The roles of intrinsic disorder-based liquid-liquid phase transitions in the "Dr. Jekyll-Mr. Hyde" behavior of proteins involved in amyotrophic lateral sclerosis and frontotemporal lobar degeneration

Abstract

Pathological developments leading to amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD) are associated with misbehavior of several key proteins, such as SOD1 (superoxide dismutase 1), TARDBP/TDP-43, FUS, C9orf72, and dipeptide repeat proteins generated as a result of the translation of the intronic hexanucleotide expansions in the C9orf72 gene, PFN1 (profilin 1), GLE1 (GLE1, RNA export mediator), PURA (purine rich element binding protein A), FLCN (folliculin), RBM45 (RNA binding motif protein 45), SS18L1/CREST, HNRNPA1 (heterogeneous nuclear ribonucleoprotein A1), HNRNPA2B1 (heterogeneous nuclear ribonucleoprotein A2/B1), ATXN2 (ataxin 2), MAPT (microtubule associated protein tau), and TIA1 (TIA1 cytotoxic granule associated RNA binding protein). Although these proteins are structurally and functionally different and have rather different pathological functions, they all possess some levels of intrinsic disorder and are either directly engaged in or are at least related to the physiological liquid-liquid phase transitions (LLPTs) leading to the formation of various proteinaceous membrane-less organelles (PMLOs), both normal and pathological. This review describes the normal and pathological functions of these ALS- and FTLD-related proteins, describes their major structural properties, glances at their intrinsic disorder status, and analyzes the involvement of these proteins in the formation of normal and pathological PMLOs, with the ultimate goal of better understanding the roles of LLPTs and intrinsic disorder in the "Dr. Jekyll-Mr. Hyde" behavior of those proteins.

Keywords: amyotrophic lateral sclerosis; frontotemporal lobar degeneration; intrinsically disordered proteins; liquid-liquid phase transition; neurodegeneration; proteinaceous membrane-less organelles.

Figure 1.

Classic and modern protein structure-function relationships. Schematic representation of the classic “one gene–one protein–one function” paradigm (top part, blue) and its modification by alternative splicing and PTMs when affected genes encode ordered proteins (middle part, pink) or intrinsically disordered and hybrid proteins containing ordered and intrinsically disordered domains (bottom part, red). Reproduced with permission from ref. .

Figure 2.

Factors causing pathogenic transformation in an IDP/IDPR. Schematic representation of different ways by which aberrant regulation at the genetic level or various posttranslational (non-genetic) mechanisms can cause pathogenic transformation in an IDP/IDPR. Reproduced with permission from ref. .

Figure 3.

Intrinsic disorder in proteins presented in this study. The intrinsic disorder predisposition of these proteins was evaluated by PONDR® VSL2. (A) SOD1; (B) TARDBP; (C) FUS; (D) C9orf72; (E) PFN1; (F) GLE1; (G) PURA; (H) FLCN; (I) RBM45; (J) SS18L1/CREST; (K) HNRNPA1; (L) HNRNPA2B1; (M) ATXN2; (N) TIA1; and (O) MAPT. In the corresponding plots, regions with disorder scores above the 0.5 threshold (shown as a thin black line) are predicted to be intrinsically disordered.

Figure 4.

Multifactorial analysis of intrinsic disorder in GLE1 (UniProt ID: Q53GS7) (A) and PURA (UniProt ID: Q00577) (B). Intrinsic disorder propensity and some important disorder-related functional information generated for the corresponding proteins by the D²P² database are shown. Here, the green-and-white bar in the middle of the plot shows the predicted disorder agreement among 9 predictors, with green parts corresponding to disordered regions by consensus. The yellow bar shows the location of the predicted disorder-based binding sites (molecular recognition features, MoRFs), whereas colored circles at the bottom of the plot show the location of various PTMs.

Figure 5.

Multifactorial analysis of intrinsic disorder in FLCN (UniProt ID: Q8NFG4) (A), SS18L1/CREST (UniProt ID: O75177) (B), and RBM45 (UniProt ID: Q8IUH3) (C). Plots (A) and (B) were generated using the D²P² database. Here, keys are similar to those described in legends to Fig. 4. Because the D²P² database does not have related information for human RBM45, plot (C) represents disorder profiles generated by PONDR® VLXT (black line), PONDR® FIT (red line), PONDR® VL3 (green line), PONDR® VSL2 (yellow line), IUPred_short (blue line) and IUPred_long (pink line). The cyan dashed line shows the mean disorder propensity calculated by averaging the disorder profiles of the individual predictors. The light pink shadow around the PONDR® FIT shows the error distribution. In these analyses, the predicted intrinsic disorder scores above 0.5 are considered to correspond to the disordered residues/regions, whereas regions with disorder scores between 0.2 and 0.5 are considered flexible.

Figure 6.

Diversity of PMLOs found in eukaryotic cells. Schematic representation of the multitude of cytoplasmic, nuclear, mitochondrial and chloroplast PMLOs.

Figure 7.

Effect of the ALS-related mutations within the C-terminal region of human TARDBP on the generation and morphology of SGs in response to osmotic stress. (A) Localization of the exogenous wild-type TARDBP, as well as transiently expressed pathological TARDBP mutants and TARDBP lacking the C-terminal tail in HEK293T cells. Exogenous TARDBP was stained with anti-MYC antibody; nuclei, with ToPro-3. Bar: 10 μm. (B) Levels of expression of proteins shown in panel (A). (C) Quantification of SG size (pixels²/granule) following 1 h of sorbitol stress. Shown are mean granule sizes ± SEM for wild-type (open bar) and mutant (filled bars) TARDBP (*, P < 0.05). Reproduced with permission from ref. .

Figure 8.

Effect of mutated forms of VCP (A232E and R155H) on the formation of constitutive SGs and the recruitment of TARDBP to those SGs. HeLa cells were transfected with plasmid expressing wild-type or mutant VCP-GFP forms and stained for the SG marker EIF3B and TARDBP. Overexpression of mutant but not wild-type VCP resulted in the formation of TARDBP-containing SGs. Scale bar: 10 µm. Reproduced with permission from ref. .