HCV IRES Captures an Actively Translating 80S Ribosome.

Translation initiation of hepatitis C virus (HCV) genomic RNA is induced by an internal ribosome entry site (IRES). Our cryoelectron microscopy (cryo-EM) analysis revealed that the HCV IRES binds to the solvent side of the 40S platform of the cap-dependently translating 80S ribosome. Furthermore, we obtained the cryo-EM structures of the HCV IRES capturing the 40S subunit of the IRES-dependently translating 80S ribosome. In the elucidated structures, the HCV IRES "body," consisting of domain III except for subdomain IIIb, binds to the 40S subunit, while the "long arm," consisting of domain II, remains flexible and does not impede the ongoing translation. Biochemical experiments revealed that the cap-dependently translating ribosome becomes a better substrate for the HCV IRES than the free ribosome. Therefore, the HCV IRES is likely to efficiently induce the translation initiation of its downstream mRNA with the captured translating ribosome as soon as the ongoing translation terminates.

Expression, purification, and crystallization of Schizosaccharomyces pombe eIF2B.

Tight control of protein synthesis is necessary for cells to respond and adapt to environmental changes rapidly. Eukaryotic translation initiation factor (eIF) 2B, the guanine nucleotide exchange factor for eIF2, is a key target of translation control at the initiation step. The nucleotide exchange activity of eIF2B is inhibited by the stress-induced phosphorylation of eIF2. As a result, the level of active GTP-bound eIF2 is lowered, and protein synthesis is attenuated. eIF2B is a large multi-subunit complex composed of five different subunits, and all five of the subunits are the gene products responsible for the neurodegenerative disease, leukoencephalopathy with vanishing white matter. However, the overall structure of eIF2B has remained unresolved, due to the difficulty in preparing a sufficient amount of the eIF2B complex. To overcome this problem, we established the recombinant expression and purification method for eIF2B from the fission yeast Schizosaccharomyces pombe. All five of the eIF2B subunits were co-expressed and reconstructed into the complex in Escherichia coli cells. The complex was successfully purified with a high yield. This recombinant eIF2B complex contains each subunit in an equimolar ratio, and the size exclusion chromatography analysis suggests it forms a heterodecamer, consistent with recent reports. This eIF2B increased protein synthesis in the reconstituted in vitro human translation system. In addition, disease-linked mutations led to subunit dissociation. Furthermore, we crystallized this functional recombinant eIF2B, and the crystals diffracted to 3.0 Å resolution.

Molecular evidence of the involvement of heat shock protein 90 in brassinosteroid signaling in Arabidopsis T87 cultured cells.

KEY MESSAGE: A closer association of HSP90s with brassinosteroid signaling is suggested by the brassinosteroid-triggered formation of an HSP90-containing macromolecular complex and the direct interaction between HSP90.3 and BES1. ABSTRACT: Heat shock protein 90 (HSP90) is a highly conserved molecular chaperone that is reportedly involved in the proper folding, stabilization, intracellular trafficking, maintenance and degradation of numerous proteins, as well as the facilitation of cellular signaling in various organisms including plants. Brassinosteroids (BRs), a class of unique steroidal hormones, play crucial roles in plant growth and development. The interaction between HSP90 proteins and BR action has been poorly understood. Here, we present molecular evidence suggesting that HSP90 proteins have a function(s) in BR signal transduction. First, blue native/sodium dodecyl sulfate-polyacrylamide gel electrophoresis linked immunoblotting demonstrated that a bioactive BR, brassinolide (BL), promotes the formation of some HSP90-containing macromolecular complexes with molecular weight more than 480 kDa in Arabidopsis T87 cultured cells. Second, HSP90.3, one of seven Arabidopsis HSP90 family proteins, was observed to interact in vitro with BRI1-EMS-SUPPRESSOR 1 (BES1), a transcription factor acting in BR signaling. Geldanamycin, an inhibitor of ATPase activity in HSP90, not only diminished HSP90.3 interaction with BES1 in vitro, but also suppressed BL-induced down-regulation of two BR biosynthesis genes, CONSTITUTIVE PHOTHOMORPHOGENESIS AND DWARFISM and DWARF4 in vivo. The results suggest the involvement of the HSP90/BES1 heterocomplexes in BR signaling-mediated feedback control in BR contents. Together, our results provide important clues to elucidate HSP90s' functions in the BR signaling pathway in Arabidopsis.

Glyceraldehyde-derived advanced glycation end-products having pyrrolopyridinium-based crosslinks.

Reducing sugars and reactive aldehydes, such as glyceraldehyde, non-enzymatically react with amino or guanidino groups of proteins to form advanced glycation end-products (AGEs) by the Maillard reaction that involves Schiff base formation followed by Amadori rearrangement. AGEs are found relatively in abundance in the human eye and to accumulate at a higher rate in diseases that impair vision such as cataract, diabetic retinopathy or age-related macular degeneration. We identified two novel AGEs of pyrrolopyridinium lysine dimer derived from glyceraldehyde, PPG1 and PPG2, in the Maillard reaction of N α-acetyl-l-lysine with glyceraldehyde under physiological conditions. Having fluorophores similar to that of vesperlysine A, which was isolated from the human lens, PPGs were found to act as photosensitizers producing singlet oxygen in response to blue light irradiation. Moreover, PPG2 interacts with receptor for AGE (RAGE) in vitro with a higher binding affinity than GLAP, a well-known ligand of the receptor. We also proposed a pathway to form PPGs and discussed how they would be formed in vitro. As glyceraldehyde-derived AGEs have been studied extensively in connection with various hyperglycemia-related diseases, further studies will be required to find PPGs in vivo such as in the lens or other tissues.

A novel crosslinked type of advanced glycation end-product derived from lactaldehyde.

Glycation of amino or guanidino groups of proteins with glucose and glucose-derived reactive aldehydes, such as α-hydroxyaldehydes, leads to accumulation of advanced glycation end-products (AGEs) in the body, resulting in diabetic complications and age-related pathology. Although molecular structures of glycolaldehyde- and glyceraldehyde-derived AGEs have been described in previous studies, little is known about lactaldehyde-derived AGEs of α-hydroxyaldehydes. Here, we report a novel crosslinked type of AGE, named as lactaldehyde-derived lysine dimer (LAK2), which is produced due to non-enzymatic glycation of N α-acetyl-L-lysine with lactaldehyde under physiological conditions. We have identified the molecular structure of LAK2 by extensive mass spectrometry and nuclear magnetic resonance analyses. Furthermore, we propose a reaction pathway to produce LAK2, in which it is formed through an intermediate common with the recently reported lactaldehyde-derived pyridinium-type lysine adduct (LAPL). Since lactaldehyde is known to be produced from L-threonine in a myeloperoxidase (MPO)-mediated reaction at sites of inflammation, LAK2 has the potential to be an oxidative stress marker of MPO-mediated reactions induced in inflammation.

Translation efficiency affects the sequence-independent +1 ribosomal frameshifting by polyamines.

Antizyme (AZ) interacts with ornithine decarboxylase, which catalyzes the first step of polyamine biosynthesis and recruits it to the proteasome for degradation. Synthesizing the functional AZ protein requires transition of the reading frame at the termination codon. This programmed +1 ribosomal frameshifting is induced by polyamines, but the molecular mechanism is still unknown. In this study, we explored the mechanism of polyamine-dependent +1 frameshifting using a human cell-free translation system. Unexpectedly, spermidine induced +1 frameshifting in the mutants replacing the termination codon at the shift site with a sense codon. Truncation experiments showed that +1 frameshifting occurred promiscuously in various positions of the AZ sequence. The probability of this sequence-independent +1 frameshifting increased in proportion to the length of the open reading frame. Furthermore, the +1 frameshifting was induced in some sequences other than the AZ gene in a polyamine-dependent manner. These findings suggest that polyamines have the potential to shift the reading frame in the +1 direction in any sequence. Finally, we showed that the probability of the sequence-independent +1 frameshifting by polyamines is likely inversely correlated with translation efficiency. Based on these results, we propose a model of the molecular mechanism for AZ +1 frameshifting.

Huntingtin Polyglutamine-Dependent Protein Aggregation in Reconstituted Cells.

One of the aims of synthetic biology is bottom-up construction of reconstituted human cells for medical uses. To that end, we generated giant unilamellar vesicles (GUVs) that contained a HeLa cell extract, which comprises a cell-free protein synthesis (CFPS) system. Then we expressed Huntingtin protein fragments that contained polyglutamine (polyQ) sequences (Htt-polyQ), a hallmark of Huntington's disease. That system produced polyQ-dependent protein aggregates, as previously demonstrated in living cells. We next simplified the system by generating GUVs that contained purified human factors, which reconstituted a CFPS system. Htt-polyQ fragments expressed in these GUVs also formed protein aggregates. Moreover, an N-terminal deletion mutant, which had failed to form protein aggregates in living cells, also failed to form protein aggregates in the reconstituted GUVs. Thus, the GUV systems that encapsulated a human CFPS system could serve as reconstituted cells for studying neurological diseases.

Dynamic interaction of poly(A)-binding protein with the ribosome.

Eukaryotic mRNA has a cap structure and a poly(A) tail at the 5' and 3' ends, respectively. The cap structure is recognized by eIF (eukaryotic translation initiation factor) 4 F, while the poly(A) tail is bound by poly(A)-binding protein (PABP). PABP has four RNA recognition motifs (RRM1-4), and RRM1-2 binds both the poly(A) tail and eIF4G component of eIF4F, resulting in enhancement of translation. Here, we show that PABP interacts with the 40S and 60S ribosomal subunits dynamically via RRM2-3 or RRM3-4. Using a reconstituted protein expression system, we demonstrate that wild-type PABP activates translation in a dose-dependent manner, while a PABP mutant that binds poly(A) RNA and eIF4G, but not the ribosome, fails to do so. From these results, functional significance of the interaction of PABP with the ribosome is discussed.

Cell-free analysis of polyQ-dependent protein aggregation and its inhibition by chaperone proteins.

Protein misfolding and aggregation is one of the major causes of neurodegenerative disorders such as Alzheimer's disease, Parkinson's disease and Huntington's disease. So far protein aggregation related to these diseases has been studied using animals, cultured cells or purified proteins. In this study, we show that a newly synthesized polyglutamine protein implicated in Huntington's disease forms large aggregates in HeLa cells, and successfully recapitulate the process of this aggregation using a translation-based system derived from HeLa cell extracts. When the cell-free translation system was pre-incubated with recombinant human cytosolic chaperonin CCT, or the Hsc70 chaperone system (Hsc70s: Hsc70, Hsp40, and Hsp110), aggregate formation was inhibited in a dose-dependent manner. In contrast, when these chaperone proteins were added in a post-translational manner, aggregation was not prevented. These data led us to suggest that chaperonin CCT and Hsc70s interact with nascent polyglutamine proteins co-translationally or immediately after their synthesis in a fashion that prevents intra- and intermolecular interactions of aggregation-prone polyglutamine proteins. We conclude that the in vitro approach described here can be usefully employed to analyze the mechanisms that provoke polyglutamine-driven protein aggregation and to screen for molecules to prevent it.

Heat shock protein 90 acts in brassinosteroid signaling through interaction with BES1/BZR1 transcription factor.

Brassinosteroids (BRs), a class of phytohormones, control various physiological and developmental processes in plants. Two highly homologous transcription factors, brassinosteroid insensitive 1-EMS-SUPRESSOR 1 (BES1) and brassinazole resistant 1 (BZR1), act downstream of BR signaling to control several thousands of putative target genes. We reported previously that BES1 forms a complex with a molecular chaperone: heat shock protein 90 (HSP90). This study demonstrates that the amino-terminal and central parts of BES1 are responsible for its physical interaction with HSP90.3 in vitro. Additionally, we present evidence that BZR1 is a novel HSP90 partner aside from two BR signaling components previously identified as its clients: BES1 and brassinosteroid insensitive 2 (BIN2). Furthermore, geldanamycin, an inhibitor of ATPase activity in HSP90, caused BES1 hyperphosphorylation and disrupted the expression of BR-responsive genes. Considered together, our results imply that HSP90 takes a part in BR-mediated gene expression through complex formation with two major transcription factors.

Does brassinosteroid function require chromatin remodeling?

The phytohormone brassinosteroid (BR) is crucial for plant growth and development. Although genetic and molecular approaches have improved understanding of the cellular BR signaling pathway, we still do not have sufficient knowledge about the function of BR. Therefore, proteomic analysis was used to elucidate BR signaling and gene expression in the nuclei of suspended Arabidopsis cells treated with brassinolide, a bioactive BR, or brassinazole, a BR biosynthesis inhibitor. Interestingly, chromatin remodeling-related proteins, the abundance of which was altered in response to cellular BR levels, were identified. This suggested that BR-induced gene expression is regulated not only by transcription factors directly binding to cis-elements, but also by chromatin remodeling in response to BR signaling. In this addendum, we summarize the functions of our identified nuclear proteins in chromatin remodeling and discuss the need for chromatin remodeling regulated by BR signal transduction for expression of BR-induced genes.

Characterization of brassinosteroid-regulated proteins in a nuclear-enriched fraction of Arabidopsis suspension-cultured cells.

To identify nuclear proteins involved in the brassinosteroid (BR) signaling pathway, a targeted proteomic approach was applied to Arabidopsis thaliana suspension-cultured T87 cells. Cell growth was promoted by 0.1 µM brassinolide (BL) and inhibited by 5 µM brassinazole (Brz). Analysis of BR-regulated proteins in nuclear-enriched fractions was carried out using two-dimensional polyacrylamide gel electrophoresis with a special fluorescent dye. Proteins of interest were identified by correlating normalized spot volume of proteins on the gels with cellular BR level (Brz-treated cells, extremely low level of BRs; control cells, normal level of BRs; BL-treated cells, high level of BRs). A number of BR-responsive proteins were detected and some of these proteins were identified by nano-liquid chromatography-tandem mass spectrometry after enzymatic digestion. Fluctuations in eight identified nuclear proteins in BL-treated cells were investigated in the first 12 h of treatment. Three nuclear BR-responsive proteins, Nucleosome Assembly Protein (NAP) 1;1, Band 7 Family Protein, and Vernalization Independence 3, significantly decreased during this time. Meanwhile, NAP1;2, S-Adenosylmethionine Synthetase 2, and 60S Ribosomal Protein L14 increased markedly over time. Since some of these proteins are reportedly related to chromosome remodeling, cell growth induced by BL may involve chromatin remodeling. Interestingly, NAP1;2 was found to be post-translationally modified in response to cellular BR levels. Our study of quantitative protein changes in the nucleus provides valuable insight into BR-induced cellular and physiological responses.

Transcription of DWARF4 plays a crucial role in auxin-regulated root elongation in addition to brassinosteroid homeostasis in Arabidopsis thaliana.

The expression of DWARF4 (DWF4), which encodes a C-22 hydroxylase, is crucial for brassinosteroid (BR) biosynthesis and for the feedback control of endogenous BR levels. To advance our knowledge of BRs, we examined the effects of different plant hormones on DWF4 transcription in Arabidopsis thaliana. Semi-quantitative reverse-transcriptase PCR showed that the amount of the DWF4 mRNA precursor either decreased or increased, similarly with its mature form, in response to an exogenously applied bioactive BR, brassinolide (BL), and a BR biosynthesis inhibitor, brassinazole (Brz), respectively. The response to these chemicals in the levels of ß-glucuronidase (GUS) mRNA and its enzymatic activity is similar to the response of native DWF4 mRNA in DWF4::GUS plants. Contrary to the effects of BL, exogenous auxin induced GUS activity, but this enhancement was suppressed by anti-auxins, such as α-(phenylethyl-2-one)-IAA and α-tert-butoxycarbonylaminohexyl-IAA, suggesting the involvement of SCF(TIR1)-mediated auxin signaling in auxin-induced DWF4 transcription. Auxin-enhanced GUS activity was observed exclusively in roots; it was the most prominent in the elongation zones of both primary and lateral roots. Furthermore, auxin-induced lateral root elongation was suppressed by both Brz application and the dwf4 mutation, and this suppression was rescued by BL, suggesting that BRs act positively on root elongation under the control of auxin. Altogether, our results indicate that DWF4 transcription plays a novel role in the BR-auxin crosstalk associated with root elongation, in addition to its role in BR homeostasis.