Two-in-One: Single Protein Untangles and Chops Up Tau Fibrils

Aggregated proteins pose a challenge for the brain’s degradation machinery because these tangled clumps often conceal the cleavage sites necessary for proteolytic digestion. Cells first have to deploy specialized proteins called disaggregases to pull apart knots and expose strands to proteases. In the October 5 Nature Chemical Biology, researchers led by Michael Ehrmann at University Duisburg-Essen, Germany, describe a single protein called high temperature requirement A protein 1 (HTRA1), which can both separate aggregates and chew them up. In cell cultures, a modified HTRA1 lacking protease activity broke apart tau fibrils within hours, speeding subsequent digestion of the pieces by wild-type HTRA1. The authors traced HTRA1’s activities to two different functional domains. Each provided clues to the protein’s mechanism of action. The protein might have potential as a therapeutic, Ehrmann suggested.

Other researchers praised the rigor of the chemical data. “They did a beautiful biophysical and biochemical characterization of how this protein functions,” said Keqiang Ye at Emory University, Atlanta. Commenters noted, however, that more work will be needed to determine what role HTRA1 plays in vivo. They also pointed out that its native disaggregase abilities appear weak compared to its proteolytic capability. “This is a promising avenue of research and an excellent proof of concept, but it needs some bioengineering to improve its disaggregase activity,” said Benjamin Wolozin at Boston University. Ehrmann wrote that he is working on optimizing the protein.

Untangling Tau. HTRA1 trimers use their flexible PDZ domains (blue) like fingers to tease apart tau fibrils (black), allowing their protease domains (orange) to chew up the strands. [Courtesy of Poepsel et al., Nature Chemical Biology.]

Powerful disaggregases exist in nature from simple organisms such as yeast on upward. Scientists have engineered one such enzyme, heat shock protein 104 (Hsp104), to break up numerous types of protein aggregate (see Aug 2014 news). However, Hsp104 is not found in mammals. HTRA1, on the other hand, is expressed in all mammalian tissues. Cells make one version that remains in the cytoplasm and a second that they secrete into the extracellular space. HTRA1, a serine protease, sports a C-terminal PDZ domain. This domain helps proteins bind to specific partners. Previously, Ehrmann and colleagues reported that HTRA1 could degrade fibrillar tau in test tubes and cell cultures. They found that among samples from 25 AD brains, those with the least HTRA1 had the most tau, neurofibrillary tangles, and neuritic plaques, hinting that the protein might affect pathology (see Tennstaedt et al., 2012). 

Because HTRA1 was able to digest these packed tau aggregates, Ehrmann wondered if the protease could pull fibrils apart. To test this, first author Simon Poepsel used a catalytically inactive version of the protein, HTRA1S328A. This form busted up tau fibrils in test tubes, slashing their number by three-quarters after 16 hours. The results indicated that HTRA1S328A was indeed a disaggregase. Switching to cultures, the authors seeded HEK293 cells that overexpressed tau with fibril fragments to induce aggregation, waited one day, then added HTRA1S328A to the media for 20 hours. Cells took up the protease, and intracellular tau aggregates dropped by two-thirds.

Two-Step Process. Tau fibrils (left) drop by half after treatment with wild-type HTRA1 (middle), but disintegrate by 80 percent when pretreated with the disaggregase form (right). [Courtesy of Poepsel et al., Nature Chemical Biology.]

In theory, breaking up tau clumps should improve subsequent digestion. The authors tested this by pre-incubating fibrils with HTRA1S328A for two hours before adding wild-type HTRA1. The amount of fibrils plummeted by almost 90 percent, compared to 50 percent for HTRA1 incubation alone (see image above). This suggested that the pretreatment exposed cleavage sites. A mass spectrometry analysis found that after pre-incubation, HTRA1 cut an average of 10 sites in the core region of tau and 16 in outer regions over three hours, as compared to cutting nowhere in the core and at only three sites in outer regions without pre-incubation. 

The authors further investigated the mechanism using electron microscopy. They found that HTRA1 bound along the whole length of tau fibrils, implying the protein can pull strands apart from any point. Since PDZ domains recognize β-strands, the authors reasoned that this region might play a role in disaggregation. HTRA1’s PDZ domain bound tau, but by itself was unable to separate fibrils. The authors next removed the PDZ domain from HTRA1S328A, and found that this protein barely budged tangles. A PDZ domain attached to HTRA1 is necessary for disaggregation, they concluded.

Because it attaches to HTRA1 via a flexible peptide linker, the authors speculated that the PDZ domain may, like fingers, grab strands and tug them apart. Notably, this process does not use cellular energy in the form of ATP, unlike other known disaggregases.

Can HTRA1 break apart other types of aggregate? To test this, the authors incubated the protein with Aβ42 deposits. They detected a boost in soluble peptide. Previously, Ehrmann and colleagues had reported that HTRA1 occurred in Aβ deposits in human brain, and that inhibiting the protease caused Aβ to accumulate in cultured astrocytes (see Grau et al., 2005). The protease may act as a general disaggregase, they suggest. 

Is busting up aggregates always good for the brain? Some studies suggest deposits can protect cells by sequestering toxic oligomers (see Oct 2004 news; Mar 2006 news; Dec 2009 news). To date, sparse data illuminate how HTRA1 expression affects particular disease states.

HTRA1 variants have been linked to conditions such as arthritis and cerebral small-vessel disease (see Milner et al., 2008; Hara et al., 2009). A 10-fold risk of wet macular degeneration is associated with the polymorphism rs11200638. This variant occurs in the HTRA1 promoter, and in retinal cells correlates with higher expression of the protein (see Dewan et al., 2006; Yang et al., 2006). 

A previous genetic study found no association between several SNPs in the HTRA1 gene and Alzheimer’s (see Turunen et al., 2011). Even so, Ehrmann wrote to Alzforum that he has unpublished data based on a collaboration with Rupert Egensperger, now at Ludwig Maximilians University, Munich, that links rs11200638 to an increased amyloid load in the brain and a three-years-earlier onset of AD. While this might suggest that elevated HTRA1 harms the brain, Ehrmann noted that the previous retinal expression data for this genetic variant was indirect and that expression has not been measured in neurons. The authors have applied for a patent on the use of this polymorphism as a screening tool, Ehrmann wrote.

Commenters noted that additional in vivo studies will help nail down the physiological effects of HTRA1. Chad Dickey at the University of South Florida in Tampa suggested looking for functional changes in cultured neurons, such as improvements in plasticity after busting up aggregated proteins. Ye recommended looking at HTRA1 knockouts and overexpression in animal models of AD. In an email, Ehrmann wrote that he has crossed HTRA1 knockouts with APPSwe mice in a small pilot study, and seen about a fivefold rise in total Aβ levels, supporting the idea that HTRA1 helps control deposits.—Madolyn Bowman Rogers

Comments

No Available Comments

Make a Comment

To make a comment you must login or register.

References

News Citations

  1. Yeast Chaperone Melts Protein Aggregates
  2. New Microscope Resolves Role of Huntington Inclusions—Neuroprotection
  3. Inclusions: Part of the Problem, or the Solution?
  4. Long Life With Tight Plaques—Repressing IGF-1 Protects AD Mice

Paper Citations

  1. Tennstaedt A, Pöpsel S, Truebestein L, Hauske P, Brockmann A, Schmidt N, Irle I, Sacca B, Niemeyer CM, Brandt R, Ksiezak-Reding H, Tirniceriu AL, Egensperger R, Baldi A, Dehmelt L, Kaiser M, Huber R, Clausen T, Ehrmann M. Human High Temperature Requirement Serine Protease A1 (HTRA1) Degrades Tau Protein Aggregates. J Biol Chem. 2012 Jun 15;287(25):20931-41. PubMed.
  2. Grau S, Baldi A, Bussani R, Tian X, Stefanescu R, Przybylski M, Richards P, Jones SA, Shridhar V, Clausen T, Ehrmann M. Implications of the serine protease HtrA1 in amyloid precursor protein processing. Proc Natl Acad Sci U S A. 2005 Apr 26;102(17):6021-6. PubMed.
  3. Milner JM, Patel A, Rowan AD. Emerging roles of serine proteinases in tissue turnover in arthritis. Arthritis Rheum. 2008 Dec;58(12):3644-56. PubMed.
  4. Hara K, Shiga A, Fukutake T, Nozaki H, Miyashita A, Yokoseki A, Kawata H, Koyama A, Arima K, Takahashi T, Ikeda M, Shiota H, Tamura M, Shimoe Y, Hirayama M, Arisato T, Yanagawa S, Tanaka A, Nakano I, Ikeda S, Yoshida Y, Yamamoto T, Ikeuchi T, Kuwano R, Nishizawa M, Tsuji S, Onodera O. Association of HTRA1 mutations and familial ischemic cerebral small-vessel disease. N Engl J Med. 2009 Apr 23;360(17):1729-39. PubMed.
  5. Dewan A, Liu M, Hartman S, Zhang SS, Liu DT, Zhao C, Tam PO, Chan WM, Lam DS, Snyder M, Barnstable C, Pang CP, Hoh J. HTRA1 promoter polymorphism in wet age-related macular degeneration. Science. 2006 Nov 10;314(5801):989-92. Epub 2006 Oct 19 PubMed.
  6. Yang Z, Camp NJ, Sun H, Tong Z, Gibbs D, Cameron DJ, Chen H, Zhao Y, Pearson E, Li X, Chien J, Dewan A, Harmon J, Bernstein PS, Shridhar V, Zabriskie NA, Hoh J, Howes K, Zhang K. A variant of the HTRA1 gene increases susceptibility to age-related macular degeneration. Science. 2006 Nov 10;314(5801):992-3. Epub 2006 Oct 19 PubMed.
  7. Turunen M, Vepsäläinen S, Mäkinen P, Helisalmi S, Haapasalo A, Soininen H, Hiltunen M. No association between high temperature requirement 1 (HTRA1) gene polymorphisms and Alzheimer's disease. Neurobiol Aging. 2011 Mar;32(3):547.e7-9. PubMed.

Further Reading

Primary Papers

  1. Poepsel S, Sprengel A, Sacca B, Kaschani F, Kaiser M, Gatsogiannis C, Raunser S, Clausen T, Ehrmann M. Determinants of amyloid fibril degradation by the PDZ protease HTRA1. Nat Chem Biol. 2015 Oct 5; PubMed.

Annotate

To make an annotation you must Login or Register.