Transition metal saccharide chemistry and biology: syntheses, characterization, solution stability and putative bio-relevant studies of iron-saccharide complexes

A number of Fe(III) complexes of saccharides and their derivatives, and those of ascorbic acid were synthesized, and characterized by a variety of analytical, spectral (FT-IR, UV-Vis, EPR, Mossbauer and EXAFS), magnetic and electrochemical techniques. Results obtained from various methods have shown good correlations. Data obtained from EPR, magnetic susceptibility and EXAFS techniques could be fitted well with the mono-, di- and trinuclear nature of the complexes. The solution stability of these complexes has been established using UV-Vis absorption and cyclic voltammetric techniques as a function of pH of the solution. Mixed valent, Fe(II,III) ascorbate complexes have also been synthesized and characterized. Reductive release of Fe(II) from the complexes using sodium dithionite has been addressed. In vitro absorption of Fe(III)-glucose complex has been studied using everted sacs of rat intestines and the results have been compared with that of simple ferric chloride. Fe(III)-saccharide complexes have shown regular protein synthesis even in hemin-deficient rabbit reticulocyte lysate indicating that these complexes play a role that is equivalent to that played by hemin in order to restore the normal synthesis of protein. These complexes have exhibited enhanced DNA cleavage properties in the presence of hydrogen peroxide with pUC-18 DNA plasmid

eIF2 independently binds two distinct eIF2b subcomplexes that catalyze and regulate guanine-nucleotide exchange

eIF2B is a heteropentameric guanine-nucleotide exchange factor essential for protein synthesis initiation in eukaryotes. Its activity is inhibited in response to starvation or stress by phosphorylation of the α subunit of its substrate, translation initiation factor eIF2, resulting in reduced rates of translation and cell growth. We have used an in vitro nucleotide-exchange assay to show that wild-type yeast eIF2B is inhibited by phosphorylated eIF2 [eIF2(αP)] and to characterize eIF2B regulatory mutations that render translation initiation insensitive to eIF2 phosphorylation in vivo. Unlike wild-type eIF2B, eIF2B complexes with mutated GCN3 or GCD7 subunits efficiently catalyzed GDP exchange using eIF2(αP) as a substrate. Using an affinity-binding assay, we show that an eIF2B subcomplex of the GCN3, GCD7, and GCD2 subunits binds to eIF2 and has a higher affinity for eIF2(αP), but it lacks nucleotide-exchange activity. In contrast, the GCD1 and GCD6 subunits form an eIF2B subcomplex that binds equally to eIF2 and eIF2(αP). Remarkably, this second subcomplex has higher nucleotide-exchange activity than wild-type eIF2B that is not inhibited by eIF2(αP). The identification of regulatory and catalytic eIF2B subcomplexes leads us to propose that binding of eIF2(αP) to the regulatory subcomplex prevents a productive interaction with the catalytic subcomplex, thereby inhibiting nucleotide exchange

Phosphorylation of serine 51 in initiation factor 2α (eIF2α) promotes complex formation between eIF2α(P) and eIF2B and causes inhibition in the guanine nucleotide exchange activity of eIF2B

Phosphorylation of serine 51 residue on the α-subunit of eukaryotic initiation factor 2 (eIF2α) inhibits the guanine nucleotide exchange (GNE) activity of eIF2B, presumably, by forming a tight complex with eIF2B. Inhibition of the GNE activity of eIF2B leads to impairment in eIF2 recycling and protein synthesis. We have partially purified the wild-type (wt) and mutants of eIF2α in which the serine 51 residue was replaced with alanine (51A mutant) or aspartic acid (51D mutant) in the baculovirus system. Analysis of these mutants has provided novel insight into the role of 51 serine in the interaction between eIF2 and eIF2B. Neither mutant was phosphorylated in vitro. Both mutants decreased eIF2α phosphorylation occurring in hemin and poly(IC)-treated reticulocyte lysates due to the activation of double-stranded RNA-dependent protein kinase (PKR). However, addition of 51D, but not 51A mutant eIF2α protein promoted inhibition of the GNE activity of eIF2B in hemin-supplemented rabbit reticulocyte lysates in which relatively little or no endogenous eIF2α phosphorylation occurred. The 51D mutant enhanced the inhibition in GNE activity of eIF2B that occurred in hemin and poly(IC)-treated reticulocyte lysates where PKR is active. Our results show that the increased interaction between eIF2 and eIF2B protein, occurring in reticulocyte lysates due to increased eIF2α phosphorylation, is decreased significantly by the addition of mutant 51A protein but not 51D. Consistent with the idea that mutant 51D protein behaves like a phosphorylated eIF2α, addition of this partially purified recombinant subunit, but not 51A or wt eIF2α, increases the interaction between eIF2 and 2B proteins in actively translating hemin-supplemented lysates. These findings support the idea that phosphorylation of the serine 51 residue in eIF2α promotes complex formation between eIF2α(P) and eIF2B and thereby inhibits the GNE activity of eIF2B

Stress-induced apoptosis in Spodoptera frugiperda (Sƒ9) cells: baculovirus p35 mitigates eIF2α phosphorylation

Spodoptera frugiperda (Sƒ9) ovarian cells, natural hosts for baculovirus, are good model systems to study apoptosis and also heterologous gene expression. We report that uninfected Sƒ9 cells readily undergo apoptosis and show increased phosphorylation of the a subunit of eukaryotic initiation factor 2 (eIF2α) in the presence of agents such as UVB light, etoposide, high concentrations of cycloheximide, and EGTA. In contrast, tunicamycin, A23187, and low concentrations of cycloheximide promoted eIF2α phosphorylation in Sƒ9 cells but without apoptosis. These findings therefore suggest that increased eIF2α phosphorylation does not always necessarily lead to apoptosis, but it is a characteristic hallmark of stressed cells and also of cells undergoing apoptosis. Cell death induced by the above agents was abrogated by infection of Sƒ9 cells with wild-type (wt) AcNPV. In contrast, Sƒ9 cells when infected with vAcδ35, a virus carrying deletion of the antiapoptotic p35 gene, showed increased apoptosis and enhanced eIF2α phosphorylation. Further, a recombinant wt virus vAcS51D expressing human S51D, a phosphomimetic form of eIF2α, induced apoptosis in UVB pretreated Sƒ9 cells. However, infection with vAcS51A expressing a nonphosphorylatable form (S51A) of human eIF2α partially reduced apoptosis. Consistent with these findings, it has been observed here that caspase activation has led to increased eIF2α phosphorylation, while caspase inhibition by z-VAD-fmk reduced eIF2α phosphorylation selectively in cells exposed to proapoptotic agents. These findings therefore suggest that the stress signaling pathway determines apoptosis, and caspase activation is a prerequisite for increased eIF2α phosphorylation in Sƒ9 cells undergoing apoptosis. The findings also reinforce the conclusion for the first time that the "pancaspase inhibitor" baculovirus p35 mitigates eIF2α phosphorylation

Serine 48 in initiation factor 2α (eIF2α) is required for high-affinity interaction between eIF2α(p) and eIF2b

Phosphorylation of the serine 51 residue in the α-subunit of translational initiation factor 2 in eukaryotes (eIF2α) impairs protein synthesis presumably by sequestering eIF2B, a rate-limiting pentameric guanine nucleotide exchange protein which catalyzes the exchange of GTP for GDP in the eIF2-GDP binary complex. To further understand the importance of eIF2α phosphorylation in the interaction between eIF2α(P) and eIF2B proteins and thereby the regulation of eIF2B activity, we expressed the wild type (wt) and a mutant eIF2α in which the serine 48 residue was replaced with alanine (48A mutant) in the baculovirus system. The findings reveal that the expression of both of these recombinant subunits was very efficient (15-20% of the total protein) and both proteins were recognized by an eIF2α monoclonal antibody and were phosphorylated to the same extent by reticulocyte eIF2α kinases. However, partially purified recombinant subunits (wt or 48A mutant) were not phosphorylated as efficiently as the eIF2 subunit present in the purified reticulocyte trimeric eIF2 complex and were also found to inhibit the phosphorylation of eIF2a of the trimeric complex. Furthermore, the extents of inhibition of eIF2B activity and formation of the eIF2α(P)-eIF2B complex that occurs due to eIF2α phosphorylation in poly(IC)-treated rabbit reticulocyte lysates were decreased significantly in the presence of insect cell extracts expressing the 48A mutant eIF2α compared to those for wt. These findings support the hypothesis that the serine 48 residue is required for high-affinity interaction between eIF2α(P) and eIF2B