Much like other RNA-binding proteins, TDP-43 sports a disordered domain at its C-terminal end. Researchers believe these floppy tails enable the proteins to move effortlessly into and out of RNA granules—membraneless organelles that package and process RNA. A paper in the August 18 Structure suggests that for TDP-43, α-helices in the otherwise disordered domain form an intricate lattice that allows RNA granules to separate from the surrounding cytoplasm in a process known as liquid-liquid phase separation. Scientists led by Nicolas Fawzi, Brown University, Providence, Rhode Island, report that mutations associated with amyotrophic lateral sclerosis disrupt this phase transition, making the protein more likely to aggregate instead.
“From people to fish, we have the same exact sequence in this helical region,” Fawzi told Alzforum. “Now we are able to provide some new insight about what that region does.”
Other scientists agreed that the findings represent a significant advance. “Since we know very little about the protein-protein interactions that underlie phase separation, characterizing the contribution of the helix is new and important,” wrote Paul Taylor, St. Jude Children's Research Hospital, Memphis, to Alzforum. Taylor was not involved in the research.
Taylor and others previously had proposed that these disordered regions—also known as low-complexity domains—at the tail end of TDP-43 and other RNA-binding proteins such as FUS and hnRNPA1 facilitate the formation of RNA-protein assemblies such as stress granules (Molliex et al., 2015). Following on that idea, Fawzi’s group proposed that these assemblies formed via transient contacts among the proteins’ C-terminal domains, which remained disordered (Oct 2015 news on Burke et al., 2015). But researchers led by Jianxing Song, National University of Singapore, reported that the C-terminal domain of TDP-43 likely contains an evolutionarily conserved α-helix that is important for function, possibly allowing the protein to embed itself in the cell membrane (Lim et al., 2016). Could that ordered structure be involved in the protein-protein interactions of this otherwise disordered domain?
To test this, first author Alexander Conicella and colleagues first used NMR spectroscopy and other biophysical techniques to determine that amino acids 321 to 330 of the TDP-43 protein do form an α-helix. Interestingly, upon interacting with a partner helix, the structure became even more structured, with the helix extending to include amino acids 331 to 340. They then demonstrated that in a test tube the TDP-43 C-terminal domain can form liquid droplets reminiscent of RNA-protein assemblies.
Next, Conicella and colleagues found that the α-helix was crucial for this liquid phase separation. If the authors deleted the helical domain or introduced mutations that left it intact but interrupted the helix-helix interactions, no droplets materialized. Likewise, ALS mutations A321G and A321V, which slightly disrupt the helix, limited droplet formation—either they failed to form or they formed less efficiently. In a matter of hours though, these mutated TDP-43 proteins started to clump together into aggregates, which could be seen by differential interference contrast microscopy.
Taken together, the data suggest that in the normal TDP 43, α-helices align precisely so that the proteins interact in an orderly way. However, if a minor change disrupts that interaction, the protein clumps up and forms aggregates.
The work supports the hypothesis that ALS mutations lead to a loss of normal protein function, said Fawzi, though he did not rule out an additional toxic gain of function for the aggregates, as has been proposed previously (see Oct 2015 news). Fawzi noted that this helical interaction might be a good therapeutic target for stabilization by a small molecule. Researchers are pursuing a similar strategy for other protein assemblies such as the tetramers normally formed by the proteins transthyretin or α-synuclein (Dec 2013 news; Apr 2015 conference news).
Fawzi is currently testing other ALS-associated TDP-43 mutations to see if they affect the helical interactions. He is also working out the protein’s structure inside the granules.
“This is a very nice paper,” wrote Clifford Brangwynne, Princeton University, New Jersey, to Alzforum. “The biophysical relationship between liquid-liquid phase separation and aggregation still remains largely unclear for TDP-43 and other disease-related proteins, but this study provides a much-needed molecular view of the protein-protein contacts that underlie these processes.”
Taylor agreed. “These authors provide novel detail into the molecular basis of phase separation,” he wrote. But he said the question of how the mutations drive disease still remains. It could be by impairing the assembly and function of membraneless organelles such as stress granules, by enhancing the formation of toxic fibrils, or both.
Fawzi cautioned that because the experiments were done in a test tube with pure TDP-43 fragments and because RNA granules in vivo contain different types of protein, it remains to be seen how these interactions play out in a cell. However, he pointed to a recent, complementary finding that was published in the August 2 Cell Reports. It detailed the importance of the same subdomain of TDP-43’s C-terminal end in phase-separated droplets of TDP-43 within HEK293T cells. Broder Schmidt and Rajat Rohatgi at the Stanford School of Medicine in California found that if they deleted the region entirely, the protein could not form phase-separated droplets in the cells. Replacing the conserved helix with a non-helical segment similar to the rest of the disordered domain made it assemble into stable filaments that impaired cell growth. In addition, the ALS-associated variants M337V, N345K, and A382T, all of which somehow change the secondary structure of the conserved region, impaired the formation or function of RNA granules in some way. They also concluded that this conserved helix enables TDP-43 to enter or form these membraneless organelles.
Scientists previously proposed that the TDP-43 helix interacts with RNA-binding domains of other proteins, or that it tethers TDP-43 to the cell membrane, Song wrote to Alzforum. “It will be interesting to figure out the interplay of these roles and their physiological and pathological consequences,” he added. It also will be important to know whether the process of TDP-43 assembly that Fawzi describes is a separate pathway from formation of amyloid fibers, or a different stage of the same pathway. To parse this out, Song proposed designing mutants with stabilized helices and testing their toxicity in neurons.—Gwyneth Dickey Zakaib
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- Conicella AE, Zerze GH, Mittal J, Fawzi NL. ALS Mutations Disrupt Phase Separation Mediated by α-Helical Structure in the TDP-43 Low-Complexity C-Terminal Domain. Structure. 2016 Sep 6;24(9):1537-49. Epub 2016 Aug 18 PubMed.
- Schmidt HB, Rohatgi R. In Vivo Formation of Vacuolated Multi-phase Compartments Lacking Membranes. Cell Rep. 2016 Aug 2;16(5):1228-36. Epub 2016 Jul 21 PubMed.