP and ADP
binding activity take place. Once ATP binds , a
structural amendment by Hsp90 induces a
conformational change from an open position to a
closed position.
D. Hsp90 cont.
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Hsp90’s contributions in signal transduction,
protein folding, and protein degradation.
Hsp90 is a phosphoprotein containing two or
three covalently bound phosphate molecules per
monomer, and the phosphorylation is thought to
enhance its function. The monomer of the Hsp90
consists of a conserved 25-kaDa N-terminal
domain and a 55-kaDa C-terminal domain linked
by a 35-kaDa charged linker region.
Together the C-terminal domain and the linker
region helps in the dimerization of the protein
D. Hsp90 cont.
Hsp90 exhibits ATPase activity which is
essential for its chaperone function.
 Hsp90 binds to an array of client proteins,
where its co-chaperone specificity varies
and depends on the actual client protein.
 There is a growing list of Hsp90 client
proteins and most of them include
molecules involved in signal transduction.
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D. Hsp90 cont.
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Hsp90 forms several discrete sub-complexes, each
containing different set of co-chaperones that
function at different steps during the folding
process of the client protein.
Unlike other chaperones, Hsp90 contains two
independent chaperone sites that differ in their
substrate specificity, probably working in the form
of a switch between Hsp90 and Hsp70 client
protein interactions.
D. Hsp90 cont.
(Multi-chaperone complex)
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The best understood molecular association of
Hsp90-multi-chaperone complexes was in
conjunction with the maturation of steroid
receptors.
The folding process of steroid receptors and their
translocation to the cell nucleus requires Hsp90.
Steroid receptors also require molecular
chaperones for their ligand binding, transcriptional
activation and repression after stimulus.
D. Hsp90 cont.
Though there are reports that some Hsp90
co-chaperones can work independently of
Hsp90, the full competence of these cochaperones requires Hsp90.
 Co-chaperones also help Hsp90-client
protein binding interactions between Hsp70
client proteins, and docking of cytoskeletal
proteins.
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Hsp90 Diagram:
III. Introduction
A. Hsp90 and Cancer Cells
Hsp90 has been implicated in the survival
of cancer cells.
 Hsp90 regulates the function and stability of
many key signal proteins that help cancer
cells to escape the inherent toxicity of their
own environment, to evade chemotherapy,
and to protect themselves from the results of
their own genetic instability.
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A. Hsp90 and Cancer Cells cont.
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Hsp90 plays a key role in the conformational
maturation of oncogenic signaling proteins,
including HER-2/ErbB2, Akt, Raf-t, Bcr-Abl and
mutated p53.
Tumor Hsp90 is present entirely in multichaperone complexes with high ATPase activity.
Tumor cells overexpress Hsp90 client proteins,
suggesting that a greater amount of Hsp90 in
tumor cells might be engaged in active
chaperoning and present in multichaperone
complexes that could modulate the binding
affinity of ligands to Hsp90.
A. Hsp90 and Cancer Cells cont.
Hsp90 regulates many signaling pathways
in cancer cells.
 Recent discoveries in cell biology have
demonstrated that many of the key signaling
molecules that are deregulated in human
cancers require the action of Hsp90
chaperone family in order to maintain their
function.(Neckers and Lee 2003)
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Signaling Molecules Influenced by
Hsp90:
A. Hsp90 and Cancer Cells cont.
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Cancer cells have numerous abnormalities that
make them more dependent upon growth and
survival which, are, in turn dependent upon
Hsp90.
Hsp90 works with other chaperone proteins and
forms a Hsp90 multichaperone complex that
maintains tumor progression, by stabilizing and
interacting with a growing list of various kinases.
Hsp90 chaperone complexes control protein
folding and influence protein degradation.
A. Hsp90 multichaperone pathway and
Ubiquitin-mediated degradation pathway:
A. Hsp90 and Cancer Cells cont.
In tumors these signaling proteins are
deregulated resulting in uncontrolled cell
growth and survival.
 Certain Hsp90 inhibitors are designed to
eliminate these deregulated signal
transduction molecules from the tumor cell
leading to death of the tumor.
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B. Hsp90 Inhibitors
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Certain Hsp90 inhibitors can bind into the ATP
binding site of Hsp90 altering the function of the
Hsp90 multichaperone complex.
These inhibitors convert the Hsp90 complex from
a catalyst for protein folding into one that induces
protein degradation.
The result is the degradation of a specific set of
cancer signaling molecules, leading to cell cycle
arrest and tumor cell death.
B. Hsp90 inhibitors cont.
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Currently used Hsp inhibitors include
geldanaymicn, herbimycin A and 17-AAG.
In 1994, certain ansamycins were found to bind to
Hsp90 and to cause the degradation of client
proteins including Src kinases.
Further efforts to develop anticancer drugs were
made using geldanamycin analogs, and 17AAG
was chosen as the best candidate for clinical trials.
B. Hsp90 Inhibitors cont.
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Geldanaymicn: is a natural Hsp90 inhibitor, that
essentially causes the complete destruction of the
receptor tyrosine kinase HER2/neu, a key driver of
breast cancer growth.
Geldanaymicn: initially thought to be due to
specific tyrosine kinase inhibition, later studies
revealed that the antitumor potential relies on the
depletion of oncogenic protein kinases via the
proteasome.
C. Geldanaymicn Structure:
C. Hsp90 inhibitors cont.
Subsequent immunoprecipitation and X-ray
crystallographic studies reveled that GA
directly binds to Hsp90, and inhibits the
formation of Hsp90 multichaperone
complexes resulting in the ubiquitinmediated degradation of Hsp90 client
proteins.
 This represented the first generation of
drugs that specifically targeted Hsp90.

C. GA cont.
GA binds to the N-terminal domain of
Hsp90 and competes with ATP binding.
 The geldanamycin-Hsp90 crystal structure
also shows that the binding inhibits
substrate protein binding.
 GA also binds to Grp94, the Hsp90
analogue in the ER
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C. GA bound to Hsp90
D. Advantages of Hsp90 inhibitors
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Preclinical trials emphasize the important role of
Hsp90 inhibitors in clinical applications.
Combination therapies, applying low doses of
these drugs together with convention
chemotherapeutic agents, seem to be an effective
way to target various cancers.
For example, in the case of Bcr/Abl-expressing
leukemias, a low dose GA is sufficient to sensitize
these cells to apoptosis.
D. Advantages of Hsp90 Inhibitors
Among the hallmarks of cancer, up
regulation of growth signals and evasion of
apoptosis are the most important.
 As most growth regulatory signals depend
on Hsp90 for their function stability, Hsp90
is an ideal molecule to intervene in complex
oncogenic pathways.
 Hence, most drugs are targeting Hsp90,
which is more beneficial than the selective
oncogene pathway inhibitors.

E. 17-Allylamino, 17Demethoxygeldanamycin (17-AAG)
An analog of GA.
 There is a correlation between downregulation of Hsp90’s client proteins and
growth inhibition caused by 17-AAG.
 17-AAG has a higher affinity to bind to
Hsp90 multichaperone complexes than
Hsp90 in normal cells. There is only
speculation of why this is true.
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E. 17-AAG Structure
IV. Paper
 “A high
affinity conformation of
Hsp90 confers tumor selectivity on
Hsp90 inhibitors”
 by: Kamal, Thao, Sensintaffar, Zhang,
Boehm, Fritz, and Burrows
Important Discoveries
“Tumour Hsp90 is present entirely in
multichaperone complexes with high
ATPase activity, whereas Hsp90 from
normal tissue is in a latent uncomplexed
state.” (Kamal 2003)
 “In vitro reconstitution of chaperone
complexes with Hsp90 resulted in increased
binding affinity to 17-AAG, and increased
ATPase activity.” (Kamal 2003)
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Important Discoveries cont.
Tumour cells contain Hsp90 complexes in
an activated, high-affinity conformation that
promotes malignant progression, and that
may represent a unique target for cancer
therapeutics
 How do we prove if Hsp90 from tumour
cells had a higher binding affinity to 17AAG than that from normal cells?
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Figure 1: Hsp90 binding affinity
to 17-AAG
Figure 1: Results
“Hsp90 in tumour cells has a significantly
higher binding affinity for 17-AAG than
does Hsp90 from normal cells.” (Kamal
2003)
 The binding affinity of 17-AAG to Hsp90
from the different cells directly correlates
with the cytotoxic/cytostatic activity of 17AAG in those cells (Fig. 1d)

Further Questions
Hsp90 interacts with many co-chaperone
proteins that assemble in multi-chaperone
complexes
 Tumour cells overexpress Hsp90 client
proteins
 How to find out whether Hsp90 in tumour
cells was present in multi-chaperone
complexes?
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Figure 2: Levels of p23 and Hop
associated with Hsp90
Figure 2a: Results
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“Co-immunoprecipitation in the normal and
tumour cell lystates with antibodies to Hsp90
revealed that more Hsp90 in tumour cell was
present in complexes with p23 and Hop compared
to to normal cells” (Kamal 2003)
Control immunoprecipitaions without antibodies
did not immunoprecipitate any Hsp90
Immunoprecipitation with antibodies to both p23
and Hop revealed that the entire tumour cell pool
of Hsp90 was present in complexes unlike normal
cells.
Figure 2a: Results cont.
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Immunoprecipitation with an antibody specific for
the uncomplexed form of Hsp90 pulled down far
more Hsp90 from normal cells than from tumour
cells.
All Hsp90 in tumour cells is in the form multichaperone complex that are actively engaged in
chaperoning client oncoproteins, and that the
heightened complex formation is not due to
increased expression of Hsp90 or co-chaperones.
Figure 2b: Results
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Since the chaperone function of Hsp90 is
dependent upon ATPase activity, they
immunopreipitated Hsp90 from cell lysates and
performed ATPase assays
“Tumour Hsp90 had markedly higher ATPase
activity compared to Hsp90 from normal cells, and
was inhibited by 17-AAG.” (Kamal 2003)
“Control immunoprecipitation in the absence of
Hsp90 antibody did not have any significant
ATPase activity (data not shown).” (Kamal 2003)
Figure 2: Combined Results
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“These results suggest that essentially all soluble
Hsp90 in tumour cells is present in fully active
multi-chaperone complexes, whereas Hsp90 in
normal cells is in an uncomplexed inactive form.”
(Kamal 2003)
Data demonstrates that the Hsp90 from tumour
cells had higher binding affinity to 17-AAG, and
this correlates with presence of increased multichaperone complexes and increased ATPase
activity.
Could there by any further tests?
Further Questions and Tests
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How to examine whether reconstitution of purified
Hsp90 with co-chaperones would result in
increased binding affinity and ATPase activity?
In vitro reconstitution, they used five proteins that
have been shown to be required for the in vitro
chaperoning activity of Hsp90. (Hsp90, 70, 40,
Hop, and p23)
Figure 3: In vitro reconstitution of
purified Hsp90 with co-chaperones
Figure 3a: Results
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Of all four co-chaperones to purified Hsp90
increased the apparent affinity of 17-AAG from
600 nM for Hsp90 alone to 12 nM in the presence
of the added proteins, where as partial complexes
did not. (Fig3a)
“Therefore, Hsp90 when reconstituted with cochaperones has nanomolar binding activity, in
concordance with that observed in tumour cell
lysates.” (Kamal 2003)
Figure 3b: Results
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Hsp90 reconstituted with the indicated cochaperones has increased ATPase activity, which
can be inhibited by 10 micromolar 17-AAG
“The ATPase activity of Hsp90 was also
significantly enhanced by all four co-chaperones
and was inhibited by the addition of 17-AAG.”
(Kamal 2003)
Hsp70 had some minimal ATPase activity, but was
not inhibited by 17-AAG
Figure 3: Combined Results
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“These results suggest that Hsp90 present in
multi-chaperone complexes not only had a
higher binding affinity for 17-AAG but was
also more biochemically active.” (Kamal
2003).
Further Experiments
How to determine if these observations in
vitro also apply to mice and to clinical
cancer?
 They examined the binding affinity of 17AAG to Hsp90 in normal and malignant
mouse and human tissue samples (Fig4a,b)
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Figure 4: Hsp90 from clinical tumour
samples
Figure 4a,b: Results
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The apparent binding affinity of 17-AAG to
Hsp90 from mouse tumours (3T3-src, B16, and
CT26) was 8-35 nM compared to 200-600 nM for
the mouse normal tissues, even though Hsp90 was
more abundantly expressed in several of the
normal tissues (Fig4a)
For the human tissues, using four samples of each
tissue type: Normal breast vs. Breast carcinoma
and Normal colon vs. colon carcinomas (Fig4b)
Figure 4c,d: Results
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“Co-immunoprecipitations and observed that there
were increased amounts of p23 with Hsp90 from
the human tumours compared to the normal
tissues (Fig4c).” (Kamal 2003)
Conversely, there was significantly more
uncomplexed Hsp90 co-immunoprecipitated with
the Hsp90* antibody from the normal tissues
compared to tumour tissues (Fig4c)
Figure 4c,d: Results cont.
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“The Hsp90 activity in both clinical tumour
samples was significantly higher relative to the
normal tissue and was inhibited by 17-AAG,
radicicol, and 0.5 M KCl (Fig.4d)” (Kamal 2003).
Resulting that the Hsp90 human clinical tumour
tissues is in a high-affinity, multi-chaperone
complex with increased ATPase activity
“The markedly higher affinity tumour Hsp90 in
vivo explains the remarkable ability of ansamycins
to accumulate progressively at tumour sites in
animals.” (Kamal 2003)
Final Results
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The data showed that Hsp90 in tumour cells
exists in a functionally distinct molecular
form, and therefore clarify three
fundamental aspects of the role of Hsp90 in
tumour biology.
Fundamental Aspects
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First, the nanomolar binding of 17-AAG to highaffinity tumour Hsp90 is now consistent with the
nanomolar anti-tumour of this family of drugs
Second, the markedly higher affinity of tumour
Hsp90 in vivo explains the remarkable ability of
ansamycins to accumulate progressively at tumour
sites in animals.
Third, the complete usage of tumour Hsp90 may
provide a selection pressure leading to further
upregulation of Hsp90 that is observed in many
advanced tumours.
Final Thoughts
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“Dependence on the activated, high-affinity
chaperone could make Hsp90 an ‘Achilles heel’ of
tumour cells, driving the selective accumulation
and bioactivity of pharmacological Hsp90
inhibitors, and making tumour Hsp90 a unique
cancer target.” (Kamal 2003)
What makes Hsp90 better able to bind 17-AAG
when the protein is part of the super-chaperone
complex? Can only speculate
References:
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Kamal and Fritz. Nature. V425. pg. 407-410. Sept,
2003.
Lee and Neckers. Nature. V425. pg. 357-359.
Sept, 2003.
Richter, K. & Buchner, J. Hsp90: Chaperoning
Signal Transduction. J. Cell. Physiol. 188,
281-290. (2001).
Stebbins, C.E., Russo, A.A. Cell V89. pg. 239248. 1997.
Yano, M; Tanakasa, S.& Ansano. Gene
Expression and Roles of Heat Shock Proteins
in Human Breast Cancer. Jpn J. Cancer Res.
87, 908-915 (1996).