Molecular chaperones involved in degradation and other processes (II) Chaperones involved in degradation - background - co-chaperones link chaperones to proteolytic degradation machineries - BAG

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 22-2

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 nucleotide- exchange 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 22-3

BAG as an ATPase activator and nucleotide exchange factor for Hsp70  a proteolytically-resistant domain of BAG-1 (aa ), 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) 22-4 stimulates ATP hydrolysis stimulates substrate release

Hsc70 substrate: denatured Ligand Binding Domain (LDB) Hsc70 +/- ATP +/- BAG pull-down HA tag beads LBD monitor release of Hsc70

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 35 S-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/Mg 2+ (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, figure legend 22-5

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 what about the ubiquitin-like domain in BAG?  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 There ’ s evidence that it links the Hsc70 chaperone to the proteasome 22-7

Thress et al. (2001) Reversible inhibition of Hsp70 chaperone function by Scythe and Reaper. EMBO J. 20, Class Presentation: Scythe & Reaper 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. 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 (His- Scythe) 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 (myc- hScythe). 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.

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? 22-19

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,

 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 T. acidophilum chaperones 22-11

 compilation of the known proteases from the Thermoplasma acidophilum archaeal genome T. acidophilum proteases note: Sampylation not listed...progress!