What is the peptide fragment from the C-terminal region of TDP-43?

In addition to TDP-432C, a peptide fragment, aa: 286-331, from the C-terminal region of TDP-43 has also been shown to be amyloidogenic and neurotoxic [33].

What is the role of AIM4 in the LLPS of TDP-43?

from the in silico studies, in the 6N3C-A315T mutant TDP-43 structure (aa: 288-319), AIM4 was predicted to interact preferentially with two amino acids, glycine and phenylalanine which are also already known to be involved in its pathogenic liquid-liquid phase separation (LLPS).

How many M of mutant TDP-432C-A315T were added?

Then the compounds were added to the aggregation solution containing 400 µM of mutant TDP-432C-A315T, at a protein: compound ratio of 1:15.

At what concentration did the mutant TDP-432C-A315T exhibit amy?

The kinetic trend was observed to flatten to baseline at 1:15 protein: AIM4 concentration, whereas at 1:15 protein: AIM1 concentration the mutant TDP-432C continued to exhibit amyloid-like kinetics behavior although with a relatively prolonged lag phase as compared to the protein sample with 1:10 ratio of AIM1.

What is the structure of the ligands that were not available?

For the test ligands and other ligands whose structures were not available, the structures were first drawn using Marvin Sketch (ChemAxon), and then hydrogen atoms were added and finally the structures were 3D-cleaned and saved in ‘.pdb’ format.

How was the aggregation of mutant TDP-432C-A315T?

Then the compounds were added to the aggregation solution containing 400 µM of mutant TDP-432C-A315T, at a protein: AIM4/AIM1 ratio of 1:10, 1:15 or 1:20.

What was used to analyze protein-ligand interactions?

Simulation interactions diagram (SID) program available with the Desmond module was used to analyze protein-ligand RMSD, protein RMSF, ligand RMSF and protein-ligand interactions [83].

What is the effect of AIM4 on the phase separation of TDP-432C?

When the presence of liquid phase-separated droplets was examined, there was no visible phase separation in the TDP-432C-A315T sample incubated along with AIM4 at 1:15 ratio of the protein to AIM4.

How long did the MD simulations take to determine the stability of the AIM4 peptid?

the authors checked the stability of the 5WHN-AIM4 peptide-ligand complex, the peptide for which AIM4 displayed maximum affinity in docking, by running MD simulations for 100 nanoseconds.

What is the mechanism of AIM4 mediated anti-aggregation of TDP-?

In their previous study, the authors tested several acridine imidazolium derivatives and found that AIM4 was the most effective ligand to inhibit the in vitro aggregation of the TDP-432C [71].

How was the temperature kept at 300K?

The temperature was kept at 300K using Nose-Hoover chain method [88] and pressure was kept at 1 bar using Martyna-Tobias-Klein method [89].

What is the kinetics of the mutant TDP-432C-A315T?

Similar to as reported for the wild-type protein [71], TDP-432C-A315T also showed ThT fluorescence kinetics akin to that of an amyloid-like aggregation (Figure 6).

What peptides can form amyloid-like steric zippers?

For the peptides capable of forming amyloid-like steric zippers, 5WKD, 6CEW, 6CB9, 5WIA, and 5WIQ, the AIM4 molecule exhibited effective docking energy over the other ligands (Figure 2b and Table 2).

Open AccessJournal Article10.1016/J.IJBIOMAC.2020.01.032

Computational insights into mechanism of AIM4-mediated inhibition of aggregation of TDP-43 protein implicated in ALS and evidence for in vitro inhibition of liquid-liquid phase separation (LLPS) of TDP-432C-A315T by AIM4

Amandeep Girdhar, +10 more

- 15 Mar 2020

- International Journal of Biological Macr...

- Vol. 147, pp 117-130

TL;DR: It is found that AIM4 has a binding site in the C-terminal amyloidogenic region (aa: 288-319), with Gly-288 & Phe-289 residues which are also important for TDP-43's LLPS, and alike to previously reported effects of RNA, A IM4 could also inhibit the in vitro LLPS of a C-Terminal fragment T DP-432C bearing an A315T familial mutation.

About: This article is published in International Journal of Biological Macromolecules. The article was published on 15 Mar 2020. and is currently open access. The article focuses on the topics: Binding site.

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Most frequently asked questions

1. What contributions have the authors mentioned in the paper "Computational insights into mechanism of aim4-mediated inhibition of aggregation of tdp-43 protein implicated in als and evidence for in vitro inhibition of liquid-liquid phase separation (llps) of tdp-432c-a315t by aim4" ?

The structure of amyloid aggregates consists predominantly of β-sheets arranged in a cross-β conformation where the β-strands are arranged perpendicular to the fiber axis this paper.

2. What structures were used to predict the binding site of AIM4?

In addition, other known structures from TDP-43 sequence namely, tandem RRMs (aa: 96-269; PDB ID: 4BS2), N-terminal domain (aa: 1-89; PDB ID: 2N4P) and ten amyloidogenic peptide fragments from the low complexity domain (LCD) of TDP43, were also used to predict the binding site of AIM4.

3. What is the role of AIM4 in the inhibition of ALS?

Owing to the importance of inhibition of TDP-43 aggregation and the current unavailability of effective drugs against ALS, a small molecule AIM4 had been previously demonstrated to be a candidate for the inhibition of the TDP-43 aggregation [71].

4. What is the role of the prion-like low complexity domains in neurodegenerative?

membrane-less liquid droplet-like organelles formed by the proteins containing prion-like low complexity domains (LCD) are being implicated in several neurodegenerative diseases including ALS [5, 53-56].

Figure 4: Conformational stability of the mutant 6N3C-A315T TDP-43 (288- 319) complexed with AIM4 as determined by 100 ns of MD simulations. a. Protein only RMSD, protein-ligand (AIM4) RMSD and ligand only RMSD of the mutant A315T TDP-43 structure. RMSD of protein-ligand during MD simulation indicates the RMSD of a ligand when the protein-ligand complex is first aligned on the protein backbone of the reference and then the RMSD of the ligand heavy atoms is measured. Ligand RMSD indicates the RMSD of the ligand that is aligned and measured on its reference conformation. Protein only RMSD indicates the RMSD of the protein alone (Cα), where protein frames are first aligned on the reference frame backbone, and then the RMSD is calculated based on the atom selection. b. Root Mean Square Fluctuations (RMSF). ‘Fit Ligand on Protein’ trend indicates the ligand fluctuations with respect to the protein. 'Ligand' trend shows the fluctuations where the ligand in each frame is aligned on the ligand in the first reference frame c. The number of total contacts (H-bonds, Hydrophobic, Ionic, Water bridges) made between the mutant A315T TDP-43 peptide and the ligand (AIM4) over the course of the simulation. d. The interactions of AIM4 with the amino acids of mutant A315T TDP-43 peptide (aa: 288-319; PDB ID: 6N3C). The stacked bar charts have been normalized over the course of the trajectory: for example, a value of 1.0 suggests that the specific interaction is maintained for 100% of the simulation time. e. A timeline representation of the interactions and contacts (H-bonds, Hydrophobic, Ionic, Water bridges). it shows the residues of mutant A315T TDP-43 interacting with the ligand (AIM4) in each trajectory frame. Some residues make more than one specific contact with the ligand, which is represented by a darker shade of orange, according to the scale to the right of the plot. Fibrillar structure of 6N3C contains several chains from A to T which are appropriately labeled for its AIM4 interaction.

Figure 5: Binding poses of AIM4 and the predicted inhibitor constants for AIM4 and other used ligands with 6N3C and 6N3C-A315T TDP-43. a. The left panel shows AIM4 (orange) docked to 6N3C TDP-43 (aa: 288-319 A315E mutant) (grey) and the right panel displays the residues involved in molecular interactions with AIM4. b. The left panel shows AIM4 (orange) docked to mutant 6N3C-A315T TDP43 (aa: 288-319) (grey) and the right panel displays the residues involved in molecular interactions with AIM4. c. The predicted inhibitor constants for different ligands as determined by AutoDock software.

Figure 7: Acridine derivative AIM4 inhibits the in vitro liquid-liquid phase separation (LLPS) of the TDP-432C-A315T protein. a. Mutant A315T TDP-432C, labelled with Alexa Fluor™ 488 C5 maleimide undergoes transition from soluble state to liquid droplets suggesting of a process termed as liquidliquid phase separation (LLPS). Mutant TDP-432C-A315T (400 µM) was incubated at 37 °C in aggregation buffer (2.5M urea-PBS), either alone or with the additions of 1:15 (protein: compound molar ratio) of AIM4, AIM3, AIM1, DPH or dimebon, for a period of 5 hours. The Alexa Fluor-bound species appear as droplets as observed under fluorescence microscopy when examined using the GFP filter. With acridine compounds, AIM3 which affected TDP-432C aggregation to the least extent exhibited the formation of irregular solid shaped aggregates, whereas AIM1 which moderately inhibits TDP-432C aggregation, the formation of these liquid droplets gradually decreased and were present in far fewer numbers in comparison with the protein sample without any inhibitor and with AIM4, which exhibits maximum inhibitory potential to TDP-432C, the liquid droplets completely disappeared thereby indicating a complete inhibition of LLPS. All the images were taken at 10X magnification. b. The total number of liquid droplets found in each frame of the Alexa Fluor labelled protein without any inhibitor as well as with 1:15 (protein: compound molar ratio) of AIM1 and AIM4 were counted. The data represents the number of droplets found in 18 frames for each of the sample.

Figure 1: Protein and ligand structures used for docking.

Table 2: Docking energies of AIM4 and the other ligands with the structure of TDP-43 short amyloidogenic peptides obtained by docking.

Figure 3: Conformational stability of 6N3C (TDP-43 aa:288-319 A315E mutant) complexed with AIM4 as determined by 300 ns of MD simulations. a. Protein only RMSD, protein-ligand (AIM4) RMSD and ligand only RMSD of the mutant A315E TDP-43 structure. RMSD of protein-ligand during MD simulation indicates the RMSD of a ligand when the protein-ligand complex is first aligned on the protein backbone of the reference and then the RMSD of the ligand heavy atoms is measured. Ligand RMSD indicates the RMSD of the ligand that is aligned and measured on its reference conformation. Protein only RMSD indicates the RMSD of the protein alone (Cα), where protein frames are first aligned on the reference frame backbone, and then the RMSD is calculated based on the atom selection. b. Root Mean Square Fluctuations (RMSF). ‘Fit Ligand on Protein’ trend indicates the ligand fluctuations with respect to the protein. 'Ligand' trend shows the fluctuations where the ligand in each frame is aligned on the ligand in the first reference frame c. The number of total contacts (H-bonds, Hydrophobic, Ionic, Water bridges) made between the mutant A315E TDP-43 peptide and the ligand (AIM4)

Citations

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- 08 Jul 2022

- Signal Transduction and Targeted Therapy

TL;DR: In this article , the authors comprehensively summarize the detailed mechanisms of biomolecular condensate formation and biophysical function and review the recent major advances toward elucidating the multiple mechanisms involved in cancer cell pathology driven by aberrant liquid-liquid phase separation (LLPS).

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Targeting TDP-43 proteinopathy with drugs and drug-like small molecules.

Emanuele Buratti

- 01 Mar 2021

- British Journal of Pharmacology

TL;DR: The aim of this review will be to provide a general overview of the compounds that have been described to alter pathological characteristics of TDP‐43, including expression levels, cytoplasmic mis‐localization, post‐translational modifications, cleavage, stress granule recruitment and aggregation.

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Cytoplasmic mislocalization of TDP-43 is toxic to neurons and enhanced by a mutation associated with familial amyotrophic lateral sclerosis (Journal of Neuroscience (2010) (639-649))

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- 17 Mar 2010

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TL;DR: Results suggest that mutant TDP-43 is mislocalized to the cytoplasm, where it exhibits a toxic gain-of-function and induces cell death, a strong and independent predictor of neuronal death.

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References

•Journal Article•10.1002/JCC.21256

AutoDock4 and AutoDockTools4: Automated docking with selective receptor flexibility

Garrett M. Morris, +6 more

- 01 Dec 2009

- Journal of Computational Chemistry

TL;DR: AutoDock4 incorporates limited flexibility in the receptor and its utility in analysis of covalently bound ligands is reported, using both a grid‐based docking method and a modification of the flexible sidechain technique.

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SWISS-MODEL and the Swiss-PdbViewer: an environment for comparative protein modeling.

Nicolas Guex, +1 more

- 01 Jan 1997

- Electrophoresis

TL;DR: An environment for comparative protein modeling is developed that consists of SWISS‐MODEL, a server for automated comparativeprotein modeling and of the SWiss‐PdbViewer, a sequence to structure workbench that provides a large selection of structure analysis and display tools.

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•Journal Article•10.1002/(SICI)1096-987X(19981115)19:14<1639::AID-JCC10>3.0.CO;2-B

Automated docking using a Lamarckian genetic algorithm and an empirical binding free energy function

Garrett M. Morris, +6 more

- 15 Nov 1998

- Journal of Computational Chemistry

TL;DR: It is shown that both the traditional and Lamarckian genetic algorithms can handle ligands with more degrees of freedom than the simulated annealing method used in earlier versions of AUTODOCK, and that the Lamarckia genetic algorithm is the most efficient, reliable, and successful of the three.

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Open Babel: An open chemical toolbox

Noel M. O'Boyle, +5 more

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Avogadro: an advanced semantic chemical editor, visualization, and analysis platform

Marcus D. Hanwell, +6 more

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TL;DR: The work presented here details the Avogadro library, which is a framework providing a code library and application programming interface (API) with three-dimensional visualization capabilities; and has direct applications to research and education in the fields of chemistry, physics, materials science, and biology.

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Computational insights into mechanism of AIM4-mediated inhibition of aggregation of TDP-43 protein implicated in ALS and evidence for in vitro inhibition of liquid-liquid phase separation (LLPS) of TDP-432C-A315T by AIM4

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AI Agents for this Paper

Most frequently asked questions

1. What contributions have the authors mentioned in the paper "Computational insights into mechanism of aim4-mediated inhibition of aggregation of tdp-43 protein implicated in als and evidence for in vitro inhibition of liquid-liquid phase separation (llps) of tdp-432c-a315t by aim4" ?

2. What structures were used to predict the binding site of AIM4?

3. What is the role of AIM4 in the inhibition of ALS?

4. What is the role of the prion-like low complexity domains in neurodegenerative?

5. What is the peptide fragment from the C-terminal region of TDP-43?

6. What is the role of AIM4 in the LLPS of TDP-43?

7. How many M of mutant TDP-432C-A315T were added?

8. At what concentration did the mutant TDP-432C-A315T exhibit amy?

9. What is the structure of the ligands that were not available?

10. How was the aggregation of mutant TDP-432C-A315T?

11. What was used to analyze protein-ligand interactions?

12. What is the effect of AIM4 on the phase separation of TDP-432C?

13. How long did the MD simulations take to determine the stability of the AIM4 peptid?

14. What is the mechanism of AIM4 mediated anti-aggregation of TDP-?

15. How was the temperature kept at 300K?

16. What is the kinetics of the mutant TDP-432C-A315T?

17. What peptides can form amyloid-like steric zippers?

Figures

Citations

Liquid–liquid phase separation in tumor biology

Combating deleterious phase transitions in neurodegenerative disease.

Targeting TDP-43 proteinopathy with drugs and drug-like small molecules.

Study liquid–liquid phase separation with optical microscopy: A methodology review

Cytoplasmic mislocalization of TDP-43 is toxic to neurons and enhanced by a mutation associated with familial amyotrophic lateral sclerosis (Journal of Neuroscience (2010) (639-649))

References

AutoDock4 and AutoDockTools4: Automated docking with selective receptor flexibility

SWISS-MODEL and the Swiss-PdbViewer: an environment for comparative protein modeling.

Automated docking using a Lamarckian genetic algorithm and an empirical binding free energy function

Open Babel: An open chemical toolbox

Avogadro: an advanced semantic chemical editor, visualization, and analysis platform

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