Could Tumor Suppressor p53 Promote Proteostasis?

The tumor suppressor p53, well known to shield cells from DNA damage, may also protect against malformed proteins, according to a paper in the April 2 PLoS Biology. First author Goran Periz and colleagues at Johns Hopkins University in Baltimore discovered this new proteostasis mechanism in a screen for mutations that protect round worms from toxic protein aggregates. The authors have not yet fleshed out details of the system, but it appears to operate in fruit flies and people as well.

Suppressor Mutations.

Mutant SOD1 accumulates to fill up the head and ventral neurons of nematodes (top), but disperses in a worm missing ufd-2 and spr-5 activity. [Image courtesy of Periz et al.]

Periz and senior author Jiou Wang were searching for genes involved in protein quality control. For their initial screen, they chose Caenorhabditis elegans expressing a mutant version of the amyotrophic lateral sclerosis gene superoxide dismutase 1 (SOD1). They treated the animals with the mutagen ethyl methanesulfonate to create random mutations. Nematodes toting mSOD1 can barely move, allowing Periz to screen for wriggly worms likely to have mutations that partially saved them from this toxicity.

Strain M1 was quite squirmy. The Wang lab quantifies this by placing a worm in a droplet of liquid medium and counting its “thrashings,” or full-body twists. Control worms with wild-type SOD1 thrash about 60 times per minute and those with mutant SOD1 about five times. M1 pulled off approximately 45. When Periz sequenced M1’s genome, he found not one but two mutations. One was a truncation to a ubiquitin ligase called ubiquitin fusion degradation 2 (ufd-2). The other was a substitution, glutamine for arginine, in a conserved residue of the demethylase known as suppressor of presenilin 5 (spr-5). Spr-5 got its name because mutations in the gene suppress an egg-laying phenotype due to mutations in a worm presenilin (Elmer et al., 2002). 

Wait—presenilin? The fact that presenilins are involved in Alzheimer’s is likely coincidental to his work, Periz said. By themselves, the mutations in ufd-2 or spr-5 made only a subtle functional difference, enabling five to10 thrashes per minute, but together they restored motility to the mSOD1 mutants. They also diminished protein levels of mutant SOD1 in the animals’ neurons (see image above). Periz and Wang christened the pair SUNS, for “spr-5- and ufd-2-dependent neurodegeneration suppressor.”

Was SUNS good only for nematodes and mutant SOD1, or more generally? Periz tested other proteins that form toxic inclusions. In worm neurons, mutant SUNS diminished the amount of aggregated TDP-43 and a peptide harboring an expanded polyglutamine repeat. Next, Periz tested SUNS in Drosophila expressing mutant TDP-43 or FUS. These ALS-linked mutations cause neurodegeneration in the fly eye, but knocking down the Drosophila homologs of ufd-2 and spr-5 rescued the eye.

The human homologs of ufd-2 and spr-5 are ubiquitination factor E4B (UBE4B) and lysine-specific demethylase 1A (LSD1), respectively. In human embryonic kidney cell HEK293T cultures expressing mutant SOD1, or mutant TDP-43, knocking down both UBE4B and LSD1 via RNA interference lessened the amount of mutant protein present by 90 percent. “We now know it is not just C. elegans; this whole pathway operates elsewhere, too,” Periz concluded.

In analyzing the transcriptome of the HEK293T cells lacking UBE4B and LSD1, Periz was struck to see a pattern of gene expression similar to that induced by the transcription factor p53. In fact, LSD1 was already known to demethylate p53, deactivating it (Huang et al., 2007). Moreover, UBE4B is required for the ubiquitination and degradation of p53 (Wu and Leng, 2011). Together, these data suggested to Periz that p53 mediates the effects of the SUNS mutations. Supporting this idea, he found that p53 was activated in the SUNS cultures.

“We think p53 is emerging as a potential regulator of both genotoxic and proteotoxic stress,” Periz concluded. It seems to be part of a proteostasis pathway, in addition to known protein-repair processes such as the heat shock response. However, Ralph Nixon of the Nathan Kline Institute in Orangeburg, New York, wondered if Periz’s new pathway might overlap with the unfolded protein response (UPR) mechanism. It might be a previously unrecognized branch of the UPR, rather than a totally new pathway, he speculated.

SUNS Settings. Mutating or knocking down UBE4B and LSD1 allows p53 to regulate genes involved in autophagy and proteasome-mediated degradation. [Image courtesy of Periz et al.] 

Periz is now investigating whether cells stressed by aggregating proteins upregulate p53, and what other molecules might participate in the cascade leading from UBE4B and LSD1 to p53 activation and degradation of misfolded proteins. Brian Kraemer of the University of Washington in Seattle, who was not involved in the study, added that he would be interested to know which of p53’s many target genes participate in protein degradation. “We may see more to come on this particular pathway and its importance in neurodegenerative disease,” Kraemer predicted. There have been hints that p53 might be linked to Alzheimer's and related diseases (see Jun 2006 news; Feb 2013 news; Jul 2005 news).

Periz suggested that p53 could protect cells from toxic proteins, and plans to test p53-activating drugs in mouse models. In addition, scientists are investigating p53 gene therapy to treat cancer, since p53 promotes DNA repair (Chen et al., 2014). 

“The study does bring to light a target that seems to be 'druggable,'” commented David Borchelt of the University of Florida in Gainesville, who trained Wang but was not involved in the current research. “The question is whether p53 is too scary of a drug target to be brought to bear for disease,” he added. Besides suppressing tumors, p53 also promotes apoptosis in cells overloaded with DNA damage.—Amber Dance

Comments

  1. Periz et al. used a classic C. elegans genetic approach as a starting point to uncover a conserved cellular pathway that can limit the aggregation and toxicity of disease-associated variants of superoxide dismutase -1 (SOD1) and other proteins associated with neurodegenerative diseases. Through a fairly heroic process, they identified a rare double mutation that suppressed the toxicity caused by transgenic expression of human disease-mutant SOD1. They then used fly and human cell models to track down how these mutations worked together to ultimately activate processes that degrade abnormal proteins. A key finding was their demonstration that p53, a well-characterized regulatory protein known as the "guardian of the genome," played a central role in this protective process. Using cell culture, they demonstrated that drugs that (indirectly) activate p53 could promote the clearance of the toxic insoluble forms of mutant SOD1. They concluded that targeting p53 could be a potential wide-spectrum anti-proteotoxicity therapeutic strategy.

    While this study gives a compelling demonstration that p53 can be activated to protect cells from toxic protein aggregation, there are caveats about this being a therapeutic route in human neurodegenerative disease. Because p53 is a multifunctional protein that can promote cell repair or cell death, calibrating its activation to be therapeutic and not deleterious seems inherently challenging. As a practical matter, activating FOXO proteins—previously well-established to combat multiple forms of cell stress—might be a better therapeutic approach. Despite an impressive array of experiments, the researchers did not introduce a loss-of-function mutation in worm p53 (in the cep-1 gene) into their double-mutant suppressed worms, so it is actually an open question whether the proposed mechanism of SOD1 toxicity suppression is at work in the worm model. As is always the case, interpretation of results from model systems is inherently limited by how closely the models reproduce human disease pathology. Many transgenic models significantly over-express disease-associated proteins, leaving it uncertain whether interventions that work in these models are targeting the relevant toxic processes, or just generic aggregating protein toxicity. Nevertheless, proteotoxicity appears to be an underlying factor in almost all age-associated neurodegenerative diseases, and understanding the cellular processes that moderate proteotoxicity is undeniably relevant to understanding these diseases. 

    View all comments by Chris Link

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References

News Citations

  1. Linking APP to Apoptosis—p53 Does the Trick
  2. Exploring Genome Fragmentation in Neurodegeneration
  3. Friend or Foe? Tumor Suppressor p53 Enhances Huntingtin Toxicity

Paper Citations

  1. Eimer S, Lakowski B, Donhauser R, Baumeister R. Loss of spr-5 bypasses the requirement for the C.elegans presenilin sel-12 by derepressing hop-1. EMBO J. 2002 Nov 1;21(21):5787-96. PubMed.
  2. Huang J, Sengupta R, Espejo AB, Lee MG, Dorsey JA, Richter M, Opravil S, Shiekhattar R, Bedford MT, Jenuwein T, Berger SL. p53 is regulated by the lysine demethylase LSD1. Nature. 2007 Sep 6;449(7158):105-8. PubMed.
  3. Wu H, Leng RP. UBE4B, a ubiquitin chain assembly factor, is required for MDM2-mediated p53 polyubiquitination and degradation. Cell Cycle. 2011 Jun 15;10(12):1912-5. PubMed.
  4. Chen GX, Zhang S, He XH, Liu SY, Ma C, Zou XP. Clinical utility of recombinant adenoviral human p53 gene therapy: current perspectives. Onco Targets Ther. 2014;7:1901-9. Epub 2014 Oct 21 PubMed.

External Citations

  1. superoxide dismutase 1
  2. ubiquitin fusion degradation 2
  3. suppressor of presenilin 5
  4. ubiquitination factor E4B
  5. lysine-specific demethylase 1A

Further Reading

Papers

  1. Nanduri P, Hao R, Fitzpatrick T, Yao TP. Chaperone-mediated 26S proteasome remodeling facilitates free K63 ubiquitin chain production and aggresome clearance. J Biol Chem. 2015 Apr 10;290(15):9455-64. Epub 2015 Feb 24 PubMed.
  2. Lim J, Yue Z. Neuronal aggregates: formation, clearance, and spreading. Dev Cell. 2015 Feb 23;32(4):491-501. PubMed.
  3. Cortes CJ, La Spada AR. The many faces of autophagy dysfunction in Huntington's disease: from mechanism to therapy. Drug Discov Today. 2014 Jul;19(7):963-71. Epub 2014 Mar 13 PubMed.
  4. Fonseca AC, Oliveira CR, Pereira CF, Cardoso SM. Loss of proteostasis induced by amyloid beta peptide in brain endothelial cells. Biochim Biophys Acta. 2014 Jun;1843(6):1150-61. Epub 2014 Feb 28 PubMed.
  5. Vilchez D, Saez I, Dillin A. The role of protein clearance mechanisms in organismal ageing and age-related diseases. Nat Commun. 2014 Dec 8;5:5659. PubMed.
  6. Osellame LD, Duchen MR. Quality Control Gone Wrong: Mitochondria, Lysosomal Storage Disorders and Neurodegeneration. Br J Pharmacol. 2013 Oct 10; PubMed.

Primary Papers

  1. Periz G, Lu J, Zhang T, Kankel MW, Jablonski AM, Kalb R, McCampbell A, Wang J. Regulation of protein quality control by UBE4B and LSD1 through p53-mediated transcription. PLoS Biol. 2015 Apr;13(4):e1002114. Epub 2015 Apr 2 PubMed.

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