R2 Subunit Research Articles

DFT calculations for intermediate and active states of the diiron center with a tryptophan or tyrosine radical in Escherichia coli ribonucleotide reductase.

Class Ia ribonucleotide reductase subunit R2 contains a diiron active site. In this paper, active-site models for the intermediate X-Trp48(•+) and X-Tyr122(•), the active Fe(III)Fe(III)-Tyr122(•), and the met Fe(III)Fe(III) states of Escherichia coli R2 are studied, using broken-symmetry density functional theory incorporated with the conductor-like screening solvation model. Different structural isomers and different protonation states have been explored. Calculated geometric, energetic, Mössbauer, hyperfine, and redox properties are compared with available experimental data. Feasible detailed structures of these intermediate and active states are proposed. Asp84 and Trp48 are most likely the main contributing residues to the result that the transient Fe(IV)Fe(IV) state is not observed in wild-type class Ia E. coli R2. Asp84 is proposed to serve as a proton-transfer conduit between the diiron cluster and Tyr122 in both the tyrosine radical activation pathway and the first steps of the catalytic proton-coupled electron-transfer pathway. Proton-coupled and simple redox potential calculations show that the kinetic control of proton transfer to Tyr122(•) plays a critical role in preventing reduction from the active Fe(III)Fe(III)-Tyr122(•) state to the met state, which is potentially the reason why Tyr122(•) in the active state can be stable over a very long period.

P53R2 Inhibits the Proliferation of Human Cancer Cells in Association with Cell-Cycle Arrest

Deregulation of the expression of p53R2, a p53-inducible homologue of the R2 subunit of ribonucleotide reductase, has been found in various human cancer tissues; however, the roles p53R2 plays in cancer progression and malignancy remain controversial. In the present study, we examined changes in gene expression profiles associated with p53R2 in cancer cells, using the analysis of cDNA microarray. Gene set enrichment analysis identified that the gene set regulating cell-cycle progression was significantly enriched in p53R2-silencing human oropharyngeal carcinoma KB cells. Attenuation of p53R2 expression significantly reduced p21 expression and moderately increased cyclin D1 expression in both wild-type p53 cancer cells (KB and MCF-7) and mutant p53 cancer cells (PC3 and MDA-MB-231). Conversely, overexpression of p53R2-GFP resulted in an increase in the expression of p21 and decrease in the expression of cyclin D1, which correlated with reduced cell population in S-phase in vitro and suppressed growth in vivo. Furthermore, the MAP/ERK kinase inhibitor PD98059 partially abolished modulation of p21 and cyclin D1 expression by p53R2. Moreover, under the conditions of nonstress and adriamycin-induced genotoxic stress, attenuation of p53R2 in KB cells significantly increased phosphorylated H2AX, which indicates that attenuation of p53R2 may enhance DNA damage induced by adriamycin. Overall, our study shows that p53R2 may suppress cancer cell proliferation partially by upregulation of p21 and downregulation of cyclin D1; p53R2 plays critical roles not only in DNA damage repair but also in proliferation of cancer cells.

GABA regulates corticotropin releasing hormone levels in the paraventricular nucleus of the hypothalamus in newborn mice

The paraventricular nucleus of the hypothalamus (PVN) is a major regulator of stress responses via release of corticotropin releasing hormone (CRH) to the pituitary gland. Dysregulation of the hypothalamic-pituitary-adrenal (HPA) axis is characteristic of individuals with major depressive disorder (MDD). Postmortem data from individuals diagnosed with MDD show increased levels of CRH mRNA and CRH immunoreactive neurons in the PVN. In the current study, an immunohistochemical (IHC) analysis revealed increased levels of CRH in the PVN of newborn mice lacking functional GABA B receptors. There was no difference in the total number of CRH immunoreactive cells. By contrast, there was a significant increase in the amount of CRH immunoreactivity per cell. Interestingly, this increase in CRH levels in the GABA B receptor R1 subunit knockout was limited to the rostral PVN. While GABAergic regulation of the HPA axis has been previously reported in adult animals, this study provides evidence of region-specific GABA modulation of immunoreactive CRH in newborns.

Mössbauer properties of the diferric cluster and the differential iron(ii)-binding affinity of the iron sites in protein R2 of class Ia Escherichia coli ribonucleotide reductase: a DFT/electrostatics study

The R2 subunit of class-Ia ribonucleotide reductase (RNR) from Escherichia coli (E. coli) contains a diiron active site. Starting from the apo-protein and Fe(II) in solution at low Fe(II)/apoR2 ratios, mononuclear Fe(II) binding is observed indicating possible different Fe(II) binding affinities for the two alternative sites. Further, based on their Mössbauer spectroscopy and two-iron-isotope reaction experiments, Bollinger et al. (J. Am. Chem. Soc., 1997, 119, 5976-5977) proposed that the site Fe1, which bonds to Asp84, should be associated with the higher observed (57)Fe Mössbauer quadrupole splitting (2.41 mm s(-1)) and lower isomer shift (0.45 mm s(-1)) in the Fe(III)Fe(III) state, site Fe2, which is further from Tyr122, should have a greater affinity for Fe(II) binding than site Fe1, and Fe(IV) in the intermediate X state should reside at site Fe2. In this paper, using density functional theory (DFT) incorporated with the conductor-like screening (COSMO) solvation model and with the finite-difference Poisson-Boltzmann self-consistent reaction field (PB-SCRF) methodologies, we have demonstrated that the observed large quadrupole splitting for the diferric state R2 does come from site Fe1(III) and it is mainly caused by the binding position of the carboxylate group of the Asp84 sidechain. Further, a series of active site clusters with mononuclear Fe(II) binding at either site Fe1 or Fe2 have been studied, which show that with a single dielectric medium outside the active site quantum region, there is no energetic preference for Fe(II) binding at one site over another. However, when including the explicit extended protein environment in the PB-SCRF model, the reaction field favors the Fe(II) binding at site Fe2 rather than at site Fe1 by ~9 kcal mol(-1). Therefore our calculations support the proposal of the previous Mössbauer spectroscopy and two-iron-isotope reaction experiments by Bollinger et al.

Metabotropic γ‐aminobutyric acid (GABAB) receptors modulate feeding behavior in the calcisponge Leucandra aspera

Here, we report the presence of the γ-aminobutyric acid (GABA)-ergic system in the calcisponge Leucandra aspera and examine the cellular localization of the components of this system, including GABA-like receptors using immunofluorescence and confocal microscopy. Furthermore, we demonstrate for the first time that GABA plays a functional role as a messenger in regulating sponge-feeding behavior. We found that both GABA(B) R1 and R2 subunits are present in the choanocytes of sponges as well as in the eso- and endopinacocytes. The functional role of GABA in the feeding behavior of this sponge was tested. The involvement of GABA receptors in the endocytic processes in L. aspera was demonstrated with dextran conjugated to Texas Red as a marker for material ingestion and by treating isolated sponge cells with a GABA(B) receptor agonist and an antagonist. The amount of dextran that was ingested increased in dissociated sponge cells when the GABA(B) receptor agonist baclofen was used, and this stimulatory effect was prevented by treatment with the GABA(B) receptor antagonist phaclofen. The baclofen effect on uptake was blocked by treatment with pertussis toxin, thus indicating a role for G proteins in modulating feeding behavior in L. aspera. Moreover, we found evidence that GABA receptors are involved in the consumption of dissolved organic matter by sponge cells. These findings suggest that GABA receptors and their functional role are highly conservative traits in the animal kingdom prenervous system evolution.

The ribonucleotide reductase R1 subunits of herpes simplex virus types 1 and 2 protect cells against TNFα- and FasL-induced apoptosis by interacting with caspase-8

We previously reported that HSV-2 R1, the R1 subunit (ICP10; UL39) of herpes simplex virus type-2 ribonucleotide reductase, protects cells against apoptosis induced by the death receptor (DR) ligands tumor necrosis factor-alpha- (TNFα) and Fas ligand (FasL) by interrupting DR-mediated signaling at, or upstream of, caspase-8 activation. Further investigation of the molecular mechanism underlying HSV-2 R1 protection showed that extracellular-regulated kinase 1/2 (ERK1/2), phosphatidylinositol 3-kinase (PI3-K)/Akt, NF-κB and JNK survival pathways do not play a major role in this antiapoptotic function. Interaction studies revealed that HSV-2 R1 interacted constitutively with caspase-8. The HSV-2 R1 deletion mutant R1(1-834)-GFP and Epstein-Barr virus (EBV) R1, which did not protect against apoptosis induced by DR ligands, did not interact with caspase-8, indicating that interaction is required for protection. HSV-2 R1 impaired caspase-8 activation induced by caspase-8 over-expression, suggesting that interaction between the two proteins prevents caspase-8 dimerization/activation. HSV-2 R1 bound to caspase-8 directly through its prodomain but did not interact with either its caspase domain or Fas-associated death domain protein (FADD). Interaction between HSV-2 R1 and caspase-8 disrupted FADD-caspase-8 binding. We further demonstrated that individually expressed HSV-1 R1 (ICP6) shares, with HSV-2 R1, the ability to bind caspase-8 and to protect cells against DR-induced apoptosis. Finally, as the long-lived Fas protein remained stable during the early period of infection, experiments with the HSV-1 UL39 deletion mutant ICP6∆ showed that HSV-1 R1 could be essential for the protection of HSV-1-infected cells against FasL.

Role of H-1 and H-2 Subunits of Soybean Seed Ferritin in Oxidative Deposition of Iron in Protein

Naturally occurring phytoferritin is a heteropolymer consisting of two different H-type subunits, H-1 and H-2. Prior to this study, however, the function of the two subunits in oxidative deposition of iron in ferritin was unknown. The data show that, upon aerobic addition of 48-200 Fe(2+)/shell to apoferritin, iron oxidation occurs only at the diiron ferroxidase center of recombinant H1 (rH-1). In addition to the diiron ferroxidase mechanism, such oxidation is catalyzed by the extension peptide (a specific domain found in phytoferritin) of rH-2, because the H-1 subunit is able to remove Fe(3+) from the center to the inner cavity better than the H-2 subunit. These findings support the idea that the H-1 and H-2 subunits play different roles in iron mineralization in protein. Interestingly, at medium iron loading (200 irons/shell), wild-type (WT) soybean seed ferritin (SSF) exhibits a stronger activity in catalyzing iron oxidation (1.10 ± 0.13 μm iron/subunit/s) than rH-1 (0.59 ± 0.07 μm iron/subunit/s) and rH-2 (0.48 ± 0.04 μm iron/subunit/s), demonstrating that a synergistic interaction exists between the H-1 and H-2 subunits in SSF during iron mineralization. Such synergistic interaction becomes considerably stronger at high iron loading (400 irons/shell) as indicated by the observation that the iron oxidation activity of WT SSF is ∼10 times larger than those of rH-1 and rH-2. This helps elucidate the widespread occurrence of heteropolymeric ferritins in plants.

Replication of the hepatitis B virus requires a calcium-dependent HBx-induced G1 phase arrest of hepatocytes

Chronic HBV infections cause hepatocellular carcinoma (HCC). Activities of the HBV HBx protein regulate HBV replication and may contribute to the development of HCC. We previously reported that HBx causes primary rat hepatocytes to exit G0 but stall in G1 phase of the cell cycle; entry into G1 stimulated HBV replication. We now report that the activity of the mitochondria permeability transition pore is required for HBx regulation of cell cycle proteins and HBV replication in primary rat hepatocytes, that progression from G0 to G1 stimulates HBV polymerase activity, and that HBV replication is facilitated by the HBx-induced G1 arrest. HBx stimulation of HBV replication was linked to elevation of the R2 subunit of ribonucleotide reductase. Our studies suggest that HBx uses mitochondrial-dependent calcium signaling to cause hepatocytes to exit G0 but stall in G1 and that this HBx activity alters the cellular environment and stimulates HBV replication.

Vaccinia Virus–Encoded Ribonucleotide Reductase Subunits Are Differentially Required for Replication and Pathogenesis

Ribonucleotide reductases (RRs) are evolutionarily-conserved enzymes that catalyze the rate-limiting step during dNTP synthesis in mammals. RR consists of both large (R1) and small (R2) subunits, which are both required for catalysis by the R12R22 heterotetrameric complex. Poxviruses also encode RR proteins, but while the Orthopoxviruses infecting humans [e.g. vaccinia (VACV), variola, cowpox, and monkeypox viruses] encode both R1 and R2 subunits, the vast majority of Chordopoxviruses encode only R2 subunits. Using plaque morphology, growth curve, and mouse model studies, we investigated the requirement of VACV R1 (I4) and R2 (F4) subunits for replication and pathogenesis using a panel of mutant viruses in which one or more viral RR genes had been inactivated. Surprisingly, VACV F4, but not I4, was required for efficient replication in culture and virulence in mice. The growth defects of VACV strains lacking F4 could be complemented by genes encoding other Chordopoxvirus R2 subunits, suggesting conservation of function between poxvirus R2 proteins. Expression of F4 proteins encoding a point mutation predicted to inactivate RR activity but still allow for interaction with R1 subunits, caused a dominant negative phenotype in growth experiments in the presence or absence of I4. Co-immunoprecipitation studies showed that F4 (as well as other Chordopoxvirus R2 subunits) form hybrid complexes with cellular R1 subunits. Mutant F4 proteins that are unable to interact with host R1 subunits failed to rescue the replication defect of strains lacking F4, suggesting that F4-host R1 complex formation is critical for VACV replication. Our results suggest that poxvirus R2 subunits form functional complexes with host R1 subunits to provide sufficient dNTPs for viral replication. Our results also suggest that R2-deficient poxviruses may be selective oncolytic agents and our bioinformatic analyses provide insights into how poxvirus nucleotide metabolism proteins may have influenced the base composition of these pathogens.

A phase II study of the antisense oligonucleotide GTI-2040 plus docetaxel and prednisone as first-line treatment in castration-resistant prostate cancer

GTI-2040 is a novel antisense oligonucleotide to the R2 subunit of ribonucleotide reductase. This phase II trial was conducted to determine the efficacy and tolerability of GTI-2040 when combined with docetaxel and prednisone for the treatment of patients with castration-resistant prostate cancer (CRPC). Chemo-naïve CRPC patients with adequate performance status and organ function were treated with docetaxel 75 mg/m(2) IV on day 1 plus GTI-2040 5 mg/kg/day by continuous intravenous infusion day 1-14 on a 21 day cycle, with prednisone 5 mg orally twice daily. The primary endpoint was PSA response rate. Pharmacokinetic studies of GTI-2040 and pharmacodynamic studies on peripheral blood mononuclear cells (PBMC) were also performed. Twenty-two patients in total (19 from this study and 3 from a prior phase I/II study at this institution) were treated at the recommended phase II dose. A confirmed PSA response was seen in 9/22 patients (41%). Of 16 patients with measurable disease, there was 1 partial response (PR) and 12 stable disease (SD) lasting 3.6 months (median), as best response. The most common toxicities were anemia, fatigue, lymphopenia, leucopenia and neutropenia. Grade 3+ toxicities included neutropenia, lymphopenia, leucopenia, fatigue, febrile neutropenia and hypophosphatemia. The PSA response rate of GTI-2040 in combination with docetaxel and prednisone just met the minimum phase II criteria for further enrollment. However, after evaluation of all the clinical data, further study of this dose and schedule of GTI-2040 in CRPC was not recommended.

Dopamine D1 but not D3 receptor is critical for spatial learning and related signaling in the hippocampus

Experimental evidence suggests the involvement of the brain dopaminergic system in learning and memory processes, although the associated molecular mechanism has yet to be fully characterized. Memory formation occurs via a number of signaling pathways associated with activation of many synaptic plasticity-related proteins, including the N-Methyl-d-aspartic acid (NMDA) receptor, Ca2+/calmodulin-dependent protein kinase II (CaMKII), mitogen-activated protein kinases (MAPKs) and the cAMP-response element binding protein (CREB). To evaluate the roles of dopamine D1 and D3 receptors in spatial learning and memory and underlying molecular events, we have used genetically modified mice carrying either the D1 or D3 receptor gene mutations to explore the intracellular signaling pathways using Morris water maze (MWM) tasks. We show that D1 receptor mutant mice do not acquire spatial memory and do not show hippocampal activation of extracellular signal-regulated kinase (ERK) compared to wild-type mice. D3 receptor mutant mice exhibit apparent normal learning abilities in the MWM test and normal activation of MAPK signaling. Furthermore, activation of the NMDA receptor R1 subunit (NR1), CaMKII and CREB in the hippocampus is also significantly lower in D1 receptor mutant mice compared to wild-type and D3 receptor mutant mice. These results suggest that dopamine D1 but not D3 receptor is critical for spatial learning. D1 receptor-mediated signaling, associated with activation of NR1, CaMKII, ERK and CREB, is highly involved in the encoding of spatial learning and memory.

ChemInform Abstract: Synthesis and Biological Activity of Cyclic Peptide Inhibitors of Ribonucleotide Reductase.

A series of lactam-bridged peptide inhibitors (2−6) of mammalian ribonucleotide reductase (mRR) has been designed and synthesized on the basis of the heptapeptide N-AcFTLDADF (1), corresponding to the C-terminus of the R2 subunit of mRR. Inhibition studies revealed a direct relation between ring size and activity, with peptide 5 being 2.5 times more potent than peptide 1.

Regulation of ribonucleotide reductase by Spd1 involves multiple mechanisms

The correct levels of deoxyribonucleotide triphosphates and their relative abundance are important to maintain genomic integrity. Ribonucleotide reductase (RNR) regulation is complex and multifaceted. RNR is regulated allosterically by two nucleotide-binding sites, by transcriptional control, and by small inhibitory proteins that associate with the R1 catalytic subunit. In addition, the subcellular localization of the R2 subunit is regulated through the cell cycle and in response to DNA damage. We show that the fission yeast small RNR inhibitor Spd1 is intrinsically disordered and regulates R2 nuclear import, as predicted by its relationship to Saccharomyces cerevisiae Dif1. We demonstrate that Spd1 can interact with both R1 and R2, and show that the major restraint of RNR in vivo by Spd1 is unrelated to R2 subcellular localization. Finally, we identify a new behavior for RNR complexes that potentially provides yet another mechanism to regulate dNTP synthesis via modulation of RNR complex architecture.

Expression, purification, crystallization and preliminary X-ray analysis of ORF60, the small subunit (R2) of ribonucleotide reductase from Kaposi's sarcoma-associated herpesvirus (KSHV).

Ribonucleotide reductase (RNR) is responsible for converting ribonucleotides to deoxyribonucleotides, which are the building blocks of DNA. The enzyme is present in all life forms as well as in some large DNA viruses such as herpesviruses. The alpha-herpesviruses and gamma-herpesviruses encode two class Ia RNR subunits, R1 and R2, while the beta-herpesvirus subfamily only encode an inactive R1 subunit. Here, the crystallization of the R2 subunit of RNR encoded by the ORF60 gene from the oncovirus Kaposi's sarcoma-associated gamma-herpesvirus (KSHV) is reported. These are the first crystals of a viral R2 subunit; the use of in situ proteolysis with chymotrypsin and the addition of hexamine cobalt(III) chloride that were necessary to obtain crystals are described. Optimization of the crystallization conditions yielded crystals that diffracted to 2.0 A resolution. The crystals belonged to space group P2(1), with unit-cell parameters a = 63.9, b = 71.2, c = 71.8 A, alpha = 90, beta = 106.7, gamma = 90 degrees. The data set collected was 95.3% complete, with an R(merge) of 9.6%. There are two molecules in the asymmetric unit, corresponding to a solvent content of 43.4%.

Roles for gamma-aminobutyric acid in the development of the paraventricular nucleus of the hypothalamus.

The development of the hypothalamic paraventricular nucleus (PVN) involves several factors that work together to establish a cell group that regulates neuroendocrine functions and behaviors. Several molecular markers were noted within the developing PVN, including estrogen receptors (ER), neuronal nitric oxide synthase (nNOS), and brain-derived neurotrophic factor (BDNF). By contrast, immunoreactive gamma-aminobutyric acid (GABA) was found in cells and fibers surrounding the PVN. Two animal models were used to test the hypothesis that GABA works through GABA(A) and GABA(B) receptors to influence the development of the PVN. Treatment with bicuculline to decrease GABA(A) receptor signaling from embryonic day (E) 10 to E17 resulted in fewer cells containing immunoreactive (ir) ERalpha in the region of the PVN vs. control. GABA(B)R1 receptor subunit knockout mice were used to examine the PVN at P0 without GABA(B) signaling. In female but not male GABA(B)R1 subunit knockout mice, the positions of cells containing ir ERalpha shifted from medial to lateral compared with wild-type controls, whereas the total number of ir ERalpha-containing cells was unchanged. In E17 knockout mice, ir nNOS cells and fibers were spread over a greater area. There was also a significant decrease in ir BDNF in the knockout mice in a region-dependent manner. Changes in cell position and protein expression subsequent to disruption of GABA signaling may be due, in part, to changes in nNOS and BDNF signaling. Based on the current study, the PVN can be added as another site where GABA exerts morphogenetic actions in development.

N‐methyl D‐aspartate (NMDA) receptors endow dendrites of gonadotropin releasing‐hormone (GnRH) neurons with additional active properties

Dendrites of GnRH neurons express tetrodotoxin‐sensitive voltage‐gated sodium channels and generate action potentials. Other dendrites exhibiting action potentials possess additional active properties such as NMDA‐mediated potentials. We tested the hypothesis that GnRH dendrites express R1 subunits of NMDA receptors and exhibit amplification of sub‐threshold, excitatory post‐synaptic potentials (EPSPs) via NMDA receptor activation. Immunochemistry indicated GnRH dendrites express R1 subunits. Stimulating living GnRH dendrites with simulated sub‐threshold EPSPs resulted in plateau potentials and individual action potentials. When GABAergic inputs were blocked with picrotoxin (100 μM), repetitive action potentials occurred. The NMDA receptor antagonist D‐2‐amino‐5‐phosphono‐valeric (50 μM) eliminated responses. In small diameter dendrites, NMDA receptors would have a pronounced impact. Activating simulated NMDA conductances (at 560 μm dendrite length) in our compartmental models of GnRH neurons resulted in repetitive action potentials. Activating proximal NMDA receptors (at 200 and 100 μm dendrite length) diminished the impact of NMDA receptors. Our findings indicate GnRH dendrites exhibit regionalized expression of the NMDA conductance thereby forming location‐dependent microdomains where estrogen‐sensitive GABAergic neurons can regulate local excitability.

Cancer Treatment with Gemcitabine Does Not Increase Fetal Hemoglobin Levels.

Abstract 5112 BackgroundPharmacological induction of fetal hemoglobin (Hb F) can benefit patients with beta-hemoglobinopathies. Hydroxyurea (HU) is the drug most commonly used to induce Hb F. HU is converted to a free radical scavenger in vivo and inhibits the R2 subunit of ribonucleotide reductase. When used in the treatment of myeloproliferative syndromes, HU has been described to increase Hb F in people without hemoglobinopathies (Alter et al, Blood 66(2) 373-5). Gemcitabine is a nucleoside analog chemotherapeutic that inhibits the large R1 subunit of ribonucleotide reductase. We hypothesized that like HU, gemcitabine treatment would cause Hb F percentage to rise. Though oncology patients are a distinct population, gemcitabine induction of Hb F during the treatment of cancer would suggest a potential role for gemcitabine in the management of beta-hemoglobinopathies such as sickle cell disease and beta-thalassemia. MethodsParticipants for this prospective observational cohort study were recruited from the Moores UCSD Cancer Center upon documentation of a cancer diagnosis and oncologist-initiated plans for treatment with single agent gemcitabine. Enrolled subjects were gemcitabine naïve and had not received any chemotherapy for the preceding 4 weeks. Serum Hb F was quantified by electrophoresis prior to each of the first 4 gemcitabine treatments. All other laboratory evaluation was standard-of-care. The primary objective was to assess changes in Hb F among cancer patients in response to their initial 3 treatments with gemcitabine. Enrollment was halted early given the absence of a substantial treatment effect among the first 6 subjects. ResultsSix subjects were eligible. Three were men. Five were Caucasian and one was an Asian/Pacific Islander. All subjects received gemcitabine monotherapy for the treatment of adenocarcinoma of the pancreas or breast at a weekly dose of 1000 mg/m2. Baseline Hb F range was 0 – 1.0%. Four of the six subjects (three women, one man; all Caucasian) experienced no change in Hb F from baseline to the final measurement. Two subjects experienced a 0.4% increase in Hb F from baseline to the final measurement 4 weeks later. Final Hb F range was 0 – 1.4%. ConclusionsWe found that gemcitabine monotherapy for the treatment of cancer did not induce Hb F in this small study cohort. Gemcitabine may lack this ability. Alternative explanations for the absence of clinically meaningful Hb F induction include the small population studied, differences between oncology patients and the general population, short duration of treatment, and intermittent dosing of gemcitabine. DisclosuresNo relevant conflicts of interest to declare.

The manganese/iron-carboxylate proteins: what is what, where are they, and what can the sequences tell us?

The manganese/iron-carboxylate proteins make up a recently discovered group of proteins that contain a heterodinuclear Mn/Fe redox cofactor. The chemical potential of the heterodinuclear metal site is just starting to be characterized, but available data suggest that it may have capabilities for similarly versatile chemistry as the extensively studied diiron-carboxylate cofactor. The presently identified members of the manganese/iron-carboxylate proteins are all sequence homologues of the radical-generating R2 subunit of class I ribonucleotide reductase, canonically a diiron protein. They are also commonly misannotated as such in databases. In spite of the sequence similarity, the manganese/iron-carboxylate proteins form at least two functionally distinct groups, radical-generating ribonucleotide reductase subunits and ligand-binding Mn/Fe proteins. Here, the presently available sequences for the manganese/iron-carboxylate proteins are gathered, grouped, and analyzed. The analysis provides sequence determinants that allow group identification of new sequences on the single-protein level. Key differences between the groups are mapped on the known representative structures, providing clues to the structural prerequisites for metal specificity, cofactor formation, and difference in function. The organisms that encode manganese/iron-carboxylate proteins are briefly discussed; their environmental preference suggests that the Mn/Fe heterodinuclear cofactor is preferred by extremophiles and pathogens with a particularly high relative presence in Archaea.

Targeted Delivery of Antisense Oligodeoxynucleotide by Transferrin Conjugated pH-Sensitive Lipopolyplex Nanoparticles: A Novel Oligonucleotide-Based Therapeutic Strategy in Acute Myeloid Leukemia

Therapeutic use of oligodeoxynucleotides (ODNs) that hybridize to and downregulate target mRNAs encoding proteins that contribute to malignant transformation has a sound rationale, but has had an overall limited clinical success in cancer due to insufficient intracellular delivery. Here we report a development of formulations capable of promoting targeted delivery and enhanced pharmacologic activity of ODNs in acute myeloid leukemia (AML) cell lines and patient primary cells. In this study, transferrin (Tf) conjugated pH-sensitive lipopolyplex nanoparticles (LPs) were prepared to deliver GTI-2040, an antisense ODN against the R2 subunit of ribonucleotide reductase that has been shown to contribute to chemoresistance in AML. LPs had an average particle size around 110 nm and a moderately positive zeta potential at approximately 10 mV. The ODN encapsulation efficiency of LPs was >90%. These nanoparticles could release ODNs at acidic endosomal pH and facilitate the cytoplasmic delivery of ODNs after endocytosis. In addition, Tf-mediated targeted delivery of GTI-2040 was achieved. R2 downregulation at both mRNA and protein levels was improved by 8-fold in Kasumi-1 cells and 2- to 20-fold in AML patient primary cells treated with GTI-2040-Tf-LPs, compared to free GTI-2040 treatment. Moreover, Tf-LPs were more effective than nontargeted LPs, with 10 to 100% improvement at various concentrations in Kasumi-1 cells and an average of 45% improvement at 3 microM concentration in AML patient primary cells. Treatment with 1 microM GTI-2040-Tf-LPs sensitized AML cells to the chemotherapy agent cytarabine, by decreasing its IC(50) value from 47.69 nM to 9.05 nM. This study suggests that the combination of pH sensitive LP formulation and Tf mediated targeting is a promising strategy for antisense ODN delivery in leukemia therapy.

A Phase I/II Study of GTI-2040 Plus Docetaxel as Second-Line Treatment in Advanced Non-small Cell Lung Cancer: A Study of the PMH Phase II Consortium

GTI-2040, an antisense oligonucleotide, targets the ribonucleotide reductase R2 subunit, critical for DNA synthesis. This study determined the recommended phase II dose (RP2D) of docetaxel plus GTI-2040, toxicity, and response rate in advanced non-small cell lung cancer (NSCLC). Advanced solid tumor patients, preferably with platinum-treated NSCLC, performance status 0 to 2, no symptomatic central nervous system metastases, adequate organ and bone marrow function, and >or=1 prior chemotherapy regimen were treated with escalating doses of GTI-2040 given by 14-day continuous intravenous infusion (CVI) plus docetaxel every 21 days. Twenty-nine patients were treated, (24 NSCLC, 3 hormone-refractory prostate cancer, 1 head and neck, and 1 small cell lung cancer). GTI-2040 5 mg/kg as CVI for 14 days plus docetaxel 75 mg/m(2) intravenously every 21days was determined as the RP2D. Dose-limiting toxicity was not seen. Two patients at RP2D developed grade 4/5 febrile neutropenia. One prostate specific antigen response was seen in phase I, but no objective tumor responses in the NSCLC patients. Median time to progression was 3.4 months, 3.2 months in the NSCLC patients treated at RP2D. Activity of the combination at RP2D, GTI-2040 5 mg/kg/d x 14 days by CVI plus docetaxel 75 mg/m(2) does not seem superior to docetaxel alone in previously treated NSCLC.