Insights into the aggregation mechanism of RNA recognition motif domains in TDP-43: a theoretical exploration.
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ABSTRACT:
SUBMITTER: Liu W
PROVIDER: S-EPMC8371369 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Many of the molecular mechanisms underlying the pathological aggregation of proteins observed in neurodegenerative diseases are still not fully understood. Among the aggregate-associated diseases, Amyotrophic Lateral Sclerosis (ALS) is of relevant importance. In fact, although understanding the processes that cause the disease is still an open challenge, its relationship with protein aggregation is widely known. In particular, human TDP-43, an RNA/DNA binding protein, is a major component of the pathological cytoplasmic inclusions observed in ALS patients. Indeed, the deposition of the phosphorylated full-length TDP-43 in spinal cord cells has been widely studied. Moreover, it has also been shown that the brain cortex presents an accumulation of phosphorylated C-terminal fragments (CTFs). Even if it is debated whether the aggregation of CTFs represents a primary cause of ALS, it is a hallmark of TDP-43 related neurodegeneration in the brain. Here, we investigate the CTFs aggregation process, providing a computational model of interaction based on the evaluation of shape complementarity at the molecular interfaces. To this end, extensive Molecular Dynamics (MD) simulations were conducted for different types of protein fragments, with the aim of exploring the equilibrium conformations. Adopting a newly developed approach based on Zernike polynomials, able to find complementary regions in the molecular surface, we sampled a large set of solvent-exposed portions of CTFs structures as obtained from MD simulations. Our analysis proposes and assesses a set of possible association mechanisms between the CTFs, which could drive the aggregation process of the CTFs. To further evaluate the structural details of such associations, we perform molecular docking and additional MD simulations to propose possible complexes and assess their stability, focusing on complexes whose interacting regions are both characterized by a high shape complementarity and involve β3 and β5 strands at their interfaces.
| S-EPMC8699346 | biostudies-literature
Project description:A common pathological hallmark of several neurodegenerative diseases, including amyotrophic lateral sclerosis, is cytoplasmic mislocalization and aggregation of nuclear RNA-binding protein TDP-43. Perry disease, which displays inherited atypical parkinsonism, is a type of TDP-43 proteinopathy. The causative gene DCTN1 encodes the largest subunit of the dynactin complex. Dynactin associates with the microtubule-based motor cytoplasmic dynein and is required for dynein-mediated long-distance retrograde transport. Perry disease-linked missense mutations (e.g., p.G71A) reside within the CAP-Gly domain and impair the microtubule-binding abilities of DCTN1. However, molecular mechanisms by which such DCTN1 mutations cause TDP-43 proteinopathy remain unclear. We found that DCTN1 bound to TDP-43. Biochemical analysis using a panel of truncated mutants revealed that the DCTN1 CAP-Gly-basic supradomain, dynactin domain, and C-terminal region interacted with TDP-43, preferentially through its C-terminal region. Remarkably, the p.G71A mutation affected the TDP-43-interacting ability of DCTN1. Overexpression of DCTN1G71A, the dynactin-domain fragment, or C-terminal fragment, but not the CAP-Gly-basic fragment, induced cytoplasmic mislocalization and aggregation of TDP-43, suggesting functional modularity among TDP-43-interacting domains of DCTN1. We thus identified DCTN1 as a new player in TDP-43 cytoplasmic-nuclear transport, and showed that dysregulation of DCTN1-TDP-43 interactions triggers mislocalization and aggregation of TDP-43, thus providing insights into the pathological mechanisms of Perry disease and other TDP-43 proteinopathies.
| S-EPMC8070438 | biostudies-literature
Project description:Several neurodegenerative diseases including amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration with ubiquitinated inclusions (FTLD-U) are characterized by inclusion bodies formed by TDP-43 (TDP). We established cell and transgenic Drosophila models expressing TDP carboxyl terminal fragment (ND251 and ND207), which developed aggregates recapitulating important features of TDP inclusions in ALS/FTLD-U, including hyperphosphorylation at previously reported serine(403,404,409,410) residues, polyubiquitination and colocalization with optineurin. These models were used to address the pathogenic role of hyperphosphorylation in ALS/FTLD-U. We demonstrated that hyperphosphorylation and ubiquitination occurred temporally later than aggregation in cells. Expression of CK2? which phosphorylated TDP decreased the aggregation propensity of ND251 or ND207; this effect could be blocked by CK2 inhibitor DMAT. Mutation of serines(379,403,404,409,410) to alanines (S5A) to eliminate phosphorylation increased the aggregation propensity and number of aggregates of TDP, but mutation to aspartic acids (S5D) or glutamic acids (S5E) to simulate hyperphosphorylation had the opposite effect. Functionally, ND251 or ND207 aggregates decreased the number of neurites of Neuro2a cells induced by retinoic acid or number of cells by MTT assay. S5A mutation aggravated, but S5E mutation alleviated these cytotoxic effects of aggregates. Finally, ND251 or ND251S5A developed aggregates in neurons, and salivary gland of transgenic Drosophila, but ND251S5E did not. Taken together, our data indicate that hyperphosphorylation may represent a compensatory defense mechanism to stop or prevent pathogenic TDP from aggregation. Therefore, enhancement of phosphorylation may serve as an effective therapeutic strategy against ALS/FTLD-U.
| S-EPMC3151276 | biostudies-literature
Project description:Post-translational modifications (PTMs) have emerged as key modulators of protein phase separation and have been linked to protein aggregation in neurodegenerative disorders. The major aggregating protein in amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), the RNA-binding protein TDP-43, is hyperphosphorylated in disease on several C-terminal serine residues, a process generally believed to promote TDP-43 aggregation. Here, we however find that Casein kinase 1 -mediated TDP-43 hyperphosphorylation or C-terminal phosphomimetic mutations reduce TDP-43 phase separation and aggregation, and instead render TDP-43 condensates more liquid-like and dynamic. Multi-scale molecular dynamics simulations reveal reduced homotypic interactions of TDP-43 low-complexity domains through enhanced solvation of phosphomimetic residues. Cellular experiments show that phosphomimetic substitutions do not affect nuclear import or RNA regulatory functions of TDP-43, but suppress accumulation of TDP-43 in membrane-less organelles and promote its solubility in neurons. We speculate that TDP-43 hyperphosphorylation may be a protective cellular response to counteract TDP-43 aggregation.
| S-SCDT-EMBOJ-2021-108443 | biostudies-other
Project description:Pathological alteration of TDP-43 (TAR DNA-binding protein-43), a protein involved in various RNA-mediated processes, is a hallmark feature of the neurodegenerative diseases amyotrophic lateral sclerosis and frontotemporal lobar degeneration. Fragments of TDP-43, composed of the second RNA recognition motif (RRM2) and the disordered C terminus, have been observed in cytoplasmic inclusions in sporadic amyotrophic lateral sclerosis cases, suggesting that conformational changes involving RRM2 together with the disordered C terminus play a role in aggregation and toxicity. The biophysical data collected by CD and fluorescence spectroscopies reveal a three-state equilibrium unfolding model for RRM2, with a partially folded intermediate state that is not observed in RRM1. Strikingly, a portion of RRM2 beginning at position 208, which mimics a cleavage site observed in patient tissues, increases the population of this intermediate state. Mutually stabilizing interactions between the domains in the tethered RRM1 and RRM2 construct reduce the population of the intermediate state and enhance DNA/RNA binding. Despite the high sequence homology of the two domains, a network of large hydrophobic residues in RRM2 provides a possible explanation for the increased stability of RRM2 compared with RRM1. The cluster analysis suggests that the intermediate state may play a functional role by enhancing access to the nuclear export signal contained within its sequence. The intermediate state may also serve as a molecular hazard linking productive folding and function with pathological misfolding and aggregation that may contribute to disease.
| S-EPMC3961654 | biostudies-literature
Project description:TAR DNA binding protein 43 kDa (TDP-43) is a ribonuclear protein regulating many aspects of RNA metabolism. Amyotrophic Lateral Sclerosis (ALS) and Frontotemporal Lobar Degeneration (FTLD) are fatal neurodegenerative diseases with the presence of TDP-43 aggregates in neuronal cells. Chaperone Mediated Autophagy (CMA) is a lysosomal degradation pathway participating in the proteostasis of several cytosolic proteins including neurodegenerative associated proteins. In addition, protein oligomers or aggregates can affect the status of CMA. In this work, we studied the relationship between CMA and the physiological and pathological forms of TDP-43. First, we found that recombinant TDP-43 was specifically degraded by rat liver's CMA+ lysosomes and that endogenous TDP-43 is localized in rat brain's CMA+ lysosomes, indicating that TDP-43 can be a CMA substrate in vivo. Next, by using a previously reported TDP-43 aggregation model, we have shown that wild-type and an aggregate-prone form of TDP-43 are detected in CMA+ lysosomes isolated from cell cultures. In addition, their protein levels increased in cells displaying CMA down-regulation, indicating that these two TDP-43 forms are CMA substrates in vitro. Finally, we observed that the aggregate-prone form of TDP-43 is able to interact with Hsc70, to co-localize with Lamp2A, and to up-regulate the levels of these molecular components of CMA. The latter was followed by an up-regulation of the CMA activity and lysosomal damage. Altogether our data shows that: (i) TDP-43 is a CMA substrate; (ii) CMA can contribute to control the turnover of physiological and pathological forms of TDP-43; and (iii) TDP-43 aggregation can affect CMA performance. Overall, this work contributes to understanding how a dysregulation between CMA and TDP-43 would participate in neuropathological mechanisms associated with TDP-43 aggregation.
| S-EPMC7040037 | biostudies-literature
Project description:Aggregation of the RNA-binding protein, TDP-43, is the unifying hallmark of amyotrophic lateral sclerosis and frontotemporal dementia. TDP-43-related neurodegeneration involves multiple changes to normal physiological TDP-43, which undergoes nuclear depletion, cytoplasmic mislocalisation, post-translational modification, and aberrant liquid-liquid phase separation, preceding inclusion formation. Along with toxic cytoplasmic aggregation, concurrent depletion and dysfunction of normal nuclear TDP-43 in cells with TDP-43 pathology is likely a key potentiator of neurodegeneration, but is not well understood. To define processes driving TDP-43 dysfunction, we used CRISPR/Cas9-mediated fluorescent tagging to investigate how disease-associated stressors and pathological TDP-43 alter abundance, localisation, self-assembly, aggregation, solubility, and mobility dynamics of normal nuclear TDP-43 over time in live cells. Oxidative stress stimulated liquid-liquid phase separation of endogenous TDP-43 into droplet-like puncta, or spherical shell-like anisosomes. Further, nuclear RNA-binding-ablated or acetylation-mimicking TDP-43 readily sequestered and depleted free normal nuclear TDP-43 into dynamic anisosomes, in which recruited endogenous TDP-43 proteins remained soluble and highly mobile. Large, phosphorylated inclusions formed by nuclear or cytoplasmic aggregation-prone TDP-43 mutants also caused sequestration, but rendered endogenous TDP-43 immobile and insoluble, indicating pathological transition. These findings suggest that RNA-binding deficiency and post-translational modifications including acetylation exacerbate TDP-43 aggregation and dysfunction by driving sequestration, mislocalisation, and depletion of normal nuclear TDP-43 in neurodegenerative diseases.
| S-EPMC10023653 | biostudies-literature
Project description:TAR DNA-binding protein 43 (TDP-43) is a major component in aggregates of ubiquitinated proteins in amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD). Here we report that lipopolysaccharide (LPS)-induced inflammation can promote TDP-43 mislocalization and aggregation. In culture, microglia and astrocytes exhibited TDP-43 mislocalization after exposure to LPS. Likewise, treatment of the motoneuron-like NSC-34 cells with TNF-alpha (TNF-?) increased the cytoplasmic levels of TDP-43. In addition, the chronic intraperitoneal injection of LPS at a dose of 1mg/kg in TDP-43(A315T) transgenic mice exacerbated the pathological TDP-43 accumulation in the cytoplasm of spinal motor neurons and it enhanced the levels of TDP-43 aggregation. These results suggest that inflammation may contribute to development or exacerbation of TDP-43 proteinopathies in neurodegenerative disorders.
| S-EPMC4596857 | biostudies-literature
Project description:TDP-43 is a nuclear RNA-binding protein that forms neuronal cytoplasmic inclusions in two major neurodegenerative diseases, ALS and FTLD. While the self-assembly of TDP-43 by its structured N-terminal and intrinsically disordered C-terminal domains has been widely studied, the mechanism by which mRNA preserves TDP-43 solubility in the nucleus has not been addressed. Here, we demonstrate that tandem RNA recognition motifs of TDP-43 bind to long GU-repeats in a cooperative manner through intermolecular interactions. Moreover, using mutants whose cooperativity is impaired, we found that the cooperative binding of TDP-43 to mRNA may be critical to maintain the solubility of TDP-43 in the nucleus and the miscibility of TDP-43 in cytoplasmic stress granules. We anticipate that the knowledge of a higher order assembly of TDP-43 on mRNA may clarify its role in intron processing and provide a means of interfering with the cytoplasmic aggregation of TDP-43.
| S-EPMC8523171 | biostudies-literature
Project description:The normally soluble TAR DNA-binding protein 43 (TDP-43) is found aggregated both in reversible stress granules and in irreversible pathogenic amyloid. In TDP-43, the low-complexity domain (LCD) is believed to be involved in both types of aggregation. To uncover the structural origins of these two modes of β-sheet-rich aggregation, we have determined ten structures of segments of the LCD of human TDP-43. Six of these segments form steric zippers characteristic of the spines of pathogenic amyloid fibrils; four others form LARKS, the labile amyloid-like interactions characteristic of protein hydrogels and proteins found in membraneless organelles, including stress granules. Supporting a hypothetical pathway from reversible to irreversible amyloid aggregation, we found that familial ALS variants of TDP-43 convert LARKS to irreversible aggregates. Our structures suggest how TDP-43 adopts both reversible and irreversible β-sheet aggregates and the role of mutation in the possible transition of reversible to irreversible pathogenic aggregation.
| S-EPMC5990464 | biostudies-literature