Analysis of glutathione mediated S-(de)nitrosylation in complex biological matrices by immuno-spin trapping and identification of two novel substrates.

The intracellular concentration of reduced glutathione (GSH) lies in the range of 1-10 mM, thereby indisputably making it the most abundant intracellular thiol. Such a copious amount of GSH makes it the most potent and robust cellular antioxidant that plays a crucial role in cellular defence against redox stress. The role of GSH as a denitrosylating agent is well established; in this study, we demonstrate GSH mediated denitrosylation of HepG2 cell-derived protein nitrosothiols (PSNOs), by a unique spin-trapping mechanism, using 5,5-dimethyl-1-pyrroline N-oxide (DMPO) as the spin trapping agent, followed by a western blot analysis. We also report our findings of two, hitherto unidentified substrates of GSH mediated S-denitrosylation, namely S-nitrosoglutaredoxin 1 (Grx1-SNO) and S-nitrosylated R1 subunit of ribonucleotide reductase (R1-SNO).

Molecular basis for AU-rich element recognition and dimerization by the HuR C-terminal RRM.

Human antigen R (HuR) is a key regulator of cellular mRNAs containing adenylate/uridylate-rich elements (AU-rich elements; AREs). These are a major class of cis elements within 3' untranslated regions, targeting these mRNAs for rapid degradation. HuR contains three RNA recognition motifs (RRMs): a tandem RRM1 and 2, followed by a flexible linker and a C-terminal RRM3. While RRM1 and 2 are structurally characterized, little is known about RRM3. Here we present a 1.9-Å-resolution crystal structure of RRM3 bound to different ARE motifs. This structure together with biophysical methods and cell-culture assays revealed the mechanism of RRM3 ARE recognition and dimerization. While multiple RNA motifs can be bound, recognition of the canonical AUUUA pentameric motif is possible by binding to two registers. Additionally, RRM3 forms homodimers to increase its RNA binding affinity. Finally, although HuR stabilizes ARE-containing RNAs, we found that RRM3 counteracts this effect, as shown in a cell-based ARE reporter assay and by qPCR with native HuR mRNA targets containing multiple AUUUA motifs, possibly by competing with RRM12.

The La-related proteins: structures and interactions of a versatile superfamily of RNA-binding proteins.

The La-related proteins (LaRPs) are an ancient superfamily of RNA-binding proteins orchestrating the major fates of RNA, from processing and maturation to regulation of mRNA translation. LaRPs are instrumental in modulating complex assemblies where the RNA is bound, folded, processed, escorted and presented to the functional effectors often through recruitment of protein partners. This intricate web of protein-RNA and protein-protein interactions is enabled by the modular nature of the LaRPs, comprising several structured domains connected by flexible linkers, and other sequences lacking recognizable folded motifs. Recent structures, together with biochemical and biophysical studies, have provided insights into how each LaRP family has evolved unique mechanisms of RNA recognition, not only through the conserved RNA-binding unit, the La-module, but also mediated by other family-specific motifs. Furthermore, in a series of unexpected twists and turns, they have revealed that the dynamic and conformational interplay of multi-structured domains and disordered regions operate in unison to achieve RNA substrate discrimination. This review proposes a perspective of our current knowledge of the structure-function relationship of the LaRP superfamily.

La proteins couple use of sequence-specific and non-specific binding modes to engage RNA substrates.

La shuttles between the nucleus and cytoplasm where it binds nascent RNA polymerase III (pol III) transcripts and mRNAs, respectively. La protects the 3' end of pol III transcribed RNA precursors, such as pre-tRNAs, through the use of a well-characterized UUU-3'OH binding mode. La proteins are also RNA chaperones, and La-dependent RNA chaperone activity is hypothesized to promote pre-tRNA maturation and translation at cellular and viral internal ribosome entry sites via binding sites distinct from those used for UUU-3'OH recognition. Since the publication of La-UUU-3'OH co-crystal structures, biochemical and genetic experiments have expanded our understanding of how La proteins use UUU-3'OH-independent binding modes to make sequence-independent contacts that can increase affinity for ligands and promote RNA remodeling. Other recent work has also expanded our understanding of how La binds mRNAs through contacts to the poly(A) tail. In this review, we focus on advances in the study of La protein-RNA complex surfaces beyond the description of the La-UUU-3'OH binding mode. We highlight recent advances in the functions of expected canonical nucleic acid interaction surfaces, a heightened appreciation of disordered C-terminal regions, and the nature of sequence-independent RNA determinants in La-RNA target binding. We further discuss how these RNA binding modes may have relevance to the function of the La-related proteins.

Subunit Interaction Dynamics of Class Ia Ribonucleotide Reductases: In Search of a Robust Assay.

Ribonucleotide reductases (RNRs) catalyze the conversion of nucleotides (NDP) to deoxynucleotides (dNDP), in part, by controlling the ratios and quantities of dNTPs available for DNA replication and repair. The active form of Escherichia coli class Ia RNR is an asymmetric α2ß2 complex in which α2 contains the active site and ß2 contains the stable diferric-tyrosyl radical cofactor responsible for initiating the reduction chemistry. Each dNDP is accompanied by disulfide bond formation. We now report that, under in vitro conditions, ß2 can initiate turnover in α2 catalytically under both "one" turnover (no external reductant, though producing two dCDPs) and multiple turnover (with an external reductant) assay conditions. In the absence of reductant, rapid chemical quench analysis of a reaction of α2, substrate, and effector with variable amounts of ß2 (1-, 10-, and 100-fold less than α2) yields 3 dCDP/α2 at all ratios of α2:ß2 with a rate constant of 8-9 s-1, associated with a rate-limiting conformational change. Stopped-flow fluorescence spectroscopy with a fluorophore-labeled ß reveals that the rate constants for subunit association (163 ± 7 µM-1 s-1) and dissociation (75 ± 10 s-1) are fast relative to turnover, consistent with catalytic ß2. When assaying in the presence of an external reducing system, the turnover number is dictated by the ratio of α2:ß2, their concentrations, and the concentration and nature of the reducing system; the rate-limiting step can change from the conformational gating to a step or steps involving disulfide rereduction, dissociation of the inhibited α4ß4 state, or both. The issues encountered with E. coli RNR are likely of importance in all class I RNRs and are central to understanding the development of screening assays for inhibitors of these enzymes.

Regulation of ribonucleotide reductase in response to iron deficiency.

Ribonucleotide reductase (RNR) is an essential enzyme required for DNA synthesis and repair. Although iron is necessary for class Ia RNR activity, little is known about the mechanisms that control RNR in response to iron deficiency. In this work, we demonstrate that yeast cells control RNR function during iron deficiency by redistributing the Rnr2-Rnr4 small subunit from the nucleus to the cytoplasm. Our data support a Mec1/Rad53-independent mechanism in which the iron-regulated Cth1/Cth2 mRNA-binding proteins specifically interact with the WTM1 mRNA in response to iron scarcity and promote its degradation. The resulting decrease in the nuclear-anchoring Wtm1 protein levels leads to the redistribution of the Rnr2-Rnr4 heterodimer to the cytoplasm, where it assembles as an active RNR complex and increases deoxyribonucleoside triphosphate levels. When iron is scarce, yeast selectively optimizes RNR function at the expense of other non-essential iron-dependent processes that are repressed, to allow DNA synthesis and repair.

TIA-1 RRM23 binding and recognition of target oligonucleotides.

TIA-1 (T-cell restricted intracellular antigen-1) is an RNA-binding protein involved in splicing and translational repression. It mainly interacts with RNA via its second and third RNA recognition motifs (RRMs), with specificity for U-rich sequences directed by RRM2. It has recently been shown that RRM3 also contributes to binding, with preferential binding for C-rich sequences. Here we designed UC-rich and CU-rich 10-nt sequences for engagement of both RRM2 and RRM3 and demonstrated that the TIA-1 RRM23 construct preferentially binds the UC-rich RNA ligand (5΄-UUUUUACUCC-3΄). Interestingly, this binding depends on the presence of Lys274 that is C-terminal to RRM3 and binding to equivalent DNA sequences occurs with similar affinity. Small-angle X-ray scattering was used to demonstrate that, upon complex formation with target RNA or DNA, TIA-1 RRM23 adopts a compact structure, showing that both RRMs engage with the target 10-nt sequences to form the complex. We also report the crystal structure of TIA-1 RRM2 in complex with DNA to 2.3 Šresolution providing the first atomic resolution structure of any TIA protein RRM in complex with oligonucleotide. Together our data support a specific mode of TIA-1 RRM23 interaction with target oligonucleotides consistent with the role of TIA-1 in binding RNA to regulate gene expression.

Structural insight into the mechanism of stabilization of the 7SK small nuclear RNA by LARP7.

The non-coding RNA 7SK is the scaffold for a small nuclear ribonucleoprotein (7SKsnRNP) which regulates the function of the positive transcription elongation factor P-TEFb in the control of RNA polymerase II elongation in metazoans. The La-related protein LARP7 is a component of the 7SKsnRNP required for stability and function of the RNA. To address the function of LARP7 we determined the crystal structure of its La module, which binds a stretch of uridines at the 3'-end of 7SK. The structure shows that the penultimate uridine is tethered by the two domains, the La-motif and the RNA-recognition motif (RRM1), and reveals that the RRM1 is significantly smaller and more exposed than in the La protein. Sequence analysis suggests that this impacts interaction with 7SK. Binding assays, footprinting and small-angle scattering experiments show that a second RRM domain located at the C-terminus binds the apical loop of the 3' hairpin of 7SK, while the N-terminal domains bind at its foot. Our results suggest that LARP7 uses both its N- and C-terminal domains to stabilize 7SK in a closed structure, which forms by joining conserved sequences at the 5'-end with the foot of the 3' hairpin and has thus functional implications.

A sliding docking interaction is essential for sequential and processive phosphorylation of an SR protein by SRPK1.

The 2.9 A crystal structure of the core SRPK1:ASF/SF2 complex reveals that the N-terminal half of the basic RS domain of ASF/SF2, which is destined to be phosphorylated, is bound to an acidic docking groove of SRPK1 distal to the active site. Phosphorylation of ASF/SF2 at a single site in the C-terminal end of the RS domain generates a primed phosphoserine that binds to a basic site in the kinase. Biochemical experiments support a directional sliding of the RS peptide through the docking groove to the active site during phosphorylation, which ends with the unfolding of a beta strand of the RRM domain and binding of the unfolded region to the docking groove. We further suggest that the priming of the first serine facilitates directional substrate translocation and efficient phosphorylation.

Semiquinone-induced maturation of Bacillus anthracis ribonucleotide reductase by a superoxide intermediate.

Ribonucleotide reductases (RNRs) catalyze the conversion of ribonucleotides to deoxyribonucleotides, and represent the only de novo pathway to provide DNA building blocks. Three different classes of RNR are known, denoted I-III. Class I RNRs are heteromeric proteins built up by α and ß subunits and are further divided into different subclasses, partly based on the metal content of the ß-subunit. In subclass Ib RNR the ß-subunit is denoted NrdF, and harbors a manganese-tyrosyl radical cofactor. The generation of this cofactor is dependent on a flavodoxin-like maturase denoted NrdI, responsible for the formation of an active oxygen species suggested to be either a superoxide or a hydroperoxide. Herein we report on the magnetic properties of the manganese-tyrosyl radical cofactor of Bacillus anthracis NrdF and the redox properties of B. anthracis NrdI. The tyrosyl radical in NrdF is stabilized through its interaction with a ferromagnetically coupled manganese dimer. Moreover, we show through a combination of redox titration and protein electrochemistry that in contrast to hitherto characterized NrdIs, the B. anthracis NrdI is stable in its semiquinone form (NrdIsq) with a difference in electrochemical potential of ∼110 mV between the hydroquinone and semiquinone state. The under anaerobic conditions stable NrdIsq is fully capable of generating the oxidized, tyrosyl radical-containing form of Mn-NrdF when exposed to oxygen. This latter observation strongly supports that a superoxide radical is involved in the maturation mechanism, and contradicts the participation of a peroxide species. Additionally, EPR spectra on whole cells revealed that a significant fraction of NrdI resides in its semiquinone form in vivo, underscoring that NrdIsq is catalytically relevant.

The conserved Lys-95 charged residue cluster is critical for the homodimerization and enzyme activity of human ribonucleotide reductase small subunit M2.

Ribonucleotide reductase (RR) catalyzes the reduction of ribonucleotides to deoxyribonucleotides for DNA synthesis. Human RR small subunit M2 exists in a homodimer form. However, the importance of the dimer form to the enzyme and the related mechanism remain unclear. In this study, we tried to identify the interfacial residues that may mediate the assembly of M2 homodimer by computational alanine scanning based on the x-ray crystal structure. Co-immunoprecipitation, size exclusion chromatography, and RR activity assays showed that the K95E mutation in M2 resulted in dimer disassembly and enzyme activity inhibition. In comparison, the charge-exchanging double mutation of K95E and E98K recovered the dimerization and activity. Structural comparisons suggested that a conserved cluster of charged residues, including Lys-95, Glu-98, Glu-105, and Glu-174, at the interface may function as an ionic lock for M2 homodimer. Although the measurements of the radical and iron contents showed that the monomer (the K95E mutant) was capable of generating the diiron and tyrosyl radical cofactor, co-immunoprecipitation and competitive enzyme inhibition assays indicated that the disassembly of M2 dimer reduced its interaction with the large subunit M1. In addition, the immunofluorescent and fusion protein-fluorescent imaging analyses showed that the dissociation of M2 dimer altered its subcellular localization. Finally, the transfection of the wild-type M2 but not the K95E mutant rescued the G1/S phase cell cycle arrest and cell growth inhibition caused by the siRNA knockdown of M2. Thus, the conserved Lys-95 charged residue cluster is critical for human RR M2 homodimerization, which is indispensable to constitute an active holoenzyme and function in cells.

E2F1 promote the aggressiveness of human colorectal cancer by activating the ribonucleotide reductase small subunit M2.

As the ribonucleotide reductase small subunit, the high expression of ribonucleotide reductase small subunit M2 (RRM2) induces cancer and contributes to tumor growth and invasion. In several colorectal cancer (CRC) cell lines, we found that the expression levels of RRM2 were closely related to the transcription factor E2F1. Mechanistic studies were conducted to determine the molecular basis. Ectopic overexpression of E2F1 promoted RRM2 transactivation while knockdown of E2F1 reduced the levels of RRM2 mRNA and protein. To further investigate the roles of RRM2 which was activated by E2F1 in CRC, CCK-8 assay and EdU incorporation assay were performed. Overexpression of E2F1 promoted cell proliferation in CRC cells, which was blocked by RRM2 knockdown attenuation. In the migration and invasion tests, overexpression of E2F1 enhanced the migration and invasion of CRC cells which was abrogated by silencing RRM2. Besides, overexpression of RRM2 reversed the effects of E2F1 knockdown partially in CRC cells. Examination of clinical CRC specimens demonstrated that both RRM2 and E2F1 were elevated in most cancer tissues compared to the paired normal tissues. Further analysis showed that the protein expression levels of E2F1 and RRM2 were parallel with each other and positively correlated with lymph node metastasis (LNM), TNM stage and distant metastasis. Consistently, the patients with low E2F1 and RRM2 levels have a better prognosis than those with high levels. Therefore, we suggest that E2F1 can promote CRC proliferation, migration, invasion and metastasis by regulating RRM2 transactivation. Understanding the role of E2F1 in activating RRM2 transcription will help to explain the relationship between E2F1 and RRM2 in CRC and provide a novel predictive marker for diagnosis and prognosis of the disease.

Structure and functional implications of a complex containing a segment of U6 RNA bound by a domain of Prp24.

U6 RNA plays a critical role in pre-mRNA splicing. Assembly of U6 into the spliceosome requires a significant structural rearrangement and base-pairing with U4 RNA. In the yeast Saccharomyces cerevisiae, this process requires the essential splicing factor Prp24. We present the characterization and structure of a complex containing one of Prp24's four RNA recognition motif (RRM) domains, RRM2, and a fragment of U6 RNA. NMR methods were used to identify the preferred U6 binding sequence of RRM2 (5'-GAGA-3'), measure the affinity of the interaction, and solve the structure of RRM2 bound to the hexaribonucleotide AGAGAU. Interdomain contacts observed between RRM2 and RRM3 in a crystal structure of the free protein are not detectable in solution. A structural model of RRM1 and RRM2 bound to a longer segment of U6 RNA is presented, and a partial mechanism for Prp24's annealing activity is proposed.

Human DND1-RRM2 forms a non-canonical domain swapped dimer.

RNA recognition motif (RRM) being the most abundant RNA binding domain in eukaryotes, is a major player in cellular regulation. Several variations in the canonical ßαßßαß topology have been observed. We have determined the 2.3 Å crystal structure of the human DND1-RRM2 domain. The structure revealed an interesting non-canonical RRM fold, which is maintained by the formation of a 3D domain swapped dimer between ß1 and ß4 strands across protomers. We have delineated the structural basis of the stable domain swapped dimer formation using the residue level dynamics of protein explored by NMR spectroscopy and MD simulations. Our structural and dynamics studies substantiate major determinants and molecular basis for domain swapped dimerization observed in the RRM domain.

mTORC2 regulates ribonucleotide reductase to promote DNA replication and gemcitabine resistance in non-small cell lung cancer.

Ribonucleotide reductase (RNR) is the key enzyme that catalyzes the production of deoxyribonucleotides (dNTPs) for DNA replication and it is also essential for cancer cell proliferation. As the RNR inhibitor, Gemcitabine is widely used in cancer therapies, however, resistance limits its therapeutic efficacy and curative potential. Here, we identified that mTORC2 is a main driver of gemcitabine resistance in non-small cell lung cancers (NSCLC). Pharmacological or genetic inhibition of mTORC2 greatly enhanced gemcitabine induced cytotoxicity and DNA damage. Mechanistically, mTORC2 directly interacted and phosphorylated RNR large subunit RRM1 at Ser 631. Ser631 phosphorylation of RRM1 enhanced its interaction with small subunit RRM2 to maintain sufficient RNR enzymatic activity for efficient DNA replication. Targeting mTORC2 retarded DNA replication fork progression and improved therapeutic efficacy of gemcitabine in NSCLC xenograft model in vivo. Thus, these results identified a mechanism through mTORC2 regulating RNR activity and DNA replication, conferring gemcitabine resistance to cancer cells.

Use of 3-aminotyrosine to examine the pathway dependence of radical propagation in Escherichia coli ribonucleotide reductase.

Escherichia coli ribonucleotide reductase (RNR), an alpha2beta2 complex, catalyzes the conversion of nucleoside 5'-diphosphate substrates (S) to 2'-deoxynucleoside 5'-diphosphates. alpha2 houses the active site for nucleotide reduction and the binding sites for allosteric effectors (E). beta2 contains the essential diferric tyrosyl radical (Y(122)(*)) cofactor which, in the presence of S and E, oxidizes C(439) in alpha to a thiyl radical, C(439)(*), to initiate nucleotide reduction. This oxidation occurs over 35 A and is proposed to involve a specific pathway: Y(122)(*) --> W(48) --> Y(356) in beta2 to Y(731) --> Y(730) --> C(439) in alpha2. 3-Aminotyrosine (NH(2)Y) has been site-specifically incorporated at residues 730 and 731, and formation of the aminotyrosyl radical (NH(2)Y(*)) has been examined by stopped-flow (SF) UV-vis and EPR spectroscopies. To examine the pathway dependence of radical propagation, the double mutant complexes Y(356)F-beta2:Y(731)NH(2)Y-alpha2, Y(356)F-beta2:Y(730)NH(2)Y-alpha2, and wt-beta2:Y(731)F/Y(730)NH(2)Y-alpha2, in which the nonoxidizable F acts as a pathway block, were studied by SF and EPR spectroscopies. In all cases, no NH(2)Y(*) was detected. To study off-pathway oxidation, Y(413), located 5 A from Y(730) and Y(731) but not implicated in long-range oxidation, was examined. Evidence for NH(2)Y(413)(*) was sought in three complexes: wt-beta2:Y(413)NH(2)Y-alpha2 (a), wt-beta2:Y(731)F/Y(413)NH(2)Y-alpha2 (b), and Y(356)F-beta2:Y(413)NH(2)Y-alpha2 (c). With (a), NH(2)Y(*) was formed with a rate constant that was 25-30% and an amplitude that was 25% of that observed for its formation at residues 731 and 730. With (b), the rate constant for NH(2)Y(*) formation was 0.2-0.3% of that observed at 731 and 730, and with (c), no NH(2)Y(*) was observed. These studies suggest the evolution of an optimized pathway of conserved Ys in the oxidation of C(439).

2.6 A X-ray crystal structure of human p53R2, a p53-inducible ribonucleotide reductase .

Human p53R2 (hp53R2) is a 351-residue p53-inducible ribonucleotide reductase (RNR) small subunit. It shares >80% sequence identity with hRRM2, the small RNR subunit responsible for normal maintenance of the deoxyribonucleotide (dNTP) pool used for DNA replication, which is active during the S phase in a cell cycle-dependent fashion. But rather than cyclic dNTP synthesis, hp53R2 has been shown to supply dNTPs for DNA repair to cells in G0-G1 in a p53-dependent fashion. The first X-ray crystal structure of hp53R2 is determined to 2.6 A, in which monomers A and B exhibit mono- and binuclear iron occupancy, respectively. The pronounced structural differences at three regions between hp53R2 and hRRM2 highlight the possible regulatory role in iron assimilation and help explain previously observed physical and biochemical differences in the mobility and accessibility of the radical iron center, as well as radical transfer pathways between the two enzymes. The sequence-structure-function correlations that differentiate hp53R2 and hRRM2 are revealed for the first time. Insight gained from this structural work will be used in the identification of biological function, regulation mechanism, and inhibitor selection in RNR small subunits.

Active nuclear import and passive nuclear export are the primary determinants of TDP-43 localization.

ALS (Amyotrophic Lateral Sclerosis) is a neurodegenerative disease characterized by the redistribution of the RNA binding protein TDP-43 in affected neurons: from predominantly nuclear to aggregated in the cytosol. However, the determinants of TDP-43 localization and the cellular insults that promote redistribution are incompletely understood. Here, we show that the putative Nuclear Export Signal (NES) is not required for nuclear egress of TDP-43. Moreover, when the TDP-43 domain which contains the putative NES is fused to a reporter protein, YFP, the presence of the NES is not sufficient to mediate nuclear exclusion of the fusion protein. We find that the previously studied "∆NES" mutant, in which conserved hydrophobic residues are mutated to alanines, disrupts both solubility and splicing function. We further show that nuclear export of TDP-43 is independent of the exportin XPO1. Finally, we provide evidence that nuclear egress of TDP-43 is size dependent; nuclear export of dTomato TDP-43 is significantly impaired compared to Flag TDP-43. Together, these results suggest nuclear export of TDP-43 is predominantly driven by passive diffusion.

NMR studies of binding of 5-FdUDP and dCDP to ribonucleoside-diphosphate reductase from Escherichia coli.

5-Fluoro-2'-deoxyuridine-5'-diphosphate (5-FdUDP) has been synthesised using an original route, previously applied to the synthesis of natural nucleoside diphosphates. The interaction between 5-FdUDP and the enzyme ribonucleoside-diphosphate reductase (EC 1.17.4.1) has been studied with 19F-NMR. The product analogue is shown to be in fast exchange with substrate binding sites on protein subunit 1 (R1) of ribonucleoside-diphosphate (NDP) reductase. The number of binding sites is reduced to half when the complete holoenzyme R1R2 is formed. The temperature dependence of the line broadening of 5-FdUDP was studied using 19F-NMR, and of dCDP and dUDP using 1H-NMR. The temperature dependences are complex and a molecular model in which R1 is in a temperature dependent equilibrium between at least two conformations is suggested in order to explain the observed behaviour. Binding of a ligand to the substrate binding sites affects the conformational equilibrium in a ligand specific way. Formation of the holoenzyme R1R2 also affects the equilibrium.

The enantioselectivities of the active and allosteric sites of mammalian ribonucleotide reductase.

Here we examine the enantioselectivity of the allosteric and substrate binding sites of murine ribonucleotide reductase (mRR). L-ADP binds to the active site and L-ATP binds to both the s- and a-allosteric sites of mR1 with affinities that are only three- to 10-fold weaker than the values for the corresponding D-enantiomers. These results demonstrate the potential of L-nucleotides for interacting with and modulating the activity of mRR, a cancer chemotherapeutic and antiviral target. On the other hand, we detect no substrate activity for L-ADP and no inhibitory activity for N3-L-dUDP, demonstrating the greater stereochemical stringency at the active site with respect to catalytic activity.