How a cell handles its garbage—meaning, misfolded or damaged proteins—can be a matter of life and death. For that reason, cells have evolved chaperones that capture denatured proteins and either refold them, or, if that fails, help initiate their degradation. Both the inducible chaperone Hsp70 (heat shock protein 70) and its partner and co-chaperone CHIP (carboxy terminus of Hsp70 binding protein) have been implicated in protecting cells against the buildup of misfolded proteins that cause neurodegenerative diseases, including Parkinson disease, Huntington disease, and ALS.

In particular, attention has focused on CHIP as a key regulator of protein fate and a link between chaperones such as Hsp70 and the proteasome protein degradation pathway. When a misfolded protein binds Hsp70, the chaperone tries to refold the protein and release it. But if the protein cannot be refolded, then the ubiquitin ligase activity of CHIP tags the abnormal protein, sending it to be destroyed.

A paper in the March 23 issue of Nature shows a new side to CHIP, as a regulator of the Hsp70 protein stability, and thus the cell stress response. The work, from the lab of Cam Patterson at the University of North Carolina at Chapel Hill, reveals that once CHIP has dispatched with abnormal proteins, it turns its ubiquitin ligase activity on Hsp70, causing destruction of the chaperone. The results show a new and unsuspected mechanism for regulating the duration of inducible Hsp expression. The central role of CHIP in the response to misfolded proteins raises the possibility that it could play a part in causing the buildup of toxic, disease-causing proteins, or might be enlisted to clear them.

CHIP, first cloned by Patterson in 1999, was known to regulate the transcriptional induction of Hsp70 after heat shock, independently of its effects on protein clearance. Efforts to study this regulation in more detail yielded a paradoxical observation: First author Shu-Bing Qian discovered that either overexpression or underexpression of CHIP in cultured cells resulted in higher levels of Hsp70 protein. The first result was explained by CHIP’s ability to enhance Hsp70 gene transcription, but the second observation was puzzling. By pulling apart CHIP function using transfection studies and CHIP-/- cells, Qian et al. showed that CHIP enhanced the ubiquitination and degradation of Hsp70. However, in the presence of a misfolded protein substrate, Hsp70 ubiquitination and degradation is inhibited. It is only when misfolded proteins are depleted that CHIP ubiquitinates Hsp70 and speeds its degradation. In this way, the authors explain, CHIP maintains homeostasis by shutting down the Hsp70 response after misfolded proteins have been cleared from cells.

In the last few years, several studies have established that Hsp70/CHIP activity affects the levels or toxicity of tau (Shimura et al., 2004; Petrucelli et al., 2004; Sahara et al., 2005), polyglutamine-containing proteins (Miller et al., 2005), α-synuclein (Shin et al., 2005), and mutant SOD1 (Urushitani et al., 2004). In addition, CHIP has been reported to be associated with parkin, and to enhance that protein’s ubiquitin ligase activity (Imai et al., 2002). Understanding how CHIP regulates the stress response and protein degradation could lead to new ways to help neuronal cells clean up their act when these toxic proteins enter the picture.—Pat McCaffrey

Q&A with Cam Patterson.

Q: CHIP activity has been shown to enhance the degradation of several mutated or misfolded proteins involved in neurodegeneration, for example, tau, α-synuclein, SOD mutants, polyglutamine-expanded proteins. Do your results tell us something new about the potential role of CHIP in neurodegeneration?
A: Our new results tell us a lot of things, and I think that in the context of neurodegenerative diseases, there are a couple of particularly salient points. First, it is clear that CHIP plays an important role in removing many of the proteins that contribute to neurodegenerative diseases— the ones you describe above—because they are prone to misfold. Our new studies point out that the stress response that depends on Hsp70 to repair and refold proteins is turned off in a coordinated fashion when damaged proteins are cleared by the ubiquitin ligase CHIP. In situations where the misfolded proteins cannot be cleared, and this may particularly apply to neurodegenerative diseases, the stress response will remain continually activated, and this will ultimately have adverse consequences at the cellular level. Thus, impairment of this new mechanism we have discovered to shut down the stress response may play an important role in perpetuating injury to neurons that express these misfolded mutant proteins. This may be one important reason why neurons die and brains atrophy in these horrible diseases.

Q: A few years ago you generated CHIP knockout mice. Do they show evidence of any kind of neurodegeneration?
A: This is an interesting question. Our mice have indeed generated a lot of attention from investigators who study mouse models of neurodegenerative disease, and we have gone out of our way to share our mice with these investigators and to collaborate with them when we can. I don't want to diminish the impact of these studies, since they haven't yet been published, but will say that there are some very fundamental and exciting observations that will soon be reported about acceleration of neurodegeneration in our mice that lack CHIP.

Q: You suggest that increasing CHIP activity might be a way to address protein misfolding diseases. Any ideas how you might do that?
A: Gene therapy would be one way, and some of our collaborators are working on this in animal models of neurodegenerative disease. Perhaps even more exciting in this regard is the recent report of the crystal structure of CHIP that was published by Laurence Pearl's group in a recent edition of Molecular Cell (Zhang et al., 2005). The structure of CHIP is beautiful and very surprising, and we are looking at clues from the structure that might help us to enhance CHIP's activity using pharmacologic approaches. This could be a practical and exciting way to modulate outcomes in individuals who are at risk for neurodegenerative diseases.

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  1. Chipping away at HSP70 regulation
    This week’s Nature presents an elegant article by Cam Patterson’s group documenting a novel regulatory axis controlling Hsp70 levels. CHIP is a ubiquitin ligase that binds to Hsp70. CHIP is known to mediate induction of Hsp70 in response to stress by inducing activity of the transcription factor HSF-1. The current manuscript describes a second point in the regulatory axis of Hsp70 where CHIP acts. Patterson’s group found that CHIP mediates degradation of Hsp70 during the recovery phase after stress has been quenched. An elegant aspect of this model is that CHIP senses the level of misfolded proteins, and as the level of misfolded proteins decreases, CHIP switches from ubiquitinating these proteins to ubiquitinating Hsp70, which targets it for degradation by the proteasome. Thus, CHIP acts at two points in the regulation of Hsp70. CHIP regulates the transcription of Hsp70 (via HSF-1) and regulates degradation of Hsp70 (via ubiquitination of Hsp70).

    The disposition of misfolded proteins is clearly one of the central issues in neurodegenerative research. Hsp70 plays a key role as a chaperone for misfolded proteins, helping either to refold them or to send them for degradation. By regulating levels of Hsp70, CHIP clearly is a primary player in this process. Interest in CHIP is heightened by the discovery that CHIP ubiquitinates tau protein in a process that shifts it to an insoluble protein fraction (1,2). CHIP also binds to parkin, which is linked to familial parkinsonism (1). Patterson’s work provides a valuable framework that greatly enhances our understanding of the ubiquitin proteasomal system.

    References:

    . CHIP and Hsp70 regulate tau ubiquitination, degradation and aggregation. Hum Mol Genet. 2004 Apr 1;13(7):703-14. PubMed.

    . CHIP-Hsc70 complex ubiquitinates phosphorylated tau and enhances cell survival. J Biol Chem. 2004 Feb 6;279(6):4869-76. PubMed.

  2. CHIPs Ahoy: Hsp70 Is Called to Serve and Then Consumed
    Almost 30 percent of all cellular proteins are formed without being properly folded. By mutation or through various protein modifications associated with neurodegenerative diseases, additional specific ones become misfolded. To maintain homeostasis in a crowded protein milieu, a quality control system engages molecular chaperones to properly (re)fold proteins at risk for aggregation or facilitate their disposal by the ubiquitin-dependent proteasome (UPS). The "C-terminus Hsc70 interacting protein" (CHIP) is a bi-functional molecular co-chaperone that links molecular chaperones to the UPS (McClellan and Frydman, 2001; McDonough and Patterson, 2003). The CHIP amino terminus has three tetratricopeptide repeat (TPR) domains, responsible for protein-protein interactions with heat shock proteins (HSP)70/90 and other molecular chaperones Hip, Hop, Cyclophilin 40, FKBP, Bag1, etc. (Connell et al., 2001; Kampinga et al., 2003; Dai et al., 2005; Hwang et al., 2005). The CHIP C-terminus contains a U-box or RING finger-like domain endowed with ubiquitin E3 ligase activity toward several substrates (Arvind et al., 2000). Since CHIP also activates heat shock transcription factor-1 (HSF-1), thus controlling Hsp70 induction (Dai et al., 2003), it plays a major role in orchestrating the protein quality control system (Jiang et al., 2001; Demand et al., 2001; Cyr et al., 2002; Hatakeyama et al., 2004; Xu et al., 2002)

    In the present paper, several hypotheses about CHIP function were tested, highlighting the regulation of Hsp70 turnover. The authors have shown that CHIP influences Hsp70 levels by another mechanism that is independent of HSF1 transcriptional activation and opposite in effect. After calling up Hsp70 to chaperone misfolded proteins for ubiquitin-dependent and CHIP-mediated proteasomal destruction, CHIP then targets the same Hsp70 to a similar fate. However, it only prepares the unneeded Hsp70 for disposal once the un- or misfolded substrate level is effectively depleted. The "stress recovery" effect is specific to Hsp70, since a slight difference of amino acid sequence with Hsc70 in the C-terminal region (EEVD motif) results in only modest proteasomal degradation of the latter. The authors elegantly show that a sort of non-mutual competition between Hsp70 and substrate exists for CHIP's ubiquitination action. Thus, higher levels of Hsp70 do not inhibit unfolded protein (BSA) turnover. CHIP is sensing only stress protein levels in its decision to terminate Hsp70. So, when CHIP is busy preparing a misfolded protein (luciferase) for degradation, Hsp70 levels are stabilized. When the substrate is either in its normal conformation or levels of its denatured form are eliminated, CHIP then turns on Hsp70 to reestablish basal levels.

    Several important implications, predictions, and questions arise from this work. The coordinated rise and rapid fall in Hsp70 levels post-stress suggest that inducibility is essential to optimal chaperone function. What happens then to the biphasic regulation of Hsp70 in neurodegenerative diseases where the misfolded protein chronically accumulates? Could chronic elevations of Hsp70 possibly be deleterious to the cell? Where confounding toxic proteins build up, such as tau and Aβ in AD, could a therapeutic elimination of p-tau (a substrate for CHIP) result in a down-regulation of Hsp70 levels, effectively removing this protective mechanism (as we have shown; Magrane et al., 2004) to neutralize intracellular Aβ? It is also possible that a defect in the Hsp response may exacerbate neurodegenerative disease. For instance, Qian et al. show that in HSF-1-null cells, CHIP expression and activity induces degeneration after heat shock stress. On the other hand, in CHIP-/- cells, where basal Hsp70 levels are higher than WT, the Hsp70 response to heat shock is still present but lessened as expected. Although cell viability is not given, one would predict such cells to also fare worse.

    There are other interesting observations from this excellent paper to digest. For instance, the question arises whether the Hsp70 that is stress-recovered is use-dependent modified in some way for recognition by CHIP to be ubiquitinated. This was partly answered when the authors showed that in HSF-1-/- cells (or in CHIP-/- cells treated with cyclohexamide), expression of CHIP in absence of stress or misfolded proteins causes basal Hsp70 degradation. Another prediction is that if CHIP activity could be shut off at the 4-hour point after heat shock, that Hsp70 levels would remain high. However, this result was essentially shown in an experiment using a mutant CHIP construct (H260Q) in which the Ubox/ligase activity is lost but HSF-1 stimulating activity is retained.

    In the past 7 years, involvement of CHIP as a molecular sensing switch in neurodegenerative disease has been increasingly recognized. CHIP appears to play a dual role in familial Parkinson disease, where it assists in the dissociation of Hsp70 from a complex with parkin and PAEL-R, facilitating parkin mediated PAEL-R ubiquitination (Imai et al., 2002). CHIP mediates the degradation of α-synuclein by triaging between proteasomal and lysosomal pathways (Shin et al., 2005). CHIP appears to play a role in mitigating tauopathies. It polyubiquitinates 4 repeat-phosphorylated tau, attenuating tau aggregation and enhancing cell survival viewed (Hatakeyama et al., 2004; Petrucelli et al., 2004; Sahara et al., 2005). Its range of substrates may be even greater, as we reported last fall that βAPP is bound to and regulated by CHIP (Kumar et al., Neurosciences Meeting, 2005). There may be something to the idea that dual-function proteins are involved in neurodegenerative disorders where loss of homeostasis and self-perpetuating mechanisms occur.

    References:

    . Molecular chaperones and the art of recognizing a lost cause. Nat Cell Biol. 2001 Feb;3(2):E51-3. PubMed.

    . CHIP: a link between the chaperone and proteasome systems. Cell Stress Chaperones. 2003 Winter;8(4):303-8. PubMed.

    . The co-chaperone CHIP regulates protein triage decisions mediated by heat-shock proteins. Nat Cell Biol. 2001 Jan;3(1):93-6. PubMed.

    . Overexpression of the cochaperone CHIP enhances Hsp70-dependent folding activity in mammalian cells. Mol Cell Biol. 2003 Jul;23(14):4948-58. PubMed.

    . CHIP activates HSF1 and confers protection against apoptosis and cellular stress. EMBO J. 2003 Oct 15;22(20):5446-58. PubMed.

    . Heat shock protein 70 participates in the neuroprotective response to intracellularly expressed beta-amyloid in neurons. J Neurosci. 2004 Feb 18;24(7):1700-6. PubMed.

    . CHIP is associated with Parkin, a gene responsible for familial Parkinson's disease, and enhances its ubiquitin ligase activity. Mol Cell. 2002 Jul;10(1):55-67. PubMed.

    . The co-chaperone carboxyl terminus of Hsp70-interacting protein (CHIP) mediates alpha-synuclein degradation decisions between proteasomal and lysosomal pathways. J Biol Chem. 2005 Jun 24;280(25):23727-34. PubMed.

    . U-box protein carboxyl terminus of Hsc70-interacting protein (CHIP) mediates poly-ubiquitylation preferentially on four-repeat Tau and is involved in neurodegeneration of tauopathy. J Neurochem. 2004 Oct;91(2):299-307. PubMed.

    . CHIP and Hsp70 regulate tau ubiquitination, degradation and aggregation. Hum Mol Genet. 2004 Apr 1;13(7):703-14. PubMed.

    . In vivo evidence of CHIP up-regulation attenuating tau aggregation. J Neurochem. 2005 Sep;94(5):1254-63. PubMed.

    View all comments by Henry Querfurth

References

Paper Citations

  1. . CHIP-Hsc70 complex ubiquitinates phosphorylated tau and enhances cell survival. J Biol Chem. 2004 Feb 6;279(6):4869-76. PubMed.
  2. . CHIP and Hsp70 regulate tau ubiquitination, degradation and aggregation. Hum Mol Genet. 2004 Apr 1;13(7):703-14. PubMed.
  3. . In vivo evidence of CHIP up-regulation attenuating tau aggregation. J Neurochem. 2005 Sep;94(5):1254-63. PubMed.
  4. . CHIP suppresses polyglutamine aggregation and toxicity in vitro and in vivo. J Neurosci. 2005 Oct 5;25(40):9152-61. PubMed.
  5. . The co-chaperone carboxyl terminus of Hsp70-interacting protein (CHIP) mediates alpha-synuclein degradation decisions between proteasomal and lysosomal pathways. J Biol Chem. 2005 Jun 24;280(25):23727-34. PubMed.
  6. . CHIP promotes proteasomal degradation of familial ALS-linked mutant SOD1 by ubiquitinating Hsp/Hsc70. J Neurochem. 2004 Jul;90(1):231-44. PubMed.
  7. . CHIP is associated with Parkin, a gene responsible for familial Parkinson's disease, and enhances its ubiquitin ligase activity. Mol Cell. 2002 Jul;10(1):55-67. PubMed.
  8. . Chaperoned ubiquitylation--crystal structures of the CHIP U box E3 ubiquitin ligase and a CHIP-Ubc13-Uev1a complex. Mol Cell. 2005 Nov 23;20(4):525-38. PubMed.

Further Reading

Papers

  1. . The triage of damaged proteins: degradation by the ubiquitin-proteasome pathway or repair by molecular chaperones. FASEB J. 2006 Apr;20(6):741-3. PubMed.
  2. . CHIP: a link between the chaperone and proteasome systems. Cell Stress Chaperones. 2003 Winter;8(4):303-8. PubMed.
  3. . Phosphorylated tau and the neurodegenerative foldopathies. Biochim Biophys Acta. 2005 Jan 3;1739(2-3):298-310. PubMed.
  4. . CHIP activates HSF1 and confers protection against apoptosis and cellular stress. EMBO J. 2003 Oct 15;22(20):5446-58. PubMed.

Primary Papers

  1. . CHIP-mediated stress recovery by sequential ubiquitination of substrates and Hsp70. Nature. 2006 Mar 23;440(7083):551-5. PubMed.