Structure and functional impact of glycosaminoglycan modification of HSulf-2 endosulfatase revealed by atomic force microscopy and mass spectrometry.

The human sulfatase HSulf-2 is one of only two known endosulfatases that play a decisive role in modulating the binding properties of heparan sulfate proteoglycans on the cell surface and in the extracellular matrix. Recently, HSulf-2 was shown to exhibit an unusual post-translational modification consisting of a sulfated glycosaminoglycan chain. This study describes the structural characterization of this glycosaminoglycan (GAG) and provides new data on its impact on the catalytic properties of HSulf-2. The unrevealed nature of this GAG chain is identified as a chondroitin/dermatan sulfate (CS/DS) mixed chain, as shown by mass spectrometry combined with NMR analysis. It consists primarily of 6-O and 4-O monosulfated disaccharide units, with a slight predominance of the 4-O-sulfation. Using atomic force microscopy, we show that this unique post-translational modification dramatically impacts the enzyme hydrodynamic volume. We identified human hyaluronidase-4 as a secreted hydrolase that can digest HSulf-2 GAG chain. We also showed that HSulf-2 is able to efficiently 6-O-desulfate antithrombin III binding pentasaccharide motif, and that this activity was enhanced upon removal of the GAG chain. Finally, we identified five N-glycosylation sites on the protein and showed that, although required, reduced N-glycosylation profiles were sufficient to sustain HSulf-2 integrity.

Using the structural diversity of RNA: protein interfaces to selectively target RNA with small molecules in cells: methods and perspectives.

In recent years, RNA has gained traction both as a therapeutic molecule and as a therapeutic target in several human pathologies. In this review, we consider the approach of targeting RNA using small molecules for both research and therapeutic purposes. Given the primary challenge presented by the low structural diversity of RNA, we discuss the potential for targeting RNA: protein interactions to enhance the structural and sequence specificity of drug candidates. We review available tools and inherent challenges in this approach, ranging from adapted bioinformatics tools to in vitro and cellular high-throughput screening and functional analysis. We further consider two critical steps in targeting RNA/protein interactions: first, the integration of in silico and structural analyses to improve the efficacy of molecules by identifying scaffolds with high affinity, and second, increasing the likelihood of identifying on-target compounds in cells through a combination of high-throughput approaches and functional assays. We anticipate that the development of a new class of molecules targeting RNA: protein interactions to prevent physio-pathological mechanisms could significantly expand the arsenal of effective therapeutic compounds.

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.

Identification of a small molecule splicing inhibitor targeting UHM domains.

Splicing factor mutations are frequent in myeloid neoplasms, blood cancers, and solid tumors. Cancer cells harboring these mutations present a particular vulnerability to drugs that target splicing factors such as SF3b155 or CAPERα. Still, the arsenal of chemical probes that target the spliceosome is very limited. U2AF homology motifs (UHMs) are common protein interaction domains among splicing factors. They present a hydrophobic pocket ideally suited to anchor small molecules with the aim to inhibit protein-protein interaction. Here, we combined a virtual screening of a small molecules database and an in vitro competition assay and identified a small molecule, we named UHMCP1 that prevents the SF3b155/U2AF65 interaction. NMR analyses and molecular dynamics simulations confirmed the binding of this molecule in the hydrophobic pocket of the U2AF65 UHM domain. We further provide evidence that UHMCP1 impacts RNA splicing and cell viability and is therefore an interesting novel compound targeting an UHM domain with potential anticancer properties.

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.

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.

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.

YB-1, an abundant core mRNA-binding protein, has the capacity to form an RNA nucleoprotein filament: a structural analysis.

The structural rearrangements accompanying mRNA during translation in mammalian cells remain poorly understood. Here, we discovered that YB-1 (YBX1), a major partner of mRNAs in the cytoplasm, forms a linear nucleoprotein filament with mRNA, when part of the YB-1 unstructured C-terminus has been truncated. YB-1 possesses a cold-shock domain (CSD), a remnant of bacterial cold shock proteins that have the ability to stimulate translation under the low temperatures through an RNA chaperone activity. The structure of the nucleoprotein filament indicates that the CSD of YB-1 preserved its chaperone activity also in eukaryotes and shows that mRNA is channeled between consecutive CSDs. The energy benefit needed for the formation of stable nucleoprotein filament relies on an electrostatic zipper mediated by positively charged amino acid residues in the YB-1 C-terminus. Thus, YB-1 displays a structural plasticity to unfold structured mRNAs into extended linear filaments. We anticipate that our findings will shed the light on the scanning of mRNAs by ribosomes during the initiation and elongation steps of mRNA translation.

Metabolomics with Nuclear Magnetic Resonance Spectroscopy in a Drosophila melanogaster Model of Surviving Sepsis.

Patients surviving sepsis demonstrate sustained inflammation, which has been associated with long-term complications. One of the main mechanisms behind sustained inflammation is a metabolic switch in parenchymal and immune cells, thus understanding metabolic alterations after sepsis may provide important insights to the pathophysiology of sepsis recovery. In this study, we explored metabolomics in a novel Drosophila melanogaster model of surviving sepsis using Nuclear Magnetic Resonance (NMR), to determine metabolite profiles. We used a model of percutaneous infection in Drosophila melanogaster to mimic sepsis. We had three experimental groups: sepsis survivors (infected with Staphylococcus aureus and treated with oral linezolid), sham (pricked with an aseptic needle), and unmanipulated (positive control). We performed metabolic measurements seven days after sepsis. We then implemented metabolites detected in NMR spectra into the MetExplore web server in order to identify the metabolic pathway alterations in sepsis surviving Drosophila. Our NMR metabolomic approach in a Drosophila model of recovery from sepsis clearly distinguished between all three groups and showed two different metabolomic signatures of inflammation. Sham flies had decreased levels of maltose, alanine, and glutamine, while their level of choline was increased. Sepsis survivors had a metabolic signature characterized by decreased glucose, maltose, tyrosine, beta-alanine, acetate, glutamine, and succinate.

Cucurbitacin I elicits the formation of actin/phospho-myosin II co-aggregates by stimulation of the RhoA/ROCK pathway and inhibition of LIM-kinase.

Cucurbitacins are cytotoxic triterpenoid sterols isolated from plants. One of their earliest cellular effect is the aggregation of actin associated with blockage of cell migration and division that eventually lead to apoptosis. We unravel here that cucurbitacin I actually induces the co-aggregation of actin with phospho-myosin II. This co-aggregation most probably results from the stimulation of the Rho/ROCK pathway and the direct inhibition of the LIMKinase. We further provide data that suggest that the formation of these co-aggregates is independent of a putative pro-oxidant status of cucurbitacin I. The results help to understand the impact of cucurbitacins on signal transduction and actin dynamics and open novel perspectives to use it as drug candidates for cancer research.

Structural basis for the association of MAP6 protein with microtubules and its regulation by calmodulin.

Microtubules are highly dynamic αß-tubulin polymers. In vitro and in living cells, microtubules are most often cold- and nocodazole-sensitive. When present, the MAP6/STOP family of proteins protects microtubules from cold- and nocodazole-induced depolymerization but the molecular and structure determinants by which these proteins stabilize microtubules remain under debate. We show here that a short protein fragment from MAP6-N, which encompasses its Mn1 and Mn2 modules (MAP6(90-177)), recapitulates the function of the full-length MAP6-N protein toward microtubules, i.e. its ability to stabilize microtubules in vitro and in cultured cells in ice-cold conditions or in the presence of nocodazole. We further show for the first time, using biochemical assays and NMR spectroscopy, that these effects result from the binding of MAP6(90-177) to microtubules with a 1:1 MAP6(90-177):tubulin heterodimer stoichiometry. NMR data demonstrate that the binding of MAP6(90-177) to microtubules involve its two Mn modules but that a single one is also able to interact with microtubules in a closely similar manner. This suggests that the Mn modules represent each a full microtubule binding domain and that MAP6 proteins may stabilize microtubules by bridging tubulin heterodimers from adjacent protofilaments or within a protofilament. Finally, we demonstrate that Ca(2+)-calmodulin competes with microtubules for MAP6(90-177) binding and that the binding mode of MAP6(90-177) to microtubules and Ca(2+)-calmodulin involves a common stretch of amino acid residues on the MAP6(90-177) side. This result accounts for the regulation of microtubule stability in cold condition by Ca(2+)-calmodulin.

Mapping the conformational stability of maltose binding protein at the residue scale using nuclear magnetic resonance hydrogen exchange experiments.

Being able to differentiate local fluctuations from global folding-unfolding dynamics of a protein is of major interest for improving our understanding of structure-function determinants. The maltose binding protein (MBP), a protein that belongs to the maltose transport system, has a structure composed of two globular domains separated by a rigid-body "hinge bending". Here we determined, by using hydrogen exchange (HX) nuclear magnetic resonance experiments, the apparent stabilization free energies of 101 residues of MBP bound to ß-cyclodextrin (MBP-ßCD) under native conditions. We observed that the last helix of MBP (helix α14) has a lower protection factor than the rest of the protein. Further, HX experiments were performed using guanidine hydrochloride under subdenaturing conditions to discriminate between local fluctuations and global unfolding events and to determine the MBP-ßCD energy landscape. The results show that helix α4 and a part of helices α5 and α6 are clearly grouped into a subdenaturing folding unit and represent a partially folded intermediate under native conditions. In addition, we observed that amide protons located in the hinge between the two globular domains share similar ΔG(gu)(app) and m values and should unfold simultaneously. These observations provide new points of view for improving our understanding of the thermodynamic stability and the mechanisms that drive folding-unfolding dynamics of proteins.

Phosphorylation controls the interaction of the connexin43 C-terminal domain with tubulin and microtubules.

Connexins are structurally related transmembrane proteins that assemble to form gap junction channels involved in the mediation of intercellular communication. It has been shown that the intracellular tail of connexin43 (Cx43) interacts with tubulin and microtubules with putative impacts on its own intracellular trafficking, its activity in channel communication, and its interference with specific growth factor signal transduction cascades. We demonstrate here that the microtubule binding of Cx43 is mainly driven by a short region of 26 amino acid residues located within the intracellular tail of Cx43. The nuclear magnetic resonance structural analysis of a peptide (K26D) corresponding to this region shows that this peptide is unstructured when free in solution and adopts a helix conformation upon binding with tubulin. In addition, the resulting K26D-tubulin molecular complex defines a new structural organization that could be shared by other microtubule partners. Interestingly, the K26D-tubulin interaction is prevented by the phosphorylation of K26D at a src kinase specific site. Altogether, the results elucidate the mechanism of the interaction of Cx43 with the microtubule cytoskeleton and propose a pathway for understanding the microtubule-dependent regulation of Cx43 gap junctional communications and the involvement of Cx43 in TGF-ß signal transduction.

The state of the guanosine nucleotide allosterically affects the interfaces of tubulin in protofilament.

The dynamics of microtubules is essential for many microtubule-dependent cellular functions such as the mitosis. It has been recognized for a long time that GTP hydrolysis in αß-tubulin polymers plays a critical role in this dynamics. However, the effects of the changes in the nature of the guanosine nucleotide at the E-site in ß-tubulin on microtubule structure and stability are still not well understood. In the present work, we performed all-atom molecular dynamics simulations of a αßα-tubulin heterotrimer harboring a guanosine nucleotide in three different states at the E-site: GTP, GDP-Pi and GDP. We found that changes in the nucleotide state is associated with significant conformational variations at the α-tubulin N- and ß-tubulin M-loops which impact the interactions between tubulin protofilaments. The results also show that GTP hydrolysis reduces αß-tubulin interdimer contacts in favor of intradimer interface. From an atomistic point view, we propose a role for α-tubulin glutamate residue 254 in catalytic magnesium coordination and identified a water molecule in the nucleotide binding pocket which is most probably required for nucleotide hydrolysis. Finally, the results are discussed with reference to the role of taxol in microtubule stability and the recent tubulin-sT2R crystal structures.

Probing interactions of tubulin with small molecules, peptides, and protein fragments by solution nuclear magnetic resonance.

The description of the molecular mechanisms of interaction between tubulin or microtubules and partners at atomic scale is expected to have critical impacts on the understanding of basic physiological processes. This information will also help the design of future drug candidates that may be used to fight various pathologies such as cancer or neurological diseases. For these reasons, this aspect of tubulin research has been tackled since the seventies using many different methods and at different scales. NMR appears as a unique approach to provide, with atomic resolution, the solution structure and dynamical properties of tubulin/microtubule partners in free and bound states. Though tubulin is not directly amenable to solution NMR, the NMR ligand-based experiments allow one to obtain valuable data on the molecular mechanisms that sustain structure-function relationship, in particular atomic details on the partner binding site. We will first describe herein some basic principles of solution NMR spectroscopy that should not be missed for a comprehensive reading of NMR reports. A series of results will then be presented to illustrate the wealth and variety of NMR experiments and how this approach enlightens tubulin/microtubules interaction with partners.

NMR characterization and molecular modeling of fucoidan showing the importance of oligosaccharide branching in its anticomplementary activity.

Fucoidan is a potent inhibitor of the human complement system whose activity is mediated through interactions with certain proteins belonging to the classical pathway, particularly the protein C4. Branched fucoidan oligosaccharides displayed a higher anticomplementary activity as compared to linear structures. Nuclear magnetic resonance (NMR) characterization of the branched oligosaccharides and saturation transfer difference-NMR experiment of the interaction with the protein C4 allowed the identification of the glycan residues in close contact with the target protein. Transferred nuclear Overhauser effect spectroscopy experiment and molecular modeling of fucoidan oligosaccharides indicated that the presence of side chains reduces the flexibility of the oligosaccharide backbone, which thus adopts a conformation which is very close to the one recognized by the protein C4. Together, these results suggest that branching of fucoidan oligosaccharides, determining their conformational state, has a major impact on their anticomplementary activity.

The stathmin-derived I19L peptide interacts with FtsZ and alters its bundling.

FtsZ is a prokaryotic tubulin-like protein. Despite a low degree of sequence identity with tubulin, it presents the same folding pattern and some similar functions, notably in cell division. Indeed, FtsZ and tubulin polymerize to form bundles and microtubules, respectively, which are essential for cell cytokinesis. We previously demonstrated that peptides derived from the N-terminal stathmin domain interact with tubulin and impede microtubule formation. We demonstrated here that I19L, the most efficient of these peptides, also alters FtsZ bundling assembly in vitro. STD-NMR and TRNOESY experiments revealed that I19L interacts with FtsZ and folds upon its binding but in a way different from what we observed with tubulin. These NMR data were used in molecular modeling calculations to propose models of the I19L-FtsZ complex. Interestingly, two models, consistent with NMR data, show an interaction of I19L near the T7 loop or near the GTP binding site of FtsZ, explaining the modifications of the bundling assembly observed with this peptide. The fine analysis of the structural differences of the complexes of I19L with FtsZ and tubulin should help for the rational development of new specific antibiotic agents.

The PN2-3 domain of centrosomal P4.1-associated protein implements a novel mechanism for tubulin sequestration.

Microtubules are cytoskeletal components involved in multiple cell functions such as mitosis, motility, or intracellular traffic. In vivo, these polymers made of alphabeta-tubulin nucleate mostly from the centrosome to establish the interphasic microtubule network or, during mitosis, the mitotic spindle. Centrosomal P4.1-associated protein (CPAP; also named CENPJ) is a centrosomal protein involved in the assembly of centrioles and important for the centrosome function. This protein contains a microtubule-destabilizing region referred to as PN2-3. Here we decrypt the microtubule destabilization activity of PN2-3 at the molecular level and show that it results from the sequestration of tubulin by PN2-3 in a non-polymerizable 1:1 complex. We also map the tubulin/PN2-3 interaction both on the PN2-3 sequence and on the tubulin surface. NMR and CD data on free PN2-3 in solution show that this is an intrinsically unstructured protein that comprises a 23-amino acid residue alpha-helix. This helix is embedded in a 76-residue region that interacts strongly with tubulin. The interference of PN2-3 with well characterized tubulin properties, namely GTPase activity, nucleotide exchange, vinblastine-induced self-assembly, and stathmin family protein binding, highlights the beta subunit surface located at the intermolecular longitudinal interface when tubulin is embedded in a microtubule as a tubulin/PN2-3 interaction area. These findings characterize the PN2-3 fragment of CPAP as a protein with an unprecedented tubulin sequestering mechanism distinct from that of stathmin family proteins.

Benomyl and colchicine synergistically inhibit cell proliferation and mitosis: evidence of distinct binding sites for these agents in tubulin.

Benomyl, a tubulin-targeted antimitotic antifungal agent, belongs to the benzimidazole group of compounds, which are known to inhibit the binding of colchicine to tubulin. Therefore, benomyl was thought to bind at or near the colchicine-binding site on tubulin. However, recent mutational studies in yeast and fluorescence studies involving competitive binding of benomyl and colchicine on goat brain tubulin suggested that benomyl may bind to tubulin at a site distinct from the colchicine-binding site. We set out to examine whether colchicine and benomyl bind to tubulin at distinct sites using a human cervical cancer (HeLa) cell line with the thinking that these agents should exert either additive or synergistic activity on cell proliferation if their binding sites on tubulin are different. We found that benomyl and colchicine synergistically inhibited the proliferation of HeLa cells and blocked their cell cycle progression at mitosis. The synergistic activity of benomyl and colchicine was also apparent from their strong depolymerizing effects on both the spindle and interphase microtubules when used in combinations, providing further evidence that these agents bind to tubulin at different sites. Using NMR spectroscopy, we finally demonstrated that benomyl and colchicine bind to tubulin at different sites and that the binding of colchicine seems to positively influence the binding of benomyl to tubulin and vice versa. Further, an analysis of the saturation transfer difference NMR data yielded an interesting insight into the colchicine-tubulin interaction. The data presented in this study provided a mechanistic understanding of the synergistic effects of benomyl and colchicine on HeLa cell proliferation.