Biology subjects

Pastre, D.

Publications and source records attributed to Pastre, D..

3 recordsLinked to original sources

bioRxiv2025

Interplay between human ribosomal proteins, PARP1, PARP2, HPF1 and histones

ADP-ribosyl-transferases (ADP-ribose polymerases) PARP1 and PARP2 are critical players in DNA damage response in the nucleus. Being activated by a genotoxic stress, these enzymes utilize NAD+ to attach ADP-ribose chains to wide variety of proteins; ribosomal proteins (RPs) have been identified among the major targets of the modification in different cell lines. However, little remained known concerning the peculiarities of the reaction of RPs ADP-ribosylation itself. Here, we study ADP-ribosylation of human RPs within the large (60S) and small (40S) ribosomal subunits and those isolated from the subunits, with PARP1 and PARP2 in vitro using radioactively labeled NAD+. We fail to detect the modification of ribosome-bound RPs but observed ADP-ribosylation of certain ribosome-free RPs when we use total protein isolated from the subunits. RPs from the 60S subunit were globally more modified than those from the 40S subunit, and ADP-ribosylation of several 60S RPs (but not 40S) was considerably enhanced in the presence of histone PARylation factor 1 (HPF1). With all kind RPs, HPF1 switches the modification preferentially to their serine/tyrosine residues. Major targets of the 60S RPs ADP-ribosylation were identified as RPL4 (uL4), RPL6 (eL6) and RPL13A/RPL15 (uL13/eL15). The modification levels of particular RPs differently depend on the concentration of total RP; the most selective HPF1-dependent ADP-ribosylation occurs in RPL6 (eL6). When present simultaneously with histones, RPs win linker histone H1 in the competition for both PARPs; in contrast, core histones strongly compete with RPs for ADP-ribosylation. Possible functional assignments of ADP-ribosylation of RPs are discussed. Bullet points- Free human ribosomal proteins are PARylated by PARP1 and PARP2; - PARylation of ribosomal 60S proteins but not 40S ones is mostly HPF1-dependent; - RPL4, RPL6 and RPL13A/RPL15 are the major targets of PARylation among 60S RPs; - Linker histone H1 is a poor competitor to ribosomal proteins for PARPs; - Core histones strongly competes with ribosomal proteins for PARPs.

molecular biology↗

bioRxiv2025

HPF1 REGULATES THE FORMATION OF FUS-DEPENDENT COMPARTMENTS BY PARP1 AND PARP2 ACTIVATION ON DAMAGED DNA

FUS participates in the formation of biomolecular condensates associated with PARP1-dependent synthesis of poly(ADP-ribose) (PAR). HPF1 regulates auto- and hetero-PARylation activities of PARP1 and PARP2 and may influence the formation of FUS compartments during PARP1 or PARP2 auto-PARylation. In this study, we used atomic force microscopy in combination with biochemical assay to investigate the formation of FUS compartments under activation of PARP1 and PARP2, when HPF1 modulates their activity. Similar to PARP1, FUS and PARylated PARP2 form DNA-rich compartments, indicating that PARP2 PARylation is sufficient for the formation of such compartments. The excess of HPF1 over PARP1 diminishes PARP1 activity and reduces the size of DNA-rich compartments. However, an excess of HPF1 over PARP2 does not significantly affect PARP2 activity and the size of compartments. Furthermore, HPF1 stimulates hetero-PARylation of FUS; this modification is stronger with PARP2 than with PARP1. HPF1-dependent intensive PARylation of FUS catalyzed by PARP1 or PARP2 impairs the assembly of DNA-rich compartment. These data provide a basis for investigating the effect of HPF1 on the formation of PAR-dependent condensates involving RNA-binding proteins like FUS, which interact effectively with PAR and show the ability to be targets of PARylation to regulate condensate formation at DNA damage sites. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=100 SRC="FIGDIR/small/673448v1_ufig1.gif" ALT="Figure 1"> View larger version (28K): org.highwire.dtl.DTLVardef@1fb3dadorg.highwire.dtl.DTLVardef@18e3b4forg.highwire.dtl.DTLVardef@d7e22forg.highwire.dtl.DTLVardef@14293af_HPS_FORMAT_FIGEXP M_FIG C_FIG

molecular biology↗

bioRxiv2022

Targeting RNA:Protein Interactions with an Integrative Approach Leads tothe Identification of Potent YB-1 Inhibitors

RNA-binding proteins are promising targets for developing new molecules of therapeutic interest. Nevertheless, targeting RNA:Protein interfaces is hampered by the lack of methods able to detect these interactions in cells while being amenable to High Content Screening. Here, we adapt the microtubule bench assay to score small molecules targeting interactions of endogenous mRNA with a specific protein in cells and demonstrate its robustness by targeting YB-1 (YBX-1 gene), a mRNA-binding protein involved in cancer progression and resistance to chemotherapy. The implementation of an integrative approach led to the identification of 22 hits validated by NMR and MD simulations of which 11 were found to significantly interfere with the binding of mRNA to YB-1 in cells at low micromolar concentrations. One of our leads is P1, an FDA-approved PARP-1 inhibitor. This work shows the potential of our integrative approach and paves the way for the development of RNA:Protein Interaction inhibitors.

biophysics↗