Peptide Bond Cleavage by Ni(II) Ions within the Nuclear Localization Signal Sequence

Nickel is harmful to humans, being both carcinogenic and allergenic. However, the mechanisms of this toxicity are still unresolved. We propose that Ni(II) ions disintegrate proteins by hydrolysis of peptide bonds preceding the Ser/Thr‐Xaa‐His sequences. Such sequences occur in nuclear localization signals (NLSs) of human phospholipid scramblase 1, Sam68‐like mammalian protein 2, and CLK3 kinase. We performed spectroscopic experiments showing that model nonapeptides derived from these NLSs bind Ni(II) at physiological pH. We also proved that these sequences are prone to Ni(II) hydrolysis. Thus, the aforementioned NLSs may be targets for nickel toxicity. This implies that Ni(II) ions disrupt the transport of some proteins from cytoplasm to cell nucleus.     

Cleavage and polyadenylation specificity factor 1 (CPSF1) regulates alternative splicing of interleukin 7 receptor (IL7R) exon 6

Interleukin 7 receptor, IL7R, is expressed exclusively on cells of the lymphoid lineage, and its expression is crucial for the development and maintenance of T cells. Alternative splicing of IL7R exon 6 results in membrane-bound (exon 6 included) and soluble (exon 6 skipped) IL7R isoforms. Interestingly, the inclusion of exon 6 is affected by a single-nucleotide polymorphism associated with the risk of developing multiple sclerosis. Given the potential association of exon 6 inclusion with multiple sclerosis, we investigated the cis-acting elements and trans-acting factors that regulate exon 6 splicing. We identified multiple exonic and intronic cis-acting elements that impact inclusion of exon 6. Moreover, we utilized RNA affinity chromatography followed by mass spectrometry to identify trans-acting protein factors that bind exon 6 and regulate its splicing. These experiments identified cleavage and polyadenylation specificity factor 1 (CPSF1) among protein-binding candidates. A consensus polyadenylation signal AAUAAA is present in intron 6 of IL7R directly downstream from the 5′ splice site. Mutations to this site and CPSF1 knockdown both resulted in an increase in exon 6 inclusion. We found no evidence that this site is used to produce cleaved and polyadenylated mRNAs, suggesting that CPSF1 interaction with intronic IL7R pre-mRNA interferes with spliceosome binding to the exon 6 5′ splice site. Our results suggest that competing mRNA splicing and polyadenylation regulate exon 6 inclusion and consequently determine the ratios of soluble to membrane-bound IL7R. This may be relevant for both T cell ontogeny and function and development of multiple sclerosis.     

Duplex Stability of Oligonucleotides Containing 7-Substituted 7-Deaza- and 8-Aza-7-Deazapurine Nucleosides

Abstract

The synthesis of 7-substituted 7-deaza- and 8-aza-7-deazapurine 2′-deoxyribonucleosides, their incorporation into oligonucleotides, and the stability of corresponding duplexes is described.     

Meeting report: Guanosines and quadruplexes (London, September 15-17, 2010)

The meeting entitled “Guanosines and quadruplexes” was held in London on September 2010. It attracted over 80 participants from all over the world, combining researchers interested in nucleoside/nucleotide self-assembly and nucleic acids structures, working in fields ranging from chemistry, biology, physics, theory to material sciences and nanotechnology.     

Oligonucleotides incorporating N7-(2'-deoxy-beta-D-erythro-pentofuranosyl)isoguanine

The H-phosphonate and the phosphoramidite of N7-2'-deoxyisoguanosine (2) were prepared and incorporated into oligonucleotide duplexes. Their base pairing properties were investigated and compared with those of the parent purine nucleosides.     

Triple-helix formation in the antiparallel binding motif of oligodeoxynucleotides containing N(9)- and N(7)-2-aminopurine deoxynucleosides

Triplex-forming oligodeoxynucleotide 15mers, designed to bind in the antiparallel triple-helical binding motif, containing single substitutions (Z) of the four isomeric αN⁷-, βN⁷-, αN⁹- and βN⁹-2-aminopurine (ap)-deoxyribonucleosides were prepared. Their association with double-stranded DNA targets containing all four natural base pairs (X-Y) opposite the aminopurine residues was determined by quantitative DNase I footprint titration in the absence of monovalent metal cations. The corresponding association constants were found to be in a rather narrow range between 1.0 × 10⁶ and 1.3 × 10⁸ M^–1. The following relative order in Z × X-Y base-triple stabilities was found: Z = αN⁷ap: T-A > A-T> C-G ~ G-C; Z = βN⁷ap: A-T > C-G > G-C > T-A; Z = αN⁹ap: A-T = G-C > T-A > C-G; and Z = βN⁹ap: G-C > A-T > C-G > T-A.     

Stabilization of the non-canonical adenine-adeninium base pair by N(7) coordination of Zn(II)

A new zinc (II) compound with 9-ethyladenine (9-EtA) of formula [Zn(9-EtA-N7)Cl(3)](9-EtAH) has been synthesized and characterized by X-ray diffraction. Its X-structure consists of an Zn(II) anionic complex and 9-ethyladeninium as counteranion. The Zn(II) complex shows a distorted tetrahedral geometry in which three Cl and an 9-EtA coordinates through N(7) position are the ligands. An indirect chelation via intramolecular H-bond between N(6)H and an Cl ligand is present in the complex. The network of [Zn(9-EtA-N7)Cl(3)](9-EtAH) shows interesting features. Thus, self-association of coordinated adenine-adeninium takes place by H-bonding of N(6)-H...N(1) and N(6)-H...N(7), leading to a polymeric ribbon-like 1D supramolecular arrangement. Ab initio calculations have been applied in order to study the stability of the adenine-adeninium interaction due to the coordination of the Zn(II) to the N(7) position and to compare experimental and theoretical structural data.     

Cleavage of automodified poly(ADP-ribose) polymerase during apoptosis. Evidence for involvement of caspase-7.

The abundant nuclear enzyme poly(ADP-ribose) polymerase (PARP) synthesizes poly(ADP-ribose) in response to DNA strand breaks. During almost all forms of apoptosis, PARP is cleaved by caspases, suggesting the crucial role of its inactivation. A few studies have also reported a stimulation of PARP during apoptosis. However, the role of PARP stimulation and cleavage during this cell death process remains poorly understood. Here, we measured the stimulation of endogenous poly(ADP-ribose) synthesis during VP-16-induced apoptosis in HL60 cells and found that PARP was cleaved by caspases at the time of its poly(ADP-ribosyl)ation. In vitro experiments showed that PARP cleavage by caspase-7, but not by caspase-3, was stimulated by its automodification by long and branched poly(ADP-ribose). Consistently, caspase-7 exhibited an affinity for poly(ADP-ribose), whereas caspase-3 did not. In addition, caspase-7 was activated and accumulated in the nucleus of HL60 cells in response to the VP-16 treatment. Furthermore, caspase-7 activation was concommitant with PARP cleavage in the caspase-3-deficient cell line MCF-7 in response to staurosporine treatment. These results strongly suggest that, in vivo, it is caspase-7 that is responsible for PARP cleavage and that poly(ADP-ribosyl)ation of PARP accelerates its proteolysis. Cleavage of the active form of caspase substrates could be a general feature of the apoptotic process, ensuring the rapid inactivation of stress signaling proteins.     

Protein arginine (N)-methyl transferase 7 (PRMT7) as a potential target for the sensitization of tumor cells to camptothecins

PRMT7 belongs to the protein arginine methyl-transferases family. We show that downregulation of PRMT7alpha and beta isoforms in DC-3F hamster cells was associated with increased sensitivity to the Top1 inhibitor camptothecin (CPT). This effect was not due to a change in Top1 contents or catalytic activity, or to a difference in the reversal of DNA breaks. Overexpression of PRMT7alpha and beta in DC-3F cells had no effect on CPT sensitivity, whereas it conferred a resistance to DC-3F/9-OH-E cells for which both isoforms are reduced by two- to three-fold as compared to DC-3F parental cells. Finally, downregulation of the human PRMT7 could also sensitize HeLa cells to CPT, suggesting that it could be used as a target to potentiate CPT derivatives.     

A 1H and 31P NMR study of cis-Pt(NH3)2[d(CpGpG)-N7(2),N7(3)]. The influence of a 5'-terminal cytosine, on the structure of the cis-Pt(NH3)2[d(GpG)-N7,N7] intrastrand cross-link

Proton NMR studies at 300 MHz and 500 MHz have been carried out on the trinucleoside bisphosphate d(CpGpG) and on cis-Pt(NH3)2[d(CpGpG)-N7(2),N7(3)] [abbreviated as d(CpGpGp) . cisPt]. For the Pt adduct, 13C and 31P NMR was also used for characterizing the oligonucleotide. d(CpGpG) appears to revert to a B-DNA-type single helix at lower temperatures. The relatively small concentration dependence of the proton chemical shifts, in comparison with shifts due to intramolecular stacking effects, indicates that the compound is essentially single-stranded. In d(CpGpGp) . cisPt, the first nucleoside, C(1), stacks well on top of the second, G(2), despite the N conformation of the G(2) sugar ring. The platinated GpG part in this trimer adopts largely the same structure as in cis-Pt(NH3)2[d(GpGpG)-N7(1),N7(2)] [den Hartog, J. H. J., et al. (1982) Nucleic Acids Res. 10, 4715-4730]. Main differences however, are changes in H8 chemical shifts and a 0.6-ppm downfield shift of the third nucleotide phosphorus, P(3), in d(CpGpGp) . cisPt with respect to P(2) in d(GpG) . cisPt. The latter shift change is likely to be induced by a structural alteration, caused by stacking of C(1) on top of G(2). Also, the large chemical shift differences between the two H8 protons in d(NpGpG) . cisPt fragments is discussed; the deviation from a mirror symmetry of the two guanine bases seems to be the main origin of this effect. The chemical shift changes, observed in the proton and phosphorus NMR chemical shift temperature and chemical shift pH profiles have been explained in terms of stack-destack equilibria changes.     

Inhibition of activity of purine nucleoside phosphorylase with N9- and N7-(beta-D-glucofuranuronosyl)purines and 8-substituted N9-(beta-D-ribofuranosyl)purines)]

In an effort to develop more potent inhibitors of purine nucleoside phosphorylase (PNP, EC 2.4.2.1) as immunosuppressive and anticancer chemotherapeutic agents, the affinity of the electrophoretically homogeneous enzyme from rabbit kidney for sixteen N9- and N7-beta-D-glucofuranuronosides and for C8-substituted beta-D-ribofuranosyl purines was determined. In all cases N7-substituted analogues of hypoxanthine and guanine were twice more active inhibitors of PNP than N9-substituted compounds. No effective inhibitors were found among the C8-substituted analogues, apparently due to the bulky C8-groups hindering rotation around the glycosidic bond and thus preventing optimal binding with the enzyme.     

N1-Substituted benzimidazole scaffold for farnesoid X receptor (FXR) agonists accompanying prominent selectivity against vitamin D receptor (VDR)

As a cellular bile acid sensor, farnesoid X receptor (FXR) participates in regulation of bile acid, lipid and glucose homeostasis, and liver protection. With respect to the bone metabolism, FXR positively regulates bone metabolism through both bone formation and resorption of the bone remodeling pathways. Some of FXR agonists possessing isoxazole moiety are undergoing clinical trials for the treatment of non-alcoholic steatohepatitis. To date, therefore, the activation of FXR leads to considerable interest in FXR as potential therapeutic targets. We have identified a series of nonsteroidal FXR agonists bearing N¹-methyl benzimidazole and isoxazole moieties that are bridged with aromatic derivatives. They showed affinity to FXR, but also weak affinity toward the vitamin D receptor (VDR) that involves regulation of calcium and phosphate homeostasis and is activated by bile acids. The deployment of FXR agonists without activity against VDR as off-target is therefore crucial in the development of FXR ligands. Our efforts focusing on increasing the agonist properties towards FXR led to the discovery of 19, which activates FXR at and below nanomolar levels (EC₅₀ = 26.5 ± 10.5 nM TR-FRET and 0.8 ± 0.2 nM luciferase, respectively) and functions as a FXR agonist: the affinity toward FXR over eight nuclear receptors, including VDR [IC₅₀ (VDR) / EC₅₀ (FXR) > 5000] and TGR5, effects FXR target genes, and activates bone morphogenetic protein-2-induced differentiation of mouse bone marrow-derived mesenchymal stem cell-like ST2 cells into osteoblast.     

Glycosidic Bond Conformation Preference Plays a Pivotal Role in Catalysis of RNA Pseudouridylation: A Combined Simulation and Structural Study

The most abundant chemical modification on RNA is isomerization of uridine (or pseudouridylation) catalyzed by pseudouridine synthases. The catalytic mechanism of this essential process remains largely speculative, partly due to lack of knowledge of the pre-reactive state that is important to the identification of reactive chemical moieties. In the present study, we showed, using orthogonal space random-walk free-energy simulation, that the pre-reactive states of uridine and its reactive derivative 5-fluorouridine, bound to a ribonucleoprotein particle pseudouridine synthase, strongly prefer the syn glycosidic bond conformation, while that of the nonreactive 5-bromouridine-containing substrate is largely populated in the anti conformation state. A high-resolution crystal structure of the 5-bromouridine-containing substrate bound to the ribonucleoprotein particle pseudouridine synthase and enzyme activity assay confirmed the anti nonreactive conformation and provided the molecular basis for its confinement. The observed preference for the syn pre-reactive state by the enzyme-bound uridine may help to distinguish among currently proposed mechanisms.     

Detection of human autoantibodies specific for 5'-m7GMP and m7G(5')ppp(5')N

An enzyme-linked immunosorbent assay was utilized for the detection of spontaneously occurring antibodies with apparent specificities for m7G, 5'-m7GMP, and m7G(5')ppp(5')C. From the sera of 50 patients containing anti-nuclear antibodies, 48 (96%) possessed antibodies which bound to one or more immobilized nucleoside-BSA antigens (A-, G-, C-, U-, and T-BSA). Additionally, 8 (16%) of these sera contained immunoglobulins that reacted with m7G-BSA antigen. In these latter sera, soluble competitors such as m7G, 5'm7GMP, and m7G(5')ppp(5')C (but not 5'-AMP, -GMP, -CMP, -UMP, and -TMP or m1G and m22G) effectively inhibited antibody-binding to immobilized m7G-BSA. These results indicate the existence of spontaneously occurring anti-m7G antibodies in autoimmune diseases which are distinct from anti-G antibody populations.     

Synthesis of N epsilon-(7-diethylaminocoumarin-3-carboxyl)- and N epsilon-(7-methoxycoumarin-3-carboxyl)-L-Fmoc lysine as tools for protease cleavage detection by fluorescence.

Two coumarin-labelled lysines were conveniently prepared as a fluorescence resonance energy transfer (FRET) pair for peptide cleavage detection. 7-Methoxy and 7-diethylamino coumarin-3-carboxylic acids were synthesized according to a modification of known procedures. Labelling at lysine was achieved in solution via the active N-hydroxysuccinimide ester of the carboxylic acid coumarin derivatives to give the target compounds in good yield. Subsequently, these modified amino acids were used in solid phase peptide synthesis (SPPS), and their potential utility in an extracellular matrix metalloprotease (MMP-1) activity measurement via FRET and/or quenching studies was demonstrated.     

Synthesis of N6-Substituted 9-[3-(Phosphonomethoxy)Propyl]Adenine Derivatives As Possible Antiviral Agents

A number of N ⁶ -substituted 9-[3-(phosphonomethoxy)propyl]adenine derivatives having hydroxymethyl at C-1′-position were prepared from the appropriate 6-chloroadenine derivative. The syntheses of the corresponding prodrugs of these compounds are also reported. These compounds showed poor activity against HCV in replicon assay.     

A Randomized Clinical Trial of an Inactivated Avian Influenza A (H7N7) Vaccine

Background

Concern for a pandemic caused by a newly emerged avian influenza A virus has led to clinical trials with candidate vaccines as preparation for such an event. Most trials have involved vaccines for influenza A (H5N1), A (H7N7) or A (H9N2).

Objective

To evaluate dosage-related safety and immunogenicity of an inactivated influenza A (H7N7) vaccine in humans.

Design

One hundred twenty-five healthy young adults were randomized to receive two doses intramuscularly of placebo or 7.5, 15, 45 or 90 µg of HA of an inactivated subunit influenza A (H7N7) vaccine (25 per group), four weeks apart. Reactogenicity was evaluated closely for one week and for any adverse effect for six months after each dose. Serum hemagglutination-inhibiting and neutralizing antibody responses were determined four weeks after each dose and at six months.

Results

Reactogenicity evaluations indicated the vaccinations were well tolerated. Only one subject developed a ≥4-fold serum hemagglutination-inhibition (HAI) antibody response and a final titer of ≥1∶40 four weeks after dose two and only five subjects developed a neutralizing antibody rise and a final titer of ≥1∶40 in tests performed at a central laboratory. Four of the five were given the 45 or 90 µg HA dosage. A more sensitive HAI assay at the study site revealed a dose-response with increasing HA dosage but only 36% in the 90 µg HA group developed a ≥4-fold rise in antibody in this test and only one of these achieved a titer of ≥1∶32.

Conclusion

This inactivated subunit influenza A (H7N7) vaccine was safe but poorly immunogenic in humans.

Trials Registration

ClinicalTrials.gov {"type":"clinical-trial","attrs":{"text":"NCT00546585","term_id":"NCT00546585"}}NCT00546585