. 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.


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  1. The paper is very interesting. As compared to our previous paper (Lim et al., 2016), two points are particularly interesting:

    1.     In 2005, we discovered that previously-claimed “insoluble proteins,” including the most hydrophobic integral membrane fragment in nature, could all be solubilized in pure water (unsalted) (Song, 2009). Now our discovery has been confirmed by many labs worldwide and, in particular, a consortium of Japanese scientists showed that almost all human proteins, including membrane proteins, could be dissolved in pure water for preparing cancer vaccines (Futami et al., 2014). With this discovery, we have successfully studied several previously thought “insoluble” ALS-causing proteins/mutants including TDP-43 N-domain (Qin et al., 2014), prion-like domain (Lim et al., 2016), and SOD1 (Lim et al., 2015; Lim and Song, 2016). 

    Very interestingly, Professor Fawzi’s group used an organic buffer MES (N-morpholinoethanesulfonic acid) of very low ionic strength to mimic pure water to successfully study TDP-43 prion-like domain in their current paper, as they previously did for the FUS prion-like domain (Burke et al., 2015). Our latest study indicates that MES buffer is indeed similar to unsalted water (Lu et al., 2016). This is a very interesting result that most likely can be generally applied to studying other “insoluble proteins” causing ALS or other diseases/aging.

    2.     The most novel discovery is that the helical region is critical to maintaining the dynamic assembly of the wild-type TDP-43 prion-like domain necessary for its functions, while the destabilization of the helix will lead to pathological aggregation. It is very important to note that the helical region is a typical hydrophobic fragment that bears no prion-like sequence, but plays a central role in forming classic amyloid fibers. Our latest results indeed show that a shorter TDP-43 C-terminal domain lacking this hydrophobic region is no longer able to form classic amyloid fibers (Lu et al., 2016). Therefore, Professor Fawzi’s current results, together with our previous (Lim et al., 2016) and latest findings (Burke et al., 2015), reveal a significant difference between the molecular mechanisms for the assembly of the WT and ALS-causing mutants of TDP-43 C-domain: Although both of them form fibrillar structures, as we found (Lim et al., 2016), in the dynamic WT assembly the helical region continues to be a helix, while in the ALS-causing mutants, the helical region transforms into amyloid structures rich in cross-β structures as judged by CD and fluorescence spectra in our previous paper (Lim et al., 2016). Furthermore, as the TDP-43 C-terminal domain contains this hydrophobic helical region and prion-like sequences, it appears that two mechanisms are simultaneously operating in the assembly/amyloid formation of the TDP-43 C-terminal domain: The hydrophobic region follows the classic mechanism for protein-protein interactions/amyloid formation, while the prion-like sequences assemble into a dynamic/reversible hydrogel with a pH-dependent transformation from the intramolecular to intermolecular backbone-sidechain hydrogen networks, as we first proposed (Lim et al., 2016) and now further enforced (Lu et al., 2016). The delineation of the different mechanisms may have important implications in understanding the molecular basis for ALS and other protein-aggregation-causing diseases/aging.

    Furthermore, I also have some thoughts for further investigations:

    1.     The helical region has now been revealed to be potentially involved in three roles: 1) mediation of the dynamic assembly of TDP-43 C-domain as shown in Professor Fawzi’s current paper; 2) interaction with the RNA-binding domains in the context of the full-length TDP-43 (Wei et al., 2016); 3) potential interaction with membranes (Lim et al., 2016). It will be extremely interesting to figure out how these roles interplay and are regulated in cells, and what their physiological and pathological consequences might be. Also, whether the potential to interact with membranes is physiological or just a pathological aberrance needs to be addressed. Recently we found documented a biophysical mechanism (Qin et al., 2013; Lim et al., 2015) whereby ALS-causing mutants of cytosolic SOD1, whose native functions have nothing to do with membranes, can, unbelievably, acquire the capacity to insert into ER membranes to initiate ALS without any detectable protein aggregation (Sun et al., 2015). We have even found that although the native functions of the E. coli protein S1 have nothing to do with membranes, cutting it to mimic the oxidation-induced fragmentation seen during aging unlocks fragments with high amphiphilicity/hydrophobicity that have characteristics of “insolubility” in buffers but suddenly acquire the novel capacity to interact with membranes (Lim et al., 2016). 

    2.     It is also very interestingly to investigate whether the dynamic assembly mediated by the helix formation and pathological formation of amyloid fibers represent two different pathways, or are different stages of the same pathway. It may be possible to clarify this by assessing if mutants designed to significantly stabilize the helix can reduce or completely remove neurotoxicity in vivo.


    . ALS-Causing Mutations Significantly Perturb the Self-Assembly and Interaction with Nucleic Acid of the Intrinsically Disordered Prion-Like Domain of TDP-43. PLoS Biol. 2016 Jan;14(1):e1002338. Epub 2016 Jan 6 PubMed.

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    . Unlocked capacity of proteins to attack membranes characteristic of aggregation: the evil for diseases and aging from Pandora's box. bioRχiv. 2016 Aug 24.

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