22-1
Molecular chaperones involved in
degradation and other processes (II)
Chaperones involved in degradation
- background
- co-chaperones link chaperones to proteolytic degradation machineries
- BAG-1
22-2
Chaperones involved in protein
degradation
 Hsp70, Hsp40 (in bacteria and eukaryotes), Hsp90/Grp94 (in the eukaryotic cytosol
and ER), trigger factor and GroEL (in bacteria) all have been implicated in facilitating
protein degradation
 presumably, these molecular chaperones bind non-native proteins and can
‘target’ them for degradation by proteases such as the proteasome in eukaryotes
 the above chaperones have effects on the turnover of certain proteins,
particularly under conditions where the proteins are destabilized (e.g., under stress
conditions)
evidence is gathering that chaperone cofactors may mediate the
critical link between various chaperones and the degradation
machinery
22-3
BAG: a molecular link between Hsp70
and the proteasome
 BAG is an acronymn for Bcl2-Associated athanoGene; it was first identified in the
mammalian cytosol by virtue of its interaction with the anti-apoptotic protein Bcl2, and
was shown to promote cell survival
 different BAG isoforms likely bind to various partners (e.g., protein kinase Raf-1,
which regulates proliferation, differentiation, and apoptosis)
 there are numerous isoforms of BAG (BAG-1 to BAG-5 in humans)
 the BAG family also interacts with and modulates the activity of Hsp70
 BAG-1 stimulates the ATPase rate of Hsp70 in an Hsp40-dependent manner
and promotes sustrate release by allowing ADP-ATP exchange
- in other words, BAG functions as an ATPase activator and nucleotideexchange factor
 BAG-1 isoforms contain a ubiquitin homology domain; BAG-3 contains a WW
domain which mediates protein-protein interactions
 the ubiquitin-like domain in BAG-1 suggests a link with the proteasome
BAG as an ATPase activator and
nucleotide exchange factor for Hsp70
stimulates
ATP hydrolysis
stimulates
substrate
release
22-4
 a proteolytically-resistant domain of BAG-1
(aa151-264), BAG-1M, was uncovered and
characterized
 this truncation mutant stimulates the ATPase
activity of ATP-bound Hsp70 approximately as
well as that of wild-type BAG
 it also stimulates the release of LBD (ligand
binding domain, i.e. a denatured hormone
binding domain) as well as wild-type BAG
 recall that substrate release from Hsp70
requires exchange of ADP with ATP (ATP is
the low-affinity binding state for this
chaperone)
 this latter activity is similar to that of GrpE
(there’s no GrpE in the eukaryotic cytosol)
LBD HA tag
Hsc70 substrate:
denatured
Ligand Binding Domain
(LDB)
Hsc70
pull-down
+/- ATP
+/- BAG
monitor release
of Hsc70
beads
22-5
figure legend
Figure 1. Functional characterization of Bag-1M and the Bag domain. (A) Bag-1M and the Bag domain stimulate the
ATPase activity of Hsc70 in a Hsp40-dependent manner. Hsc70 (3 µM) was incubated at 30°C with Hsp40 (3 µM)
and Bag-1M or isolated Bag domain (3 µM) as indicated. The amount of ATP hydrolyzed was quantitated (9, 25). (B)
Bag-1M and the Bag domain stimulate Hsc70 release from substrate polypeptide in a nucleotide-dependent manner.
Release of 35S-methionine-labeled Hsc70 from partially denatured immobilized LBD of the glucocorticoid receptor
was measured upon incubation with ovalbumin (Ova), Bag-1M, or Bag domain (5 µM each) for 10 min at 25°C in
the presence or absence of ATP/Mg2+ (2 mM) (26, 33). S, supernatant fractions containing released Hsc70; P, pellet
fractions containing LBD-bound Hsc70. Supernatants and pellets were analyzed by SDS-polyacrylamide gel
electrophoresis, followed by phosphoimaging. The bar diagram shows the amounts of Hsc70 released from LBD
expressed in percentage of total bound Hsc70. Error bars in (A) and (B) indicate SD of three independent
experiments. Sondermann et al. (2001) Science 291, 1553-1557
Structure of BAG /Hsc70 ATPase
 BAG opens the ATP-binding cleft in the Hsc70 ATPase domain
 the ATPase domain is homologous to that of actin, which also binds ATP, and
whose proper folding requires ATP binding
22-6
Comparison of Hsc70 BAG/GrpE structures
22-7
what about the ubiquitin-like
domain in BAG?
There’s evidence that it links the
Hsc70 chaperone to the proteasome
 BAG and GrpE have completely different
structures, but have undergone convergent
functional evolution to serve similar (yet different)
purposes
 the opening of the cleft allows for the
efficient release of ADP and binding of ATP
 substrate is released under these conditions
Class Presentation: Scythe & Reaper
Thress et al. (2001)
Reversible inhibition of
Hsp70 chaperone
function by Scythe and
Reaper. EMBO J.
20,1033-41.
Fig. 1. Scythe structurally resembles BAG family
proteins. (A) The domains of several BAG family
members along with Xenopus and human Scythe
showing the relative positions of the ubiquitin-like
motif (black) and C-terminal ‘BAG’ domain (striped).
The complete open reading frame of BAG-3 has yet to
be fully sequenced. C.e., Caenorhabditis elegans; S.p.,
Schizosaccharomyces pombe.
22-8
Fig. 5. Reaper specifically inhibits the
physical association of Scythe and
Hsp70. (A) His-Scythe (1 µM) or GST–
BAG-1 (1 µM) was incubated with
Hsp70 (1 µM) in refolding buffer. After
complex formation, increasing
concentrations (0, 2, 4, 8, 10 µM) of
Reaper were added. Bound proteins were
precipitated with either Ni+-agarose (HisScythe) or glutathione–Sepharose (GST–
BAG-1), washed, resolved by SDS–
PAGE and processed for western blotting
using Hsp70 monoclonal antibody 5a5.
(B) 293T cells were transfected with 5 µg
of myc-tagged human Scythe (mychScythe). Thirty-six hours after
transfection, cells were lysed and
centrifuged, and supernatants were
incubated with recombinant GST or
GST–Reaper (GST–Rpr) for 30 min at
4°C. Subsequently, the lysates were
incubated with a monoclonal myc
antibody for 1 h at 4°C. PAS beads were
then added and, after an additional 1 h
incubation, the beads were pelleted,
washed three times in lysis buffer, and
bound proteins were resolved by SDS–
PAGE. After western transfer, the blots
were probed with an anti-Hsc70
monoclonal antibody.
22-19
Quality control
- various chaperones with links to protein degradation are part of
quality control mechanisms: e.g., Hsp70, Hsp40, BiP, Hsp90, Grp94,
and especially AAA ATPases
- co-chaperones also participate in the so-called protein triage
folding?
degradation?
22-10
Putting it all together:
chaperones and
proteases
 sequence of the archaeum,
Thermoplasma acidophilum, showing
the major molecular chaperones and
proteolytic systems
 grows at 60ºC, pH2; has ~1500
proteins
 this schematic is highly
simplified; there are many, many
more proteins involved in protein
biogenesis and degradation (e.g.,
small Hsp, proteins involved in
transport and translocation,
PPIases, PDIases, etc. are not
shown but are in the genome)
 picture would be even more
complex in eukaryotes
Ruepp et al. (2000)
Nature 407, 508-513
T. acidophilum chaperones
 compilation of the known chaperones from the Thermoplasma
acidophilum archaeal genome
 note: many archaea do not contain an Hsp70 system, while most others
contain PAN, the AAA ATPase associated with the proteasome
22-11
T. acidophilum proteases
22-12
 compilation of the known proteases from the Thermoplasma
acidophilum archaeal genome
note: Sampylation not listed...progress!