Ribosomal Subunit Protein Research Articles

HectD1 controls hematopoietic stem cell regeneration by coordinating ribosome assembly and protein synthesis.

Impaired ribosome function is the underlying etiology in a group of bone marrow failure syndromes called ribosomopathies. However, how ribosomes are regulated remains poorly understood, as are approaches to restore hematopoietic stem cell (HSC) function loss because of defective ribosome biogenesis. Here we reveal a role of the E3 ubiquitin ligase HectD1 in regulating HSC function via ribosome assembly and protein translation. Hectd1-deficient HSCs exhibit a striking defect in transplantation ability and exvivo maintenance concomitant with reduced protein synthesis and growth rate under stress conditions. Mechanistically, HectD1 ubiquitinates and degrades ZNF622, an assembly factor for the ribosomal 60S subunit. Hectd1 loss leads to accumulation of ZNF622 and the anti-association factor eIF6 on 60S, resulting in 60S/40S joining defects. Importantly, Znf622 depletion in Hectd1-deficient HSCs restored ribosomal subunit joining, protein synthesis, and HSC reconstitution capacity. These findings highlight the importance of ubiquitin-coordinated ribosome assembly in HSC regeneration.

Narrow Leaf21, encoding ribosomal protein RPS3A, controls leaf development in rice.

Leaf morphology influences photosynthesis, transpiration, and ultimately crop yield. However, the molecular mechanism of leaf development is still not fully understood. Here, we identified and characterized the narrow leaf21 (nal21) mutant in rice (Oryza sativa), showing a significant reduction in leaf width, leaf length and plant height, and increased tiller number. Microscopic observation revealed defects in the vascular system and reduced epidermal cell size and number in the nal21 leaf blade. Map-based cloning revealed that NAL21 encodes a ribosomal small subunit protein RPS3A. Ribosome-targeting antibiotics resistance assay and ribosome profiling showed a significant reduction in the free 40S ribosome subunit in the nal21 mutant. The nal21 mutant showed aberrant auxin responses in which multiple auxin response factors (ARFs) harboring upstream open-reading frames (uORFs) in their 5'-untranslated region were repressed at the translational level. The WUSCHEL-related homeobox 3A (OsWOX3A) gene, a key transcription factor involved in leaf blade lateral outgrowth, is also under the translational regulation by RPS3A. Transformation with modified OsARF11, OsARF16, and OsWOX3A genomic DNA (gDNA) lacking uORFs rescued the narrow leaf phenotype of nal21 to a better extent than transformation with their native gDNA, implying that RPS3A could regulate translation of ARFs and WOX3A through uORFs. Our results demonstrate that proper translational regulation of key factors involved in leaf development is essential to maintain normal leaf morphology.

Improving the Methanol Tolerance of an Escherichia coli Methylotroph via Adaptive Laboratory Evolution Enhances Synthetic Methanol Utilization.

There is great interest in developing synthetic methylotrophs that harbor methane and methanol utilization pathways in heterologous hosts such as Escherichia coli for industrial bioconversion of one-carbon compounds. While there are recent reports that describe the successful engineering of synthetic methylotrophs, additional efforts are required to achieve the robust methylotrophic phenotypes required for industrial realization. Here, we address an important issue of synthetic methylotrophy in E. coli: methanol toxicity. Both methanol, and its oxidation product, formaldehyde, are cytotoxic to cells. Methanol alters the fluidity and biological properties of cellular membranes while formaldehyde reacts readily with proteins and nucleic acids. Thus, efforts to enhance the methanol tolerance of synthetic methylotrophs are important. Here, adaptive laboratory evolution was performed to improve the methanol tolerance of several E. coli strains, both methylotrophic and non-methylotrophic. Serial batch passaging in rich medium containing toxic methanol concentrations yielded clones exhibiting improved methanol tolerance. In several cases, these evolved clones exhibited a > 50% improvement in growth rate and biomass yield in the presence of high methanol concentrations compared to the respective parental strains. Importantly, one evolved clone exhibited a two to threefold improvement in the methanol utilization phenotype, as determined via 13C-labeling, at non-toxic, industrially relevant methanol concentrations compared to the respective parental strain. Whole genome sequencing was performed to identify causative mutations contributing to methanol tolerance. Common mutations were identified in 30S ribosomal subunit proteins, which increased translational accuracy and provided insight into a novel methanol tolerance mechanism. This study addresses an important issue of synthetic methylotrophy in E. coli and provides insight as to how methanol toxicity can be alleviated via enhancing methanol tolerance. Coupled improvement of methanol tolerance and synthetic methanol utilization is an important advancement for the field of synthetic methylotrophy.

Innate immunity to yeast prions: Btn2p and Cur1p curing of the [URE3] prion is prevented by 60S ribosomal protein deficiency or ubiquitin/proteasome system overactivity.

[URE3] is an amyloid-based prion of Ure2p, a negative regulator of poor nitrogen source catabolism in Saccharomyces cerevisiae. Overproduced Btn2p or its paralog Cur1p, in processes requiring Hsp42, cure the [URE3] prion. Btn2p cures by collecting Ure2p amyloid filaments at one place in the cell. We find that rpl4aΔ, rpl21aΔ, rpl21bΔ, rpl11bΔ, and rpl16bΔ (large ribosomal subunit proteins) or ubr2Δ (ubiquitin ligase targeting Rpn4p, an activator of proteasome genes) reduce curing by overproduced Btn2p or Cur1p. Impaired curing in ubr2Δ or rpl21bΔ is restored by an rpn4Δ mutation. No effect of rps14aΔ or rps30bΔ on curing was observed, indicating that 60S subunit deficiency specifically impairs curing. Levels of Hsp42p, Sis1p, or Btn3p are unchanged in rpl4aΔ, rpl21bΔ, or ubr2Δ mutants. Overproduction of Cur1p or Btn2p was enhanced in rpn4Δ and hsp42Δ mutants, lower in ubr2Δ strains, and restored to above wild-type levels in rpn4Δ ubr2Δ strains. As in the wild-type, Ure2N-GFP colocalizes with Btn2-RFP in rpl4aΔ, rpl21bΔ, or ubr2Δ strains, but not in hsp42Δ. Btn2p/Cur1p overproduction cures [URE3] variants with low seed number, but seed number is not increased in rpl4aΔ, rpl21bΔ or ubr2Δ mutants. Knockouts of genes required for the protein sorting function of Btn2p did not affect curing of [URE3], nor did inactivation of the Hsp104 prion-curing activity. Overactivity of the ubiquitin/proteasome system, resulting from 60S subunit deficiency or ubr2Δ, may impair Cur1p and Btn2p curing of [URE3] by degrading Cur1p, Btn2p or another component of these curing systems.

Expression and affinity purification of recombinant mammalian mitochondrial ribosomal small subunit (MRPS) proteins and protein-protein interaction analysis indicate putative role in tumourigenic cellular processes.

Mitochondrial ribosomal small subunit (MRPS) group of proteins is structural constituents of the small subunit of mitoribosomes involved in translation. Recent studies indicate role in tumourigenic process, however, unlike cytosolic ribosomal proteins, knowledge on the role of MRPS proteins in alternate cellular processes is very limited. Mapping protein-protein interactions (PPIs) onto known cellular processes can be a valuable tool to identify novel protein functions. In this study, to identify PPIs of MRPS proteins, we have constructed 31 glutathione-S-transferase (GST)/MRPS fusion clones. GST/MRPS fusion proteins were confirmed by MALDI-TOF analysis. GST pull-downs were performed using eight GST/MRPS proteins (MRPS9, MRPS10, MRPS11, MRPS18B, MRPS31, MRPS33, MRPS38 and MRPS39), GST alone as pull-down control and HEK293 cell lysate as the source for anchor proteins followed by nLC/MS/MS analysis and probable PPIs of eight MRPS proteins were identified. Three PPIs from GST pull-downs and interaction between six MRPS proteins and p53 previously reported in PPI database were validated. The PPI network analysis revealed putative role in cellular processes with implications for tumourigenesis. Gene expression screening of a cancer cell line panel indicated overexpression of MRPS10 and MRPS31 in breast cancer. Co-expression module identification tool analysis of breast cancer gene expression and MRPS10 and MRPS31 PPIs revealed putative role for PPI with acyl-CoA dehydrogenase in fatty acid oxidation process regulated by brain-derived neurotrophic factor signalling pathway.

DIAPH3 promotes pancreatic cancer progression by activating selenoprotein TrxR1-mediated antioxidant effects.

Pancreatic cancer is a highly malignant tumour of the digestive tract which is difficult to diagnose and treat. Approximately 90% of cases arise from ductal adenocarcinoma of the glandular epithelium. The morbidity and mortality of the disease have increased significantly in recent years. Its 5‐year survival rate is <1% and has one of the worst prognoses amongst malignant tumours. Pancreatic cancer has a low rate of early‐stage diagnosis, high surgical mortality and low cure rate. Selenium compounds produced by selenoamino acid metabolism may promote a large amount of oxidative stress and subsequent unfolded reactions and endoplasmic reticulum stress by consuming the NADPH in cells, and eventually lead to apoptosis, necrosis or necrotic cell death. In this study, we first identified DIAPH3 as a highly expressed protein in the tissues of patients with pancreatic cancer, and confirmed that DIAPH3 promoted the proliferation, anchorage‐independent growth and invasion of pancreatic cancer cells using overexpression and interference experiments. Secondly, bioinformatics data mining showed that the potential proteins interacted with DIAPH3 were involved in selenoamino acid metabolism regulation. Selenium may be incorporated into selenoprotein synthesis such as TrxR1 and GPX4, which direct reduction of hydroperoxides or resist ferroptosis, respectively. Our following validation confirmed that DIAPH3 promoted selenium content and interacted with the selenoprotein RPL6, a ribosome protein subunit involved in selenoamino acid metabolism. In addition, we verified that DIAPH3 could down‐regulate cellular ROS level via up‐regulating TrxR1 expression. Finally, nude mice xenograft model experimental results demonstrate DIAPH3 knock down could decrease tumour growth and TrxR1 expression and ROS levels in vivo. Collectively, our observations indicate DIAPH3 could promote pancreatic cancer progression by activating selenoprotein TrxR1‐mediated antioxidant effects.

Ribosomal protein synthesis is decreased in tauopathy as revealed by noncanonical amino acid labelling

AbstractBackgroundTau is a scaffolding protein which serves multiple cellular functions that are perturbed in neurodegenerative diseases, including Alzheimer’s disease (AD) and frontotemporal dementia (FTD). Recently we demonstrated that amyloid‐b, the second hallmark of AD, induces de novo protein synthesis of tau, with this activation being found to be tau‐dependent. This raised the question of whether FTD‐tau by itself affects protein synthesis.MethodIn order to examine protein synthesis, we optimised non‐canonical amino acid (NCAA) labelling to label newly synthesised proteins in vivo in the K369I tau transgenic K3 mouse model of FTD. Combining this technique with either fluorescent non‐canonical amino acid tagging (FUNCAT) or bio‐orthogonal non‐canonical amino acid tagging (BONCAT), we were able to visualise and purify newly synthesised proteins from mouse brain tissue. We also used polysome profiling in primary neurons to further explore the effect of tau on ribosome biogenesis.ResultOur analysis reveals massively decreased protein synthesis in neurons containing pathologically phosphorylated tau, a finding we confirmed in P301L mutant tau transgenic rTg4510 mice. Using quantitative SWATH‐MS proteomics, we identify alterations in the synthesis of 247 proteins in de novo proteome of K3 mice, including the decreased synthesis of the ribosomal subunit proteins RPL23, RPLP0, RPL19 and RPS16, a finding that was validated in both K3 and rTg4510 mice. We also demonstrate that there is a decrease in overall abundance of one of these proteins, RPL23, and that this decrease correlated with the levels of both total and AT8 phosphorylated tau. Lastly, we recapultate these findings in human tissue, with FTD‐brains showing significantly lower levels of RPL23 than healthy controls.ConclusionTogether, our findings present a potential pathomechanism by which pathological tau interferes with cellular functions through the dysregulation of ribosomal protein synthesis.

Lefamulin: A Novel Oral and Intravenous Pleuromutilin for the Treatment of Community-Acquired Bacterial Pneumonia.

Lefamulin is a novel oral and intravenous (IV) pleuromutilin developed as a twice-daily treatment for community-acquired bacterial pneumonia (CABP). It is a semi-synthetic pleuromutilin with a chemical structure that contains a tricyclic core of five-, six-, and eight-membered rings and a 2-(4-amino-2-hydroxycyclohexyl)sulfanylacetate side chain extending from C14 of the tricyclic core. Lefamulin inhibits bacterial protein synthesis by binding to the 50S bacterial ribosomal subunit in the peptidyl transferase center (PTC). The pleuromutilin tricyclic core binds to a pocket close to the A site, while the C14 side chain extends to the P site causing a tightening of the rotational movement in the binding pocket referred to as an induced-fit mechanism. Lefamulin displays broad-spectrum antibacterial activity against Gram-positive and Gram-negative aerobic and anaerobic bacteria as well as against atypical bacteria that commonly cause CABP. Pleuromutilin antibiotics exhibit low rates of resistance development and lack cross-resistance to other antimicrobial classes due to their unique mechanism of action. However, pleuromutilin activity is affected by mutations in 23S rRNA, 50S ribosomal subunit proteins rplC and rplD, ATP-binding cassette (ABC)-F transporter proteins such as vga(A), and the methyltransferase cfr. The pharmacokinetic properties of lefamulin include: volume of distribution (Vd) ranging from 82.9 to 202.8 L, total clearance (CLT) of 19.5 to 21.4 L/h, and terminal elimination half-life (t1/2) of 6.9-13.2 h; protein binding of lefamulin is high and non-linear. The oral bioavailability of lefamulin has been estimated as 24% in fasted subjects and 19% in fed subjects. A single oral dose of lefamulin 600 mg administered in fasted patients achieved a maximum plasma concentration (Cmax) of 1.2-1.5 mg/L with a time of maximum concentration (Tmax) ranging from 0.8 to 1.8 h, and an area under the plasma concentration-time curve from 0 to infinity (AUC0-∞) of 8.5-8.8 mgh/L. The pharmacodynamic parameter predictive of lefamulin efficacy is the free plasma area under the concentration-time curve divided by the minimum inhibitory concentration (fAUC24h/MIC). Lefamulin efficacy has been demonstrated using various animal models including neutropenic murine thigh infection, pneumonia, lung infection, and bacteremia. Lefamulin clinical safety and efficacy was investigated through a Phase II clinical trial of acute bacterial skin and skin structure infection (ABSSSI), as well as two Phase III clinical trials of CABP. The Phase III trials, LEAP 1 and LEAP 2 established non-inferiority of lefamulin to moxifloxacin in both oral and IV formulations in the treatment of CABP. The United States Food and Drug Administration (FDA), European Medicines Agency (EMA), and Health Canada have each approved lefamulin for the treatment of CABP. A Phase II clinical trial has been completed for the treatment of ABSSSI, while the pediatric program is in Phase I. The most common adverse effects of lefamulin include mild-to-moderate gastrointestinal-related events such as nausea and diarrhea. Lefamulin represents a safe and effective option for treating CABP in cases of antimicrobial resistance to first-line therapies, clinical failure, or intolerance/adverse effects to currently used agents. Clinical experience and ongoing clinical investigation will allow clinicians and antimicrobial stewardship programs to optimally use lefamulin in the treatment of CABP.

Modified Linggui Zhugan Decoction () Ameliorates Glycolipid Metabolism and Inflammation via PI3K-Akt/mTOR-S6K1/AMPK-PGC-1 α Signaling Pathways in Obese Type 2 Diabetic Rats.

To investigate the protective effects of modified Linggui Zhugan Decoction (, MLZD), a traditional Chinese medicine formula, on obese type 2 diabetes mellitus (T2DM) rats. Fifty Sprague-Dawley rats were randomly divided into 5 groups by a random number table, including normal, obese T2DM (ob-T2DM), MLZD low-dose [MLDZ-L, 4.625 g/(kg·d)], MLZD middle-dose [MLD-M, 9.25 g/(kg·d) ] and MLZD high-dose [MLD-H, 18.5 g/(kg·d)] groups, 10 rats in each group. After 4-week intervention, blood samples and liver, pancreas, muscle tissues were collected to assess the insulin resistance (IR), blood lipid, adipokines and inflammation cytokines. The alteration of phosphatidylinositol 3 kinase (PI3K)-protein kinase B (PKB or Akt)/the mammalian target of rapamycin (mTOR)-ribosome protein subunit 6 kinase 1 (S6K1 )/AMP-activated protein kinase (AMPK)-peroxisome proliferator-activated receptor gamma coactivator 1 alpha (PGC-1 α) pathways were also studied. MLZD dose-dependently reduced fasting blood glucose, fasting insulin, homeostasis model of assessment for IR index and increased insulin sensitive index compared with ob-T2DM rats (P<0.05). Similarly, total cholesterol, triglyceride, low-density lipoprotein cholesterol and free fatty acids were also decreased compared with ob-T2DM rats after 4-week treatment (P<0.05 or P<0.01). Improvements in adipokines and inflammatory cytokines were observed with a raised level of adiponectin and a reduced level of leptin, resistin, tumor necrosis factor-α and interleukin-6 (P<0.05 or P<0.01). MLZD regulated the PI3K-Akt/mTOR-S6K1/AMPK-PGC-1 α pathways and restored the tissue structure of liver and pancreas (P<0.05 or P<0.01). MLZD ameliorated glycolipid metabolism and inflammation, which may be attributed to the regulation of PI3K-Akt/mTOR-S6K1/AMPK-PGC-1 α pathways.

First report of Fusarium lacertarum causing Fusarium Head Blight on sorghum in North Carolina.

In August 2018, sorghum plants (Sorghum bicolor (L.) Moench) from research field plots in Wake County, North Carolina were observed with head blight symptoms including panicles with red lesions, visible mycelium, and necrosis. At the time of collection, all plants in research plots displayed symptoms of Fusarium head blight and panicles averaged 33% area affected by symptoms and signs. From these affected plants, samples (n = 5) were collected for further identification. Symptomatic grains were surface sterilized for one minute in 0.825% sodium hypochlorite solution and rinsed for one minute in sterile, deionized water. After drying on sterile paper towels, grains were plated onto water agar. Resulting fungal hyphal tips were then transferred to antibiotic-amended potato dextrose agar (PDA) and incubated at 25oC. Cultures were incubated for 3 to 5 days. Isolates had abundant white hyphae accompanied with peach-colored pigment production. Macroconidia with 5-6 septations were 23.47 ± 7.74 µm long and 3.47 ± 0.66 µm wide with foot-shaped basal cells, tapering to hooked apical cells. Chlamydospores were present in chains but microconidia were not present. Morphological species recognition (MSR) criteria tentatively identified the isolate as Fusarium lacertarum Subrahm., in the Fusarium incarnatum-equiseti species complex (FIESC) using characteristics described by Leslie and Summerell 2006. Molecular characterization using translation elongation factor 1α (TEF-1 α, primers EF1 and EF2 from O'Donnell et al. 1998), β tubulin (TUB2, primers T1 and T22 from O'Donnell and Cigelnik 1997), and ribosomal protein subunit II (RPB2, primers 5F2 and 11AR from Cerón-Bustamante et al. 2018) was conducted to confirm morphological identification. DNA from the hyphae of pure cultures was extracted using the DNeasy PowerSoil DNA extraction kit according to manufacturer's guidelines. DNA amplification conditions followed the protocols for each primer set (O'Donnell et al. 1998; O'Donnell and Cigelnik 1997; Cerón-Bustamante et al. 2018). BLASTn analysis of TEF-1α (Isolate Accession MT149915, 573bp) alignment had 99.8% identity to F. lacertarum (NCBI accession: JF740828), TUB2 (Isolate Accession MT149914, 1,183bp) alignment had 99.3% identity to F. equiseti (NCBI accession: KJ396338), and RPB2 (Isolate Accession MT184173, 1,538bp) concatenated sequences had 95.3% identity to F. lacertarum (NCBI accession: MH582185). The TUB2 region most closely aligns to F. equiseti, which is likely due to an absence of TUB2 sequences labeled for F. lacertarum in the NCBI database. Pathogenicity was confirmed by spray-inoculating Southern Harvest 80G4 sorghum panicles (n = 9) at anthesis with four ml of conidial suspension (3.3×104 conidia/ml). Control plants (n = 9) were sprayed with sterile water. Plastic bags were placed around panicles for 24 hours to ensure moist conditions during the infection period. Plants were maintained in a greenhouse under a 12-hour light cycle and fertilized bi-weekly with 20-20-20 fertilizer. Symptoms were observed on inoculated panicles after 14 days, and the F. lacertarum isolate was recovered from inoculated plants and confirmed using methods described above. Fusarium spp. were not re-isolated from non-inoculated control plants. Members of FIESC are known to contribute to the Fusarium Head Blight disease complex and may be capable of producing mycotoxins associated with infections (Lincy et al. 2011; Marin et al. 2012; Moretti 2017); however, mycotoxin characterization in F. lacertarum has not been characterized. To our knowledge, this is the first report of F. lacertarum causing disease to sorghum in North Carolina and the United States. Fusarium lacertarum may cause impactful losses to sorghum producers due to direct yield and quality losses by the pathogen as well as the potential for mycotoxins to impact trade.

Calmodulin inhibitors improve erythropoiesis in Diamond-Blackfan anemia.

Diamond-Blackfan anemia (DBA) is a rare hematopoietic disease characterized by a block in red cell differentiation. Most DBA cases are caused by mutations in ribosomal proteins and characterized by higher than normal activity of the tumor suppressor p53. Higher p53 activity is thought to contribute to DBA phenotypes by inducing apoptosis during red blood cell differentiation. Currently, there are few therapies available for patients with DBA. We performed a chemical screen using zebrafish ribosomal small subunit protein 29 (rps29) mutant embryos that have a p53-dependent anemia and identified calmodulin inhibitors that rescued the phenotype. Our studies demonstrated that calmodulin inhibitors attenuated p53 protein amount and activity. Treatment with calmodulin inhibitors led to decreased p53 translation and accumulation but does not affect p53 stability. A U.S. Food and Drug Administration-approved calmodulin inhibitor, trifluoperazine, rescued hematopoietic phenotypes of DBA models in vivo in zebrafish and mouse models. In addition, trifluoperazine rescued these phenotypes in human CD34+ hematopoietic stem and progenitor cells. Erythroid differentiation was also improved in CD34+ cells isolated from a patient with DBA. This work uncovers a potential avenue of therapeutic development for patients with DBA.

Comprehensive proteomic analysis revealing multifaceted regulatory network of the xero-halophyte Haloxylon salicornicum involved in salt tolerance

Haloxylon salicornicum is a xero-halophyte which grow predominantly in dry saline areas. However, the proteomic approach for revealing the regulatory network involved in salt adaptation of this important xerohalophyte has not been studied so far. In the present investigation, the label-free quantitative proteomic analysis was carried out in shoot of H. salicornicum to get an insight into the functional network of proteins involved in salt tolerance. Comparative proteomic analysis in control and salt treated plants of H. salicornicum by nano-ESI-LC–MS and MS/MS, and data base searching led to the identification of 723 proteins. Pathway enrichment analysis by KEGG uncovered various biological pathways to which salinity induced differentially regulated proteins are involved. In H. salicornicum, out of 723 identified proteins, 187 proteins were differentially regulated in response to salinity. In addition to significant up-regulation of stress responsive proteins, other proteins involved in carbohydrate metabolism, TCA cycle, protein synthesis, antioxidative defense systems, energy transfer, ion transport, nucleotide binding, and proteosomal proteins also significantly up-regulated under salinity in H. salicornicum. The major photosynthetic proteins up-regulated were RuBisCo, D1 protein, photosystem II-CP47, and cytochrome b599. TCA cycle component proteins such as citrate synthase, succinate dehydrogenase, and malate dehydrogenase upregulated indicating their significant roles in providing vital energy for salinity tolerance. Salinity induced higher expressions of ion transporters in H. salicornicum suggest efficient compartmentalization of toxic sodium ions. In addition, up-regulation of antioxidative defense system can be correlated with effective scavenging of salinity induced ROS, hence imparting salt tolerance. In H. salicornicum, protein synthesis was boosted under salinity as confirmed from the salinity-induced up-regulation of the ribosome associated proteins. Salinity induced significantly changed proteins of the ribosomal pathway include ribosomal protein components such as elongation factor-Tu (EF-Tu), initiation factor 1 and 2 (IF1, 2), Rpo cluster C and B, etc. Functional integrity of protein synthesis machinery in H. salicornicum is maintained under high salinity by higher abundance of ribosomal subunit proteins in NaCl-treated plants. We assume that consistent energy supply by the up-regulation of TCA cycle along with uninterrupted protein synthesis and maintenance of structural integrity of the photosynthetic machinery are the primary mechanism of salinity tolerance of H. salicornicum. In the present study, we comprehensively elucidated possible mechanisms associated with systematic salt tolerance of H. salicornicum employing proteomic approach. The information from this study will contribute to the genetic improvement of crop plants that can be grown in saline marginal lands.

Cryo-electron microscopy structure of the 70S ribosome from Enterococcus faecalis

Enterococcus faecalis is a gram-positive organism responsible for serious infections in humans, but as with many bacterial pathogens, resistance has rendered a number of commonly used antibiotics ineffective. Here, we report the cryo-EM structure of the E. faecalis 70S ribosome to a global resolution of 2.8 Å. Structural differences are clustered in peripheral and solvent exposed regions when compared with Escherichia coli, whereas functional centres, including antibiotic binding sites, are similar to other bacterial ribosomes. Comparison of intersubunit conformations among five classes obtained after three-dimensional classification identifies several rotated states. Large ribosomal subunit protein bL31, which forms intersubunit bridges to the small ribosomal subunit, assumes different conformations in the five classes, revealing how contacts to the small subunit are maintained throughout intersubunit rotation. A tRNA observed in one of the five classes is positioned in a chimeric pe/E position in a rotated ribosomal state. The 70S ribosome structure of E. faecalis now extends our knowledge of bacterial ribosome structures and may serve as a basis for the development of novel antibiotic compounds effective against this pathogen.

Metformin-induced suppression of Nemo-like kinase improves erythropoiesis in preclinical models of Diamond–Blackfan anemia through induction of miR-26a

Diamond-Blackfan anemia (DBA) results from haploinsufficiency of ribosomal protein subunits in hematopoietic progenitors in the earliest stages of committed erythropoiesis. Nemo-like kinase (NLK) is chronically hyperactivated in committed erythroid progenitors and precursors in multiple human and murine models of DBA. Inhibition of NLK activity and suppression of NLK expression both improve erythroid expansion in these models. Metformin is a well-tolerated drug for type 2 diabetes with multiple cellular targets. Here we demonstrate that metformin improves erythropoiesis in human and zebrafish models of DBA. Our data indicate that the effects of metformin on erythroid proliferation and differentiation are mediated by suppression of NLK expression through induction of miR-26a, which recognizes a binding site within the NLK 3' untranslated region (3'UTR) to facilitate transcript degradation. We propose that induction of miR-26a is a potentially novel approach to treatment of DBA and could improve anemia in DBA patients without the potentially adverse side effects of metformin in a DBA patient population.

Site-specific and substrate-specific control of accurate mRNA editing by a helicase complex in trypanosomes.

Trypanosome U-insertion/deletion RNA editing in mitochondrial mRNAs involves guide RNAs (gRNAs) and the auxiliary RNA editing substrate binding complex (RESC) and RNA editing helicase 2 complex (REH2C). RESC and REH2C stably copurify with editing mRNAs but the functional interplay between these complexes remains unclear. Most steady-state mRNAs are partially edited and include misedited “junction” regions that match neither pre-mRNA nor fully edited transcripts. Editing specificity is central to mitochondrial RNA maturation and function, but its basic control mechanisms remain unclear. Here we applied a novel nucleotide-resolution RNA-seq approach to examine ribosomal protein subunit 12 (RPS12) and ATPase subunit 6 (A6) mRNA transcripts. We directly compared transcripts associated with RESC and REH2C to those found in total mitochondrial RNA. RESC-associated transcripts exhibited site-preferential enrichments in total and accurate edits. REH2C loss-of-function induced similar substrate-specific and site-specific editing effects in total and RESC-associated RNA. It decreased total editing primarily at RPS12 5′ positions but increased total editing at examined A6 3′ positions. REH2C loss-of-function caused site-preferential loss of accurate editing in both transcripts. However, changes in total or accurate edits did not necessarily involve common sites. A few 5′ nucleotides of the initiating gRNA (gRNA-1) directed accurate editing in both transcripts. However, in RPS12, two conserved 3′-terminal adenines in gRNA-1 could direct a noncanonical 2U-insertion that causes major pausing in 3′–5′ progression. In A6, a noncanonical sequence element that depends on REH2C in a region normally targeted by the 3′ half of gRNA-1 may hinder early editing progression. Overall, we defined transcript-specific effects of REH2C loss.

Transcriptomic comparison reveals modifications in gene expression, photosynthesis, and cell wall in woody plant as responses to external pH changes

Soil acidification is one of the crucial global environmental problems, affecting sustainable land use, crop yield, and ecosystem stability. Previous research reported the tolerance of crops to acid soil stress. However, the molecular response of woody plant to acid conditions remains largely unclear. Rhododendron L. is a widely distributed woody plant genus and prefers to grow in acidic soils. Herein, weighted gene coexpression network analysis was performed on R. protistum var. giganteum seedlings subjected to five pH treatments (3.5, 4.5, 5.5, 6.0, 7.0), and their ecophysiological characteristics were determined for the identification of their molecular responses to acidic environments. Through pairwise comparison, 855 differentially expressed genes (DEGs) associated with photosynthesis, cell wall, and phenylpropanoid metabolism were identified. Most of the DEGs related to photosynthesis and cell wall were up-regulated after pH 4.5 treatment. Results implied that the species improves its photosynthetic abilities and changes its cell wall characteristics to adapt to acidic conditions. Weighted gene co-expression network analyses showed that most of the hub genes were annotated to the biosynthetic pathways of ribosomal proteins and photosynthesis. Expression pattern analysis showed that genes encoding subunit ribosomal proteins decreased at pH 7.0 treatment, suggesting that pH 7.0 treatment led to cell injury in the seedlings. The species regulates protein synthesis in response to high pH stress (pH 7.0). The present study revealed the molecular response mechanism of woody plant R. protistum var. giganteum to acid environments. These findings can be useful in enriching current knowledge of how woody species adapt to soil acidification under global environmental changes.

Heavy ion radiation-induced DNA damage mediates apoptosis via the Rpl27a-Rpl5-MDM2-p53/E2F1 signaling pathway in mouse spermatogonia.

The risk of exposure to ionizing radiation (IR) environments has increased with the development of nuclear technology. IR exposure induces excessive apoptosis of the spermatogonia, which leads to male infertility. Spermatogonia apoptosis may be involved in ribosomal stress triggered by DNA damage following exposure to IR because ribosomal proteins (RPs) directly interact with mouse double minute 2 homolog (MDM2) to induce apoptosis. This study aimed to use comparative proteomics and transcriptomics approach to screen the differential RPs and ribosomal mRNAs in mouse testes following high linear energy transfer (LET) carbon ion radiation (CIR). The expression of ribosomal large subunit protein 27a (Rpl27a) decreased at both protein and mRNA levels in the spermatogonia in vivo. After 6h of CIR, the immunofluorescence signal of 8-oxo-dG and phosphorylated ataxia-telangiectasia-mutated protein (ATM)/histone H2Ax increased, but that of Rpl27a decreased in the spermatogonia of p53 wild-type and knockout mouse testes. Moreover, the nucleolin was scattered throughout the nucleoplasm after CIR. These results suggested that CIR-induced DNA damage might trigger ribosomal stress, and the reduction in the expression of Rpl27a was associated with DNA damage in the spermatogonia. Similarly, in vitro, the immunofluorescence signal of 8-oxo-dG increased in the GC-1cells after CIR. Moreover, the expression of Rpl27a was regulated by DNA damage because the co-transfection of ATM and Rpl27a or inhibition of ATM-treated CIR could restore the expression of Rpl27a. Furthermore, the reduction in the expression of Rpl27a led to weakened binding of E2F transcription factor 1 (E2F1) and p53 to MDM2, causing p53 activation and E2F1 degradation in p53 wild-type and knockdown GC-1cells. This study proposed that heavy ion radiation-induced DNA damage mediated spermatogonia apoptosis via the Rpl27a-Rpl5-MDM2-p53/E2F1 signaling pathway. The results provided the underlying molecular mechanisms of spermatogonia apoptosis following exposure to high LET radiation.

Ribosomal protein S11 influences glioma response to TOP2 poisons.

Topoisomerase II poisons are one of the most common class of chemotherapeutics used in cancer. We and others had shown that a subset of glioblastomas (GBM), the most malignant of all primary brain tumors in adults, are responsive to TOP2 poisons. To identify genes that confer susceptibility to this drug in gliomas, we performed a genome-scale CRISPR knockout screen with etoposide. Genes involved in protein synthesis and DNA damage were implicated in etoposide susceptibility. To define potential biomarkers for TOP2 poisons, CRISPR hits were overlapped with genes whose expression correlates with susceptibility to this drug across glioma cell lines, revealing ribosomal protein subunit RPS11, 16, 18 as putative biomarkers for response to TOP2 poisons. Loss of RPS11 led to resistance to etoposide and doxorubicin and impaired the induction of pro-apoptotic gene APAF1 following treatment. The expression of these ribosomal subunits was also associated with susceptibility to TOP2 poisons across cell lines from gliomas and multiple other cancers.

The Progression of Acute Myeloid Leukemia from First Diagnosis to Chemoresistant Relapse: A Comparison of Proteomic and Phosphoproteomic Profiles.

Acute myeloid leukemia (AML) is an aggressive hematological malignancy. Nearly 50% of the patients who receive the most intensive treatment develop chemoresistant leukemia relapse. Although the leukemogenic events leading to relapse seem to differ between patients (i.e., regrowth from a clone detected at first diagnosis, progression from the original leukemic or preleukemic stem cells), a common characteristic of relapsed AML is increased chemoresistance. The aim of the present study was to investigate at the proteomic level whether leukemic cells from relapsed patients present overlapping molecular mechanisms that contribute to this chemoresistance. We used liquid chromatography–tandem mass spectrometry (LC–MS/MS) to compare the proteomic and phosphoproteomic profiles of AML cells derived from seven patients at the time of first diagnosis and at first relapse. At the time of first relapse, AML cells were characterized by increased levels of proteins important for various mitochondrial functions, such as mitochondrial ribosomal subunit proteins (MRPL21, MRPS37) and proteins for RNA processing (DHX37, RNA helicase; RPP40, ribonuclease P component), DNA repair (ERCC3, DNA repair factor IIH helicase; GTF2F1, general transcription factor), and cyclin-dependent kinase (CDK) activity. The levels of several cytoskeletal proteins (MYH14/MYL6/MYL12A, myosin chains; VCL, vinculin) as well as of proteins involved in vesicular trafficking/secretion and cell adhesion (ITGAX, integrin alpha-X; CD36, platelet glycoprotein 4; SLC2A3, solute carrier family 2) were decreased in relapsed cells. Our study introduces new targetable proteins that might direct therapeutic strategies to decrease chemoresistance in relapsed AML.

A simple, rapid typing method for Streptococcus agalactiae based on ribosomal subunit proteins by MALDI-TOF MS

Streptococcus agalactiae (Group B Streptococcus, GBS), is a frequent human colonizer and a leading cause of neonatal meningitis as well as an emerging pathogen in non-pregnant adults. GBS possesses a broad animal host spectrum, and recent studies proved atypical GBS genotypes can cause human invasive diseases through animal sources as food-borne zoonotic infections. We applied a MALDI-TOF MS typing method, based on molecular weight variations of predefined 28 ribosomal subunit proteins (rsp) to classify GBS strains of varying serotypes into major phylogenetic lineages. A total of 249 GBS isolates of representative and varying capsular serotypes from patients and animal food sources (fish and pig) collected during 2016–2018 in Hong Kong were analysed. Over 84% (143/171) noninvasive carriage GBS strains from patients were readily typed into 5 globally dominant rsp-profiles. Among GBS strains from food animals, over 90% (57/63) of fish and 13% (2/15) of pig GBS matched with existing rsp-profiles, while the remainder were classified into two novel rsp-profiles and we failed to assign a fish strain into any cluster. MALDI-TOF MS allowed for high-throughput screening and simultaneous detection of novel, so far not well described GBS genotypes. The method shown here is rapid, simple, readily transferable and adapted for use in a diagnostic microbiology laboratory with potential for the surveillance of emerging GBS genotypes with zoonotic potential.