Ribosomal Protein Levels Research Articles

Response of the biomining Acidithiobacillus ferrooxidans to high cadmium concentrations

Cadmium is a heavy metal present in contaminated soils. It has no biological role but when entering cells generates DNA damage, overexpression of stress response proteins and misfolded proteins, amongst other deleterious effects. Acidithiobacillus ferrooxidans is an acidophilic bacterium resisting high concentrations of heavy metals such as cadmium. This is important for industrial bioleaching processes where Cd+2 concentrations can be 5–100 mM. Cadmium resistance mechanisms in these microorganisms have not been fully characterized. A. ferrooxidans ATCC 53993 contains genes coding for possible metal resistance determinants such as efflux systems: P-type ATPases, RND transporters and cation diffusion facilitators. In addition, it has extra copies of these genes in its exclusive genomic island (GI). Several of these putative genes were characterized in the present report by determining their transcriptional expression profiles and functionality. Moreover, an iTRAQ proteomic analysis was carried out to explore new cadmium resistance determinants in this bacterium. Changes in iron oxidation components, upregulation of transport proteins and variations in ribosomal protein levels were seen. Finally, increased concentrations of exclusive putative cadmium ATPases present in strain ATCC 53993 GI and other non-identified proteins such as Lferr_0210, forming part of a possible operon, could explain its extreme cadmium resistance. SignificanceCadmium is a very toxic heavy metal present in mining operations and contaminated environments, it can affect all living organisms, including humans. Therefore, it is important to know the resistance mechanisms of bacteria highly resistant to this metal. These microorganisms in turn, can be used to bioremediate more efficiently environments highly polluted with metals. The results obtained suggest A. ferrooxidans strain ATCC 53993 can be an efficient bacterium to remove cadmium, copper and other metals from contaminated sites.

Small and Large Ribosomal Subunit Deficiencies Lead to Distinct Gene Expression Signatures that Reflect Cellular Growth Rate

Levels of the ribosome, the conserved molecular machine that mediates translation, are tightly linked to cellular growth rate. In humans, ribosomopathies are diseases associated with cell-type-specific pathologies and reduced ribosomal protein (RP) levels. Because gene expression defects resulting from ribosome deficiency have not yet been experimentally defined, we systematically probed mRNA, translation, and protein signatures that were either unlinked from or linked to cellular growth rate in RP-deficient yeast cells. Ribosome deficiency was associated with altered translation of gene subclasses, and profound general secondary effects of RP loss on the spectrum of cellular mRNAs were seen. Among these effects, growth-defective 60S mutants increased synthesis of proteins involved in proteasome-mediated degradation, whereas 40S mutants accumulated mature 60S subunits and increased translation of ribosome biogenesis genes. These distinct signatures of protein synthesis suggest intriguing and currently mysterious differences in the cellular consequences of deficiency for small and large ribosomal subunits.

Ribosomal flavours: an acquired taste for specific mRNAs?

The regulation of translation is critical in almost every aspect of gene expression. Nonetheless, the ribosome is historically viewed as a passive player in this process. However, evidence is accumulating to suggest that variations in the ribosome can have an important influence on which mRNAs are translated. Scope for variation is provided via multiple avenues, including heterogeneity at the level of both ribosomal proteins and ribosomal RNAs and their covalent modifications. Together, these variations provide the potential for hundreds, if not thousands, of flavours of ribosome, each of which could have idiosyncratic preferences for the translation of certain messenger RNAs. Indeed, perturbations to this heterogeneity appear to affect specific subsets of transcripts and manifest as cell-type-specific diseases. This review provides a historical perspective of the ribosomal code hypothesis, before outlining the various sources of heterogeneity, their regulation and functional consequences for the cell.

Critical Role for Saccharomyces cerevisiae Asc1p in Translational Initiation at Elevated Temperatures.

The eukaryotic ribosomal protein RACK1/Asc1p is localized to the mRNA exit channel of the 40S subunit but lacks a defined role in mRNA translation. Saccharomyces cerevisiae deficient in ASC1 exhibit temperature-sensitive growth. Using this null mutant, potential roles for Asc1p in translation and ribosome biogenesis are evaluated. At the restrictive temperature the asc1Δ null mutant has reduced polyribosomes. To test the role of Asc1p in ribosome stability, cryo-EM is used to examine the structure of 80S ribosomes in an asc1Δ yeast deletion mutant at both the permissive and nonpermissive temperatures. CryoEM indicates that loss of Asc1p does not severely disrupt formation of this complex structure. No defect is found in rRNA processing in the asc1Δ null mutant. A proteomic approach is applied to survey the effect of Asc1p loss on the global translation of yeast proteins. At the nonpermissive temperature, the asc1Δ mutant has reduced levels of ribosomal proteins and other factors critical for translation. Collectively, these results are consistent with recent observations suggesting that Asc1p is important for ribosome occupancy of short mRNAs. The results show the Asc1 ribosomal protein is critical in translation during heat stress.

ITRAQ-Based Global Proteomic Analysis of Salmonella enterica Serovar Typhimurium in Response to Desiccation, Low Water Activity, and Thermal Treatment.

In this study, the changes in the global proteome of Salmonella in response to desiccation and thermal treatment were investigated by using an iTRAQ multiplex technique. A Salmonella enterica serovar Typhimurium strain was dried, equilibrated at high (1.0) and low (0.11) water activity (aw), and thermally treated at 75°C. The proteomes were characterized after every treatment. The proteomes of the different treatments differed in the expression of 175 proteins. On the basis of their proteomic expression profiles, the samples were clustered into two major groups, namely, "dry" samples and "moist" samples. The groups had different levels of proteins involved in DNA synthesis and transcription and in metabolic reactions, indicating that cells under either of the aw conditions need to strictly control energy metabolism, the rate of replication, and protein synthesis. The proteins with higher expression levels in moist samples were flagellar proteins (FlgEFGH), membrane proteins, and export systems (SecF, SecD, the Bam complex), as well as stress response proteins, suggesting that rehydration can trigger stress responses in moist cells. Dry samples had higher levels of ribosomal proteins, indicating that ribosomal proteins might be important for additional regulation of the cellular response, even when the synthesis of proteins is slowed down. At both aws, no differences in protein expression were observed between the thermally treated samples and the nonheated cells. In conclusion, our study indicates that the preadaptation to a dry condition was linked to increased thermal tolerance, while reversion from a dry state to a moist state induced a significant change in protein expression, possibly linked to the observed loss of thermal tolerance.IMPORTANCESalmonella enterica is able to survive in dry environments for very long periods. While it is well known that the initial exposure to desiccation is fundamental to trigger thermal tolerance in this organism, the specific physiological and molecular processes involved in this cross-protection phenomenon have not been fully characterized. Several studies have focused on the low-aw transcriptome of this pathogen when inoculated in different food matrices or on abiotic surfaces, but proteomic analyses have not been reported in the literature. Our study investigated the changes in proteomic expression in Salmonella enterica serovar Typhimurium during desiccation, exposure to low aw, and thermal treatment. A better knowledge of the systems involved in the response to desiccation and thermal tolerance, as well as a better understanding of their interplay, is fundamental to identify the most effective combination of interventions to prevent Salmonella's contamination of foods.

Lysine acetylation stoichiometry and proteomics analyses reveal pathways regulated by sirtuin 1 in human cells

Lysine acetylation is a widespread posttranslational modification affecting many biological pathways. Recent studies indicate that acetylated lysine residues mainly exhibit low acetylation occupancy, but challenges in sample preparation and analysis make it difficult to confidently assign these numbers, limiting understanding of their biological significance. Here, we tested three common sample preparation methods to determine their suitability for assessing acetylation stoichiometry in three human cell lines, identifying the acetylation occupancy in more than 1,300 proteins from each cell line. The stoichiometric analysis in combination with quantitative proteomics also enabled us to explore their functional roles. We found that higher abundance of the deacetylase sirtuin 1 (SIRT1) correlated with lower acetylation occupancy and lower levels of ribosomal proteins, including those involved in ribosome biogenesis and rRNA processing. Treatment with the SIRT1 inhibitor EX-527 confirmed SIRT1's role in the regulation of pre-rRNA synthesis and processing. Specifically, proteins involved in pre-rRNA transcription, including subunits of the polymerase I and SL1 complexes and the RNA polymerase I-specific transcription initiation factor RRN3, were up-regulated after SIRT1 inhibition. Moreover, many protein effectors and regulators of pre-rRNA processing needed for rRNA maturation were also up-regulated after EX-527 treatment with the outcome that pre-rRNA and 28S rRNA levels also increased. More generally, we found that SIRT1 inhibition down-regulates metabolic pathways, including glycolysis and pyruvate metabolism. Together, these results provide the largest data set thus far of lysine acetylation stoichiometry (available via ProteomeXchange with identifier PXD005903) and set the stage for further biological investigations of this central posttranslational modification.

Humanized mutant FUS drives progressive motor neuron degeneration without aggregation in 'FUSDelta14' knockin mice.

Mutations in FUS are causative for amyotrophic lateral sclerosis with a dominant mode of inheritance. In trying to model FUS-amyotrophic lateral sclerosis (ALS) in mouse it is clear that FUS is dosage-sensitive and effects arise from overexpression per se in transgenic strains. Novel models are required that maintain physiological levels of FUS expression and that recapitulate the human disease-with progressive loss of motor neurons in heterozygous animals. Here, we describe a new humanized FUS-ALS mouse with a frameshift mutation, which fulfils both criteria: the FUS Delta14 mouse. Heterozygous animals express mutant humanized FUS protein at physiological levels and have adult onset progressive motor neuron loss and denervation of neuromuscular junctions. Additionally, we generated a novel antibody to the unique human frameshift peptide epitope, allowing specific identification of mutant FUS only. Using our new FUSDelta14 ALS mouse-antibody system we show that neurodegeneration occurs in the absence of FUS protein aggregation. FUS mislocalization increases as disease progresses, and mutant FUS accumulates at the rough endoplasmic reticulum. Further, transcriptomic analyses show progressive changes in ribosomal protein levels and mitochondrial function as early disease stages are initiated. Thus, our new physiological mouse model has provided novel insight into the early pathogenesis of FUS-ALS.

High MRPS23 expression contributes to hepatocellular carcinoma proliferation and indicates poor survival outcomes.

Hepatocellular carcinoma is one of the most prevalent neoplasms and the leading cause of cancer-related mortality worldwide. Mitochondrial ribosomal protein S23 is encoded by a nuclear gene and participates in mitochondrial protein translation. Mitochondrial ribosomal protein S23 overexpression has been found in many types of cancer. In this study, we explored mitochondrial ribosomal protein S23 expression in primary hepatocellular carcinoma tissues compared with matched adjacent non-tumoral liver tissues using mitochondrial ribosomal protein S23 messenger RNA and protein levels collected from public databases and clinical samples. Immunohistochemistry was performed to analyze the relationship between mitochondrial ribosomal protein S23 and various clinicopathological features. The results indicated that mitochondrial ribosomal protein S23 was significantly overexpressed in hepatocellular carcinoma. High mitochondrial ribosomal protein S23 expression was correlated with the tumor size and tumor-metastasis-node stage. Moreover, patients with high mitochondrial ribosomal protein S23 expression levels presented poorer survival rates. Mitochondrial ribosomal protein S23 was an independent prognostic factor for survival, especially at the early stage of hepatocellular carcinoma. In addition, the downregulation of mitochondrial ribosomal protein S23 decreased the proliferation of hepatocellular carcinoma in vitro and in vivo. In conclusion, we verified for the first time that mitochondrial ribosomal protein S23 expression was upregulated in hepatocellular carcinoma. High mitochondrial ribosomal protein S23 levels can predict poor clinical outcomes in hepatocellular carcinoma, and this protein plays a key role in tumor proliferation. Therefore, mitochondrial ribosomal protein S23 may be a potential therapeutic target for hepatocellular carcinoma.

Heterogeneous Ribosomes Preferentially Translate Distinct Subpools of mRNAs Genome-wide

Emerging studies have linked the ribosome to more selective control of gene regulation. However, an outstanding question is whether ribosome heterogeneity at the level of core ribosomal proteins (RPs) exists and enables ribosomes to preferentially translate specific mRNAs genome-wide. Here, we measured the absolute abundance of RPs in translating ribosomes and profiled transcripts that are enriched or depleted from select subsets of ribosomes within embryonic stem cells. We find that heterogeneity in RP composition endows ribosomes with differential selectivity for translating subpools of transcripts, including those controlling metabolism, cell cycle, and development. As an example, mRNAs enriched in binding to RPL10A/uL1-containing ribosomes are shown to require RPL10A/uL1 for their efficient translation. Within several of these transcripts, this level of regulation is mediated, at least in part, by internal ribosome entry sites. Together, these results reveal a critical functional link between ribosome heterogeneity and the post-transcriptional circuitry of gene expression.

Genome-wide mRNA processing in methanogenic archaea reveals post-transcriptional regulation of ribosomal protein synthesis.

Unlike stable RNAs that require processing for maturation, prokaryotic cellular mRNAs generally follow an ‘all-or-none’ pattern. Herein, we used a 5΄ monophosphate transcript sequencing (5΄P-seq) that specifically captured the 5΄-end of processed transcripts and mapped the genome-wide RNA processing sites (PSSs) in a methanogenic archaeon. Following statistical analysis and stringent filtration, we identified 1429 PSSs, among which 23.5% and 5.4% were located in 5΄ untranslated region (uPSS) and intergenic region (iPSS), respectively. A predominant uridine downstream PSSs served as a processing signature. Remarkably, 5΄P-seq detected overrepresented uPSS and iPSS in the polycistronic operons encoding ribosomal proteins, and the majority upstream and proximal ribosome binding sites, suggesting a regulatory role of processing on translation initiation. The processed transcripts showed increased stability and translation efficiency. Particularly, processing within the tricistronic transcript of rplA-rplJ-rplL enhanced the translation of rplL, which can provide a driving force for the 1:4 stoichiometry of L10 to L12 in the ribosome. Growth-associated mRNA processing intensities were also correlated with the cellular ribosomal protein levels, thereby suggesting that mRNA processing is involved in tuning growth-dependent ribosome synthesis. In conclusion, our findings suggest that mRNA processing-mediated post-transcriptional regulation is a potential mechanism of ribosomal protein synthesis and stoichiometry.

Abstract B43: Global upregulation of ribosomal protein gene pathway in normal cerebellar progenitor development and in pathogenesis of sonic hedgehog subtype medulloblastomas

Abstract Co-regulated transcription of ribosomal protein genes (RPGs) is at the heart of efficient ribosome biogenesis; however, mechanisms that regulate RPG pathways in relation to normal vertebrate development and disease are not well understood. Granule progenitors in the external granule layer (EGL) of the developing mouse cerebellum show a sharp proliferative spike during the first two weeks after birth in a sonic hedgehog (Shh)-dependent manner. Medulloblastomas (MBs) constitute the most common malignant pediatric brain tumor originating in the cerebellum. In particular, Shh subtype MBs result from unregulated proliferation of granule progenitors, and understanding mechanisms underlying their proliferative capacity can provide insights into MB pathogenesis and therapeutics. Interestingly, increased ribosomal protein (RP) levels in MBs have been associated with poor patient survival. Here, we describe an unexpected role of global upregulation of the RPG pathway in normal cerebellar granule progenitor development and in pathogenesis of Shh subtype MBs. Using unbiased RNA-seq analysis, we detected global transcriptional upregulation of RPG pathway in mouse cerebellum during postnatal development compared to adults. In situ hybridization and immunohistochemistry of individual RPs demonstrated increased transcript and protein levels of individual RPs specifically in the EGL, respectively. Increased RP levels in EGL progenitors resulted predominantly by transcriptional mechanisms, as opposed to post-mitotic purkinje neurons showing selective upregulation of RPG protein but not transcript levels. Translational analysis of nascent polypeptide chains by incorporation of O-propargyl-puromycin by flow cytometry and immunofluorescence revealed increased protein translation in EGL with respect to rest of the neonatal cerebellum, suggesting functional ribosomal biogenesis in addition to global upregulation of RPG pathway. To examine if reducing RP levels affect normal tissue homeostasis, we analyzed brain and cerebellum development in a hypomorphic allele of an individual RP (Rpl24 Bst/+). Although RPG pathway was transcriptionally downegulated in Rpl24 Bst/+ neonatal cerebellum with respect to wild type, gross cerebellar morphology and granule neuron numbers in adult animals were not affected. In addition, neurogenesis in the subventricular zone of the lateral ventricle and subgranular zone of the dentate gyrus of Rpl24 Bst/+ mice were not affected. Thus, partial reduction of RPs does not impact upon normal brain development. Interestingly, Shh subtype MBs in mice demonstrated global upregulation of RPG transcripts and increased individual protein levels in comparison to postnatal cerebellum, which already has higher levels of RPG pathway compared to adults. Human Shh-MB samples also demonstrated higher RP levels with respect to normal cerebellar tissue. Analysis of RNA-seq datasets in TCGA suggested that global transcriptional upregulation of the RPG pathway is not a common feature of all human tumors, in particular only ~50% of tumor subtypes showed global upregulation. However, global upregulation of RPG pathway in Shh-MBs is remarkably significant both in the extent and severity of the increase. Interestingly, we observed reduced tumor size in mice with Rpl24 hypomorphism compared with control mice expressing only constitutively active Smoothened (SmoM2) in neural stem/progenitors, suggesting that the RPG pathway determines tumorigenic burden in Shh-MBs. Thus, unlike cerebellar progenitor development, Shh-MBs show dependency on global RPG transcription. Characterizing the RPG pathway in human MB subtypes with respect to patient prognosis and tumor recurrence, and/or therapeutic strategies targeting ribosomal biogenesis might be transformative in developing effective Shh-MB therapies. Citation Format: Issei Shimada, Somatilaka Bandarigoda, John Shelton, Zhenyu Xuan, Veena Rajaram, Saikat Mukhopadhyay. Global upregulation of ribosomal protein gene pathway in normal cerebellar progenitor development and in pathogenesis of sonic hedgehog subtype medulloblastomas. [abstract]. In: Proceedings of the AACR Special Conference on Translational Control of Cancer: A New Frontier in Cancer Biology and Therapy; 2016 Oct 27-30; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2017;77(6 Suppl):Abstract nr B43.

Proteomic Profiling of the Microsomal Root Fraction: Discrimination of Pisum sativum L. Cultivars and Identification of Putative Root Growth Markers.

Legumes are a large and economically important family, containing a variety of crop plants. Alongside different cereals, some fruits, and tropical roots, a number of leguminosae evolved for millennia as crops with human society. One of these legumes is Pisum sativum L., the common garden pea. In the past, breeding has been largely selective on improved above-ground organs. However, parameters, such as root-growth, which determines acquisition of nutrients and water, have largely been underestimated. Although the genome of P. sativum is still not fully sequenced, multiple proteomic studies have been published on a variety of physiological aspects in the last years. The presented work focused on the connection between root length and the influence of the microsomal root proteome of four different pea cultivars after five days of germination (cultivar Vroege, Girl from the Rhineland, Kelvedon Wonder, and Blauwschokker). In total, 60 proteins were identified to have significantly differential abundances in the four cultivars. Root growth of five-days old seedlings and their microsomal proteome revealed a similar separation pattern, suggesting that cultivar-specific root growth performance is explained by differential membrane and ribosomal protein levels. Hence, we reveal and discuss several putative root growth protein markers possibly playing a key role for improved primary root growth breeding strategies.

Impact of carbohydrate restriction in the context of obesity on prostate tumor growth in the Hi-Myc transgenic mouse model.

IntroductionPreviously, we showed that carbohydrate restriction with calorie restriction slowed tumor growth in xenograft mouse prostate cancer models. Herein, we examined the impact of carbohydrate restriction without calorie restriction on tumor development within the context of diet-induced obesity in the Hi-Myc transgenic mouse model of prostate cancer.MethodsMice were randomized at 5 weeks of age to ad libitum Western diet (WD; 40% fat, 42% carbohydrate; n=39) or ad libitum no carbohydrate ketogenic diet (NCKD; 82% fat, 1% carbohydrate; n=44). At age 3 or 6 months, mice were sacrificed, prostates weighed and prostate histology, proliferation, apoptosis, and macrophage infiltration evaluated by H&E, Ki67, TUNEL and F4/80 staining, respectively. Body composition was assessed by DEXA, serum cytokines measured using multiplex, and Akt/mTOR signaling assessed by Western.ResultsCaloric intake was higher in the NCKD group, resulting in elevated body weights at 6 months of age, relative to the WD group (45g vs. 38g; p=0.008). Despite elevated body weights, serum MCP-1 and IL-1α levels were lower in NCKD versus WD mice (p=0.046 and p=0.118, respectively), and macrophage infiltration was reduced in prostates of NCKD versus WD mice (p=0.028). Relative Akt phosphorylation and phospho-S6 ribosomal protein levels were reduced in prostates of NCKD versus WD mice. However, while mice randomized to NCKD had smaller prostates after adjustment for body weight at 3 and 6 months (p=0.004 and p=0.002, respectively), NCKD mice had higher rates of adenocarcinoma at 6 months compared to WD mice (100% vs. 80%, p=0.04).ConclusionsDespite higher caloric intake and elevated body weights, carbohydrate restriction lowered serum MCP-1 levels, reduced prostate macrophage infiltration, reduced prostate weight, but failed to slow adenocarcinoma development. Together, these data suggest that although carbohydrate restriction within the context of obesity may reduce obesity-associated systemic inflammation, it is not sufficient to counteract obesity-associated tumor growth.

A RACK1-like protein regulates hyphal morphogenesis, root entry and in vivo virulence in Verticillium dahliae

To identify key genes expressed in Verticillium dahliae in early stages of infection of cotton roots, spore suspensions of eight V. dahliae isolates with different virulence levels were induced by cotton roots and genes expressed in these isolates during the early stages of infection were profiled. A gene that was differentially expressed between highly and less virulent strains was identified. Cloning and bioinformatics analysis of the gene suggested that it belongs to the putative Gβ-like/RACK1 protein family, and has seven WD40 domains. Targeted deletion of the gene revealed that it controls a number of growth-related phenotypes, including conidia and microsclerotia production, normal spore germination and hyphal development. RACK1 is a component of eukaryotic ribosomes, and here we found by qRT-PCR that disruption of RACK1 in V. dahliae (designated VdRACK1) significantly altered the transcriptional levels of other ribosomal proteins, suggesting possible global effects of VdRACK1 deletion on the protein translation of other genes. VdRACK1-null mutants lost the ability to penetrate intact cotton roots. However, the mutant strain was able to infect root-wounded cotton plants and, intriguingly, resulted in a hypervirulent phenotype, implicating a role for VdRACK1 in the restriction of rampant growth within the plant.

PP2A Balances Glucose Metabolism and Foxo Activation to Maintain Cellular Redox Homeostasis in Acute Lymphoblastic Leukemia

Introduction: Protein phosphatase 2A (PP2A) is a Ser/Thr phosphatase negatively regulates a diverse set of signaling pathways promoting tumor growth. While PP2A functions as tumor suppressor in multiple types of cancer, here, we demonstrate an unexpected pro-survival role of PP2A in pre-B acute lymphoblastic leukemia (ALL) cells. By calibrating PI3K-AKT-mTOR signaling strength, PP2A regulates glycolysis rate and thereby balances energy demands against anti-oxidant protection of pre-B ALL cells. In addition, PP2A reinstates activity of FOXO factors by dephosphorylation and thereby enables the anti-oxidant function of FOXO1 and FOXO3. Consistent with previous studies, we find PP2A is dispensable for the survival of myeloid leukemia cells which indicates a lineage-specific role of PP2A. Combined with genetic study and small molecule inhibitor, we verify that regulation in patient-derived xenografts and highlight PP2A as a therapeutic target in pre-B ALL.Results: Consistent with a divergent role of PP2A in pre-B ALL (compared to CML), we found that high mRNA levels of PP2A subunits at the time of diagnosis predict poor outcome of children (COG P9906; n=207) and adults (ECOG 2993; n=215) with ALL. Consistent with these findings, mutations in PP2A subunits are extremely rare in B cell malignancies but relatively common in solid tumors and myeloid malignancies (COSMIC). We therefore, studied the function of PP2A in a genetic mouse model for Cre-induced deletion of Ppp2r1a in BCR-ABL1 (Ph+) ALL. Inducible activation of Cre reduced protein expression of the targeted PP2A subunit A and the catalytic subunit C, which leads to near-complete loss of PP2A phosphatase activity. Conversely, Cre-mediated deletion increased phosphorylation levels of FoxO1, FoxO3a, p70S6K and S6 ribosomal protein, which indicated elevated PI3K-Akt-mTOR signaling. Acute deletion of Ppp2r1afl/fl in B cell-lineage ALL cells dramatically affected survival and colony formation, both of which could be rescued by overexpression of wildtype PP2A. PI3K and mTOR inhibitors also have rescue effect on PP2A deficient ALL cells in growth-competition assay. Luciferase-labeled PP2A-deleted ALL cells showed reduced cell growth and leukemia progression after being transplanted into recipient mice. However, Cre-mediated deletion had no deleterious effects in a Ppp2r1afl/fl CML model. This lineage-specific role of PP2A was verified by inducible CEBPα expression to reprogram B cell lineage ALL cells into myeloid cells.Interestingly, inducible deletion of PP2A caused profound imbalances of glucose metabolism in Ph+ ALL but not in CML cells. Upon PP2A-deletion, ALL cells showed higher glycolytic flux shunted into lactate rather than NADPH production. By employing glucose flux metabolic profiling assay using [1,2-13C2]-D-glucose tracer, we found elevated glycolysis and repressed pentose phosphate pathway (PPP) flux in PP2A-deleted pre-B ALL cells. Lower NADPH/NADP ratio and higher reactive oxygen species level in PP2A-deleted pre-B ALL cells, together with decreased anti-oxidant gene expression, increased DNA damage, including H2AX phosphorylation and p53 expression. The mechanistic role of ROS downstream of PP2A was supported by a strong rescue effect of overexpression of the antioxidant catalase in PP2A-deleted cells. The unexpected role of PP2A in Ph+ ALL was further validated by CRISPR-Cas9 mediated disruption of PPP2R1A in ALL xenografts derived from three patients. In addition, a PP2A specific inhibitor LB-100 (in clinical trial for solid tumors) was employed to pharmacologically inhibit PP2A activity. Low micromolar concentrations of LB-100 induced cell death in patient-derived ALL xenografts in parallel with ROS-accumulation and increased S6 and H2AX phosphorylation.Conclusion: Here we revealed an unexpected role of PP2A in maintaining redox homeostasis in pre-B ALL cells. By regulating AKT-mTOR signaling, PP2A keeps the balance of glycolysis and PPP to meet the energy demands of pre-B ALL cells and avoids extreme levels of oxidative stress. We confirmed this pro-survival role of PP2A in both genetic mouse ALL model and human Ph+ ALL-patients derived leukemia cells. These findings highlight PP2A as a therapeutic target and suggest that agents like the PP2A inhibitor LB-100 may be of interest for pre-clinical development and testing. DisclosuresNo relevant conflicts of interest to declare.

Distinct Metabolic Properties of MDS Stem Cells Provide Novel Opportunities for Therapeutic Intervention

This project is focused on characterizing the malignant stem cells that drive the pathogenesis of MDS, with the goal of developing improved therapeutic strategies. To this end, we have established methods by which MDS stem cells can be identified, isolated and characterized in xenograft models. These approaches have been employed to permit global metabolomic and transcriptomic analyses, which have subsequently led to modeling novel therapeutic regimens.Initial studies exploited previous work in acute myelogenous leukemia to identify candidate phenotypic markers of stem cell malignancy. These efforts demonstrated that up-regulation of CD123 in the hematopoietic stem cell compartment identifies MDS stem cells as they transition from low risk to high risk stages of pathogenesis. Thus, using cell sorting, we are able to isolate early (CD123-) vs. late (CD123+) stage stem cells from MDS patient specimens and subject them to the experimental analyses outlined below. We first performed a transcriptomic study that demonstrated a massive increase in cellular protein translation machinery through significant increases in ribosomal proteins as stem cells progress to advanced stages of MDS. To functionally validate these findings, MDS stem cell populations were cultured with the fluorescent protein substrate OP-puro, which detects newly synthesized polypeptide chains. CD123+ stem cells strongly increase protein synthesis levels (~13-fold increase). Given the established role of protein translation homeostasis in hematopoietic stem cells [Signer et al., 2014], this finding indicates a major change in cellular metabolism as well as a potential therapeutic entry point.To further characterize cellular changes occurring as MDS stem cells evolve, we performed global mass spectrometry-based metabolomic analyses which further demonstrates increased protein biosynthesis as well as altered glutathione metabolism (elevated oxidized glutathione). Taken together, these findings indicate major metabolic changes in MDS stem cells as they acquire increasing malignant phenotypes.Next, we investigated signaling related to increased ribosomal protein production. Specifically, we examined the hypothesis that ribosomal subunit binding of MDM2, may play a role in pathogenesis. Intriguingly, our data show that the specific subunits known to bind MDM2 are increased in CD123+ MDS stem cells. Consistent with this observation, MDM2 is elevated as well. This finding indicates that increased ribosomal protein levels may function to inhibit p53 activity, thereby enhancing pathogenic outgrowth of MDS stem cells.Using the mechanistic insights outlined above, we explored novel therapeutic strategies. First, we examined drugs known to selectively target metabolism through altering protein synthesis and targeting ribosomal proteins (e.g. homoharringtonine, HHT)). In addition, given the prevalent role of hypomethylating agents in current MDS treatment regimens, we also examined how these inhibitors interact with 5-azacytidine (5-aza). In vitro studies indicate that multiple protein synthesis inhibitors selectively eradicate MDS stem cells (CD123+). In addition, combination with 5-aza yielded additive to synergistic eradication of MDS stem cells. We have demonstrated that high risk MDS specimens effectively engraft the NSG-S strain of immune deficient mice, when T cell depletion and a busulfan conditioning regimen are employed. Using this model, we transplanted primary MDS specimens and challenged using the regimens above. Treatment with HHT demonstrated selective eradication of MDS stem cells, with a significant differential toxicity observed in multiple samples (50-60% selective ablation). Finally, analysis of protein synthesis inhibitors in combination with the hypomethylating agent 5-aza demonstrated potent efficacy in targeting the MDS stem cell population with greater then additive toxicity when compared to single agent treatment(70-80% selective ablation).Taken together, these data show that changes in metabolic properties represent a critical inflection point in the pathogenesis and progression of the MDS. Focusing on such changes, we have identified novel pharmacological approaches that may effectively target the MDS stem cell population. Importantly, these approaches function well in conjunction with commonly used agents used in the treatment of MDS. DisclosuresPollyea:Alexion: Other: advisory board; Celgene: Other: advisory board, Research Funding; Glycomimetics: Other: DSMB member; Ariad: Other: advisory board; Pfizer: Other: advisory board, Research Funding.

Up-regulation of ribosome biogenesis by MIR196A2 genetic variation promotes endometriosis development and progression.

Aberrant miRNA expression has been reported in endometriosis and miRNA gene polymorphisms have been linked to cancer. Because certain ovarian cancers arise from endometriosis, we genotyped seven cancer-related miRNA single nucleotide polymorphisms (MiRSNPs) to investigate their possible roles in endometriosis. Genetic variants in MIR196A2 (rs11614913) and MIR100 (rs1834306) were found to be associated with endometriosis development and related clinical phenotypes, such as infertility and pain. Downstream analysis of the MIR196A2 risk allele revealed upregulation of rRNA editing and protein synthesis genes, suggesting hyper-activation of ribosome biogenesis as a driving force for endometriosis progression. Clinical studies confirmed higher levels of small nucleolar RNAs and ribosomal proteins in atypical endometriosis lesions, and this was more pronounced in the associated ovarian clear cell carcinomas. Treating ovarian clear cells with CX5461, an RNA polymerase I inhibitor, suppressed cell growth and mobility followed by cell cycle arrest at G2/M stage and apoptosis. Our study thus uncovered a novel tumorigenesis pathway triggered by the cancer-related MIR196A2 risk allele during endometriosis development and progression. We suggest that anti-RNA polymerase I therapy may be efficacious for treating endometriosis and associated malignancies.

Changes in the Cytoplasmic Composition of Amino Acids and Proteins Observed in Staphylococcus aureus during Growth under Variable Growth Conditions Representative of the Human Wound Site.

Staphylococcus aureus is an opportunistic pathogen responsible for a high proportion of nosocomial infections. This study was conducted to assess the bacterial responses in the cytoplasmic composition of amino acids and ribosomal proteins under various environmental conditions designed to mimic those on the human skin or within a wound site: pH6-8, temperature 35–37°C, and additional 0–5% NaCl. It was found that each set of environmental conditions elicited substantial adjustments in cytoplasmic levels of glutamic acid, aspartic acid, proline, alanine and glycine (P< 0.05). These alterations generated characteristic amino acid profiles assessed by principle component analysis (PCA). Substantial alterations in cytoplasmic amino acid and protein composition occurred during growth under conditions of higher salinity stress implemented via additional levels of NaCl in the growth medium. The cells responded to additional NaCl at pH 6 by reducing levels of ribosomal proteins, whereas at pH 8 there was an upregulation of ribosomal proteins compared with the reference control. The levels of two ribosomal proteins, L32 and S19, remained constant across all experimental conditions. The data supported the hypothesis that the bacterium was continually responding to the dynamic environment by modifying the proteome and optimising metabolic homeostasis.

The role of ribosomal proteins in the regulation of cell proliferation, tumorigenesis, and genomic integrity.

Ribosomal proteins (RPs), the essential components of the ribosome, are a family of RNA-binding proteins, which play prime roles in ribosome biogenesis and protein translation. Recent studies revealed that RPs have additional extra-ribosomal functions, independent of protein biosynthesis, in regulation of diverse cellular processes. Here, we review recent advances in our understanding of how RPs regulate apoptosis, cell cycle arrest, cell proliferation, neoplastic transformation, cell migration and invasion, and tumorigenesis through both MDM2/p53-dependent and p53-independent mechanisms. We also discuss the roles of RPs in the maintenance of genome integrity via modulating DNA damage response and repair. We further discuss mutations or deletions at the somatic or germline levels of some RPs in human cancers as well as in patients of Diamond-Blackfan anemia and 5q- syndrome with high susceptibility to cancer development. Moreover, we discuss the potential clinical application, based upon abnormal levels of RPs, in biomarker development for early diagnosis and/or prognosis of certain human cancers. Finally, we discuss the pressing issues in the field as future perspectives for better understanding the roles of RPs in human cancers to eventually benefit human health.

Adaptive Remodeling of the Bacterial Proteome by Specific Ribosomal Modification Regulates Pseudomonas Infection and Niche Colonisation.

Post-transcriptional control of protein abundance is a highly important, underexplored regulatory process by which organisms respond to their environments. Here we describe an important and previously unidentified regulatory pathway involving the ribosomal modification protein RimK, its regulator proteins RimA and RimB, and the widespread bacterial second messenger cyclic-di-GMP (cdG). Disruption of rimK affects motility and surface attachment in pathogenic and commensal Pseudomonas species, with rimK deletion significantly compromising rhizosphere colonisation by the commensal soil bacterium P. fluorescens, and plant infection by the pathogens P. syringae and P. aeruginosa. RimK functions as an ATP-dependent glutamyl ligase, adding glutamate residues to the C-terminus of ribosomal protein RpsF and inducing specific effects on both ribosome protein complement and function. Deletion of rimK in P. fluorescens leads to markedly reduced levels of multiple ribosomal proteins, and also of the key translational regulator Hfq. In turn, reduced Hfq levels induce specific downstream proteomic changes, with significant increases in multiple ABC transporters, stress response proteins and non-ribosomal peptide synthetases seen for both ΔrimK and Δhfq mutants. The activity of RimK is itself controlled by interactions with RimA, RimB and cdG. We propose that control of RimK activity represents a novel regulatory mechanism that dynamically influences interactions between bacteria and their hosts; translating environmental pressures into dynamic ribosomal changes, and consequently to an adaptive remodeling of the bacterial proteome.