AS A THERAPEUTIC TARGET Arrigo André-Patrick Stress, Chaperons and Cell Death

519 Pages·2007·8.08 MB·English

by  Helen Neumann

Module 1 – Oral lectures:

1A_01_S

HSP27 (HSPB1) AS A THERAPEUTIC TARGET
Arrigo André-Patrick
Stress, Chaperons and Cell Death Laboratory, CGMC, CNRS UMR 5534,
Claude Bernard University LYON 1, 16 Rue Dubois, Bat. Gregor Mendel,
69622 Villeurbanne, France, [email protected]

Human Hsp27(HspB1) is a molecular chaperone which is constitutively
expressed in several mammalian cells, particularly in pathological conditions.
This protein has functions as diverse as protection against toxicity mediated
by aberrantly folded proteins or oxidative-inflammation conditions. In
addition, it has anti-apoptotic properties and is tumorigenic when expressed
in cancer cells. Hsp27(HspB1) has implications, either positive or deleterious,
in pathologies such as neurodegenerative diseases, asthma, and cancers.
Moreover, mutations in hsp27 gene have been detected which are responsive
of hereditary motor neuropathies. Hsp27(HspB1) is therefore an active
determinant in health and disease and not just a passive storage device.
Approaches as well as preliminary results towards therapeutic strategies
aimed at modulating the expression and/or the activities of Hsp27(HspB1) will
be presented.

1A_02_S

HSPB8 FORMS WITH BAG3 A CHAPERONE COMPLEX STIMULATING
MACROAUTOPHAGY: POSSIBLE INVOLVEMENT OF SHSP IN
PROTEIN QUALITY CONTROL

Jacques Landry

Centre de recherche en cancérologie de l’Université Laval, L’Hôtel-Dieu de
Québec, Québec, Canada, G1R 2J6, [email protected]

Intracellular protein aggregation can occur upon proteotoxic stress or
genetic mutations and represents a major threat in the crowded
environment of the cell. The Hsp70/Hsp90 proteins and their associated
co-chaperones form a protein quality control system which can recognize
such proteins and either assist them in renaturation or target them for
degradation. Proteins from the small Hsp family, and in particular HspB1
(Hsp27) and HspB8, may similarly act as molecular chaperones as
evidenced by their association with human diseases featuring protein
conformation defects. Diverse types of activities are associated with each
of these proteins. On one hand, HspB1 (Hsp27) but not HspB8 potentiates
the renaturation of damaged proteins in the cells. In this activity, HspB1
recognizes the substrate and then recruits a renaturating machinery likely
involving Hsp70 family members. On the other hand, HspB8, but not
HspB1, forms in cells a stable protein complex with Bag3, a Bag family
member previously identified as a Hsp70 co-chaperone. In association
with Bag3 but independently Bag3 binding to Hsp70, HspB8 efficiently
limits the accumulation of aggregation-prone protein substrates by
targeting them to destruction by macroautophagy, a lysosome-based
protein degradation system capable of degrading large structures and
insoluble protein aggregates. In this complex HspB8 likely acts as a
molecular chaperone responsible for the recognition the damaged
substrates whereas Bag3 is responsible for recruiting and stimulating the
macroautophagy machinery. Accordingly, HspB1 engineered to interact
with Bag3 can also target unstable proteins to degradation. Hence HspB
proteins can, like Hsp70/Hsp90, function in protein quality control by
recognizing protein substrates, the fate of which being determined by
associated co-chaperones that are specific to individual chaperones.

1A_03_S

RECOVERY OF MACROMOLECULAR SYNTHESIS AFTER STRESS: THE
ROLE OF SMALL HEAT SHOCK PROTEIN

Nicolette H. Lubsen, [email protected]

Cells stressed by heat downregulate protein synthesis. During recovery
from heat stress protein synthesis slowly recovers. In the presence of
either αB-crystallin or Hsp27, both small heat shock proteins, the rate of
recovery is enhanced. Using fluorescence recovery after photobleaching
we showed that Hsp27, but not αB-crystallin, increased the pool of mobile
stress granule-associated EGFP-eIF4E in heat shocked cells. Hsp27 also
partially prevented the sharp decrease in the pool of mobile cytoplasmic
EGFP-eIF4G, supporting other evidence for a direct interaction between
eIF4G and Hsp27. sHsps did not prevent the phosphorylation of eIF2α by
a heat shock, but promoted dephosphorylation during recovery. Blocking
the endogenous heat shock response by expressing a dominant negative
HSF1 mutant during recovery from a heat shock slows the rate of
recovery of protein synthesis and blocks the restorative effect of sHsps,
showing that sHsps need to cooperate with other Hsps of which the
synthesis is induced by heat shock. Translational recovery 24 hours after
heat shock does not differ between cells expressing dnHSF1 and control
cells. However, if cells express dnHSF1 as well as the C-terminal fragment
of GADD34, which causes constitutive dephosphorylation of eIF2α,
translation does not recover. These data show that two partially
redundant pathways are involved in translational recovery from a heat
shock.

1A_04_S

REGULATION OF SMALL HEAT SHOCK PROTEINS IN AGING AND
RESISTANCE TO STRESS

Robert M. Tanguay, Geneviève Morrow, Hyun-Ju Kim, Sébastien Michaud

Lab Cellular Developmental Genetics, CREFSIP, Dept Medecine, Pav.
Marchand, Université Laval, Québec, Canada G1K 7P4. E-mail:
[email protected]

Small HSP are involved in the refolding and/or disposal of protein
aggregates, a feature of many age-associated diseases. In Drosophila
melanogaster, there are 4 main small Hsps each residing in a different
intracellular compartment. Targeting the expression of the mitochondrial
Hsp22, to different cell types can increase lifespan by more than 30%.
Long-lived flies expressing Hsp22 in motorneurons have an increased
resistance to oxidative stress and maintain their locomotor activity longer.
Over expressing the cytosolic Hsp23 in a pan-neuronal fashion (transgenic
GAL4/UAS system) also increases longevity by 15%. Conversely, a strain
carrying an insertion in the promoter of hsp23 which downregulates its
expression in specific cells of the embryonic CNS and in adults has a
decreased lifespan. The action of these chaperones on lifespan likely
involves different pathways as suggested by the longevity curves, and
their respective site and developmental pattern of expression. Microarrays
analysis was used to unveil the mechanisms involved in lifespan. The
transcriptional changes brought by Hsp22 overexpression occur early in
adulthood and are associated with genes involved in energy metabolism,
protein biosynthesis, and protein folding. The relation between the
insulin/IGF signaling pathway and the heat shock response has also been
examined using flies with mutations in the heat shock factor HSF and
dFOXO. Altogether, these results confirm a beneficial role of the
expression of small chaperones and corroborate the pivotal role of the
nervous system and the insulin/IGF pathway in the ageing process.
Supported by CIHR (Canada) and the EU 6th Framework Programme
MiMage.

1B_01_S

FUNCTIONAL INTERPLAY AMONG THE MAJOR CHAPERONE
MACHINES OF E. COLI
Ronald S. Ullers1, Debbie Ang1, Françoise Schwager1, Pierre Genevaux2,
and Costa Georgopoulos1

1Département de Microbiologie et Médecine Moléculaire, Centre Médical
Universitaire, 1, Rue Michel-Servet, CH-1211 Geneva, Switzerland; and
2Laboratoire de Microbiologie et Génétique Moléculaires, Institut de
Biologie Cellulaire et de Génétique, Centre National de la Recherche
Scientifique, Université Paul-Sabatier, 118 Route de Narbonne, 31062
Toulouse Cedex 09, France

Polypeptides emerging from the ribosome are assisted by a pool of
molecular chaperones and targeting factors, which enable them to
efficiently partition as cytoplasmic, integral membrane, or exported
proteins. In Escherichia coli, the chaperones SecB, Trigger Factor (TF),
and DnaK are key players in this process. Here, we report that, as with
dnaK or dnaJ mutants, a secB null strain exhibits a strong cold-sensitive
(Cs) phenotype. Through suppressor analyses, we found that inactivating
mutations in the tig gene encoding TF fully relieve both the Cs phenotype
and protein aggregation observed in the absence of SecB. This
antagonistic effect of TF depends on its ribosome-binding and chaperone
activities but unrelated to its peptidyl-prolyl cis/trans isomerase’s (PPIase)
activity. Furthermore, in contrast to the previously known synergistic
action of TF and the DnaK/DnaJ chaperone machine above 30°C, a tig null
mutation partially suppresses the Cs phenotype exhibited by a
compromised DnaK/DnaJ chaperone machine. The antagonistic role of TF
is further exemplified by the fact that the secB dnaJ double mutant is
viable only in the absence of TF. Finally, we show that, in the absence of
TF, more SecA and ribosomes are associated with the inner membrane,
suggesting that the presence of TF directly or indirectly somehow
interferes with the process of cotranslational protein targeting to the Sec
translocon. Various models to explain our results (occasionally seemingly
contradictory) will be offered.

1B_02_S

ROLE OF HSP110 IN THE FUNCTIONAL NETWORK OF HSP70
CHAPERONES

Jocelyne Fiaux, Claes Andréasson, Heike Rampelt, Heather Sadlish,
Matthias Mayer and Bernd Bukau

ZMBH, University of Heidelberg, Im Neuenheimer Feld 282, D-69120
Heidelberg, Germany, [email protected]
Eukaryotic cells express Hsp110 proteins that constitute a diverged branch
of the Hsp70 molecular chaperone superfamily. Recently, both the yeast
Hsp110 member Sse1p and the mammalian orthologue Hsp105 were
found to act as potent nucleotide exchange factors for cytosolic Hsp70
chaperones. We set out to characterize the steps of the nucleotide
exchange cycle of the yeast Hsp70, Ssa1p, catalyzed by Sse1p using H/D
exchange and mass spectrometry. The mechanism was found to involve
formation of a stable but nucleotide-sensitive complex. Furthermore,
Sse1p itself displays nucleotide binding and hydrolysis and adopts
conformations dependent on the nucleotide binding status. Interestingly,
nucleotide binding by Sse1p appears to regulate its activity as a
nucleotide exchange factor. We also used H/D exchange and mass
spectrometry to map the interaction surface of the Sse1p-Ssa1p complex.
Based on these results, we can now propose a detailed model for the
Sse1p-catalyzed nucleotide exchange cycle.

1B_03_S

NETWORKS OF HSP70 AND J-PROTEIN MOLECULAR CHAPERONES

Elizabeth A. Craig, Alison Meyer and Chandan Sahi

Department of Biochemistry, University of Wisconsin, Madison WI, 53706,
USA [email protected]

Highly conserved molecular chaperones function in a wide variety of
cellular processes, including protein folding, translocation of proteins
across membranes and remodeling of protein complexes. J-proteins are
obligate partners of Hsp70s that act via their J-domains to stimulate
Hsp70’s ATPase activity, stabilizing their interactions with client proteins.
In addition many J-proteins contain additional domains, some of which are
capable of binding client proteins and allowing J-proteins to “deliver” them
to their partner Hsp70. Both Hsp70s and J-proteins are encoded by large
multigene families. Our results indicate that certain Hsp70s and J-proteins
have evolved to function in specialized cellular processes and/or have
“nontraditional” functions. For example, Ssz1 forms a stable heterodimer
with the ribosome-associated J-protein Zuo1 and is required for Zuo1’s
efficient stimulation of the ATPase activity of its partner Hsp70 Ssb. Jjj1, is
an example of a specialized J-protein. It plays an important role in the
biogenesis of 60S ribosomal subunits, likely facilitating the dissociation of
factors from preribosomes to generate subunits competent for translation.
In contrast, other J-proteins are multifunctional. The most abundant J-
protein of the yeast cytosol, Ydj1, functions in multiple cellular processes.
Surprisingly, however, expression of only a J-domain at normal levels is
capable of rescuing the severe growth defect caused by the absence of
Ydj1. Thus many functions carried out by this highly conserved and
complex J-protein require only the capacity to stimulate Hsp70s ATPase
activity, not the “delivery” of client proteins.

1B_04_S

HSP110 PROTEIN CHAPERONE FUNCTION IN YEAST

Kevin Morano

University of Texas Medical School, Micro and Molecular Genetics, 6431
Fannin, JFB 1.765, Houston, TX 77030, USA
[email protected]

SSE1 and SSE2 encode the essential yeast members of the Hsp70-related
Hsp110 molecular chaperone family. Both mammalian Hsp110 and the
Sse proteins functionally interact with cognate cytosolic Hsp70s as
nucleotide exchange factors, and in yeast Sse1 is required for Hsp90
chaperoning. We demonstrate that Sse1 forms high affinity heterodimeric
complexes with both yeast Ssa and mammalian Hsp70 chaperones, and
that ATP binding to Sse1 is required for binding to Hsp70s. The nucleotide
binding domains (NBD) of both Sse1/2 and the Hsp70s dictate interaction
specificity, and are sufficient to mediate heterodimerization with no
discernable contribution from the peptide binding domains (PBD).
However, the PBD is required for NEF activity. To better understand the
roles of the Hsp110 chaperones, we are investigating the participation of
Sse1 in cellular processes requiring Hsp70. To that end, we have
generated a novel temperature sensitive allele of SSE1 that should shed
light on the essential functions of this intriguing chaperone family.

1C_01_S

MULTI-SITE POST-TRANSLATIONAL MODIFICATIONS AND
FUNCTIONAL INTERPLAY OF HSF1 AND HSF2

Lea Sistonen

Turku Centre for Biotechnology and Department of Biology, Åbo Akademi
University, BioCity, Tykistökatu 6, 20520 Turku, Finland. Email:
[email protected]

Heat shock factors, HSFs, are specific transcriptional regulators of heat
shock genes encoding heat shock proteins, Hsps, that function as
molecular chaperones in protecting cells against proteotoxic stress. The
activity of HSFs is under stringent, mainly post-translational control, e.g.
acetylation, phosphorylation, sumoylation, and ubiquitylation. Among the
functional domains of HSFs, the amino-terminal helix-turn-helix DNA-
binding domain is the most conserved and it also designates membership
to the HSF family. The activation-induced trimeric assembly of HSFs is
mediated by hydrophobic heptad repeats and is unusual, as proteins
containing leucine zippers often form dimers. Four HSFs have been
identified in vertebrates, HSF1 and HSF2 being ubiquitously expressed and
well conserved throughout evolution, whereas HSF3 has been found only
in avian species and HSF4 only in mammals. The different members of the
mammalian HSF family have been considered to be functionally distinct;
HSF1 is essential for the heat shock response, whereas HSF2 and HSF4
are refractory to stress stimuli but are important for differentiation and
development, including corticogenesis, spermatogenesis, and maintenance
of sensory organs, e.g. lens and olfactory epithelium. We have, however,
evidence for a functional interplay between HSF1 and HSF2. These factors
can interact through their trimerization domains, and in response to
stress, they can bind to hsp promoters as well as to satellite III repeats at
locus 9q12. In both cases, an intact HSF1 is required, suggesting that
HSF1 influences the DNA-binding activity of HSF2. At the transcriptional
level, HSF2 is able to modulate HSF1-mediated expression of the target
genes in a gene-specific manner.

1C_02_S

HSF1 ACTIVATION AND THE CONTROL OF APOPTOSIS IN
CHEMORESISTANT CANCERS

G. Belardo, A. Rossi, S. Ciafre’, A. Ciucci, P. Gianferretti, S. Roberts and
M.G. Santoro

Department of Biology, University of Rome Tor Vergata and Institute of
Neurobiology and Molecular Medicine, CNR, Rome, Italy; Department of
Chemistry, University of Manchester, Manchester, UK.

Activation of the heat shock response (HSR) via HSF1 contributes to
preserve cellular function and homeostasis under stress conditions, and
to establish a cytoprotective state in several human diseases. In cancer,
however, HSR activation has been associated with both anti- and pro-
apoptotic responses. Heat-induced expression of cytoprotective and
antiapoptotic heat shock proteins (HSP) is a known complication of
hyperthermia, resulting in cancer cell thermotolerance and
chemoresistance. In some instances, however, HSF1 activation may
result in apoptosis induction. We have developed a library of novel potent
inducers of HSF1 which are characterized by pro-apoptotic activity in
several types of chemoresistant cancers. We have previously shown that
HSF1 induction prevents TNFα- and mitogen-induced activation of NF-κB,
a nuclear factor playing an important role in promoting inflammation, as
well as cell proliferation and survival. NF-κB has been found to be
constitutively activated in several types of chemoresistant cancers where
it suppresses cell death pathways by switching on genes that dampen
pro-apoptotic signals. We now show that activation of HSF1 by diverse
chemical inducers, as well as by hyperthermia itself, results in inhibition
of constitutive NF-κB activity and rapid down-regulation of the expression
of NF-κB-dependent survival genes, triggering apoptosis in aggressive
cancers presenting aberrant NF-κB regulation. The results suggest that
the block of anti-apoptotic signaling pathways utilizing the IκB kinase IKK
may play an important role in modulating HSR pro-apoptotic effects in
chemoresistant cancers.

1C_03_S

ROLES OF HSF1 IN INFLAMMATORY AND IMMUNE RESPONSE

Akira Nakai

Department of Biochemistry and Molecular Biology, Yamaguchi University
School of Medicine, Ube, Japan, e-mail: [email protected]

Inflammatory cytokines such as IL-1, IL-6, and TNF-α are induced in
response to bacterial infection and diseases. These cytokines elicit the
febrile response that is a complex physiological reaction to disease
including cytokine-mediated rise in body temperature and activation of
inflammatory systems. Fever plays beneficial roles on disease prognosis
clinically. Experimentally, pretreatment of animals with heat shock also
increases survival in a LPS-injected endotoxic model. These beneficial
roles of fever are mediated partly by suppressing pyrogenic and
inflammatory cytokines as expression of TNF-α, IL-1β, and IL-6 reduces in
heat-shocked cells and in whole body exposed to high temperature. It was
shown that HSF1 inhibits expression of cytokines by binding directly to
TNF-α promoter, or by physically interacting with NF-IL6, an activator for
IL-1β. However, molecular mechanisms underlining fever-mediated
suppression of cytokine gene expression are uncovered yet. Here we show
that heat shock suppresses LPS-induced induction of IL-6 by activating
HSF1 that induces ATF3, a negative regulator of IL-6. In vivo analysis
using HSF1-null and ATF3-null mice reveals that HSF1 as well as ATF3
acts as a negative regulator of IL-6 expression in whole body and is
required to inhibit fever. Unexpectedly, overexpression of ATF3 into cells
has no effect on IL-6 expression in the absence of HSF1. HSF1 also binds
directly to IL-6 promoter, and is required not only for a repressor ATF3,
but also an activator NF-kB to bind to IL-6 promoter by partially opening
chromatin structure. Taken together with the effects on expression of
TNF-α and IL-1β, these results indicate that HSF1 plays a major role in
feedback regulation of the febrile response.

1C_04_S

HSF2 INFLUENCES THE DECISION BETWEEN PROLIFERATION AND
MIGRATION FOR NEURAL CORTICAL PROGENITORS

Diane Trouillet, Anne Le Mouël, Rachid El Fatimy, Laurence Denis and
Valérie Mezger

CNRS UMR8541, Ecole Normale Supérieure, 46 rue d’Ulm 75005 Paris

Heat Shock Factors (HSFs) are not only responsible for response to
environmental stress, but are involved in developmental processes. We
showed in collaboration with Lea Sistonen’s lab that HSF2 is involved in
meiosis in both genders and in brain development (Kallio et al 2002;
Chang et al 2006). We will describe how HSF2 influences the radial
migration of young postmitotic neurons during cortical development, but
also the proliferation of their neural progenitors (NPCs), using loss and
gain of function models : Hsf2-/- mice and the overexpression of HSF2 in
the chick neural tube, by in ovo electroporation. The identification of new
HSF2 target genes suggests us that HSF2 directly regulates genes that are
involved in the control of microtubule dynamics, either in mitosis or during
migration. The question is how HSF2, which is active in NPCs as well as in
migrating postmitotic neurons, might differentially regulate distinct pools
of target genes in these cell populations. The ability of HSF2 to
differentially recruit transcription factors and chromatin modifiers as well
as its distinct biochemical properties in NPCs versus postmitotic neurons
has been investigated by biochemical and chromatin immunoprecipitation
(ChIP) analyses and will be discussed. Such a combination of events and
properties, coupled with the geography of HSF2 binding sites and of other
transcription factors sites –characteristic of a given target gene – should
allow HSF2 to induce or repress transcription and therefore to allow the
decision for a NPC to continue to divide or to exit the cell cycle and start
to migrate. Grant supports : ARC (Association pour la Recherche contre le
Cancer) and ANR Neurosciences (Agence nationale pour la Recherche).
1D_01_S

HSP RELEASE: PASSIVE VS ACTIVE RELEASE MECHANISMS

Alexzander Asea
Division of Investigative Pathology, Scott & White Clinic and Texas A&M
University System Health Science Center College of Medicine, 2401 South
31st Street, Temple, TX 76508 United States
E-mail: [email protected]

Thus far, two mechanisms are recognized by which heat shock proteins
(HSP) are released from cells into the extracellular milieu and
subsequently into systemic circulation; a passive release mechanism as a
result of necrotic cell death, severe blunt trauma, surgery and following
infection with lytic viruses, and an active release mechanism which
involves the non classical protein release pathway by which HSP70 is
released as free HSP72 and within highly immunologically potent
exosomes. This presentation covers the most recent findings on the
mechanisms by which stress induces the release of HSP72 into the
systemic circulation and addresses the biological significance of circulating
HSP72 to host defense against disease.

1D_02_S

HSP70 SECRETION AND BINDING: ITS PLACE IN THE IMMUNE
RESPONSE

Stuart K. Calderwood, Salamatu S Mambula, Jimmy Theriault and Jianlin
Gong

Beth Israel Deaconess Medical center, Harvard Medical School and Center
for Molecular Stress Response, Boston University Medical School.

Hsp70 plays a significant extracellular role within the immune response
and can carry out both pro-inflammatory / pro-immune functions and
anti-inflammatory effects depending on cellular and tissue context.
However, understanding its place in the immune response requires
deciphering the mechanisms by which it is released and the cell surface
structures which it binds on target cells in the immune system.
We have examined mechanisms of Hsp70 release from tumor cells and
macrophages and have found evidence for an active secretion mechanism.
Our experiments indicate that hsp70 secretion employs a similar pathway
to that used by other “leaderless” proteins such as interleukin 1β (IL-1β)
involving the entry of Hsp70 into secretory lysosomes and ATP dependent
release. Our evidence suggest however, that the trigger for hsp70 release