Characterization of the potential role for RNA-binding protein FUS in DNA damage response: A quantitative proteomic approach

FUS/TLS (fused in sarcoma/translocated in liposarcoma) is a ubiquitously expressed protein of the hnRNP family, that has been discovered as fused to transcription factors in several human sarcomas and found in protein aggregates in neurons of patients with an inherited form of Amyotrophic Lateral Sclerosis [Vance C. et al., 2009]. FUS is a 53 kDa nuclear protein that contains structural domains, such as a RNA Recognition Motif (RRM) and a zinc finger motif, that give to FUS the ability to bind to both RNA and DNA sequences. It has been implicated in a variety of cellular processes, such as pre-mRNA splicing, miRNA processing, gene expression control and transcriptional regulation [Fiesel FC. and Kahle PJ., 2011]. Moreover, some evidences link FUS to genome stability control and DNA damage response: mice lacking FUS are hypersensitive to ionizing radiation (IR) and show high levels of chromosome instability and, in response to double-strand breaks, FUS is phosphorylated by the protein kinase ATM [Kuroda M. et al., 2000; Hicks GG. et al., 2000; Gardiner M. et al., 2008]. Furthermore, preliminary results of mass spectrometric identification of FUS interacting proteins in HEK293 cells, expressing a recombinant flag-tagged FUS protein, highlighted the interactions with proteins involved in DNA damage response, such as DNA-PK, XRCC-5/-6, and ERCC-6, raising the possibilities that FUS is involved in this pathway, even though its role still needs to be clarified. This study aims to investigate the biological roles of FUS in human cells and in particular the putative role in DNA damage response through the characterization of the proteomic profile of the neuroblastoma cell line SH-SY5Y upon FUS inducible depletion, by a quantitative proteomic approach. The SH-SY5Y cell line that will be used in this study expresses, in presence of tetracycline, a shRNA that targets FUS mRNA, leading to FUS protein depletion (SH-SY5Y FUS iKD cells). To quantify changes in proteins expression levels a SILAC strategy (Stable Isotope Labeling by Amino acids in Cell culture) will be conducted on SH-SY5Y FUS iKD cells and a control SH-SY5Y cell line (that expresses a mock shRNA) and the relative changes in proteins levels will be evaluated after five and seven days upon FUS depletion, by nanoliquid chromatography coupled to tandem mass spectrometry (nLC-MS/MS) and bioinformatics analysis. Preliminary experiments demonstrated that the SH-SY5Y FUS iKD cells, when subjected to genotoxic stress (high dose of IR), upon inducible depletion of FUS, showed a increased phosphorylation of gH2AX with respect to control cells, suggesting an higher activation of the DNA damage response.

The involvement of miRNA Dysregulation in Amyotrophic Lateral Sclerosis

ALS is a neurodegenerative disease that specifically affects upper and lower motor neurons leading to progressive paralysis and death. There is currently no effective treatment. Thus, identification of the signaling pathways and cellular mediators of ALS remains a major challenge in the search for novel therapeutic approaches. Recent studies have shown that non-coding RNAs have a significant impact on normal CNS development and onset and progression of neurological disorders. Based on this evidence we specifically test the hypothesis that misregulation of miRNA expression is a common feature in familiar ALS. Hence, we are exploiting human neuroblastoma cell lines either expressing the SOD1(G93A) mutation or depleted from Fused in Sarcoma (FUS) as tools to investigate the role of miRNAs in familiar ALS. To this end we performed a genome-wide scale miRNA expression on these cells, using whole-genome small RNA deep-sequencing followed by quantitative real time validation (qPCR). This strategy allowed us to find a group of dysregulated miRNAs, which are predicted to play a role in the motorneurons physiology and pathology. We verified our data on cDNA derived from SOD1-ALS mice models at early stage of the disease and on cDNA derived from lymphocytes from a small group of ALS patients. In the future, we plan to define the mechanisms responsible for the miRNA dysregulation, by silencing or stimulating the signal transduction pathways putatively involved in miRNA expression and regulation.

FUS protein interacts with U7 snRNP and plays a role in replication-dependant histone genes expression

The U7 small nuclear ribonucleoprotein (U7 snRNP) is an essential factor mediating the unique 3’end processing of non-polyadenylated, replication-dependent histone mRNAs in metazoans. These histone genes expression and processing of their transcripts are cell cycle-regulated mechanisms that recruit a number of specific proteins as well as common factors required for expression and maturation of polyadenylated mRNAs. However, despite all the knowledge we have so far, there are still gaps in understanding of core histone RNA 3’ end processing, its coupling to transcription and regulation during cell cycle. To further elucidate this phenomena we used affinity chromatography based on tagged version of U7 snRNA molecule to identify proteins associated with U7 snRNP/U7 snRNA that could be potentially involved in core histone genes expression in human cells. Mass spectrometric analysis of affinity-purified fraction revealed, among others, multifunctional RNA/DNAbinding protein FUS/TLS (fused in sarcoma/translocated in liposarcoma) as a new factor interacting with U7 snRNA/RNP. Co-immunoprecipitation and RIP experiments confirmed the binding between FUS and the U7 RNA/snRNP. Interestingly, FUS:U7 snRNA interaction seems to be activated in S phase where the core histone genes are expressed. Moreover, FUS co-fractionates in 10-50% continuous glycerol gradient with other factors involved in histone premRNAs 3’end processing. However, this unique 3’end maturation was not disturbed upon FUS knockdown. Instead, we found that FUS depletion leads to a de-regulation of expression from selected histone promoters, suggesting that FUS is rather involved in regulation of core histone genes transcription. Thus, FUS bound to U7 snRNP can play a role in coupling between transcription and 3’end processing of replication dependant histone mRNAs.

Synthesis and testing of photoaffinity probes for the site-selectivechemical modifications of the 5-HT3 receptor

The 5-HT3 receptor (5-HT3R) is an important ion channel responsible for the transmission of nerve impulses in the central nervous system.1 It is difficult to characterize transmembrane dynamic receptors with classical structural biology approaches like crystallization and x-ray. The use of photoaffinity probes is an alternative approach to identify regions in the protein that are important for the binding of small molecules. Therefore we synthesized a small library of photoaffinity probes by conjugating photophores via various linkers to granisetron which is a known antagonist of the 5-HT3R. We were able to obtain several compounds with diverse linker lengths and different photolabile moieties that show nanomolar binding affinities for the orthosteric binding site. Furthermore we established a stable h5-HT3R expressing cell line and a purification protocol to yield the receptor in a high purity. Currently we are investigating the photo crosslinking of these ligands with the 5-HT3R.

Synthesis and testing of photoaffinity probes for the site-selective chemical modifications of the 5-HT3 receptor

The 5-HT3 receptor (5-HT3R) is an important ion channel responsible for the transmission of nerve impulses in the central nervous system.[1] It is difficult to characterize transmembrane dynamic receptors with classical structural biology approaches like crystallization and x-ray. The use of photoaffinity probes is an alternative approach to identify regions in the protein that are important for the binding of small molecules. Therefore we synthesized a small library of photoaffinity probes by conjugating photolabile building blocks via various linkers to granisetron which is a known antagonist of the 5-HT3R. We were able to obtain several compounds with diverse linker lengths and different photo-labile moieties that show nanomolar binding affinities for the orthosteric binding site. Further on we developed a stable 5-HT3R overexpressing cell line and a purification method to yield the receptor in a high purity. Currently we are investigating crosslinking experiments and subsequent MS – analysis.

Synthesis and testing of photoaffinity probes for the site-selectivechemical modifications of the 5-HT3 receptor

The 5-HT3 receptor (5-HT3R) is an important ion channel responsible for the transmission of nerve impulses in the central nervous system.1 It is difficult to characterize transmembrane dynamic receptors with classical structural biology approaches like crystallization and x-ray. The use of photoaffinity probes is an alternative approach to identify regions in the protein that are important for the binding of small molecules. Therefore we synthesized a small library of photoaffinity probes by conjugating photophores via various linkers to granisetron which is a known antagonist of the 5-HT3R. We were able to obtain several compounds with diverse linker lengths and different photolabile moieties that show nanomolar binding affinities for the orthosteric binding site. Furthermore we established a stable h5-HT3R expressing cell line and a purification protocol to yield the receptor in a high purity. Currently we are investigating the photo crosslinking of these ligands with the 5-HT3R.

Synthesis and testing of photoaffinity probes for the site-selective chemical modifications of the 5-HT3 receptor

The 5-HT3 receptor (5-HT3R) is an important ion channel responsible for the transmission of nerve impulses in the central nervous system.1 It is difficult to characterize transmembrane dynamic receptors with classical structural biology approaches like crystallization and x-ray. The use of photoaffinity probes is an alternative approach to identify regions in the protein that are important for the binding of small molecules. Therefore we synthesized a small library of photoaffinity probes by conjugating photophores via various linkers to granisetron which is a known antagonist of the 5-HT3R. We were able to obtain several compounds with diverse linker lengths and different photolabile moieties that show nanomolar binding affinities for the orthosteric binding site. Furthermore we established a stable h5-HT3R expressing cell line and a purification protocol to yield the receptor in a high purity. Currently we are investigating the photo crosslinking of these ligands with the 5-HT3R.

Ice-Core Based Assessment of Historical Anthropogenic Heavy Metal (Cd, Cu, Sb, Zn) Emissions in the Soviet Union

The development of strategies and policies aiming at the reduction of environmental exposure to air pollution requires the assessment of historical emissions. Although anthropogenic emissions from the extended territory of the Soviet Union (SU) considerably influenced concentrations of heavy metals in the Northern Hemisphere, Pb is the only metal with long-term historical emission estimates for this region available, whereas for selected other metals only single values exist. Here we present the first study assessing long-term Cd, Cu, Sb, and Zn emissions in the SU during the period 1935–1991 based on ice-core concentration records from Belukha glacier in the Siberian Altai and emission data from 12 regions in the SU for the year 1980. We show that Zn primarily emitted from the Zn production in Ust-Kamenogorsk (East Kazakhstan) dominated the SU heavy metal emission. Cd, Sb, Zn (Cu) emissions increased between 1935 and the 1970s (1980s) due to expanded non-ferrous metal production. Emissions of the four metals in the beginning of the 1990s were as low as in the 1950s, which we attribute to the economic downturn in industry, changes in technology for an increasing metal recovery from ores, the replacement of coal and oil by gas, and air pollution control.

A new sensitive method for the quantification of glyoxal and methylglyoxal in snow and ice by stir bar sorptive extraction and liquid desorption-HPLC-ESI-MS

In this study, the development of a new sensitive method for the analysis of alpha-dicarbonyls glyoxal (G) and methylglyoxal (MG) in environmental ice and snow is presented. Stir bar sorptive extraction with in situ derivatization and liquid desorption (SBSE-LD) was used for sample extraction, enrichment, and derivatization. Measurements were carried out using high-performance liquid chromatography coupled to electrospray ionization tandem mass spectrometry (HPLC-ESI-MS/MS). As part of the method development, SBSE-LD parameters such as extraction time, derivatization reagent, desorption time and solvent, and the effect of NaCl addition on the SBSE efficiency as well as measurement parameters of HPLC-ESI-MS/MS were evaluated. Calibration was performed in the range of 1–60 ng/mL using spiked ultrapure water samples, thus incorporating the complete SBSE and derivatization process. 4-Fluorobenzaldehyde was applied as internal standard. Inter-batch precision was <12 % RSD. Recoveries were determined by means of spiked snow samples and were 78.9 ± 5.6 % for G and 82.7 ± 7.5 % for MG, respectively. Instrumental detection limits of 0.242 and 0.213 ng/mL for G and MG were achieved using the multiple reaction monitoring mode. Relative detection limits referred to a sample volume of 15 mL were 0.016 ng/mL for G and 0.014 ng/mL for MG. The optimized method was applied for the analysis of snow samples from Mount Hohenpeissenberg (close to the Meteorological Observatory Hohenpeissenberg, Germany) and samples from an ice core from Upper Grenzgletscher (Monte Rosa massif, Switzerland). Resulting concentrations were 0.085–16.3 ng/mL for G and 0.126–3.6 ng/mL for MG. Concentrations of G and MG in snow were 1–2 orders of magnitude higher than in ice core samples. The described method represents a simple, green, and sensitive analytical approach to measure G and MG in aqueous environmental samples.

Effects of sources and meteorology on particulate matter in the Western Mediterranean Basin: An overview of the DAURE campaign

DAURE (Determination of the Sources of Atmospheric Aerosols in Urban and Rural Environments in the Western Mediterranean) was a multidisciplinary international field campaign aimed at investigating the sources and meteorological controls of particulate matter in the Western Mediterranean Basin (WMB). Measurements were simultaneously performed at an urban-coastal (Barcelona, BCN) and a rural-elevated (Montseny, MSY) site pair in NE Spain during winter and summer. State-of-the-art methods such as 14C analysis, proton-transfer reaction mass spectrometry, and high-resolution aerosol mass spectrometry were applied for the first time in the WMB as part of DAURE. WMB regional pollution episodes were associated with high concentrations of inorganic and organic species formed during the transport to inland areas and built up at regional scales. Winter pollutants accumulation depended on the degree of regional stagnation of an air mass under anticyclonic conditions and the planetary boundary layer height. In summer, regional recirculation and biogenic secondary organic aerosols (SOA) formation mainly determined the regional pollutant concentrations. The contribution from fossil sources to organic carbon (OC) and elemental carbon (EC) and hydrocarbon-like organic aerosol concentrations were higher at BCN compared with MSY due to traffic emissions. The relative contribution of nonfossil OC was higher at MSY especially in summer due to biogenic emissions. The fossil OC/EC ratio at MSY was twice the corresponding ratio at BCN indicating that a substantial fraction of fossil OC was due to fossil SOA. In winter, BCN cooking emissions were identified as an important source of modern carbon in primary organic aerosol.

Structure/affinity studies in the bicyclo-DNA series: Synthesis and properties of oligonucleotides containing bcen-T and iso-tricyclo-T nucleosides

We present the synthesis of the two novel nucleosides iso-tc-T and bcen-T, belonging to the bicyclo-/tricyclo-DNA molecular platform. In both modifications the torsion around C6’–C7’ within the carbocyclic ring is planarized by either the presence of a C6’–C7’ double bond or a cyclopropane ring. Structural analysis of these two nucleosides by X-ray analysis reveals a clear preference of torsion angle γ for the gauche orientation with the furanose ring in a near perfect 2’-endo conformation. Both modifications were incorporated into oligodeoxynucleotides and their thermal melting behavior with DNA and RNA as complements was assessed. We found that the iso-tc-T modification was significantly more destabilizing in duplex formation compared to the bcen-T modification. In addition, duplexes with complementary RNA were less stable as compared to duplexes with DNA as complement. A structure/affinity analysis, including the already known bc-T and tc-T modifications, does not lead to a clear correlation of the orientation of torsion angle γ with DNA or RNA affinity. There is, however, some correlation between furanose conformation (N- or S-type) and affinity in the sense that a preference for a 3’-endo like conformation is associated with a preference for RNA as complement. As a general rule it appears that Tm data of single modifications with nucleosides of the bicyclo-/tricyclo-DNA platform within deoxyoligonucleotides are not predictive for the stability of fully modified oligonucleotides.

Unraveling the Conformational Determinants of Peptide Dendrimers Using Molecular Dynamics Simulations

Peptide dendrimers are synthetic tree-like molecules composed of amino acids. There are at least two kinds of preferential structural behaviors exhibited by these molecules, which acquire either compact or noncompact shapes. However, the key structural determinants of such behaviors remained, until now, unstudied. Herein, we conduct a comprehensive investigation of the structural determinants of peptide dendrimers by employing long molecular dynamics simulations to characterize an extended set of third generation dendrimers. Our results clearly show that a trade-off between electrostatic effects and hydrogen bond formation controls structure acquisition in these systems. Moreover, by selectively changing the dendrimers charge we are able to manipulate the exhibited compactness. In contrast, the length of branching residues does not seem to be a major structural determinant. Our results are in accordance with the most recent experimental evidence and shed some light on the key molecular level interactions controlling structure acquisition in these systems. Thus, the results presented constitute valuable insights that can contribute to the development of truly tailor-made dendritic systems.

Impaired genome encapsidation restricts the in vitro propagation of human parvovirus B19.

The lack of a permissive cell culture system hampers the study of human parvovirus B19 (B19V). UT7/Epo is one of the few established cell lines that can be infected with B19V but generates none or few infectious progeny. Recently, hypoxic conditions or the use of primary CD36+ erythroid progenitor cells (CD36+ EPCs) have been shown to improve the infection. These novel approaches were evaluated in infection and transfection experiments. Hypoxic conditions or the use of CD36+ EPCs resulted in a significant acceleration of the infection/transfection and a modest increase in the yield of capsid progeny. However, under all tested conditions, genome encapsidation was impaired seriously. Further analysis of the cell culture virus progeny revealed that differently to the wild-type virus, the VP1 unique region (VP1u) was exposed partially and was unable to become further externalized upon heat treatment. The fivefold axes pore, which is used for VP1u externalization and genome encapsidation, might be constricted by the atypical VP1u conformation explaining the packaging failure. Although CD36+ EPCs and hypoxia facilitate B19V infection, large quantities of infectious progeny cannot be generated due to a failure in genome encapsidation, which arises as a major limiting factor for the in vitro propagation of B19V.

Analyzing diffuse scattering with supercomputers

Two new approaches to quantitatively analyze diffuse diffraction intensities from faulted layer stacking are reported. The parameters of a probability-based growth model are determined with two iterative global optimization methods: a genetic algorithm (GA) and particle swarm optimization (PSO). The results are compared with those from a third global optimization method, a differential evolution (DE) algorithm [Storn & Price (1997). J. Global Optim. 11, 341–359]. The algorithm efficiencies in the early and late stages of iteration are compared. The accuracy of the optimized parameters improves with increasing size of the simulated crystal volume. The wall clock time for computing quite large crystal volumes can be kept within reasonable limits by the parallel calculation of many crystals (clones) generated for each model parameter set on a super- or grid computer. The faulted layer stacking in single crystals of trigonal three-pointedstar- shaped tris(bicylco[2.1.1]hexeno)benzene molecules serves as an example for the numerical computations. Based on numerical values of seven model parameters (reference parameters), nearly noise-free reference intensities of 14 diffuse streaks were simulated from 1280 clones, each consisting of 96 000 layers (reference crystal). The parameters derived from the reference intensities with GA, PSO and DE were compared with the original reference parameters as a function of the simulated total crystal volume. The statistical distribution of structural motifs in the simulated crystals is in good agreement with that in the reference crystal. The results found with the growth model for layer stacking disorder are applicable to other disorder types and modeling techniques, Monte Carlo in particular.