Crystal structure and biochemical characterization of the transmembrane PAP2 type phosphatidylglycerol phosphate phosphatase from Bacillus subtilis.

Type 2 phosphatidic acid phosphatases (PAP2s) can be either soluble or integral membrane enzymes. In bacteria, integral membrane PAP2s play major roles in the metabolisms of glycerophospholipids, undecaprenyl-phosphate (C55-P) lipid carrier and lipopolysaccharides. By in vivo functional experiments and biochemical characterization we show that the membrane PAP2 coded by the Bacillus subtilis yodM gene is the principal phosphatidylglycerol phosphate (PGP) phosphatase of B. subtilis. We also confirm that this enzyme, renamed bsPgpB, has a weaker activity on C55-PP. Moreover, we solved the crystal structure of bsPgpB at 2.25 Å resolution, with tungstate (a phosphate analog) in the active site. The structure reveals two lipid chains in the active site vicinity, allowing for PGP substrate modeling and molecular dynamic simulation. Site-directed mutagenesis confirmed the residues important for substrate specificity, providing a basis for predicting the lipids preferentially dephosphorylated by membrane PAP2s.

FUS fibrillation occurs through a nucleation-based process below the critical concentration required for liquid-liquid phase separation.

FUS is an RNA-binding protein involved in familiar forms of ALS and FTLD that also assembles into fibrillar cytoplasmic aggregates in some neurodegenerative diseases without genetic causes. The self-adhesive prion-like domain in FUS generates reversible condensates via the liquid-liquid phase separation process (LLPS) whose maturation can lead to the formation of insoluble fibrillar aggregates in vitro, consistent with the appearance of cytoplasmic inclusions in ageing neurons. Using a single-molecule imaging approach, we reveal that FUS can assemble into nanofibrils at concentrations in the nanomolar range. These results suggest that the formation of fibrillar aggregates of FUS could occur in the cytoplasm at low concentrations of FUS, below the critical ones required to trigger the liquid-like condensate formation. Such nanofibrils may serve as seeds for the formation of pathological inclusions. Interestingly, the fibrillation of FUS at low concentrations is inhibited by its binding to mRNA or after the phosphorylation of its prion-like domain, in agreement with previous models.

Targeting RNA:protein interactions with an integrative approach leads to the identification of potent YBX1 inhibitors.

RNA-protein interactions (RPIs) are promising targets for developing new molecules of therapeutic interest. Nevertheless, challenges arise from the lack of methods and feedback between computational and experimental techniques during the drug discovery process. Here, we tackle these challenges by developing a drug screening approach that integrates chemical, structural and cellular data from both advanced computational techniques and a method to score RPIs in cells for the development of small RPI inhibitors; and we demonstrate its robustness by targeting Y-box binding protein 1 (YB-1), a messenger RNA-binding protein involved in cancer progression and resistance to chemotherapy. This approach led to the identification of 22 hits validated by molecular dynamics (MD) simulations and nuclear magnetic resonance (NMR) spectroscopy of which 11 were found to significantly interfere with the binding of messenger RNA (mRNA) to YB-1 in cells. One of our leads is an FDA-approved poly(ADP-ribose) polymerase 1 (PARP-1) inhibitor. This work shows the potential of our integrative approach and paves the way for the rational development of RPI inhibitors.

FUS RRM regulates poly(ADP-ribose) levels after transcriptional arrest and PARP-1 activation on DNA damage.

PARP-1 activation at DNA damage sites leads to the synthesis of long poly(ADP-ribose) (PAR) chains, which serve as a signal for DNA repair. Here we show that FUS, an RNA-binding protein, is specifically directed to PAR through its RNA recognition motif (RRM) to increase PAR synthesis by PARP-1 in HeLa cells after genotoxic stress. Using a structural approach, we also identify specific residues located in the FUS RRM, which can be PARylated by PARP-1 to control the level of PAR synthesis. Based on the results of this work, we propose a model in which, following a transcriptional arrest that releases FUS from nascent mRNA, FUS can be recruited by PARP-1 activated by DNA damage to stimulate PAR synthesis. We anticipate that this model offers new perspectives to understand the role of FET proteins in cancers and in certain neurodegenerative diseases such as amyotrophic lateral sclerosis.

The cooperative binding of TDP-43 to GU-rich RNA repeats antagonizes TDP-43 aggregation.

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.