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Title: Heat Shock Proteins, Hsp90 Inhibitors, and Protein Degradation


1
Heat Shock Proteins, Hsp90 Inhibitors, and
Protein Degradation
  • By Vince Centioni
  • Paper A high affinity conformation of Hsp90
    confers tumour selectivity on Hsp90 inhibitors

2
I. BackgroundA. Protein Degradation
  • 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.

3
A. Protein Degradation cont.
  • General principles Peptide bond Planar amide
    bond between a-carboxyl group a-amino group of
    2 adjacent amino acids.
  • Proteolysis Biochemical degradation of protein
    through hydrolysis of peptide bonds.

4
A. General Principle Diagram
Peptide Water Acid Amine
                                         
                                    
5
A. Protein Degradation cont. (Intracellular
Proteolytic Systems)
  • 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

6
Ubiquitin-Proteasome Degradation

7
A. Protein Degradation cont.
  • Cells which are subject to stress such as
    starvation, heat-shock, chemical insult or
    mutation respond by increasing the rates of
    degradation.
  • Selective degradation of particular proteins may
    occur in response to internal and external
    signals.

8
B. Heat Shock Proteins
  • 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

9
B. Heat Shock Proteins cont.
  • 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 cells 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.

10
B. Heat Shock Proteins cont.
  • 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 cells 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.

11
C. Hsp-danger signal to the immune system
  • A sick cell dies and ruptures, spilling the
    Hsp-peptide 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.

12
C. Hsp-danger signal cont.
  • 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
    cancers fingerprint.

13
D. Heat Shock Protein 90
  • Cellular chaperone protein required for the
    activation of several eukaryotic protein kinases,
    including the cyclin-dependant kinase (CDK4).
  • Geldanamycin, and inhibitor of the
    protein-refolding activity of Hsp90, has been
    shown to have antitumor activities.
  • Hsp90 is the most abundant heat shock protein
    under normal conditions.

14
D. Hsp90 cont.
  • The protein exists as two major isoforms,
    Hsp90-alpha and Hsp90-beta.
  • The proteins activity has been shown to be ATP
    dependant with a unique pocket located in the
    N-terminal 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.

15
D. Hsp90 cont.
  • Hsp90s 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

16
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.

17
D. Hsp90 cont.
  • 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.

18
D. Hsp90 cont. (Multi-chaperone complex)
  • 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.

19
D. Hsp90 cont.
  • Though there are reports that some Hsp90
    co-chaperones can work independently of Hsp90,
    the full competence of these co-chaperones
    requires Hsp90.
  • Co-chaperones also help Hsp90-client protein
    binding interactions between Hsp70 client
    proteins, and docking of cytoskeletal proteins.

20
Hsp90 Diagram
21
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.

22
A. Hsp90 and Cancer Cells cont.
  • 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
    multi-chaperone 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.

23
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)

24
Signaling Molecules Influenced by
Hsp90
25
A. Hsp90 and Cancer Cells cont.
  • 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.

26
A. Hsp90 multichaperone pathway and
Ubiquitin-mediated degradation pathway
27
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.

28
B. Hsp90 Inhibitors
  • 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.

29
B. Hsp90 inhibitors cont.
  • 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.

30
B. Hsp90 Inhibitors cont.
  • 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.

31
C. Geldanaymicn Structure
32
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
    ubiquitin-mediated degradation of Hsp90 client
    proteins.
  • This represented the first generation of drugs
    that specifically targeted Hsp90.

33
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

34
C. GA bound to Hsp90
35
D. Advantages of Hsp90 inhibitors
  • 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.

36
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.

37
E. 17-Allylamino, 17-Demethoxygeldanamycin
(17-AAG)
  • An analog of GA.
  • There is a correlation between down-regulation of
    Hsp90s 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.

38
E. 17-AAG Structure
39
IV. Paper
  • A high affinity conformation of Hsp90 confers
    tumor selectivity on Hsp90 inhibitors
  • by Kamal, Thao, Sensintaffar, Zhang, Boehm,
    Fritz, and Burrows

40
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)

41
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 17-AAG than that from
    normal cells?

42
Figure 1 Hsp90 binding affinity to 17-AAG

43
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 17-AAG in those
    cells (Fig. 1d)

44
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?

45
Figure 2 Levels of p23 and Hop associated with
Hsp90
46
Figure 2a Results
  • 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.

47
Figure 2a Results cont.
  • 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
    multi-chaperone 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.

48
Figure 2b Results
  • 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)

49
Figure 2 Combined Results
  • 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
    multi-chaperone complexes and increased ATPase
    activity.
  • Could there by any further tests?

50
Further Questions and Tests
  • 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)

51
Figure 3 In vitro reconstitution of purified
Hsp90 with co-chaperones
52
Figure 3a Results
  • 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
    co-chaperones has nanomolar binding activity, in
    concordance with that observed in tumour cell
    lysates. (Kamal 2003)

53
Figure 3b Results
  • Hsp90 reconstituted with the indicated
    co-chaperones 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

54
Figure 3 Combined Results
  • 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).

55
Further Experiments
  • How to determine if these observations in vitro
    also apply to mice and to clinical cancer?
  • They examined the binding affinity of 17-AAG to
    Hsp90 in normal and malignant mouse and human
    tissue samples (Fig4a,b)

56
Figure 4 Hsp90 from clinical tumour samples
57
Figure 4a,b Results
  • 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)

58
Figure 4c,d Results
  • 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)

59
Figure 4c,d Results cont.
  • 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)

60
Final Results
  • 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.

61
Fundamental Aspects
  • First, the nanomolar binding of 17-AAG to
    high-affinity 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.

62
Final Thoughts
  • 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

63
References
  • 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.
    239- 248. 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).
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