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.

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.

YB-1 unwinds mRNA secondary structures in vitro and negatively regulates stress granule assembly in HeLa cells.

In the absence of the scanning ribosomes that unwind mRNA coding sequences and 5'UTRs, mRNAs are likely to form secondary structures and intermolecular bridges. Intermolecular base pairing of non polysomal mRNAs is involved in stress granule (SG) assembly when the pool of mRNAs freed from ribosomes increases during cellular stress. Here, we unravel the structural mechanisms by which a major partner of dormant mRNAs, YB-1 (YBX1), unwinds mRNA secondary structures without ATP consumption by using its conserved cold-shock domain to destabilize RNA stem/loops and its unstructured C-terminal domain to secure RNA unwinding. At endogenous levels, YB-1 facilitates SG disassembly during arsenite stress recovery. In addition, overexpression of wild-type YB-1 and to a lesser extent unwinding-defective mutants inhibit SG assembly in HeLa cells. Through its mRNA-unwinding activity, YB-1 may thus inhibit SG assembly in cancer cells and package dormant mRNA in an unfolded state, thus preparing mRNAs for translation initiation.

Lin28, a major translation reprogramming factor, gains access to YB-1-packaged mRNA through its cold-shock domain.

The RNA-binding protein Lin28 (Lin28a) is an important pluripotency factor that reprograms translation and promotes cancer progression. Although Lin28 blocks let-7 microRNA maturation, Lin28 also binds to a large set of cytoplasmic mRNAs directly. However, how Lin28 regulates the processing of many mRNAs to reprogram global translation remains unknown. We show here, using a structural and cellular approach, a mixing of Lin28 with YB-1 (YBX1) in the presence of mRNA owing to their cold-shock domain, a conserved ß-barrel structure that binds to ssRNA cooperatively. In contrast, the other RNA binding-proteins without cold-shock domains tested, HuR, G3BP-1, FUS and LARP-6, did not mix with YB-1. Given that YB-1 is the core component of dormant mRNPs, a model in which Lin28 gains access to mRNPs through its co-association with YB-1 to mRNA may provide a means for Lin28 to reprogram translation. We anticipate that the translational plasticity provided by mRNPs may contribute to Lin28 functions in development and adaptation of cancer cells to an adverse environment.

Bacterial Lipid II Analogs: Novel In Vitro Substrates for Mammalian Oligosaccharyl Diphosphodolichol Diphosphatase (DLODP) Activities.

Mammalian protein N-glycosylation requires the transfer of an oligosaccharide containing 2 residues of N-acetylglucosamine, 9 residues of mannose and 3 residues of glucose (Glc3Man9 GlcNAc2) from Glc3Man9GlcNAc2-diphospho (PP)-dolichol (DLO) onto proteins in the endoplasmic reticulum (ER). Under some pathophysiological conditions, DLO biosynthesis is perturbed, and truncated DLO is hydrolyzed to yield oligosaccharyl phosphates (OSP) via unidentified mechanisms. DLO diphosphatase activity (DLODP) was described in vitro, but its characterization is hampered by a lack of convenient non-radioactive substrates. Our objective was to develop a fluorescence-based assay for DLO hydrolysis. Using a vancomycin-based solid-phase extraction procedure coupled with thin layer chromatography (TLC) and mass spectrometry, we demonstrate that mouse liver membrane extracts hydrolyze fluorescent bacterial lipid II (LII: GlcNAc-MurNAc(dansyl-pentapeptide)-PP-undecaprenol) to yield GlcNAc-MurNAc(dansyl-pentapeptide)-P (GM5P). GM5P production by solubilized liver microsomal proteins shows similar biochemical characteristics to those reported for human hepatocellular carcinoma HepG2 cell DLODP activity. To conclude, we show, for the first time, hydrolysis of lipid II by a eukaryotic enzyme. As LII and DLO are hydrolyzed by the same, or closely related, enzymes, fluorescent lipid II analogs are convenient non-radioactive substrates for investigating DLODP and DLODP-like activities.

PARP-1 Activation Directs FUS to DNA Damage Sites to Form PARG-Reversible Compartments Enriched in Damaged DNA.

PARP-1 synthesizes long poly(ADP-ribose) chains (PAR) at DNA damage sites to recruit DNA repair factors. Among proteins relocated on damaged DNA, the RNA-binding protein FUS is one of the most abundant, raising the issue about its involvement in DNA repair. Here, we reconstituted the PARP-1/PAR/DNA system in vitro and analyzed at the single-molecule level the role of FUS. We demonstrate successively the dissociation of FUS from mRNA, its recruitment at DNA damage sites through its binding to PAR, and the assembly of damaged DNA-rich compartments. PARG, an enzyme family that hydrolyzes PAR, is sufficient to dissociate damaged DNA-rich compartments in vitro and initiates the nucleocytoplasmic shuttling of FUS in cells. We anticipate that, consistent with previous models, FUS facilitates DNA repair through the transient compartmentalization of DNA damage sites. The nucleocytoplasmic shuttling of FUS after the PARG-mediated compartment dissociation may participate in the formation of cytoplasmic FUS aggregates.

Nanoscale Analysis Reveals the Maturation of Neurodegeneration-Associated Protein Aggregates: Grown in mRNA Granules then Released by Stress Granule Proteins.

TDP-43 and FUS are two mRNA-binding proteins associated with neurodegenerative diseases that form cytoplasmic inclusions with prion-like properties in affected neurons. Documenting the early stages of the formation of TDP-43 or FUS protein aggregates and the role of mRNA stress granules that are considered as critical intermediates for protein aggregation is therefore of interest to understand disease propagation. Here, we developed a single molecule approach via atomic force microscopy (AFM), which provides structural information out of reach by fluorescence microscopy. In addition, the aggregation process can be probed in the test tube without separating the interacting partners, which would affect the thermodynamic equilibrium. The results demonstrate that isolated mRNA molecules serve as crucibles to promote TDP-43 and FUS multimerization. Their subsequent merging results in the formation of mRNA granules containing TDP-43 and FUS aggregates. Interestingly, TDP-43 or FUS protein aggregates can be released from mRNA granules by either YB-1 or G3BP1, two stress granule proteins that compete for the binding to mRNA with TDP-43 and FUS. Altogether, the results indicate that age-related successive assembly/disassembly of stress granules in neurons, regulated by mRNA-binding proteins such as YB-1 and G3BP1, could be a source of protein aggregation.

Catalytic mechanism of MraY and WecA, two paralogues of the polyprenyl-phosphate N-acetylhexosamine 1-phosphate transferase superfamily.

The MraY transferase catalyzes the first membrane step of bacterial cell wall peptidoglycan biosynthesis, namely the transfer of the N-acetylmuramoyl-pentapeptide moiety of the cytoplasmic precursor UDP-MurNAc-pentapeptide to the membrane transporter undecaprenyl phosphate (C55P), yielding C55-PP-MurNAc-pentapeptide (lipid I). A paralogue of MraY, WecA, catalyzes the transfer of the phospho-GlcNAc moiety of UDP-N-acetylglucosamine onto the same lipid carrier, leading to the formation of C55-PP-GlcNAc that is essential for the synthesis of various bacterial cell envelope components. These two enzymes are members of the polyprenyl-phosphate N-acetylhexosamine 1-phosphate transferase superfamily, which are essential for bacterial envelope biogenesis. Despite the availability of detailed biochemical information on the MraY enzyme, and the recently published crystal structure of MraY of Aquifex aeolicus, the molecular basis for its catalysis remains poorly understood. This knowledge can contribute to the design of potential inhibitors. Here, we report a detailed catalytic study of the Bacillus subtilis MraY and Thermotoga maritima WecA transferases. Both forward and reverse exchange reactions required the presence of the second substrate, C55P and uridine monophosphate (UMP), respectively. Both enzymes did not display any pyrophosphatase activity on the nucleotide substrate. Moreover, we showed that the nucleotide substrate UDP-MurNAc-pentapeptide, as well as the nucleotide product UMP, can bind to MraY in the absence of lipid ligands. Therefore, our data are in favour of a single displacement mechanism. During this "one-step" mechanism, the oxyanion of the polyprenyl-phosphate attacks the ß-phosphate of the nucleotide substrate, leading to the formation of lipid product and the liberation of UMP. The involvement of an invariant aspartyl residue in the deprotonation of the lipid substrate is discussed.

Membrane Topology and Biochemical Characterization of the Escherichia coli BacA Undecaprenyl-Pyrophosphate Phosphatase.

Several integral membrane proteins exhibiting undecaprenyl-pyrophosphate (C55-PP) phosphatase activity were previously identified in Escherichia coli that belonged to two distinct protein families: the BacA protein, which accounts for 75% of the C55-PP phosphatase activity detected in E. coli cell membranes, and three members of the PAP2 phosphatidic acid phosphatase family, namely PgpB, YbjG and LpxT. This dephosphorylation step is required to provide the C55-P carrier lipid which plays a central role in the biosynthesis of various cell wall polymers. We here report detailed investigations of the biochemical properties and membrane topology of the BacA protein. Optimal activity conditions were determined and a narrow-range substrate specificity with a clear preference for C55-PP was observed for this enzyme. Alignments of BacA protein sequences revealed two particularly well-conserved regions and several invariant residues whose role in enzyme activity was questioned by using a site-directed mutagenesis approach and complementary in vitro and in vivo activity assays. Three essential residues Glu21, Ser27, and Arg174 were identified, allowing us to propose a catalytic mechanism for this enzyme. The membrane topology of the BacA protein determined here experimentally did not validate previous program-based predicted models. It comprises seven transmembrane segments and contains in particular two large periplasmic loops carrying the highly-conserved active site residues. Our data thus provide evidence that all the different E. coli C55-PP phosphatases identified to date (BacA and PAP2) catalyze the dephosphorylation of C55-PP molecules on the same (outer) side of the plasma membrane.

Efficient access to peptidyl-RNA conjugates for picomolar inhibition of non-ribosomal FemX(Wv) aminoacyl transferase.

Peptidyl-RNA conjugates have various applications in studying the ribosome and enzymes participating in tRNA-dependent pathways such as Fem transferases in peptidoglycan synthesis. Herein a convergent synthesis of peptidyl-RNAs based on Huisgen-Sharpless cycloaddition for the final ligation step is developed. Azides and alkynes are introduced into tRNA and UDP-MurNAc-pentapeptide, respectively. Synthesis of 2'-azido RNA helix starts from 2'-azido-2'-deoxyadenosine that is coupled to deoxycytidine by phosphoramidite chemistry. The resulting dinucleotide is deprotected and ligated to a 22-nt RNA helix mimicking the acceptor arm of Ala-tRNA(Ala) by T4 RNA ligase. For alkyne UDP-MurNAc-pentapeptide, meso-cystine is enzymatically incorporated into the peptidoglycan precursor and reduced, and L-Cys is converted to dehydroalanine with O-(mesitylenesulfonyl)hydroxylamine. Reaction of but-3-yne-1-thiol with dehydroalanine affords the alkyne-containing UDP-MurNAc-pentapeptide. The Cu(I)-catalyzed azide alkyne cycloaddition reaction in the presence of tris[(1-hydroxypropyl-1H-1,2,3-triazol-4-yl)methyl]amine provided the peptidyl-RNA conjugate, which was tested as an inhibitor of non-ribosomal FemX(Wv) aminoacyl transferase. The bi-substrate analogue was found to inhibit FemX(Wv) with an IC(50) of (89±9) pM, as both moieties of the peptidyl-RNA conjugate contribute to high-affinity binding.

Mechanistic analysis of muraymycin analogues: a guide to the design of MraY inhibitors.

The systematic structure-activity relationship (SAR) of the muraymycins (MRYs) using an Ugi four-component reaction (U4CR) was investigated. The impact of the lipophilic substituent on antibacterial activity was significant, and the analogues 8 and 9 having a lipophilic side chain exhibited good activity against a range of Gram-positive bacterial pathogens, including MRSA and VRE. Further investigation of compounds 8 and 9 revealed these analogues to be selective inhibitors of the MraY transferase and nontoxic to HepG2 cells. The SAR of the accessory urea-peptide moiety indicated that it could be simplified. Our SAR study of the MRYs suggests a probable mechanism for inhibition of the MraY, where the inner moiety of the urea-dipeptide motif interacts with the carbohydrate recognition domain in the cytoplasmic loop 5. The predicted binding model would provide further direction toward the design of potent MraY inhibitors. This study has set the stage for the generation of novel antibacterial "lead" compounds based on MRYs.

X-ray structure and site-directed mutagenesis analysis of the Escherichia coli colicin M immunity protein.

Colicin M (ColM), which is produced by some Escherichia coli strains to kill competitor strains from the same or related species, was recently shown to inhibit cell wall peptidoglycan biosynthesis through enzymatic degradation of its lipid II precursor. ColM-producing strains are protected from the toxin that they produce by coexpression of a specific immunity protein, named Cmi, whose mode of action still remains to be identified. We report here the resolution of the crystal structure of Cmi, which is composed of four ß strands and four α helices. This rather compact structure revealed a disulfide bond between residues Cys31 and Cys107. Interestingly, these two cysteines and several other residues appeared to be conserved in the sequences of several proteins of unknown function belonging to the YebF family which exhibit 25 to 35% overall sequence similarity with Cmi. Site-directed mutagenesis was performed to assess the role of these residues in the ColM immunity-conferring activity of Cmi, which showed that the disulfide bond and residues from the C-terminal extremity of the protein were functionally essential. The involvement of DsbA oxidase in the formation of the Cmi disulfide bond is also demonstrated.

Preparative enzymatic synthesis of polyprenyl-pyrophosphoryl-N-acetylglucosamine, an essential lipid intermediate for the biosynthesis of various bacterial cell envelope polymers.

The WecA transferase is an integral membrane protein and a member of the polyprenyl phosphate N-acetylhexosamine-1-phosphate transferase superfamily. It initiates the biosynthesis of various bacterial cell envelope components such as the lipopolysaccharide O-antigen. We report on the first large-scale enzymatic synthesis, purification, and characterization of the undecaprenyl-pyrophosphoryl-N-acetylglucosamine product of the WecA transferase. This is an essential lipid intermediate for the biosynthesis of various bacterial cell envelope components. Its availability in a pure form will allow the biochemical and structural characterization of the various enzymes requiring it as a substrate for the synthesis of cell wall polymers.

Purification and characterization of the bacterial UDP-GlcNAc:undecaprenyl-phosphate GlcNAc-1-phosphate transferase WecA.

To date, the structural and functional characterization of proteins belonging to the polyprenyl-phosphate N-acetylhexosamine-1-phosphate transferase superfamily has been relentlessly held back by problems encountered with their overexpression and purification. In the present work and for the first time, the integral membrane protein WecA that catalyzes the transfer of the GlcNAc-1-phosphate moiety from UDP-GlcNAc onto the carrier lipid undecaprenyl phosphate, yielding undecaprenyl-pyrophosphoryl-GlcNAc, the lipid intermediate involved in the synthesis of various bacterial cell envelope components, was overproduced and purified to near homogeneity in milligram quantities. An enzymatic assay was developed, and the kinetic parameters of WecA as well as the effects of pH, salts, cations, detergents, and temperature on the enzyme activity were determined. A minimal length of 35 carbons was required for the lipid substrate, and tunicamycin was shown to inhibit the enzyme at submicromolar concentrations.

Active site mapping of MraY, a member of the polyprenyl-phosphate N-acetylhexosamine 1-phosphate transferase superfamily, catalyzing the first membrane step of peptidoglycan biosynthesis.

The MraY transferase is an integral membrane protein that catalyzes an essential step of peptidoglycan biosynthesis, namely the transfer of the phospho-N-acetylmuramoyl-pentapeptide motif onto the undecaprenyl phosphate carrier lipid. It belongs to a large superfamily of eukaryotic and prokaryotic prenyl sugar transferases. No 3D structure has been reported for any member of this superfamily, and to date MraY is the only protein that has been successfully purified to homogeneity. Nineteen polar residues located in the five cytoplasmic segments of MraY appeared as invariants in the sequences of MraY orthologues. A certain number of these invariant residues were found to be conserved in the whole superfamily. To assess the importance of these residues in the catalytic process, site-directed mutagenesis was performed using the Bacillus subtilis MraY as a model. Fourteen residues were shown to be essential for MraY activity by an in vivo functional complementation assay using a constructed conditional mraY mutant strain. The corresponding mutant proteins were purified and biochemically characterized. None of these mutations did significantly affect the binding of the nucleotidic and lipidic substrates, but the k cat was dramatically reduced in almost all cases. The important residues for activity therefore appeared to be distributed in all the cytoplasmic segments, indicating that these five regions contribute to the structure of the catalytic site. Our data show that the D98 residue that is invariant in the whole superfamily should be involved in the deprotonation of the lipid substrate during the catalytic process.

Purification and characterization of the bacterial MraY translocase catalyzing the first membrane step of peptidoglycan biosynthesis.

The MraY translocase catalyzes the first membrane step of bacterial cell wall peptidoglycan synthesis (i.e. the transfer of the phospho-N-acetylmuramoyl-pentapeptide motif onto the undecaprenyl phosphate carrier lipid), a reversible reaction yielding undecaprenylpyrophosphoryl-N-acetylmuramoyl-pentapeptide (lipid intermediate I). This essential integral membrane protein, which is considered as a very promising target for the search of new antibacterial compounds, has thus far been clearly underexploited due to its intrinsic refractory nature to overexpression and purification. We here report conditions for the high level overproduction and for the first time the purification to homogeneity of milligram quantities of MraY protein. The kinetic parameters and effects of pH, salts, cations, and detergents on enzyme activity are described, taking the Bacillus subtilis MraY translocase as a model.