HEAT SHOCK

PROTEINS
What is Protein folding ?
Structural Levels of Proteins
Primary Secondary
 Protein stability
The situation becomes more complicated
since this stability must be ascertained in
a certain range of environmental conditions

pH Protein Temp.
Salt Conc.

a certain range of environmental conditions.The native

conformation of a protein is stable in a narrow range of
temperature, pH, chemical composition of solvent, etc.
 Forces involved in Protein stabilisation

Hydrogen Bonding
Vander Waals interactions
Ionic strengths
Disulfide bonds
Hydrophobicity: the dominant
force in protein folding
 A stably folded proteins has..

Hydrophobic side chains buried

Charged side chains on the surface
Cysteines form Covalent disulfide bonds
Pack as close together as possible
Minimize contacts between hydrophobic
groups and water
All these features will contribute to Minimum
energy state
 Reasons for protein misfolding

Mutations
Premature termination of Translation
Fault in post-translational modifications
Strong Promoters
High Inducer concentrations
Loss of conformation due to stress`
ALL CELLS
ALL ORGANISMS
Living in the World
Must cope with
Stress !!!
 What is stress?

In biology, stress is the driving force behind the

process of adaptation and evolution.
 Cell

Loss of Protein Network

Function failure

Death

Unfolded
Proteins Aggregates

Temp
cell
Folded
Proteins
Temp
environ
 Interesting story
F. Ritossa 1960 discovered the heat shock
(HS) response while observing the salivary
cells of Drosophila and named them HSPs

My name is
Chaperone
 HEAT SHOCK PROTEINS
Cover and back image
Conceptual image depicting
Hsps as protein-folding
chaperones in the
mitochondria. As nascent
mitochondrial peptides (light
blue) emerge from the
ribosome (purple), they are
bound by the mitochondrial
Hsp70 (orange) via its peptide
binding domain (revealed
schematic ribbon structure) to
prevent mis folding and
aggregation. Some proteins
require further folding
assistance by the Hsp60/Hsp10
(yellow) chaperonin
complexes. A single Hsp60
subunit of the chaperonin
complex is shown as a ribbon
structure.
 DEFINITION
Heat shock proteins are a group of highly conserved proteins
found in both eukaryotic and prokaryotic cells. They are involved in
a wide range of cellular processes such as assisting protein folding
and degradation of misfolded proteins, modulating signaling
pathways and regulating immune responses.
The multi-functional nature of heat shock proteins enables them to
play critical roles in the regulation of protein homeostasis and cell
survival.
 How do HSPs work?
One major function of chaperones is to prevent both newly
synthesised polypeptide chains and assembled subunits from
aggregating into non functional structures
High temperatures and other stresses, such as altered pH and oxygen
deprivation, make it more difficult for proteins to form their proper
structures and cause some already structured proteins to unfold
Heat Shock Proteins are induced rapidly at high levels to deal
with this problem
 Heat shock factor 1
MASTER REGULATOR OF HEAT SHOCK PROTEIN EXPRESSION
INACTIVE STATE-HSF1 monomers are held in complex with Hsp
70/hsp 90
ACTIVE STATE-That is on the onset of stress, 5 MAJOR steps occur-
1.Hsf 1 is released from the complex
2.It homo trimerizes
3.Translocates to nucleus
4.And activates the transcription of its downstream targets i.e Hsp 72
and Hsp 27
5.By binding to the HSE(heat shock elements) in the promoter
regions of target proteins.
 Different Types of Heat Shock Proteins
Heat Shock Proteins are classified by their molecular
weight, size, structure, and function.
They are divided into several families, namely -
1. HSP100
2. HSP90
3. HSP70
4. HSP60 (chaperonin)
5. Small Heat Shock Proteins/ (alpha)-crystalline
proteins
 Functions of HSP families
Family Major Functions
Hsp 100 Stress tolerance, Protein disaggregation, thermo tolerance

Regulatory interactions with signaling proteins,

Hsp 90 stabilization of misfolded proteins

Hsp 70 Protein folding, membrane transport of proteins,

Auto regulation in heat shock response, anti apoptotic
Protein folding (limited substrates in eukaryotic
Hsp 60 cytoplasm)

Hsp 40 Protein folding, co-chaperone for Hsp70

Small Stabilization of misfolded proteins, thermotolerance,

Hsps eye lens structural proteins
 Why Don't Heat Shock Proteins
Denature?

Better Hydrogen Bonds

Better Hydrophobic Internal Packing
Enhanced Secondary Structure
Helix Dipole Stabilization
 INTRODUCTION
All organisms exhibit homeostatic-like responses
when subjected to rapid changes in their
environment.
The ability of the organism to successfully adapt or
acclimate to its new environment is critical to its
survival, and likely represents an integral driving force
in evolution.
One well studied response to sudden adverse
environmental changes is the so-called heat shock or
stress response.
The resultant increase and accumulation of the Hsps
now gives the stressed cell added protection, thereby
allowing for continued cell survival.
In addition to increased temperatures, other insults
also result in increased Hsp expression.
These include exposure of cells to various metals,
amino acid analogues, hypoxia, and a large number
of agents/treatments which result in reduced ATP
levels. Because so many adverse conditions lead to
increased Hsp expression, the heat shock response
now is commonly referred to as the stress
response.
 HSP 90
Dimer

ATP dependent
HoP and p23
stabilizes proteins prior to complete folding or activation
forms stable complexes with inactive glucocorticoid receptor and other
transcription factors
most abundant non-ribosomal protein (cytosolic version)
most abundant protein in endoplasmic reticulum (ER version)
Represents almost 1% of total cellular proteins in unstressed cells.
Bacterial homologue: HTpG family (typically non essential proteins) ,
A functional Hsp90 is required for viability under all conditions in
eukaryotic cells.
HSP90 interacts with HSP40, HSC70/HSP90 organizing protein
(HOP), and co-chaperones to bind and stabilize newly synthesized substrate/client proteins. This ATPregulated
cycle of substrate binding is critical to the activation of many oncogenic signaling molecules.
 Members of hsp 90 family
Hsp 90a Cytosolic form, induced by elevated temperature; ATPase activity;
three splice variants
Hsp 90 b Cytosolic form, constitutively expressed; ATPase activity; three splice
variants
p23 Binds to telomerase and progesterone receptor; also functions as a
cytosolic prostaglandin E2 synthase; phosphorylated at Ser113, 118,
148 and 151 and acetylated at Lys33
P50/cdc37 A chaperone that binds Hsp90 and is required for the activity of
numerous protein kinases
TRAP1 Mitochondrial form; has ATPase activity that is inhibited by both
geldanamycin and radicicol; highly conserved through evolution;
phosphorylation by PINK1 prevents oxidative-stressinduced
apoptosis; four splice variants

Grp 94 ER form; glucose regulated and induced by glucose starvation;

participates in protein folding and assembly, protein secretion,
protecting cells from apoptosis, and mediating immunogenicity; C-
terminal sequence KDEL mediates retention in the ER; two splice
variants
Hsp90 is involved in the regulation of many of the hallmarks of
cancer.
namely sustaining proliferative signalling, resisting cell death,
evading growth suppressors, inducing angiogenesis, enabling
replicative immortality, invasion and metastasis, and emerging
hallmarks including deregulating cellular energetic and avoiding
immune destruction .
In addition, high expression of Hsp90 is an independent prognostic
marker in a number of cancers.
In breast cancer, it is associated with decreased survival.
in gastric cancer, high Hsp90 expression is linked to poor
prognosis and tumour aggressiveness .
In CML, Hsp90 correlates with disease state and high levels are
associated with resistance to therapy
Hsp90 proteins exist as homodimers of subunits consisting of an N-terminal ATPase domain,
a C-terminal dimerisation / protein interaction domain, and a middle domain associated with
client protein binding.
ATPase activity is essential for the chaperoning activity of Hsp90. Following the addition of
ATP, Hsp90 undergoes a conformational change, which induces an open to shut
conformation shift , with transient dimerisation of the N-terminal domains and N-M domain
association.
The middle segment of Hsp90 has been identified as the binding site for protein kinase
PKB/Akt and is implicated as the main site for client protein interactions . This segment can
also interact with cochaperones and is required for N-terminal ATPase activity.
The C-terminal domain is involved in dimerisation and contains a highly conserved EEVD
sequence which is required for the binding of tetratricopeptide repeat (TPR) containing
family of cofactors, such as HOP.
.
 Functions of hsp 90
Hsp90 along with one set of its co-chaperones (and the Hsp70/Hsp40 chaperone
machinery) binds to and stabilizes steroid hormone receptors in their inactive
state within the cytosol.
Upon subsequent binding to the appropriate steroid hormone ligand, the
receptor undergoes a conformational change resulting in its acquisition of DNA
binding and transcriptional activity.
In a similar scenario, Hsp90 along with another set of co-chaperones binds to
and stabilizes the newly synthesized forms of various protein kinases,
maintaining them in a folding-competent conformation.
Thus, via its utilization of numerous co-chaperones and ATP, the very abundant
Hsp90 chaperone functions in unstressed cells to regulate client proteins
important for growth and development.
Altering the levels of Hsp90 (via genetic means or manipulations with Hsp90
inhibitory drugs) leads to rapid alterations in cell signaling pathways and the
adaptation of new cellular phenotypes.
 Elevated levels of Hsp70 proteins have been linked with
inhibition of apoptosis as well as the resistance of cells
to various chemotherapeutic agents.
In addition, numerous studies continue to demonstrate
that changes in the levels of the different Hsp70 family
members may prove clinically useful for the diagnosis of
many important human diseases.
Purified Hsp70 proteins are available for biochemical
and immunological studies.
 HSP100
6-7 monomer
ATP
no co-chaperon is required
Functions
-solubilize protein aggregates thereby dissociating them
-facilitates proteolysis
-essential in yeast for acquired thermo tolerance
-essential for yeast prion propagation
 Members of the Hsp family participate in the early stages
of protein synthesis, protein folding, and the transport
of newly synthesized proteins from the cytoplasm into
different intracellular compartments.
Under conditions of stress, where protein
folding/assembly events may be compromised, the
increased expression and accumulation of the stress
proteins facilitates the ability of cells to both repair and
synthesize new proteins to replace those that were
damaged after the particular metabolic insult.
 Various medical conditions, including fever, ischemia,
hemodynamic overload or neurological injuries are well
known activators of the stress response in vivo.
The ability of the affected tissue or organ to mount a
robust stress response is thought to be important for its
survival and recovery.
In infectious diseases, stress proteins present within
different pathogens are known to be major targets of
our immune system.
Hsp 70/40 chaperone machinery

One of the largest stress protein families

Its bacterial counter part Dna K
Set of genes for both phage and host DNA
replication and therefore referred to as Dna J
and Grp E.
Three above genes work together in
facilitating the disassembly of large protein
complex necessary for commencement of
DNA replication.
Dna K function as a type of MOLECULAR
CROWBAR/DETERGENT.
 Dna J co chaperones family facilitates subtrate binding as well as
stimulate Dna K and ATP hydrolysis.
Grp E protein facilitates nucleotide exchange reaction needed to
allow for new reaction cycle.
Thus through repeated cycles of binding and releasing the Dna K
chaperone machinery helps prevent premature folding/
aggregation, therefore facilitating high fidelity protein maturation
throughout the cell.
This cycling of DnaK chaperone between the open and closed
states is regulated by the co-chaperones DnaJ (or Hsp40) and GrpE
(or BAG-1).
 MEMBERS OF Hsp 70

1.cytosolic/nuclear Hsp 70
2.Hsc 70/hsp 72
3.Hsp 70/hsp 72
4.Grp 78/Bip present in lumen of ER
5.Grp 75/MORTALIN present in mitochondria
Bag 1
Bag 2
hip
Co Chaperons-for Hsp 70 family

1.Hsp 40 -co chaperone for Hsc/Hsp 70

2.cytosolic Dna J homologue- co chaperone for
Hsc/Hsp 70
3.Dna J homologue(ER)- Bip
4.Dna J like protein- mortalin/Grp 75
5.hip,hop,CHIP- also influence chaperone machinery.
 Role in diseases
Hsp70 has been implicated as a potential autoantigen in MS.
In IDDM, the preferential expression of Hsp70 by cells, but not
cells, in the islets of Langerhans might be important for the
understanding of autoimmune destruction of cells in this disease.
Autoantibodies to the constitutive form of Hsp70 (Hsc70) have
been identified in a proportion of patients with primary biliary
cirrhosis (45.7%) and patients with autoimmune hepatitis patients
(52.9%), but not in patients with chronic hepatitis B or C infection.
Reactivity to Hsp70 has also been implicated in the induction of
disease in toxin induced interstitial nephritis.
 Role of hsp 70 in diseases
The cytoplasmic Hsp70s regulate the apoptosis pathway at multiple levels, for
example,
Hsp70s have been shown to protect Bcl-2 from proteasomal degradation,
block Bax translocation to the mitochondria thereby preventing cytochrome c
release
bind Apaf-1 and prevent the recruitment of caspase-9 to the apoptosome
and to prevent AIF translocation to the nucleus to cause chromatin condensation
and DNA degradation.
It is interesting to note that the function of Hsp70s do not always rely on their
ATPase activity, for instance it has been shown that Hsp72 inhibits JNK activation
independently of its chaperoning activity.
The Hsp70s also play a protective role against senescence. Hsp72 knock down
induces senescence in a variety of cancer cell lines , and Hsp72 controls Her-2-
induced senescence by regulating p21 and survivin in a mouse breast tumour
model
Evidence also suggests that Hsp70 supports autophagy by maintaining protein
homeostasis and supporting cancer cell survival. Hsp70 localises at the
autophagosome/ lysosomal membrane compartments and inhibits lysosomal
permeabilisation.
In addition, Hsp70 participates in chaperone mediated autophagy by delivering
target proteins to the lysosome surface receptor LAMP-2A, where it enables their
translocation into the lysosomal lumen
 Chaperone mediated autophagy
Evidence also suggests that Hsp70 supports autophagy by
maintaining protein homeostasis and supporting cancer cell survival.
Hsp70 localises at the autophagosome/ lysosomal membrane
compartments and inhibits lysosomal permeabilisation.
In addition, Hsp70 participates in chaperone mediated autophagy by
delivering target proteins to the lysosome surface receptor LAMP-
2A, where it enables their translocation into the lysosomal lumen
Hsp70 Cytoplasm/nucleus All: bind to extended
polypeptides; prevent
aggregation of
unfolded peptides;
dissociate some
oligomers; bind ATP
and show ATPase
activity, Mitochondria
Hsp70 is involved in
regulation of HSF1
activity and the
repression of heat
shock protein gene
transcription
Grp 78/Bip ER
Mt hsp70/Grp 75 mitochondria
 Hsp 110
Distant relative to Hsp 70
FUNCTION- ACTS AS NUCLEOTIDE exchanger factor for
the cytosolic Hsc/Hsp 70 proteins.
As an independent chaperone, unlike Hsp70, Hsp110
cannot assist protein folding, but acts to prevent protein
aggregation of denatured proteins with higher efficiency
compared to Hsp70
It also exhibits differential substrate binding properties
to Hsp70s with preference for substrates with aromatic
residues, and this may account for the different
chaperone activities of Hsp110 and Hsp70 .
 Hsp 60/10 family ( the chaperonins)
Participate in protein maturation events and have been given
special name CHAPERONINS.
Molecular weight 60 kDa
STRUCTURE: oligomer
Gro EL:
bacterial equivalent of HSP 60,has role in bacteriophage growth,
exists as a large homo oligomeric complex (approx 800 kDa),
Binds to unfolded proteins and can discriminate between folded and
unfolded proteins.
Co factor- Hsp 10(eukaryotes) and Gro ES (bacteria)
Function: bind newly synthesized polypeptides and facilitates
their folding to the nature state in an ATP dependent cycle.
 Functions
Specifically, binding and sequestration of the substrate polypeptide occurs within
the large central cavity of the chaperonin complex.
Thereby, reduce the probability of misfolding and aggregation of the target
protein with other polypeptides.
GroEL proteins from different pathogens elicit strong humoral and cellular
immune responses.
Finally, chaperonins are now proving useful as it leads to the in vitro folding of
recombinant proteins important for clinical medicine and therapeutic purposes.
 Structure of hsp 60
3 domains of hsp 60:
1. apical domain :
2. equatorial domain :binding site for ATP
3.intermediate domain : binds equatorial domain and apical
domain
induces conformational change when ATP is bound allowing for an
alternation between hydrophilic and hydrophobic substrate binding
sites.
In inactive state, the protein is in a hydrophobic state.
When activated by ATP-intermediate domain undergoes
conformational change and exposes the hydrophilic region.
Hsp 10-acts like a lid or dome like cover on the ATP active form of
HSP60.this causes the central cavity to enlarge and helps in protein
folding.
 Hsp60/Hsp10 and TRiC/CCT chaperonins. The group I (mitochondrial) and group II
(cytosolic) chaperones are large oligomeric complexes, Hsp60/Hsp10 and TriC/ CCT, involved
in ATP-dependent folding of client proteins such as aconitase and actin, respectively.
 GroEL protein
Most notable is the bacterial GroEL protein, the
so called common antigen, which elicits both a
strong humoral and cellular immune response
whenever animals are infected with different
microbes.
Oligomer formed by monomers.
Monomers are arranged in 2 stacked heptameric
rings
These rings form a barrel like cavity.
In this cavity misfolded/unfolded substrate
proteins are folded.
GroES(hsp 10),forms a single heptameric ring,acts
as a lid to the chamber and can bind to either end
of double GroEL rings.
A) Reconstruction of the
GroEL structure with and
without the GroES
lid from cryoelectron
microscopy pictures.
B) Model of the GroEL
chaperone cycle. Two
misfolded proteins (green
and blue) are simultaneously
folded in a phase-shifted
manner. The red circles
symbolize the hydrophobic
substrate binding sites of
GroEL
 How GroEL works
ATP and polypeptide binds to one GroEL ring
followed by GroES capping, resulting in the encapsulation of
polypeptide in a hydrophilic cavity which promotes protein folding
conditions.
Once the substrate is inside the chamber, ATP is hydrolysed slowly,
allowing time for the protein to fold.
The two rings of GroEL act in an alternate fashion, with ATP
hydrolysis in one ring resulting in a structural transition in the
opposite ring making it available for ATP binding.
Which in turn triggers the release of GroES and substrate protein
from the original ring
A substrate protein may go through multiple binding and release
cycles to reach its folded state.
 Members of chaperonin family

Hsp 60 Mitochondrial protein, essential for folding and

assembly of newly imported proteins; also a signaling
molecule in the innate immune system; mutations
associated with autosomal recessive spastic paraplegia

Hsp 10 Closely linked to the Hsp60 gene (HspD1); forms

chaperonin 'cap' structure

Tcp 1 Member of the chaperonin containing TCP1 complex

(CCT), also known as the TCP1 ring complex (TRiC),
consisting of two identical stacked rings, each containing
eight different proteins; the complex folds various
proteins, including actin and tubulin, in an ATP-
dependent manner; two alternative splice forms; unlike
Hsp60, no known associated Hsp10/GroES cofactor

CCT2,CCT3,CCT4,CCT5,CCT6A,CCT Components of TRiC complex

6B,CCT7
Hsp 60 as immuno modulators and
intercellular signaling molecules

Human Hsp60 activates CD45RA+RO (naive) T cells,

bacterial-specific peptides activate CD45RARO+
(memory) T cells and bacterial Hsp60 activates both
CD45RA+RO and CD45RARO+ T cells . The
observation that both types of T-cell subset are
activated by bacterial Hsp60 indicates that T cells can
recognise and respond to conserved (self) epitopes
on the whole bacterial molecule
 Role of hsp 60 in diseases
1. rheumatoid arthiritis

Hsp60 is expressed in the synovial tissue of patients with rheumatoid arthritis

(RA) and juvenile chronic arthritis , and T cells derived from the synovial fluid are
activated by mycobacterial Hsp65 .
T-cell reactivity to self-Hsp60 has been reported in patients with RA
immortalised B cells from the synovial tissue of RA patients show specificity for
bacterial Hsp60 and elevated levels of circulating antibodies to Hsp60 are
present in children with juvenile chronic arthritis
. T-cell-mediated responses to mycobacterial Hsp65 have also been implicated in
experimental models of arthritis, and disease can be initiated in rats by the
transfer of T-cell clones specific for mycobacterial Hsp65
In addition, antibodies to Hsp65 are elevated in mice with pristane-induced
arthritis
 2. Diabetes mellitus
Evidence of a role for Hsp60 in type 1 diabetes [insulin-dependent
diabetes mellitus (IDDM)] is somewhat equivocal .
Supporting such a role is evidence that naive T cells from non-obese
diabetic (NOD) mice can be activated by both self-Hsp60 and
mycobacterial Hsp60 ,
that anti-Hsp60 T cells can mediate insulitis and hyperglycaemia in the
NOD mouse ,
and that peripheral blood T cells from patients with IDDM demonstrate a
heightened proliferative response to human Hsp60 and Hsp60 peptides.
However, in NOD mice, immunity to autoantigens other than heat shock
proteins, such as glutamic acid decarboxylase 65 (GAD), appears much
earlier than responsiveness to mycobacterial Hsp65 , thereby arguing
against an essential role for heat shock proteins in disease induction in
this model.
In addition, no evidence for serological immunity to islet cell heat shock
proteins has been reported in IDDM .
 3. Multiple sclerosis
Evidence of a role for Hsp60 in the pathogenesis of multiple
sclerosis (MS) is less apparent.
Hsp60 expression has been identified in chronic MS plaques , and a
humoral response to Hsp60 has been detected in the cerebrospinal
fluid of patients with MS;
however, the latter is not specific for MS and is also present in a
number of chronic degenerative conditions .
Nor is peripheral blood lymphocyte reactivity to Hsp60 altered in
MS patients.
 sHsp
most widespread
Most conserved

8-24 monomer
exhibit chaperone activity in vitro
thermo protection in vivo
produced at significant levels in cells experiencing heat stress
most are heat inducible, but some are synthesized in unstressed
conditions-such as for cell development.
Mol wt: 14-45 kDa with most in the 20 kDa range
While bacteria and single-cell eukaryotes express only one or two
members, Drosophila melanogaster expresses 16, humans 10, and
plants as many as 19
Denatured or unfolded substrates bind to the hydrophilic surface of small HSP complexes and prevent the substrate
from aggregating.The substrate either stays sequestered or is released to be refolded or degraded.
 Members of small hsp family
The best characterized member of the family, a-crystallin is
abundant in the lens where the overall protein concentration is
quite high.
In such a crowded environment the a-crystallins (A and B) are
thought to help prevent protein aggregation resulting from light
damage and/or other metabolic insults.
Similarly, other members of the low molecular weight Hsps are
now thought to function as ATP-independent molecular
chaperones.
Via their large surface and potential to recognize and bind exposed
hydrophobic patches, Hsp27 and its counterparts may act to bind
unfolded proteins and then present their substrates to the other
ATPdependent molecular chaperone machineries (e.g. Hsp60,
Hsp70 or Hsp90) for subsequent re-folding
 Hsp 27
Functions:
to prevent protein aggregation by directly binding misfolded substrates,
promoting protein refolding by interaction with the Hsp70 chaperone
complex.
In addition, Hsp27 can directly prevent cell death by interfering with key
components of the apoptosis pathway, such as blocking the formation
of the apoptosome by binding to cytochrome c released from the
mitochondria and by interacting with Daxx, a mediator of Fas-induced
apoptosis
Under stress conditions, Hsp27 is also directly involved in the ubiquitin-
proteasome pathway by binding to the 26S proteasome and multi-
ubiquitin chains, to facilitate the degradation of a selective range of
target proteins. By doing so, Hsp27 can mediate its cytoprotective effect
at multiple levels by facilitating the degradation of various apoptotic and
cell cycle proteins. For example,
Hsp27 can enhance the anti-apoptotic activtity of the transcription factor
NF-B, as the presence of Hsp27 in the proteasome-protein substrate
complex is required for the degradation of I-B, the inhibitor of NF-B.
Hsp27 can also promote the degradation of the cell cycle inhibitor p27,
thereby avoiding cell cycle arrest during stress.
 sequence homology with the a-crystalline proteins;
In the case of the human low molecular weight Hsp,
collectively termed the Hsp27 family, the proteins are
found in complexes of 400 to 500 k Da.
Phosphorylation of Hsp27, in response to different
stimuli, may play a role in the oligomeric dynamics of
the protein.
Crystallin alpha-A Expression restricted to the lens
Crystallin alpha-B Broad tissue expression; elevated expression in many neurological
diseases; a missense mutation associated with a desmin-related
myopathy
Crystallin beta-A1 Mutation causes the autosomal dominant disease 'zonular cataract
with sutural opacities'; member of the acidic group of b crystallins;
b-crystallins form aggregates of different sizes and are able to self-
associate to form dimers or to form heterodimers with other b-
crystallins.
Crystallin, gamma- N b-g hybrid crystallin; expressed in retina and lens nuclear fibers in
rodents
Heat shock 22kDa Charcot-Marie-Tooth disease type 2L; Hereditary motor
protein 8 neuropathy type II
Heat shock 27kDa Associates with myotonic dystrophy protein kinase (DMPK)
protein 2
Heat shock protein, a- Structural component of eye lens
crystallin-related, B6
Heat shock protein, a- Testes specific
crystallin-related, B9
 Other stress proteins :Hsp 47

ER-localized
member of the serpin family of serine protease
inhibitors;
expression induced by heat shock;
binds collagen and thought to be a chaperone
involved in the maturation of collagen;
auto-antibodies found in patients with rheumatoid
arthritis
 Protein Disulfide Isomerases (PDI)
Protein Disulfide Isomerases (PDI) are numerous, with
the different family members likely acting on both
common and distinct protein targets. Finally, because
many secreted or membrane localized proteins are
modified by glycosylation, the ER contains a number of
lectin-like chaperones including calnexin and calreticulin.
These latter chaperones recognize carbohydrate
moieties and therefore ensure that proteins being read
for secretion are properly glycosylated and folded prior
to their transport out of the ER. It is believed that all of
the ER localized chaperones together provide for a type
of cellular quality control.
 calnexin

Calcium-binding ER protein
interacts transiently with newly synthesized N-linked
glycoproteins,
facilitating protein folding and assembly;
it may also play a central role in the quality control of
protein folding by retaining incorrectly folded protein
subunits within the ER for degradation.
Transmembrane protein
 calreticulin
Calcium-binding ER protein;
also found in the nucleus;
can bind and inhibit nuclear hormone receptors.
Non- transmembrane protein.
Function:Calreticulin binds to misfolded proteins and
prevents them from being exported from ER to the Golgi
apparatus.
 Haeme oxygenase(decyclizing)
HMOX1, Hsp Haeme oxygenase (decyclizing) 32
Highly inducible by heavy metals, endotoxin, oxidizing agents, UVA;
cleaves heme ring at the a methene bridge to form biliverdin
Haeme oxygenase (decyclizing) 2, HMOX2
Non-inducible;
cleaves heme ring at the a methene bridge to form biliverdin.
Pathogenic Role of hsps in diseases

Heat shock proteins might provide a link between

infection and autoimmunity, either through
recognition of conserved epitopes or via cross-
reactivity/molecular mimicry .
Evidence for a link between heat shock protein
reactivity and disease pathogenesis, particularly
autoimmune disease, vascular disease and organ
allograft rejection, has arisen from several studies.
 Hsp role in neurodegenerative
diseases-ALZHEIMERS DISEASE
The histopathological hallmark of ND diseases is the accumulation
of inclusions of disease causing proteins in residual neurons in
targeted regions.
The inclusions combine with many components of molecular
chaperone pathways and ubiquitin proteasome, raising the
possibility that mis folding and altered degradation of mutant
proteins may be involved in pathogenesis of ND diseases
Overexpression of Hsps have been reported to reduce the number
and size of inclusions and accumulation of disease causing proteins.
With the use of pathophysiology of Hsps and using animal models
has led to the development of disease modifying drugs, i.e. Hsp90
inhibitors and Hsp inducers which inhibit the pathogenic process
of ND. Hsp90 inhibitors also exert therapeutic effects through
selective proteasome degradation of its client protein.
 Kakimura found that extracellular hsps, such as Hsp90,Hsp70 and
Hsp 32, facilitate A clearance by activation of microglial
phagocytosis and A degradation.
High affinity Hsp-CHIP complex recognizes and selectively degrades
phosphorylated tau proteins in AD. A CRITICAL MEDIATOR OF THIS
MECHANISM IS CARBOXY TERMINUS OF HSP70 INTERACTING
PROTEIN(CHIP) ,A TAU UBIQUITIN LIGASE.
Co chaperones were involved in Hsp90 mediated removal of p-
tau,while the maure Hsp90 refolding complex prevented this effect.
THIS MAY PROVIDE A RATIONALE FOR DEVELOPMENT OF NOVEL
HSP90 BASED THERAPEUTIC STRATEGIES.
 PARKINSONS DISEASE
Lewi bodies with aberrant misfolding and aggregation are common
pathologic hallmark of PD, again due to abnormality in protein
homeostasis.
It was found that Hsp70 could inhibit SN (alpha synuclein) fibril
formation through preferential binding to prefibrillar species to
change the characteristics of toxic SN aggregates
SN plays a critical role in regulating synaptic vesicle size with
particular relevance to dopamine storage.
SN at nanomolecular concentration is able to increase Hsp70
protein level in cells, which can reduce SN aggregation and
toxicity.
 Potential role of hsps reactivity in
transplantation
Heat shock proteins and reactivity to heat shock proteins have been associated
with allograft rejection.
Heat shock proteins are induced during graft preservation, ischemia
reperfusion and surgery , and by the inflammatory process of the rejection
response, including the localized production of cytokines by infiltrating
leukocytes .
Studies In rats,
Hsp70 gene and protein expression are increased in rejecting cardiac allografts,
and graft-infiltrating lymphocytes proliferate in response to recombinant
mycobacterial Hsp65 and Hsp71 .
Heat shock protein expression is also induced in the intestinal epithelium and
lamina propria after rat small-bowel transplantation and appears, in part at least,
to be resistant to immunosuppression with tacrolimus
Studies In humans, heat shock protein expression is increased in rejecting lungs
T cells from rejected renal grafts respond to Hsp72
and mycobacterial Hsp65-induced growth of graft-infiltrating lymphocytes from
endomyocardial biopsies correlates with cardiac graft rejection
 On the basis of above findings

These findings have led to the proposition that hsp expression in

allograft tissue induces heat shock protein reactivity, thereby
promoting the development of acute and chronic graft rejection.
However, heat shock proteins are cytoprotective molecules and
their induction in the peri and immediate post-transplantation
periods is likely to be a protective response targeted towards the
maintenance of cell and tissue integrity.
This is supported by reports that heat shock proteins attenuate
preservation and ischaemiareperfusion injury and that they
protect endothelial cells from neutrophil-mediated necrosis and a
variety of cell types from oxidative injury.
In addition, lower levels of Hsp70 in pre-liver-transplant biopsies
and organ perfusates are associated with early graft loss.
Continued-

The precise influence of heat shock proteins on

allograft survival is currently unclear.
A direct involvement of Hsp60 in the rejection
process has been suggested by the observations that
skin from transgenic mice overexpressing Hsp60
transplanted into allogeneic recipients is rejected
more rapidly than skin transplanted from wild-type
donors.
By contrast, skin transplanted into Hsp60-transgenic
mice, in which spontaneous autoimmunity to Hsp60 is
reduced, is rejected more slowly than skin grafted
into wild-type recipients.
 Role of hsp reactivity and vascular
disease
First, the intensity of heat shock protein expression positively
correlates with the severity of atherosclerosis.
Second, there is a localized enrichment of T cells in the lesion ,
and this is of particular interest given the capacity of T cells to
directly recognize and respond to autologous heat shock proteins.
Third, immunization with recombinant mycobacterial Hsp65 can
induce atherosclerotic lesions in normo cholesterolaemic rabbits.
 Continued-

The inflammatory response associated with atherosclerosis might

in part be promoted by the activation of T cells reactive with the
Hsp60 that is expressed on monocytes within the lesion or released
locally.
Localized expression of heat shock proteins might also be
influenced by hemodynamic factors, as raised blood pressure has
direct effects on the vasculature and vessels subjected to greater
mechanical and shear stress express heat shock proteins and are
more prone to the development of atherosclerosis.
 Humoral responses to heat shock proteins have also been
implicated in vascular disease. Elevated levels of circulating
antibody to the mycobacterial 65 kDa heat shock protein have
been reported in carotid atherosclerosis, coronary heart disease
and borderline hypertension.
Levels of antibodies to human Hsp60 are also raised in peripheral
vascular disease .
However, hsps have been shown to mediate endothelial cell
cytotoxicity
Levels of anti-Hsp65 antibodies shown to have diagnostic value as
titres have recently been shown to predict the 5-year mortality of
patients with carotid atherosclerosis.
 Hsp in aging
With increasing age there is progressive decline in hsp levels.
Therefore, studies have suggested that an attenuated heat shock protein
response could contribute the increased susceptibility to environmental
challenges and the more prevalent morbidity and mortality seen in aged
individuals .
In vitro studies have shown that Hsp70 expression in heat-stressed lung
cells, hepatocytes, myocardium and mononuclear cells is reduced with
increasing age, as is the induction of Hsp70 expression in response to
ischemia and mitogenic stimulation.
Hsp70 gene expression declines during normal aging in human retina, and
heat shock-induced Hsp70 expression is decreased in senescent and late-
passage cells, both of which suggest that the process of aging itself might
be associated with reduced Hsp70 production to the increased
susceptibility to environmental challenges and the more prevalent
morbidity and mortality seen in aged individuals .
 Hsp 72 protects against obesity
induced insulin resistance

Patients with DM-II have decreased expression of Hsp 72,

which leads to decreased insulin sensitivity.
On heat therapy ,Hsp 72 activation leads to increased insulin
sensitivity.
Activation of several inflammatory signalling proteins such as
c-jun amino terminal kinase(JNK), of kappa B kinase and
TNF-, induce insulin resistance,BUT HSP72 CAN BLOCK THE
INDUCTION OF THESE MOLECULES IN VITRO.
THEREFORE, heat shock therapy was applied there was
elevation of Hs
 Autoimmune diseases
de Graeff-Meeder and co-workers observed in patients with
juvenile chronic arthritis, in whom the disease follows a relapsing
remitting rather than progressive course,
the presence of circulating T cells responsive to human (self)
Hsp60 was beneficial.
These T cells were of the regulatory T helper 2 (Th2) phenotype,
whereas T cells reactive with the 65 kDa mycobacterial antigen
Hsp65 displayed the inflammatory Th1 phenotype and their
presence correlated with disease severity .
The ability of Hsp60 peptides to modulate adjuvant arthritis
appears to reside in the capacity of induced regulatory T cells to
produce IL-10, as well as IL-4 and interferon (IFN-)n of T cells
from the synovial fluid of RA patients with humans.
 Development of HSP as a vaccine
vehicle
Two Hsp 70 genes are located within the MHC class III region
between complement and TNF genes, similarly, Hsp 90 is linked to
minor histocompatibility locus, therefore Hsps appear to play
variety of important roles in TNF genes.

It has been found that immunization with antigens genetically

fused with Hsp 70 elicits strong and long lived humoral and
cellular immune responses in the absence of adjuvant.(in mice)
It has been seen that recombinant protein consisting of HIV-1 p24
Ag fused to amino terminus of mycobacterial Hsp 70 (producing
fusion protein) elicits both humoral and cellular responses in mice.
These antibody responses were stronger and more persistant upto
68 wks.
Splenocytes from mice immunized with fusion protein
proliferated and produced IFN , IL-2 and IL-5 in response to in
vitro stimulation.
Later they fused oval albumin(ova), a well characterized T cell Ag,
to the amino terminus of mycobacterial Hsp 70
Mice were immunized twice with OVA produced OVA specific
cytotoxic T cells (CTL) with lysed cells expressing the immuno
dominant OVA MHC class 1 epitope; (SINFEKL)
THIS WAS AN UNEXPECTED RESULT, as immunization with soluble
proteins especially in the absence of adjuvant,rarely elicit CTL
responses which is CD4 independent.
 Chaperone function of hsps delivers the fusion protein to
intracellular compartments of APCs for processing into short
peptides and loading onto MHC class 1.
CTLs were also induced when CD4 knock out mice was immunized
with OVA mycobacterium Hsp 70
A 200 a.a domain of Hsp 70 is sufficient to elicit CTL
Therefore,above mechanism suggests tht hsp 70 may be useful
vehicle for development of prophylaxis and therapy of HIV 1 and
oppurtunistic infections.
Thank you..

Dr. Shahar Bano Khan

P.G.1 Dept of Pathology