Oxidative Folding of Proteins: The “Smoking Gun” of Glutathione

Glutathione has long been suspected to be the primary low molecular weight compound present in all cells promoting the oxidative protein folding, but twenty years ago it was found “not guilty”. Now, new surprising evidence repeats its request to be the “smoking gun” which reopens the criminal trial revealing the crucial involvement of this tripeptide.     

The DEAD-Box Protein Rok1 Coordinates Ribosomal RNA Processing in Association with Rrp5 in Drosophila

Ribosome biogenesis and processing involve the coordinated action of many components. The DEAD-box RNA helicase (Rok1) is essential for cell viability, and the depletion of Rok1 inhibits pre-rRNA processing. Previous research on Rok1 and its cofactor Rrp5 has been performed primarily in yeast. Few functional studies have been performed in complex multicellular eukaryotes. Here, we used a combination of genetics and developmental experiments to show that Rok1 and Rrp5, which localize to the nucleolus, play key roles in the pre-rRNA processing and ribosome assembly in D. melanogaster. The accumulation of pre-rRNAs caused by Rok1 depletion can result in developmental defects. The loss of Rok1 enlarged the nucleolus and led to stalled ribosome assembly and pre-rRNA processing in the nucleolus, thereby blocking rRNA maturation and exacerbating the inhibition of mitosis in the brain. We also discovered that rrp5^4-2/4-2 displayed significantly increased ITS1 signaling by fluorescence in situ hybridization, and a reduction in ITS2. Rrp5 signal was highly enriched in the core of the nucleolus in the rok1^167/167 mutant, suggesting that Rok1 is required for the accurate cellular localization of Rrp5 in the nucleolus. We have thus uncovered functions of Rok1 that reveal important implications for ribosome processing in eukaryotes.     

Phylogenetic relationships among species of Saccharomyces, Schizosaccharomyces, Debaryomyces and Schwanniomyces determined from partial ribosomal RNA sequences

Species of the genera Saccharomyces, Schizosaccharomyces, Debaryomyces and Schwanniomyces were compared from their extent of divergence in three regions from small (18S) and large (25S) subunit ribosomal RNAs comprising a total of 900 nucleotides. With the exception of the closely related Saccharomyces bayanus and S. pastorianus, which appear to have identical sequences, all other species could be distinguished by nucleotide differences in a variable region of the large subunit, and genus-specific nucleotides were discernible in all three regions. The taxon D. tamarii differed markedly from other species and is excluded from Debaryomyces. By contrast, Schwanniomyces occidentalis showed few nucleotide differences with Debaryomyces spp. and its transfer to Debaryomyces is proposed. Schizosaccharomyces proved to be somewhat more divergent than Saccharomyces and Debaryomyces, but species differences appear insufficient for dividing the genus. Some of the factors influencing estimates of phylogenetic distances from rRNA sequences are discussed.     

Identification of p90 Ribosomal S6 Kinase 2 as a Novel Host Protein in HBx Augmenting HBV Replication by iTRAQ‐Based Quantitative Comparative Proteomics

Purpose

The aim of this study was to screen for novel host proteins that play a role in HBx augmenting Hepatitis B virus (HBV) replication.

Experimental design

Three HepG2 cell lines stably harboring different functional domains of HBx (HBx, HBx‐Cm6, and HBx‐Cm16) were cultured. ITRAQ technology integrated with LC‐MS/MS analysis was applied to identify the proteome differences among these three cell lines.

Results

In brief, a total of 70 different proteins were identified among HepG2‐HBx, HepG2‐HBx‐Cm6, and HepG2‐HBx‐Cm16 by double repetition. Several differentially expressed proteins, including p90 ribosomal S6 kinase 2 (RSK2), were further validated. RSK2 was expressed at higher levels in HepG2‐HBx and HepG2‐HBx‐Cm6 compared with HepG2‐HBx‐Cm16. Furthermore, levels of HBV replication intermediates were decreased after silencing RSK2 in HepG2.2.15. An HBx‐minus HBV mutant genome led to decreased levels of HBV replication intermediates and these decreases were restored to levels similar to wild‐type HBV by transient ectopic expression of HBx. After silencing RSK2 expression, the levels of HBV replication intermediates synthesized from the HBx‐minus HBV mutant genome were not restored to levels that were observed with wild‐type HBV by transient HBx expression.

Conclusion and clinical Relevance

Based on iTRAQ quantitative comparative proteomics, RSK2 was identified as a novel host protein that plays a role in HBx augmenting HBV replication.

     

Structures and Functions of Qβ Replicase: Translation Factors beyond Protein Synthesis

Qβ replicase is a unique RNA polymerase complex, comprising Qβ virus-encoded RNA-dependent RNA polymerase (the catalytic β-subunit) and three host-derived factors: translational elongation factor (EF) -Tu, EF-Ts and ribosomal protein S1. For almost fifty years, since the isolation of Qβ replicase, there have been several unsolved, important questions about the mechanism of RNA polymerization by Qβ replicase. Especially, the detailed functions of the host factors, EF-Tu, EF-Ts, and S1, in Qβ replicase, which are all essential in the Escherichia coli (E. coli) host for protein synthesis, had remained enigmatic, due to the absence of structural information about Qβ replicase. In the last five years, the crystal structures of the core Qβ replicase, consisting of the β-subunit, EF-Tu and Ts, and those of the core Qβ replicase representing RNA polymerization, have been reported. Recently, the structure of Qβ replicase comprising the β-subunit, EF-Tu, EF-Ts and the N-terminal half of S1, which is capable of initiating Qβ RNA replication, has also been reported. In this review, based on the structures of Qβ replicase, we describe our current understanding of the alternative functions of the host translational elongation factors and ribosomal protein S1 in Qβ replicase as replication factors, beyond their established functions in protein synthesis.     

Comparative Evolution of S7 Intron 1 and Ribosomal Internal Transcribed Spacer in Coilia nasus (Clupeiformes: Engraulidae)

Coilia nasus is widely distributed in the Yangtze River, the coastal waters of China, Korea and the Ariake Sound of Japan. Several ecotypes exist and this provides a useful model for the study of comparative diversity between molecular markers. Here we analyze and compare the nucleotide sequences between single-copy ribosomal protein S7 gene intron 1 (rpS7) and multiple-copy ribosomal internal transcribed spacer 1 (ITS1) in this species to compare the phylogenetic signal of the two nuclear genes. Nucleotide substitutions among the two gene sequences and partial sequence of mitochondrial cytochrome c oxidase subunit I (COI) gene were also analyzed. A total of 115 clones for rpS7 and 122 clones for ITS1 were obtained from 37 specimens. The nucleotide sequence length is 741 to 743 bp for rpS7 and 334 to 348 bp for ITS1. Intra- and inter-specimen variation in rpS7 results from nucleotide substitution, while such variation in ITS1 is mainly due to different numbers of short base repeats. The content of G + C is lower in rpS7 (43.5%) than in ITS1 (68.2%). Our results indicate that the proportion of the sequence variable sites is higher in rpS7 (61) than in ITS1 (23); the informative parsimony of rpS7 is evidently higher than that of ITS1 (26 vs. 2); the overall ratio between transitions and transversions in ITS1 is slightly lower than in rpS7, but remarkably lower than in COI. These results suggest that rpS7 is more suitable than ITS1 as a marker for genetic divergence of this group. Furthermore, gene flow is observed between the different geographic populations of C. nasus from the phylogeny of this species based on rpS7, showing that rpS7 has more evolutionary characteristics for understanding the processes of genomic evolution at the intraspecific level.     

In vivo formation of a stable pentameric (P2α/P1β)–P0–(P1α/P2β) ribosomal stalk complex in Saccharomyces cerevisiae

Heterodimers of acidic proteins P1α/P2β and P1β/P2α bind to P0 and are fundamental for the assembly of the ribosomal stalk. However, different inconsistencies are found in the literature regarding additional P protein heterodimer formations and their individual interactions with P0. Using the two‐hybrid approach, we have found results that help to clarify these interactions. Thus, we have found that neither P1 nor P2 directly interact with P0 unless the endogenous heterodimer partner is being expressed in the cell. In addition, a P2‐free amino end is a requisite in these heterodimers for binding to P0. With regard to the two‐hybrid interactions between P1 and P2, the known canonical P1α–P2β and P1β–P2α interactions do not depend on either a free amino end or the presence of endogenous P0, P1 or P2 proteins. Furthermore, the non‐canonical P1β–P2β pair also behaves similarly, although this interaction is weaker. Interestingly, P1α–P2α, P1α–P1β and P2α–P2β two‐hybrid interactions were also detected, although in these cases the endogenous P proteins were involved. Thus, these positive interactions are the consequence of the interaction between two canonical heterodimers. As the ribosome anchorage protein P0 is also necessary, the results suggest that, in vivo, all five P proteins form a complex, independent of the ribosome, containing the two canonical heterodimers and P0. This complex has been isolated in cells expressing a P0 protein unable to bind to the ribosome. Copyright © 2010 John Wiley & Sons, Ltd.     

Inhibitors of the Large Ribosomal Subunit from Haloarcula marismortui

The crystal structures that have been obtained for 23 different inhibitors bound to the large ribosomal subunit from Haloarcula marismortui are reviewed here. These structures provide important insights into how anti-ribosomal antibiotics inhibit protein synthesis, how species specificity arises, and the relationship between ribosomal mutations and antibiotic resistance. These structural studies also provide compelling evidence that the conformation of the peptidyl transferase center of the large ribosomal subunit is intrinsically variable, and that conformational equilibria play a role in determining its functional properties.     

Molecular cloning of the RPS0 gene from Candida tropicalis.

The Saccharomyces cerevisiae RPS0 A and B genes encode proteins essential for maturation of the 40S ribosomal subunit precursors. We have isolated a homologue of the RPS0 gene from Candida tropicalis, which we named CtRPS0. The C. tropicalis RPS0 encodes a protein of 261 amino acid residues with a predicted molecular weight of 28.65 kDa and an isoelectric point of 4.79. CtRps0p displays significant amino acid sequence homology with Rps0p from C. albicans, S. cerevisiae, Neurospora crassa, Schizosaccharomyces pombe, Pneumocystis carinii and higher organisms, such as human, mouse and rat. CtRPS0 on a high copy number vector can complement the lethal phenotype linked to the disruption of both RPS0 genes in S. cerevisiae. Southern blot analysis suggests that CtRPS0 is present at a single locus within the C. tropicalis genome.     

IV. XV. Yeast mapping reports. RPL44 and RPL44′, encoding acidic ribosomal phosphoproteins YP2α(l44) and YP1β(l44′), are adjacent to rig and STF1 on Saccharomyces cerevisiae chromosomes XV and IV respectively

The RPL44′ gene from Saccharomyces cerevisiae encoding the ribosomal protein YP1β(L44′) has been found to be linked to the STF1 gene, encoding a stabilizing factor of the F₁F₀-ATPase inhibitor protein from mitochondria. Evidence of this linkage comes from results obtained from Northern hybridization using a DNA probe that contains a complementary region to the 5′ end of the mRNA of RPL44′. Similarly, a data bank search has shown that RPL44, encoding ribosomal protein YP2α(L44) is linked to the rig gene that encodes ribosomal protein S21.