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ACS Infectious Diseases Jun 2024Shiga toxins are the main virulence factors of Shiga toxin producing (STEC) and . There is no effective therapy to counter the disease caused by these toxins. The A1...
Shiga toxins are the main virulence factors of Shiga toxin producing (STEC) and . There is no effective therapy to counter the disease caused by these toxins. The A1 subunits of Shiga toxins bind the C-termini of ribosomal P-stalk proteins to depurinate the sarcin/ricin loop. The ribosome binding site of Shiga toxin 2 has not been targeted by small molecules. We screened a fragment library against the A1 subunit of Shiga toxin 2 (Stx2A1) and identified a fragment, , which bound at the ribosome binding site and mimicked the binding mode of the P-stalk proteins. We synthesized analogs of and identified a series of molecules with similar affinity and inhibitory activity. These are the first compounds that bind at the ribosome binding site of Stx2A1 and inhibit activity. These compounds hold great promise for further inhibitor development against STEC infection.
PubMed: 38873918
DOI: 10.1021/acsinfecdis.4c00224 -
Molecular and Cellular Biology Jun 2024The human Origin Recognition Complex (ORC) is required not only for the initiation of DNA replication, but is also implicated in diverse cellular functions, including...
The human Origin Recognition Complex (ORC) is required not only for the initiation of DNA replication, but is also implicated in diverse cellular functions, including chromatin organization, centrosome biology, and cytokinesis. The smallest subunit of ORC, Orc6, is poorly conserved amongst eukaryotes. Recent studies from our laboratory have suggested that human Orc6 is not required for replication licensing, but is needed for S-phase progression. Further, ATR-dependent phosphorylation of Orc6 at T229 is implicated in DNA damage response during S-phase. In this study, we demonstrate that the CDK-dependent phosphorylation of Orc6 at T195 occurs during mitosis. While the phosphorylation at T195 does not seem to be required to exit mitosis, cells expressing the phosphomimetic T195E mutant of Orc6 impede S-phase progression. Moreover, the phosphorylated form of Orc6 associates with ORC more robustly, and Orc6 shows enhanced association with the ORC outside of G1, supporting the view that Orc6 may prevent the role of Orc1-5 in licensing outside of G1. Finally, Orc6 and the phosphorylated Orc6 localize to the nucleolar organizing centers and regulate ribosome biogenesis. Our results suggest that phosphorylated Orc6 at T195 prevents replication.
PubMed: 38867464
DOI: 10.1080/10985549.2024.2356880 -
RNA (New York, N.Y.) Jun 2024Cap-independent, or eukaryotic initiation factor (eIF) 4E-independent, translation initiation in eukaryotes requires scaffolding protein eIF4G or its homolog,...
Cap-independent, or eukaryotic initiation factor (eIF) 4E-independent, translation initiation in eukaryotes requires scaffolding protein eIF4G or its homolog, death-associated protein 5 (DAP5). eIF4G associates with the 40S ribosomal subunit, recruiting the ribosome to the RNA transcript. A subset of RNA transcripts, such as fibroblast growth factor 9 (FGF-9), contain 5' untranslated regions (5' UTRs) that directly bind DAP5 or eIF4GI. Internal-ribosome-entry-site (IRES)-like cap-independent translation initiation does not require an unpaired 5' end for eIF binding, as these eIFs recruit the 40S ribosome at or near the start codon. For viral mRNA, eIF recruitment usually utilizes RNA structure, such as a pseudoknot or stem loops, and the RNA helicase eIF4A is required for DAP5- or 4G-mediated translation, suggesting these 5' UTRs are structured. However, for cellular IRES-like translation, no consensus RNA structures or sequences have yet been identified for eIF binding. FGF-9 is a member of a subset of mRNAs that are cap-independently upregulated in breast and colorectal cancer cells, likely using an IRES-like mechanism. However, the DAP5 binding site within the FGF-9 5' UTR is unknown. Moreover, DAP5 binds to other, dissimilar 5' UTRs, some of which require proximity to an unpaired, accessible 5' end to stimulate cap-independent translation. Using SHAPE-seq, we modeled the 186-nt FGF-9 5' UTR RNA's complex secondary structure in vitro. Further, DAP5 footprinting, toeprinting, and UV-crosslinking experiments identify DAP5-RNA interactions. Modeling of FGF-9 5' UTR tertiary structure aligns DAP5-interacting nucleotides on one face of the predicted structure. We propose that RNA structure involving tertiary folding, rather than a conserved sequence or secondary structure, acts as a DAP5 binding site. DAP5 appears to contact nucleotides near the start codon. Our findings offer a new perspective in the hunt for cap-independent translational enhancers. Structural, rather than sequence-specific, eIF binding sites may act as attractive chemotherapeutic targets or as dosage tools for mRNA-based therapies.
PubMed: 38866431
DOI: 10.1261/rna.080013.124 -
Life Science Alliance Aug 2024Multispanning membrane proteins are inserted into the endoplasmic reticulum membrane by the ribosome-bound multipass translocon (MPT) machinery. Based on cryo-electron...
Multispanning membrane proteins are inserted into the endoplasmic reticulum membrane by the ribosome-bound multipass translocon (MPT) machinery. Based on cryo-electron tomography and extensive subtomogram analysis, we reveal the composition and arrangement of ribosome-bound MPT components in their native membrane environment. The intramembrane chaperone complex PAT and the translocon-associated protein (TRAP) complex associate substoichiometrically with the MPT in a translation-dependent manner. Although PAT is preferentially part of MPTs bound to translating ribosomes, the abundance of TRAP is highest in MPTs associated with non-translating ribosomes. The subtomogram average of the TRAP-containing MPT reveals intermolecular contacts between the luminal domains of TRAP and an unknown subunit of the back-of-Sec61 complex. AlphaFold modeling suggests this protein is nodal modulator, bridging the luminal domains of nicalin and TRAPα. Collectively, our results visualize the variability of MPT factors in the native membrane environment dependent on the translational activity of the bound ribosome.
Topics: Ribosomes; Membrane Proteins; Endoplasmic Reticulum; Protein Biosynthesis; Cryoelectron Microscopy; SEC Translocation Channels; Molecular Chaperones; Protein Transport; Models, Molecular
PubMed: 38866426
DOI: 10.26508/lsa.202302496 -
Frontiers in Cell and Developmental... 2024Mitochondria play a central role in cellular metabolism producing the necessary ATP through oxidative phosphorylation. As a remnant of their prokaryotic past,... (Review)
Review
Mitochondria play a central role in cellular metabolism producing the necessary ATP through oxidative phosphorylation. As a remnant of their prokaryotic past, mitochondria contain their own genome, which encodes 13 subunits of the oxidative phosphorylation system, as well as the tRNAs and rRNAs necessary for their translation in the organelle. Mitochondrial protein synthesis depends on the import of a vast array of nuclear-encoded proteins including the mitochondrial ribosome protein components, translation factors, aminoacyl-tRNA synthetases or assembly factors among others. Cryo-EM studies have improved our understanding of the composition of the mitochondrial ribosome and the factors required for mitochondrial protein synthesis and the advances in next-generation sequencing techniques have allowed for the identification of a growing number of genes involved in mitochondrial pathologies with a defective translation. These disorders are often multisystemic, affecting those tissues with a higher energy demand, and often present with neurodegenerative phenotypes. In this article, we review the known proteins required for mitochondrial translation, the disorders that derive from a defective mitochondrial protein synthesis and the animal models that have been established for their study.
PubMed: 38855161
DOI: 10.3389/fcell.2024.1410245 -
FEBS Open Bio Jun 2024Mitoribosome biogenesis is a complex process involving RNA elements encoded in the mitochondrial genome and mitoribosomal proteins typically encoded in the nuclear... (Review)
Review
Mitoribosome biogenesis is a complex process involving RNA elements encoded in the mitochondrial genome and mitoribosomal proteins typically encoded in the nuclear genome. This process is orchestrated by extra-ribosomal proteins, nucleus-encoded assembly factors, which play roles across all assembly stages to coordinate ribosomal RNA processing and maturation with the sequential association of ribosomal proteins. Both biochemical studies and recent cryo-EM structures of mammalian mitoribosomes have provided insights into their assembly process. In this article, we will briefly outline the current understanding of mammalian mitoribosome biogenesis pathways and the factors involved. Special attention is devoted to the recent identification of iron-sulfur clusters as structural components of the mitoribosome and a small subunit assembly factor, the existence of redox-sensitive cysteines in mitoribosome proteins and assembly factors, and the role they may play as redox sensor units to regulate mitochondrial translation under stress.
PubMed: 38849194
DOI: 10.1002/2211-5463.13844 -
Molecular Cell Jun 2024In response to stress, eukaryotes activate the integrated stress response (ISR) via phosphorylation of eIF2α to promote the translation of pro-survival effector genes,...
In response to stress, eukaryotes activate the integrated stress response (ISR) via phosphorylation of eIF2α to promote the translation of pro-survival effector genes, such as GCN4 in yeast. Complementing the ISR is the target of rapamycin (TOR) pathway, which regulates eIF4E function. Here, we probe translational control in the absence of eIF4E in Saccharomyces cerevisiae. Intriguingly, we find that loss of eIF4E leads to de-repression of GCN4 translation. In addition, we find that de-repression of GCN4 translation is accompanied by neither eIF2α phosphorylation nor reduction in initiator ternary complex (TC). Our data suggest that when eIF4E levels are depleted, GCN4 translation is de-repressed via a unique mechanism that may involve faster scanning by the small ribosome subunit due to increased local concentration of eIF4A. Overall, our findings suggest that relative levels of eIF4F components are key to ribosome dynamics and may play important roles in translational control of gene expression.
Topics: Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins; Phosphorylation; Stress, Physiological; Basic-Leucine Zipper Transcription Factors; Eukaryotic Initiation Factor-4F; Protein Biosynthesis; Gene Expression Regulation, Fungal; Eukaryotic Initiation Factor-4E; Eukaryotic Initiation Factor-2; Signal Transduction; Ribosomes; Eukaryotic Initiation Factor-4A
PubMed: 38848692
DOI: 10.1016/j.molcel.2024.04.016 -
Nature Structural & Molecular Biology Jun 2024The ribosomal RNA of the human protein synthesis machinery comprises numerous chemical modifications that are introduced during ribosome biogenesis. Here we present the...
The ribosomal RNA of the human protein synthesis machinery comprises numerous chemical modifications that are introduced during ribosome biogenesis. Here we present the 1.9 Å resolution cryo electron microscopy structure of the 80S human ribosome resolving numerous new ribosomal RNA modifications and functionally important ions such as Zn, K and Mg, including their associated individual water molecules. The 2'-O-methylation, pseudo-uridine and base modifications were confirmed by mass spectrometry, resulting in a complete investigation of the >230 sites, many of which could not be addressed previously. They choreograph key interactions within the RNA and at the interface with proteins, including at the ribosomal subunit interfaces of the fully assembled 80S ribosome. Uridine isomerization turns out to be a key mechanism for U-A base pair stabilization in RNA in general. The structural environment of chemical modifications and ions is primordial for the RNA architecture of the mature human ribosome, hence providing a structural framework to address their role in healthy states and in human diseases.
PubMed: 38844527
DOI: 10.1038/s41594-024-01274-x -
Journal of Bacteriology Jun 2024RsgA (small ribosomal subunit, 30S, GTPase), a late-stage biogenesis factor, releases RbfA from 30S-RbfA complex. Δ (deleted for ) shows a slow growth phenotype and an...
UNLABELLED
RsgA (small ribosomal subunit, 30S, GTPase), a late-stage biogenesis factor, releases RbfA from 30S-RbfA complex. Δ (deleted for ) shows a slow growth phenotype and an increased accumulation of 17S rRNA (precursor of 16S rRNA) and the ribosomal subunits. Here, we show that the rescue of the Δ strain by multicopy (IF2) is enhanced by simultaneous overexpression of initiator tRNA (i-tRNA), suggesting a role of initiation complex formation in growth rescue. The synergistic effect of IF2/i-tRNA is accompanied by increased processing of 17S rRNA (to 16S), and protection of the 16S rRNA 3'-minor domain. Importantly, we show that an IF2-binding anticonvulsant drug, lamotrigine (Ltg), also rescues the Δ strain growth. The rescue is accompanied by increased processing of 17S rRNA, protection of the 3'-minor domain of 16S rRNA, and increased 70S ribosomes in polysome profiles. However, Ltg becomes inhibitory to the strain whose growth was already rescued by an L83R mutation in . Interestingly, like wild-type , overproduction of Ltg alleles (having indel mutations in their domain II) also rescues the Δ strain (independent of Ltg). Our observations suggest the dual role of IF2 in rescuing the Δ strain. First, together with i-tRNA, IF2 facilitates the final steps of processing of 17S rRNA. Second, a conformer of IF2 functionally compensates for RsgA, albeit poorly, during 30S biogenesis.
IMPORTANCE
RsgA is a late-stage ribosome biogenesis factor. Earlier, (IF2) was isolated as a multicopy suppressor of the Δ strain. How IF2 rescued the strain growth remained unclear. This study reveals that (i) the multicopy -mediated growth rescue of Δ and the processing of 17S precursor to 16S rRNA in the strain are enhanced upon simultaneous overexpression of initiator tRNA and (ii) a conformer of IF2, whose occurrence increases when IF2 is overproduced or when Δ is treated with Ltg (an anticonvulsant drug that binds to domain II of IF2), compensates for the function of RsgA. Thus, this study reveals yet another role of IF2 in ribosome biogenesis.
PubMed: 38837341
DOI: 10.1128/jb.00119-24 -
Plant Disease Jun 2024Maxim. (), one of the Chinese herbal medicines, is an economically important crop in Anhui Province, China. In recent years, gummy stem blight disease, a major disease...
Maxim. (), one of the Chinese herbal medicines, is an economically important crop in Anhui Province, China. In recent years, gummy stem blight disease, a major disease of cucurbits, was widespread in many plantations. The initial symptoms on the naturally infected stems appeared as dark brown water-soaked lesions, and as the disease progressed, vines of gradually withered. On leaves, brown water-soaked lesions were visible initially, and then lesions enlarged and coalesced, resulting in extensive necrosis of leaves. On fruit, lesions covered with the white mycelium were nearly circular and tan to brown initially. Subsequently, the diseased fruit turned black and rotten commonly known as fruit rot or black rot. A -like organism was consistently isolated from symptomatic stems, leaves and fruits. Fungal isolates were initially white and later turned dark grey or black with woolly to floccose aerial mycelium on PDA medium. Twenty-four isolates from different plantations were selected for further morphological studies. Pycnidia and conidia were formed after inoculating on cucumber fruit for 3 days. Pycnidia were globose to sub-globose, brown, ostiolate and 106.7 to 213.6 μm (average 160.1 μm, n = 50) in diameter. Conidia were hyaline, ellipsoidal, aseptate or one-septate, slightly constricted at the septa, 6.1 to 13.6 × 3.5 to 4.8 μm (average 9.9 × 4.1 μm, n = 50), and contained two or more oil drops. Three different loci of the genomic DNA, including the nuclear ribosome DNA internal transcribed spacer (), RNA polymerase II second-largest subunit (), and β-tubulin () genes., were amplified using primers ITS1/ITS4 (White et al. 1990), RBP2DF/RBP2DR (Lawrence et al. 2013), and T1/β-Sandy-R (O' Donnell and Cigelnik 1997; Stukenbrock et al. 2012), respectively and sequenced. A phylogenetic tree was built based on analysis of , , and sequences that deposited in GenBank (MW485497-MW485502 for ITS, MW531661-MW531666 for RPB2, and MW531667-MW531672 for TUB2), using the maximum likelihood method. The phylogenetic tree showed that the isolates fell into a single clade with . On the basis of morphological and molecular characteristics, the isolates obtained from were identified as . Pathogenicity tests were carried out on stems and leaves of 4-week-old seedlings and on immature fruit collected from adult plants. The epidermis, previously injured with a syringe needle, was inoculated with 5-mm-diameter mycelial plugs, and the inoculated areas were then wrapped in water-soaked cotton. Controls were similarly inoculated with agar plugs. The diameters of lesions were measured in two perpendicular directions. Re-isolations from the stem and leaf lesions were performed on the PDA medium. , was re-identified based on its colony and conidial characteristics and, therefore, completed Koch's postulates. Gummy stem blight caused by has been reported in a wide range of hosts, including cucumber, luffa, pumpkin, gourd, muskmelon, cantaloupe, and watermelon (Jiang et al. 2015; Keinath 2011; Zhao et al. 2019). To our knowledge, this is the first report of gummy Stem blight disease on caused by in China. The research provides a basis for the development and implementation of effective management strategies. Pathogenicity tests were carried out on stems and leaves of 4-week-old seedlings and on immature fruits collected from adult plants. The epidermis, previously injured with a syringe needle, was inoculated with 5-mm-diameter mycelial plugs, and the inoculated areas were then wrapped in water-soaked cotton. Controls were treated similarly but inoculated with agar plugs. Diameters of lesions were measured in two mutually perpendicular directions. Reisolations from the lesions were performed on PDA medium, and was re-identified based on its colony and conidial characteristics to complete Koch's postulates. Gummy stem blight caused by have been reported in a wide range of hosts, including cucumber, luffa, pumpkin, gourd, muskmelon, cantaloupe, and watermelon (Jiang et al. 2015; Keinath 2011; Zhao et al. 2019). To our knowledge, this is the first report of gummy Stem blight disease on caused by in China. The research provides a basis for the development and implementation of effective management strategies.
PubMed: 38831589
DOI: 10.1094/PDIS-09-23-1782-PDN