Novel mechanisms of eIF2B action and regulation by eIF2α phosphorylation.

Eukaryotic translation initiation factor 2 (eIF2) is a heterotrimeric GTPase, which plays a critical role in protein synthesis regulation. eIF2-GTP binds Met-tRNAi to form the eIF2-GTP•Met-tRNAi ternary complex (TC), which is recruited to the 40S ribosomal subunit. Following GTP hydrolysis, eIF2-GDP is recycled back to TC by its guanine nucleotide exchange factor (GEF), eIF2B. Phosphorylation of the eIF2α subunit in response to various cellular stresses converts eIF2 into a competitive inhibitor of eIF2B, which triggers the integrated stress response (ISR). Dysregulation of eIF2B activity is associated with a number of pathologies, including neurodegenerative diseases, metabolic disorders, and cancer. However, despite decades of research, the underlying molecular mechanisms of eIF2B action and regulation remain unknown. Here we employ a combination of NMR, fluorescence spectroscopy, site-directed mutagenesis, and thermodynamics to elucidate the mechanisms of eIF2B action and its regulation by phosphorylation of the substrate eIF2. We present: (i) a novel mechanism for the inhibition of eIF2B activity, whereby eIF2α phosphorylation destabilizes an autoregulatory intramolecular interaction within eIF2α; and (ii) the first structural model for the complex of eIF2B with its substrate, eIF2-GDP, reaction intermediates, apo-eIF2 and eIF2-GTP, and product, TC, with direct implications for the eIF2B catalytic mechanism.

Human eIF5 and eIF1A Compete for Binding to eIF5B.

Eukaryotic translation initiation is a multistep process requiring a number of eukaryotic translation initiation factors (eIFs). Two GTPases play key roles in the process. eIF2 brings the initiator Met-tRNAi to the preinitiation complex (PIC). Upon start codon selection and GTP hydrolysis promoted by the GTPase-activating protein (GAP) eIF5, eIF2-GDP is displaced from Met-tRNAi by eIF5B-GTP and is released in complex with eIF5. eIF5B promotes ribosomal subunit joining, with the help of eIF1A. Upon subunit joining, eIF5B hydrolyzes GTP and is released together with eIF1A. We found that human eIF5 interacts with eIF5B and may help recruit eIF5B to the PIC. An eIF5B-binding motif was identified at the C-terminus of eIF5, similar to that found in eIF1A. Indeed, eIF5 competes with eIF1A for binding and has an ∼100-fold higher affinity for eIF5B. Because eIF5 is the GAP of eIF2, the newly discovered interaction offers a possible mechanism for coordination between the two steps in translation initiation controlled by GTPases: start codon selection and ribosomal subunit joining. Our results indicate that in humans, eIF5B displacing eIF2 from Met-tRNAi upon subunit joining may be coupled to eIF1A displacing eIF5 from eIF5B, allowing the eIF5:eIF2-GDP complex to leave the ribosome.

eIF2B Mechanisms of Action and Regulation: A Thermodynamic View.

Eukaryotic translation initiation factor 2B (eIF2B) is the guanine nucleotide exchange factor of the GTPase eIF2, which brings the initiator Met-tRNAi to the ribosome in the form of the eIF2-GTP·Met-tRNAi ternary complex (TC). The activity of eIF2B is inhibited by phosphorylation of its substrate eIF2 by several stress-induced kinases, which triggers the integrated stress response (ISR). The ISR plays a central role in maintaining homeostasis in the cell under various stress conditions, and its dysregulation is a causative factor in the pathology of a number of neurodegenerative disorders. Over the past three decades, virtually every aspect of eIF2B function has been the subject of uncertainty or controversy: from the catalytic mechanism of nucleotide exchange, to whether eIF2B only catalyzes nucleotide exchange on eIF2 or also promotes binding of Met-tRNAi to eIF2-GTP to form the TC. Here, we provide the first complete thermodynamic analysis of the process of recycling of eIF2-GDP to the TC. The available evidence leads to the conclusion that eIF2 is channeled from the ribosome (as an eIF5·eIF2-GDP complex) to eIF2B, converted by eIF2B to the TC, which is then channeled back to eIF5 and the ribosome. The system has evolved to be regulated by multiple factors, including post-translational modifications of eIF2, eIF2B, and eIF5, as well as directly by the energy balance in the cell, through the GTP:GDP ratio.

eIF1A/eIF5B interaction network and its functions in translation initiation complex assembly and remodeling.

Eukaryotic translation initiation is a highly regulated process involving multiple steps, from 43S pre-initiation complex (PIC) assembly, to ribosomal subunit joining. Subunit joining is controlled by the G-protein eukaryotic translation initiation factor 5B (eIF5B). Another protein, eIF1A, is involved in virtually all steps, including subunit joining. The intrinsically disordered eIF1A C-terminal tail (eIF1A-CTT) binds to eIF5B Domain-4 (eIF5B-D4). The ribosomal complex undergoes conformational rearrangements at every step of translation initiation; however, the underlying molecular mechanisms are poorly understood. Here we report three novel interactions involving eIF5B and eIF1A: (i) a second binding interface between eIF5B and eIF1A; (ii) a dynamic intramolecular interaction in eIF1A between the folded domain and eIF1A-CTT; and (iii) an intramolecular interaction between eIF5B-D3 and -D4. The intramolecular interactions within eIF1A and eIF5B interfere with one or both eIF5B/eIF1A contact interfaces, but are disrupted on the ribosome at different stages of translation initiation. Therefore, our results indicate that the interactions between eIF1A and eIF5B are being continuously rearranged during translation initiation. We present a model how the dynamic eIF1A/eIF5B interaction network can promote remodeling of the translation initiation complexes, and the roles in the process played by intrinsically disordered protein segments.

Effects of polyallylamine-coated nanoparticles on the optical and photochemical properties of rose bengal.

BACKGROUND: Inasmuch as optical and photochemical properties of a photosensitizer can be modified upon association with the nanoparticle (NP), we wondered whether the effectiveness of phototherapeutic rose bengal (RB) was affected upon tethering to the sodium lanthanide fluoride NP with an outer polyallylamine (PAH) coat. METHODS: RB molecules were electrostatically bound to the NaYF 4 :Gd 3+ :Nd 3+ NPs with inner silica and outer PAH coats. The products were analyzed for their size, shape and zeta potential using transmission electron microscopy and dynamic light scattering instrument. Ultraviolet-visible absorption spectrometry and fluorescence spectrometry were used to examine the spectral properties. Photodynamic effect in terms of singlet oxygen generation was quantitatively determined using the indicator 1,3-diphenylisobenzofuran (DPBF). Photocytotoxicity mediated by NP-bound RB was tested using A549 cells (Student's t test was used for statistical evaluation). RESULTS: NP-bound RB had the major absorbance peak at 561 nm, in comparison with 549 nm for free RB, accompanied with a significant decrease in absorptivity. The molar extinction coefficient becomes 36 000 M -1 cm -1 , only ~35% of that for free RB. Fluorescence spectral analyses showed a paradoxical decrease in the emission with higher NP concentrations even at very low dilutions. Most importantly, the association of RB with these NPs drastically increased its singlet oxygen production upon irradiation. The interaction of RB with PAH coat could partly account for this enhancement, given our finding that PAH in solution also caused a drastic rise in DPBF reactivity by free RB. These NPs exhibited strong photocytotoxic effects, and their promise in photodynamic therapy was addressed. CONCLUSION: Our findings provide evidence that the PAH coat plays a key role in enhanced biological activities of RB delivered via NPs, including the increase in singlet oxygen production and photocytotoxic effects.