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ROLES OF MOLECULAR CHAPERONES IN PROTEIN FOLDING, TRAFFICKING, AND STRESS SENSORS IN CELL GROWTH AND DEATH

Molecular chaperones have an essential role in the regulation of protein conformation states -- the process during which transient or stable interactions with client proteins affects their conformation and activity. Chaperones capture unfolded polypeptides, stabilize intermediates, and prevent misfolded species from accumulating in stressed cells. The capacity of the Hsp70 and Hsp90 chaperones to regulate these processes involves a constellation of positive and negative co-chaperones that function in various combinations to interact with chaperones to release folded proteins, to facilitate the assembly or disassembly or chaperone-containing heteromeric complexes, to confer substrate specificities, and to affect subcellular trafficking. We are interested in the role of the J-domain, TPR-domain, immunophilins, Bag1, Hip, and CHIP co-chaperones in the regulation of Hsp70 and Hsp90 using biochemical and biophysical approaches with purified recombinant proteins, cell biological methods using GFP-fusion proteins and dynamic image analysis, and the use of conditional cell lines expressing altered levels of wild type and mutant proteins.

Neurodegenerative disease can originate from the misfolding and aggregation of proteins, such as Amyloid-ß, SOD1, or Huntingtin. Fortunately, all cells possess protein quality control machinery that sequesters misfolded proteins, either refolding or degrading them, before they can self-associate into proteotoxic oligomers and aggregates. This activity is largely performed by the stress response chaperones (i.e. Hsp70). However, the expression level of molecular chaperones varies widely among cell types. To understand the potential consequence of this variation, we have studied the process of protein aggregation in the presence of molecular chaperones using mathematical modeling. We find that protein aggregation, in the presence of molecular chaperones, is a bistable process. Bistability in protein aggregation offers an explanation for threshold transitions to high aggregate concentration, which are observed both in vitro and in vivo. Additionally, we find that slight variations in chaperone concentration, due to natural fluctuations, have important consequences in a bistable system for the onset of protein aggregation. Therefore, our results offer a possible theoretical explanation for neuronal vulnerability observed in vivo and the onset of neurodegenerative phenotypes in neurons lacking an effective heat shock response. Our current studies include an experimental test of bistability and chaperone concentrations to obtain a mechanistic understanding of chaperone function with various aggregation-prone proteins.

Survival following stress requires a precise orchestration of cell signaling events to ensure that biosynthetic processes are immediately alerted and cell survival pathways are initiated and executed. Common to a variety of stress conditions is the activation of the heat shock response and elevated expression of heat shock proteins and molecular chaperones such as Hsp70. Hsp70 has been shown to be a regulator of multiple components of the apoptotic machinery and functions in cell stress to transduce a negative signal directly to regulators of cell growth.

We are interested in the role of the co-chaperone Bag1, an Hsp70-binding protein with anti-apoptotic properties, to coordinate signals for cell growth by regulating the activity of Raf-1 kinase. In vitro, Raf1 and Hsp70 compete for binding to Bag1 and in vivo, Bag1 associates with Raf1 to activate the downstream Erk kinases and DNA synthesis. Enhanced expression of Hsp70, to levels achieved during cell stress, negatively regulates Bag1. Consequently, during heat shock Bag1-Raf-1 complexes are displaced by the appearance of Bag1-Hsp70 complexes.

To address this, we have generated a collection of point mutants in Bag1 and Hsp70 to identify the sites of interaction; moreover cell lines expressing these mutant proteins have been invaluable in establishing the importance of Bag1-Hsp70 interaction. To establish a role for Bag1 in the cellular response to stress, we have shown that the arrest of DNA synthesis following heat shock did not occur in cells expressing Bag1 mutants that did not interact with Hsp70. These results reveal that molecular chaperones function as sensors in stress signaling and establish a novel role for Bag1 in the crosstalk between cell stress and cell growth.

Our current studies are to elucidate the various functions of Bag1-Hsp70 in signaling via Raf1 and as well as effects on other regulatory proteins including Bcl2 and steroid aporeceptor complexes.

References

  1. Westerheide, S.D., J. Anckar, S.M. Stevens, Jr., L. Sistonen, and R.I. Morimoto. Stress-Inducible Regulation of Heat Shock Factor 1 by the Deacetylase SIRT1. Science 20: 1063-1066 (2009).
  2. Prahlad, V., T. Cornelius and R.I. Morimoto, . Regulation of the Cellular Heat Shock Response in Caenorhabditis elegans by Thermosensory Neurons. Science 320: 811-814 (2008).
  3. Prahlad, V., and R.I. Morimoto. Integrating the Stress Response: Lessons for Neurodegenerative Diseases from C. elegans. Trends in Cell Biology 19: 52-61 (2009). PMID: 19112021 [PubMed - as supplied by publisher]
  4. Matsumoto, G., S. Kim, and R.I. Morimoto. Huntingtin and mutant SOD1 form aggregate structures with distinct molecular properties in human cells. J Biol Chem. 281: 4477-4485 (2006).
  5. Rieger TR, Morimoto RI, and V. Hatzimanikatis. Bistability explains threshold phenomena in protein aggregation both in vitro and in vivo. Biophys J. 90: 886-95 (2006).
  6. Mosser DD, Morimoto RI. Molecular chaperones and the stress of oncogenesis. Oncogene, 23(16):2907-18 (2004).
  7. Nollen EA, Morimoto RI. Chaperoning signaling pathways: molecular chaperones as stress-sensing 'heat shock' proteins. J Cell Sci. 115: 2809-16 (2002).
  8. Song, J, Takeda M and Morimoto RI. Hsp70-Bag1 complex mediates a physiological stress signaling pathway that regulates Raf-1/ERK and cell growth. Nature Cell Biology 3: 276-282 (2001).
  9. Thress K, Song J, Morimoto RI, Kornbluth S. Reversible inhibition of Hsp70 chaperone function by Scythe and Reaper. EMBO J 1:1033-1041 (2001).
  10. Nollen EA. Brunsting JF. Song J. Kampinga HH. Morimoto RI. Bag1 functions in vivo as a negative regulator of Hsp70 chaperone activity. Mol Cell Biol:20(3):1083-8, 2000.
  11. Beere H, Wolf B, Mosser R, Klein K, Kuwano T, Morimoto R, Cohen G and Green D. Heat shock protein 70 (Hsp70) inhibits apoptosis by preventing recruitment of procaspase-9 to aggregated Apaf-1. Nature Cell Bio. 2; 469-475, 2000.
  12. Montgomery DL, Morimoto RI, Gierasch LM Mutations in the substrate binding domain of the Escherichia coli 70 kDa molecular chaperone, DnaK, which alter substrate affinity or interdomain coupling. J Mol Biol:286(3):915-32, 1999.
  13. Bimston D, Song J, Winchester D, Takayama S, Reed JC, Morimoto RI. BAG-1, a negative regulator of Hsp70 chaperone activity, uncouples nucleotide hydrolysis from substrate release EMBO 17: 6871-6878, 1998
  14. Takayama S. Bimston DN. Matsuzawa S. Freeman BC. Aime-Sempe C. Xie Z. Morimoto RI. Reed JC. BAG-1 modulates the chaperone activity of Hsp70/Hsc70. EMBO Journal. 16(16):4887-96, 1997
  15. Freeman BC. Morimoto RI. The human cytosolic molecular chaperones hsp90, hsp70 (hsc70) and hdj-1 have distinct roles in recognition of a non-native protein and protein refolding. EMBO Journal. 15(12):2969-79, 1996.
  16. Freeman BC, Toft DO and Morimoto RI. Molecular chaperone machines: chaperone activites of the cyclophilin Cyp-40 and the steroid aporeceptor-associated protein p23. Science 274: 1718-20, 1996.
  17. Freeman BC. Myers MP. Schumacher R. Morimoto RI. Identification of a regulatory motif in Hsp70 that affects ATPase activity, substrate binding and interaction with HDJ-1. EMBO Journal. 14(10):2281-92, 1995
Transcriptional regulation of heat shock response
Roles of Molecular Chaperones in Protein Folding, Trafficking, and Stress Sensors in Cell Growth and Death
All Chaperome Project
Misfolded and aggregation prone proteins in neu
C elegans as a model system for analysis of stress response and diseases of protein misfolding
Small molecule screen for the stress response
Systems Approach to Stress Biology