1. Cell
Stress
and
Chaperones:
basic
concepts
-­‐
Harm
H.
Kampinga
Cell
Stress
and
Chaperones:
basic
concepts
Harm H. Kampinga
Dept. of Cell Biology, UMC Groningen, The Netherlands
1

2. Cell
Stress
and
Chaperones:
basic
concepts
-­‐
Harm
H.
Kampinga
Originally,
heat
shock
genes
(Ritossa,
1962)
and
later
heat
shock
proteins
(Tissieres
et
al
1974),
were
described
to
be
genes/proteins
of
which
the
expression
increased
aBer
an
heat
shock.
Ritossa,
1962
Tissieres
et
al
.,
1974
2

3. Cell
Stress
and
Chaperones:
basic
concepts
-­‐
Harm
H.
Kampinga
Originally,
heat
shock
genes
(Ritossa,
1962)
and
later
heat
shock
proteins
(Tissieres
et
al
1974),
were
described
to
be
genes/proteins
of
which
the
expression
increased
aBer
an
heat
shock.
Heat
shock
proteins
(HSP)
were
defined
as
proteins
encoded
by
genes
that
contain
so-­‐called
“heat
shock
elements
(HSE)
in
their
genes
that
are
regulated
by
the
“heat
shock
transcripNon
factor-­‐1
(HSF-­‐1)”
3

4. Cell
Stress
and
Chaperones:
basic
concepts
-­‐
Harm
H.
Kampinga
Originally,
heat
shock
genes
(Ritossa,
1962)
and
later
heat
shock
proteins
(Tissieres
et
al
1974),
were
described
to
be
genes/proteins
of
which
the
expression
increased
aBer
an
heat
shock.
Heat
shock
proteins
(HSP)
were
defined
as
proteins
encoded
by
genes
that
contain
so-­‐called
“heat
shock
elements
(HSE)
in
their
genes
that
are
regulated
by
the
“heat
shock
transcripNon
factor-­‐1
(HSF-­‐1)”
Akerfelt
et
al.,
2011
3

5. Cell
Stress
and
Chaperones:
basic
concepts
-­‐
Harm
H.
Kampinga
Originally,
heat
shock
genes
(Ritossa,
1962)
and
later
heat
shock
proteins
(Tissieres
et
al
1974),
were
described
to
be
genes/proteins
of
which
the
expression
increased
aBer
an
heat
shock.
Heat
shock
proteins
(HSP)
were
defined
as
proteins
encoded
by
genes
that
contain
so-­‐called
“heat
shock
elements
(HSE)
in
their
genes
that
are
regulated
by
the
“heat
shock
transcripNon
factor-­‐1
(HSF-­‐1)”
Nowadays,
we
know
that
many
situaNons,
both
physiologically
and
stressful
condiNons
can
acNvate
HSF-­‐1
Morimoto
1998
4

6. Cell
Stress
and
Chaperones:
basic
concepts
-­‐
Harm
H.
Kampinga
Originally,
heat
shock
genes
(Ritossa,
1962)
and
later
heat
shock
proteins
(Tissieres
et
al
1974),
were
described
to
be
genes/proteins
of
which
the
expression
increased
aBer
an
heat
shock.
Heat
shock
proteins
(HSP)
were
defined
as
proteins
encoded
by
genes
that
contain
so-­‐called
“heat
shock
elements
(HSE)
in
their
genes
that
are
regulated
by
the
“heat
shock
transcripNon
factor-­‐1
(HSF-­‐1)”
Nowadays,
we
know
that
many
situaNons,
both
physiologically
and
stressful
condiNons
can
acNvate
HSF-­‐1
There
are
many
members
in
at
least
5
different
heat
shock
proteins
families
(HSP90,
HSP70,
HSP40,
chaperonins,
and
small
HSP).
sHsp Hsp40 Hsp90
Chaperonins
(HspB: 10 members) (DnaJ: 50 members) (HspC: 5 members)
(HspD/E+ CCT: 2+12 members)
Hsp70
(HspA+H: 13+4 members)
Kampinga
et
al.,
Cell
Stress
Chaperones.
(2009)
5

7. Cell
Stress
and
Chaperones:
basic
concepts
-­‐
Harm
H.
Kampinga
HSP
belong
to
the
group
of
Molecular
Chaperones
6

8. Cell
Stress
and
Chaperones:
basic
concepts
-­‐
Harm
H.
Kampinga
HSP
belong
to
the
group
of
Molecular
Chaperones
A
chaperone
is
an
adult
or
typically
older
person
who
accompanies
or
supervises
one
or
more
young,
unmarried
men
or
women
during
social
occasions
usually
with
the
specific
intent
of
prevenNng
inappropriate
social
or
sexual
interacNons
or
illegal
behavior
(e.g.,
underage
drinking,
drug
use).
6

9. Cell
Stress
and
Chaperones:
basic
concepts
-­‐
Harm
H.
Kampinga
HSP
belong
to
the
group
of
Molecular
Chaperones
A
chaperone
is
an
adult
or
typically
older
person
who
accompanies
or
supervises
one
or
more
young,
unmarried
men
or
women
during
social
occasions
usually
with
the
specific
intent
of
prevenNng
inappropriate
social
or
sexual
interacNons
or
illegal
behavior
(e.g.,
underage
drinking,
drug
use).
6

10. Cell
Stress
and
Chaperones:
basic
concepts
-­‐
Harm
H.
Kampinga
HSP
belong
to
the
group
of
Molecular
Chaperones
A
chaperone
is
an
adult
or
typically
older
person
who
accompanies
or
supervises
one
or
more
young,
unmarried
men
or
women
during
social
occasions
usually
with
the
specific
intent
of
prevenNng
inappropriate
social
or
sexual
interacNons
or
illegal
behavior
(e.g.,
underage
drinking,
drug
use).
6

11. Cell
Stress
and
Chaperones:
basic
concepts
-­‐
Harm
H.
Kampinga
HSP
belong
to
the
group
of
Molecular
Chaperones
A
chaperone
is
an
adult
or
typically
older
person
who
accompanies
or
supervises
one
or
more
young,
unmarried
men
or
women
during
social
occasions
usually
with
the
specific
intent
of
prevenNng
inappropriate
social
or
sexual
interacNons
or
illegal
behavior
(e.g.,
underage
drinking,
drug
use).
6

12. Cell
Stress
and
Chaperones:
basic
concepts
-­‐
Harm
H.
Kampinga
HSP
belong
to
the
group
of
Molecular
Chaperones
DefiniNon:
any
protein
(HSP
being
the
largest
group),
which
interacts,
stabilizes
or
helps
a
non-­‐naNve
protein
to
acquire
its
na?ve
conforma?on
but
is
not
present
in
the
final
funcNonal
structure
7

13. Cell
Stress
and
Chaperones:
basic
concepts
-­‐
Harm
H.
Kampinga
HSP
belong
to
the
group
of
Molecular
Chaperones
DefiniNon:
any
protein
(HSP
being
the
largest
group),
which
interacts,
stabilizes
or
helps
a
non-­‐naNve
protein
to
acquire
its
na?ve
conforma?on
but
is
not
present
in
the
final
funcNonal
structure
7

14. Cell
Stress
and
Chaperones:
basic
concepts
-­‐
Harm
H.
Kampinga
HSP
belong
to
the
group
of
Molecular
Chaperones
DefiniNon:
any
protein
(HSP
being
the
largest
group),
which
interacts,
stabilizes
or
helps
a
non-­‐naNve
protein
to
acquire
its
na?ve
conforma?on
but
is
not
present
in
the
final
funcNonal
structure
7

15. Cell
Stress
and
Chaperones:
basic
concepts
-­‐
Harm
H.
Kampinga
HSP
belong
to
the
group
of
Molecular
Chaperones
DefiniNon:
any
protein
(HSP
being
the
largest
group),
which
interacts,
stabilizes
or
helps
a
non-­‐naNve
protein
to
acquire
its
na?ve
conforma?on
but
is
not
present
in
the
final
funcNonal
structure
7

16. Cell
Stress
and
Chaperones:
basic
concepts
-­‐
Harm
H.
Kampinga
HSP
belong
to
the
group
of
Molecular
Chaperones
DefiniNon:
any
protein
(HSP
being
the
largest
group),
which
interacts,
stabilizes
or
helps
a
non-­‐naNve
protein
to
acquire
its
na?ve
conforma?on
but
is
not
present
in
the
final
funcNonal
structure
8

17. Cell
Stress
and
Chaperones:
basic
concepts
-­‐
Harm
H.
Kampinga
HSP
belong
to
the
group
of
Molecular
Chaperones
DefiniNon:
any
protein
(HSP
being
the
largest
group),
which
interacts,
stabilizes
or
helps
a
non-­‐naNve
protein
to
acquire
its
na?ve
conforma?on
but
is
not
present
in
the
final
funcNonal
structure
8

18. Cell
Stress
and
Chaperones:
basic
concepts
-­‐
Harm
H.
Kampinga
HSP
belong
to
the
group
of
Molecular
Chaperones
DefiniNon:
any
protein
(HSP
being
the
largest
group),
which
interacts,
stabilizes
or
helps
a
non-­‐naNve
protein
to
acquire
its
na?ve
conforma?on
but
is
not
present
in
the
final
funcNonal
structure
The
basic
chaperone
acNon
comprise
an
iteraNve
cycles
of
client
binding
to
and
release
from
HSPs
in
which
clients
may
fold
or
may
be
degraded
if
folding
is
not
successful
Kampinga & Craig - Nature Reviews | Molecular Cell Biology
9

19. Cell
Stress
and
Chaperones:
basic
concepts
-­‐
Harm
H.
Kampinga
HSP
belong
to
the
group
of
Molecular
Chaperones
DefiniNon:
any
protein
(HSP
being
the
largest
group),
which
interacts,
stabilizes
or
helps
a
non-­‐naNve
protein
to
acquire
its
na?ve
conforma?on
but
is
not
present
in
the
final
funcNonal
structure
The
basic
chaperone
acNon
comprise
an
iteraNve
cycles
of
client
binding
to
and
release
from
HSPs
in
which
clients
may
fold
or
may
be
degraded
if
folding
is
not
successful
Such
acNon
is
required
for
assisted
folding
of
nascent
proteins
and
for
protein
translocaNon
(consNtuNvely
expressed
HSP)
and
for
refolding
of
stress-­‐unfolded
proteins
(consNtuNve
and
heat-­‐inducible
HSP).
Kampinga & Craig - Nature Reviews | Molecular Cell Biology
9

20. Cell
Stress
and
Chaperones:
basic
concepts
-­‐
Harm
H.
Kampinga
HSP
belong
to
the
group
of
Molecular
Chaperones
DefiniNon:
any
protein
(HSP
being
the
largest
group),
which
interacts,
stabilizes
or
helps
a
non-­‐naNve
protein
to
acquire
its
na?ve
conforma?on
but
is
not
present
in
the
final
funcNonal
structure
The
basic
chaperone
acNon
comprise
an
iteraNve
cycles
of
client
binding
to
and
release
from
HSPs
in
which
clients
may
fold
or
may
be
degraded
if
folding
is
not
successful
Such
acNon
is
required
for
assisted
folding
of
nascent
proteins
and
for
protein
translocaNon
(consNtuNvely
expressed
HSP)
and
for
refolding
of
stress-­‐unfolded
proteins
(consNtuNve
and
heat-­‐inducible
HSP).
nascent
Kampinga & Craig - Nature Reviews | Molecular Cell Biology
9

21. Cell
Stress
and
Chaperones:
basic
concepts
-­‐
Harm
H.
Kampinga
HSP
belong
to
the
group
of
Molecular
Chaperones
DefiniNon:
any
protein
(HSP
being
the
largest
group),
which
interacts,
stabilizes
or
helps
a
non-­‐naNve
protein
to
acquire
its
na?ve
conforma?on
but
is
not
present
in
the
final
funcNonal
structure
The
basic
chaperone
acNon
comprise
an
iteraNve
cycles
of
client
binding
to
and
release
from
HSPs
in
which
clients
may
fold
or
may
be
degraded
if
folding
is
not
successful
Such
acNon
is
required
for
assisted
folding
of
nascent
proteins
and
for
protein
translocaNon
(consNtuNvely
expressed
HSP)
and
for
refolding
of
stress-­‐unfolded
proteins
(consNtuNve
and
heat-­‐inducible
HSP).
Native stress
nascent
Kampinga & Craig - Nature Reviews | Molecular Cell Biology
9

22. Cell
Stress
and
Chaperones:
basic
concepts
-­‐
Harm
H.
Kampinga
HSP
belong
to
the
group
of
Molecular
Chaperones
DefiniNon:
any
protein
(HSP
being
the
largest
group),
which
interacts,
stabilizes
or
helps
a
non-­‐naNve
protein
to
acquire
its
na?ve
conforma?on
but
is
not
present
in
the
final
funcNonal
structure
The
basic
chaperone
acNon
comprise
an
iteraNve
cycles
of
client
binding
to
and
release
from
HSPs
in
which
clients
may
fold
or
may
be
degraded
if
folding
is
not
successful
Such
acNon
is
required
for
assisted
folding
of
nascent
proteins
and
for
protein
translocaNon
(consNtuNvely
expressed
HSP)
and
for
refolding
of
stress-­‐unfolded
proteins
(consNtuNve
and
heat-­‐inducible
HSP).
Heat-­‐induced
HSP
up-­‐regulaNon
(e.g.
Hsp70)
thus
protects
cells
against
the
toxicity
of
subsequent
heaNng
(thermotolerance).
10

23. Cell
Stress
and
Chaperones:
basic
concepts
-­‐
Harm
H.
Kampinga
HSP
belong
to
the
group
of
Molecular
Chaperones
DefiniNon:
any
protein
(HSP
being
the
largest
group),
which
interacts,
stabilizes
or
helps
a
non-­‐naNve
protein
to
acquire
its
na?ve
conforma?on
but
is
not
present
in
the
final
funcNonal
structure
The
basic
chaperone
acNon
comprise
an
iteraNve
cycles
of
client
binding
to
and
release
from
HSPs
in
which
clients
may
fold
or
may
be
degraded
if
folding
is
not
successful
Such
acNon
is
required
for
assisted
folding
of
nascent
proteins
and
for
protein
translocaNon
(consNtuNvely
expressed
HSP)
and
for
refolding
of
stress-­‐unfolded
proteins
(consNtuNve
and
heat-­‐inducible
HSP).
Heat-­‐induced
HSP
up-­‐regulaNon
(e.g.
Hsp70)
thus
protects
cells
against
the
toxicity
of
subsequent
heaNng
(thermotolerance)
and
this
correlates
with
increased
refolding
capacity.
Luciferase
100
% luciferase activity + HSP
90
60
40 - HSP
20
0
HS 0 1 2
hrs after HS
11

24. Cell
Stress
and
Chaperones:
basic
concepts
-­‐
Harm
H.
Kampinga
HSP
belong
to
the
group
of
Molecular
Chaperones
DefiniNon:
any
protein
(HSP
being
the
largest
group),
which
interacts,
stabilizes
or
helps
a
non-­‐naNve
protein
to
acquire
its
na?ve
conforma?on
but
is
not
present
in
the
final
funcNonal
structure
The
basic
chaperone
acNon
comprise
an
iteraNve
cycles
of
client
binding
to
and
release
from
HSPs
in
which
clients
may
fold
or
may
be
degraded
if
folding
is
not
successful
Such
acNon
is
required
for
assisted
folding
of
nascent
proteins
and
for
protein
translocaNon
(consNtuNvely
expressed
HSP)
and
for
refolding
of
stress-­‐unfolded
proteins
(consNtuNve
and
heat-­‐inducible
HSP).
Heat-­‐induced
HSP
up-­‐regulaNon
(e.g.
Hsp70)
thus
protects
cells
against
the
toxicity
of
subsequent
heaNng
(thermotolerance)
and
this
correlates
with
increased
refolding
capacity.
toxicity
protec*on chaperone
ac*vity
HSP70 HSP70
Nollen et al | Mol Cell Biol 1999
12

25. Cell
Stress
and
Chaperones:
basic
concepts
-­‐
Harm
H.
Kampinga
CHAPERONE
ACTIVITY
in
vitro
assays
13

26. Cell
Stress
and
Chaperones:
basic
concepts
-­‐
Harm
H.
Kampinga
CHAPERONE
ACTIVITY
in
vitro
assays
1.
heat
substrate
+/-­‐
HSP
2.
measure
substrate
aggrega*on
13

27. Cell
Stress
and
Chaperones:
basic
concepts
-­‐
Harm
H.
Kampinga
CHAPERONE
ACTIVITY
in
vitro
assays
1.
heat
substrate
+/-­‐
HSP
2.
measure
substrate
aggrega*on
light
sca<ering client:
HSP
1:0
1:1
1:2
1:5
13

28. Cell
Stress
and
Chaperones:
basic
concepts
-­‐
Harm
H.
Kampinga
CHAPERONE
ACTIVITY
in
vitro
assays
1.
heat
substrate
+/-­‐
HSP 1.
heat
substrate
+/-­‐
HSP
2.
measure
substrate
aggrega*on 2.
reincubate
at
37oC
+/-­‐
HSP
and
+/-­‐
ATP
3.
measure
ac*vity
light
sca<ering client:
HSP
1:0
1:1
1:2
1:5
13

29. Cell
Stress
and
Chaperones:
basic
concepts
-­‐
Harm
H.
Kampinga
CHAPERONE
ACTIVITY
in
vitro
assays
1.
heat
substrate
+/-­‐
HSP 1.
heat
substrate
+/-­‐
HSP
2.
measure
substrate
aggrega*on 2.
reincubate
at
37oC
+/-­‐
HSP
and
+/-­‐
ATP
3.
measure
ac*vity
light
sca<ering ac=vity
assays client:
HSP
/
ATP
client:
HSP
1:0 1:5
/
with
ATP
1:2
/
with
ATP
1:1
1:1
/
with
ATP
1:2
1:5
/
no
ATP
1:5
1:0
/
no
ATP
13

30. Cell
Stress
and
Chaperones:
basic
concepts
-­‐
Harm
H.
Kampinga
CHAPERONE
ACTIVITY
in
vitro
assays
1.
heat
substrate
+/-­‐
HSP 1.
heat
substrate
+/-­‐
HSP
2.
measure
substrate
aggrega*on 2.
reincubate
at
37oC
+/-­‐
HSP
and
+/-­‐
ATP
3.
measure
ac*vity
light
sca<ering ac=vity
assays client:
HSP
/
ATP
client:
HSP
1:0 1:5
/
with
ATP
1:2
/
with
ATP
1:1
1:1
/
with
ATP
1:2
1:5
/
no
ATP
1:5
1:0
/
no
ATP
aggrega?on
preven?on
(“holdase”)
13

31. Cell
Stress
and
Chaperones:
basic
concepts
-­‐
Harm
H.
Kampinga
CHAPERONE
ACTIVITY
in
vitro
assays
1.
heat
substrate
+/-­‐
HSP 1.
heat
substrate
+/-­‐
HSP
2.
measure
substrate
aggrega*on 2.
reincubate
at
37oC
+/-­‐
HSP
and
+/-­‐
ATP
3.
measure
ac*vity
light
sca<ering ac=vity
assays client:
HSP
/
ATP
client:
HSP
1:0 1:5
/
with
ATP
1:2
/
with
ATP
1:1
1:1
/
with
ATP
1:2
1:5
/
no
ATP
1:5
1:0
/
no
ATP
aggrega?on
preven?on suppor?ng
refolding
(“holdase”) (“foldase”)
13

32. Cell
Stress
and
Chaperones:
basic
concepts
-­‐
Harm
H.
Kampinga
CHAPERONE
ACTIVITY
sHsp Hsp40 Hsp60/10 Hsp90
(HspB) (DnaJ) (HspD/E) (HspC)
Hsp70
(HspA)
14

33. Cell
Stress
and
Chaperones:
basic
concepts
-­‐
Harm
H.
Kampinga
CHAPERONE
ACTIVITY
ATP-independent
‘holders’
sHsp Hsp40 Hsp60/10 Hsp90
(HspB) (DnaJ) (HspD/E) (HspC)
Hsp70
(HspA)
14

34. Cell
Stress
and
Chaperones:
basic
concepts
-­‐
Harm
H.
Kampinga
CHAPERONE
ACTIVITY
ATP-independent
‘holders’
sHsp Hsp40 Hsp60/10 Hsp90
(HspB) (DnaJ) (HspD/E) (HspC)
ATP-dependent
‘holders & folders’
Hsp70
(HspA)
14

35. Cell
Stress
and
Chaperones:
basic
concepts
-­‐
Harm
H.
Kampinga
CHAPERONE
ACTIVITY
ATP-independent
‘holders’
sHsp Hsp40 Hsp60/10 Hsp90
(HspB) (DnaJ) (HspD/E) (HspC)
ATP-dependent
‘holders & folders’
Hsp70
(HspA)
The Hsp70 machine
14

36. Cell
Stress
and
Chaperones:
basic
concepts
-­‐
Harm
H.
Kampinga
CHAPERONE
ACTIVITY
The
Hsp70
machine
15

37. Cell
Stress
and
Chaperones:
basic
concepts
-­‐
Harm
H.
Kampinga
CHAPERONE
ACTIVITY
The
Hsp70
machine
Kampinga & Craig - Nature Reviews | Molecular Cell Biology
16

38. Cell
Stress
and
Chaperones:
basic
concepts
-­‐
Harm
H.
Kampinga
CHAPERONE
ACTIVITY
Hsp90
Mayer
et
et
al.
2002
17

39. Cell
Stress
and
Chaperones:
basic
concepts
-­‐
Harm
H.
Kampinga
CHAPERONE
ACTIVITY
Hsp90
Mayer
et
et
al.
2002
18

40. Cell
Stress
and
Chaperones:
basic
concepts
-­‐
Harm
H.
Kampinga
CHAPERONE
ACTIVITY
GroEL/ES
(Hsp60/10)
Ranson
et
al.
2001
Ranford
et
al.
2000
19

41. Cell
Stress
and
Chaperones:
basic
concepts
-­‐
Harm
H.
Kampinga
CHAPERONE
ACTIVITY
GroEL/ES
(Hsp60/10)
Tang
et
al.
2006
20

42. Cell
Stress
and
Chaperones:
basic
concepts
-­‐
Harm
H.
Kampinga
CHAPERONE
ACTIVITY
small
Hsp
Kim
et
al
1998 Jaya
et
al.
20069
21

43. Cell
Stress
and
Chaperones:
basic
concepts
-­‐
Harm
H.
Kampinga
CHAPERONE
ACTIVITY
small
Hsp
Teydmer
et
al
2011
22

44. Cell
Stress
and
Chaperones:
basic
concepts
-­‐
Harm
H.
Kampinga
CHAPERONE
NETWORK
HSPC
Kampinga & Craig - Nature Reviews | Molecular Cell Biology
23

45. Cell
Stress
and
Chaperones:
basic
concepts
-­‐
Harm
H.
Kampinga
CHAPERONE
NETWORK
HSPC
HSPD/E
Kampinga & Craig - Nature Reviews | Molecular Cell Biology
24

46. Cell
Stress
and
Chaperones:
basic
concepts
-­‐
Harm
H.
Kampinga
CHAPERONE
NETWORK
HSPC
HSPD/E
HSPB
Kampinga & Craig - Nature Reviews | Molecular Cell Biology
25

47. Cell
Stress
and
Chaperones:
basic
concepts
-­‐
Harm
H.
Kampinga
Originally,
heat
shock
genes
(Ritossa,
1962)
and
later
heat
shock
proteins
(Tissieres
et
al
1974),
were
described
to
be
genes/proteins
of
which
the
expression
increased
aBer
an
heat
shock.
Heat
shock
proteins
(HSP)
were
defined
as
proteins
encoded
by
genes
that
contain
so-­‐called
“heat
shock
elements
(HSE)
in
their
genes
that
are
regulated
by
the
“heat
shock
transcripNon
factor-­‐1
(HSF-­‐1)”
Nowadays,
we
know
that
many
situaNon,
both
physiologically
and
stressful
condiNons
can
acNvate
HSF-­‐1
There
are
many
members
in
at
least
5
different
heat
shock
proteins
families
(HSP90,
HSP70,
HSP40,
chaperonins,
and
small
HSP)
that
work
in
a
cellular
network.
sHsp Hsp40 Hsp90
Chaperonins
(HspB: 10 members) (DnaJ: 50 members) (HspC: 5 members)
(HspD/E+ CCT: 2+12 members)
Hsp70
(HspA+H: 13+4 members)
Kampinga
et
al.,
Cell
Stress
Chaperones.
(2009)
26

48. Cell
Stress
and
Chaperones:
basic
concepts
-­‐
Harm
H.
Kampinga
ButOriginally,
heat
shock
genes
(Ritossa,
have so many proteins
why do humans 1962)
and
later
heat
shock
HSPs?
(Tissieres
et
al
1974),
were
described
to
be
genes/proteins
of
which
the
expression
increased
aBer
an
heat
shock.
Heat
shock
proteins
(HSP)
were
defined
as
proteins
encoded
by
genes
that
contain
so-­‐called
“heat
shock
elements
(HSE)
in
their
genes
that
are
regulated
by
the
“heat
shock
transcripNon
factor-­‐1
(HSF-­‐1)”
Nowadays,
we
know
that
many
situaNon,
both
physiologically
and
stressful
condiNons
can
acNvate
HSF-­‐1
There
are
many
members
in
at
least
5
different
heat
shock
proteins
families
(HSP90,
HSP70,
HSP40,
chaperonins,
and
small
HSP)
that
work
in
a
cellular
network.
sHsp Hsp40 Hsp90
Chaperonins
(HspB: 10 members) (DnaJ: 50 members) (HspC: 5 members)
(HspD/E+ CCT: 2+12 members)
Hsp70
(HspA+H: 13+4 members)
Kampinga
et
al.,
Cell
Stress
Chaperones.
(2009)
26

49. Cell
Stress
and
Chaperones:
basic
concepts
-­‐
Harm
H.
Kampinga
ButOriginally,
heat
shock
genes
(Ritossa,
have so many proteins
why do humans 1962)
and
later
heat
shock
HSPs?
1. compartmentalization escribed
to
be
genes/proteins
of
which
Per-UPR?)
(Tissieres
et
al
1974),
were
d (cyt/nuc-HSR; ER-UPR; mit-UPR; the
expression
increased
aBer
an
heat
shock.
Heat
shock
proteins
(HSP)
were
defined
as
proteins
encoded
by
genes
that
contain
so-­‐called
“heat
shock
elements
(HSE)
in
their
genes
that
are
regulated
by
the
“heat
shock
transcripNon
factor-­‐1
(HSF-­‐1)”
Nowadays,
we
know
that
many
situaNon,
both
physiologically
and
stressful
condiNons
can
acNvate
HSF-­‐1
There
are
many
members
in
at
least
5
different
heat
shock
proteins
families
(HSP90,
HSP70,
HSP40,
chaperonins,
and
small
HSP)
that
work
in
a
cellular
network.
sHsp Hsp40 Hsp90
Chaperonins
(HspB: 10 members) (DnaJ: 50 members) (HspC: 5 members)
(HspD/E+ CCT: 2+12 members)
Hsp70
(HspA+H: 13+4 members)
Kampinga
et
al.,
Cell
Stress
Chaperones.
(2009)
26

50. Cell
Stress
and
Chaperones:
basic
concepts
-­‐
Harm
H.
Kampinga
ButOriginally,
heat
shock
genes
(Ritossa,
have so many proteins
why do humans 1962)
and
later
heat
shock
HSPs?
1. compartmentalization escribed
to
be
genes/proteins
of
which
Per-UPR?)
(Tissieres
et
al
1974),
were
d (cyt/nuc-HSR; ER-UPR; mit-UPR; the
expression
increased
aBer
an
heat
shock.
2. Heat
shock
proteins
(HSP)
were
defined
as
proteins
encoded
by
genes
that
contain
differential regulation (e.g., many HSPs are NOT heat-/HSF-1 regulated)
so-­‐called
“heat
shock
elements
(HSE)
in
their
genes
that
are
regulated
by
the
“heat
shock
transcripNon
factor-­‐1
(HSF-­‐1)”
Nowadays,
we
know
that
many
situaNon,
both
physiologically
and
stressful
condiNons
can
acNvate
HSF-­‐1
There
are
many
members
in
at
least
5
different
heat
shock
proteins
families
(HSP90,
HSP70,
HSP40,
chaperonins,
and
small
HSP)
that
work
in
a
cellular
network.
sHsp Hsp40 Hsp90
Chaperonins
(HspB: 10 members) (DnaJ: 50 members) (HspC: 5 members)
(HspD/E+ CCT: 2+12 members)
Hsp70
(HspA+H: 13+4 members)
Kampinga
et
al.,
Cell
Stress
Chaperones.
(2009)
26

51. Cell
Stress
and
Chaperones:
basic
concepts
-­‐
Harm
H.
Kampinga
But why do humans have so many HSPs?
1. compartmentalization (cyt/nuc-HSR; ER-UPR; mit-UPR; Per-UPR?)
2. differential regulation (e.g., many HSPs are NOT heat-/HSF-1 regulated)
-HS
DNAJB12 = 0.1%
DNAJB11 = 2.0% +HS
DNAJB9 = 0.3%
DNAJB8 = 0.0%
DNAJB7 = 0.1%
DNAJB6 = 2.4%
HS DNAJB5 = 0.0%
DNAJB4 = 0.6%
DNAJB2 = 0.0% GAPD = 29.0%
GAPD = 82.9% DNAJA1 = 2.5%
DNAJA1 = 4.2% DNAJA2 = 0.4%
DNAJA3 = 0.4%
DNAJA2 = 1.4% DNAJA4 = 0.0%
DNAJA3 = 2.3%
DNAJA4 = 0.0%
DNAJB1 = 2.8%
DNAJB2 = 0.0% DNAJB1 = 62.1%
DNAJB4 = 0.3%
DNAJB5 = 0.1%
DNAJB6 = 2.3%
DNAJB7 = 0.3%
DNAJB8 = 0.0%
DNAJB9 = 0.2%
DNAJB11 = 2.4%
DNAJB12 = 0.7%
Hageman
et
al
2011
.
27

52. Cell
Stress
and
Chaperones:
basic
concepts
-­‐
Harm
H.
Kampinga
But why do humans have so many HSPs?
1. compartmentalization (cyt/nuc-HSR; ER-UPR; mit-UPR; Per-UPR?)
2. differential regulation (e.g., many HSPs are NOT heat-/HSF-1 regulated)
3. functional specificity (structural differences)
sHsp Hsp40 Hsp90
Chaperonins
(HspB: 10 members) (DnaJ: 50 members) (HspC: 5 members)
(HspD/E+ CCT: 2+12 members)
Hsp70
(HspA+H: 13+4 members)
Kampinga
et
al.,
Cell
Stress
Chaperones.
(2009)
28

53. Cell
Stress
and
Chaperones:
basic
concepts
-­‐
Harm
H.
Kampinga
But why do humans have so many HSPs?
1. compartmentalization (cyt/nuc-HSR; ER-UPR; mit-UPR; Per-UPR?)
2. differential regulation (e.g., many HSPs are NOT heat-/HSF-1 regulated)
3. functional specificity (structural differences)
sHsp Hsp40 Hsp90
Chaperonins
(HspB: 10 members) (DnaJ: 50 members) (HspC: 5 members)
(HspD/E+ CCT: 2+12 members)
Hsp70
(HspA+H: 13+4 members)
Kampinga
et
al.,
Cell
Stress
Chaperones.
(2009)
28

54. Cell
Stress
and
Chaperones:
basic
concepts
-­‐
Harm
H.
Kampinga
FUNCTIONAL
SPECIFICITY
OF
THE
HSP70
MACHINE
Kampinga & Craig - Nature Reviews | Molecular Cell Biology
29

55. Cell
Stress
and
Chaperones:
basic
concepts
-­‐
Harm
H.
Kampinga
FUNCTIONAL
SPECIFICITY
OF
THE
HSP70
MACHINE
The
Hsp70
machine
is
involved
in
many
different
biochemical
and
biological
funcNons
ranging
from
co-­‐translaNonal
folding,
to
protein
translocaNon
across
membranes,
from
protein
remodeling
to
protein
degradaNon.
Kampinga & Craig - Nature Reviews | Molecular Cell Biology
29

56. Cell
Stress
and
Chaperones:
basic
concepts
-­‐
Harm
H.
Kampinga
FUNCTIONAL
SPECIFICITY
OF
THE
HSP70
MACHINE
The
Hsp70
machine
is
involved
in
many
different
biochemical
and
biological
funcNons
ranging
from
co-­‐translaNonal
folding,
to
protein
translocaNon
across
membranes,
from
protein
remodeling
to
protein
degradaNon.
1.
Role
expansion
HSP70
family
Kampinga & Craig - Nature Reviews | Molecular Cell Biology
29

57. Cell
Stress
and
Chaperones:
basic
concepts
-­‐
Harm
H.
Kampinga
FUNCTIONAL
SPECIFICITY
OF
THE
HSP70
MACHINE
The
Hsp70
machine
is
involved
in
many
different
biochemical
and
biological
funcNons
ranging
from
co-­‐translaNonal
folding,
to
protein
translocaNon
across
membranes,
from
protein
remodeling
to
protein
degradaNon.
1.
Role
expansion
HSP70
family
Hsp70
Yeast Human 0 200 400 600 800 1000
Ssa1-4; Ssb1,2 HSPA1,2,6,7,8 Cytosol
Kar2 HSPA5 ER
Ssc1;SSq1;Emc10 HSPA9 Mitochondria
HSPA12A,B Cytosol
Ssz1 HSPA14 Cytosol
Kampinga & Craig - Nature Reviews | Molecular Cell Biology
29

58. Cell
Stress
and
Chaperones:
basic
concepts
-­‐
Harm
H.
Kampinga
FUNCTIONAL
SPECIFICITY
OF
THE
HSP70
MACHINE
The
Hsp70
machine
is
involved
in
many
different
biochemical
and
biological
funcNons
ranging
from
co-­‐translaNonal
folding,
to
protein
translocaNon
across
membranes,
from
protein
remodeling
to
protein
degradaNon.
1.
Role
expansion
HSP70
family
Hsp70
Yeast Human 0 200 400 600 800 1000
Ssa1-4; Ssb1,2 HSPA1,2,6,7,8 Cytosol
Kar2 HSPA5 ER
Ssc1;SSq1;Emc10 HSPA9 Mitochondria
HSPA12A,B Cytosol
Ssz1 HSPA14 Cytosol
Hsp70
are
structural
almost
idenNcal
(same
domain
structure)
Hsp70
show
all
similar
biochemical
acNvity
(an
ATP-­‐dependent
client
protein
binding/release
cycle).
Except
HSPA14
that
lacks
substrate
binding
Kampinga & Craig - Nature Reviews | Molecular Cell Biology
29

59. Cell
Stress
and
Chaperones:
basic
concepts
-­‐
Harm
H.
Kampinga
FUNCTIONAL
SPECIFICITY
OF
THE
HSP70
MACHINE
The
Hsp70
machine
is
involved
in
many
different
biochemical
and
biological
funcNons
ranging
from
co-­‐translaNonal
folding,
to
protein
translocaNon
across
membranes,
from
protein
remodeling
to
protein
degradaNon.
Kampinga & Craig - Nature Reviews | Molecular Cell Biology
30

60. Cell
Stress
and
Chaperones:
basic
concepts
-­‐
Harm
H.
Kampinga
FUNCTIONAL
SPECIFICITY
OF
THE
HSP70
MACHINE
The
Hsp70
machine
is
involved
in
many
different
biochemical
and
biological
funcNons
ranging
from
co-­‐translaNonal
folding,
to
protein
translocaNon
across
membranes,
from
protein
remodeling
to
protein
degradaNon.
2.
Role
expansion
DNAJ
protein
family
Kampinga & Craig - Nature Reviews | Molecular Cell Biology
30

61. Cell
Stress
and
Chaperones:
basic
concepts
-­‐
Harm
H.
Kampinga
FUNCTIONAL
SPECIFICITY
OF
THE
HSP70
MACHINE
The
Hsp70
machine
is
involved
in
many
different
biochemical
and
biological
funcNons
ranging
from
co-­‐translaNonal
folding,
to
protein
translocaNon
across
membranes,
from
protein
remodeling
to
protein
degradaNon.
2.
Role
expansion
DNAJ
protein
family
Yeast Human 0 100 200 300 400 500 600 Yeast Human 0 100 200 300 400 500 600
Promiscous client binding Promiscous client binding
Ydj1 DNAJA1 Ydj1 DNAJA1
Xdj1 DNAJA2;A4 Xdj1 DNAJA2;A4
Apj1 Apj1
Scj1 Scj1
Mdj1 DNAJA3 Mdj1 DNAJA3
Sis1 DNAJB1 Sis1 DNAJB1
DNAJB4;B5 DNAJB4;B5
DNAJB11 DNAJB11
DNAJB9 DNAJB9
DNAJB2a,2b DNAJB2a,2b
Djp1 Djp1
Caj1 Caj1
DNAJB6a,6b DNAJB6a,6b
DNAJB8 DNAJB8
DNAJB7 DNAJB7
Erj5 Erj5
DNAJB12a,12b DNAJB12a,12b
DNAJB14a,14b DNAJB14a,14b
DNAJC18 DNAJC18
Selective client binding Selective client binding
Jjj1 DNAJC21 590/531 Jjj1 DNAJC21 590/531
Jjj3 DNAJC24 Jjj3 DNAJC24
DNAJC5,5b,5g
FuncNonal
classificaNon
DNAJ
proteins
DNAJC5,5b,5g
Jac1 DNAJC20 Jac1 DNAJC20
Cwc23 DNAJC17 Cwc23 DNAJC17
DNAJC10
DNAJC16
793
782
1:
Client
binding
and
client
delivery
to
Hsp70
DNAJC10
DNAJC16
793
782
Swa2
DNAJC6
668
913
Swa2
a)
promiscuous
client
binding
DNAJC6
668
913
b)
selecNve
client
binding
DNAJC26 1311
DNAJC26 1311
DNAJC27 DNAJC27
DNAJC3
DNAJC3
DNAJC7 2:
No
client
binding,
just
ATPase
sNmulaNon
Jem1
DNAJC7
Jem1
DNAJC29
4306
4579 a)
tethering
Hsp70
to
a
locaNon/
a
posiNon
DNAJC29
DNAJC14
4306
702
4579
DNAJC14 702
DNAJC22
Client binding unclear
b)
no
tethering
DNAJC22
Client binding unclear Kampinga & Craig - Nature Reviews | Molecular Cell Biology
DNAJB13 DNAJB13
31

62. Cell
Stress
and
Chaperones:
basic
concepts
-­‐
Harm
H.
Kampinga
FUNCTIONAL
SPECIFICITY
OF
THE
HSP70
MACHINE
The
Hsp70
machine
is
involved
in
many
different
biochemical
and
biological
funcNons
ranging
from
co-­‐translaNonal
folding,
to
protein
translocaNon
across
membranes,
from
protein
remodeling
to
protein
degradaNon.
2.
Role
expansion
DNAJ
protein
family
Yeast Human 0 100 200 300 400 500 600 Yeast Human 0 100 200 300 400 500 600
Promiscous client binding Promiscous client binding
Ydj1 DNAJA1 Ydj1 DNAJA1
Xdj1 DNAJA2;A4 Xdj1 DNAJA2;A4
Apj1 Apj1
Scj1 Scj1
Mdj1 DNAJA3 Mdj1 DNAJA3
Sis1 DNAJB1 Sis1 DNAJB1
DNAJB4;B5 DNAJB4;B5
DNAJB11 DNAJB11
DNAJB9 DNAJB9
DNAJB2a,2b DNAJB2a,2b
Djp1 Djp1
Caj1 Caj1
DNAJB6a,6b DNAJB6a,6b
DNAJB8 DNAJB8
DNAJB7 DNAJB7
Erj5 Erj5
DNAJB12a,12b DNAJB12a,12b
DNAJB14a,14b DNAJB14a,14b
DNAJC18 DNAJC18
Selective client binding Selective client binding
Jjj1 DNAJC21 590/531 Jjj1 DNAJC21 590/531
Jjj3 DNAJC24 Jjj3 DNAJC24
DNAJC5,5b,5g
FuncNonal
classificaNon
DNAJ
proteins
DNAJC5,5b,5g
Jac1 DNAJC20 Jac1 DNAJC20
Cwc23 DNAJC17 Cwc23 DNAJC17
DNAJC10
DNAJC16
793
782
1:
Client
binding
and
client
delivery
to
Hsp70
DNAJC10
DNAJC16
793
782
Swa2
DNAJC6
668
913
Swa2
a)
promiscuous
client
binding
DNAJC6
668
913
b)
selecNve
client
binding
DNAJC26 1311
DNAJC26 1311
DNAJC27 DNAJC27
DNAJC3
DNAJC3
DNAJC7 2:
No
client
binding,
just
ATPase
sNmulaNon
Jem1
DNAJC7
Jem1
DNAJC29
4306
4579 a)
tethering
Hsp70
to
a
locaNon/
a
posiNon
DNAJC29
DNAJC14
4306
702
4579
DNAJC14 702
DNAJC22
Client binding unclear
b)
no
tethering
DNAJC22
Client binding unclear Kampinga & Craig - Nature Reviews | Molecular Cell Biology
DNAJB13 DNAJB13
31

63. Cell
Stress
and
Chaperones:
basic
concepts
-­‐
Harm
H.
Kampinga
FUNCTIONAL
SPECIFICITY
OF
THE
HSP70
MACHINE
The
Hsp70
machine
is
involved
in
many
different
biochemical
and
biological
funcNons
ranging
from
co-­‐translaNonal
folding,
to
protein
translocaNon
across
membranes,
from
protein
remodeling
to
protein
degradaNon.
2.
Role
expansion
DNAJ
protein
family
Yeast Human 0 100 200 300 400 500 600 Yeast Human 0 100 200 300 400 500 600
Promiscous client binding Promiscous client binding
Ydj1 DNAJA1 Ydj1 DNAJA1
Xdj1 DNAJA2;A4 Xdj1 DNAJA2;A4
Apj1 Apj1
Scj1 Scj1
Mdj1 DNAJA3 Mdj1 DNAJA3
Sis1 DNAJB1 Sis1 DNAJB1
DNAJB4;B5 DNAJB4;B5
DNAJB11 DNAJB11
DNAJB9 DNAJB9
DNAJB2a,2b DNAJB2a,2b
Djp1 Djp1
Caj1 Caj1
DNAJB6a,6b DNAJB6a,6b
DNAJB8 DNAJB8
DNAJB7 DNAJB7
Erj5 Erj5
DNAJB12a,12b DNAJB12a,12b
DNAJB14a,14b DNAJB14a,14b
DNAJC18 DNAJC18
Selective client binding Selective client binding
Jjj1 DNAJC21 590/531 Jjj1 DNAJC21 590/531
Jjj3 DNAJC24 Jjj3 DNAJC24
DNAJC5,5b,5g
FuncNonal
classificaNon
DNAJ
proteins
DNAJC5,5b,5g
Jac1 DNAJC20 Jac1 DNAJC20
Cwc23 DNAJC17 Cwc23 DNAJC17
DNAJC10
DNAJC16
793
782
1:
Client
binding
and
client
delivery
to
Hsp70
DNAJC10
DNAJC16
793
782
Swa2
DNAJC6
668
913
Swa2
a)
promiscuous
client
binding
DNAJC6
668
913
b)
selecNve
client
binding
DNAJC26 1311
DNAJC26 1311
DNAJC27 DNAJC27
DNAJC3
DNAJC3
DNAJC7 2:
No
client
binding,
just
ATPase
sNmulaNon
Jem1
DNAJC7
Jem1
DNAJC29
4306
4579 a)
tethering
Hsp70
to
a
locaNon/
a
posiNon
DNAJC29
DNAJC14
4306
702
4579
DNAJC14 702
DNAJC22
Client binding unclear
b)
no
tethering
DNAJC22
Client binding unclear Kampinga & Craig - Nature Reviews | Molecular Cell Biology
DNAJB13 DNAJB13
31